AU2008201545B2 - Glycoprotein antigen SIMA135 expressed in metastatic human tumor cells - Google Patents

Abstract

Abstract The invention provides a protein that is a tumor marker protein. This protein can be used to prepare antibodies that bind to the tumor marker protein. These antibodies can be used to reduce, or eliminate metastasis by cancer cells that produce the tumor marker protein. In addition, the invention provides methods that can be used to diagnose cancer, and metastasis by cancer cells.

Description

Australian Patents Act 1990 - Regulation 3.2 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title "Glycoprotein antigen SIMA 135 expressed in metastatic human tumor cells" The following statement is a full description of this invention, including the best method of performing it known to us: AOPFR\MJC\30 8653 new div 095-de GLYCOPROTEIN ANTIGEN SIMA135 EXPRESSED IN METASTATIC HUMAN TUMOR CELLS This application is a divisional of Australian Patent Application No. 2004213568, the entire content of which is incorporated herein by reference. Field of the Invention 5 The invention relates generally to methods to diagnose cancer and to decrease metastasis by cancer cells in a manual, such as a human. More specifically, the invention relates to the use of a tumor marker protein to create antibodies that can be used to detect increased production of the protein in a cell, and use of the antibodies to decrease metastasis by cancer cells that produce the tumor marker protein. 10 Background of the Invention A malignant tumor sheds cells which migrate to new tissues and create secondary tumors while a benign tumor does not generate secondary tumors. The process of generating secondary tumors is called metastasis and is a complex process in which tumor 15 cells colonize sites distant from the primary tumor. Tumor metastasis remains the major cause of deaths in cancer patients, yet the molecular mechanisms underlying tumor cell dissemination are not clearly understood. Metastasis is a multi-step process in which tumor cells must detach from the primary tumor, invade the cellular matrix, penetrate through blood vessels, thus enter the 20 circulatory system (intravasate), arrest at a distant site, exit the blood stream (extravasate), and grow. See, e.g., G. L. Nicolson (1982) Biochim. Biophis. Acta. 695: 113-176; G. L. Nicolson and G. Poste (1983) In. Rev. Exp. Pathol. 25: 77-181; G. Poste and I. J. Fidler (1980) Nature 283: 139-145; and E. Roos (1984) Biochim. Biophis. Acta. 738: 263-284. Given the complexity of the process, it is thought that numerous genes mediate tumor cell 25 metastasis. Indeed, the metastatic phenotype has been correlated with expression of a variety of proteins, including proteases, adhesion molecules, and the like. However, evidence that a given protein is directly involved in dissemination is often lacking, or difficult to prove. L. A. Liotta and W. Stetler-Stevenson (1989) J. Natl. Cancer Inst. 81: 556-557. 1 The human epidermoid carcinoma, HEp-3, provides a unique system that can be used to detect and characterize genes which effect metastatic dissemination. HEp-3 cells, propagated by serial passage on the chick chorioallantoic membrane (CAM), are both tumorigenic and spontaneously metastatic (T+M+). L. Ossowski and E. Reich (1980a) 5 Cancer Res. 40: 2300-2309. However, when such cells are grown continuously in vitro, they readily form primary tumors, but progressively become non-metastatic (T+M-) 'with time. L. Ossowski and E. Reich (1980b) Cancer Res. 40: 2310-2315. With prolonged cultivation in vitro, they eventually become non-tumorigenic also (T-M-). The loss of metastatic ability is reversible. T+M- cells carried on the chorioallantoic membrane for 10 two to three passages regain the ability to form spontaneous metastases. Thus, by altering growth conditions, the metastatic potential of these cells can be manipulated by the investigator. Human urokinase-type plasminogen activator (uPA) was shown to be directly involved in dissemination of HEp-3, as spontaneous metastasis of HEp-3 cells in the chick 15 embryo was inhibited by antibodies that were specific for human uPA. L. Ossowski and E. Reich (1983b) Cell 35: 611-619. Subsequently, it was observed that inhibition of uPA activity blocked infiltration of the CAM mesenchyme by individual HEp-3 cells. L. Ossowski (1988a) Cell 52: 321-328. However, active uPA appeared to be required for tumor cell intravasation but not extravasation. L. Ossowski (1988a). Thus, some other 20 factor(s) must be also involved in HEp-3 dissemination and in dissemination of cancer cells in general. J. P. Quigley et al. (1988) Ciba Foundation Symposium 141: 22-47, Brooks et al. (1993) J. Cell Biol. 122 (6): 1351-1359 and Testa, et al. in U.S. Patent No's 6,245,898 and 6,498,014 describe the generation of monoclonal antibodies (mAbs), using "subtractive immunization", which recognize cell surface antigens expressed on HEp-3 cells and inhibit 25 tumor metastasis in the chorioallantoic membrane model. Nevertheless, these antigens have not been correlated to in vivo metastasis nor shown to be directly involved in the process of in vivo metastasis. Consequently, there is a need to identify biological molecules that are functionally involved in cancer cell dissemination in order to develop therapies that can be used to 30 inhibit the migration of tumor cells to new tissues. Also, methods to inhibit tumor cell metastasis and to diagnose cancer are needed to help in the battle to control cancer by reducing or eliminating the spread of cancer cells throughout the body of a mammal afflicted with cancer. 2 Summary of the Invention The present invention concerns the identification and characterization of a protein (SIMAI 35)(SEQ ID NO: 1) that is produced in metastatic cells. Accordingly, the invention 5 provides SIMA1I35 in glycosylated and non-glycosylated form. The invention also provides antibodies that specifically and selectively bind to SIMA135, and fragments of SIMA135. These antibodies can bind to SIMA135, or fragments of SIMA135, that are glycosylated or non-glycosylated. Accordingly, the invention provides methods to prevent, reduce, or eliminate metastasis of cancer cells through use of antibodies that specifically 10 bind to SIMA135. The invention also provides methods to diagnose cancer, and to determine the presence of cancer cells in a test sample. These methods may be used in association with a mammal, such as a human. The invention also provides pharmaceutical compositions and kits that contain antibodies that specifically and selectively bind to SIMA135 and fragments of SIMA135. The invention also provides a method to screen for 15 agents that modulate production of SIA135 by a cell. The invention provides SIMA135, and fragments of SIMA135. Preferably SIMA135 or a fragment of SIMA135 is not glycosylated. More preferably the SIMA135 or the fragment of SIMA135 is glycosylated. Preferably fragments of SIMA135 are antigenic and are able to elicit an immune response when administered to an organism, such as a mammal 20 or an avian, Preferably the antigenic fragments of SIMA135 are glycosylated. The invention provides antibodies that bind to SEIMA135 or fragments of SIMA135 such as monoclonal antibody 41-2. Preferably, the antibody is not monoclonal antibody 41 2 as the monoclonal antibody preferably selectively and specifically binds with SIMA135. Preferably the antibodies are recombinant antibodies. More preferably the antibodies are 25 polyclonal antibodies. Even more preferably the antibodies are humanized antibodies. Most preferably the antibodies are monoclonal antibodies. Preferably the antibodies bind to non-glycosylated SIMA135 or to a non-glycosylated fragment of SIMA135. More preferably the antibodies bind to glycosylated SIMA135, or a glycosylated fragment of SIMA135. 30 The invention provides a method to prevent, reduce, or eliminate metastasis of a cancer cell. The method involved administering an antibody that binds to SIMA135 to an organism in need thereof. Monoclonal antibody 41-2 can bind SIMA 135 and also with other antigens involved in metastasis but it is preferred that the antibody of the method 3 should be other than monoclonal antibody 41-2. Such other antibody will selectively and specifically bind with SIMA 135. Preferably the organism is a mammal. More preferably the organism is a human. Preferably the antibody binds to SIMA135, or to a fragment thereof, non-specifically. More preferably the antibody binds to SIMA135, or a fragment 5 thereof, specifically. Preferably the antibody is a polyclonal antibody. More preferably the antibody is a recombinant antibody. Even more preferably the antibody is a monoclonal antibody. Most preferably the antibody is a humanized antibody. Preferably the antibodies bind to non-glycosylated SIMA135 or to a non-glycosylated fragment of SIMA135. More preferably the antibodies bind to glycosylated SIMA135, or a glycosylated fragment of 10 SIMA135. Preferably the antibody is administered to the organism is need thereof in as a pharmaceutical composition. The invention also provides methods to diagnose cancer in an organism. In one embodiment, antibodies that bind to SIMA135 can be contacted with a test sample obtained from the organism, and then the relative amount of antibodies that bind to the test sample 15 are compared to the relative amount of antibodies that bind to a non-cancerous control sample. Increased antibody binding to the test sample relative to the control sample indicates that the organism has cancer. In another embodiment, the invention provides an immunohistochemical method to diagnose cancer in an organism wherein antibodies are contacted with a test sample obtained from the organism, and the antibody binding pattern 20 exhibited by the test sample is compared to an antibody binding pattern produced through use of a control sample. If the antibody binding pattern produced using the test sample matches an antibody pattern produced through use of a cancerous control sample, the organism is diagnosed as having cancer. Alternatively, if the antibody binding pattern to the test sample is different than an antibody binding pattern produced through use of a non 25 cancerous control sample, then the organism is diagnosed as having cancer. Preferably the antibody binds non-specifically to SIMA1 35, or a fragment thereof. More preferably the antibody binds specifically to SIMAI 35, or a fragment thereof. The invention also provides pharmaceutical compositions that contain an antibody that binds to SIMvA135, or a fragment thereof, and a pharmaceutical carrier provided that 30 the antibody is not monoclonal antibody 41-2. The invention also provides kits that contain an antibody that binds to SIMA135, or a fragment of SIMA135, and packaging material. 