WO2008103327A2

WO2008103327A2 - Mn/ca ix and bladder cancer

Info

Publication number: WO2008103327A2
Application number: PCT/US2008/002131
Authority: WO
Inventors: Jan Zavada
Original assignee: Bayer Pharmaceuticals Corporation; Institute Of Organic Chemistry And Biochemistry; Institute Of Molecular Genetics
Priority date: 2007-02-20
Filing date: 2008-02-19
Publication date: 2008-08-28
Also published as: WO2008103327A3

Abstract

The present invention is directed to methods to detect the presence or recurrence of urinary tract cancer, particularly bladder cancer, comprising the use of an immunoassay to detect soluble MN/CA IX (s-CA IX) in a patient urine sample. The disclosed methods are diagnostic/prognostic for urinary tract diseases, and useful to monitor the status of a patient's disease, and/or to monitor how a patient is responding to an anticancer therapy. The disclosed methods are further useful to select therapies for patients with urinary tract diseases. MN/CA IX is particularly identified as a sensitive and specific urinary marker for surveillance of urinary tract cancer recurrence, more particularly bladder cancer recurrence.

Description

MN/CA IX AND BLADDER CANCER

FIELD OF THE INVENTION

The present invention is in the general area of medical genetics and in the fields of biochemical engineering, immunochemistry and oncology. More specifically, it relates to the MN gene - a cellular gene considered to be an oncogene, known alternatively as MWCA9, CA9, or carbonic anhydrase 9, which gene encodes the oncoprotein now known alternatively as the MN protein, the MN/CA IX isoenzyme, MN/CA IX, carbonic anhydrase IX, CA IX or the MN/G250 protein.

More specifically, the instant invention concerns diagnostic/prognostic methods for urinary tract cancer, particularly bladder cancer, comprising detecting or detecting and quantifying soluble MN/CA IX antigen (s-CA IX) in a patient urine sample. In particular, such diagnostic/prognostic and therapeutic methods can help clinicians to monitor recurrence of urinary tract cancer.

BACKGROUND OF THE INVENTION

As indicated above, the MN gene and protein are known by a number of alternative names, which names are used herein interchangeably. The MN protein was found to bind zinc and have carbonic anhydrase (CA) activity and is now considered to be the ninth carbonic anhydrase isoenzyme - MN/CA IX or CA IX [Opavsky et al. 1996]. According to the carbonic anhydrase nomenclature, the abbreviated names for human CA isoenzymes are written in capital letters and Roman numbers, whereas those of the corresponding genes are written in italic letters and Arabic numbers. Alternatively, "MN" is used herein to refer either to carbonic anhydrase isoenzyme IX (CA IX) proteins/polypeptides, or carbonic anhydrase isoenzyme 9 {CA9) gene, nucleic acids, cDNA, mRNA etc. as indicated by the context.

The MN protein has also been identified with the G250 antigen. Uemura et al., "Expression of Tumor-Associated Antigen MN/G250 in Urologic Carcinoma: Potential Therapeutic Target, " J. Urol.. 157 (4 Suppl.): 377 (Abstract 1475; 1997) states: "Sequence analysis and database searching revealed that G250 antigen is identical to MN, a human tumor-associated antigen identified in cervical carcinoma (Pastorek et al., 1994)."

CA IX is a cancer-related carbonic anhydrase identified by Zavada, Pastorekova, Pastorek (U.S. Patent 5,387,676) using the M75 monoclonal antibody first reported by Pastorekova et al. [Virology 187: 620-626 (1992)]. That antibody was employed in cloning of cDNA encoding CA IX [Pastorek et al., Oncogene, 9: 2788-2888 (1994)], in assessment of CA IX expression in tumors and normal tissues [Zavada et al., lnt J Cancer. 54: 268-274, (1993) and many other references], in study of CA IX regulation by cell density [Lieskovska et al., Neoplasma, 46: 17-24, (1999), Kaluz et al., Cancer Research. 62: 4469-4477, (2002)] as well in demonstration of CA IX induction by hypoxia [Wykoff et al., Cancer Research. 60: 7075-7083 (2000), and many other references]. All those studies supported the assumption made in the original U. S Patent 5,387,676 that CA IX can be used diagnostically and/or prognostically as a preneoplastic/neoplastic tumor marker and therapeutically as a target, and showed that the M75 monoclonal antibody is a valuable CA IX-specific reagent useful for different immunodetection methods and immunotargeting approaches.

Zavada et al., International Publication Number WO 93/18152 (published 16 September 1993) and U.S. Patent No. 5,387,676 (issued February 7, 1995), describe the discovery and biological and molecular nature of the MN gene and protein. The MN gene was found to be present in the chromosomal DNA of all vertebrates tested, and its expression to be strongly correlated with tumorigenicity. The MN protein was first identified in HeLa cells, derived from a human carcinoma of cervix uteri. It is found in many types of human carcinomas (notably uterine cervical, ovarian, endometrial, renal, bladder, breast, colorectal, lung, esophageal, and prostate, among others). Very few normal tissues have been found to express MN protein to any significant degree. Those MN-expressing normal tissues include the human gastric mucosa and gallbladder epithelium, particularly in the large bile ducts, and some other normal tissues of the alimentary tract. Paradoxically, MN gene expression has been found to be lost or reduced in carcinomas and other preneoplastic/neoplastic diseases in some tissues that normally express MN, e.g., gastric mucosa. MN is manifested in HeLa cells by a twin protein, p54/58N. lmmunoblots using a monoclonal antibody reactive with p54/58N (Mab M75) revealed two bands at 54 kDa and 58 kDa. Those two bands may correspond to one type of protein that most probably differs by post-translational processing. It is N- glycosylated with a single 3 kDa carbohydrate chain and under non-reducing conditions forms S-S-linked oligomers [Pastorekova et al., Virology, 187: 620-626 (1992); Pastorek et al., Oncogene. 9: 2788-2888 (1994)]. MN/CA IX is a transmembrane protein located at the cell surface, although in some cases it has been detected in the nucleus [Zavada et al., Int. J. Cancer. 54: 268-274 (1993); Pastorekova et al., supral.

In general, oncogenesis may be signified by the abnormal expression of MN protein. For example, oncogenesis may be signified: (1) when MN protein is present in a tissue which normally does not express MN protein to any significant degree; (2) when MN protein is absent from a tissue that normally expresses it; (3) when MN gene expression is at a significantly increased level, or at a significantly reduced level from that normally expressed in a tissue; or (4) when MN protein is expressed in an abnormal location within a cell.

Zavada et al., WO 93/18152 and Zavada et al., WO 95/34650 (published 21 December 1995) disclose how the discovery of the MN gene and protein and the strong association of MN gene expression and tumorigenicity led to the creation of methods that are both diagnostic/prognostic and therapeutic for cancer and precancerous conditions. Methods and compositions were provided therein for identifying the onset and presence of neoplastic disease by detecting or detecting and quantitating abnormal MN gene expression in vertebrates. Abnormal MN gene expression can be detected or detected and quantitated by a variety of conventional assays in vertebrate samples, for example, by immunoassays using MN-specific antibodies to detect or detect and quantitate MN antigen, by hybridization assays or by PCR assays, such as RT-PCR, using MN nucleic acids, such as, MN cDNA, to detect or detect and quantitate MN nucleic acids, such as, MN mRNA.

Zavada et al., WO 93/18152 and/or WO 95/34650 disclose the MN cDNA sequence (SEQ ID NO: 1) shown herein in Figure 1A-1C, the MN amino acid sequence (SEQ ID NO: 2) also shown in Figure 1A-1C, and the MN genomic sequence (SEQ ID NO: 3) shown herein in Figure 2A-2F. The MN gene is organized into 11 exons and 10 introns. The human MN cDNA sequence of SEQ ID NO: 1 contains 1522 base pairs (bp).

The first thirty seven amino acids of the MN protein shown in Figure 1A-1C (SEQ ID NO: 2) is the putative MN signal peptide (SP) [SEQ ID NO: 4]. The MN protein has an extracellular (EC) domain [amino acids (aa) 38-414 of Figure 1A- 1C (SEQ ID NO: 5)], a transmembrane (TM) domain [aa 415-434 (SEQ ID NO: 6)] and an intracellular (IC) domain [aa 435-459 (SEQ ID NO: 7)]. The extracellular domain contains the proteoglycan-like (PG) domain at about amino acids (aa) 53- 111 (SEQ ID NO. 8) or preferably at about aa 52-125 (SEQ ID NO: 25), and the carbonic anhydrase (CA) domain at about aa 135-391 (SEQ ID NO: 9) or preferably, at about aa 121-397 (SEQ ID NO: 26).

Zavada et al, WO 93/18152 and WO 95/34650 describe the production of MN-specific antibodies. A representative and preferred MN-specific antibody, the monoclonal antibody M75 (Mab M75), was deposited at the American Type Culture Collection (ATCC) in Manassus, VA (USA) under ATCC Number HB 11128. The M75 antibody was used to discover and identify the MN protein and can be used to identify readily MN antigen in Western blots, in radioimmunoassays and immunohistochemically, for example, in tissue samples that are fresh, frozen, or formalin-, alcohol-, acetone- or otherwise fixed and/or paraffin-embedded and deparaffinized. Another representative and preferred MN-specific antibody, Mab MN12, is secreted by the hybridoma MN 12.2.2, which was deposited at the ATCC under the designation HB 11647. Example 1 of Zavada et al., WO 95/34650 provides representative results from immunohistochemical staining of tissues using Mab M75, which results support the designation of the MN gene as an oncogene.

Immunodominant epitopes are considered to be essentially those that are within the PG domain of MN/CA IX, including the repetitive epitopes for the M75 Mab, particularly the amino acid sequence PGEEDLP (SEQ ID NO: 11 ), which is 4X identically repeated in the N-terminal PG region (Zavada et al. 2000). The epitope for the MN12 Mab is also immunodominant.

The M75 Mab was first reported in Pastorekova et al., Virology, 187: 620-626 (1992) and is claimed specifically, as well as generically with all MN/CA IX- specific antibodies, polyclonal and monoclonal as well as fragments thereof, in a number of U.S. and foreign patents, including, for example, Zavada et al., U.S. Patent No. 5,981 ,711 and EP 0 637 336 B1. [See also, Zavada et al., U.S. Patent Nos. 5,387,676; 5,955,075; 5,972,353; 5,989,838; 6,004,535; 6,051 ,226; 6,069,242; 6,093,548; 6,204,370; 6,204,887; 6,297,041 ; and 6,297,051 ; and Zavada et al., AU 669694; CA 2,131 ,826; DE 69325577.3; and KR 282284.] Those Zavada et al. U.S. and foreign patents are herein incorporated by reference.

CA IX is a highly active member of α carbonic anhydrase family of zinc metalloenzymes that catalyze the reversible conversion between carbon dioxide and bicarbonate [Pastorek et al. (1994); Opavsky et al. (1996); Chegwidden et al. (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), mitochondrion (CA VA, VB), secretory vesicles (CA Vl) and plasma membrane (CA IV, IX, XII, XIV). Some of the isozymes are distributed over broad range of tissues (CA I, II, CA IV), others are more restricted to particular organs (CA Vl in salivary glands) and two isoforms have been linked to cancer tissues (CA IX, XII) [reviewed in Chegwidden (2000); Pastorekova and Pastorek (2004)]. Enzyme activity and kinetic properties, as well as sensitivity to sulfonamide inhibitors vary 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, Xl) are acatalytic due to incompletely conserved active site. This extraordinary variability among the genetically related members of the same family of proteins creates a basis for their employment in diverse physiological and pathological processes. The catalytic activity is of fundamental relevance for the maintenance of acid-base balance and exchange of ions and water in metabolically active tissues. Via this activity, CAs substantially contribute to respiration, production of body fluids (vitreous humor, gastric juice, cerebrospinal fluid), bone resorption, renal acidification etc. (Chegwidden 2000).

