WO2009031151A2 - Antibodies and methods for diagnosing and treating cancer - Google Patents

Abstract

A monoclonal antibody capable of interacting with a FXYD5 polypeptide of about 24 kDa and a FXYD5 polypeptide of about 55 kDa is disclosed. An exemplary monoclonal antibody is disclosed having a deposit number of CNCM I-4069. Use of same for diagnosing and treating cancer is also disclosed.

Description

ANTIBODIES AND METHODS FOR DIAGNOSING AND TREATING CANCER

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to methods of treating and diagnosing cancer and, more particularly, to antibodies capable of same.

FXYD is a family of proteins named after an invariant extracellular motif (1,2). The 7 family members specifically interact with the Na⁺/K⁺-ATPase and modulate its kinetic properties. The tissue distribution and kinetic effects of each FXYD protein are different. Therefore, it is generally assumed that FXYD proteins are tissue-specific auxiliary subunits or regulators of the Na⁺/K⁺-ATPase, which adjust its kinetic properties to specific needs of the cells in which they are expressed, without affecting it elsewhere (1,3,4). Accumulating data suggest additional roles for some FXYD proteins, and it is possible that they also regulate other transporters (1). In particular, data has been accumulated suggesting that FXYDl (phospholemman) also modulates activity of the cardiac Na⁺/Ca²⁺ exchanger (5-8). FXYD proteins are type I membrane proteins with an intracellular C-terminus, a single transmembrane domain, and an extracellular N-terminal that may have a signal peptide. Usually the extracellular N-terminal is shorter than 40 amino-acids. The only exception is FXYD5 which has an atypically long extracellular domain of more than 140 amino acids. The biochemical and functional properties of FXYD5 have previously been studied (9). Using polyclonal antibodies, the cellular and tissue distribution of FXYD5 as well as its interaction with the Na⁺/K⁺-ATPase were elucidated. FXYD5 was found to be a ~24 kDa protein that is particularly expressed in the basolateral membrane of epithelial cells in kidney, intestine and lung. It is specifically immunoprecipitated by antibodies to the α subunit of the Na⁺/K⁺-ATPase and vice versa. Co-expressing FXYD5 with the Na⁺/K⁺-ATPase in Xenopus oocytes elicits more than a 2-fold increase in the V_max of the pump, without affecting the K_{0 5} for external K⁺ (9).

Different functional and structural properties of FXYD5 have been reported by Hirohashi and co-workers (10-12). This group cloned FXYD5 as the antigen of a monoclonal antibody that stains cancer cells, but not normal cells (10). The expression of FXYD5 (termed in these studies dysadherin) was associated with down-regulation of E-cadherin, increased cell motility, decreased aggregation, and metastasis (12,13). In a series of clinical studies correlation was demonstrated between expression of this antigen and survival chances in various human cancers (14-17). The polypeptide detected in these studies had an apparent molecular weight of 50-55 kDa, much higher than the calculated value (-17 kDa) and the size observed in previous studies (9) (24 kDa). Such an abnormally high MW was explained by excessive glycosylation of FXYD5 (11).

Additional background art includes Cancer Res. 2004 Oct l;64(19):6989-95.

SUMMARY OF THE INVENTION

According to one aspect, there is provided a monoclonal antibody capable of interacting with a FXYD5 polypeptide of about 24 kDa and a FXYD5 polypeptide of about 55 kDa.

According to another aspect, there is provided a method of diagnosing cancer, the method comprising analyzing a glycosylation of FXYD5, wherein an alteration in the glycosylation of the FXYD5 in cancerous versus non-cancerous cells is indicative of the cancer.

According to yet another aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the humanized monoclonal antibody of the present invention, thereby treating the cancer. According to yet another aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an siRNA comprising a polynucleotide sequence as set forth by SEQ ID NO: 11, thereby treating the cancer.

According to still another aspect, there is provided a monoclonal antibody having the deposit number CNCM 1-4069.

According to still another aspect, there is provided an aptamer which binds the extracellular portion of FXYD5.

According to an additional aspect, there is provided a method of identifying a cancer cell, the method comprising analyzing a glycosylation of FXYD5, wherein an increase in the glycosylation of the FXYD5 in a cell as compared to a non-cancerous cell is indicative of the cancer cell.

According to further features in embodiments of the invention described below, the antibody interacts with an extracellular portion of the FXYD5 polypeptide. According to still further features in the described embodiments, the FXYD5 polypeptide is O-glycosylated.

According to still further features in the described embodiments, the FXYD5 polypeptide is expressed in a non-cancerous cell. According to still further features in the described embodiments, the FXYD5 polypeptide is expressed in a cancerous cell.

According to still further features in the described embodiments, the noncancerous cell is selected from the group consisting of spleen, lung, kidney, colon and heart. According to still further features in the described embodiments, the antibody is humanized.

According to still further features in the described embodiments, the antibody is a neutralizing antibody.

According to still further features in the described embodiments, the analyzing is effected using a monoclonal antibody capable of interacting with a FXYD5 polypeptide of about 24 kDa and a FXYD5 polypeptide of about 55 kDa.

According to still further features in the described embodiments, the antibody has a deposit number CNCM 1-4069.

According to still further features in the described embodiments, the analyzing is further effected using an antibody capable of recognizing a 55kDa FXYD5 and not a 24 kDa FXYD5. According to still further features in the described embodiments, the cancer is a metastatic cancer.

The present invention successfully addresses the shortcomings of the presently known configurations by providing a novel antibody capable of recognizing the extracellular domain of FXYD5. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings:

FIG's. IA-B are sequences and photographs illustrating the existence of FX YD 5 splice variants. (A). Partial sequences of three FXYD5 splice variants. The shaded area marks the transmembrane domain and splice variant unique residues are shown in bold. (B). Agarose gel of RT-PCR products amplified from RNA of mouse kidney cortex, medulla, and mouse dendritic cells. Arrows and asterisk mark three different FXYD5 sequences amplified using primer pairs I/II and I/IV, listed in Table 1.

FIG's. 2A-C are photographs of Western blots illustrating FXYD5 antibody specificity. Xenopus oocytes were injected with cRNA mixtures coding for αβ Na⁴VK⁺- ATPase and either FXYD5 or different FXYD5/FXYD4 chimera. Each chimera is identified by a three letter code corresponding to the origin of the extracellular, transmembrane and intracellular domain, respectively. R stands for FXYD5 (RIC) and C for FXYD4 (CHIF). Thus, RRC marks a chimera whose extracellular N terminal and transmembrane domains come from FXYD5 and the C-terminal sequence from FXYD4, etc. Membrane proteins were resolved electrophoretically and transferred to blotting membrane. (A) High, medium and low MW segments of the gel were blotted with antibodies to α, FXYD4 (polyclonal anti C-terminal peptide) and FXYD5 (polyclonal anti-C tail). Each of the two FXYD antibodies recognized only constructs whose C-terminus originates from the corresponding FXYD. The size of these proteins was either 24 or 7 kDa depending on the origin of the N-terminal. (B) The same membrane preparations were blotted with the monoclonal anti-FXYD5 antibody. This antibody recognized only constructs whose N terminal originates from FXYD5. (C). E. CoIi was transformed with plasmid DNA having the extracellular domain of FXYD5 down stream the lac operon. Cells were incubated for 1 h in the presence and absence of 0.1 mM IPTG which should stimulate translation of the FXYD5 construct, and bacterial lysates were prepared and assayed by Western blotting with the monoclonal anti FXYD5 antibody. The antibody recognized a 20 kDa band that was specific to IPTG stimulated cells.

FIG. 3 is a photograph of a Western blot illustrating the effects of FXYD5 on β glycosylation. Xenopus oocytes were injected with cRNA mixtures coding for αβ Na⁺/K⁺-ATPase and either FXYD4, or FXYD5 or different FXYD5/FXYD4 chimera. Microsomes were extracted three days later and their proteins were resolved electrophoretically and transferred to blotting membrane. The membrane was cut into high, medium and low MW segments and blotted with antibodies to α, β and FXYD5 (monoclonal). The asterisks mark the chimera with increased mobility of β.

