PROGNOSIS AND THERAPY OF MALIGNANT DISEASES BY ANTIBODIES WHICH BIND PHOSPHORYLATED MET PROTEIN

Abstract: Antibodies which bind phosphorylated Met protein and do not bind non-phosphorylated Met protein are provided as well as hybridomas secreting such antibodies. The antibodies are useful as prognostic agents for malignant diseases involving the expression of pMet and are used in a method for determining in an individual having such a disease the probability of having or developing a metastatic disease and for determining the individual's survival expectancy. In addition, a novel method which enables to provide an accurate prognosis for an individual having a malignant disease involving the expression of Met is provided which is based on two novel quantitive prognostic indices (De and DI). The antibodies of the invention are also suitable for treatment of various diseases which involve the expression of Met and pharmaceutical compositions comprising these antibodies are provided for the treatment of such malignant diseases.

Description: POGNOSIS AND THERAPY OF MALIGNANT DISEASES BY ANTIBODIES WHICH BIND PHOSPHORYLATED MET PROTEIN FIELD OF THE INVENTION The present invention concerns novel monoclonal antibodies (mAbs), hybridomas producing the mAbs, peptidic sequences of antigens to which the monoclonal antibodies bind, use of the monoclonal antibodies for prognosis and 5 therapy of malignant diseases and a novel prognostic method for prognosis of malignant diseases. PRIOR ART The following is a list of references which are intended for better l o understanding of the background of the present invention: Beviglia, L., Matsumoto, K., Lin, C. S., Ziober, B. L., and Kramer, R. H. Expression of the c-Met/HGF receptor in human breast carcinoma: correlation with tumor progression. Int J Cancer, 74: 301-9, 1997. 15 Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A. M., Kmiecik, T. E., Vande, W.G., and Aaronson, S.A. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science, 251:802-4. 1991. 0 Ghoussoub, R. A. D., Dillon, D. A., DAquila, T., Rimm, E. B., Fearon, E. R., and Rimm, D. L. Expression of c-met is a strong independent prognostic factor in breast carcinoma. Cancer, 82: 1513-1520, 1998. 5 Jin, L., Pang, Y. Y., Joseph, A., Li, Y., Rosen, E. M., Bhargava, M. M., and Goldberg, I. D. Rat placental hepatocyte growth factor/scatter factor: purification, characterization, and developmental regulation. Proc. Soc. Exp. Biol. Med., 204: 75-80, 1993. Nagy, J., Clark, J. S., Cooke, A., Campbell, A. M., Connor, J. M., Purushotham, A. D., and George, W. D. Expression and loss of heterozygosity of c-met proto-oncogene in primary breast cancer. J. Surg. Oncol., 60: 95-9, 1995. Ponzetto, C, Bardelli, A., Zhen, Z., Maina, F., Dalla, Z.P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P.M., A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell, 77:261-71, 1994. Soriano, J. V., Pepper, M. S., Nakamura, T., Orci, L., and Montesano, R. Hepatocyte growth factor stimulates extensive development of branching duct-like structures by cloned mammary gland epithelial cells. J. Cell Sci., 108: 413-30, 1995. Tsarfaty, I., Resau, J. H., Rulong, S., Keydar, I., Faletto, D. L., and Vande Woude, G. F. The met proto-oncogene receptor and lumen formation. Science, 257: 1258-1261, 1992. Tsarfaty, I., Alvord, W.G., Resau, J., Altstock, R.T., Lidereau, R, Bieche, I, Bertrand, F., Horev, J., Klabansky, R.L., Keydar, I., and Vande Woude, G.F. Alteration of Met Expression and Prognosis in Breast Carcinomas. Analytical and Quantitative Cytology and Histology, Anal Quant Cytol. 21: 397-408, (1999). Vande Woude, G.F., Jeffers, M., Cortner, J., Alvord, G., Tsarfaty, I., and Resau, J., Met-HGF/SF: tumorigenesis, invasion and metastasis. Proceedings of the Ciba Symposium, 1997. Yamashita, J., Ogawa, M., and Beppu, T. Immunoreactive hepatocyte growth factor is present in tissue extracts from human breast cancer but not in conditioned medium of human breast cancer cell lines. Res. Commun. Chem. Pathol. Pharmacol, 82:249-52, 1993. Yang, Y., Spitzer, E., Meyer, D., Sachs, M., Niemann, C, Hartmann, G., Weidner, K. M., Birchmeier, C, and Birchmeier, W. Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland. J. Cell Biol., 131: 215-26, 1995. BACKGROUND OF THE INVENTION The Met proto-oncogene encodes the Met protein which is a member of the tyrosine kinase growth factor receptor family and a receptor for hepatocyte growth factor/scatter factor (HGF/SF) (Bottaro, D.P., et al, 1991). Met and HGF/SF have been shown to be involved in breast cancer, although there are contradicting publications regarding the expression of Met in breast tumors as compared to benign tissues. In some cases, it has been shown that Met is expressed and activated in normal breast ducts, but reduced in certain breast tumors (Tsarfaty, I.,). Others have reported differences in Met expression between cancer and benign diseased breast tissues, which could, however, not be correlated with prognosis (Nagy, J., 1995). Overexpression of Met in breast tumors and co-expression of Met and HGF/SF in breast tumors was also reported (Jin, L., et al, 1993). The Met protein has been proposed as a prognostic factor in breast cancer (Ghoussoub, R.A.D., et al, 1998 and Beviglia, L., 1997) and elevated levels of HGF/SF were shown to be a predictor of recurrence and reduced survival in human breast cancer (Yamashita, J., 1993). Upon binding of its HGF/SF ligand, the Met receptor is phosphorylated. Phosphorylated Met (pMet) elicits specific signal transduction cascades that induce cell proliferation, movement and was shown to be involved in metastasis (Vande Woude, 1997). The biological effect of Met signal transduction was shown to be dependent on phosphorylation of specific tyrosines in the human Met