CA IX isozyme integrates several properties that make it an important subject of basic as well as clinical research. First of all, expression of CA IX is very tightly associated with a broad variety of human tumors, while it is generally absent from the corresponding normal tissues [Zavada et al. (1993); Liao et al. (1994); Turner et al., 1997; Liao et al., 1997; Saarnio et al., 1998; Vermylen et al., 1999; Ivanov et al. (2001 ); Bartosova et al. (2002)]. Tumor hypoxia strongly activates transcription of CA9 gene via a hypoxia-inducible transcription factor (HIF), which binds to a hypoxia-response element (HRE) localized in the minimal CA9 promoter proximal to the transcription start site at the -10/-3 position [Wykoff et al. (2000)]. The HIF transcription factor significantly changes the expression profile of weakly oxygenated tumor cells by activation of genes that either support their survival and adaptation to hypoxic stress or lead to their death. As a result, hypoxia selects for more aggressive tumor cells with increased capability to invade and metastasize and is therefore inherently associated with bad prognosis and poor response to anticancer therapy [Harris (2002)].

Since tumor hypoxia is an important phenomenon with dramatic implications for cancer development and therapy [Hockel and Vaupel (2001 )], CA IX bears a significant potential as an intrinsic hypoxic marker with a prognostic/predictive value and as a promising therapeutic target [Wykoff et al. (2000); Wykoff et al. (2001 ); Beasley et al. (2001 ); Giatromanolaki et al. (2001 ); Koukourakis et al. (2001 ); Potter and Harris (2003)]. In favor of the proposed clinical applications, CA IX is an integral plasma membrane protein with a large extracellular part exposed at the surface of cancer cells and is thus accessible by the targeting tools, including the specific monoclonal antibodies. Furthermore, CA IX differs from the other CA isozymes by the presence of a unique proteoglycan-related region (PG) that forms an N-terminal extension of the extracellular CA domain and allows for elimination of cross-recognition with other isozymes [Opavsky et al. (1996)].

Moreover, CA IX appears to play an active role in tumor biology both via modulation of cell adhesion and control of pH [Svastova et al. (2003), Svastova et al. (2004), Swietach et al. (2007)]. CA IX participates in bicarbonate transport metabolon and contributes to acidification of extracellular microenvironment in response to hypoxia [Morgan et al. (2007), Svastova et al. (2004)]. In addition, CA IX's intracellular domain (IC) has a potential third tumorigenic role, at least in renal cell carcinoma: tyrosine-phosphorylated CA IX (mediated via EGFR) interacts with the regulatory subunit of PI-3K (p85), resulting in activation of Akt [Dorai et al. (2005)]. Because of its many potential activities contributing to oncogenesis, targeting the CA IX protein for abrogation of its function is expected to have therapeutic effects. Soluble MN/CA IX (s-CA IX) as a Cancer Marker

So far, most of the basic CA IX-related studies were performed using a single mouse monoclonal antibody M75 that recognizes the N-terminal PG region of CA IX [Pastorekova (1992), Zavada (2000)]. The M75 Mab proved to be highly specific and perfectly suitable for certain purposes including immunohistochemical analyzes of cancer tissue sections [Liao et al. (1994); Ivanov et al. (2001 ) and references therein], targeting hypoxic tumor cells in animal models [Chrastina et al. (2003)], CA IX immunodetection in vitro, and molecular characterization [Pastorek et al. (1994); Lieskovska et al. (1999); Kaluz et al. (2002)]. Zavada et al. WO2004/005348 describes immunoassays to detect/quantitate soluble MN/CA IX (s-CA IX) in human body fluids. CA IX-specific monoclonal antibodies such as the Mab V/10, with epitope specificity different from that of M75, were used to detect s-CA IX in the sera and urine of renal cell carcinoma (RCC) patients, based on capture-detection principle. One obstacle to detecting s-CA IX in blood and urine samples from RCC patients is that, whereas permanent tumor cell lines or short-term tumor explants shed an easily detectable amount of s-CA IX in vitro, the soluble antigen is rapidly cleared from the blood in vivo by absorption to unidentified deposits or by excretion in urine. As a consequence, s-CA IX concentration in the serum or urine of RCC patients is only a few picograms per ml, which is not readily detectable by current methods. In many patients with RCC, s-CA IX was undetectable in blood or urine in spite of the fact that almost 100% of the tumors expressed high levels of MN/CA IX. In contrast, other tumor markers like PSA or CEA reach in the sera of patients with carcinoma of prostate or colon, concentrations of several nanograms per ml and can be easily detected and quantitated.

The underlying hypothesis for the instant invention concerned the possibility that urine samples from patients with bladder carcinomas, particularly superficial transitional cell carcinomas (TCC), could be more suitable for assaying s- CA IX. Superficial TCC tumors are directly in contact with urine, and the s-CA IX shed therefrom should remain within the confines of the bladder until excreted with the urine.

TCC accounts for more than 9 out of 10 cases of bladder cancer. Many cases of TCC tend to remain confined to the lining of the bladder for long periods of time only superficially affecting the function of the bladder. A more invasive stage occurs when TCC spreads to the deeper layers of the bladder. Metastases can then follow. TCC is very treatable when it is diagnosed at the stage where it is still isolated in the bladder lining [http://www.ricancercouncil.org/cancer- info/bladder-cancer-facts. php]. TCC can be multifocal, intraepithelial, and sometimes very small; therefore, some of the tumors can escape endoscopic detection and surgical removal, increasing the danger of recurrence. Those characteristics of TCC warrant the monitoring of a marker protein in urine.

Bladder Cancer Markers

Bladder cancer is a common disease, being the second most common genitourinary cancer, with over 60,000 new cases in 2006 [Jemal et al., CA Cancer J Clin, 56: 106-130 (2006)]. Staging and grading currently are the most reliable variables for recurrence and progression [Burchardt et al. (2000)]. Clinical staging (T category) is established by bimanual examination at cystoscopy and biopsy under anaesthetic. Pathological staging (denoted by the prefix "p") is determined by the histopathologist after examination of the biopsy. Biopsies can be staged only up to stage pT2a, as staging higher than this requires a whole specimen from cystectomy. [D. Fawcett, "Bladder Cancer," in ABC of Urology, C. Dawson and Hugh N. Whitfield (eds.); Blackwell BMJ Books, 2^nd Edition (2006); pp. 29-33.]

Transitional cell carcinoma [TCC] accounts for >90% of bladder cancers. TCC is classified histopathologically into three types: superficial diseases (papillary tumors), superficially invasive tumors (pT1 , pTa tumors) and deep muscle invasive tumors (stages pT1-pT4) [Xie et al., 2005]. The majority (65-89%) of bladder cancer is superficial [stage Ta/T1/Tis] at presentation. Following transurethral resection (TUR or TURBT), anywhere from 50-90% of nonmuscle- invasive bladder cancer [NMIBC] will eventually recur during the course of a patient's life; however, only 10-30% of cases progress to a more advanced stage. The 5-year survival rates for noninvasive, locally invasive, and metastatic disease are roughly 94, 50 and 6%, respectively, driving the search for markers for the early detection of disease, either primary or recurrent [Nielsen et al., Curr Opin Urol, 16: 350-355 (2006)]. Treatment: Superficial tumors are easily treated by transurethral resection. Tumors that are considered to be high grade and/or invade the deeper layers of the bladder wall have a much greater potential for metastatic spread. Tumors invading into the detrusor muscle are treated with either radical cystectomy or concomitant chemotherapy and radiation. The benefit of neoadjuvant or adjuvant chemotherapy for locally advanced tumors remains unproven and controversial. [Kufe et al. (eds.); Cancer Medicine 6. BC Decker Inc. (2003); Section 29 (pt.110); available online at www.ncbi.nlm.nih.qov/books/bv.fcgi?rid^:=cmed61.

Detection: A combination of methods has become the established means of detecting bladder cancer, as no single procedure is 100% sensitive.

Exophytic lesions are readily identified with cystoscopy, but flat lesions, in particular carcinoma in situ (CIS), can be more difficult to detect. In addition, upper-tract urothelial cancers in the ureter or renal pelvis elude detection by cystoscopic examination alone. Since its introduction in 1945 by Papinicolau and Marshall, the microscopic examination of urinary cytology has been used as an adjunct to cystoscopy. Urinary cytology is highly specific for bladder cancer, with overall specificities of more than 90%. The sensitivity of cytology for low-grade lesions is much less, in the range of 40-60%.

Due to the expenses associated with lifelong monitoring and treatment of recurrences, bladder cancer is considered to be the most costly tumor type per patient in adults [Nielsen et al., 2006]. The discomfort and inconvenience to patients from frequent cystoscopies, and the low sensitivity and specificity of urine cytology for low-grade lesions, has driven an intensive search for urinary markers of recurrent bladder cancer. Dozens of candidate markers are being investigated for possible use in screening for, and surveillance of, bladder cancer. A recent International Consensus Panel on bladder tumor markers concluded that the most practical application of urine-based tests is in surveillance for recurrence [Lokeshwar et al., Urology. 66(6 Suppl. 1 ): 35-63 (2005)]. Potential cancer markers and assays include, among others, telomerase, bladder tumor antigen [BTA], bladder cancer antigen 4 [BCL4], cytokeratin 20, ImmunoCyt (a panel of three monoclonal antibodies to mucin and carcinoembryonic antigens associated with bladder cancer), nuclear matrix protein 22 [NMP22], microsatellite instability assays, fluorescent in- situ hybridization [FISH], fibrinogen degradation products, hyaluronic acid, etc. [recently reviewed in Mohammed et al., 2008; Nielsen et al., 2006; van Rhijn et al. 2005; Burchardt et al., 2000]. However, variable results are seen for the sensitivity and specificity of any given marker, and many show lower specificity than cytology when using controls with benign genitourinary conditions. Therefore, there is need in the art for a novel sensitive and specific urinary marker for surveillance of bladder cancer recurrence.

SUMMARY OF THE INVENTION

The instant invention is based on the discovery that some human patients with urinary tract cancer have been found to have elevated levels of soluble MN/CA IX (s-CA IX) in their urine prior to cancer recurrence. In one aspect, then, the instant invention is directed to the use of an MN immunoassay to screen for urinary cancer recurrence, preferably at an early stage of recurrence, comprising monitoring s-CA IX levels in patient urine samples. Exemplary of urinary tract cancers that are subject to the methods of the instant invention are the following: superficial bladder cancer, invasive bladder cancer, transitional cell carcinoma (TCC), squamous cell carcinoma (SCC), adenocarcinoma, sarcoma and small cell carcinoma, TCC of the urinary bladder, TCC of the renal pelvis, TCC of the ureter and TCC of the urethra. Levels of s-CA IX found in patient urine will be used to detect urinary tract cancer recurrence, assess prognosis of patients, guide therapies, predict the clinical outcomes of patients, and/or monitor the efficacy of therapies. MN-positive urinary tract cancer patients may receive conventional therapy, such as surgery, immunotherapy, chemotherapy and radiotherapy; MN-targeted monotherapy (such as Rencarex®, Wilex, Munich, Germany); or MN-targeted therapy combined with conventional therapies.

The immunoassays that are used according to the methods of the invention can be in standard formats, as for example, by an immunoassay that is selected from the group consisting of Western blots, enzyme-linked immunosorbent assays, radioimmunoassays, competition immunoassays, dual antibody sandwich assays, immunohistochemical staining assays, agglutination assays, and fluorescent immunoassays. A preferred format is a sandwich immunoassay, preferably a sandwich enzyme-linked immunosorbent assay (ELISA) or an equivalent assay. The immunoassays used according to the methods of the invention include CA IX- specific antibodies. Preferably, at least one of the CA IX-specific antibodies used in the immunoassays of the invention is labeled. A preferred immunoassay format to detect s-CA IX in human urine is a double antibody sandwich assay, using CA IX- specific antibodies directed to different regions of CA IX, preferably directed to the PG and/or CA domains. A preferred combination of CA IX-specific antibodies would be, for example, the M75 Mab to the PG domain and the V/10 Mab to the CA domain.