FIG's. 4A-B are photographs of Western blots illustrating the expression and tissue distribution of FXYD5. (A) Membrane proteins were isolated from various mouse organs. They were resolved electrophoretically, transferred to blotting membranes, and blotted with the monoclonal anti FXYD5 antibody. (B) HeLa cells were transfected with the FXYD5 variant with ten extra C-terminal residues in which amino-acids 106-109 of the extracellular domain were HA tagged. Membranes were prepared from transfected (+) and non-transfected (-) cells, proteins were resolved electrophoretically, transferred to blotting membrane, and blotted with antibodies to the α subunit of the Na⁺/K⁺-ATPase and with anti HA.

FIG's. 5A-D are photographs of Western blots illustrating the binding of FXYD5 to wheat germ agglutinin. (A) Xenopus oocytes were injected with cRNA mixtures coding for α, lOXHis tagged β Na⁴VK⁺- ATPase and FXYD5. Microsomes were extracted three days later, dissolved in a buffer containing 1 % Triton-X-100, and proteins associated with the lOXhis tag were precipitated using Ni-NTA beads. 5 % of the total Triton X-100 soluble fraction (tot.) and the whole volume of pulled down proteins (pd) were resolved electrophoretically and transferred to blotting membrane. The membrane was cut into high, medium and low MW segments that were blotted with antibodies to α, β and FXYD5. (B) Triton solubilized oocyte membranes were incubation with agarose wheat germ agglutinin beads with and without 200 mM GlcNac as described under General Materials and Methods. 10 % of the total detergent solubilized fraction (total) and the whole volume of proteins eluted from the beads (bound) were resolved electrophoretically, transferred to blotting membrane, and blotted with anti-FXYD5 antibody. (C). Pig kidney microsomes (Mic.) and purified Na⁴VK⁺- ATPase (Pur.) were resolved electrophoretically, transferred to blotting membrane, and the membrane was cut to high and low MW regions and blotted with antibodies to α and FXYD5, respectively. (D) Purified Na⁴VK⁺- ATPase was dissolved in 1% Triton-X- 100 and incubated with agarose wheat germ agglutinin beads with and without 20OmM GlcNac. Lectin bound proteins were resolved electrophoretically, transferred to blotting membrane, and blotted with anti C-terminus FXYD5 antibody. FIG's. 6A-B are photographs of Western blots following co-precipitation of αβ with FXYD constructs. Xenopus oocytes were injected with cRNA mixtures coding for α, lOXHis tagged β NaVK⁺- ATPase and either FXYD4 or FXYD4/FXYD5 chimera (A), or FXYD5 and various mutants (B). One group of oocytes was injected with FXYD5 but no αβ (Con). Microsomes were extracted three days later and solubilized in 3mg/ml C₁₂Ei₀. Pump complexes were precipitated using Ni-NTA beads. The pulled down proteins (pd) and 5 % of the total Ci₂Ei₀ soubilized proteins (tot.) were resolved electrophoretically, transferred to blotting membrane, cut to low, medium and high MW segments, and assayed by blotting with antibodies to α, β and either FXYD4 (Figure 6A) or FXYD5 (Figure 6B). FIG's. 7A-C are graphs and photographs illustrating the effects of FXYD5 and

FXYD5/FXYD4 chimera on Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity was measured as ouabain sensitive ⁸⁶Rb⁺ uptake in Na⁺ loaded Xenopus oocytes, as described under General Materials and Methods. (A). ⁸⁶Rb⁺ uptake was measured in groups of oocytes expressing the α and β subunits of the pump with and without different FXYD constructs. Measurements were done in the presence of 2 mM (dotted bars) or 10 μM (hatched bars) ouabain. Means ± SEM of values obtained in 7-8 oocytes are depicted. The flux mediated by the pump is shown in filled bars. (B). Ouabain sensitive fluxes in oocytes injected with αβ with and without different FXYD constructs are shown. The figure depicts means ± SEM of the pump mediated fluxes from 3-5 experiments (numbers shown in brackets) using 7-8 oocytes in each experiment for each condition. Data are expressed as % of the pump activity in oocytes injected with αβ alone. In FXYD5 and CRC the flux measured was significantly higher than the control (P<0.01, asterisk). (C): A representative Western blot comparing the amount of surface biotinylated α and β in oocytes injected with different FXYD constructs.

FIG. 8 is a photograph of a Western blot of H 1299 lysates with anti FXYD5 antibody. Lanes 1, 2 represent wild type cells. Lanes 3, 4 represent two different clones of siRNA (SEQ ID NO: 11) transfected cells. In wild type cells the antibody recognizes a ~55 kDa band (arrow) that is much weaker in the siRNA silenced cells.

FIG. 9 is a photograph of an ethidium bromide stained gel, illustrating the results of an RT-PCR of H1299 RNA using FXYD5 specific primers (SEQ ID NOs: 9 and 10).

After 20 cycles the expected product is seen in wild type (WT) but not siRNA transfected cells (clones 1 and 2). After 25 cycles the product is amplified from silenced

RNA too but its abundance is much lower than that amplified from WT RNA.

FIG's. 10A-D are images of wild-type (Figures 1 OC-D) H 1299 cells and siRNA treated (Figures 1 OA-B) H 1299 cells. Images were taken shortly after seeding (0 h) and 10 hours later (10 h). The wild type cells remained round and less adhered to the surface while the FXYD silenced cells adapted polar structure and had pods.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of an antibody capable of recognizing both the 24 kDa and the 55 kDa form of FXYD5.

Specifically, the present invention can be used to diagnose and/or treat cancer.

The principles and operation of the antibody according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. FXYD5 is known to be expressed in at least two forms - a 24 kDa polypeptide expressed in normal cells (9) and a 50-55 kDa polypeptide, the latter of which was shown to be differentially expressed in cancer cells (10). In an attempt to interpret the discrepancies between the apparent sizes of FXYD5, the present inventors generated a monoclonal antibody capable of interacting with the extracellular portion of FXYD5.

Whilst reducing the invention to practice, the present inventors showed that the major FXYD5 polypeptide in normal cells recognized by the novel antibody was the 24 kDa FXYD5 polypeptide. This polypeptide was shown to be O-glycosylated, but not to an extent that significantly changed the size of the polypeptide. The present inventors thus deduced that the 24 kDa polypeptide only undergoes extensive glycosylation in cancerous cells and that glycosylation of the polypeptide may serve as a marker for cancer. It has been proposed that FXYD5 plays a role in neoplastic transformation since it has been shown to downregulate E-cadherin expression in tumors. It has been shown recently that normal glycosylation of the β subunit is essential for production of stable association of cells with the adherens junction and plays an important role in cell-cell contact formation [23]. Thus, interference of FXYD5 with glycosylation of the β subunit might destabilize its interactions with E-cadherin and the adherens junction, and thus account for the lowered expression of E-cadherin, decreased cell-cell contact, increased motility and metastasis. The present inventors have shown that the N terminal portion of FXYD5 reduces glycosylation of NaVK⁺- ATPase. Therefore, without being bound to theory, it is suggested that an antibody which interacts with the N-terminal portion of FXYD5 as described herein will be capable of neutralizing FXYD5 activity, and more specifically activity associated with the N terminal, and as such may also serve as a therapeutic tool for the treatment of cancer and more specifically metastatic cancer.

Thus, according to one aspect, there is provided a method of diagnosing cancer, the method comprising analyzing a glycosylation of FXYD5, wherein an alteration in the glycosylation of said FXYD5 in cancerous versus non-cancerous cells is indicative of the cancer.

As used herein, the term "diagnosing" refers to determining the presence of a cancer, classifying a cancer, determining a severity of cancer (grade or stage), monitoring cancer progression, forecasting an outcome of the cancer and/or prospects of recovery.

The subject may be a healthy animal or human subject undergoing a routine well-being check up. Alternatively, the subject may be at risk of having cancer (e.g., a genetically predisposed subject, a subject with medical and/or family history of cancer, a subject who has been exposed to carcinogens, occupational hazard, environmental hazard] and/or a subject who exhibits suspicious clinical signs of cancer [e.g., blood in the stool or melena, unexplained pain, sweating, unexplained fever, unexplained loss of weight up to anorexia, changes in bowel habits (constipation and/or diarrhea), tenesmus (sense of incomplete defecation, for rectal cancer specifically), anemia and/or general weakness). According to another embodiment, the subject may be a diagnosed cancer patient and is performing a routine check-up, in-between treatments.

