MXPA00009153A - Urokinase plasminogen activator receptor as a target for diagnosis of metastases - Google Patents

Abstract

The present invention relates to the use of molecules capable of specifically binding a urokinase plasminogen activator receptor (uPAR) as diagnostic reagents for the detection of metastases in vivo. Such metastases can include, but are not limited to, micrometastases.

Description

ACTIVATOR RECEPTOR OF PLASMINOGENO OF UROQUINASE AS WHITE FOR THE DIAGNOSIS OF METASTASIS • 1. INTRODUCTION The present invention relates to the use of molecules capable of specifically binding with a urokinase plasminogen activator receptor (uPAR) as diagnostic reagents for the detection of metastases. Such metastases may include, but are not limited to, micrometastases. • 10 2. BACKGROUND Invasion of cancer cells and metastases is a multi-stage process involving several interdependent processes (Liotta, 1986, Cancer Res. 46: 1-7, Liotta et al., 1991, Cell 64: 327-336; Mundy, 1997, Cancer 15 80 (9): 1546-1556). Metastasis, the growth of secondary tumors at sites distant from a primary tumor is the leading cause of failure in the treatment of cancer. 2.1. THE METASTATIC PROCESS The regulatory mechanisms involved in metastasis 20 differ from the regulatory mechanisms that cause tumor formation. In fact, metastatic cells appear to be physiologically different from tumor cells. For example, metastatic cells differ in the expression of genes such as oncogen ras, serine-threonine kinases, tyrosine kinases, and p53 and also differ in signal transmission (for review see, Liotta et al., 1991, Cell 64: 327-336). Before metastasis, the expansion of a tumor involves angiogenesis, the formation of new blood vessels (Folkman et al., 1989, Nature 339: 58-61). It has been shown that tumors induce angiogenesis through several soluble factors (Folkman et al., 1987, Science 235: 442-447, Pepper et al., 1990, J. Cell Biol. 111: 743-755). Angiogenesis is a multi-step process that emanates from microvascular endothelial cells. Endothelial cells that are resting in vessels of origin are stimulated to degrade the endothelial base membrane, migrate in the perivascular stroma, and initiate a capillary outbreak (Liotta et al., 1991, Cell 64: 327-336). The capillary bud subsequently expands and assumes a tubular structure. Endothelial proliferation causes the extension of the microvascular tubules that develop in loops and then in the functioning circulatory network. The exit of the endothelial cells from the source vessel includes cell migration and degradation of the extracellular matrix (ECM) in a manner similar to the invasion of EMC cancerous cells (Liotta et al., 1991, Cell 64: 327-336). The invasion of cancer cells includes interactions of cancer cells with the ECM, a dense cross-linking of collagen and elastin integrated into a gel-like substance composed of proteoglycans and glycoproteins. The EMC consists of the base membrane and its underlying interstitial stroma. Tumor invasion includes: (1) detachment of cancer cells from their original location; (2) fixation 5 on the extracellular matrix; (3) degradation of the extracellular matrix; (4) locomotion in the extracellular matrix (for review, see Liotta, 1986, Cancer Res. 46: 1-7). After the detachment of the cancer cells, the cells migrate in the extracellular matrix and adhere to components of • the extracellular matrix such as, for example, laminin, type IV collagen and fibronectin through cell surface receptors. Cell adhesion molecules such as, for example, integrin, measured in the attachment of cancer cells on vascular endothelial cells and on matrix proteins (Mundy, 1997, Cancer 80 (9): 1546-1556). The fixed cancer cell then secretes hydrolytic enzymes or induces host cells to secrete enzymes that locally degrade the matrix. Lysis of the matrix occurs in a highly localized region near the surface of cancer cell, where the amount of active enzyme exceeds the natural proteinase inhibitors present in the serum, in the matrix, or those secreted by normal cells in the vicinity (Liotta et al., 1991, Cell 64: 327- 336) . A positive association with tumor aggressiveness has been observed for several classes of degrading enzymes, which - «Mn? -i? Í-i.MH i y -? I i iii i ?? i 'i. ^. ^^ - ^ - .. ^. ^ - ^. ^ jtjy ^ jM ^ - ^^^^, ^ - ^ - ^, ^ - ^ - ^ - ^^^^^ - ^ ^ - ^ - ^ include: heparinases, thiol-proteinases (including cathepsins B and L), metalloproteinases (including collagenases, gelatinases and stromelysins), and serine proteinases (including plasminogen activator of urokinase and plasmin). During the locomotion step of the invasion, the cancer cells migrate through the base membrane and stroma through the matrix proteolysis zone. The cancer cells then penetrate the tumor capillaries (which arise as a result of specific angiogenic factors) and reach the general circulation through these capillaries. After moving to distant sites of the organism, the intravasated cancer cells adhere to the vascular endothelium and extravasate through the vascular endothelium, and initiate the formation of a new tumor, that is, they first form a mass of cells that, Through the process of angiogenesis, it becomes a vascularized tumor. Thus, metastasis is not a simple random process but rather a multi-stage process that depends on the specific properties of the tumor cells and supporting factors in the environment of the metastatic site. 2.2. IMPLICATION OF uPA AND uPAR IN THE METASSTASIC PROCESS ON THE PRIMARY TUMOR SITE Numerous different molecules are involved in the metastatic process. Two examples of such molecules are uPA J. ^^^^ and its uPAR receptor, which have been implicated in the invasion aspect of tumor cells of the metastatic process. ^ fc During the invasion of cancer, uPAR binds to uPA released from surrounding cancer or stromal cells. The binding of uPA on its receptor focuses the proteolytic action towards the surface of the cancer cells. uPA converts an enzymatically inactive plasminogen into serine protease, plasmin. Plasmin degrades many extracellular matrix proteins such as, for example, fibronectin, vitronectin and fibrin facilitating the degradation of the extracellular matrix, proliferation of cancer cells, invasion and metastasis (Schmitt et al., 1997, Thrombosis and Haemostasis 78 (1): 285-296). Plasmin can also catalyze the activation of the zymogenic forms of several metalloproteinases. Studies have shown that anti-uPA antibodies decrease the invasion of tumor cells and / or metastasis of cells from cultured cell lines transplanted in animal models (for review see Andreasen et al., 1997, Int. J. Cancer 72: 1-22). Several studies have been conducted to examine the therapeutic effect of substances that interact with components of the plasminogen activation pathway. The manipulation of the plasminogen activation pathway resulted in rates of decreased tumor growth (Jankun et al., Patent - * - - «» - * »« »North American No. 5,679,350 (injection of a drug coupled to PAI-1 or PAI-2); Damage et al., North American Patent No. 5,519,120 (injection of anti-uPA or anti-uPAR antibodies); and Xing and Rabbani, 1996, Proc. Amer. Assoc. 5 Cancer Res. 37:90 (abstract # 626) (injection of anti-uPAR antibodies)). These studies indicate that uPAR plays a role in the early stages of metastasis, i.e., tumor cell invasion. Clinical findings have shown that elevated levels of 10 uPA, and the plasminogen activator, PAI-1, in primary tumor tissue are related to a guarded prognosis of several cancers including breast cancer, cervical cancer, ovarian cancer, stomach cancer, colon cancer, lung cancer, brain cancer, kidney cancer, bladder cancer, and soft tissue cancer (for a review, see Schmitt et al., 1997, Thrombosis and Haemostasis 78 (1) : 285-296, Andreasen et al., 1997, Int. J. Cancer 72: 1- 22). To a lesser extent, high levels of uPAR may also indicate a guarded prognosis (Schmitt et al., Thrombosis 20 and Haemostasis 78 (1): 285-296). 