4 The invention provides a method to identify agents that modulate production of SIMA 135 by a cell, The invention as well includes the cell line operable in the method as well as the assay method itself. Preferably an agent identified according to the method increases production of SIMA135 by a cell. Such an identification will demonstrate 5 carcinogenicity of such an agent and thus can be used as a rapid test for cancer-causing agents. More preferably an agent identified according to the method decreases production of SIMA135 by a cell. Such an identification will demonstrate the anti-carcinogenicity of such an agent. In one embodiment, a candidate agent is contacted with a test cell and production of SIMA 135 by the test cell is compared to a control cell that was not contacted 10 with the candidate agent. An increase or decrease in SIMA135 production by the test cell as compared to the control cell indicates that the candidate agent modulates production of SIMA135 by a cell. Preferably the cell is a mammalian cell. More preferably the cell is a human cell. Even more preferably the cell is a non-metastatic HEp3 cell. Most preferably the cell is a metastatic HEp3 cell. 15 In one aspect the present invention provides a tumor marker protein comprising amino acid sequence SEQ ID NO: 1 wherein the protein is glycosylated, In one aspect the present invention provides a protein comprising amino acid sequence SEQ ID NO: 1, wherein the protein is a phosphotyrosine protein. In one aspect the present invention provides an antigenic fragment of SEQ ID 20 NO: 1, wherein the fragment has at least 5 amino acids of SEQ ID NO: 1, and wherein the fragment is glycosylated. In one aspect the present invention provides an antigenic fragment of SEQ ID NO: 1, wherein the fragment contains Aug-525 of SEQ ID NO: 1 and/or Ser-827 of SEQ ID NO: 1. 25 In one aspect the present invention provides a soluble immunoreactive fragment of SEQ ID NO: I with a molecular weight of approximately 110 kDa by reducing SDS PAGE. In one aspect the present invention provides a monoclonal antibody that binds specifically to a protein consisting of amino acid sequence SEQ ID NO: 1, wherein the 30 antibody is a Fab' fragment, a F(ab')2 fragment, a Fv fragment, or a sFv fragment, 5 C:\NRPrblDCC\REC425046 I DOC-2 a62l 12 - 5A In one aspect the present invention provides an isolated protein comprising amino acid sequence SEQ ID NO: 1, wherein the protein is a phosphotyrosine protein. In one aspect the present invention provides a monoclonal antibody that binds specifically to: (i) a tumor marker protein comprising amino acid sequence SEQ ID NO: 1; or (ii) the fragment of claim 2 or claim 3, wherein said antibody is not monoclonal antibody 41-2. Brief Description of the Drawing Figure 1 illustrates the amino acid sequence of SIMA 135. The signal sequence is in lower case lettering and the putative transmembrane domain is boxed. Twelve consensus N glycosylation motifs are indicated with filled triangles. Cytoplasmic tyrosine residues are circled. CUB domains that are thought to span residues 221 to 348 and 417 to 544 are underlined. The three peptides identified from trypsin digestion and sequencing are overlined. The Arg residue preceding peptide 2 and the Lys preceding peptide 3 are boxed to highlight the consistency with trypsin specificity for Arg/Lys containing substrates. Cytoplasmic domain PXXP sequences are underlined. A consensus palmitylation motif, following the putative transmembrane domain, is indicated by filled circles. A consensus palmitylation motif, following the putative transmembrane domain, is indicated by filled circles. Detailed Description of the Invention The invention relates to the discovery of a glycosylated protein that was purified from metastatic HEp3 cells through subtractive immunization using a monoclonal antibody designated 41-2. The protein is designated SIMA135 (subtractive immunization M+ Ep3 associated 135 kDa protein). SIMAl 35 refers to a protein that can be physically isolated from cells, as opposed to being a punitive protein predicted from translation of a nucleic acid sequence. Physical 5 isolation of the SIMA135 protein is significant for a number of reasons. One reason is that isolation of the protein indicates that the mRNA is actually translated into a polypeptide. Secondly, isolation and characterization of the protein as reported herein indicates that the protein is glycosylated. Physical isolation of the glycosylated protein confirms that glycosylation sites within the polypeptide are available for glycosylation and are not buried 10 within the folded protein to become inaccessible to glycosyltransferases. Such conformation is important due to the known role that glycosylation plays in protein folding and immunogenicity. Therefore, isolation of the SIMA135 protein is a significant advance when compared to a theoretical polypeptide sequence predicted from a nucleic acid sequence. 15 The SLIA135 cDNA is shown herein to encode a 135 kDa type I transmembrane cell surface protein that specifically immunoreacts with mAb 41-2. Immunopurification and amino acid sequencing confirms that the mature protein commences at Phe30 following removal of a 29 amino acid signal peptide. Immunocytochemical analysis confirms localization of the protein to the cell surface and the type I orientation of this protein. In 20 addition, consistent with the presence of 12 potential extracellular glycosylation sites, Western blot analysis of deglycosylated cell lysates indicates that up to 40 kDa of the difference between the apparent (-135 kDa) and theoretical (-90 kDa) molecular weight of mature SIMA135 is due to N-linked glycans. Western blot analysis demonstrates that SIMA135 is a phosphotyrosine protein, consistent with the presence of 5 intracellular 25 tyrosine residues. In addition, the inhibitor PP2 has been used to demonstrate that a Src kinase family member acts to phosphorylate tyrosines of SIMA135 in HEp3 cells. The domain structure of SIMA135 indicates that it may interact with extracellular proteins such as soluble ligands, other cell surface proteins and/or matrix components; potentially via putative CUB domains present within its amino terminal region. These 30 structures are thought to mediate binding to a variety of protein ligands. For example, homodimerization of the MASP serine proteases acting within the lectin branch of the complement cascade is stabilized through interactions involving CUB domains (Chen and Wallis, 2001). Also a number of the type II transmembrane seine proteases contain CUB 6 domains thought to mediate enzyme-substrate interactions (Hooper et al., 2001). In addition, CUB domains of cubilin mediate binding to both the intrinsic factor-cobalarnin as well as albumin (Yammani et al., 2001). As SIMA135 is heavily glycosylated within its extracellular domain, it is thought that ligand binding will be, at least partially, dependent 5 on carbohydrate moieties as has been demonstrated for various isoforms of the cell surface glycoprotein CD44 (Bajorath, 2000). Glycosylation is also thought to contribute to SIMA 135 protein folding, and trafficking to and maintenance at the cell surface (Gorelik et al., 2001; Grogan et al., 2002). SIMA135 displayed differences in amino acid sequence from other proteins 10 associated with signet ring carcinoma (GenBank entry AK026622) and the non small lung cell carcinoma cell line Calu 6 (GenBank entry AY026461) (Scherl-Mostageer et al., 2001). These differences are thought to affect the ability of SIMA135 to interact with other molecules, as compared to previously known proteins. The first amino acid change, 525Arg-+Gln, occurs within an extracellular potential ligand binding domain; the second 15 of the potential CUB domains of SIMA135. The second amino acid change, 709Gly->Asp, is located 2 residues after a tyrosine residue. This change from a non-polar amino acid to a charged residue could be expected to have a significant impact on the ability of the proximal tyrosine to be phosphorylated, and therefore is thought to have an impact on the capacity of SIMA135 to bind to, for example, SH2 domains. The last change, 20 827Ser-Asn, is located 4 residues from a PXXP motif. Accordingly, this change may also impact on the ability of SIMA135 to interact with other proteins; in this case SH3 domain containing proteins. In normal colon tissue, SIMA135 protein is observed on basal and apical surfaces of epithelial cells lining the colon lumen and on the apical surface of crypt epithelial cells. In 25 contrast to its distinct localization in normal colon, SIMA135 distribution in colon tumor tissue is disarrayed and heterogeneous, appearing dysregulated with both plasma membrane and cytoplasmic staining. It appears that expression of SIMA135 is more intense in invading glands deeper in the colonic serosa and within draining blood vessels. These results indicate that increased SIMA1 35 protein expression is associated more with later 30 stages of carcinogenesis, such as local invasion and metastasis. This proposal is partly supported by Western blot analysis of pairs of human tumor cell lines originating from the same tissue. For example, SIMA-135 levels were much higher in highly-metastatic M+ HEp3 cells compared to the congenic and low metastatic variant, M- HEp3. In addition, the 7 noncongenic prostate cancer cell lines PC-3 and LNCaP showed a similar trend; the former, a metastatic cell line, showing much higher levels of SIMA1 35 compared to the latter, a low metastatic cell type (Soos et al., 1997). The observation of apparently free SIMA135 in glandular mucus of both normal and 5 malignant glands is consistent with the observation that a 110 kDa soluble form of this protein is released in vitro by HEp3 cells. The distinct loss of glandular tissue ultrastructure that is apparent during tumorigenesis may permit the release of the soluble form of SIMA135/CDCP1 into the fluid and vascular system of the colon cancer patient. Accordingly, SIMA135 is thought to have utility as a serum or tissue fluid marker as has 10 been proposed for the transmembrane proteins MUCI (Rye and McGuckin, 2001), CD44 (Adham et al., 1990) and ICAM-1 (Maruo et al., 2002). I. SIMA135, fragments, and variants thereof that are glycosylated or non glycosylated. 