The term "cancer" as used herein, refers to a disease or disorder resulting from the proliferation of oncogenically transformed cells. Examples of particular cancers that may be diagnosed according to the method of the present invention include but are not limited to: thyroid carcinoma, colorectal carcinoma, pancreatic ductal adenocarcinoma, gastric cancer, squamous cell carcinoma of the tongue, esophageal squamous cell carcinoma, testicular tumours or cutaneous malignant melanoma. These tumors were shown to express the 55 kDa form of FXYD5 and the expression level correlated tumor aggressivness and poor prognosis.

As used herein, the term "FXYD5" refers to the 178 amino acid transmembrane polypeptide such as set forth by Genbank accession number NP_054883 and NP 659003 and variants thereof, including, but not limited to that set forth by Swissprot number P97808-1, a sequence predicted by a single mouse EST entry BC031112 and other variants described herein.

The term "glycosylation" as used herein refers to the process by which a polypeptide (i.e. FXYD5) is covalently linked with one or more oligosaccharide chains (carbohydrates containing two or more simple sugars linked together e.g. from two to about twelve simple sugars linked together). The oligosaccharide side chains are typically linked to the backbone of the polypeptide through either N- or O-linkages. However, since FXYD5 does not comprise N-glycosylation sites, typically the glycosylation is O-glycosylation.

Various methods may be effected in order to analyze the glycosylation of the FXYD5 polypeptide. Such methods include high-performance liquid chromatography (HPLC) method, immunoassay, chemical method (NBT test), dye-binding methods, enzymatic methods (e.g. employing an Amadori compound oxidoreductase such as fructosyl amino acid oxidase (FAOD)), and lectin binding assays as described in the Examples section herein below. Another method for analyzing the glycosylation of the FXYD5 polypeptide involves analyzing the expression of FXYD5 using an antibody that specifically recognizes the minimally-glycosylated form of FXYD5 (the 24 kDa FXYD5) and not the 55 kDa form. Alternatively, or additionally the expression of FXYD5 may be analyzed using an antibody that specifically recognizes the hyper-glycosylated form of FXYD5 (the 55 kDa FXYD5) and not the 24 kDa form.

In addition, the glycosylation status of FXYD5 may be analyzed using an antibody that is capable of recognizing both the 24 kDa polypeptide and the 55 kDa polypeptide, such as the antibody generated by the present inventors. Analysis of the size of the polypeptide recognized by the antibody is predictive of its glycosylation status. Such analysis may be effected using methods known in the art such as Western blotting and immunoprecipitation.

According to one embodiment the antibody is a capable of interacting with an extracellular portion of FXYD5 polypeptide such as the antibody generated by the present inventions. Exemplary sequences of the extracellular portion of FXYD5 are set forth in SEQ ID NO: 1 and SEQ ID NO: 2. The present inventors have shown that such an antibody is capable of binding to both the 24 kDa FXYD5 polypeptide (minimally glycosylated form) in non-cancer cells including, but not limited to spleen, lung, kidney, colon and heart cells and to the 55 kDa FXYD5 (hyper-glycosylated form) in cancer cells (e.g. non-small lung carcinoma cells).

As mentioned herein above, the antibody may be one that is capable of interacting with the 55 kDa form (hyperglycosylated form) of the FXYD5 polypeptide only and not the 24 kDa form. Such an antibody has been described by Hirohashi and co-workers (10-12) and was shown to interact with the glycosylated form of FXYD5. It will be appreciated that the above described antibody may be used in conjunction with the antibody of the present invention or, alternatively in lieu of the antibody of the present invention.

The term "antibody" as used in this invention includes intact molecules (e.g., monoclonal or polyclonal) as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen- binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab¹ fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference). Thus, for example, for generating FXYD5, the antigenic protein may be the complete FXYD5 protein (e.g., recombinant) or an antigenic portion thereof which is derived from the ectopic domain of the protein.

Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5 S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (1972)]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97- 105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11 :1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Such antibodies are important for clinical and especially therapeutic applications as will be further described hereinbelow. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported

CDR or framework sequences. 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 CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)]. Methods for humanizing non-human antibodies are well known in the art.

Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al., J. MoI. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

By analyzing the size of the FXYD5 polypeptides which interact with the above described antibodies, it is possible to determine its glycosylation status.

As mentioned, the method of the present invention is effected by analyzing the glycosylation status of the FXYD5 in normal cells (i.e. control) and comparing its glycosylation status in potentially cancerous cells, wherein a change (e.g. up-regulation of glycosylation of FXYD5) is indicative of the cancer.

The control cells may be taken from a healthy subject preferably from the same tissue which is being analyzed for the presence of cancerous cells. Preferably, the subject is of the same species e.g. human, preferably matched with the same age, weight, sex etc. It will be appreciated that the control sample may also be of the same subject from a healthy tissue, prior to disease progression or following disease remission.

Typically, the cells are removed (e.g. by a biopsy) and the FXYD5 glycosylation status is examined ex-vivo. It will be appreciated that the method of the present invention may also be effected in vitro (e.g. in a cell culture) in order to distinguish a cancer cell from a noncancerous cell, wherein a presence of the hyperglycosylated FXYD5 is indicative of a cancerous cell.

As mentioned, down-regulation of FXYD5 has also been postulated as a method for treating cancer e.g. a metastatic cancer since it has been shown to downregulate E- cadherin expression in tumors or by some other mechanism associated with reduced glycosylation of the β subunit of the NaVK⁺- ATPase. The present inventors have shown that down-regulation of FXYD5 (using siRNA) reduces cell adherence and motility in lung cancer cells. Thus, the present inventors envisage treating cancer with an agent capable of downregulating FXYD5. Such an agent may be a polynucleotide agent, such as an siRNA (e.g. as set forth by SEQ ID NO: 11).

A small interfering RNA (siRNA) molecule is an example of an nucleic acid agents agent capable of downregulating Hl 9RNA. RNA interference is a two-step process. During the first step, which is termed the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which cleaves dsRNA (introduced directly or via an expressing vector, cassette or virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each strand with 2-nucleotide 3' overhangs [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature 409:363-366 (2001)].

In the effector step, the siRNA duplexes bind to a nuclease complex to form the RNA- induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); Hammond et al, (2001) Nat. Rev. Gen. 2: 110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although the mechanism of cleavage is still to be elucidated, research indicates that each RISC contains a single siRNA and an RNase [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].

It is possible to eliminate the "intiation step" by providing a priori siRNA. Because of the remarkable potency of RNAi, an amplification step within the

RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs, which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC [Hammond et al., Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev. 15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)]. For more information on RNAi see the following reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat. Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575: 15-25 (2002).

Synthesis of RNAi molecules suitable for use with the present invention can be effected as follows. First, the Hl 9 nucleic acid sequence target is scanned downstream for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Second, potential target sites are compared to an appropriate genomic database

(e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server

(wwwdotncbidotnlmdotnihdotgov/BLAST/). Putative target sites that exhibit significant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene. Alternatively, the capable of downregulating FXYD5 may be a polypeptide agent, such as an antibody capable of neutralizing an activity of FXYD5 - (e.g. the antibody of the present invention). According to one embodiment antibodies capable of restoring the glycosylation of the Na⁺/K⁺-ATPase may be used. Such antibodies may be screened using the methods provided in the example section. Alternatively, or additionally aptamers capable interacting with an extracellular portion of FXYD5 may be used as agents for treating cancer.

As used herein, the term "aptamer" refers to an oligonucleic acid or peptide molecule that binds a specific target molecule i.e. the extracellular portion of FXYD5, by adopting a particular three dimensional shape. Aptamers are generally produced through an in vitro evolutionary process called

"systematic evolution of ligands by exponential enrichment" (SELEX). The method is an iterative process based on selection and amplification of the anticipated tight binding aptamer. The start library for selection of aptamers contains single stranded DNA oligonucleotides with a central region of randomized sequences (up to 10¹⁵ different sequences) which are flanked by constant regions for subsequent transcription, reverse transcription and DNA amplification. The start library is amplified by PCR and transcribed to an RNA start pool by T7 transcription. Target specific RNA is selected from the pool by allowing the pool to interact with the target molecule, only tight binding RNA molecules with high affinity are removed from the reaction cycle, the tight binding RNA molecules are reverse transcribed to cDNA and amplified to double stranded DNA by PCR. These enriched binding sequences are transcribed back to RNA which is the source for the next selection and amplification cycle. Such selection cycles are usually repeated 5-12 times in order to obtain only sequences with highest binding affinities against the target molecule.