2.3. DIAGNOSIS AND STAGE OF THE DISEASE While the detection of metastatic disease markers at the primary tumor site may be useful for the prognosis and design of therapeutic modalities, there is currently no reliable system for detecting «^^ ¡^^^ X & ^ ^ twi B" "^ s • * - - - * * '* - •••" - "' **" "- micrometastasis in a patient - information that could be extremely important to determine the stage of the disease and design an appropriate clinical approach Even though metastatic tumors are derived from cells of the primary tumor, metastatic tumors are significantly altered in their physiological and growth characteristics, and do not necessarily express the same surface markers as In fact, the inability to diagnose and represent metastases, especially micrometastases, in vivo, remains a major obstacle to the successful treatment of cancer.The current surgical practice usually resorts to sight and touch in combination with locally determined protocols that dictate the magnitude of tissue resection, so the tissue removed during surgery includes not only e the tissue that is suspected of being neoplastic, but includes the amount of healthy tissue taken because the precise margins of tumor and areas of micrometastases can not be easily determined by the surgeon. In addition, an isolated metastatic tissue distant from the primary tumor frequently can not be easily detected by the methods usually employed. Accordingly, there is a great need, in the art, for having sensitive methods for detecting and locating • "" • - * - - * reliable way in vivo metastases. 3. COMPENDIUM OF THE INVENTION The present invention relates to methods for the diagnosis and metastasis imaging using 5 labeled molecules that specifically bind to the plasminogen activator receptor of urokinase, to detect and image in vivo metastases. The present invention is based, in part, on the unexpected discovery of the applicant in the sense that antibodies directed against • 10 uPAR can be used to detect not only primary tumors in vivo but can also be used to detect or represent micrometastases and metastases in sites .distinators of the primary tumor. Metastatic tumors, while derived from cells of the primary tumor, are significantly altered in terms of their physiological characteristics and in terms of their growth and do not necessarily express the same surface markers as primary tumors of origin. Prior to the discovery of the applicant, uPA and uPAR had been associated only with primary tumors that have metastatic properties. In addition, uPA and uPAR are considered to be involved in the initial stages of the metastatic process - that is, the mobilization of cells outside the primary tumor. Thus, it was quite surprising to discover that cells distant from the primary tumor, which participated in the establishment of * - - - »'- • -' M? new tumors (ie, by fixation - no cell expansion mobilization, angiogenesis, etc.) can be detected using uPA or uPAR as a marker. In a preferred embodiment of the invention, metastases in a subject are detected by: (a) administration of labeled molecules that specifically bind to uPAR; (b) allowing the labeled molecules to be preferentially concentrated in one or more metastatic lesions in the subject and that the unbound labeled molecule be depurated at the background level; (c) determine the background level; and (d) detecting the labeled molecule in such a way that detection of the labeled molecule above the background level indicates the presence of a metastatic lesion. In another preferred embodiment, the tagged molecule of the invention can be detected in a subject wherein the subject has received the tagged molecule at a sufficient time interval prior to detection to allow the tagged molecule to be preferentially concentrated in the lesions metastatic In specific modalities, the labeled molecule is a labeled anti-uPAR antibody or fragments that contain the binding domain of uPAR or peptides that mimic uPAR. In another specific embodiment, the labeled molecule is a peptide or derivative thereof that binds to uPAR, for example peptides having the amino acid sequence SEQ ID NO: 1 (FIG. 1) and SEQ ID NO: 2 (FIG. 2) , but not limited to them. The principle of the present invention is illustrated by working examples demonstrating the biodistribution of 5 uPAR in vivo, and showing the preferential accumulation of antibodies to uPAR in metastatic lesions in animal models. 4. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Amino acid sequence of the binding domain of • Residue receptor 7-33 of human uPA (Appella et al., 1987, J. Biol. Chem. 262 (10): 4437-4440). Figure 2. Amino acid sequence of receptor binding domain of residues 12-32 of human uPA (Appella et al., 1987, J. Biol. Chem. 262 (10): 4437-4440). 15 Figures 3A, B. Characterization of ruPAR by immunofluorescence. (A) Mat B-III cells were cultured at 70% confluency in glass dishes and incubated with 10 μg / ml pre-immune rabbit IgG or (B) with 10 μg / ml ruPAR IgG. After incubation with a secondary antibody of anti-rabbit IgG conjugate with FITC (X 20), the cells were analyzed for their immunofluorescence. Figure 4. Inhibition dependent on the invasion dose of Mat Ly Lu and Mat B-III cells by anti-ruPAR IgG. Mat 25 cells Ly Lu and Mat B-III were cultured in culture and added to L.-MaO-a-.J! '»> . »». ,., , , , ...... Y, . , Y . , «JÜ? . upper compartment of a Boyden chamber with 50 or 100 μg / ml ruPAR IgG. After 24 hours the number of cells that migrated to the lower aspect of the Boyden chamber filter was counted. Percent inhibition 5 of cell invasion was calculated by taking the number of cells that invaded after treatment with 50 or 100 μg / ml of pre-immune IgG as 100%. The results are the mean +/- standard error of four experiments of this type. The significant inhibition in invasion of cells from cells of • 10 control is represented by asterisks (* P <0.05). Figures 5A, B. Effect of ruPAR IgG and uPAR on the ruPAR IgG linkage labeled with 125 I in Mat Ly Lu and Mat B-III-uPAR cells. (A) Mat Ly Lu and (B) Mat 'B-III-uPAR are incubated with 125 I ruPAR-IgG, with or without increasing the ruPAR protein concentrations. The percentage change in binding of 125 I ruPAR-IgG after incubation with different concentrations of recombinant rat uPAR compared to control cells is shown. The results are the mean +/- standard error of four experiments. A significant inhibition of binding compared to control cells is represented by asterisks (* P <0.05). Figure 6. Temporal course of IgG absorption of ruPAR labeled with 125 I by primary tumors in vivo. 'Absorption of radioactivity in Mat B-III-Upar tumors in Fischer rats 25 females was monitored at several time points after - • • • *. • i i tt-Wfia * of intravenous injection of ruPAR IgG pre-immune labeled with 125I. The data represent the average IgG percentage of five animals +/- standard error of two experiments of this type. Figure 7. Absorption of ruPAR IgG labeled with 125I from the site of primary and metastatic Mat B-III tumors. On day 10 after inoculation with Mat B-III-uPAR cells in Fischer rats, the animals received an injection of ruPAR IgG labeled with 125 I pre-immune. After twelve hours, the biodistribution of ruPAR IgG labeled with 125 I in different normally unaffected tissues (adrenal glands, muscle, heart) was determined; sites of tumor metastasis (liver, spleen, kidney, lungs, lymph nodes); and primary tumors. The results represent percentage% ID / g of six animals in each group +/- standard error of three of these experiments. Figure 8. Absorption of ruPAR IgG labeled with 125I in primary tumors and metastatic lesions in mice bearing tumors. Tumor xenografts of prostate cells were established in Balb / c nu / nu mice. Five weeks after inoculation with tumor cells, the animals received an injection with 125 I-labeled pre-immune ruPAR IgG. After 12 hours the biodistribution of ruPAR IgG labeled with 125I was examined in different tissues including: normal tissue (heart), sites of metastasis ... .. A u »^.