The s-CA IX is predominantly a twin band of 50/54 kDa on Western blot, and is considered to represent only the extracellular part of CA IX [EC domain]. The s-CA IX that can be detected or detected and quantitated can be bound by CA IX-specific antibodies to the EC domain of CA IX, preferably to the PG or CA domains, such as the M75 Mab or the V/10 Mab, respectively. The smaller form of the s-CA IX with a molecular weight of about 20 to about 30 kDa, is considered to lack the PG domain, so that form would preferably be detected and quantitated by CA IX-specific antibodies directed to CA domain, preferably to epitopes that are substantially separated on that domain, which separation may well depend on the conformational nature of the epitopes and the protein/polypeptide 3-D structure, that may differ from that of the full-length CA IX or EC domain thereof. It is soluble MN/CA IX (s-CA IX) which the exemplary assays of the instant invention are mainly detecting and quantifying (whether semi-quantitatively or quantitatively). However, one of skill in the art would know that the methods of the instant invention can also be adapted to detect/quantitate insoluble MN/CA IX, for example, that is bound to cells, present in a patient urine sample. Alternatively, the methods can be adapted and used to detect both s-CA IX and insoluble MN/CA IX present in a patient urine sample. The instant invention encompasses any method that detects s-CA IX and/or insoluble MN/CA IX in a urine sample of a urinary tract cancer patient. Further one of skill in the art could use conventional knowledge and routine experimentation to modify and optimize the exemplary methods of the invention.

In one aspect, the invention concerns diagnostic/prognostic methods to detect recurrence of bladder cancer in a human patient who has had an at least preliminary diagnosis of, and treatment for bladder cancer. One method of detecting said recurrence of bladder cancer in a human patient comprises the steps of:

(a) obtaining a urine sample from said patient, wherein said sample is collected after treatment for bladder cancer; (b) contacting said patient sample with one or more antibodies which specifically bind to MN/CA IX's extracellular domain; and

(c) detecting and quantifying binding of said one or more antibodies to MN/CA IX in said patient sample, and comparing the patient sample MN/CA IX level to MN/CA IX levels found in urine samples from normal humans, wherein if the level of MN/CA IX in said patient sample is above 95% of the MN/CA IX levels in the urine samples from said normal humans, that MN/CA IX level in said patient sample is considered to be elevated and to indicate recurrence of bladder cancer. Preferably, said patient urine sample and/or said normal urine samples are concentrated. Preferably said bladder cancer is selected from the group consisting of TCC, squamous cell carcinoma (SCC), adenocarcinoma, sarcoma and small cell carcinoma.

Said one or more antibodies are selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antigen binding antibody fragments comprising an MN/CA IX extracellular domain binding region, and recombinant antibodies. Preferably, said one or more antibodies are monoclonal antibodies.

More preferably, the antibodies are monoclonal, wherein one monoclonal antibody is specific for the carbonic anhydrase (CA) domain of MN/CA IX, and wherein the other monoclonal antibody is directed to the proteoglycan-like (PG) domain of MN/CA IX. Still more preferably, said one or more monoclonal antibodies are selected from the group consisting of the monoclonal antibodies designated M75 which are secreted by the hybridoma VU-M75, which was deposited at the American Type Culture Collection under ATCC No. HB 11128, and monoclonal antibodies that are not directed to the M75 epitope. Most preferably, the antibody directed to the CA domain is the V/10 monoclonal antibody which is produced by the hybridoma VU- V/10, which was deposited at BCCM™/LMBP in Ghent, Belgium under Accession No. LMBP 6009CB, and the antibody directed to the PG domain is the M75 monoclonal antibody that is secreted from the hybridoma VU-M75, which was deposited at the American Type Culture Collection under ATCC No. HB 11128. A second aspect of the invention concerns an immunoassay to detect recurrence of transitional cell carcinoma (TCC) of the urinary tract in a human patient who has had an at least preliminary diagnosis of, and treatment for TCC. One such method comprises the steps of: (a) obtaining a urine sample from said patient, wherein said sample is collected after treatment for TCC;

(b) contacting said patient sample with one or more antibodies which specifically bind to MN/CA IX's extracellular domain; and

(c) detecting and quantifying binding of said one or more antibodies to MN/CA IX in said patient sample, and comparing the patient sample MN/CA IX level to MN/CA IX levels found in urine samples from normal humans, wherein if the level of MN/CA IX in said patient sample is above 95% of the MN/CA IX levels in the urine samples from said normal humans, that MN/CA IX level in said patient sample is considered to be elevated and to indicate recurrence of TCC. Preferably, said TCC is selected from the group consisting of TCC of the urinary bladder, TCC of the renal pelvis, TCC of the ureter and TCC of the urethra.

Another embodiment of the invention concerns a method of detecting recurrence of bladder cancer in a human patient who has had an apparent spontaneous remission of bladder cancer. Said method of detecting recurrence of bladder cancer in a human patient who has had an apparent spontaneous remission of bladder cancer comprises the steps of:

(a) obtaining a urine sample from said patient;

(b) contacting said patient sample with one or more antibodies which specifically bind to MN/CA IX's extracellular domain; and (c) detecting and quantifying binding of said one or more antibodies to

MN/CA IX in said patient sample, and comparing the patient sample MN/CA IX level to MN/CA IX levels found in urine samples from normal humans, wherein if the level of MN/CA IX in said patient sample is above 95% of the MN/CA IX levels in the urine samples from said normal humans, that MN/CA IX level in said patient sample is considered to be elevated and to indicate recurrence of bladder cancer.

Still another aspect of the invention concerns a method of detecting recurrence of bladder cancer in a human patient who has had an at least preliminary diagnosis of, and treatment for bladder cancer. Said method of detecting bladder cancer recurrence in a bladder cancer patient comprises the steps of:

(c) detecting and quantifying binding of said one or more antibodies to MN/CA IX in said patient sample, and comparing the patient sample MN/CA IX level to MN/CA IX levels found in urine samples from normal humans, wherein if the level of MN/CA IX in said patient sample is above the upper limit of normal, that MN/CA IX level in said patient sample is considered to be elevated and to indicate recurrence of bladder cancer.

As indicated above, the methods of the invention can be detecting/quantifying MN/CA IX level(s) that is/are predominantly or solely soluble MN/CA IX (s-CA IX) level(s), but alternatively can also detect/quantify insoluble CA IX, for example, cell-bound MN/CA IX, in urine samples. In a preferred aspect, the methods of the invention detect/quantify s-CA IX levels.

In another embodiment, the invention concerns a method of monitoring the status of bladder cancer in a patient, and/or monitoring how a patient with said bladder cancer is responding to a therapy, comprising immunologically detecting and quantifying serial changes in soluble MN/CA IX (s-CA IX) levels in urine samples taken from said patient over time; wherein increasing urine levels of s-CA IX over time indicate disease progression or a negative response to said therapy, and wherein decreasing urine levels of s-CA IX over time indicate disease remission or a positive response to said therapy. In an alternative embodiment, said method of monitoring the status and/or response to therapy of a bladder cancer patient comprises immunologically detecting and quantifying serial changes in soluble MN/CA IX (s-CA IX) levels in patient urine samples relative to s-CA IX levels in urine samples taken from normal humans; wherein patient urine levels of s-CA IX above 95% of the s-CA IX levels in the urine samples from said normal humans indicate disease progression or a negative response to said therapy, and wherein patient urine levels of s-CA IX at or below 95% of the s-CA IX levels in the urine samples from said normal humans indicate disease remission or a positive response to said therapy. Exemplary therapies include those selected from the group consisting of surgery, immunotherapy, chemotherapy and radiotherapy.

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The above-listed articles and the references cited therein are hereby incorporated by reference.

Abbreviations The following abbreviations are used herein: aa - amino acid

AP - alkaline phosphatase

ATCC - American Type Culture Collection

BCL4 - bladder cancer antigen 4 BL - bioluminescence bp - base pairs

BTA - bladder tumor antigen

CA - carbonic anhydrase

⁰C - degrees centigrade CL - chemiluminescence

EC - ectodomain

ECL - enhanced chemiluminescence

EGFR - epidermal growth factor receptor

ELISA - enzyme-linked immunosorbent assay FISH - fluorescent in-situ hybridization

FR - framework region

GST-MN - fusion protein MN glutathione S-transferase h - hour he-eo hematoxylin-eosin

HIF hypoxia-inducible factor

HRE hypoxia response element

HRP horseradish peroxidase

IC intracellular

IHC immunohistochemistry kDa or kd kilodaltons

M molar

Mab monoclonal antibody min. minute(s) mg milligram ml milliliter ng nanogram nm nanometer

NMIBC nonmuscle-invasive bladder cancer

NMP22 nuclear matrix protein 22 nt nucleotide

ORF open reading frame

PCR polymerase chain reaction

PG proteoglycan

PI-3K phosphotidylinositol-3-kinase pNPP paranitrophenyl phosphate pg picogram

Pl isoelectric point

PT pathological staging pTa pathological stage of bladder cancer; papillary noninvasive carcinoma (superficial) pT1 pathological stage of bladder cancer; invasion of the suburothelial stroma (superficial/invasive) pTis pathological stage of bladder cancer; flat carcinoma in situ

PVDF polyvinylidene difluoride

RCC renal cell carcinoma

RT-PCR reverse transcription polymerase chain reaction S-CA IX soluble form of MN/CA IX

SCC squamous cell carcinoma

SD standard deviation(s)

SDS sodium dodecyl sulfate

SP signal peptide

TC tissue culture

TCC transitional cell carcinoma

TM transmembrane

TMB tetramethyl benzidine

TUR or TURBT - transurethral resection μg microgram

ULN upper limit of normal

VEGF vascular endothelial growth factor

WB Western blot

Cell Lines

CGL3 tumorigenic HeLa x normal fibroblast hybrid cells (HeLa D98/AH.2 derivative; express CA9, but level increased by both high density and hypoxia);

HeLa aneuploid, epithelial-like cell line isolated from a human cervical adenocarcinoma [Gey et al., Cancer Res., 12: 264 (1952); Jones et al., Obstet. Gynecol.. 38: 945-949 (1971 )] obtained from Professor B. Korych, [Institute of Medical Microbiology and Immunology, Charles University; Prague, Czech Republic]; and

HT29 A cell line derived from colorectal carcinoma. (ATCC No. HBT-38; DSMZ ACC299).

Nucleotide and Amino Acid Sequence Symbols

The following symbols are used to represent nucleotides herein: Base

Symbol Meaning

A adenine

C cytosine G guanine

T thymine

U uracil

I inosine

M A or C R A or G

W A or T/U

S C or G

Y C or T/U

K G or T/U V A or C or G

H A or C or T/U

D A or G or T/U

B C or G or T/U

N/X A or C or G or T/U

There are twenty main amino acids, each of which is specified by a different arrangement of three adjacent nucleotides (triplet code or codon), and which are linked together in a specific order to form a characteristic protein. A three- letter or one-letter convention is used herein to identify said amino acids, as, for example, in Figure 1 as follows:

3 Ltr. 1 Ltr.

Amino acid name Abbrev. Abbrev

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic Acid Asp D

Cysteine Cys C

Glutamic Acid GIu E Glutamine GIn Q

Glycine GIy G

Histidine His H lsoleucine Ne I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine VaI V

Unknown or other X

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A-C provides the nucleotide sequence for a MN cDNA [SEQ ID NO: 3] clone. Figure 1A-C also sets forth the predicted amino acid sequence [SEQ ID NO: 4] encoded by that MN cDNA.

Figure 2A-F provides a 10,898 bp complete genomic sequence of MN [SEQ ID NO: 5]. The base count is as follows: 2654 A; 2739 C; 2645 G; and 2859 T. The 11 exons are in general shown in capital letters, but exon 1 is considered to begin at position 3507 as determined by RNase protection assay.

Figure 3 schematically represents the MN protein structure. The abbreviations of individual MN/CA IX protein domains are as follows: PG (proteoglycan-like domain), CA (carbonic anhydrase domain), TM (transmembrane anchor) and IC (intracytoplasmic tail). The scale indicates the number of amino acids.

Figure 4 shows detection of s-CA IX in urine and sera by Western blot analysis. Lanes A, B, C, E, F, 35, 36, 37, 38, 49, 50, 51 , 55, 56, 57 = urine of TCC patients; C1 , C2, C3 = urine of healthy controls; 51 , 52, 53, 54 = serum of RCC patients. (Patient 51 had a combination of both RCC and TCC).