15 The invention provides the SIMA135 protein (SEQ ID No:1), fragments of SIMA135, and variants of SIMA135 that can be glycosylated or non-glycosylated. These proteins, fragments, and variants of SIMA135 can be used as antigens to induce production of antibodies that bind to SIMA135, e.g. antibodies that bind specifically and/or selectively to SIMA135. Such selectively binding antibodies include those that bind to SIIMA135, or a 20 portion of SIMA135, but that do not bind to proteins and fragments of proteins that are not SIMA135, or a fragment of SIMA135. These proteins, fragments, and variants can also be used to select for antibodies that specifically and selectively bind to SIMA135. Such specifically and selectively binding antibodies include those that bind to SIMA135, or a portion of SIMA135, but that do not bind to proteins and fragments of proteins that are not 25 SIMA135, or a fragment of SIMA135. In particular, the selectivity of such antibodies means that they bind to SIMA 135 or a portion of SIMAI35 but do not also bind to the 180kD protein produced from metastatic Hep-3 cell lysate as described in US Pat No. 6,498,014 with a dissociation constant of the same order of magnitude as that resulting from binding to SIMA1 35 or a fragment thereof, although binding of such antibodies with 30 the 18OkD protein may occur at a dissociation constant at least two orders of magnitude greater than that for binding with SIMA35 or a fragment thereof. The specificity of such antibodies means that the immunogenic binding is the result of epitopal - hypervariable region interaction and not the result of non-specific protein - protein interaction. Non 8 specific protein - protein interaction typically will have a dissociation constant at least 3 orders of magnitude greater than the dissociation constant for the specific binding of an epitopal - hypervariable region interaction. The dissociation constant for an antibody antigen immunobinding pair can be measured according to the techniques described in 5 Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Pub. 1988)), which is hereby incorporated by reference. A fragment of a SIMA135 protein as used herein, refers to a peptide fragment of a sufficient length to be antigenic. Generally speaking, the fragment includes at least 5 amino acids. The invention pertains to a fragment of SIMA135, e.g. a fragment of SEQ ID No:1, 10 glycosylated or non-glycosylated wherein said fragment contains amino acid 525 and or amino acid 827. The present invention pertains to a fragment of SIMA1 35, e.g. a fragment of SEQ ID No: 1, wherein in said fragment the amino acid 525 is not a Glutamine and /or amino acid 827 is not an Asparagine. The present invention pertains to a fragment of SIMA135, e.g. a fragment of SEQ ID No: 1, wherein in said fragment the amino acid 525 is 15 Arginine and /or amino acid 827 is Serine. Variant proteins include proteins having amino acid substitutions that are biologically active, or that elicit antibody production when used as an antigen. A variant of SIMAl35 is intended to include a protein derived from native SIMA135 by deletion (so called truncation) or addition of one or more amino acids to the N-terminal and/or C 20 terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Preferably, the variant at position 525 is not Glutamine, the variant at position 827 is not Asparagine and preferably the amino acid at position 709 Glycine. Such variants may result from, for example, genetic polymorphism or from human 25 manipulation. Methods for such manipulations are generally known in the art. Thus, the SIMA135 proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in DNA encoding SIMA135. Methods 30 for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA, 82, 488 (1985); Kunkel et al., Methods in Enzmol., 154:367 (1987); U. S. Patent No. 4,873,192; Walker and Gaastra, eds., Techniques in Molecular biology MacMillan Publishing Company, New York (1983) and 9 the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., Atlas of Protein Sequence and Structure, Natl. Biomed. Res. Found., Washington, C.D. (1978), herein incorporated by reference. Conservative substitutions, 5 such as exchanging one amino acid with another having similar properties, are preferred. Conservative amino acid substitutions are preferred and include, for example; aspartic glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids. Conservative amino acid 10 substitution also includes groupings based on side chains. Members in each group can be substituted for one another. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine. These may be substituted for one another. A group of amino acids having aliphatic-hydroxyl side chains is serine and threonine. A group of amino acids having amide-containing side chains is asparagine and 15 glutamine. A group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan. A group of amino acids having basic side chains is lysine, arginine, and histidine. A group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino 20 acid with a structurally related amino acid may be accomplished to produce a variant polypeptide of the invention. The proteins of the invention may be glycosylated or not glycosylated. The proteins may be glycosylated in vivo by expressing the proteins in a cell that is able to glycosylate the recombinant protein. Alternatively, the proteins of the invention can be glycosylated in 25 vitro through use of sugar transferases. The proteins may be treated to cleave any linked glycans through use of commercially available enzymes, for example PNGase F (New England Biolabs, Beverly, MA). Accordingly, the proteins of the invention can be used to produce antibodies that bind to native SIMA135, denatured SIMA135, specific portions of SIMA135, glycosylated SIMA135, and to non-glycosylated SIMAl35. 30 II. An antibody that selectively binds to SIMAl35, or to a fragment of SIMAI35, The invention provides antibodies that bind to SIMAI 35. Preferred antibodies to be used in pharmaceutical compositions include those antibodies that inhibit tumor metastasis. 10 Inhibition of tumor metastasis can be determined by a number of assays, such as the migration assay, the invasion assay or the chick chorioallantoic membrane assay. Antibodies can be prepared that recognize natively folded SIMA135, or denatured SUMA135 by immunizing an animal with native SIMA13 Idenatured SIMA135 5 respectively. In addition, antibodies can be prepared that recognize SIMA135 that is glycosylated, or SIMA135 that is not glycosylated by immunizing an animal with SIMA135 that is glycosylated or non-glycosylated respectively. Antibodies that recognize various forms of SIMA135 (for example, native vs. denatured, and glycosylated vs. non glycosylated) are useful for determining if a cell is able to properly fold and glycosylate 10 SIMA135. Such antibodies are useful for determining if a candidate agent is able to interfere with cellular actions that process SIMA135 during metastasis. Accordingly, such antibodies may be used to identify the action of agents that can be used to inhibit metastasis by cancer cells. Antibodies that bind to SLMA135, fragments of SIMA135, and variants of 15 SIMA 135, can be prepared using an intact protein or fragment containing small peptides of interest as the immunizing antigen. Fragments of SIMA135 that can be used as antigens include those that produce an immune response in an animal. These fragments will generally be five amino acids or greater in length. The protein or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis which can 20 be conjugated to a carrier protein, if desired. Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled protein or peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit). The monoclonal antibody 41-2 is an antibody that was initially employed to isolate SIMAl 35. 25 It recognizes SIMA135 and in addition several other metastasis proteins including the 180 kD protein described in U.S Patent No's. 6,245,898 and 6,498,014. For this reason, a preferred antibody according to the invention is a monoclonal antibody that binds, e.g. selectively and/or specifically, with SIMAI35, e.g. said antibody does not bind with other metastasis proteins, e.g. said antibody recognizes an epitope of SIMA135. 30 If desired, polyclonal or monoclonal antibodies can be purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal 11 antibodies, as well as monoclonal antibodies (Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, incorporated by reference). An antibody suitable for binding to a protein of the invention is specific for at least one portion of a region of the protein. For example, one of skill in the art can use a protein 5 or peptide to generate appropriate antibodies of the invention. Antibodies of the invention include polyclonal antibodies, monoclonal antibodies, and fragments of polyclonal and monoclonal antibodies. The present invention pertains to an antibody according to the invention for use as a therapeutic treatment of the human and animal body, the invention also relates to an 10 antibody according to the invention for the preparation of a medicament for use to inhibit metastasis by a cancer cell in a mammal. The preparation of polyclonal antibodies is well-known to those skilled in the art (Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al., Production of Polyclonal Antisera in 15 Rabbits, Rats, Mice and Hamsters, in Current Protocols in Immunology, section 2.4.1 (1992), which are hereby incorporated by reference). The preparation of monoclonal antibodies likewise is conventional (Kohler & Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988)), which are 20 hereby incorporated by reference. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and 25 isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size exclusion chromatography, and ion-exchange chromatography (Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G 30 (IgG), in Methods in Molecular Biology, Vol. 10, pages 79-104 (Humana Press 1992)). Methods of in vitro and in vivo multiplication of monoclonal antibodies is well-known to those skilled in the art. Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished 12 by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation 5 can be carried out by homogenous suspension culture in an air reactor, in a continuous stirrer reactor, or immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g., osyngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristine 10 tetramethylpentadecane prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal. An antibody of the invention may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse 15 immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described (Orlandi et al., Proc. Natl. Acad. Sci. 20 USA, L6:3833 (1989) which is hereby incorporated in its entirety by reference). Techniques for producing humanized monoclonal antibodies are described (Jones et al., Nature, 321:522 (1986); Riechmann et al., Nature, 332:323 (1988); Verhoeyen et al, Science, 239:1534 (1988); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Sandhu, Crit. Rev. Biotech., 12:437 (1992); and Singer et al., J. Immunol., 150:2844 25 (1993), which are hereby incorporated by reference). In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains 30 of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic 13 mice are described (Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature, 368:856 (1994); and Taylor et al., Int. Immunol., 6:579 (1994), which are hereby incorporated by reference). Antibody fragments of the invention can be prepared by proteolytic hydrolysis of 5 the antibody or by expression in E. coli 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 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the 10 sulfbydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described (Goldenberg, U.S. patents No. 4,036,945 and 4,331,647; and references contained therein; Porter, Biochem. J., 73:119 (1959); Edelman et al., Methods in Enzymology, Vol. 1, page 15 422 (Academic Press 1967); and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4). 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. 20 For example, Fv fragments include, an association of VH and VL chains. This association may be noncovalent (Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659 (1972)). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (Sandhu, supra). Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain 25 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 30 described (Whitlow et al., Methods: A Companion to Methods in Enzymology, Vol. 2, page 97 (1991); Bird et al., Science, 242:423 (1988), Ladner et al., U.S. patent No. 4,946,778; Pack et al., Bio/Technology, 11:1271 (1993); and Sandhu, supra). 14 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 5 variable region from RNA of antibody-producing cells (Larrick et al., Methods: A Companion to Methods in Enzymology, Vol. 2, page 106 (1991)). III. A method to treat a metastatic tumor and to inhibit metastasis by a tumor cell. The invention also includes methods of treating metastatic tumors. "Treating a 10 metastatic tumor" means that the metastasis of the tumor is prevented, delayed, or inhibited. Metastatic tumors include both tumors at the primary site capable of metastasizing and metastasized tumors at a secondary site. Such metastatic tumors can be of a tissue origin of the lung, liver, kidney, mammary gland, epithelial, thyroid, leukemic, pancreatic, endometrial, ovarian, cervical, skin, colon and lymphoid tissue. A subject which can be 15 treated can be any mammalian subject, including humans, dogs, monkeys, cows and the like, with the exception of mice. One embodiment of the present invention provides methods of treating a metastatic tumor in a subject by administering to the subject a therapeutically effective amount of a tumor metastasis-inhibiting antibody of the present invention. Tumor metastasis-inhibiting 20 antibodies of the present invention have been described hereinabove. Preferred tumor metastasis-inhibiting antibodies include those antibodies that selectively bind to SIMA135, or a fragment thereof. A tumor metastasis-inhibiting antibody can be administered alone or together with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier includes all 25 solvents, such as fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, dispersion media, cell culture media, and the like, or combinations thereof, that are non toxic to the recipient subject. In accordance with the present invention, the active ingredients can be combined with the carrier in any convenient and practical manner, e.g., by solution, suspension, 30 emulsification, admixture, encapsulation, absorption and the like, and if necessary, by shaping the combined compositions into pellets or tablets. Such procedures are routine for those skilled in the art. 15 Dosages of an antibody to be therapeutically effective depend on the disease state and other clinical factors, such as weight and condition of the subject, the subject's response to the therapy, the type of formulations and the route of administration. The precise dosage of a compound to be therapeutically effective can be determined by those 5 skilled in the art. As a general rule, the therapeutically effective dosage of an antibody can be in the range of about 0.5 pg to about 2 grams per unit dosage form. A unit dosage form refers to physically discrete units suited as unitary dosages for mammalian treatment: each unit containing a predetermined quantity of the active material calculated to produce the desired therapeutic effect in association with any required pharmaceutical carrier. The 10 methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time. The administration of a tumor metastasis-inhibiting antibody may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. Preferably, the antibodies of the present invention are 15 administered to a patient by subcutaneous (s.c.), intraperitoneal (i.p.), intra-arterial (i.a.), or intravenous (i.v.) injection. IV. A method to diagnose cancer in a mammal. The invention also includes methods of diagnosing metastatic tumors in a subject by 20 detecting the expression of SIMA135. Metastatic tumors include both tumors at the primary site capable of metastasizing and metastasized tumors at a secondary site. Such metastatic tumors can be of a tissue origin of the lung, liver, kidney, mammary gland, epithelial, thyroid, leukemic, pancreatic, endometrial, ovarian, cervical, skin, colon and lymphoid tissues. 25 The expression of SIMA135 can be detected by using an antibody that binds to SIMAl35, or a fragment of SIMA135. Both polyclonal antibodies and monoclonal antibodies can be employed. In one embodiment, a sample is taken from the subject, e.g., a biopsy specimen taken from tissue suspected of having a metastatic tumor. Generally, the sample is treated before 30 an assay is performed. Assays which can be employed include ELISA, RIA, EIA, Western Blot analysis, immunohistological staining and the like. Depending upon the assay used, the antigens or the antibodies can be labeled by an enzyme, a fluorophore or a radioisotope. 16 See, e.g., Coligan et al. Current Protocols in Immunology, John Wiley & Sons Inc., New York, N.Y. (1994); and Frye et al., Oncogen 4:1153-1157, 1987. The treatment of the sample may vary depending on the assay that is used to detect SIMA135. For example, cells of tissue biopsy can be lysed and the cell lysates are used in 5 e.g., Western Blot analysis. For assays such as the Whole Cell ELISA assay, cells can be washed with, e.g., PBS, and then fixed with 0.25% glutaraldehyde in PBS before the assay. The expression of SIMA135, or a fragment of SIMA135, detected by using any of the above-described methods, is compared with the expression of the same antigen in the normal part of the tissue. A substantial increase in the level of expression of the antigen 10 when compared with the expression in the normal tissue, is indicative of a metastatic tumor. A substantial increase means an increase of at least about 20%, preferably, at least about 25%, more preferably, at least about 35%. In another embodiment, immunohistochemistry can be used to diagnose a metastatic tumor in an organism. In this embodiment, a sample is taken from an organism, e.g., a 15 biopsy specimen taken from tissue suspected of having a metastatic tumor. The sample can be affixed to a slide and contacted with antibodies, as disclosed herein, that bind to SIMA135. The antibodies can be labeled by an enzyme, a fluorophore or a radioisotope. See, e.g., Coligan et al. Current Protocols in Immunology, John Wiley & Sons Inc., New. Following binding of the antibodies to SIMAl 35, the position of the antibodies is 20 determined through use of known techniques. Heterologous staining, extensive expression of SIMA 135 throughout the tissue sample, and staining within malignant glands in the colonic serosa indicate metastatic cancer. V. A kit containing an antibody that selectively binds to S1MA135. or fragments 25 thereof, and packaging material. The invention provides a kit containing an antibody that binds to SIMA 135 and packaging material. Such kits are useful for shipping and storage of antibodies that can be used for treating and detecting cancer. Specifically, such kits may be used by medical personal in a laboratory for detecting metastatic cancer in a tissue sample obtained from an 30 organism. Furthermore, such kits may be useful for medical personal for the formulation of pharmaceutical compositions that contain an antibody of the invention. 17 The packaging material will provide a protected environment for the antibody. For example, the packaging material may keep the antibody from being contaminated. In addition, the packaging material may keep an antibody in solution from becoming dry. Examples of suitable materials that can be used for packaging materials include 5 glass, plastic, metal, and the like. Such materials may be silanized to avoid adhesion of an antibody to the packaging material. VI. A pharmaceutical composition containing an antibody that selectively binds to SIMA135, or to a fragment of SIMA135, and a pharmaceutically acceptable 10 carrier. A pharmaceutical composition of the invention includes an antibody that binds to SIMA135 that is formulated as a pharmaceutical composition and administered to an animal host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or 15 subcutaneous routes. Thus, an antibody may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral 20 therapeutic administration, the antibody may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the antibody. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given 25 unit dosage form. The amount of the one or more antibodies in such therapeutically useful compositions is such that an effective dosage level will be obtained. When administered orally, the compositions of the invention can preferably be administered in a gelatin capsule. The tablets, troches, pills, capsules, and the like may also contain the following: 30 binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, 18 or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or 5 capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the antibody or antibodies, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the antibody 10 or antibodies may be incorporated into sustained-release preparations and devices. The antibody or antibodies of the invention may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the antibody or antibodies can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. 15 Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the antibody or antibodies which are adapted for the extemporaneous preparation of sterile injectable or 20 infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and 25 suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be 30 preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. 19 Sterile injectable solutions are prepared by incorporating an antibody or antibodies in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. 