docking site (Ponzetto, 1994). GENERAL DESCRIPTION OF THE INVENTION In the context of the present invention, two explicitly described, calculable or measurable variations are considered equivalent to each other when the two variables are proportional to each other. In accordance with the invention, it was realized that the phosphorylated Met protein (pMet) may be useful as a prognostic factor in various metastatic cancers and preferably in the prognosis of breast cancer. Thus, monoclonal antibodies which bind the phosphorylated Met receptor (hereinafter "pMet") and do not bind the non-phosphorylated Met receptor (hereinafter "Met") were developed. In accordance with the invention, it has been found that by comparing the level of binding of anti-pMet mAbs to the malignant regions of human breast adeno carcinoma biopsy specimens to the level of binding of the same mAbs to the non-malignant regions of the same tested specimens, it is possible to provide an accurate prognosis for the individual from which the biopsy was taken from. The present invention thus provides by a first of its aspects hybridoma cell lines which produce monoclonal antibodies that bind to a phosphorylated Met peptide and do not bind to non-phosphorylated Met peptide. Such mAbs will be referred to hereinafter as "antipMet mAbs ". Furthermore, it was realized that, for prognosis of such diseases, it would be preferable that the anti pMet antibodies are such which bind the phosphorylated form of the Met docking site. Thus, in accordance with a preferred embodiment of this aspect of the invention, there are provided hybridoma cell lines which produce anti-pMet mAbs which bind to one or more phosphorylated tyrosines present in the Met docking site. The major tyrosine phosphorylation site in the human Met docking site is Tyr . Most preferably, there are provided hybridomas which produce mAbs which bind the phosphorylated tyrosine in the Met docking site. An example of a hybridoma cell line in accordance with the invention is the hybrodima cell line, which was deposited on April 12, 2000, in the Centre for Applied Microbiology and Research (CAMR), European Collection of Cell Cultures (ECACC) depository under Accession No. 00041122. In accordance with an additional aspect of the present invention, mAbs produced by the above mentioned hybridoma cell lines are provided. Such mAbs are selected from the group consisting of: (a) mAbs which bind to pMet and do not bind to Met; (b) Fragments of the antibodies of (a) substantially retaining the antigen binding characteristics of the whole antibody; and (c) Antibodies that bind to an antigenic epitope bound by any one of the antibodies of (a) and (b) above. By one embodiment, the mAbs of the invention are such which bind to the pMet docking site, fragments of such antibodies which substantially retain the antigen binding characteristics of the whole antibody and antibodies which bind to the antigenic epitope bound by such antibodies or their fragments. By a preferred embodiment, the mAbs of the invention are such which bind to one or more of the autophosphorylation sites of Met, fragments of these antibodies substantially retaining the antigen binding characteristics of the whole antibody and antibodies capable of binding to the antigenic epitope bound by such antibodies and their fragments. Most preferred are mAbs which bind to the major tyrosine phosphorylation site of Met Tyr 1356 , fragments of these antibodies substantially retaining the antigen binding characteristics of the whole antibody and antibodies which bind to the antigenic epitope bound by such antibodies and their fragments. In accordance with the invention, it has also been found that anti-pMet mAbs may be used for prognosis of various diseases involving the expression of Met and preferably for prognosis of breast cancer. The anti-pMet mAbs may be used for this purpose in vitro, ex vivo or in vivo. For in vivo (in situ) prognosis, the invention therefore provides, by one of its further aspects, a prognostic composition comprising one or more of the above mAbs together with a suitable carrier. By an additional aspect of the invention, it has been found that anti-pMet mAbs may be useful in the treatment of various diseases involving the expression of Met and preferably for the treatment of breast cancer. The mAbs may, for example, be administered to an individual in order to block activation or down-regulate a portion of the Met receptor level of expression or activity in the tumor cells. Compositions comprising the mAbs of the invention may be used for the treatment of various malignant diseases, particularly breast cancer. Thus, in accordance with an additional aspect of the invention, pharmaceutical compositions comprising, as an active ingredient, an effective amount of one or more of the mAbs of the invention together with a pharmaceutically acceptable carrier are 5 provided. The "effective amount" is an amount of the mAbs or mAbs which upon administration to the individual in need results in the reduction of the symptoms related with the disease and/or inhibition or prevention of the development of the disease into its next stages, e.g. inhibition or prevention of metastasis. The effective l o amount of the mAb will depend on a number of parameters such as the type of disease, the stage of the disease, physiological parameters of the individual to be treated, the nature of the mAb, etc. and may be deteπnined by a person skilled in the art. Methods for the treatment of such diseases comprising administering to an 1 5 individual in need a therapeutically effective amount of the mAbs of the invention are also within the scope of the invention. In addition, a kit for use in the prognosis of diseases, involving expression of Met and especially breast