Figure 5 provides a graph showing distribution of s-CA IX level in urine of TCC patients and of controls.

DETAILED DESCRIPTION

The present invention is directed to immunoassays that measure the levels of MN/CA IX, particularly soluble CA IX (s-CA IX) in human urine, and/or serial changes in the levels of MN/CA IX, particularly s-CA IX in human urine, which assays are useful diagnostically/prognostically to detect or monitor a urinary tract preneoplastic/neoplastic disease in a human, particularly to detect recurrence of such a disease, or to select a therapy for a patient with such a urinary tract preneoplastic/neoplastic disease. Exemplary of urinary tract neoplastic diseases are the following, as well as precancers leading to the following: superficial bladder cancer, invasive bladder cancer, transitional cell carcinoma (TCC), squamous cell carcinoma (SCC), adenocarcinoma, sarcoma and small cell carcinoma, TCC of the urinary bladder, TCC of the renal pelvis, TCC of the ureter and TCC of the urethra. In particular, the levels of MN/CA IX, particularly s-CA IX in patient urine can be used to predict clinical outcome and/or as an aid in therapy selection.

The assays of this invention can be diagnostic and/or prognostic, i.e., diagnostic/prognostic. The term "diagnostic/ prognostic" is herein defined to encompass the following processes either individually or cumulatively depending upon the clinical context: determining the presence of disease, determining the nature of a disease, distinguishing one disease from another, forecasting as to the probable outcome of a disease state, determining the prospect as to recovery from a disease as indicated by the nature and symptoms of a case, monitoring the disease status of a patient, monitoring a patient for recurrence of disease, and/or determining the preferred therapeutic regimen for a patient. The diagnostic/prognostic methods of this invention are useful, for example, for screening populations for the presence of neoplastic or preneoplastic disease, determining the risk of developing neoplastic disease, diagnosing the presence of neoplastic and/or preneoplastic disease, monitoring the disease status of patients with neoplastic disease, and/or determining the prognosis for the course of neoplastic disease.

The present invention is useful for screening for the presence of a variety of urinary tract preneoplastic/neoplastic diseases. Such an assay can be used to detect tumors, detect recurrence of tumors, monitor their growth, and help in the diagnosis and prognosis of disease. The assays can also be used to detect potentially the presence of cancer metastasis, as well as confirm the absence of tumor tissue following cancer treatment, such as TUR (TURBT), chemotherapy and/or radiation therapy. The assays can further be used to monitor cancer chemotherapy and tumor reappearance.

The methods include quantifying MN/CA IX, particularly s-CA IX, if any, present in serial urine samples taken from a human subject diagnosed with, or suspected of having, a urinary tract preneoplastic/neoplastic disease. The quantified MN/CA IX, particularly s-CA IX levels are compared with the average levels in urine samples taken from individuals of a control population, wherein an above average level of MN/CA IX, particularly s-CA IX is indicative of abnormal MN/CA IX expression. As used herein, an "above average" level of MN/CA IX, particularly s- CA IX indicates a level higher than 95% of such levels in the urine samples from said control population, or alternatively, a level higher than two standard deviations (SD) above the mean level found in control samples (Upper Limit of Normal, or ULN). The individuals of the control population can be of either gender, or can be restricted to those who are of the same gender as the subject.

As used herein, "cancerous" and "neoplastic" have equivalent meanings, and "precancerous" and "preneoplastic" have equivalent meanings. The use of detection of gene expression products of oncogenes as diagnostic/prognostic indicators for preneoplastic/neoplastic diseases is considered conventional by those of skill in the art. However, the application of such approaches to measure MN/CA IX, particularly s-CA IX levels in urine, or serial changes in MN/CA IX, particularly s-CA IX levels in urine, to detect or monitor a urinary tract preneoplastic/neoplastic disease, particularly bladder cancer, and more particularly the recurrence of bladder cancer is new. Urinary Tract Preneoplastic/Neoplastic Tissues

The preneoplastic/neoplastic diseases that are the subject to the methods of the invention comprise any urinary tract preneoplastic/neoplastic disease characterized by abnormal MWCA9 gene expression. Exemplary preneoplastic/neoplastic diseases include at the least urinary tract preneoplastic/neoplastic diseases selected from the group consisting of superficial bladder cancer or invasive bladder cancer, and preneoplastic/neoplastic diseases of the renal pelvis, ureter, and urethra. Preferably said bladder cancer is selected from the group consisting of transitional cell carcinoma (TCC), squamous cell carcinoma (SCC), adenosquamous cell, adenocarcinoma, sarcoma and small cell carcinoma. Preferably, said TCC is selected from the group consisting of TCC of the urinary bladder, TCC of the renal pelvis, TCC of the ureter and TCC of the urethra.

MN Gene and Protein The terms "CA IX" and "MWCA9" are herein considered to be synonyms for MN. Also, the G250 antigen is considered to refer to MN protein/polypeptide [Jiang et al., PNAS (USA) 97: 1749-173 (2000)].

Zavada et al., WO 93/18152 and/or WO 95/34650 disclose the MN cDNA sequence shown herein in Figures 1A-1C [SEQ ID NO: 1], the MN amino acid sequence [SEQ ID NO: 2] also shown in Figures 1A-1 C, and the MN genomic sequence [SEQ ID NO: 3]. The MN gene is organized into 11 exons and 10 introns. The ORF of the MN cDNA shown in Figure 1 has the coding capacity for a 459 amino acid protein with a calculated molecular weight of 49.7 kDa. The overall amino acid composition of the MN protein is rather acidic, and predicted to have a pi of 4.3. Analysis of native MN protein from CGL3 cells by two-dimensional electrophoresis followed by immunoblotting has shown that in agreement with computer prediction, the MN is an acidic protein existing in several isoelectric forms with pis ranging from 4.7 to 6.3.

The first thirty seven amino acids of the MN protein shown in Figures 1A-1 C is the putative MN signal peptide [SEQ ID NO: 4]. The MN protein has an extracellular domain [amino acids (aa) 38-414 of Figures 1A-1 C [SEQ ID NO: 5], a transmembrane domain [aa 415-434; SEQ ID NO: 6] and an intracellular domain [aa 435-459; SEQ ID NO: 7]. The extracellular domain contains the proteoglycan-like domain [aa 53-111 : SEQ ID NO: 8; or preferably, aa 52-125: SEQ ID NO: 25] and the carbonic anhydrase (CA) domain [aa 135-391 ; SEQ ID NO: 9; or preferably, aa 121-397: SEQ ID NO: 26].

MN Gene -- Cloning and Sequencing

Figure 1A-C provides the nucleotide sequence for a full-length MN cDNA clone [SEQ ID NO: 1]. A complete MN genomic sequence is represented by SEQ ID NO: 3, and the nucleotide sequence for a proposed MN promoter is represented by SEQ ID NO: 24. It is understood that because of the degeneracy of the genetic code, that is, that more than one codon will code for one amino acid [for example, the codons TTA, TTG, CTT, CTC, CTA and CTG each code for the amino acid leucine (leu)], that variations of the nucleotide sequences in, for example, SEQ ID NOS: 1 and 3 wherein one codon is substituted for another, would produce a substantially equivalent protein or polypeptide according to this invention. All such variations in the nucleotide sequences of the MN cDNA and complementary nucleic acid sequences are included within the scope of this invention.

It is further understood that the nucleotide sequences herein described and shown in Figure 1 represent only the precise structures of the cDNA, genomic and promoter nucleotide sequences isolated. It is expected that slightly modified nucleotide sequences will be found or can be modified by techniques known in the art to code for substantially similar or homologous MN proteins and polypeptides, for example, those having similar epitopes, and such nucleotide sequences and proteins/ polypeptides are considered to be equivalents for the purpose of this invention.

DNA or RNA having equivalent codons is considered within the scope of the invention, as are synthetic nucleic acid sequences that encode proteins/polypeptides homologous or substantially homologous to MN proteins/polypeptides, as well as those nucleic acid sequences that would hybridize to said exemplary sequences [SEQ. ID. NOS. 1 , 3 and 24] under stringent conditions, or that, but for the degeneracy of the genetic code would hybridize to said cDNA nucleotide sequences under stringent hybridization conditions. Modifications and variations of nucleic acid sequences as indicated herein are considered to result in sequences that are substantially the same as the exemplary MN sequences and fragments thereof.

Only very closely related nt sequences having a homology of at least 80-90%, or at least 90%, would hybridize to each other under stringent conditions. A sequence comparison of the MN cDNA sequence shown in Figure 1 and a corresponding cDNA of the human carbonic anhydrase Il (CA II) showed that there are no stretches of identity between the two sequences that would be long enough to allow for a segment of the CA Il cDNA sequence having 25 or more nucleotides to hybridize under stringent hybridization conditions to the MN cDNA or vice versa. Stringent hybridization conditions are considered herein to conform to standard hybridization conditions understood in the art to be stringent. For example, it is generally understood that stringent conditions encompass relatively low salt and/or high temperature conditions, such as provided by 0.02 M to 0.15 M NaCI at temperatures of 5O⁰C to 7O⁰C. Less stringent conditions, such as, 0.15 M to 0.9 M salt at temperatures ranging from 2O⁰C to 55⁰C can be made more stringent by adding increasing amounts of formamide, which serves to destabilize hybrid duplexes as does increased temperature.

Exemplary stringent hybridization conditions are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, pages 1.91 and 9.47-9.51 [Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N. Y.; (1989)]; Maniatis et al., Molecular Cloning: A Laboratory Manual, pages 387-389 [Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1982)]; Tsuchiya et al., Oral Surgery. Oral Medicine. Oral Pathology. 71(6): 721-725 (June 1991 ); and in U.S. Pat. No. 5,989,838, U.S. Pat. No. 5,972,353, U.S. Pat. No. 5,981 ,711 , and U.S. Pat. No. 6,051 ,226.

Plasmids containing the MN genomic sequence (SEQ ID NO: 3) - the A4a clone and the XE1 and XE3 subclones -- were deposited at the American Type Culture Collection (ATCC) on June 6, 1995, respectively under ATCC Deposit Nos. 97199, 97200, and 97198.

MN Proteins and Polypeptides

The phrase "MN proteins and/or polypeptides" (MN proteins/ polypeptides) is herein defined to mean proteins and/or polypeptides encoded by an MN gene or fragments thereof. An exemplary and preferred MN protein according to this invention has the deduced amino acid sequence shown in Figure 1. Preferred MN proteins/polypeptides are those proteins and/or polypeptides that have substantial homology with the MN protein shown in Figure 1. For example, such substantially homologous MN proteins/polypeptides are those that are reactive with the MN-specific antibodies, preferably the Mab M75 or its equivalent, and/or the V/10 Mab or its equivalent.

A "polypeptide" or "peptide" is a chain of amino acids covalently bound by peptide linkages and is herein considered to be composed of 50 or less amino acids. A "protein" is herein defined to be a polypeptide composed of more than 50 amino acids. The term polypeptide encompasses the terms peptide and oligopeptide.

It can be appreciated that a protein or polypeptide produced by a neoplastic cell in vivo could be altered in sequence from that produced by a tumor cell in cell culture or by a transformed cell. Thus, MN proteins and/or polypeptides which have varying amino acid sequences including without limitation, amino acid substitutions, extensions, deletions, truncations and combinations thereof, fall within the scope of this invention. It can also be appreciated that a protein extant within body fluids is subject to degradative processes, such as, proteolytic processes; thus, MN proteins that are significantly truncated and MN polypeptides may be found in body fluids, such as, sera. The phrase "MN antigen" is used herein to encompass MN proteins and/or polypeptides.

It will further be appreciated that the amino acid sequence of MN proteins and polypeptides can be modified by genetic techniques. One or more amino acids can be deleted or substituted. Such amino acid changes may not cause any measurable change in the biological activity of the protein or polypeptide and result in proteins or polypeptides which are within the scope of this invention, as well as, MN muteins.