5 V1I. A method to identify an agent that modulates production of SIMA135 by a cell The invention provides a method to identify an agent that increases or decreases production of SIMA135 by a cell. Generally, the method involves contacting a test cell with a candidate agent and determining if production of SIMA135 by the test cell is increased or decreased relative to a control cell that was not contacted with the candidate 10 agent. SIMA135 production by the test cell and the control cell can be detected through use of many art recognized methods. Such methods are exemplified by immunological methods that include radioimmunoassay (RIA), enzyme-linked immunosorbant assay (ELISA), use of fluorescently labeled antibodies, and the like. 15 Many examples of candidate agents that can be screened according to the method are described in the official United States Pharmacopeia, official National Formulary, or any supplement to them. Briefly, examples of candidate agents include, hydrocarbons, cyclic organic molecules, bicyclic organic molecules, aryl organic molecules, alkyl organic molecules, and the like. Merck Manual, Merck Research Laboratories, Whitehouse 20 Station, N.J. 17d edition, eds, Beers and Berkow 1999; Merck Index, Merck Research Laboratories, Whitehouse Station, N.J., 1 3 * ed., 2001. The metastatic HEp3 cells such as those exemplified in the following section may be used as test cells and control cells in the method of the invention. However, the method may also be practiced with cells that produce SIMA135 normally or through recombinant 25 methods. For example, an expression construct that provides for the production of SlMA135 may be introduced into a cell that does not produce SIMA135 prior to the introduction of the expression cassette. The transformed cell may then be used within the method of the invention to identify agents that modulate SIMA135 production. The diagnostic methods described above for detection of the presence and quantity of SIMA 30 135 can be used with such cell systems to assay the SIMA 135 production by test and control cells. The method of the invention may also be practiced in vivo. As exemplified in the following section, a candidate agent may be administered to a test animal. A tissue sample 20 may be obtained from the test animal and SIMA135 production by cells in the tissue sample may be compared to SIMA135 production by cells in a tissue sample obtained from a control animal. An increase in SIMA135 production by the test animal relative to the control animal indicates that the candidate agent increases SIMA135 production. A 5 decrease in SIMA135 production by the test animal relative to the control animal indicates that the candidate agent decreases SIMA135 production. The assay of the SIMA 135 may be determined by any of the analytic methods given in the diagnosis section above. Numerous animals may be used within the method of the invention. Examples of such animals include rabbits, rats, mice, monkeys, and the like. 10 The method of the invention may be practiced in vitro. For example, test cells and control cells may be grown in tissue culture. This allows the candidate agent to be contacted with a test cell in vitro. SIMA135 production by the test cells can then be compared to SIMA135 production by control cells as described above to determine if the candidate agent increases or decreases SIMA135 production by a cell. 15 The in vitro and in vivo methods will determine the ability of a test agent to promote and to minimize or prevent metastasis. Determination of promotion will identify the agent as a cancer causing or enhancing agent. This determination has practical application for the rapid identification of cancer causing agents. Determination of minimization or prevention will identify the agent as a cancer inhibiting agent. This determination has practical 20 application for the identification of anti-cancer agents. Example I Cell Lines and Hybridomas Human cervical adenocarcinoma HeLa, fibrosarcoma HT1080, colon adenocarcinoma 25 DLD-1 and SW480, breast adenocarcinoma MCF7, prostate adenocarcinoma PC-3, prostate carcinoma lymph node metastasis LNCaP, lung carcinoma A549 and kidney rhabdoid tumor G401 cells were obtained from the American Type Culture Collection (Rockville, MD). Human liver cancer HuH7 and HLE, and gastric cancer MKN45 and STKM-1 cells were provided by Dr. Peter Vogt (The Scripps Research Institute, La Jolla, CA) and breast 30 adenocarcinoma MDA-MB-231 cells by Dr. Liliana Ossowski (Mount Sinai School of Medicine, NY). Human epidermoid carcinoma HEp3 cells, were obtained from solid tumors serially passaged on the chorioallantoic membrane (CAM) of chicken embryos (Testa, 1992; Brooks et al., 1993). The metastatic variant of HEp3 cells, M+ HEp3, was 21 cultured for less than 20 days before use. The low metastatic variant, M- HEp3, was maintained in culture for at least 80 days before use. Human nicrovascular endothelial cells (HEC) and dermal fibroblasts (HDF) were obtained from Clonetics (San Diego, CA) and maintained in EGM-2 MV and FGM-2 media (Clonetics) respectively. Cancer cell 5 lines were maintained as monolayer cultures in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (HyClone, Logan, UT), sodium pyruvate, penicillin/streptomycin and non-essential amino acids (Invitrogen) and grown in a humidified 5% CO 2 atmosphere at 37'C. Hybridomas producing MoAb 41-2 were generated by a previously described subtractive immunization approach (Brooks et al., 10 1993). Hybridoma culturing and purification of mAbs were performed by the Protein and Nucleic Acids Core Facility of The Scripps Research Institute using standard procedures. Example II Reagents 15 Protease inhibitors, normal mouse IgG, anti-FLAG M2 mAb, DAB reagent and Gill hematoxylin were purchased from Sigma (St. Louis, MO). Reverse transcription and PCR reagents and the pCR-II Topo vector were from Invitrogen. PP2 was obtained from Calbiochem (La Jolla, CA). 20 Example II Protein Purification, Peptide Sequencing and Protein Analysis Irnmunoprecipitations were performed on lysates from either unlabelled or 5 S-labelled HEp3 cells (5x10 7 ). Metabolic labeling was performed overnight in methionine/cysteine free DMEM containing Tran "S-label (100 pCi/ml; ICN, Costa Mesa, CA). Cells were 25 washed thoroughly with PBS then lysed in a buffer containing 0.1 M Tris (pH 8.0), 0.1% Triton X-1 00, 150 mM NaCl, 5 mlM EDTA, 10 pM trans-epoxysuccinyl-L-leucylamido (4 guanidino) butane, 20 pg/ml soybean trypsin inhibitor and 25 pg/nl aprotinin. Lysates were pre-cleared against protein G-Sepharose (Pharmacia Biotech, Piscataway, NJ) at 4"C for 30 minutes then incubated overnight at 4"C with 20 pLg of either mAb 41-2 or, as control, 30 nmIgG. Immunocomplexes was precipitated using protein G-Sepharose and complexes were denatured by boiling in reducing SDS loading buffer before analysis by polyacrylamide gel electrophoresis. For 35S-labelled proteins, the gel was dried and exposed to film at -80"C. Otherwise proteins were transferred to polyvinylidine difluoride 22 (PVDF) membranes (Millipore, Bedford, MA). The predominant coomassie stained band, at 135 kDa, was excised then digested with trypsin. The resulting peptides were separated by high pressure liquid chromatography and sequenced on a Procise 494 protein sequencer (Applied Biosystems, Inc., Foster City, CA). Trypsin digestion and peptide sequencing 5 were performed by the Protein and Nucleic Acids Core Facility of The Scripps Research Institute. Peptide sequences were used to search the GenBank database using algorithms available at the National Center for Biotechnology Information (NCBI) website. The complete SIMA 135 protein sequence was analyzed for structural domains, cellular processing signals and consensus post-translational modification motifs using the Prosite 10 database (Falquet et al., 2002), the SMART algorithm (Schultz et al., 1998), the PSORT algorithm (Nakai and Kanehisa, 1992) and the NetPhos 2.0 algorithm (Blom et al., 1999). Example IV Expression Constructs and Transient Transfections 15 SIMA135 cDNA in the eukaryotic expression vector pME18S-FL3 (GenBank accession number AK026622) was generated as part of the Japanese NEDO human cDNA sequencing project and kindly provided by Dr. Hiroko Hata (Dept. of Virology, Institute of Medical Science, University of Tokyo). The SIMAI 35FLAGin construct was generated by PCR placing sequences encoding the FLAG epitope (DYKDDDDK) immediately before 20 the stop codon of the parent construct. Both constructs were sequenced. HeLa cells (4x1 05) were transiently transfected with either the SIMA1 35 or SIMAl 35FLAGin expression constructs using Superfect reagent (Qiagen, Valencia, CA) as described by the manufacturer. Cells were lysed in ice cold buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, 1% Triton X-100, 5 mM EDTA and 1x Complete mini EDTA-free protease 25 inhibitor cocktail (Roche, Indianapolis, IN). Insoluble material was removed by centrifugation at 14000 rpm for 10 min. Example V Cloning of the SIMA135 cDNA from HEp3 Cells 30 Total RNA was isolated using an RNeasy kit (Qiagen) and 2 [Ig served as template in a reverse transcription reaction using Superscript II reverse transcriptase. PCR was performed on 1 pl of the resulting cDNA using primers TCCCCACCGTCGTTTCC (SEQ ID NO:2) and GGTTAGGAACACGGACGGGTG (SEQ ID NO:3)(designed based upon 23 GenBank accession AK026622) and the proof reading enzyme Platinum Pfx DNA polymerase. PCR cycling conditions were 94"C for 3 min, 30 cycles of 94*C for 30 sec, 55*C for 30 sec and 72*C for 150 sec followed by a final 72*C extension for 10 min. PCR products were gel purified (Qiagen) adenosine tailed using Platinum Taq DNA polymerase 5 then cloned in the pCR-II Topo vector and sequenced. Example VI Immunofluorescence HeLa cells transiently transfected with the SIMA135FLAGin expression construct and 10 HEp3 cells were plated on coverslips. After incubation for 48 hr at 37*C cells were washed with PBS then fixed in 2% formaldehyde. HeLa cells to be incubated with anti-FLAG mAb were either not permeabilized or permeabilized by incubating in 0.5% Triton X-100 in PBS for 5 min at room temperature. Both cell types were blocked in 5% BSA in PBS. Following overnight incubation at 4*C with either mAb 41-2 (5 pg/ml) or anti-FLAG M2 15 mAb (4 Lg/ml) in blocking buffer, cells were washed with PBS then incubated with Alexa Fluor 546 conjugated goat anti-mouse IgG (2 pg/ml) (Molecular Probes). Labeled cells were visualized and photographed using a BioRad 1024 MRC2 scanning confocal imaging system. 20 Example VII Northern Blot Analysis A human 12 lane multiple tissue Northern blot (Clontech) was hybridized with [a- 2 P]dCTP labeled (Ambion) EcoRI/HincII DNA insert fragments of the SIMA135 cDNA overnight in UltraHyb solution (Ambion) at 68*C. The blot was washed to a final stringency of 0.1 x 25 SSC, 0.1% SDS at 68*C then exposed to film at -80"C. Blots were reprobed with B-actin cDNA to determine consistency of RNA loading in each lane. Example VIII Western Blot Analysis 30 Cell lysates, serum free conditioned media and immunoprecipitated proteins were separated by electrophoresis through 8% SDS-polyacrylamide gels then transferred to nitrocellulose membranes (Millipore). Membranes were blocked in 5% non-fat skim milk powder in PBS then incubated overnight at 40C with either mAb 41-2 (2 p/ml), anti-FLAG M2 mAb (0.8 24 pg/ml) or anti-phosphotyrosine mAb (1 pg/ml; Upstate Biotechnology, Lake Placid, NY). Following extensive washing membranes were incubated for 2 hr at room temperature with goat anti-mouse IgG (0.16 4g/ml, Pierce, Rockford, Il) and immunoreactive bands detected by enhanced chemiluminescence (Pierce). 5 Example IX Biochemical Characterization Procedures For removal of N-linked glycans, lysates (50 gl) from M+ HEp3 cells and HeLa cells transiently transfected with the SIMAl135FLAGin expression construct were denatured and 10 reduced in 0.5% SDS, 1% p-mercaptoethanol for 10 minutes at 100*C then incubated with PNGase F (New England Biolabs, Beverly, MA) at 37"C for 45 minutes. For analysis of the basal level of tyrosine phosphorylation of SIMA135, subconfluent cultures of HEp3 and HeLa (as negative control) cells were incubated at 37*C for 30 min with serum free DMEM containing 50 mM NaF and 1 mM Na 3 V0 4 then washed with ice cold PBS. For 15 inhibition of Src kinase family phosphorylation, HEp3 cells were cultured in serum free DMEM without NaF and Na 3 V0 4 for 30 minutes at 37*C with PP2 (50 iM). Cells were then lysed in ice cold buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X 100, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 25 ig/ml aprotinin, 25 pg/mi leupeptin, 50 mM NaF and 1 mM Na 3