Tumors (liver, spleen, kidney, lung, lymph nodes); and primary tumors. Biodistribution was calculated and expressed as percentage of injected dose / gram (% ID / g). Figure 9. Effect of anti-uPAR IgG on the volume of primary tumors in vivo. Rat B-III-uPAR rat breast adenocarcinoma cells were implanted in the mammary fat of female Fischer rats. From day 1 to day 7 after inoculation of tumor cells, the animals received pre-immune rabbit IgG (50-100 μg / ml / day) or IgG ANTI-ruPAR (100 μg / day). For two or three weeks after the inoculation of tumor cells, the size of the primary tumor was measured in two dimensions with calibrators and the volume of the tumor was calculated. 5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to peptide-derived antibodies, and analogs, peptides and mimetics that specifically bind to a urokinase plasminogen activator receptor (uPAR). The use of the term "a uPAR" indicates that even though the uPAR polypeptide portion in one species may be the same for all uPARs, there are several uPARs. For example, the carbohydrate part of the surface fixation mechanism of the uPAR may be different. In addition some cells such as, for example, cancer cells may have different uPARs. The invention further relates to the use of molecules that have binding specificity for uPAR for the detection, diagnosis, or in vivo monitoring of metastases, preferably micrometastases. In the embodiment of the invention the subject is injected with a molecule that has binding specificity for uPAR. After a sufficient time to allow distribution and accumulation in vivo, an image of the subject can be formed. Several methods can be employed to detect the accumulated labeled material in vivo, including, without limitation, radio-formative techniques. of image, such as, for example, X-rays, CAT scan, magnetic resonance imaging (M I), sonography, and positron emission tomography (PET). 5.1. URQQUINA5A RECEIVER LINK MOLECULES Methods for the production of molecules capable of specifically recognizing one or more uPAR epitopes or epitopes of conserved variants or uPAR peptide fragments are described herein, including, without being limited to, antibodies, derivatives (including fragments, but not limited to them) and analogs thereof, as well as peptides and peptide mimetics. Such uPAR binding molecules can be used, for example, in the detection of uPAR in a biological sample and can therefore be used as part of a diagnostic technique whereby tests can be performed on patients to detect abnormal levels of uPAR. In accordance with one embodiment of the invention, a uPAR binding molecule binds specifically with the human uPAR. 5.1.1. UPAR, DERIVATIVE AND ANALOGUE ANTIBODIES Such uPAR binding molecules may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') 2 fragments. / fragments produced by expression library, anti-idiotypic antibodies (anti-Id), and epitope binding fragments of any of the foregoing. Various methods known in the art can be used for the production of polyclonal antibodies to a uPAR protein or fragments thereof for the production of polyclonal antibody, several host animals can be immunized by injection with a native uPAR protein, or a synthetic version or fragment thereof, including, but not limited to, rabbits, mice, rats, chickens, etc. Various adjuvants can be used to increase the immune response, depending on the host species, and including, but not limited to, Freund activators (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols , polyanions, peptides, emulsions in oil, tin hemocyanins, dinitrophenol, and adjuvants potentially useful for humans such as BCG (Bacillus Calmette-Guerin) and corynebacterium parvum. For the preparation of monoclonal antibodies directed towards a uPAR protein sequence, it can be used • any technique that provides for the production of antibody molecules by continuous cell lines in culture. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256, 495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4 , 72), and the • 10 EBV-hybridoma technique to produce human monoclonal antibodies (Colé et al., 1985 in Monoclonal Antibodies and Cancer Therapy (Monoclonal Antibodies and Cancer Therapy), Alan R. Liss, Inc., pages 77-96). In addition, techniques developed for the production of "chimeric antibodies" can be employed (Morrison, et al., 1984, Proc. Nati, Acad. Sci., 81, 6851-6855; Neuberger, et al., 1984, Nature 312 , 604-608; Takeda, et al., 1985, Nature, 314, 452-454) by splicing the genes of a mouse antibody molecule of appropriate antigenic specificity together with genes from an antibody molecule to be human of appropriate biological activity. A chimeric antibody is a molecule in which different portions are derived from different animal species such as, for example, those having the region derived from a - • "'" - * * * -' * - "* • ^ IWaliá ^ ^^^ V ^^^^^^^^ Hj ^^^^ mAb and a constant region derived from human immunoglobulin. , for example, Cabilly et al., U.S. Patent No. 4,816,567, and Boss et al., U.S. Patent No. 4, 816397, which are hereby incorporated by reference in their entirety.) In addition, techniques for production have been developed. of humanized antibodies (See, for example, Queen, U.S. Patent No. 5,585,089 and U.S. Patent No. 5,225,539, which are incorporated herein by reference in their entirety.) A variable region of light or heavy immunoglobulin chain consists of a region of "structure" interrupted by three hypervariable regions known as complementarity determining regions (CDRs) .The magnitudes of the structural region and the CDRs have been defined with precision (See "Sequences of Proteins of Immunological Interest" (Sequence of Proteins of Immunological Interest). ), Kabat, E et al., Dep American Department of Health and Human Services (1983)). In short, humanized antibodies are antibody molecules from non-human species that have one or more CDRs from the non-human species and a structural region that comes from a human immunoglobulin molecule. Alternatively, the techniques described for the production of single chain antibodies can be adapted (US Patent No. 4,946,778; Bird, 1998, Science 242, 423-426; Houston, et al., 1988, Proc. Nati. Acad. Sci. USA 85, 5879-5883; and Ward, et al., 1989, Nature 334, 544-546) to produce single chain antibodies against uPAR. Single chain antibodies are formed by binding the heavy and light chain fragments of the FV region through an amino acid bridge, resulting in a single chain polypeptide. Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include, but are not limited to: F (ab ') 2 fragments, which can be produced by digestion of pepsin from the antibody molecule and Fab antibodies, which can be generated by reducing the disulfide bridges of the fragments of F (ab ') 2. Alternatively, Fab expression libraries can be constructed (Huse et al., 1989, Science, 246, 1275-1281) to allow rapid and easy identification of the monoclonal Fab fragments with the desired specificity. 