Soluble CA IX (s-CA IX)

The soluble CA IX (s-CA IX) found in vertebrate body fluids, preferably mammalian body fluids, more preferably human body fluids, has a molecular weight of from about 10 kilodaltons (kDa) to about 65 kDa, preferably from about 15 kDa to about 54 kDa, more preferably from about 20 kDa to about 54 kDa; still more preferably said s-CA IX has a molecular weight of either from about 15 kDa to about 35 kDa or from about 45 kDa to about 54 kDa, more preferably either from about 20 kDa to about 30 kDa or from about 50 kDa to about 54 kDa, and most preferably said s-CA IX has a molecular weight predominantly as a twin protein having a molecular weight of about 50/54 kDa as approximated from Western blotting.

The s-CA IX found in body fluids and in the culture medium of tumor cell lines, e.g., HT29, is primarily seen on Western blots as a twin band of 50/54 kDa. The other major form of CA IX is the cell associated, transmembrane protein seen on Western blot as a twin bond of 54/58 kDa. The s-CAIX found in body fluids and tissue culture (TC) media is considered to be the CA IX extracellular portion released by proteolytic cleavage from the transmembrane (TM) and intracellular (IC) domains. The s-CA IX is predominantly a twin band of 50/54 kDa on Western blot, is considered to represent only the extracellular part of CA IX, comprising the proteoglycan-like (PG) domain and the carbonic anhydrase (CA) domain (see Figure 3). The complete cell-associated CA IX, predominantly a twin band of 54/58 kDa on Western blot, further comprises a transmembrane (TM) region and an intracellular (IC) anchor.

The s-CA IX also exists in other smaller forms, preferably 20-30 kDa, which is considered to comprise the CA domain or parts thereof. Higher molecular weight species of the s-CA IX of about 62 kDa have been seen in the body fluids of cancer patients, but such species are considered to be rare and perhaps aberrant, as conjoined with other molecular species, in view of the theoretical molecular weight of the CA IX extracellular (EC) domain being about 50/54 kDa. The s-CA IX is considered to be a diagnostic/prognostic marker of many different cancers. A preferred diagnostic/prognostic use for s-CA IX is considered to be to monitor patients after surgical removal of a tumor, and to make decisions about the optimal method for therapy. The CA IX-specific antibodies that are directed to the non-immunodominant epitopes alone or in combination with CA IX-specific antibodies to the immunodominant epitopes, such as that of the M75 Mab, are considered important to detect all forms of s-CA IX, which are at low concentrations in body fluids. Immunoassay Test Kits

The above outlined assays can be embodied in test kits to detect and/or quantitate soluble CA IX antigen. Kits to detect and/or quantitate soluble CA IX antigen can comprise CA IX protein(s)/polypeptides(s). Such diagnostic/prognostic test kits can comprise one or more sets of antibodies, polyclonal and/or monoclonal, for a sandwich format wherein antibodies recognize epitopes on the soluble CA IX antigen, and one set is appropriately labeled or is otherwise detectable.

Test kits for an assay format wherein there is competition between a labeled (or otherwise detectable) CA IX protein/polypeptide and CA IX antigen in the sample, for binding to an antibody, can comprise the combination of the labeled protein/polypeptide and the antibody in amounts which provide for optimum sensitivity and accuracy.

A kit for use in an enzyme-immunoassay typically includes an enzyme- labelled reagent and a substrate for the enzyme. The enzyme can, for example, bind either a CA IX-specific antibody of this invention or to an antibody to such an CA IX-specific antibody. Test kits may comprise other components as necessary, for example, to perform a preferred assay as outlined in the Examples below. Such test kits can have other appropriate formats for conventional assays.

MN-Specific Antibodies

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. Antibodies useful according to the methods of the invention may be prepared by conventional methodology and/or by genetic engineering. Preferred antibodies according to the invention are pairs of antibodies that bind to different domains of MN/CA IX, preferably to the CA and PG domains, or preferably to sufficiently spatially separated regions on said domains or domain, for example, on the CA domain. Most preferred are the antibodies used in the exemplary immunoassay of the invention, the V/10 and M75 antibodies, or antibodies produced by subclones of the V/10-VU and VU-M75 hybridomas, so long as they exhibit the desired biological activity.

"Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or variable domain thereof. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; biospecific antibodies; and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature. 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-628 (1991 ) and Marks et al.. J. MoI. Biol.. 222: 581-597 (1991 ), for example.

The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity [U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. ScL USA, 81.: 6851-6855 (1984)].

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. Such modifications are made to refine further antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 :522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Qp. Struct. Biol.. 2: 593-596 (1992).

"Single-chain Fv" or "sFv" antibody fragments comprise the V_H and V_L domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V_H and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V_H) connected to a light chain variable domain (V_L) in the same polypeptide chain (V_H- VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161 ; and Hollinger et al., Proc. Natl. Acad. ScL, USA, 90: 6444-6448 (1993).

The expression "linear antibodies" refers to the antibodies described in Zapata et al., Protein Enq.. 8(10): 1057-1062 (1995). Briefly, such antibodies comprise a pair of tandem Fd segments (V_H-CH1 -VH-C_H1 ) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. Representative monoclonal antibodies useful according to this invention include Mabs M75, MN9, MN12 and MN7 described in earlier Zavada et al. patents and patent applications. [US Pat. No. 6,297,041 ; US Pat. No. 6,204,370; US Pat. No. 6,093,548; US Pat. No. 6,051 ,226; US Pat. No. 6,004,535; US Pat. No. 5,989,838; US Pat. No. 5,981 ,711 ; US Pat. No. 5,972,353; US Pat. No. 5,955,075; US Pat. No.5,387,676; US Application Nos: 20050031623, 20030049828, and 20020137910; and International Publication No. WO 03/100029]. Monoclonal antibodies useful according to this invention serve to identify MN proteins/ polypeptides in various laboratory prognostic tests, for example, in clinical samples. For example, monoclonal antibody M75 (Mab M75) is produced by mouse lymphocytic hybridoma VU-M75, which was deposited under ATCC designation HB 1 1128 on September 17, 1992 at the American Tissue Type Culture Collection [ATCC]. The production of hybridoma VU-M75 is described in Zavada et al., International Publication No. WO 93/18152. Mab M75 recognizes both the nonglycosylated GST-MN fusion protein and native MN protein as expressed in CGL3 cells equally well. The M75 Mab recognizes both native and denatured forms of the MN protein [Pastorekova et al., Virology. 187: 620-626 (1992)]. General texts describing additional molecular biological techniques useful herein, including the preparation of antibodies include Berger and Kimmel, Guide to Molecular Cloning Techniques. Methods in Enzvmoloqy. Vol. 152, Academic Press, Inc., Sambrook et al., Molecular Cloning: A Laboratory Manual, [Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y. (1989)] Vol. 1-3; Current Protocols in Molecular Biology. F. M. Ausabel et al. [Eds.], Current Protocols, a joint venture between Green Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 2000), Harlow et al., Monoclonal Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press (1988), Paul [Ed.]; Fundamental Immunoloqy.Lippincott Williams & Wilkins (1998), and Hariow et al., Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press (1998).

Labels

The antibodies useful according to this invention to identify MN/CA IX proteins can be labeled in any conventional manner. A preferred label, according to this invention is horseradish peroxidase, and a preferred method of labeling the antibodies is by using biotin-strepavidin complexes.

As appropriate, antibodies used in the immunoassays of this invention that are used as tracers may be labeled in any manner, directly or indirectly, that results in a signal that is visible or can be rendered visible. Detectable marker

3 125 131 substances include radionuclides, such as H, I, and I; fluorescers, such as, fluorescein isothiocyanate and other fluorochromes, phycobiliproteins, phycoerythin, rare earth chelates, Texas red, dansyl and rhodamine; colorimetric reagents (chromogens); electron-opaque materials, such as colloidal gold; bioluminescers; chemiluminescers; dyes; enzymes, such as, horseradish peroxidase, alkaline phosphatases, glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesteMN/CA IXe, alpha -, beta-galactosidase, among others; coenzymes; enzyme substrates; enzyme cofactors; enzyme inhibitors; enzyme subunits; metal ions; free radicals; or any other immunologically active or inert substance which provides a means of detecting or measuring the presence or amount of immunocomplex formed. Exemplary of enzyme substrate combinations are horseradish peroxidase and tetramethyl benzidine (TMB), and alkaline phosphatases and paranitrophenyl phosphate (pNPP).

Another preferred detection, or detection and quantitation systems according to this invention produce luminescent signals, bioluminescent (BL) or chemiluminescent (CL). In chemiluminescent (CL) or bioluminescent (BL) assays, the intensity or the total light emission is measured and related to the concentration of the unknown analyte. Light can be measured quantitatively using a luminometer (photomultiplier tube as the detector) or charge-coupled device, or qualitatively by means of photographic or X-ray film. The main advantages of using such assays is their simplicity and analytical sensitivity, enabling the detection and/or quantitation of very small amounts of analyte.

Exemplary luminescent labels are acridinium esters, acridinium sulfonyl carboxamides, luminol, umbelliferone, isoluminol derivatives, photoproteins, such as aequorin, and luciferases from fireflies, marine bacteria, Varqulla and Renilla.

Luminol can be used optionally with an enhancer molecule, preferably selected from the group consisting of 4-iodophenol or 4-hydroxy-cinnamic acid. Acridinium esters are one of the preferred types of CL labels according to this invention. Typically, a CL signal is generated by treatment with an oxidant under basic conditions. Also preferred luminescent detection systems are those wherein the signal (detectable marker) is produced by an enzymatic reaction upon a substrate. CL and BL detection schemes have been developed for assaying alkaline phosphatases (AP), glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase (HRP), and xanthine-oxidase labels, among others. AP and HRP are two preferred enzyme labels which can be quantitated by a range of CL and BL reactions. For example, AP can be used with a substrate, such as an adamantyl 1 ,2-dioxetane aryl phosphate substrate (e.g. AMPPD or CSPD; [Kricka, L. J., "Chemiluminescence and Bioluminescence, Analysis by," at p. 167, Molecular Biology and Biotechnology: A Comprehensive Desk Reference (ed. R.A. Meyers) (VCH Publishers; N.Y., N.Y.; 1995)]; preferably a disodium salt of

4-methoxy-4-(3-phosphatephenyl) spiro [1 ,2-dioxetane-3,2'-adamantane], with or without an enhancer molecule, preferably, 1-(trioctylphosphonium methyl)-4- (tributylphosphonium methyl) benzene diochloride. HRP is preferably used with substrates, such as, 2',3',6'- trifluorophenyl 3-methoxy- 10-methylacridan-9-carboxylate.

CL and BL reactions can be adapted for analysis not only of enzymes, but also of other substrates, cofactors, inhibitors, metal ions and the like. For example, luminol, firefly luciferase, and marine bacterial luciferase reactions are indicator reactions for the production or consumption of peroxide, ATP, and NADPH, respectively. They can be coupled to other reactions involving oxidases, kinases, and dehydrogenases, and can be used to measure any component of the coupled reaction (enzyme, substrate, cofactor). The detectable marker may be directly or indirectly linked to an antibody used in an assay of this invention. Exemplary of an indirect linkage of the detectable label is the use of a binding pair between an antibody and a marker or the use of a signal amplification system. Exemplary of binding pairs that can be used to link antibodies of assays of this invention to detectable markers are biotin/avidin, streptavidin, or anti-biotin; avidin/anti-avidin; thyroxine/thyroxine-binding globulin; antigen/antibody; antibody/ anti-antibody; carbohydrate/lectins; hapten/anti-hapten antibody; dyes and hydrophobic molecules/hydrophobic protein binding sites; enzyme inhibitor, coenzyme or cofactor/enzyme; polynucleic acid/homologous polynucleic acid sequence; fluorescein/anti- fluorescein; dinitrophenol/anti-dinitrophenol; vitamin B12/intrinsic factor; cortisone, cortisol/cortisol binding protein; and ligands for specific receptor protein/membrane associated specific receptor proteins. Preferred binding pairs according to this invention are biotin/avidin or streptavidin, more preferably biotin/ streptavidin.