VO

4 . Insoluble material was removed by 20 centrifugation at 14000 rpm for 10 min. Immunoprecipitation was performed as described above on 300 pg of cell lysates using 1 jig of either mAb 41-2 or nmIgG (as negative control). For assays for the presence of soluble SIMA135, HEp3 cells approaching confluence, were washed three times with PBS then incubated in serum free conditioned media for 24 hr. The media was collected and centrifuged at 4"C and 10,000g then 25 concentrated 10 fold using micron centrifugal filters with a molecular weight cut off of 30,000 kDa (Millipore). Cells lysates were collected as described above. Example X Immunohistochenistry 30 Cryostat sections (6gm) from archival human adenocarcinoma colon tissue samples from three patients were fixed in zinc-formalin for 15 min, rinsed briefly with PBS then non specific binding sites blocked by incubating in PBS containing 3% BSA. mAb 41-2 (5 gg/ml) was applied at 4*C overnight. Specific antibody binding was detected by the 25 addition of biotin conjugated anti-mouse antibodies (Pierce) followed by peroxidase conjugated neutravidin (Pierce) which was visualized using DAB reagent. Sections were counterstained using Gill hematoxylin. 5 Example XI mAb 41-2 Recognizes a 135 kDa Antigen Expressed at Elevated Levels in Highly Metastatic Human Tumor HEp3 Cells The antigen recognized by the antibody mAb 41-2 was identified and characterized. As an 10 initial step in determining the significance of the antigen recognized by mAb 41-2, Western blot analysis was performed using mAb 41-2 on lysates (20 4g) prepared from high metastatic (M+) and low metastatic (M~) HEp3 cells electrophoresed under non-reducing conditions. Monoclonal antibody 41-2 (mAb), generated by subtractive immunization, detected a single band of approximately 135 kDa in both cell types. Consistent with the 15 subtractive immunization approach taken in generating mAb 41-2, the immunoreactive protein was expressed at higher levels in M* than in M HEp3 cells. Parallel Coomassie stained gels of the lysates prepared from high metastatic (M+) and low metastatic (M~) HEp3 cells demonstrate that the overall protein pattern and content was indistinguishable for M+ and M~ HEp3 cell extracts. The difference in mAb 41-2 immunoreactivity shows a 20 significant difference in the level of expression of the cognate antigen between the two cell lines. Example XII Identification of the Antigen Recognized by mAb 41-2 from Metastatic HEp3 Cells 25 Purified mAb 41-2 was used to immunoprecipitate the antigen of Example XI from M* HEp3 cells. 35 S labeled proteins immunoprecipitated from M+ HEp3 cells with mAb 41-2 or normal mouse IgG were analyzed on SDS-PAGE under reducing conditions. The gel was dried and exposed to film at -80*C overnight. The major protein immunoprecipitated from radiolabeled HEp3 cells had a molecular weight of approximately 135 kDa. This was 30 consistent with the molecular weight of the antigen detected in Example XI. In a parallel experiment using unlabeled HEp3 cells, in which immunoprecipitated proteins were transferred to a PVDF membrane, the 135 kDa protein band was excised, subjected to 26 trypsin digestion and the separated fragments sequenced from the N-terminus. Three major peptide sequences were obtained. Searches of the GenBank non-redundant protein database indicated that each of the peptide sequences had exact or near exact matches with the theoretical sequence of an unpublished entry with accession number BAB15511, 5 translated from unpublished cDNA entry AK026622. Peptide 1 FEIALPRESQITVLG(I)KXGT SEQ ID No: 4 BAB15511 FEIALPRESNITVLIKXGT SEQ ID No:5 10 Peptide 2 XXXXIPGSTTNPE SEQ ID No:6 BAB15511 VEYYIPGSTTNPE SEQ ID No:7 Peptide 3 XYXLQVPSDILH SEQ ID No:8 BAB15511 SYSLQVPSDILH SEQ ID No:9 15 The complete sequence of the identified protein, which was designated subtractive immunization M*HEp3 associated 135 kDa protein (SIMA135), is shown in Figure 1. To confirm that SIMA 135 was the same protein specifically recognized by mAb 41-2, Western blot was performed on lysates from HEp3 cells, untransfected HeLa cells and HeLa cells 20 transiently transfected with the SIMAl 35 cDNA. Western blot analysis probing with mAb 41-2 of total cell lysates (25 Rg) electrophoresed under non-reducing conditions from HEp3 cells, mock transfected HeLa and HeLa cells transiently transfected with the SIMA135 cDNA. mAb 41-2 reacted with the same 135 kDa protein band that is present in HEp3 cells and in HeLa cells transiently transfected with the SIMA135 cDNA, but is absent in the 25 untransfected HeLa cells. To provide additional confirmation, the protein encoding region of the SIMA 35 mRNA was cloned from HEp3 cells by reverse transcription-PCR. DNA sequence analysis of two clones generated by this approach confirmed that SIMA135 mRNA is expressed by these cells. Four nucleotide differences were identified between GenBank entry AK026622 and SIMA1 35 sequence obtained from HEp3 cells: nucleotide 30 16 8 4 G-A, 18 47T-C 2 2 3 6 G-A and 259 0 G-A. The second transition is silent and the others result in amino acid changes 525 Ag-GIn, 70 gGly-AP and 827S-A" respectively. Example XIII 27 SIMA135 Structural Features The SIMA135 protein sequence includes 836 amino acids (SEQ ID No.1 - Figure 1) and has a deduced molecular weight of 92.9 kDa. Sequence analysis identified the following structural features. A putative amino terminal signal peptide with cleavage predicted to 5 occur following Ala 29 . This feature is consistent with the sequence of peptide 1 indicating that mature SIMA135, with a predicted molecular weight of 90.1 kDa, starts at Phe". A potential transmembrane domain, spanning residues 666 to 686 (boxed in Figure 1), is predicted (Hartmann et al., 1989) to orient SIMA135 with its carboxy terminus located intracellularly. Twelve consensus motifs for N-glycosylation are indicted in Figure 1. A 10 consensus type 1 palmitylation motif (IICCV) (Hansen et al., 1999) is located at residues 687 to 691. Five PXXP motifs are present (Figure 1) which in other proteins have been shown to mediate binding to Src homology (SH) 3 domains (Pawson 1995; Mayer, 2001). Five tyrosine residues (circled in Figure 1) may be phosphorylation sites. Two closely spaced tyrosine residues (Tyr 734 and Tyr 743 ) are present in consensus motifs (YXXL/I) for 15 SH2 domain binding (Songyang et al., 1993). SIMA135 lacked homology with other confirmed proteins in the GenBank non-redundant database. However, SIMA135 did have high homology to the theoretical protein CDCP 1 as determined by translation of the reported nucleic acid sequence (Scherl-Mostageer et al., 2001). In addition, two regions of the SIMA135 protein, spanning residues 221 to 348 and 417 to 544, were identified that 20 had low homology to CUB (complement protein subcomponents _Cr/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains (Bork and Beckmann, 1993). These domains have been reported to act in mediating protein-protein interactions (Chen and Wallis, 2001; Sieron et al., 2000). A third putative CUB domain described by Scherl-Mostageer and co-workers spanning residues 545 to 660 (Scherl 25 Mostageer et al., 2001) was below the homology detection threshold of the search algorithms used by us to scan the S5MA135 amino acid sequence and the theoretical amino acid sequence for CDCP 1. Example XIV 30 Expression Pattem of SIMA135 in Normal and Malignant Cells and Tissues Northern blot analysis of poly A RNA (1 gg per lane) from 12 normal human tissues probed with a 3 P labeled SIMA1 35 cDNA probe was performed.. Levels of P-actin mRNA 28 are shown as a measure of loading. The expression pattern of SIMA135 mRNA in 12 normal human tissues was examined by Northern blot analysis by hybridizing with a 3p labeled 2.8 kb SIMA135 cDNA probe. A band of approximately 6.0 kb was detected at highest levels in skeletal muscle and colon with lower levels of expression in kidney, small 5 intestine, placenta and lung. A barely detectable signal at -6.0 kb also was present in peripheral blood leukocytes. In addition, a much weaker signal at approximately 3.3 kb was present in skeletal muscle, colon, placenta and lung. SIMA135 mRNA was not detected in brain, heart, thymus, spleen or liver. Based upon alignment of SIMA135 with CDCPI cDNA 7 and genomic sequences (data not shown), it appears most likely that the 10 two SIMA1 35 transcripts detected by Northern blot analysis result from use of alternate polyadenylation signals within the SIMA-135 3' UTR. The longer, more highly expressed transcript is thought to have resulted from use of a more 3' consensus poladenylation signal (at nucleotide 5950 '), whereas the shorter, more lowly expressed transcript likely results from use of a variant, less efficient polyadenylation signal located at nucleotide 3186 7. It is 15 also possible that these variant transcripts result from alternate splicing of the SIMAI 35 pre-mRNA. SIMA135 protein expression in 16 human cell lines, 14 human tumor cell lines and two normal human cell lines probing with mAb 41-2, was analyzed by Western blot analysis under non-reducing conditions in which equal amounts of cell lysate protein (20gg) were 20 electrophoresed for each cell line. SIMA135 was most highly expressed in metastatic HEp3 cells, with the prostate cancer cell line PC3, and the colon cancer cell line DLD-1 also manifesting high levels of expression. Moderate levels of the antigen were detected in the fibrosarcoma cell line HT1080, the gastric cancer cell lines IKN45 and STKM-1, the colon cancer cell line SW480 and the non-metastatic prostate cancer cell line LNCaP. Low 25 levels of SIMA135 were detected in 2 liver cancer cell lines, 2 breast cancer cell lines, the lung cancer cell line A549 and the kidney rhabdoid tumor cell line G401. SIMA135 was not detectable in normal human microvascular endothelial cells and dermal fibroblast cells. Varying levels of SIMA135 protein was expressed in a number of human tumor cell lines, while two normal human cell types did not express the protein. 30 Example XV SIMA135 is a Cell Surface. Phosphorylated Glycoprotein. 29 Immunocytochemistry was used to determine the cellular location of SIMA135 in HEp3 cells, and also in HeLa cells that were transiently transfected with a SIMA1 35 expression construct. The expression construct contained a FLAG epitope fused after the carboxy terminus of the SJIMA135 protein. HEp3 cells incubated with mAb 41-2 showed strong 5 staining on the plasma membrane. HeLa cells transiently transfected with FLAG tagged S[MAI 35 also showed similar strong membrane staining when incubated with mAb 41-2. In addition, the SIMIA 135 carboxy terminus was determined to be intracellular as transiently transfected HeLa cells permeabilized using 0.5% Triton X-100 to permit access of the antibody to the intracellular located FLAG tag and incubated with anti-FLAG mAb 10 showed strong membrane staining when incubated with an anti-FLAG epitope mAb, while non-permeabilized cells exhibited low or near background staining with anti-FLAG nAb. Untransfected HeLa cells were essentially free of staining when incubated with either mAb 41-2 or an anti-FLAG epitope mAb. These data confirmed the predicted cell surface location as well as the type I orientation of SIMA135. Also, the coincidence of staining 15 observed with mAb 41-2 and anti-FLAG mAb in HeLa cells transiently transfected with the SIMA135-FLAG tag expression construct, additionally confirmed that SIMA135 is the target antigen for mAb 41-2. The theoretical molecular weight of mature SIMA135 is 90.1 kDa. However, the apparent 20 molecular weight of the protein detected by mAb 41-2 was 135 kDa.. Cell lysates from HeLa cells transiently transfected with a SIMA135 FLAG tag expression construct were treated with N-glycosidase F under conditions optimal for enzyme activity to determine if the difference in molecular weight was due to N-glycosylation. Proteins were examined by Western blot analysis under reducing conditions using an anti-FLAG epitope mAb of 25 untreated (-) and N-glycosidase F (PNGase F) treated (+) lysates from HeLa cells transiently transfected with the SIMA135FLAGin expression construct. Bands due to SIMA135 are indicated. A non-specific cross reacting band is apparent at 80 kDa. N glycosidase F treatment resulted in the disappearance of the SIMA135 protein band at 135 kDa and replacement with a broad lower molecular weight band of approximately 95 to 105 30 kDa (data not shown). Lysates of M* HEp3 cells were also immunoprecipitated with mAb 41-2 and treated with N-glycosidase F. The proteins detected according to this method also manifested a similar diminished molecular weight. Therefore, up to 30-40 kDa of the apparent molecular weight of SIMA 135 is due to N-glycosylation, consistent with the large 30 number of consensus glycosylation sites in the extracellular region of this protein (Figure 1, SEQ ID No.1). The intracellular region of SIMA135 contained 5 tyrosine residues (Figure 1). Western blot analysis under reducing conditions with an anti-phosphotyrosine antibody was 5 performed on proteins immunoprecipitated from HEp3 cell lysates with mAb 41-2 to determine whether any of these residues are phosphorylated. immunoprecipitations that were performed with either normal mouse IgG (nmIgG) or mAb 41-2 from HEp3 and HeLa (negative control) cell lysates. Immunoprecipitated proteins were probed with an anti phosphotyrosine antibody (Anti-P-Tyr) and with mAb 41-2 to indicate the presence of 10 phosphorylated and total SIMA135, respectively. Cell lysate proteins were immunoprecipitated with mAb 41-2. Lysates were prepared from HEp3 cells either untreated (-) or (+) incubated for 30 minutes at 37* with PP2 (50 gM). The anti phosphotyrosine antibody detected a protein of 135 kDa that immunoprecipitated from HEp3 cells with mAb 41-2. The same protein