5.1.2 PEPTIDES, DERIVATIVES, ANALOGUES, AND PEPTIDE MIMETHICS In one embodiment of the invention, uPAR binding molecules include peptide derivatives and analogs thereof, and peptide mimetics. In particular embodiments of the invention, Peptide peptides or mimetics are selected to mimic the following human uPA sequences: VPSNCDCLNGGTCVSNKYFSNIHWCNC (SEQ ID NO: l) and DCLNGGTCVSNKYFSNIHWCN (SEQ ID NO: 2). In the specific embodiment, the methods of the present invention employ uPA derivatives and analogues, particularly uPA fragments and derivatives of such fragments, which comprise one or more domains of a uPA protein. In another specific embodiment, the methods of the present invention employ a protein, fragment, analog, or uPA derivative that is expressed as a chimeric or fusion protein product (comprising the protein, fragment, analog, or derivative linked through a link on a heterologous protein sequence (of a different protein)). A specific embodiment relates to a chimeric protein comprising a uPA fragment of at least 6 amino acids. Peptides, derivatives and analogs thereof, and peptide mimetics that bind especially with uPAR can be produced by various methods known in the art, including, but not limited to, solid phase synthesis or solution (Nakanishi et al. , 1993, Gene 137: 51-56; Merrifield, 1963, J. Am. Chem. Soc. 15: 2149-2154; Neurath, H. et al., Eds., The Proteins, Vol II, 3d Ed., P. 105-237, Academic Press, New York, NY (1976)). For example, a peptide corresponding to a portion of a uPA protein comprising the desired domain that binds to a receptor, can be synthesized by the use of a synthesizer. • peptides. In addition, if desired, non-classical amino acids or chemical amino acid analogues can be introduced as a substitution or addition in a uPA sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, acid cysteic, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, β-methyl amino acids, Ca-methylamino acids, and Na-methyl amino acids. The uPA peptides can be isolated and purified by standard methods including chromatography (e.g. ion exchange, affinity and column chromatography according to size), centrifugation, differential solubility or through any other standard technique for the purification of peptides. The functional properties can be evaluated using Any suitable assay, including, but not limited to, competitive and non-competitive assay systems employing techniques such as radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, immunoassays in situ (using colloidal gold markers, enzyme or radioisotopes, for example), Western Blot, immunofluorescence, as well as immunoelectrophoresis assays, etc. For example, to select antibodies that recognize a specific domain of a uPAR, hybridomas generated for a product that binds to a uPAR fragment containing said domain can be tested. In one embodiment, antibody binding is detected by detecting a marker in the antibody. Numerous techniques are known to detect binding in an immunoassay and are within the scope of the present invention. The production and use of derivatives and analogs related to a uPA are within the scope of the present invention. In a specific embodiment, the analogous derivative is functionally active, that is, capable of exhibiting one or more functional activities associated with a full-length wild-type uPA prctein. As an example, derivatives or analogs having the desired antigenicity can be employed, for example, in diagnostic immunoassays in accordance with the writing in Section 5.2. UPA derivatives or analogues can be tested for the desired activity by methods known in the art, including essays below, without being limited to them. In a specific embodiment, peptide libraries can be screened to select a peptide with the desired activity; such screening can be carried out by means of the uPAR binding assay, for example. Particularly, uPA derivatives can be made by altering uPA sequences by substitutions, additions, or deletions that provide functionally equivalent molecules. The uPA derivatives of the present invention include, but are not limited to, the derivatives that contain, as the primary amino acid sequence, all or a portion of the amino acid sequence of a uPA peptide that includes altered sequences wherein amino acid residues functionally equivalent are replaced by residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence may be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence can be selected from among other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid. UPA derivatives or analogs include, but are not limited to, peptides substantially homologous with uPA or fragments thereof. Within the scope of the invention are fragments of uPA protein or other differentially modified analog derivatives during or after the translation such as, for example, by glycosylation, acetylation, phosphorylation, amidation, derivatization by known blocking / blocking groups, proteolytic dissociation, binding with an antibody molecule or another cellular ligand, etc. Any of several chemical modifications can be carried out by known techniques, including, but not limited to, the specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tumicamycin; etc. 5.2. MARKING Methods are described for detectably labeling molecules capable of specifically recognizing one or more uPAR epitopes. The marking and detection methods employees here may be, for example, the methods described Bi - d¡ -? I _ ^ _ ^ _-_ ^ _-_ ^^^^^^^.-_-_ ^^^^^ »- ^» - ^ »- ^» «^ - ^ - ^ _ ^ _- ^ - ^ - ^ - ^ - ^ - ^ _ ^ _ ^ _ ^ _ ^ _ ^ _ ^ _ ^ _ ^ _ ^ _ ^ _ ^^^^^^^^^^^^ ^^ ¿¿? ^^^^ ^^ jgg ^ in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual" (Antibodies: A Laboratory Manual), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference in its entirety. One of the ways in which a specific antibody for uPAR or peptide mimetic can be detectably labeled is by linking it with an enzyme, such labeled molecules can be used in an enzyme immunoassay such as for example ELISA (test in an enzyme-linked absorber). The bound enzyme on the antibody reacts with an appropriate substrate, preferably a chromogenic substrate in such a way that a chemical portion is produced which can be detected as, for example, by spectrophotometric, fluorimetric, or visual means. Enzymes that can be used to detectably label the antibodies, derivatives and analogs thereof, and peptides include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate, dehydrogenase, triosophosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection can be achieved by calorimeter methods that employ a chromogenic substrate for the enzyme. Detection can also be achieved by visual comparison of the magnitude of the enzymatic reactions of a substrate compared to standards prepared in a similar manner. For use in the detection methods of the invention, the molecules are preferably labeled with an isotope radio, including, but not limited to: 125I, 131I or 99mTc. Such peptides and antibodies can be detected in in vitro assays using radioimmunoassay (RIA) or radiosonde. The radioactive isotope can be detected by means such as the use of a gamma counter or a scintillation counter or by autoradiography. It is also possible to label antibodies, derivatives and analogs thereof, and peptides with a fluorescent compound. When the fluorescently labeled peptide is exposed to a light having an appropriate wavelength, its presence can be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Derivative antibodies and analogs thereof, and peptides can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals ^^ g ^^ - can be affixed on the antibodies derived and analogues thereof, and peptides using metal chelation groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). 5 Antibodies, derivatives and analogs thereof, and peptides can also be detectably labeled by coupling with a chemiluminescent compound. The presence of the chemiluminescent-labeled peptides is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of especially useful chemiluminescent labeling compounds are luminol, isoluminol, acrominic ester, imidazole, acridinium salt and oxalate ester. In the same way, a luminescent compound can be used to label antibodies, derivatives and analogs thereof, and peptides of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a protein catalytic increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined through the detection of the presence of luminescence. Luminescent compounds important for marking purposes are luciferin, luciferase and aequorin. . 