The antibodies or aptamers of the present invention may be provided per se or may be administered as a pharmaceutical composition.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the antibodies of the present invention accountable for the biological effect. Hereinafter, the phrases "physiologically acceptable carrier" and

"pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.

Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in

"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections. Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (nucleic acid construct) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., ischemia) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.

For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Dosage amount and interval may be adjusted individually to provide plasma or brain levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Animal models may be used for selecting optimal antibodies and aptamers for treating cancer metastasis may be used such as those described in Cancer Research 47, 1398-1406, March 1, 1987, Cancer. 2003 Feb 1;97(3 Suppl):748-57 and US Patent 5643551.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above. As used herein the term "about" refers to ± 10 %. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes MII Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

GENERAL MATERIALS AND METHODS cDNA clones: cDNAs of mouse FXYD5, rat αl and pig βl subunits of the Na⁺/K⁺-ATPase were described previously (9). For co-precipitation experiments the βl subunit was tagged in its N-terminus with 10 Histidines. FXYD5 mutants in which residues Al 50, 1160 and L161 were replaced by the corresponding FXYD4 residues (G, M, and A, respectively) and FXYD5/FXYD4 chimera were prepared by standard recombinant DNA techniques using overlapping oligonucleotides. In these chimera the extracellular, transmembrane and cytoplasmic segments of mouse FXYD5 were defined as: M1-K144, R145-S163 and G164-R178, respectively. The extracellular domain of FXYD5 (M1-R145) was also subcloned into pET28 vector upstream and in frame with a 6XHis tag and expressed in E. CoIi. For expression in Xenopus oocytes cDNAs were subcloned between 5' and 3' sequences of Xenopus β globin in pGEM or pBluescript derived vectors (18). cRNAs were synthesized from linearized plasmids using T7 RNA polymerase. All constructs were verified by sequencing.

Antibodies: Polyclonal antibodies against C-terminal peptides of FXYD4 and FXYD5 were described before (9,19). In addition, a monoclonal antibody was prepared against a GST-FXYD5 fusion protein expressed in E. CoIi. The antibody was found to react with an N-terminus epitope on FXYD5. Antibody to the N terminus of the αl subunit of Na⁺/K⁺-ATPase (6H) was kindly provided by Dr. M.J. Caplan, Yale University School of Medicine. Antibody to the β subunit was described in (20). Monoclonal anti Hemagglutinin A (HA)¹ antibody was purchased from Santa Cruz

Tissue and cell preparations: Mice (ICR) were euthanized using CO₂ gas and various organs were excised and rinsed in ice cold HSE buffer composed of: 250 mM sucrose, 25 mM histidine, 1 mM EDTA, pH 7.2 and a cocktail of protease inhibitors (1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin A). They were cut into small pieces and homogenized using a Polytron PT 2100 (Kinematica Switzerland, 4X6 sec pulses at setting 31). Tissue homogenates were sedimented for 15 min at 4000 g at 4 ⁰C to remove cell debris. The supernatants were further centrifuged for 90 min at 20,00Og at 4 ⁰C. The supernatants (cytosol) were saved and the pellets (membranes) were suspended in HSE buffer + protease inhibitors. Protein content was determined by the method of Lowry.

HeLa cells cultured under standard conditions, were transiently transfected with FXYD5 using polyfect (QIAGEN) and assayed for protein expression after reaching confluency. NaVK⁺- ATPase was purified from pig kidney medulla as described in (21). A cell suspension highly enriched in dendritic cells was obtained from spleens of C57B1/6 mice using monoclonal antibodies to CDl Ic attached to beads (Miltenyi Biotec, Bergisch Gladbach) according to the manufacturer's protocol.

RT-PCR: RNA was isolated from mouse kidney medulla and cortex or dendritic cells using TRI Reagent (Molecular Research Center, inc.). Reverse transcription was performed with the Super-Script™ II Reverse Transcriptase kit for RT-PCR (Invitrogen) according to the manufacturer's instructions using 1.0 μg of total RNA. Two sense primers denoted I and III were used as summarized in Table 1 herein below.

Table 1

The first corresponds to a coding sequence of mouse FXYD5 upstream the FXYD motif and the second to a 5'FXYD5 sequence. Antisense primers (II and IV in Table 1) correspond to the 3' sequences of different transcripts predicted by est entries. PCR products were ligated into pGEM^R-T Easy vector (Promega) and sequenced from the vector ends.

Heterologous expression in Xenopus oocytes: Batches of stage V-VI oocytes were injected with aliquots of 50 nl containing 10 ng rat αl cRNA, 7 ng pig lOXHis tagged βl cRNA and 3 ng cRNA transcribed from different FXYD constructs. The oocytes were incubated for 3 days at 20 ⁰C and used for assaying surface expression of the pump, co-precipitation of pump/FXYD complexes and Na⁴VK⁺- ATPase activity. For co-precipitation assays, oocytes were homogenized in a glass Teflon homogenizer in a buffer containing 10 mM HEPES pH 7.9, 83 niM NaCl, 1 mM MgCl₂, and protease inhibitor cocktail (ImM PMSF, 20 μg/ml Leupeptin, and 20 μg/ml Pepstatin A). Homogenates were first centrifuged twice at 1,000 g for 10 min at 4⁰C for yolk removal and then at 10,000 g for 20 min to yield a microsomal pellet that contained ~90 % of the heterologously expressed proteins. Membranes were stored at -80 °C in 10 mM MOPS- Tris (pH 7.2), 1 mM EDTA, 25 % glycerol and protease inhibitor cocktail.

Co-precipitation of FXYD proteins and the Na⁺ZK⁺-ATPaSe: Co-precipitation assays were performed in oocytes expressing various FXYD constructs together with αl and lOXHis tagged βl. Unless otherwise indicated, membranes were first solubilized in a buffer containing 5 mM Tris pH 7.6, 10 mM RbCl and 1 mg/ml C₁₂E₁₀. The detergent solubilized membranes were centrifuged for 30 min at 50,000xg, the supernatant collected and RbCl and Imidazol were added to final concentrations of 100 mM and 20 mM, respectively. For co-precipitation, the detergent solubilized proteins were incubated overnight under swirling at 4 ⁰C with Ni⁺² -NTA beads (lOμl beads/ lmg protein). Beads were sedimented and washed 3 times in solubilization buffer containing 1 mg/ml Ci₂E)₀, 20 mM Imidazole, 5 mM Tris pH 7.6 and 100 mM RbCl. Bound proteins were eluted with 250 mM imidazole and dissolved in SDS sample buffer. The eluted proteins and an aliquot of the total detergent solubilized membranes were resolved on either 10 % or 7.5 % Acrylamide Tris/Tricine/SDS gels. Proteins were transferred to PVDF membranes in CAPS buffer plus 10 % methanol at 13V for 90 min. The blots were blocked in 5 % milk for Ih at room temperature and cut to several pieces according to predicted sizes of the bands of interest. These were incubated overnight at 4 ⁰C with one of the following antibodies: anti FXYD5 (either polyclonal or monoclonal, 1:500), anti FXYD4 (1 :500), anti α (1:1000), anti β (1:4000), or anti HA (1 :1000). Bound antibodies were visualized by ECL following binding with HRP coupled goat anti rabbit or goat anti mouse IgG (Ih, RT, 1 :5000). Each observation was confirmed in at least three independent experiments.

Biotinylation of surface expressed proteins: Groups of 15-20 oocyte were incubated under gentle rotation for 1 hr. at 4 ⁰C with 1 mM of freshly made sulfo-NHS- SS-biotin (Pierce). Incubation was performed in ND94 medium composed of: 94 mM

NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES pH 7.4. They were washed 4 times with cold ND94 and homogenized in a buffer composed of: 20 mM Tris, 5 mM MgCl₂, 5 mM Na₂HPO₄, ImM EDTA, 80 mM sucrose and protease inhibitors (ImM PMSF, 20 μg/ml Leupeptin, and 20 μg/ml Pepstatin A) pH 7.4. Homogenization was performed on ice by passing oocytes 6 times through a 21 gauge needle, and another 6 times through a 27 gauge needle. Homogenates were centrifuged at 200 g for 5 min at 4 ⁰C to pellet the yolk and the supernatants were then centrifuged for 20 min at 14,000 g at 4 ⁰C to separate membranes and cytosol. The membrane fractions were dissolved in TBS-Triton buffer composed of: 150 mM NaCl, 10 mM Tris pH 7.5, 1 % Triton X-100, incubated on ice for 30 min and centrifuged for 20 min at 14,000 g at 4⁰C. The Triton solubilized membrane proteins obtained from 15-20 oocytes were incubated under gentle rotation for Ih at 4⁰C with 50 μl streptavidin agarose beads (Pierce). The beads were precipitated by brief centrifugation, washed 3 times with TBS- Triton buffer, suspended in SDS sample buffer and heated for 5 min at 90 ⁰C to release the biotinylated proteins. These proteins were resolved electrophoretically and analyzed on Western blot as above.