band was detected when the 15 immunoprecipitated proteins were probed with m.Ab 41-2. Western blot analysis was also performed on proteins immunoprecipitated from HeLa cell lysates with mAb 41-2, and proteins immunoprecipitated from HEp3 cell lysates with normal mouse IgG as controls. Both immunoprecipitations were free of immunoreactivity when probed with either the anti-phosphotyrosine antibody or mAb 41-2, demonstrating the specificity of the 20 immunoreactions observed with HEp3 cells. The involvement of a Src kinase family member in SIMA135 tyrosine phosphorylation was examined using PP2, a Src family selective tyrosine Icinase inhibitor (Hanke et al., 1996). Western blot analysis with an anti phosphotyrosine antibody of proteins immunoprecipitated from HEp3 cell lysates with mAb 41-2, showed that HEp3 cells treated with PP2 for 30 minutes had a significant 25 reduction (-75%) in the level of SIMA135 tyrosine phosphorylation compared to protein from untreated HEp3 cells. Western blot analysis, using mAb 41-2, of the same immunoprecipitated proteins, indicated that approximately equal amounts of SIMA135 protein were present in both lanes on the membrane. These data suggested that a Src 30 kinase family member acts during tyrosine phosphorylation of SIMA 135 in HEp3 cells. A number of integral cell surface proteins, such as c-met (Wajih et al., 2002) and CD44 (Goebeler et al., 1996), are also produced as soluble molecules. Western blot analysis, probing with mAb 41-2, was employed to examine whether HEp3 cells produce a soluble 31 form of SIMA135. HEp3 cell cultures were washed extensively with PBS then incubated for 20 hr with serum-free (SF) medium. The conditioned medium (CM) was harvested and cellular material was removed by centrifugation and the media then concentrated 10 fold. The antibody mAb 41-2 detected an immunoreactive band of approximately 110 kDa in 5 HEp3 SFCM. The cell-associated SIMA135 from HEp3 lysates was detected at 135 kDa. In contrast, untransfected HeLa cells, which do not produce SIMA135, yielded no immunoreactive bands in either the lysate or concentrated SFCM. These data indicate that HEp3 cells release a soluble form of SIMA135 and the soluble form presents as a lower molecular weight immunoreactive protein. 10 Example XVI Expression of SIMA135 in Normal and Cancerous Colon Immunohistochemical analysis was performed to determine the in vivo localization of SIMAl35 in normal and cancerous colon. Sections (6 pm) were stained with mAb 41-2 as 15 primary antibody. Representative normal colonic mucosa shows epithelial expression of SIMA135 (red-brown in color photograph, not shown). Colon carcinomas display heterogeneous and extensive expression of SIMA135. SIMA135 is expressed by cells within invading malignant glands in the colonic serosa. SIMA 35 is expressed by malignant epithelial cells within the lumen of a draining blood vessel in the serosa of the 20 colon. In normal colonic mucosa, SIMA135 was expressed exclusively by epithelial cells where it was present uniformly on the luminal and basal surfaces of cells lining the colonic lumen, and on the apical surfaces of cells lining the glandular crypts (data not shown). The presence of intense staining in the contents of goblet cells of the crypts and in the mucus in the lumen of glands supports the thought that SIMA135 is produced in a soluble form by 25 colonic epithelial cells. In colon carcinoma specimens SIMA135 was extensively and heterogeneously expressed with some focal accentuation in the mucus within malignant glands. Some groups of invading cancer cells, were heavily stained showing the presence of SIMAl 35 on the basal, apical and lateral membranes as well as within the glandular mucus. There was a definite trend towards an association of intense staining with more 30 malignant, invading glands as carcinoma cells deeper in the colonic serosa and within draining blood vessels were often strongly positive for the SIMA135 antigen. Control sections that were incubated with the secondary but not the primary antibody were free of staining. 32 Example XVII Following the transfection procedures described above for HeLa cells, 7 different cell lines were used to study and monitor expression of SIMA-135 mRNA. Of these cell lines, clone 5 no. 3 produced a significant, appropriate base pair signal. The ability of these cell lines to transport in an in vitro study, to metastasize and to form tumors in SCID mice and to colonize secondary organs in SCIP mice was also studied. It was determined that clone 3 was able to detach and migrate through porous membranes and in vivo and to colonize secondary organs. These results demonstrate that SIMA 135 is a 10 key, central factor managing metastasis. The transformed clone no. 3 cells are also useful as the cellular system for identification of agents that promote or inhibit/minimize metastasis of malignant cells. Pursuant to the transfection procedures outlined above, 7 different cell lines were employed to study the effects of SIMA-135 expression. These cell lines are illustrated in Figure 8. 15 Following the analytic procedures for SIMA-135 RMA detection given above, these 7 cell lines were analyzed. Figure 7 shows the results of the RT-PCR analysis of these 7 different cell lines monitoring SIMA-135 mRNA. Controls and irrelevant RT-PCR data is in the left-hand part of Figure 1. The right-hand part of the figure shows a 600 bp amplified signal generated from SIMA-135 specific primers. HeLa untransfected yields no signal, 20 HeLa no. 4 which also yields no signal and Eff (2) no. 3 which is now called clone no.3 and generates a substantial 600 bp signal. These data indicate that clone no. 3 cells have elevated levels of SIMA-135 mRNA. Samples of these 7 cell lines were lysed and the cellular contents analyzed according to a standard Western blot procedure to determine the presence of SIMA- 135 through binding 25 with MoAb 41-2. Western blot on lysates prepared from the 7 different cell lines probed with MoAb 41-2. The results demonstrate that clone no.3 (Eff (2) no.3) produces substantial levels of the SIMA-135 immuno-reactive protein. This cloned cell line produces more SIMA-135 protein then even our highly metastatic HEp-3 cell line, M+. Clone #4 and parental HeLa cells produce no detectable SIMA-135 protein. 30 Once it was confirmed that SIMA-1 35 negative cells and SIMA-1 35 overexpressors had been obtained, these cells were tested for their malignant potential i.e. their ability to grow and form tumors in SCID mice and their ability to colonize secondary organs in SCID mice. Table I is a summary of two separate types of assays: Panel A shows the results of an 33 experimental metastasis assay where the cells of interest are inoculated (i.v.) directly into the tail vein of the mice. A few weeks later, selected organs of the inoculated mice were analyzed for the presence of human cells (human DNA) in the background of total organ mouse DNA. The analysis was performed by real time PCR using human specific primers 5 based on alu repeat sequences as described in the following reference (A. Zijlstra, et al., Cancer Research, 2002). The results shown in panel A indicate that clone no.3 cells colonize and/or grow in mouse lung and bone marrow at levels substantially over that of similarly inoculated clone no.4 cells. It is also apparent that clone no.3 does not just spread all over the inoculated mice since another organ shown here, the pancreas, only contains 10 near background levels of both clone no.3 and clone no.4 cells. Panel B of Table 1 contains the results of a standard spontaneous metastasis assay where cells are inoculated subcutaneously in the flanks of SCID mice, tumors are allowed to develop to over 100 mg and then selected organs, usually the lungs, are analyzed for 15 secondary metastatic deposits. The secondary metastases were measured by the same real time alu PCR procedures that are specific for human DNA. The results indicate that clone no.3 and clone no.4 form primary tumors of approximately equal size (weight in milligrams-mg). However, clone no.3 appears to have metastasized to the lungs at a level that is at least 10 times greater than clone no. 4. It may be more than 10 times greater since 20 the level of cells in the clone no.4 lung is close to background (50-100 cells) at barely detectable levels. The results demonstrate that the introduction or expression of the SIMA-135 protein into cells that normally do not produce it, conveys malignant properties to those cells. In order to characterize these two clones for properties that might indicate why they have gained 25 malignant potential we carried out a few cell biological assays on the clones. A cell growth or proliferation assay was carried out in cell culture according to the procedures given above. The growth in vitro of these two clones is similar and also similar to the parental HeLa cells. Thus, simple proliferation rate is not the reason for their differential malignant potential. 30 A trans-well migration assay whereby cells of interest are forced to migrate across a porous filter inserted between two chambers was also carried out. The assay was conducted with culture medium. The results are shown in Figure 10. The upper chamber contains the cells in medium while the lower chamber contains enriched medium with fetal calf serum (FCS) 34 to attract the migrating cells from the upper chamber. The results show that the clone no.3 cells are much more migratory than clone #4 cells. This could be one of the acquired properties of SIMA-1 35 overexpressors that aid them in their malignant potential. Preliminary data also indicate that clone no.3 cells appear to be more resistant than clone 5 no.4 cells to apoptosis induced chemically by the compound ara C. Table I In .ivo malignancy of two HeLa clones; Clone #4 (SIMA-135 negative) and clone #3 (SIMA-135 overexpressors) inoculated into SCID mice. A. Experimental Metastasis (i.v. inoculation into tail vein). Calculated number of human cells/organ* #4 #3 Lung 150 2300 Bone Marrow <100 9,500 Pancreas 150 200 B. Spontaneous Metastasis (subcutaneous inoculation) #4 inoculated mice #3 inoculated mice Primary Tumor Weight (mg) 227.5 +/- 68 (n=8) 184.1 +/- 26 (n=7) Human Cells in Lung (# cells)* 250* 2800* *based on real time alu PCR performed on total DNA extracted from organ excised 2 10 weeks after i.v. inoculation and 4 weeks after s.c. inoculation. Documents Adham Iv, Klemm U, Maier WM and Engel W. (1990). Hum. Genet., 84, 125-8. Bajorath J. (2000). Proteins, 39, 103-111. 15 Blom N, Gammeltoft S and Brunak S. (1999). J. Mol. Biol., 294,1351-1362. Bork P and Beckmann G. (1993). J Mol. Biol., 231, 539-545. Briner TJ, Kuo MC, Keating KM, Rogers BL and Greenstein JL. (1993). Proc. Nat. Acad. Sci. USA, 90, 7608-7612. Brooks PC, Lin JM, French DL and Quigley JP. (1993). J. Cell Biol., 122, 1351-1359. 20 Chen CB and Wallis R. (2001). J. Biol. Chem., 276, 25894-25902. 35 Falquet L, P agni M, Bucher P, H ulo N, S igrist C J, H ofmann K and Bairoch A. (2002). Nucleic Acids Res., 30, 235-238. Goebeler M, Kaufmann D, Brocker EB and Klein CE. (1996). J. Cell Sci., 109 ( Pt 7), 1957-1964. 5 Gorelik E, Galili U and Raz A. (2001). Cancer Metastasis Rev., 20, 245-277. Grogan MJ, Pratt MR, Marcaurelle LA and Bertozzi CR. (2002). Annu. Rev. Biochem., 71, 593-634. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA and Connelly PA. (1996). J. Biol. Chem., 271, 695-701. 10 Hansen SG, Grosenbach DW and Hruby DE. (1999). Virology, 254, 124-137. Hartmann E, Rapoport TA and Lodish HF. (1989) Proc. Nati. Acad. Sci. USA, 86, 5786-90. Hooper JD, Clements JA, Quigley JP and Antalis TM. (2001). J. Biol. Chem., 276, 857-60. King SW and Morrow KJ, Jr. (1988). Biotechniques, 6, 856-861. Martin GS. (2001). Nat. Rev. Mol. Cell Biol., 2, 467-475. 15 Maruo Y, Gochi A, Kaihara A, Shimamura H, Yamada T, Tanaka N and Orita K. (2002). Int. J. Cancer, 100, 486-490. Matthew WD and Patterson PH. (1983). Cold Spring Harb. Symp. Quant. Biol., 48 Pt 2, 625-631. Mayer BJ. (2001). J. Cell Sci., 114, 1253-1263. 20 Nakai K and Kanehisa M. (1992). Genomics, 14, 897-911. Nielsen-Preiss, SM and Quigley JP. (1993). J Cell Biochem 51:219-235 Ossowski Land Reich E. (1983). Cell, 33, 323-333. Pawson T. (1995). Nature, 373, 573-580. Resh MD. (1994) Cell, 76,411-413. 25 Riggott MJ and Matthew WD. (1996). J. Neurosci. Methods, 68, 235-245. Rye PD and McGuckin MA. (2001). Tumour. Biol., 22, 269-272. Scherl-Mostageer M, Sommergruber W, Abseher R, Hauptmann R, Ambros P and Schweifer N. (2001). Oncogene, 20, 4402-4408. Schultz J, Milpetz F, Bork P and P onting C P. (1998). P roc. Natl. A cad. Sci. USA, 9 5, 30 5857-5864. Sieron AL, Tretiakova A, Jameson BA, Segall ML, Lund-Katz S, Khan MT, Li S and Stocker W. (2000). Biochemistry, 39, 3231-3239. Sleister HM and Rao AG. (2002). J. Immunol. Methods, 261, 213-220. 36 Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, Neel BG, Birge RB, Fajardo JE, Chou MM, Hanafusa H, Schaffhausen B and Cantley LC. (1993). Cell, 72, 767-778. Soos G, Jones RF, Haas GP, Wang CY. (1997). Anticancer Res., 17, 4253-4258. 5 Stocker JW and Nossal GJ. (1976). Contemp. Top. Immunobiol., 5, 191-210. Testa JE .(1992). Cancer Res., 52, 5597-5603. Testa JE, Brooks PC, Lin JM and Quigley JP. (1999). Cancer Res., 59, 3812-3820. Wajih N, Walter J and Sane DC. (2002). Circ. Res., 90, 46-52. Williams CV, Stechmann CL and McLoon SC. (1992). Biotechniques, 12, 842-847. 10 Yammani RR, Seetharam S and Seetharam B. (2001). J. Biol. Chem., 276, 44777-44784. Zhang W, Trible RP and Samelson LE. (1998). Immunity, 9, 239-246. U.S. Patent No: 6,245,898. A. ZijIstra, et al., Cancer Research, 2002. 15 All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may 20 be varied considerably without departing from the basic principles of the invention. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 37