3. METHODS OF ADMINISTRATION Molecules determined by specifically binding with uPAR can be administered to a patient in diagnostically effective doses to detect metastases. A diagnostically effective dose refers to the amount of the molecule sufficient to target a diagnosis to a cell containing uPAR on its surface such that the cell can be detected using methods commonly available in the art such as, for example, with what is described in section 5.4.1 above. 5.3.1. EFFECTIVE DOSE The toxicity and diagnostic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD = (the lethal dose for 50% of the population). The data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. For example, the animal model systems described in Examples 7 and 8 can be used to test effective doses to visualize metastatic lesions using the labeled molecules. The dosage of such compounds is preferably within the range of circulating concentrations with little or no toxicity. The precise dose to be used in the formulation will depend on the route of administration and the severity of the disease, and will be decided according to the discretion of the doctor and the circumstances of the individual patient. However, suitable dosage ranges for intravenous administration are generally between 1.0 and 20 micrograms of compound per kilogram of body weight. Plasma levels can be measured, for example, by high performance liquid chromatography. 5.3.2. FORMULATIONS AND USE Pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner employing one or more physiologically accepted carriers or excipients. Methods of administration include, but are not limited to, intravenous, subcutaneous, intraperitoneal, and intradermal routes. The administration can be systemic or local. In the specific embodiment, it is desirable to administer the pharmaceutical compositions of the invention locally with direct injection at the site (or pre-site) of a malignant tumor or metastatic tissue. In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, the compositions for administration n-ifet »intravenous are solutions in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and an anesthetic • local, such as, for example, lignocaine, in order to mitigate pain at the site of the injection. In general, the ingredients are supplied either separately or as a mixture. When the composition is to be administered by infusion, it can be supplied with an infusion bottle containing sterile pharmaceutical grade water or a saline solution. When the composition is administered by injection, a sterile water vial or saline for injection may be provided in such a manner that the ingredients can be mixed prior to administration. The invention also offers a pharmaceutical package or kit comprising one or more containers filled with one or several of the ingredients of the pharmaceutical compositions of the invention. 5.4. DIAGNOSIS AND IMAGING OF METASTASIS 20 The labeled antibodies, derivatives and analogs thereof, and peptides and mimetics of peptides that specifically bind with uPAR can be used for diagnostic purposes for the purpose of detecting, diagnosing or monitoring metastases. In the preferred modality, the molecules of the invention can be used for the purposes of diagnosis in order to detect, diagnose or monitor micrometastasis. In one embodiment, metastases are detected in patient samples. In a preferred embodiment, metastases 5 are detected in the patient. The patient is an animal and is preferably a human being. In one embodiment, the diagnosis is carried out in the following manner: a) administration to a patient of an effective amount of a labeled molecule that binds < ^ 10 specifically as a urokinase receptor; b) detection delay during a time interval after administration to allow the labeled molecule to be concentrated preferably in metastatic lesions in the patient and to allow labeled unbound molecules be purified to the background level; c) determination of background level; d) detection of the marked molecule in the patient, in such a way that the detection of the marked molecule above the background level indicates the presence of a metastatic lesion. The background level can be determined by various methods, including: the measurement of labeled molecule in tissue that does not normally express uPAR, eg, muscle, either in the patient under diagnostic or in a second subject of which no metastatic tissue is suspected; or comparing the amount of labeled molecule detected with a standard value previously determined for a particular system. According to different variables, including the type of marker used and the mode of administration, the time interval after administration to allow the labeled molecule to be concentrated preferably in metastatic lesions in the subject and for the labeled non-bound molecule to be depure until the background level is from 6 to 48 hours or bine from 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval after administration is 5 to 20 fl 10 days or 5 to 10 days. In one modality, metastasis monitoring is carried out by repeating the metastasis diagnosis method, for example, one month after the initial diagnosis, six months after the initial diagnosis, one year after the initial diagnosis, etc. 5.4.1. METHODS OF DETECTION AND FORMATION OF IMAGE The presence of the labeled molecule can be detected in the patient using known methods for in vivo exploration. These methods depend on the type of marker used. The experts in the field will be able to determine the appropriate method for detecting a particular marker. Methods and devices that can be employed in the diagnostic methods of the invention include, but are not limited to: computed tomography (CT), whole body scan, as per example positron emission tomography (PET), magnetic resonance imaging (MRI), and sonography. In a specific embodiment, the molecule is labeled with an isotope radio and detected in the patient using a surgical instrument that responds to radiation 5 (Thurston et al., US Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a scanning instrument that responds to fluorescence. 5.5 THERAPEUTIC USES A The invention provides for the treatment of various cancers by the administration of a therapeutic compound (known here as "Therapeutic Agent"). Such therapeutic agents include, but are not limited to: antibodies, derivatives and analogs thereof, and peptides and mimetics of peptides that bind specifically with uPAR (as described above). As an illustrative example see Section 9 and Table 1. In a preferred embodiment, a cytotoxic or cytostatic compound, including, but not limited to: saporin, chain A ricin, cholera A chain toxin, antibiotic, antimetabolite, is coupled to the therapeutic agent. 6. EXAMPLE: ANTIBODIES FOR UPLOAD BLOCKING THE INVASION OF TUMOR CELLS THROUGH THE BASE MEMBRANE The experiments described below demonstrate the ruPAR antibody capacity to specifically bind uPAR, and block the invasion of cancer cells through base membrane. 