Lectin binding assay: Purified pig kidney Na⁺/K⁺-ATPase and FXYD5 expressing oocytes were homogenized in 1 % Triton X-100, 150 mM NaCl and 50 mM Hepes (pH 7.4) with or without 200 mM N-Acetylglucosamine (Vector Laboratories). The insoluble fraction was removed by centrifugation, and the soluble proteins were incubated with agarose wheat germ agglutinin beads (Vector Laboratories) overnight at 4 ⁰C with rotation. Beads were washed 3 times with the incubation buffer and another 3 times with water. Immobilized proteins were then eluted with 10OmM acetic acid and analyzed by probing with anti FXYD5 antibody.

Na⁺ZX⁺-ATPaSe mediated ⁸⁶Rb⁺ fluxes in oocytes: Effects of FXYD proteins on the pump activity were measured as the initial rate of the ouabain sensitive ⁸⁶Rb⁺ uptake as detailed in (9). Measurements were performed in the presence of either 10 μM ouabain, which fully blocks the endogenous Xenopus pump but not the expressed rat Na⁺/K⁺-ATPase, or 2 mM ouabain which blocks both orthologs. Oocytes were first loaded with Na⁺ for 2 hours by incubation in a K⁺ free medium composed of: 80 mM Na-gluconate, 0.82 mM MgCl₂, 0.41 mM CaCl₂, 10 mM NMDG-HEPES pH 7.4, 5 mM BaCl₂ and 10 mM tetraethyl ammonium chloride. Na⁺ loaded oocytes were divided into two groups of 7-8 oocytes and incubated for an additional 15 min at 25 ⁰C in a solution containing 90 mM NaCl, 1 niM CaCl₂, 1 mM MgCl₂, 5mM BaCl₂, 10 mM HEPES pH 7.4 and either 10 μM or 2 mM ouabain. ⁸⁶Rb⁺ uptake was then initiated by the addition of 5 mM KCl + 5 μCi/ml ⁸⁶RbCl. The uptake was stopped 12 min later by a 15 fold dilution and 5 washings in an ice-cold incubation solution containing 5 mM nonradioactive RbCl. Oocytes were then counted individually for ⁸⁶Rb⁺ uptake.

Separation of membrane from soluble fraction: Confluent cells, grown on Petri dishes, were washed with cold PBS, scraped and centrifuged at 200 xg 5 minutes at 4 ⁰C. After resuspention in Lysis buffer: 50 mM TrisHCl pH 8, 2.5 mM MgCl₂, ImM EDTA+ mixture of protease inhibitors (ImMPMSF, 10mg/ml aprotinin, 10 mg/ml leupeptin, 2 mg/ml pepstatin A) cells were homogenized using a Polytron homogenizer (Kinematica Switzerland, four times with 6-s pulses at setting 10). Cell homogenates were sedimented for 5 minutes at 200 xg at 4 ⁰C. The supernatants were further centrifuged for 20 min at 60,000 rpm at 4 °C. The pellets (membranes) were suspended in lysis buffer with protease inhibitors. Protein content was determined by the method of Lowry.

For Western blotting, membrane protein samples (15 μg) were suspended in

SDS sample buffer and resolved on 7.5 % Tricine gel. Proteins were transferred by semi-dry blotting onto PVDF membranes in CAPS buffer plus 10 % methanol at 13 V for 90 minutes. The blots were blocked in 5 % milk for 1 hour at room temperature and then incubated overnight at 4 °C with anti-FΛΥD5 (1 :500). Protein bands were visualized by ECL using horseradish peroxidase-coupled goat anti-mouse IgG.

RNA Interference: The siRNA sequence (SEQ ID NOs: 11) chosen to target human FXYD5 were at position 30-51 in the nucleotide sequence. The basic strategy for design of oligo's was based on using pSUPER RNAi System according to the manufacture instructions.

H 1299 cells were transfected using jetPEI DNA transfection reagent with sequences as set forth in SEQ ID NOs: 7 and 8. Positive clones were selected using G- 418 antibiotics at the concentration of 1 mg/ml.

EXAMPLE 1

FXYD5 splice variants

FXYD5 sequences deposited in public databases indicate the possible existence of at least two FXYD5 isoforms which differ in their carboxy tails. The previously reported FXYD5 sequence is characterized by a very short cytoplasmic C-terminal sequence of only 15 amino acids (upper sequence in Figure IA). At least 15 mouse EST entries from different cDNA libraries predict a 92 nucleotide deletion within the last exon, which will eliminate the original stop codon and produce a longer protein with ten additional C-terminal residues (middle sequence of Figure IA). Such an isoform however is not observed in publicly available human EST entries. RT-PCR has been used to confirm expression of this new variant. Primers I and II in Table I should amplify 396 and 307 base pair products corresponding to the shorter and longer C- terminal proteins, respectively. Amplifying mouse kidney RNA resulted in a 396 bp species only (left arrow in Figure IB). Since some of the est entries predicting the long form came from a dendritic cell library, amplification of this form from dendritic cell RNA was also attempted. In this case, a minor smaller fragment was observed in addition to the predominant 396 bp species (right arrow in Figure IB). It was sequenced and found to correspond to the new variant with ten extra C-terminal amino acids. The fact that this species was amplified from dendritic but not kidney RNA does not necessarily indicate differential expression of the two forms. It may be due to the large heterogeneity of the kidney and a much lower fraction of FXYD5 -expressing cells, resulting in a lower abundance of this species in the total RNA preparation. Since none of the EST entries predicting the new species contains the whole coding region, it is in principle possible that this isoform differs also in its N-terminus. To test for this possibility RT-PCR of dendritic cells was repeated using primer III corresponding to a sequence that is upstream of the AUG start codon of FXYD5. The product sequence showed no N-terminal differences and confirmed that the additional C-terminal residues are the only difference between the two isoforms. An FXYD5 protein with an entirely different C-terminus is reported in

Swissprot (P97808-1, bottom sequence in Figure IA). This sequence is predicted by a single mouse EST entry (BC031112). This species was amplified from dendritic cell RNA using primers I and IV (asterisk in Figure IB). These primers amplified a very low abundance 413 bp product with a sequence identical to that of BC031112 and P97808-1. Aligning BC031112 with the genomic sequence of FXYD5 indicates that this species is likely to represent an incompletely spliced mRNA in which the intron between exons 8 and 9 is still present, and not necessarily a different protein. EXAMPLE 2

Monoclonal anti-FXYDS antibody

Previously, it was shown that FXYD5 is expressed in a variety of tissues as a ~24 kDa polypeptide that can be co-precipitated with the α and β subunits of Na⁺/K⁺- ATPase (9). These data differ from other reports suggesting that FXYD5 is a heavily O- glycosylated polypeptide of 50-55 kDa (10-13). Since the data suggesting that FXYD5 in native tissue is a ~24 kDa polypeptide was obtained using a polyclonal antibody, the present inventors raised a second, monoclonal antibody against recombinant FXYD5.

The hybridoma producing monoclonal antibodies specific for FXYD5 prepared on the 6, 2006, at a cell concentration of 10⁶ was sent to the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25, Re de Docteur Roux, F- 75724 Paris, CEDEX on Sept. 1, 2008. It was registered on September 2, 2008 with the name and address of Professor Haim Garty, Dept Biological Chemistry of the Weizmann Institute of Science, Rehovot, 76100, Israel, with the following reference number: 4Bl 1. The registration number given by the CNCM of the biological material was CNCM 1-4069.