Claims (20)

1. An isolated protein comprising amino acid sequence SEQ ID NO: 1, wherein the protein is a phosphotyrosine protein.

2. A soluble immunoreactive fragment of SEQ ID NO: I with a molecular weight of approximately 110 kDa.

3. The fragment of claim 2, starting at Phe-30.

4. A monoclonal antibody that binds specifically to: (i) a tumor marker protein comprising amino acid sequence SEQ ID NO: I; or (ii) the fragment of claim 2 or claim 3, wherein said antibody is not monoclonal antibody 41-2.

5. The monoclonal antibody of claim 4, wherein the antibody recognizes a glycosylated protein consisting of amino acid sequence SEQ ID NO: 1.

6. The monoclonal antibody of claim 5 which does not recognize a non-glycosylated protein consisting of amino acid sequence SEQ ID NO: 1.

7. The antibody according to any one of claims 4 to 6 for use as a therapeutic treatment of the human and animal body.

8. Use of the antibody according to any one of claims 4 to 6 for the preparation of a medicament to inhibit metastasis by a cancer cell in a mammal.

9. Method of inhibiting metastasis by a cancer cell in a mammal comprising administering an effective amount of an antibody according to any one of claims 4 to 6. C NRPorblDCC\REC\42546_ I DOC-21MW2012 -39

10. The use according to claim 8 or method according to claim 9, wherein the cancer cell is epidermoid carcinoma cell, a prostate cancer cell, a colon cancer cell, a fibrosarcoma, a gastric cancer cell, a liver cancer cell, a breast cancer cell, a lung cancer cell, or a kidney rhabdoid cancer cell.

11. The use according to claim 8 or claim 10 or method according to claim 9 or claim 10, wherein the cancer cell is HEp3 cell.

12. A pharmaceutical composition comprising the antibody according to any one of claims 4 to 6 and a pharmaceutically acceptable carrier.

13. A kit comprising the antibody according to any one of claims 4 to 6 and packaging material.

14. The use of the antibody according to any one of claims 4 to 6 in a diagnostic method of cancer which comprises: (a) contacting the antibody according to any one of claims 4 to 6 with a test sample obtained from a mammal; and (b) determining if the antibody binds to the test sample to a greater extent than the antibody binds to a control sample of non-cancerous tissue.

15. The use of the antibody according to any one of claims 4 to 6 in a diagnostic method of cancer which comprises: (a) visualizing binding of an antibody according to any one of claims 4 to 6 with a tissue section; and (b) determining if the antibody causes heterologous staining of the tissue sample, indicates extensive expression of SIMA135 throughout the sample, or stains malignant glands in the colonic serosa.

16. The use of the antibody according to any one of claims 4 to 6 to determine if a test sample contains metastatic cells comprising: (a) contacting the test sample with the antibody according to any one of claims 4 to 6; and (b) comparing binding of the antibody to the test sample to binding of the antibody to a control sample containing non-metastatic HEp3 cells; wherein increased binding of the antibody to the test sample as compared to C:\NRPertbl\DCC\REC.42504I6.1.DOC-21/)6rt202 -40 binding of the antibody to the control sample indicates that the test sample contains metastatic HEp3 cells.

17. The use of the antibody according to any one of claims 4 to 6 to determine the metastatis modulating ability of an agent, comprising: combining the agent with a cell that expresses SIMA135 of SEQ ID NO: I to produce a tested cell, determining whether the expression of SIMA135 of SEQ ID NO: 1 from the tested cell is greater or lesser than the amount expressed by the cell before its combination with the agent.

18. The use of the antibody according to any one of claims 4 to 6 to determine if a candidate agent modulates SIMA 135 production by a cell comprising: (a) contacting a test cell with the candidate agent; and (b) determining if SIMA 135 of SEQ ID NO: I production by the test cell is increased or decreased relative to SIMAI 35 of SEQ ID NO: 1 production by a control.

19. Protein according to claim 1, fragment according to claim 2 or claim 3, or antibody according to any one of claims 4 to 6, substantially as hereinbefore described with reference to any one of the examples.

20. Use according to any one of claims 8 or 14 to 18 or method according to claim 9, substantially as hereinbefore described with reference to any one of the examples.