Various means for linking labels directly or indirectly to antibodies are known in the art. For example, labels may be bound either covalently or non-covalently. Exemplary antibody conjugation methods are described in: Avarmeas et al., Scan. J. Immunol.. 8 (Suppl. 7): 7 (1978); Bayer et al., Meth. EnzymoL 62: 308 (1979); Chandler et al., J. Immunol. Meth.. 53: 187 (1982); Ekeke and Abuknesha, J. Steroid Biochem., 11 : 1579 (1979); Engvall and Perlmann. J. Immunol.. 109: 129 (1972); Geoqheqan et al.. Immunol. Comm., 7: 1 (1978); and Wilson and Nakane, Immunofluorescence and Related Techniques, p. 215 [Elsevier/North Holland Biomedical Press; Amsterdam (1978)]. Depending upon the nature of the label, various techniques can be employed for detecting and quantitating the label. For fluorescers, a large number of fluorometers are available. For chemiluminescers, luminometers or films are available. With enzymes, a fluorescent, chemiluminescent, or colored product can be determined or measured fluorometrically, luminometrically, spectrophotometrically or visually.

Various types of chemiluminescent compounds having an acridinium, benzachdinium, or acridan type of heterocyclic ring systems are preferred labels. Acridinium and benzacridinium esters are currently the more preferred chemiluminescent compounds, with preferred acridinium esters including those compounds having heterocyclic rings or ring systems that contain the heteroatom in a positive oxidation state including such ring systems as acridinium, benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a benzimidazole cation, quinolinium, isoquinolinium, quinolizinium, a cyclic substituted quinolinium, phenanthridinium, and quinoxalinium, as are well-known in the art.

The tracer may be prepared by attaching to the selected antibody either directly or indirectly a reactive functional group present on the acridinium or benzacridinium ester, as is well known to those skilled in the art, e.g. Weeks et al., Clinical Chemistry. 29(8). 1474-1479, (1983). Particularly preferred compounds are acridinium and benzacridinium esters with an aryl ring leaving group and the reactive functional group present in either the para or the meta position of the aryl ring. [See, U.S. Patent No. 4,745,181 and WO 94/21823.]

Epitopes

The affinity of a Mab to peptides containing an epitope depends on the context, e.g. on whether the peptide is a short sequence (4-6 aa), or whether such a short peptide is flanked by longer aa sequences on one or both sides, or whether in testing for an epitope, the peptides are in solution or immobilized on a surface. Therefore, it would be expected by ones of skill in the art that the representative epitopes described herein for the MN-specific Mabs would vary in the context of the use of those Mabs.

The term "corresponding to an epitope of an MN protein/polypeptide" will be understood to include the practical possibility that, in some instances, amino acid sequence variations of a naturally occurring protein or polypeptide may be antigenic and confer protective immunity against neoplastic disease and/or anti- tumorigenic effects. Possible sequence variations include, without limitation, amino acid substitutions, extensions, deletions, truncations, interpolations and combinations thereof. Such variations fall within the contemplated scope of the invention provided the protein or polypeptide containing them is immunogenic and antibodies elicited by such a polypeptide or protein cross-react with naturally occurring MN proteins and polypeptides to a sufficient extent to provide protective immunity and/or anti-tumorigenic activity when administered as a vaccine. Immunodominant Epitopes in PG Domain and In Neighboring Regions

As indicated above, the extracellular domain of the full-length CA IX comprises the PG and CA domains as well as some spacer or perhaps hinge regions. The CA IX immunodominant epitopes are primarily in the PG domain at about aa 53-111 [SEQ ID NO: 8] or at about aa 52-125 [SEQ ID NO: 25], preferably now considered to be at about aa 52-125 [SEQ ID NO: 25]. The immunodominant epitopes of CA IX may be located in regions neighboring the PG domain. For example, the epitope for aa 36-51 [SEQ ID NO: 21] would be considered an immunodominant epitope.

The main CA IX immunodominant epitope is that for the M75 Mab. The M75 monoclonal antibody is considered to be directed to an immunodominant epitope in the N-terminal, proteoglycan-like (PG) region of CA IX. The MN/CA IX PG region (aa 53-111 ) [SEQ ID NO: 8] shows some homology to the human aggrecan (aa 781-839) [SEQ ID NO: 10]. The epitope of M75 has been identified as amino acid sequence PGEEDLP (SEQ ID NO: 11 ), which is 4x identically repeated in the N-terminal PG region of CA IX [Zavada et al. (2000)]. Closely related epitopes to which the M75 Mab may also bind, which are also exemplary of immunodominant epitopes include, for example, the immunodominant 6X tandem repeat that can be found at amino acids (aa) 61-96 (SEQ ID NO. 12) of Figure 1A-1 C, showing the predicted CA IX amino acid sequence. Variations of the immunodominant tandem repeat epitopes within the PG domain include GEEDLP (SEQ ID NO: 13) (aa 61-66, aa 79-84, aa 85-90 and aa 91-96), EEDL (SEQ ID NO: 14) (aa 62-65, aa 80-83, aa 86-89, aa 92-95), EEDLP (SEQ ID NO: 15) (aa 62-66, aa 80-84, aa 86-90, aa 92- 96), EDLPSE (SEQ ID NO: 16) (aa 63-68), EEDLPSE (SEQ ID NO: 17) (aa 62-68), DLPGEE (SEQ ID NO: 18) (aa 82-87, aa 88-98), EEDLPS (SEQ ID NO: 19) (aa 62- 67) and GEDDPL (SEQ ID NO: 20) (aa 55-60). Other immunodominant epitopes could include, for example, aa 68-91 (SEQ ID NO: 22).

The monoclonal antibodies MN9 and MN 12 are considered to be directed to immunodominant epitopes within the N-terminal PG region, represented by SEQ ID NOS: 19 and 20, respectively. The MN7 monoclonal antibody could be directed to an immunodominant epitope neighboring the PG region at aa 127-147 (SEQ ID NO: 23) of Figure 1A-1 C. An epitope considered to be preferred within the CA domain (SEQ ID NO: 9) is from about aa 279-291 (SEQ ID NO: 27). An epitope considered to be preferred within the intracellular domain (IC domain) (SEQ ID NO: 7) is from about aa 435-450 (SEQ ID NO: 28). An exemplary preferred MN-specific antibody that specifically binds the carbonic anhydrase domain of MN protein is the V/10 Mab, which is produced by the hybridoma VU-V/10, deposited at BCCM™/LMBP in Ghent, Belgium under Accession No. LMBP 6009CB. As described in International Publication No. WO 03/100029, five additional Mabs (designated V/12, VII/20, VII/28, VII/32 and VII/38) have been found that are specific for the CA domain, as confirmed by their capacity to immunoprecipitate the full-length CA IX protein, but not its deletion variant lacking a large portion of CA domain.

Serial Samples of Body Fluids

In a preferred embodiment of the invention, MN/CA IX, particularly s- CA IX is quantitated in human urine samples drawn serially over time. Such urine specimens can be taken pretreatment, during treatment, or post-treatment, or can be taken from a patient who is not responding to therapy. As used herein, "serial changes over time" or "serial samples" denotes sequential testing of samples taken over time periods which would be considered relevant for the subject, depending on the context and the circumstances. For example, for cancer patient screening, serial samples might be drawn upon a patient's initial visit, after diagnosis, pre-surgery and/or post-surgery; whereas for population screening for a preneoplastic disease, serial samples might be drawn on a yearly basis.

Immunoassays

An exemplary and preferred immunoassay according to the methods of the invention is a sandwich ELISA described below in the Materials and Methods section, and was used to obtain some of the data in Example 2. It can be appreciated that alternate methods, in addition to those disclosed herein, can be used to detect and quantify MN/CA IX, preferably soluble MN/CA IX₁ in human urine. Other preferred sandwich assays could be used with other visualizing means, such as luminescent labels. Other labels are detailed below under the subsection labeled Labels. Many formats can be adapted for use with the methods of the present invention. The detection and quantitation of MN/CA IX₁ particularly s-CA IX in human urine can be performed, for example, by enzyme-linked immunosorbent assays, radioimmunoassays, dual antibody sandwich assays, agglutination assays, fluorescent immunoassays, immunoelectron and scanning microscopy using immunogold, among other assays commonly known in the art. The quantitation of MN/CA IX, particularly s-CA IX in such assays can be adapted by conventional methods known in the art. In preferred embodiments, MN/CA IX levels in urine are detected and quantified by a sandwich assay in which the capture antibody has been immobilized, using conventional techniques, on the surface of the support.

Suitable supports used in assays include among other supports, synthetic polymer supports, such as polypropylene, polystyrene, substituted polystyrene, polyacrylamides (such as polyamides and polyvinylchloride), glass beads, agarose, and nitrocellulose, among other supports.

Exemplary Immunoassay

An exemplary and preferred ELISA sandwich immunoassay is described in the Materials and Methods section and in Example 2. That exemplary ELISA uses Mab V/10 as the capture antibody and labeled Mab M75 as the detector antibody. In one embodiment, the capture Mab V/10 is immobilized on microtiter plate wells; diluted human serum/plasma samples or MN/CA IX standards (recombinant MN/CA IX) are incubated at 37°C in the wells to allow binding of MN/CA IX antigen by Mab V/10. After washing of wells, the immobilized MN/CA IX antigen is exposed to detector antibody Mab M75 (directly or indirectly linked to a label) at room temperature (20-27⁰C), after which the labeled antigen-antibody complexes are detected, e.g., by absorbance. Correlating the absorbance values of samples with the MN/CA IX standards allows the determination of a quantitative value of MN/CA IX, particularly s-CA IX, in pg/ml of urine.

Prognosis

Monitoring the levels of MN/CA IX, preferably s-CA IX, alone or in conjunction with other proteins, can be indicative of clinical outcomes for preneoplastic/neoplastic diseases. A preferred method of evaluating a clinical outcome is one based on response rate (RR)₁ clinical benefit [including complete response (CR), partial response (PR), and stable disease (SD)], time to progression (TTP), and time to death (TTD). Other methods of evaluating prognosis are known in the art and can be used.

MN/CA IX-directed Therapies

According to the methods of the invention, therapy selection for a patient with recurrent urinary tract cancer may comprise not only conventional therapies, but also MN/CA IX-directed therapies. Because of the MN protein's unique characteristics, it is an attractive candidate target for cancer therapy. In comparison to other tumor-related molecules (e.g. growth factors and their receptors), MN has the unique property of being differentially expressed in preneoplastic/neoplastic and normal tissues. Because of the extremely limited expression of MN protein in normal tissues, chemotherapeutic agents that target its expression would be expected to have reduced side effects, relative to agents that target proteins more extensively found in normal tissues (e.g., tamoxifen which binds the estrogen receptor, and finasteride which binds the androgen receptor). Phase I and Il clinical trials of an MN-specific drug, Rencarex®, have shown that at least one MN-specific agent is well-tolerated, with no serious drug-related side effects, further supporting MN as a possible target for cancer chemotherapy.

As used herein, "MN/CA IX-directed therapies" include any therapies that are targeted to MN/CA IX, or pathways that affect MN/CA IX levels or activity. Many MN-directed therapies may be useful according to the methods of the present invention, to treat preneoplastic/neoplastic diseases associated with abnormal MN expression. Preferred therapies comprise therapies selected from the group consisting of MN-specific antibodies, MN-preferential carbonic anhydrase inhibitors, MN antisense nucleic acids, MN RNA interference, and MN gene therapy vectors; some of which preferred therapies are described in greater detail below. Particularly, the MN/CA IX specific therapy may comprise using MN/CA IX-specific antibodies and/or MN/CA IX-specific antibody fragments, preferably humanized

MN/CA IX-specific antibodies and/or biologically active fragments thereof, and more preferably fully human MN/CA IX-specific antibodies and/or fully human MN/CA IX- specific biologically active antibody fragments. Said MN/CA IX-specific antibodies and biologically active MN/CA IX-specific antibody fragments, preferably humanized and more preferably fully human, may be conjugated to a cytotoxic entity, for example, a cytotoxic protein, such as ricin A, among many other cytotoxic entities.