To confirm its specificity and identify the protein region interacting with this antibody, the present inventors constructed various FXYD5/FXYD4 chimera, expressed them in Xenopus oocytes, and probed them with different antibodies. All FXYD constructs were successfully expressed as evidenced by their labeling by polyclonal antibodies directed to the C -terminal sequences of either FXYD4 or FXYD5 (Figure 2A). The size of the expressed proteins was either 24 kDa or 7kDa depending on the origin of the ecto domain. Blotting the same preparations with the new monoclonal anti- FXYDS antibody demonstrates that this antibody is directed to an extracellular N- terminal sequence (Figure 2B). It recognizes all FXYD5/FXYD4 chimera, of which the N-terminal sequences originate from FXYD5 (i.e. RRC, RCC, and RCR), but none of the other chimera. The fact that the monoclonal antibody recognizes an extracellular epitope was further confirmed by Western blotting of lysates from E. CoIi transformed with the extracellular domain of FXYD5. In this case the antibody recognized a -2OkDa band in IPTG stimulated but not in non-stimulated cells (Figure 2C). The new antibody interacts weaker with FXYD5 than the previously described polyclonal anti-C tail antibody (cf Figure 2A vs. 2B). Yet, since it recognizes an extracellular epitope it should cross-react with all three isoforms depicted in Figure IA. Another observation in these experiments was that the co-expression of αβ with FXYD5 and some FXYD5/FXYD4 chimera tends to reduce glycosylation of the β subunit. This is apparent from the increased electrophoretic mobility as compared to the fully glycosylated form of β, and is particularly notable when FXYD constructs with extracellular and transmembrane segments originating from FXYD5 are co-expressed with αβ (Figure 3, asterisks).

EXAMPLE 3 Tissue distribution and glycosylation ofFXYDS The present inventors next tested expression of FXYD 5 in different tissues using the new antibody. The antibody labeled mainly a -24 kDa polypeptide whose electrophoretic mobility and tissue distribution are similar to those reported with the polyclonal antibody (9) (Figure 4A). In addition, a faint high MW species was observed in some, but not all membrane preparations. This could, in principle, be a highly glycosylated form. The possibility cannot be excluded that since the monoclonal antibody is directed to the ecto domain of FXYD5 it does not, or only poorly, cross- reacts with the glycosylated form of the protein. However, since the same results were obtained with an antibody to the intracellular C-terminal sequence, this possibility cannot account for the lack of a major 50-55 kDa band. Another possibility considered is that the heavily glycosylated species is specific to the splice variant with ten extra C- terminal residues. Such species may fail to be detected also by the anti C-terminus antibody since the additional amino-acids are at the immediate vicinity of the sequence against which the antibody was raised. Accordingly, the present inventors have transfected HeLa cells with the long form of FXYD5 that was tagged in its extracellular domain by an HA epitope, and assayed for the size of the protein expressed by Western blot with an anti HA antibody. In this case too only a 24 kDa species was detected (Figure 4B). Moreover, it was found that the anti-C terminus antibody does cross-react with this protein, which argues against the possibility that this isoform escapes detection by the anti C-terminus antibody in native membranes. These results convincingly demonstrate that normal tissue lacks the 55 kDa highly glycosylated form of FXYD5 and that this form is specific to metastatic cells.

While the above experiments show no major 50-55 kDa FXYD5 species in normal tissue or transfected cells, some O-glycosylation of this protein is likely due to the high abundance of S, T, and P residues and its weak homology to mucins.

Glycosylation may also account for the difference between the apparent MW of the protein (24 kDa) and the calculated value of 17 kDa (assuming cleavage of the signal peptide). FXYD5 lacks N-glycosylation sites and its electrophoretic mobility is not affected by treatment with peptide 7V-glycosidase. (9). Another way to test for the presence of sugar moieties is to assay for lectin binding. In this case detergent solubilized membrane proteins are incubated with lectins covalently attached to agarose beads in the presence and absence of competing sugars, and the beads are assayed for immobilized protein. Such an assay requires dissociation between FXYD5 and the NaVK⁺- ATPase in order to avoid FXYD5 binding through the heavily glycosylated β subunit of the pump. The present inventors therefore first tested for stability of the αβ/FXYD5 complex in various detergents. It was found that while FXYD5 effectively co-precipitates with αβ when membranes are solubilized in C₁₂Ei₀ (see (9) and Figure 6) no such co-precipitation is apparent in Triton X-IOO (Figure 5A). Accordingly, lectin binding experiments were done in Triton X-100 solubilized membrane. Incubating Triton X-100 solubilized membranes from oocytes expressing αβ and FXYD5 with agarose wheat germ agglutinin beads resulted in binding of FXYD5 to the beads (Figure 5B). Such binding could be prevented by the presence of 200 mM N-Acetylglucosamine (GlcNac), suggesting that it is mediated by a specific interaction of the protein sugar moieties with the beads. To test for glycosylation in native tissue purified pig kidney Na⁺/K⁺- ATPase was prepared as described in (21). This preparation contains considerable amount of FXYD5 although it is more enriched with α (Figure 5C). In this case too FXYD5 could be immobilized on lectin beads in the absence but not in the presence of GlcNac (Figure 5D). Thus, FXYD5 is indeed glycosylated, but this presumably O-glycosylation does not evoke a large increase in size.

EXAMPLE 4 FXYD 5-Na⁺ZK" -ATPase interactions

The above FXYD5/FXYD4 chimeras have also been used to identify FXYD5 domains participating in the interaction with the αβ subunits of the Na⁺/K⁺- ATPase. It has been shown before that the detergent solubilized αβ/FXYD5 complex is more stable than the corresponding αβ/FXYD4 oligomer, and efficient α-FXYD5 co- immunoprecipitation is observed under more stringent conditions than those which preserve interactions between αβ and FXYD4 (9). Thus, comparing co-precipitation efficiencies of different FXYD4/FXYD5 chimera with αβ should identify the FXYD5 domain responsible for the extra stability of the complex in detergent. Accordingly, FXYD4 and FXYD4/FXYD5 chimera were expressed in Xenopus oocytes together with α and lOXHis tagged β. Microsomes were extracted and dissolved in Ci₂Ei₀ and pump complexes were isolated using Ni-NTA beads. These were assayed for the relative amounts of α, β and FXYD on Western blots. The data summarized in Figure 6 A demonstrate that the high co-precipitation efficiency of FXYD5 is determined by its transmembrane domain. Thus, chimera with transmembrane domains originating from FXYD5 (RRC and CRC) co-precipitate with αβ at high efficiency, irrespective of the origin of the N-terminus. A similar result was obtained before by comparing co- immunoprecipitation efficiencies of αβ/FXYD4 and αβ/FXYD2 (22). In this case it was established that the extra stability of αβ/FXYD2 is due to three transmembrane residues. In FXYD4 these residues are G41, M55 and A56. Mutating them to the corresponding FXYD2 residues (A, I and L) resulted in an αβ/FXYD4 complex with stability comparable to that of αβ/FXYD2. In FXYD5, the equivalent positions are A150 1160 and L161 i.e. the same amino acids found in FXYD2. Accordingly, the present inventors have mutated them to the FXYD4 residues (A150G, I160M and Ll 61 A) and assayed for effects on complex stability. As seen in Figure 6B a FXYD5 construct carrying these mutations alone or in combination, could still be precipitated by αβ but the co-precipitation efficiency was considerably lower than that of the wild type. Thus, the structural interactions between FXYD5 and the NaVK⁺- ATPase appears to be similar to that of FXYD2 and 4.

EXAMPLE 5

Structure-function relationships of FXYDS

FXYD4/FXYD5 chimeras have also been used to identify the domain(s) involved in the functional effects of FXYD5. Previously, it was shown that expressing FXYD5 in Xenopus oocytes together with αβ increases the V_max of the pump, an effect not seen for FXYD4 (9). This is further demonstrated in Figure 7A which depicts the initial rates of ⁸⁶Rb⁺ uptake measured in the presence of 10 μM or 2 mM ouabain in oocytes expressing Na⁺/K⁺-ATPase and different FXYD proteins. Since under the experimental conditions, intracellular Na⁺ and extracellular K⁺ are much higher than the pump Ki_/2 values for these ions, changes in the initial rate of ouabain- sensitive ⁸⁶Rb⁺ uptake reflect changes in V_max. Figure 7A also demonstrates that a chimera in which the transmembrane domain originates from FXYD5 and the extracellular and intracellular domains come from FXYD4, has a ouabain sensitive ⁸⁶Rb⁺ uptake like that of FXYD5 itself. This is further demonstrated in Figure 7B which compares pump activities in oocytes injected with chimera in which the extracellular, transmembrane, and intracellular domains of FXYD4 were replaced by the corresponding FXYD5 domain. Again, the transmembrane domain of FXYD5 evokes the increase in pump V_max, while the cytoplasmic or extracellular domains have no effect. Interpretation of these data depends however on the assumption that the different FXYD constructs have no effect on the pump surface expression so that a change in the pump mediated ⁸⁶Rb⁺ uptake does not stem from a change in the number of surface expressed pump units. To confirm this assumption the surface expression of αβ was determined by biotinylating the surface of intact oocytes and quantifying the amount of extracted α and β proteins that are immobilized on streptavidin beads. As demonstrated at the bottom of Figure 7B, the surface expression of NaVK⁺- ATPase was not affected by the co-expression of various FXYD proteins and the amount of α and β bound to streptavidin were similar or even lower in oocytes expressing RCR. Thus, FXYD5 does indeed increase V_max and the effect is mediated by its transmembrane domain.