6.1. MATERIALS AND METHODS Cell and Cell Culture The Rat Lymphoma Dunning R3227 rat prostate cancer cell line was obtained from Dr. T. J. Isaacs (Johns Hopkins School of Medicine, Baltimore, MD). The rat cancer cell line Mat B-III was obtained from the American Type Culture Collection (Rockville, MD). MAT B-III cells that overexpress uPAR (Mat B-III uPAR) were developed in accordance with the writing in Xing and Rabbani, 1996, Inc. J. Cancer 67: 423-429, which is incorporated herein by reference in its whole. Cells were maintained in RPMI 1640 or in 5A McCoy medium supplemented with 10% fetal bovine serum (FDS), 2mM glutamine, 100 units / ml penicillin and 100 ng / ml streptomycin (Gibco, Grand Island, NY) : Cells were cultured under standard tissue culture conditions at a temperature of 37 ° C in a humidified atmosphere containing 5% C02 in 75 cm2 flasks or six-well tissue culture plates (Archbarou et al., 1994, Cancer Res. 54: 2372-2377; Xing and Rabbani, 1996, Int. J. Cancer 67: 423-429). Rat Anti-uPAR Antibody Incomplete cDNA encoding rat uPAR (r) was isolated from a rat osteoblast cDNA library (Rabbani et al., * - * "-" - '• »1994, FEBS Letters 338: 69-74). A Pst I restriction digest of ruPAR resulted in the release of a 271 base pair cDNA encoding amino acids 25-114 of rat uPAR that was subcloned in sense orientation into the expression vector pTrcHis A ( Invitrogen, San Diego, CA). The orientation and insertion in ruPAR cDNA box was further confirmed with nucleotide sequence analysis. The recombinant ruPAR protein was expressed and then purified on the Protein G column, commercially available as per the manufacturer's instructions. The amino acid sequence of recombinant ruPAR was confirmed using the integrated microsequencing system Pl 2090E (Backman Instruments, Mississauga, ONT.) At Sheldon Biotechnology Center, McGill University. Rabbits were immunized with ruPAR at several sites (d-10) subcutaneously using incomplete Freund's adjuvant (Sigma, St. Louis, MO) at 4 week intervals and bleeding 10 days after each immunization. The antiserum used in this study was obtained after the third boost. An immunoglobulin fraction (IgG) was purified from antiserum against ruPAR using Protein A Sepharose CL-4B (Pharmacia, LKB Bale D'Urfe, Quebec) in accordance with the manufacturer's instructions. Indirect immunofluorescence 25 The capacity of this specific species of ruPAR IgG in the • * ~ - • "•" "- • * - sense of recognizing the endogenous ruPAR protein was examined in Mat LY Lu cancer cells and in Mat B-III cells 5xl04 cells were plated in tissue culture chambers Lab Tek (Nunc, Naperville, IL) and allowed to grow to confluence of 70-80% The cells were then incubated with 30% goat serum (Sigma, St. Louis MO) for one hour at room temperature and washed with PBS containing 1% BSA Sequentially, the cells were incubated with ruPAR primary rabbit IgG and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (FITC). Images were taken at 25-fold magnification using a Zeiss microscope MC-63. Matrigel invasion and receptor binding assay The ruPAR IgG capacity to block the invasion capacity of Mat Ly Lu and Mat B-III-uPAR cells was tested through two-compartment Boyden chambers (Transwell, Costar, United States of America) and Matrigel base membrane (Becton Dikinson Labware) (Xing and Rabbani, 1996, Int. J. Cancer 67: 423-429). Polycarbonate filters with a size of 8 μm were coated with a Matrigel base membrane (45 μg / filter) and dried under a tissue culture hood. Matrigel was rebuilt after the addition of 0.1 ml of serum-free culture medium in the upper chamber and incubated for 90 minutes. After the removal of the medium, cells (5xl04) in 0.1 ml of culture medium supplemented with 10 μg / ml of anti ruPAR IgG or 10 μg / ml of pre-immune IgG were added to the upper chamber and placed in a pre-filled chamber with 0.1 ml of serum-free medium supplemented with 5 μg / ml fibronectin (Sigma, St Louis, MO), and incubated at a temperature of 37 ° C for 24 hours. At the end of the incubation, the medium was removed and the cells were fixed in 2% paraformaldehyde, 0.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.4 at room temperature for 30 minutes. After washing with PBS, all the filters were stained with 0.05% toluidine blue. The filters were mounted on glass plates and the cells were examined under a light microscope. Ten fields with a 100-fold amplification were randomly selected and the average number of cells was calculated. One hundred micrograms of ruPAR IgG were labeled with 1 MCi or 125 I using the chloramine T method, which provides a specific activity of 0.8-1.0 μCi / μg of protein (Rabbani et al., 1992, J. Biol. Chem. 267: 14151-14156). Free 125 I was separated from labeled IgGs on a Sephadex G-25 gel filtration column (Pharmacia, Uppsala, Sweden) ie it was equilibrated and eluted with a phosphate buffered saline solution containing 0.1% bovine serum albumin ( BSA). The Mat Ly Lu and Mat B-III-uPAR cells were placed in 24-well plates (30,000 and 80,000 cells, respectively) and allowed to grow at a confluence of 70%. After serum deprivation for one hour, the cells • were treated with 50 mM glycine and 10 mM NaCl, pH 0 5 for 3 minutes. The cells were incubated for one hour at a temperature of 37 ° C in a final volume of 300 ml containing serum-free medium, 1 mg / ml BSA, 20 mM Hepes pH 7.4, ruPar IgG labeled with 125 I ( 100,000 cpm), with or without increasing the concentrations of competitor 10 (ruPAR protein). The binding reaction was suspended by washing four times with a balanced salt solution of Hanks at ice temperature, and the cells were removed with 1 ml of 0.6 N NaOH for subsequent determination of radioactivity (Rabbani et al., 1992, J. 15 Biol. Chem. 267: 14151-14156; Xing and Rabbani, 1996, Int. J. Cancer 67: 423-429). Statistical analysis The statistical analysis was carried out by means of an analysis of a sense of variance or through the Student t test. 6.2. RESULTS Characterization of rat anti uPAR IgG The ruPAR IgG ability to recognize the cell surface receptor for uPA, abundantly expressed by 25 MatB-III cells, was examined by immunofluorescence. - i. '.t. nMü? frtir? - ?, r fu • .- > -..-,. . -. - a - ^^ ..., -, Control cells incubated with 10 μg / ml of pre-immune rabbit IgG did not show any linkage with ruPAR according to what was evaluated by immunofluorescence (figure • 3A). In contrast, 10 μg / ml of ruPAR IgG showed fluorescence reaction 5 in MatB-III-uPAR cells (Figure 3B). This antibody-receptor complex was observed on the cell surface where the expression of uPAR is reported. Similar results were obtained with ruPAR IgG in Mat Ly Lu cells. The effects of anti-ruPAR IgG on invasion of tumor cells in vitro The function of the NH2-terminal region of uPAR in ligand binding in the invasion capacity of cells was further examined in Mat Ly Lu and MatB-III-uPAR cells using 15 Matrigel invasion assay. After 24 hours of incubation, both the Mat Ly Lu cells and the MatB-III cells were able to penetrate the base membrane. Incubation of these cells in the presence of 50-100] iq / L of preimmune rabbit IgG did not cause any significant inhibition of the invasiveness of these rat prostate and breast cancer cells (Figure 4). In contrast, the number of cells that invade through the base membrane was significantly reduced compared to the control cells treated with preimmune rabbit IgG (FIG. 4). It was found that these effects were dependent on the dose in J ^ ^^^^ w ^^^^^^^^^ jj * ^ * ^^^ where 50 μg / ml or 100 μg / ml of IgG inhibited cell invasion in 40% and 80%, respectively. 7. EXAMPLE: LINK WITH RECEIVER AND BIODISTRIBUTION OF RAT UPARATED IgG WITH 125I The binding specificity and biodistribution of ruPAR antibody labeled in vitro are described in the following subsections. These results showed that the labeled anti-uPAR antibody accumulated in tissues that are common sites of tumor metastasis before metastases could be detected through macroscopic investigation. These experiments also demonstrated that unlabeled ruPAR inhibits the binding of ruPAR IgG labeled with 125 I in a dose-dependent manner. The labeled ruPAR antibody is concentrated in primary tumors in a time-dependent manner. In addition, the ability of raPAR antibody to bind preferentially in the primary tumor and, surprisingly, in metastatic lesions was demonstrated. 