A preferred MN/CA IX-directed therapy according to the invention is the bis-aryl urea sorafenib (BAY 43-9006) [Onyx Pharmaceuticals, Richmond, CA (USA), and Bayer Corporation, West Haven, CT (USA); Lyons et al., Endocrine- Related Cancer. 8: 219-225 (2001 )], a small molecule which inhibits the enzyme Raf kinase.

MN-Preferential Carbonic Anhvdrase Inhibitors

The novel methods of the present invention comprise inhibiting the growth of preneoplastic/neoplastic cells with compounds that preferentially inhibit the enzymatic activity of MN protein. Said compounds are organic or inorganic, preferably organic, more preferably sulfonamides. Still more preferably, said compounds are pyridinium derivatives of aromatic or heterocyclic sulfonamides.

These preferred pyridinium derivatives of sulfonamides are likely to have fewer side effects than other compounds in three respects: they are small molecules, they are membrane-impermeant, and they are specific potent inhibitors of the enzymatic activity of the tumor-associated MN protein. The pyridinium derivatives of sulfonamides useful according to the present invention can be formed, for example, by creating bonds between pyrylium salts and aromatic or heterocyclic sulfonamide reagents, as described in U.S. Patent Application No. 2004/0146955. The aromatic or heterocyclic sulfonamide portion of a pyridinium salt of a sulfonamide compound can be called the "head," and the pyridinium portion can be called the "tail." It can be appreciated by those of skill in the art that various other MN- preferential carbonic anhydrase inhibitors can be useful according to the present invention.

EXAMPLES The following examples are for purposes of illustration only and are not meant to limit the invention in any way.

The inventor investigated expression of cell-associated MN/CA IX in the transitional cell carcinoma (TCC) of the urinary tract and of soluble MN/CA IX (s- CA IX) shed by the tumor into the serum and urine of the patients. Paraffin sections of tumor tissue from all patients were stained with hematoxylin-eosin; parallel sections were examined by indirect immunohistochemical (IHC) staining with M75 monoclonal antibody. A total of 32 patients were enrolled in the pilot study and 16 healthy individuals served as controls. Membrane-bound MN/CA IX was present in the tumor cells near the endoluminal surface. Necrosis was observed in only four samples. On Western blots, s-CA IX concentrated from urine was visualized as a double band at 50 and 54 kDa. In most cases, the presence of s-CA IX in urine correlated with MN/CA IX expression in the tumor. On the other hand, no s-CA IX exceeding the normal level was detected in the serum of TCC patients. Urine from the patients with TCC of the urinary bladder and renal pelvis contains s-CA IX in amounts allowing the detection of tumors in approximately 70% of the patients. The results indicate that a test monitoring s-CA IX levels in urine may be useful for early detection of relapse or tumor recurrence in patients following transurethral resection or another therapy to treat urinary tract tumors, particularly TCC.

Materials and Methods Patients and controls

The pilot study included 32 patients (24 men and eight women, mean age 67.9 years, range 24-88) with endoscopic suspicion of TCC mainly of the urinary bladder, in one case of the renal pelvis. All patients routinely underwent intravenous pyelography, cystoscopy, or uretheroscopy before surgery to obtain information for the standard staging protocol. Urine and serum samples were taken before surgery and stored at -80 ⁰C until further processing. Surgically removed tissue samples from all the patients were fixed in 10% buffered formalin, embedded in paraffin, and processed according to standard histopathological procedures with hematoxylin- eosin staining. In total, 16 individuals served as controls; they were healthy volunteers or patients admitted to the hospital for other urological complaints (infections, urinary stones, etc). The study was carried out with the approval of the Departmental Ethics Committee, Charles University 2^nd School of Medicine and Hospital Motol, Prague, Czech Republic. Immunohistochemistrv (IHC)

Representative tissue sections of 4 μm thickness were deparaffinized in xylene and rehydrated through decreasing concentrations of ethanol to water. After standard blocking of endogenous peroxidase activity for 20 min at room temperature, the tissue sections were incubated overnight at 4 ⁰C with M75 anti- MN/CA IX antibody, hybridoma TC fluid diluted 1 :50 [Liao et al., 1994]. The antigen- antibody complexes were visualized with universal immunoperoxidase polymer detection kit N-Histofine, Simple Stain MAX PO multi ©(Nichirei Biosciences, Tokyo, Japan) with 3,3'-diaminobenzidine (Fluka Chemie, Buchs, Switzerland) as chromogen. Only membranous staining was considered a positive result. The MN/CA IX immunostaining was scored under low magnification using a semiquantitative method, and a 4-level evaluation system of the percentage of MN/CA IX positive tumor cells was applied (score 0 - 0%, score 1 - <10%, score 2 - 10-50%, score 3 - >50%). The distribution and extent of MN/CA IX positivity was noted with an emphasis on the assessment of perinecrotic areas of tumors.

Western blot analysis

For Western blot (WB) analysis, an extremely sensitive method was used [described previously in Zavada et al., Br J Cancer, 89: 1067-71 (2003)]. No modification was introduced; thus, the present results with TCC patient samples are comparable to previous findings with RCC patient samples. Briefly, 10 ml samples of urine (or 1 ml of serum) were clarified by centrifugation, and s-CA IX was concentrated by precipitation with Mab V10 followed by anti-mouse IgG linked to Sepharose beads. All of antigen from individual specimens was eluted from the beads with sample buffer at 100 ⁰C and loaded onto single lanes of an SDS polyacrylamide gel. The proteins were separated by SDS PAGE and transferred to PVDF membrane. The blots were developed with M75 IgG directly conjugated to peroxidase and visualized by ECL (enhanced chemiluminiscence). For calibration purposes, each blot included four standard amounts of purified complete MN/CA IX or s-CA IX. M75 [Pastorekova et al. (1992)] and V10 [Zatovicova et al., (2003)] were the MN/CA IX specific Mabs used. ELISA

The procedure was described previously [Zavada et al., Br J Cancer, 89: 1067-71 (2003)]. In brief, the ELISA was a sandwich method employing VIO-IgG as capture antibody and peroxidase-conjugated M75 IgG as detector antibody.

EXAMPLE 1 lmmunohistochemical staining

Out of a group of 32 patients, seven had undergone radical cystectomy, one had undergone nephroureterectomy for TCC of the renal pelvis and the rest had undergone transurethral resection (TURBT). Histopathological analysis confirmed the diagnosis of TCC in 22 specimens. Eighteen tumors displayed a papillary growth pattern; a flat lesion was found in one case, and in three cases the type of growth could not be evaluated exactly. In one case, squamous cell carcinoma (SCC) of the urinary bladder was identified. The pT stage, the expression of MN/CA IX antigen by IHC, and the detection of s-CA IX in urine by WB are listed below in Table 1.

Table 1

Human TCC and Expression of MN/CA IX Antigen (semiquantitative) by IHC and Detection of s-CA IX in Urine by WB

Patient Tumor stage/grade IHC WB

1. T1G1 +++ -

2. TXG 1 + -

3. T4G4 + -

4. T1G3 + ++

5. T2bG2 +++ +++

6. T1G2 ++ +++

7. T1G2 ++ +

8. T3bG3 ++ ++

9. T4G3

10. TaG2 ++

11. T1G2

12. TaG2

13. TXG2 + +

14. TXG2 - ++

15. T3aG2 - ++

16. T1 G3 +++ +++

17. Ta,TisG2 nt ++

18. T1 G2 + +++

19. T1G2 ++ +++

20. TXG2 ++ -

21. T3aG2 + ++

22. TXG2 ++ ++

23. TXG3 + ++

No tumor tissue was detected in nine specimens. Inflammatory changes were present in four, spinocelular metaplasia in two, and normal finding in three specimens. The M75 antibody bound to MN/CA IX on the tumor cell membranes, and a clear signal was observed upon addition of the chromogenic substrate. Variation in staining intensity between particular cases was minimal and differences were seen primarily in the extent and distribution of immunopositivity (data not shown). The luminal epithelial cells of the neoplastic papillary structures bound M75 antibody more effectively than the basal areas of the epithelium. IHC analysis of tumors with a score 1 staining pattern revealed focal, superficial localization of MN/CA IX. In contrast, analysis of lesions classified as score 3 revealed a diffuse luminal localization of MN/CA IX as well as immunopositivity for MN/CA IX in deeper layers of the epithelium.

Necrosis was found in only three TCCs and one SCC. The necrotic area in no case exceeded 10% of the evaluated tissue section of the tumor. One TCC with focal necrosis was completely negative for MN/CA IX; the other three tumors containing necrotic areas displayed a score 3 or 2 staining pattern. No significant change in MN/CA IX immunostaining was observed in the perinecrotic tumor zones. In normal areas of urothelium MN/CA IX staining by IHC was negative.

EXAMPLE 2 Western blots

Soluble MN/CA IX (s-CA IX) concentrated from the urine and sera of 32 patients enrolled in the study (22 identified as TCC, one as SCC and 9 as non- tumor cases) and from 16 control healthy individuals was analyzed by Western blotting (WB) with M75 Ab (shown in Figure 4). The antigen was detected in 20 out of 32 urine samples from the patients (62.5%). Two samples (No. 57 and F) from TCC patients and one from a SCC patient (No. 50) displayed very high levels of s- CA IX. The amount of s-CA IX in these samples far exceeds the calibration standards. The urine samples contained the following concentrations of s-CA IX as measured by ELISA: No. 50 = 325 ng/ml, No. 57 = 6.2 ng/ml, and sample F = 595 ng/ml. The concentration of s-CA IX in the other urine samples could be visually assessed by comparison with the calibration standards. In most of the urine specimens from TCC patients it was 50 - 400 pg/10ml (= 5 - 40 pg/ml). These were scored as "positive." In seven TCC patients s-CA IX was not detectable; three of them were IHC positive, and one was not tested by IHC because the tissue sample was too small. Distribution of s-CA IX level in urine of TCC patients and of controls is indicated in Figure 5.

Remarkably, serum from TCC patients was s-CA IX negative in all instances, even the serum from patients with MN/CA IX-positive urine. Figure 4 presents randomly selected samples of s-CA IX analyses performed on urine samples from bladder carcinoma patients and from controls. Control urine samples were all MN/CA IX negative, with the exception of one positive sample obtained from an apparently healthy young male.

Summary of Results:

The materials obtained from 32 individuals, subjected to surgery (mostly to TURBT) on the ground of endoscopic findings and of other clinical tests were examined by three methods: by histology (he-eo staining), by IHC of surgically removed tissues, and by Western blotting of urine obtained before resection. Urine only was examined from 16 control subjects. Based on the results of the three tests, the patients were divided into eight groups (see Table 2).

TABLE 2

An overview of the results obtained by histology (hematoxylin-eosin staining), MN/CA IX antigen detection by immunohistochemistry (IHC) and Western blotting (WB)

Method Group Histology IHC WB No. of cases

1. + + + 13^*

2. + + - 4

3. + - + 2

4. + - - 3

5. + ND + 1

6. - + + 3

7. - - + 4

8. - - - 2

Total 32

Controls

9. - ND - 15

10. - ND + 1

Total 16

^* 9 - TCC and 1 - SCC ND - not done

Histology Positive. MN/CA IX Positive: Group 1 - all three tests positive - was represented by thirteen (56.5%) patients. Groups 2 and 3 comprise another six patients (26.1 %) with histologically confirmed carcinoma, but with MN/CA IX antigen detected only either by IHC or by WB. In all of those cases, the antigen level was low. Group 5 comprises one tumor-positive patient, for whom IHC was not done, because of a very small tissue sample sufficient only for hematoxylin-eosin staining; MN/CA IX antigen was found by WB in that patient's urine. Histology Positive. MN/CA IX Negative: In Group 4, three patients had histologically confirmed carcinoma, but antigen was not detected by either method. These are simply patients with antigen-negative tumors.