EXAMPLE 6 Analysis ofFXYDS in the human non-small lung carcinoma cell line H1299

FXYD5 is known to be expressed in human non-small lung carcinoma cell line [J Thorac Cardiovasc Surg. 2005 Sep;130(3):740-5]. Further it is known that FXYD5 is abundant in normal lung (Figure 4A). Blotting H 1299 cell lysate with the anti FXYD5 monoclonal showed a predominant 50 kDa band (Figure 8 lanes 1,2 marked by arrow). To verify that this 5OkDa band is indeed FXYD5, FXYD5 was silenced using siRNA primers as set forth by SEQ ID NOs: 7 and 8 and the pSUPER RNAi system. Two cell clones in which FXYD5 mRNA were much diminished were isolated (Figure 9). As seen in lanes 3, 4 of Figure 8, the 50 kDa band recognized by the antibody is much weaker in these cell clones. Thus the antibody does indeed recognize 50 kDa polypeptide in malignant cells and 24 kDa species in normal cell. EXAMPLE 7 Silencing FXYD5 in H1299 cells

The present inventors also tested whether silencing FXYD5 in Hl 299 cells affected their ability to adhere to the surface. Accordingly, wild type and FXYD5 silenced cells were seeded and cell adhesion and propagation was followed overnight using delta- vision microscope. In the first 10 hours after seeding, cells in which FXYD5 was silenced remained mostly round while those expressing FXYD5 converted from round to polar morphology and expressed many more pod like projections (Figure 10). Thus, silencing or inhibiting FXYD5 affects cell adherence and motility.

DISCUSSION

The current study provides further characterization of FXYD5 in native tissue and expression systems. Using a monoclonal antibody to an extracellular epitope the present inventors confirmed that in normal tissue FXYD5 exists as a -24 kDa polypeptide that is particularly expressed in spleen, lung, kidney and heart. These findings are different from previous reports by Hirohashi and co-workers (10-13) who reported that FXYD5 is a heavily O-glycosylated 50-55 kDa polypeptide that is expressed in metastatic but not normal cells. In principle, it is possible the monoclonal antibody used by Hirohashi and co-workers detects a 50-55 kDa cancer-related glycoprotein that is different from FXYD5. However, some of the data reported were obtained using siRNA making a strong link between the FXYD5 related sequence and the 50-55 kDa protein (12). The most likely explanation for the above discrepancy is that FXYD5 has different degrees of O-glycosylation in normal and cancer cells. It is well known that transformation to malignancy is associated with a change in O- glycosylation of adhesion molecules and in particular mucins (e.g. (23-26)). Also the excessive glycosylation of mucins is known to block binding to adhesion proteins and hence, down-regulate E-cadherin. Some glycosylation of the 24 kDa species is apparent since this protein specifically associates with wheat germ agglutinin beads. Previously it was shown that treating FXYD5 containing membranes with peptide N-glycosidase has no effect on the electrophoretic mobility of this protein (9). In addition, the ecto domain of FXYD5 lacks consensus Ν-glycosylation sites. Taken together with the fact that 50 out of the 122 extracellular residues of this domain are serines, threonines and prolines, it is likely that some of these residues are O-glycosylated. Such glycosylation may account at least for part of the difference between the apparent molecular weight of 24 kDa and the calculated value of 17 kDa (assuming cleavage of the signal peptide).

FXYD5/FXYD4 chimera expressed in Xenopus oocytes have been used to study structure-function relationships and structural interactions between FXYD5 and the αβ subunits of NaVK⁺- ATPase. Differences in stabilities of αβ/FXYD5 and αβ/FXYD4 complexes in detergent have been used to identify domains and residues involved in the above interactions. As observed before for FXYD2, FXYD4 and FXYD7 (22,27,28) transmembrane interactions play a key role in the association of FXYD5 with αβ. It was further demonstrated that three transmembrane residues shown before to determine stability of the αβ/FXYD4 complex in detergent play a similar role in the interaction of FXYD5 with the pump. Taken together the data suggest that different FXYD proteins interact similarly with the Na⁺/K⁺-ATPase and their different functional effects are likely to stem from a small number of key residues. It was of interest that co-expression with FXYD5 decreases to some extent glycosylation of the β subunit. The somewhat lower broad band seen under these conditions is similar to that observed following sialidase treatment (data not shown) Such an effect may indicate higher ER retention of the β subunit when co-expressed with FXYD5. However, experiments determining the amount of surface biotinylated β seem to argue against such a possibility. Another possibility is a direct FXYD5-β interaction which hinders glycosylation to some extent. FXYD5 might interfere with addition of sialic acid resides at all sites or interfere with glycosylation at one or another of the three sites. A direct FXYD5-β interaction would fit well with the previous finding that both FXYD2 and FXYD4 can be specifically crosslinked to the extracellular domain of β (29). Interference with glycosylation of the β subunit might also explain correlation between the expression of FXYD5 and metastasis. Epithelial cell-cell adhesion and cell polarity is known to require E-cadherin (30) and the β subunit of the Na⁺/K⁺-ATPase is also known to be essential for E-cadherin mediated cell-cell adhesion, polarity and also suppression of invasiveness of cancer cells (31). It has been shown recently that normal glycosylation of the β subunit is essential for production of stable association of the pump with the adherens junction and play an important role in cell-cell contact formation (32). Thus, interference of FXYD5 with glycosylation of the β 1 subunit might destabilize its interactions with E-cadherin and the adherens junction, and thus account for the lowered expression of E-cadherin, decreased cell-cell contact, increased motility and metastasis. By the same token, an antibody to the extracellular domain of FXYD5 which will impair FXYD5-β interaction is expected to inhibit metastasis.

As before (9), it was found that co-expression of FXYD5 with the NaVK⁺- ATPase increases the V_max of the pump by about two fold. In principle, the increase in V_max may stem from a higher cell surface expression of αβ. This possibility was however excluded by quantifying the amount of surface biotinylated pump units in intact oocytes. It is therefore apparent that FXYD5 increases the turnover rate of the pump. Such an effect can be the result of an increase in the rate of one of the rate limiting conformational changes i.e. E₁P-^E₂P or E₂(K)ATP->E₁ATP. Such effects are usually accompanied by changes in the apparent affinity to ligands. However, FXYD5 was found to have no effect on Ki_/2 to external K^+> and effects on affinities to Na⁺ and ATP cannot be readily measured in the current system. It was further demonstrated that the increase in V_max is mediated by the transmembrane domain of FXYD5. This is similar to the influence of the transmembrane domains of FXYD2 and 4 on the apparent affinity to cell Na⁺ (22,28,34). On the other hand the effect on the pump's affinity to ATP, involves the carboxy terminal of FXYD2 (35). Finally, at least one FXYD5 splice variant was identified with ten extra residues in the carboxy tail of the protein. Functional effects of these residues have not been tested since the increase of V_max is evoked by the transmembrane helix. Yet, it is possible that the new variant affects affinity to ATP. FXYD splice variants have been reported before for FXYD2 and FXYD3 (36,37). In both cases differential expression of the two isoforms were observed, and in the case of FXYD3 different functional effects were noted as well.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications and GenBank Accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application or GenBank Accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

REFERENCES

1. Garty, H., and Karlish, S. J. (2006) Annu Rev Physiol 68, 431 -459

2. Sweadner, K. J., and Rael, E. (2000) Genomics 68, 41-56.

3. Cornelius, F., and Mahmmoud, Y. A. (2003) News Physiol Sci 18, 119-124.

4. Geering, K. (2006) Am J Physiol Renal Physiol 290, F241 -F250

5. Song, J., Zhang, X. Q., Carl, L. L., Qureshi, A., Rothblum, L. I., and Cheung, J. Y. (2002) Am J Physiol Heart Circ Physiol 283, H576-H583.