7.1. MATERIALS AND METHODS Protocols for animals Inbred female Fischer rats weighing 200-250 g were obtained from Charles River, Inc. (St. Constant, Canada). Before inoculation, Mat B-III-uPAR tumor cells cultured in medium containing serum were washed with Hank's buffer and trypsinized for 5 minutes. The cells were then harvested in Hank's buffer and centrifuged at 1500 revolutions per minute for 5 minutes. Cell pellets (1 x 106 cells) were resuspended in 200 μl of saline and injected • using syringes of 1 ml of insulin in the mammary fat of 5 rats anesthetized with ethanol / Somnotal (MTC Pharmaceuticals, Cambridge, Ontario). For biodistribution studies, on day 10 after inoculation of tumor cells, the animals received injections of pre-immune IgG labeled with 125I or ruPAR (25 ^ fc 10 μg, 25 μCi) via tail vein injection. In a separate experiment, on day 15 after the inoculation of tumor cells, the animals received injections of labeled material through the tail vein. The animals were sacrificed 0.5-96 hours after the injection. HE removed the primary tumors from the site of inoculation of tumor cells (mammary fat) and the radioactivity of said tumors was determined. Alternatively, 12 hours after the injection of radiolabeled pre-immune IgG or ruPAR, the primary tumors and various organs were removed. (heart, liver, spleen, lungs, kidneys and lymph nodes), and the absorption of total radioactivity in these organs was monitored using a gamma counter. The biodistribution of ruPAR IgG labeled with 125I was calculated and expressed as percentage of injected dose / gram of tissue (% ID / g) of ruPAR IgG labeled with 125I-IgG • - * * - > preimmune labeled with 125I (Folli et al., 19984, Cancer Res. 54: 2643-2649). 7.2. RESULTS Linkage with receptor and biodistribution of ruPAR IgG labeled with 125I The ability of ruPAR IgG to interfere with the functional ability of uPAR was evaluated in a receptor binding assay. The total binding of ruPAR IgG labeled with 125 I was determined on Mat Ly Lu and Mat B-III-uPAR cells. The addition of different concentrations (0.1-2.0 mg) of unlabeled recombinant rat uPAR inhibited the binding of 125 I ruPAR IgG in a dose-dependent manner in both Mat Ly Lu cells (Figure 5A) and Mat B-III cells (FIG. 5B). A dose-dependent decrease in the binding of 125 I ruPAR IgG was observed after the addition of different concentrations of unlabeled ruPAR IgG compared to preimmune rabbit IgG to their tumor cells. To examine the specificity and time course of ruPAR IgG labeled with 125 I, both preimmune IgG and ruPAR IgG were labeled with 125 I and injected via the tail vein in female Fischer rats bearing Mat B-III tumors. uPAR (at 15 days after the inoculation of tumor cells). The animals were sacrificed at intervals (0.5-96 hours). The primary tumors were removed and the absorption of 125I was counted. % ID / g of ruPAR IgG was higher in animals bearing tumors 12 hours after the injection of 125I-labeled IgG after • from this time the percentage of% ID / g decreased until 96 5 hours (figure 6). Animals with tumors (10 days after the inoculation of tumor cells) received injections through the tail vein of uPAR IgG labeled with 125 I and said animals were sacrificed 12 hours after injection with the • 10 marked uPAR material. Marker accumulation was examined in: (1) normal tissues (muscle, heart); (2) tissues that are common sites of tumor metastasis (liver, spleen, kidney, lungs, lymph nodes); and (3) primary tumors. Minimal amounts of radioactivity were observed in the muscles, while levels of 125I were slightly higher in the heart due to the presence of blood that showed a high absorption of radioactivity. In contrast to this, significantly higher levels of ruPAR IgG / ID percentage were observed in liver, spleen, kidney, lungs, lymph nodes and in primary tumor (FIG. 7). Even when marked material was detected in the primary tumor and in metastatic lesions of the sacrificed animals on the 10th day after the inoculation of tumor cells (Figure 7), 25 no macroscopic metastases were observed. However, it • "- * - - *» "^? NJtirT-observed macroscopic tumor metastasis in animals sacrificed on day 15 after inoculation of tumor cells. These results suggest that the specific IgG uptake of ruPAR labeled with 125 I by Mat B-III-uPAR tumor cells, already present at these sites on day 10, subsequently developed into macroscopic metastases by day 15. 8. EXAMPLE: BIQDISTRIBUCION DE HUMAN UPGRADED IgG WITH 125I The following example demonstrates the ability of human uPAR IgG (h) labeled with 125I to recognize uPAR from cell surface and preferably concentrate on primary tumor and metastatic lesions in vivo. 8.1 MATERIALS AND METHODS Radiolabelling of human uPAR IgG: The monoclonal human uPAR IgG (# 3936, American Diagnostica Inc., Greenwich, CT) or non-specific mouse IgG were labeled using the Iodogen method providing a specific activity of 0.6 -0.9 mCi / mg. Briefly, 100 μg of IgG was added to a container precoated with 10 μg of Iodogen (Pierce Chemical Co., Rockford, II.) In accordance with the manufacturer's instructions. The reaction proceeded for 15 minutes at room temperature. Free 125 I was separated from labeled IgG using a Sephadex G25 gel filtration column (Pharmacia, Uppsula, Sweden) -L pre-equilibrated with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). In vivo biodistribution studies: Tumor xenografts were established in nude mice (Balb / c nu / nu) from 4 to 6 weeks of age by subcutaneous injection of 2 x 10 6 cells of human prostate cancer (PC-3) per mouse. Prior to injection, cells cultured in medium containing serum were washed with HBSS and trypsinized for 5 minutes. The cells were then collected in medium and centrifuged at 1500 revolutions per minute for 5 minutes and resuspended in 200 μl of saline. Five weeks after the inoculation of tumor cells, 125I-labeled IgGs were injected intravenously through the lateral tail vein in mice with tumors. The animals were sacrificed after 12 to 24 hours, and the primary tumor and several organs (heart, liver, spleen, lungs, kidney, lymph nodes, and blood) were removed. The total bioactivity was determined using a gamma counter. The biodistribution was calculated and expressed as percentage of injected dose / gram of tissue (% ID / g). 8.2.RESULTS To examine the specificity of huPAR IgG, preimmune IgG and the monoclonal human uPAR antibody, 3936, were labeled with 125 I and injected into the tail vein of normal Balb / c nu / nu mice and Balb / mice. c nu / nu carrying xenografts of human prostate cell tumors (PC-3). 5 The percentage ID / g of huPAR IgG labeled with L25I (1) normal tissue (heart) was examined; (2) tissues that are common sites of metastasis (liver, spleen, kidney, lungs, lymph nodes); and (3) primary tumors. The levels of radioactivity were slightly high in the heart due to the presence of blood that showed a high absorption of the radioactive molecule (Figure 8A). In contrast, significantly higher levels of IgG percentage were observed in the primary tumor and in the metastatic tissues, especially in the lung. 15 9. EXAMPLE: THERAPEUTIC EFFECTS OF uPAR ON ANIMALS THAT PRESENTED TUMOR The following example demonstrates the ability of anti-uPAR IgG to inhibit the growth rate of primary tumors, as well as the ability to inhibit the formation and growth of metastatic lesions. 