Histology Negative. MN/CA IX Positive: Somewhat puzzling are the seven patients in Groups 6 and 7: the pathologist described their histological samples as "no tumor cells found," but s-CA IX in patient urine (detected by WB) signaled the possible presence of a tumor. In two cases, antigen level was scored as "strong." In three patients (Group 6), IHC staining was also positive. Hematoxylin-eosin staining revealed inflammatory changes in two samples and spinocellular metaplasia in one sample. This latter group may have several interpretations: "false positivity" of the s-CA IX test, "false negativity" of histological examination, or a combination of the two. Two of the three patients in Group 6 (IHC +, s-CA IX +), one "strongly" and the other "weakly" s-CA IX -positive based on urine analysis, with no tumor cells detected by the pathologist, returned to the hospital with recurrence of TCC within six months. Pathologists confirmed that some of these samples were too small, nonrepresentative or with only small parts of transitional epithelium. Possibly this was due to imprecise TURBT performed by an inexperienced urologist. The cuts during TURBT were far too superficial and samples were partly destroyed by heat. Histology Negative. MN/CA IX Negative: Group 8 comprises two patients with negative histology and antigen-negative in IHC and urine, who remain healthy and are kept under observation.

Controls: Out of 16 urine samples obtained from healthy donors (Groups 9 and 10), a significant level of s-CA IX was detected in only one (Group 10). The cause of the antigen presence in that apparently healthy donor is unknown.

Discussion

How do the present observations of s-CA IX in body fluids of TCC patients compare with previous findings for RCC patients? There are several important differences between the two types of tumors, which may explain the distribution of s-CA IX in patient body fluids. TCCs of the urinary bladder are usually multifocal and relatively discrete neoplasms with papillary architecture. Another common feature is their superficial growth, affecting the mucosa and submucosa (Tis, Ta, T1 ). Expression of MN/CA IX in neoplastic cells is considered to be a consequence of focal hypoxia which induces ectopic MN/CA IX synthesis [Pastorekova et al. (2006); Harris (2002)]. In our IHC experiments, MN/CA IX was frequently observed in the superficial parts of neoplastic papillary structures. This staining pattern could then be interpreted as a result of low oxygen tension in areas relatively distant from the central vascular tree of the papillae. Since the three tumors containing necrosis and tested for MN/CA IX were clearly positive, an eventual increase of MN/CA IX expression in the perinecrotic tumor zones may not have been detectable by the semiquantitave method used for evaluation. The complete IHC negativity of another TCC case with focal necrosis might reflect a diverse biological nature of this tumor with constitutional inability of MN/CA IX expression. RCC, on the other hand, grows deep in the renal parenchyma. Tumor size can be massive, even over 1000 g. MN/CA IX stains as a diffuse membranous antigen, not induced by hypoxia, but by the loss or inactivation of the VHL (Von Hippel Lindau) gene, which has been identified as a tumor suppressor gene [Ivanov et al. (1998)]. The differences between TCC and RCC provide a plausible explanation for the subject observations of s-CA IX in body fluids: in RCC patients the concentration of s-CA IX in blood and in the urine is extremely low, even below the detection limit of a highly sensitive test. The concentration of s-CA IX in parallel samples of blood and urine from individual patients was very similar. Extremely high s-CA IX was found in only a single RCC patient who was already ante finem.

In contrast, as described herein, s-CA IX was detected in the urine of 69.6% of histologically confirmed TCC patients. The concentration was relatively high, and in three instances it was extremely high. However, the inventors did not detect s-CA IX in the serum of any of TCC patients. In seven patients, the s-CA IX test was positive, but TCC was not found by microscopic examination. The reason for this could be a carcinoma in situ that was not macroscopically recognized, or a small tumor not identified or damaged during transurethral resection. The recurrence of TCC was observed within 6 months in two of the 7 patients. In another patient, the pathologist did not find epithelium in the TUR sample.

The inventor proposes that, in patients with RCC, s-CA IX is first shed into the blood and circulates there for some time. Since MN/CA IX is a cell adhesion protein [Zavada et al. (2000); Zavadova and Zavada (2005)], s-CA IX is absorbed by tissues expressing putative MN/CA IX-specific receptor(s) [or MN/CA IX-binding protein(s)]. The absorbed protein is eventually internalized by the cells and destroyed. A fraction of s-CA IX is filtered through the kidneys and eliminated in urine. Conceivably, the RCC patient previously observed to have a very high concentration of s-CA IX in both blood and urine already had all of the cell surface receptors in his body saturated with s-CA IX and therefore, the newly synthesized s-

CA IX remained in the blood and some was eliminated in the urine.

A different situation was observed in TCC patients, in whom apparently most of the s-CA IX is shed directly into the urine. Shedding of the antigen into the blood is prevented by the basal membrane, which separates the tumor from the circulating blood. Consequently, the antigen was undetectable in the serum, even when it was observed in urine.

In conclusion, the present data support the view that measuring s-CA

IX levels in urine may be a useful tool for monitoring urinary tract cancer patients, particularly bladder cancer patients, more particularly TCC patients, after therapy

(such as, resection), for tumor recurrence.

Budapest Treaty Deposits

The materials listed below were deposited with the American Type Culture Collection (ATCC) now at 10810 University Blvd., Manassus, Virginia 20110- 2209 (USA). The deposits were made under the provisions of the Budapest Treaty on the International Recognition of Deposited Microorganisms for the Purposes of Patent Procedure and Regulations thereunder (Budapest Treaty). Maintenance of a viable culture is assured for thirty years from the date of deposit. The hybridomas and plasmids will be made available by the ATCC under the terms of the Budapest Treaty, and subject to an agreement with the ATCC which assures unrestricted availability of the deposited hybridomas and plasmids to the public upon the granting of patent from the instant application. Availability of the deposited strains is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any Government in accordance with its patent laws.

Hvbridoma Deposit Date ATCC #

VU-M75 September 17, 1992 HB 11128

MN 12.2.2 June 9, 1994 HB 11647

Plasmid Deposit Date ATCC #

A4a June 6, 1995 97199

XE1 June 6, 1995 97200

XE3 June 6, 1995 97198

Similarly, the hybridoma cell line V/10-VU which produces the V/10 monoclonal antibodies was deposited on February 19, 2003 under the Budapest Treaty at the International Depository Authority (IDA) of the Belgian Co-ordinated Collections of Micro-organisms (BCCM) at the Laboratorium voor Moleculaire Biologie-Plasmidencollectie (LMBP) at the Universeit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium [BCCM/LMBP] under the Accession No. LMBP 6009CB. The description of the foregoing embodiments of the invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teachings. The embodiments were chosen and described in order to explain the principles of the invention and its practical application to enable thereby others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated.

All references cited herein are hereby incorporated by reference.

Claims

CLAIMS 1. A method of detecting recurrence of bladder cancer in a human patient who has had an at least preliminary diagnosis of, and treatment for bladder cancer, said method comprising:

(c) detecting and quantifying binding of said one or more antibodies to MN/CA IX in said patient sample, and comparing the patient sample MN/CA IX level to MN/CA IX levels found in urine samples from normal humans, wherein if the level of MN/CA IX in said patient sample is above 95% of the MN/CA IX levels in the urine samples from said normal humans, that MN/CA IX level in said patient sample is considered to be elevated and to indicate recurrence of bladder cancer.

2. The method of claim 1 wherein said patient urine sample and/or said normal urine samples are concentrated.

3. The method of claim 1 wherein said treatment is selected from the group consisting of surgery, immunotherapy, chemotherapy and radiotherapy.

4. The method of claim 1 wherein said bladder cancer is superficial bladder cancer or invasive bladder cancer.

5. The method of claim 1 wherein said bladder cancer is selected from the group consisting of transitional cell carcinoma (TCC), squamous cell carcinoma (SCC), adenocarcinoma, sarcoma and small cell carcinoma.

6. The method according to claim 1 wherein said method is in a format that is selected from the group consisting of Western blots, enzyme-linked immunosorbent assays, radioimmunoassays, competition immunoassays, dual antibody sandwich assays, immunohistochemical staining assays, agglutination assays, and fluorescent immunoassays.

7. The method according to claim 1 wherein said one or more antibodies are selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antigen binding antibody fragments comprising an MN/CA IX extracellular domain binding region, and recombinant antibodies.

8. The method according to claim 1 wherein said one or more antibodies are monoclonal antibodies.

9. The method according to claim 8 wherein said one or more monoclonal antibodies are selected from the group consisting of the monoclonal antibodies designated M75 which are secreted by the hybridoma VU-M75, which was deposited at the American Type Culture Collection under ATCC No. HB 11128, and monoclonal antibodies that are not directed to the M75 epitope.

10. The method according to claim 8 wherein the antibodies are monoclonal, and wherein one monoclonal antibody is specific for the carbonic anhydrase (CA) domain of MN/CA IX, and wherein the other monoclonal antibody is directed to the proteoglycan-like (PG) domain of MN/CA IX.

11. The method according to claim 10 wherein the antibody directed to the CA domain is the V/10 monoclonal antibody which is produced by the hybridoma VU-V/10, which was deposited at BCCM™/LMBP in Ghent, Belgium under Accession No. LMBP 6009CB, and wherein the antibody directed to the PG domain is the M75 monoclonal antibody that is secreted from the hybridoma VU- M75, which was deposited at the American Type Culture Collection under ATCC No. HB 11128.

12. A method of detecting recurrence of transitional cell carcinoma (TCC) of the urinary tract in a human patient who has had an at least preliminary diagnosis of, and treatment for TCC, said method comprising: (a) obtaining a urine sample from said patient, wherein said sample is collected after treatment for TCC;

MN/CA IX in said patient sample, and comparing the patient sample MN/CA IX level to MN/CA IX levels found in urine samples from normal humans, wherein if the level of MN/CA IX in said patient sample is above 95% of the MN/CA IX levels in the urine samples from said normal humans, that MN/CA IX level in said patient sample is considered to be elevated and to indicate recurrence of TCC.

13. The method of claim 12 wherein said TCC is selected from the group consisting of TCC of the urinary bladder, TCC of the renal pelvis, TCC of the ureter and TCC of the urethra.

14. A method of detecting recurrence of bladder cancer in a human patient who has had an apparent spontaneous remission of bladder cancer, said method comprising:

(a) obtaining a urine sample from said patient; (b) contacting said patient sample with one or more antibodies which specifically bind to MN/CA IX's extracellular domain; and

15. A method of detecting recurrence of bladder cancer in a human patient who has had an at least preliminary diagnosis of, and treatment for bladder cancer, said method comprising:

16. The method of claims 1 , 12, 14 or 15 wherein said recurrence is detected at an early stage.

17. The method of claims 1 , 12, 14 or 15 wherein at least one of said one or more antibodies is labeled.

18. A method of monitoring the status of bladder cancer in a patient, and/or monitoring how a patient with said bladder cancer is responding to a therapy, comprising immunologically detecting and quantifying serial changes in soluble MN/CA IX (s-CA IX) levels in urine samples taken from said patient over time; wherein increasing urine levels of s-CA IX over time indicate disease progression or a negative response to said therapy, and wherein decreasing urine levels of s-CA IX over time indicate disease remission or a positive response to said therapy.

19. A method of monitoring the status of bladder cancer in a patient, and/or monitoring how a patient with said bladder cancer is responding to a therapy, comprising immunologically detecting and quantifying soluble MN/CA IX (s-CA IX) levels in urine samples taken from said patient over time, relative to s-CA IX levels in urine samples taken from normal humans; wherein patient urine levels of s-CA IX above 95% of the s-CA IX levels in the urine samples from said normal humans indicate disease progression or a negative response to said therapy, and wherein patient urine levels of s-CA IX at or below 95% of the s-CA IX levels in the urine samples from said normal humans indicate disease remission or a positive response to said therapy.

20. The method of claims 18 or 19, wherein said immunological detection and quantitation is by an immunoassay in a format that is selected from the group consisting of Western blots, enzyme-linked immunosorbent assays, radioimmunoassays, competition immunoassays, dual antibody sandwich assays, immunohistochemical staining assays, agglutination assays, and fluorescent immunoassays.

21. The method of claims 18 or 19, wherein said therapy is selected from the group consisting of surgery, immunotherapy, chemotherapy and radiotherapy.

22. The method of claims 1-17 wherein the MN/CA IX level and

MN/CA IX levels are the soluble MN/CA IX (s-CA IX) level and s-CA IX levels.