6. Zhang, X. Q., Qureshi, A., Song, J., Carl, L. L., Tian, Q., Stahl, R. C, Carey, D. J., Rothblum, L. L, and Cheung, J. Y. (2003) Am J Physiol Heart Circ Physiol 284, H225-H233.

7. Ahlers, B. A., Zhang, X. Q., Moorman, J. R., Rothblum, L. L, Carl, L. L., Song, J., Wang, J., Geddis, L. M., Tucker, A. L., Mounsey, J. P., and Cheung, J. Y. (2005) J Biol. Chem. 280, 19875-19882

8. Wang, J., Zhang, X. Q., Ahlers, B. A., Carl, L. L., Song, J., Rothblum, L. L, Stahl, R. C, Carey, D. J., and Cheung, J. Y. (2006) J Biol Chem 281, 32004- 32015

9. Lubarski, L, Pihakaski-Maunsbach, K., Karlish, S. J., Maunsbach, A. B., and Garty, H. (2005) J Biol. Chem. 280, 37717-37724

10. Ino, Y., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc Natl Acad Sci USA 99, 365-370.

11. Tsuiji, H., Takasaki, S., Sakamoto, M., Irimura, T., and Hirohashi, S. (2003) Glycobiology 13, 521-527.

12. Shimamura, T., Yasuda, J., Ino, Y., Gotoh, M., Tsuchiya, A., Nakajima, A., Sakamoto, M., Kanai, Y., and Hirohashi, S. (2004) Cancer Res 64, 6989-6995

13. Shimamura, T., Sakamoto, M., Ino, Y., Sato, Y., Shimada, K., Kosuge, T., Sekihara, H., and Hirohashi, S. (2003) J Clin Oncol 21, 659-667.

14. Batistatou, A., Scopa, C. D., Ravazoula, P., Nakanishi, Y., Peschos, D., Agnantis, N. J., Hirohashi, S., and Charalabopoulos, K. A. (2005) Br J Cancer 93, 1382-1387

15. Sato, H., Ino, Y., Miura, A., Abe, Y., Sakai, H., Ito, K., and Hirohashi, S. (2003) JCHn Endocrinol Metab 88, 4407-4412.

16. Shimada, Y., Yamasaki, S., Hashimoto, Y., Ito, T., Kawamura, J., Soma, T., Ino, Y., Nakanishi, Y., Sakamoto, M., Hirohashi, S., and Imamura, M. (2004) Clin Cancer Res 10, 2818-2823.

17. Aoki, S., Shimamura, T., Shibata, T., Nakanishi, Y., Moriya, Y., Sato, Y., Kitajima, M., Sakamoto, M., and Hirohashi, S. (2003) Br J Cancer 88, 726-732.

18. Liman, E. R., Tytgat, J., and Hess, P. (1992) Neuron 9, 861-871

19. Garty, H., Lindzen, M., Scanzano, R., Aizman, R., Fuzesi, M., Goldshleger, R., Farman, N., Blostein, R., and Karlish, S. J. (2002) Am J Physiol Renal Physiol 283, F607-F615.

20. Shimon, M. B., Goldshleger, R., and Karlish, S. J. (1998) J Biol Chem 273, 34190-34195

21. Jorgensen, P. L. (1988) Methods Enzymol 156, 29-43

22. Lindzen, M., Aizman, R., Lifshitz, Y., Lubarski, L, Karlish, S. J., and Garty, H. (2003) J Biol Chem 278, 18738-18743

23. Burchell, J. M., Mungul, A., and Taylor-Papadimitriou, J. (2001) J Mammary Gland Biol Neoplasia 6, 355-364

24. Burdick, M. D., Harris, A., Reid, C. J., Iwamura, T., and Hollingsworth, M. A. (1997) J Biol Chem 272, 24198-24202 25. Lloyd, K. O., Burchell, J., Kudryashov, V., Yin, B. W., and Taylor- Papadimitriou, J. (1996) J Biol Chem 111, 33325-33334

26. Brockhausen, L, Yang, J. M., Burchell, J., Whitehouse, C, and Taylor- Papadimitriou, J. (1995) Eur JBiochem 233, 607-617

27. Li, C, Crambert, G., Thuillard, D., Roy, S., Schaer, D., and Geering, K. (2005) J Biol Chem 280, 42738-42743

28. Li, C, Grosdidier, A., Crambert, G., Horisberger, J. D., Michielin, O., and Geering, K. (2004) J Biol Chem 279, 38895-38902

29. Fuzesi, M., Gottschalk, K. E., Lindzen, M., Shainskaya, A., Kuster, B., Garty, H., and Karlish, S. J. (2005) J Biol Chem 280, 18291-18301

30. Piepenhagen, P. A., and Nelson, W. J. (1998) MoI Biol. Cell 9, 3161-3177

31. Rajasekaran, S. A., Palmer, L. G., Quan, K., Harper, J. F., Ball, W. J., Jr., Bander, N. H., Peralta Soler, A., and Rajasekaran, A. K. (2001) MoI. Biol. Cell 12, 279-295

32. Vagin, O., Tokhtaeva, E., and Sachs, G. (2006) J. Biol. Chem. 281, 39573- 39587

33. Crambert, G., Li, C, Claeys, D., and Geering, K. (2005) MoI Biol Cell 16, 2363- 2371

34. Zouzoulas, A., Therien, A. G., Scanzano, R., Deber, C. M., and Blostein, R. (2003) J Biol Chem 278, 40437-40441

35. Therien, A. G., Karlish, S. J., and Blostein, R. (1999) J Biol Chem 274, 12252- 12256.

36. Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. (2000) J Biol Chem 275, 18441-18446

37. Bibert, S., Roy, S., Schaer, D., Felley-Bosco, E., and Geering, K. (2006) J Biol Chem 281, 39142-39151

Claims

WHAT IS CLAIMED IS:

1. A monoclonal antibody capable of interacting with a FXYD5 polypeptide of about 24 kDa and a FXYD5 polypeptide of about 55 kDa.

2. The monoclonal antibody of claim 1, wherein the antibody interacts with an extracellular portion of said FXYD5 polypeptide.

3. The monoclonal antibody of claim 2, wherein said FXYD5 polypeptide is O-glycosylated.

4. The monoclonal antibody of claim 1, wherein said FXYD5 polypeptide is expressed in a non-cancerous cell.

5. The monoclonal antibody of claim 1, wherein said FXYD5 polypeptide is expressed in a cancerous cell.

6. The monoclonal antibody of claim 4, wherein said non-cancerous cell is selected from the group consisting of spleen, lung, kidney, colon and heart.

7. The monoclonal antibody of claim 1, being humanized.

8. The monoclonal antibody of claim 1, being a neutralizing antibody.

9. A monoclonal antibody having the deposit number CNCM 1-4069.

10. A method of diagnosing cancer, the method comprising analyzing a glycosylation of FXYD5, wherein an alteration in said glycosylation of said FXYD5 in cancerous versus non-cancerous cells is indicative of the cancer.

11. The method of claim 10, wherein said analyzing is effected using the antibody of claim 1.

12. The method of claim 10, wherein said analyzing is effected using the antibody of claim 9.

13. The method of claim 11, wherein said analyzing is further effected using an antibody capable of recognizing a 55kDa FXYD5 and not a 24 kDa FXYD5.

14. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the monoclonal antibody of claim 8, thereby treating the cancer.

15. The method of claim 14, wherein the antibody has a deposit number CNCM 1-4069.

16. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an siRNA comprising a polynucleotide sequence as set forth by SEQ ID NO: 11, thereby treating the cancer.

17. The method of claim 14, wherein the cancer is a metastatic cancer.

18. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an aptamer capable of interacting with an extracellular portion of FXYD5, thereby treating the cancer.

19. The method of claim 18, wherein the cancer is a metastatic cancer.

20. An aptamer which binds the extracellular portion of FXYD5.

21. A method of identifying a cancer cell, the method comprising analyzing a glycosylation of FXYD5, wherein an increase in said glycosylation of said FXYD5 in a cell as compared to a non-cancerous cell is indicative of the cancer cell.