9.1 MATERIALS AND METHODS Protocols for animals Inbred female Fischer rats weighing 200-250 g were obtained from Charles River, Inc. (St. Constant, Canada). Before inoculation, tumor cells Mat B- . ^ .i, ¿._ .. i. iEÉ III-uPAR cultured in medium containing serum were washed with Hank's buffer and trypsinized for 5 minutes. The cells were then harvested in Henk's buffer and centrifuged at 1500 revolutions per minute for 5 minutes. The cell pellets (1 x 106 cells) were resuspended in 200 μl of saline and injected into 1 ml syringes of insulin in the mammary fat of rats anesthetized with ethanol / Somnotal (MTC Pharmaceuticals, Cambridge, Ontario). Animals with tumors received injections of 50-100 μg / day of ruPAR IgG subcutaneously in mammary fat from day 1 to day 7 after inoculation of tumor cells. Control groups of animals carrying tumors received only normal saline or 50-100 μg / day of pre-immune rabbit IgG as control. All animals were monitored to determine tumor development for 2-3 weeks after inoculation of tumor cells. The size of the tumor in control and experimental animals was measured in two dimensions by a calibrator and the volume of the tumor was calculated (Haq et al., 1993, J. Clin Invest. 91: 2416-2422). The control animals receiving preimmune IgG and the experimental animals, which received ruPAR IgG were sacrificed on day 10 and day 15 after the inoculation of tumor cells and were evaluated for the presence of macroscopic metastasis in various tissues. 9.2. RESULTS Effect of anti-ruPAR IgG on tumor volume The ability of anti-ruPAR IgG to inhibit the growth rate of primary tumors was evaluated. Injection of preimmune rabbit IgG in animals with tumors did not result in any significant difference in tumor growth. In contrast, anti-ruPAR IgG injection from day 1 to day 7 after inoculation of tumor cells resulted in a significant decrease in tumor volume in these experimental animals (FIG. 9). This decrease in tumor volume was more noticeable in the later stages (day 15-day 21), when the control animals continued to show a linear increase in tumor growth while the experimental animals receiving ruPAR IgG not only showed a decrease of the tumor volume but also showed a regression of the tumor growth in comparison with the previous stages (day 9-day 14) of the tumor development (figure 9). Effect of ruPAR IgG on tumor metastasis To determine the effects of ruPAR IgG on tumor metastasis, control animals with tumor received injections of preimmune rabbit IgG and experimental animals received ruPAR IgG and these animals were sacrificed 15 days after the inoculation of tumor cells. Control animals reproducibly developed large macroscopic tumor metastases in lymph nodes, axillary, retroperitoneal, and mesenteric nodes. Evidence of occasional tumor metastasis was also observed in the liver and spleen (Table 1). In contrast, animals with tumors that received ruPAR IgG had significantly lower metastatic foci in retroperitoneal and mesenteric lymph nodes, without any evidence of tumor metastases in the liver or spleen (Table 1). TABLE 1 EFFECT OF ANTI-RAT ON TUMOR METASTASES Preimmune IgG ruPAR IgG Axillary lymphatic lymph nodes 2 ± 1 lrl Retroperitoneal lymph nodes 3 ± 2 l_tl Mesenteric lymph nodes 3 ± 1 l ± l Lungs l 0 l Liver l ± l 0 Spleen l ± l 0 The present invention is not limited to as to its scope to the specific embodiments described herein, which are intended merely to illustrate individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. In fact, various modifications to the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications fall within the scope of the appended claims. Several publications are mentioned, their disclosures are incorporated here by reference in their totalities.

"- '•« - »'» - * LIST OF SEQUENCES <110> Rabbani and Hart <120> RECEIVER OF PLASMINOGENIC ACTIVATOR OF UROCHINASE AS A WHITE FOR THE DIAGNOSIS OF MET STASIS < 130 > 9471-003-228 < 140 > PCT / US99 / 06588 < 141 > 1999-03-23 < 150 > 09 / 046,106 < 151 > 1998-03-23 < 160 > 2 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 27 < 212 > PRT < 213 > Homo sapiens < 400 > 1 Val Pro Ser Asn Cys Asp Cys Leu Asn Gly Gly Thr Cys Val Ser 1 5 10 15 Asn Lys Tyr Phe Ser Asn lie His Trp Cys Asn Cys 20 25 < 210 > 2 < 211 > 21 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Asp Cys Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser 1 5 10 15 Asn lie His Trp Cys Asn 20 fifteen twenty

Claims (17)

1. CLAIMS A method for detecting one or more metastatic lesions, comprising: a) administering to a subject an effective amount of a labeled molecule that specifically binds with a urokinase plasminogen activator receptor; b) delayed detection during a time interval after administration to allow the labeled molecule to be concentrated preferably in a possible metastatic lesion in the subject and to allow the unbound labeled molecule to be removed to the background level; c) the determination of the background level; and d) the detection of the labeled molecule in the subject, where the detection of the labeled molecule above the background level indicates the presence of a metastatic lesion.
2. The method according to claim 1 wherein the subject is a human being.
3. The method according to claim 1 wherein the molecule is an antibody to a urokinase plasminogen activator receptor or a portion of said antibody containing the binding domain thereof.
4. The method according to claim 1 wherein the molecule is a humanized antibody.
5. The method according to claim 1 wherein the molecule comprises the amino acid sequence illustrated in Figure 1 (SEQ ID N0: 1) or Figure 2 (SEQ ID NO: 2).
6. The method according to claim 1 wherein the labeled molecule is labeled with a radioisotope.
7. The method according to claim 1 wherein the labeled molecule is detected in vivo.
8. The method according to claim 1 wherein the time interval is from 6 hours to 48 hours.
9. The method according to claim 1 wherein the labeled molecule is administered intravenously.
10. The method according to claim 1 further comprising repeating steps (a) to (d) at monthly intervals.
11. A method for detecting one or more metastatic lesions in a subject, comprising the image formation of said subject in a time interval after the administration to said subject of an effective amount of a labeled molecule that specifically binds to a urokinase plasminogen activator receptor, said time interval it is sufficient to allow the labeled molecule to be concentrated preferably in any eventual metastatic lesion in said subject and to allow the unbound labeled molecule to be purified to the background level 5, where the detection of the labeled molecule above the background level indicates the presence of a metastatic lesion.
12. 12. The method according to claim 11 wherein the subject is a human being.
13. 13. The method according to claim 11 wherein the molecule is an antibody to a urokinase plasminogen activator receptor or a portion of said antibody containing the binding domain thereof.
14. 14. The method according to claim 11 wherein the molecule is a humanized antibody.
15. 15. The method according to claim 11 in • where the molecule comprises the amino acid sequence presented in Figure 1 (SEQ ID NO: 1) or 20 in Figure 2 (SEQ ID NO: 2).
16. 16. The method according to claim 11 wherein the labeled molecule is labeled with a radioisotope.
17. 17. The method according to claim 11 wherein the time interval is from 6 hours to 48 hours. * - «-? Itt-¡ate? Ai (á-Wií-. *? Í-U '- • * - *