cancer is also within the scope of the present invention, the kit comprising anti-pMet mAbs, reagents required for determining 0 the level of binding of said mAbs to said sample, predetermined cut-off values (see below) to which the value calculated for the tested sample may be compared and instructions for use. In addition, in accordance with the invention, the antigen of the mAbs of the invention being a phosphotyrosine peptide corresponding to the Met 5 phosphorylation site Tyr was synthesized. Thus, in accordance with an additional embodiment of the invention, there is provided a peptide selected from the group consisting of: (a) a peptide having the amino acid (a.a) sequence: Val His Val Asn Ala Thr Tyr Val Asn Val Lys Cys of Seq. ID NO. 1 ; 0 (b) a peptide having at least 85% identity to the peptide of (a) above; and (c) a protein or peptide comprising the peptide of (a) or (b) above. The letters used above (and hereinafter) to denote specific amino acids (aa) are in accordance with the 1 -letter amino acid symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. 5 The term "having at least X% identity " refers to the percent of amino acid residues that are identical in the two compared sequences when the sequences are optimally aligned. Thus, 85% amino acid sequence identity means that 85% of the amino acids in two or more optimally aligned polypeptide sequences are identical. The peptide of the invention may be used, for example, for eliciting l o antibodies which may then be used as described above and below. A composition comprising the peptide of the invention may be useful for the treatment of various diseases which involve the expression of Met. Thus the present invention yet further provides a phaπnaceutical composition comprising as an active ingredient an effective amount (as defined above for the mAbs of the invention) of a peptide 15 of the invention. Furthermore, the present invention provides Abs which bind to the peptide of the invention and hybridoma cell lines which product mAbs that bind the peptide of the invention. DNA sequences encoding any of the amino acid sequences of the above 0 peptides are also encompassed within the scope of the invention. In addition, cells transfected with one or more of such DNA sequences, are also included within the scope of the invention. The term "malignant region " is defined in accordance with the invention as an area in the sample in which the malignant cells have clearly invaded muscle or 5 surrounding connective tissue but which are still within the primary tumor parenchyma. The non-malignant regions are defined as whole histologic areas in the sample adjacent to the malignant tissue but which appear to be uninvolved. Furthermore, it has been found in accordance with the invention that where the level of binding of the anti-pMet mAbs to the malignant region of the sample is 0 higher than the level of binding of the same mAbs to the non-malignant region in the same sample, there is a higher risk of developing a metastatic disease in the individual from which the sample was taken as well as a poor survival rate in the same individual. Where the level of binding of the anti-pMet mAbs to the malignant and non-malignant regions of the sample are equal, or where the level of 5 binding of the anti-pMet mAbs in the malignant region is lower than that in the non-malignant region, the prognosis of the tested individual is more positive. Thus, by a further aspect of the present invention, a method is provided for deteπnining in an individual having a primary cancer the probability of having or developing a metastatic disease and for determining said individual's survival l o expectancy comprising : (a) obtaining a sample from the tested individual, said sample comprising a malignant region and a non-malignant region; (b) identifying in the sample one or more malignant regions and one or more non-malignant regions; 1 5 (c) contacting said one or more malignant regions of the sample with one or more of the anti-pMet mAbs of the invention under conditions enabling binding of the mAbs to the sample; (d) contacting said one or more non-malignant regions of the sample with said one or more of the anti-pMet mAbs under conditions 0 enabling binding of the mAbs to the sample; (e) comparing the extent of binding of said mAbs in (c) and (d) above, a substantially higher level of binding in (c) above as compared to (d) above indicating a high risk of having or developing a metastatic disease and a poor survival rate in said tested individual. 5 By a preferred embodiment, the above method is provided for determining the probability of having or developing a metastatic disease and for determining the life expectancy in an individual having a primary breast cancer. Most prior art methods which are based on comparing the level of expression of a certain protein in two parts of a sample obtained from a tested 0 individual in order to provide an evaluation of the individual's prognosis are based on qualitative or "semi-quantitative " results (using indices such as 2+, 3+, +/-, etc.). In accordance with yet another aspect of the present invention novel quantitative prognostic indices (De and DI) were developed. The DI is derived from both the difference and the ratio of staining intensity of the antibodies of the 5 invention between malignant and non-malignant regions in a tested sample while the DE takes into consideration the divergence in entropy (e) levels between the two regions of an assessed image. In addition, on the basis of these novel indices, a novel method was developed which enables to provide an accurate prognosis for an individual having a primary cancer involving the expression and/or activation of i o Met and preferably for an individual having a primary breast cancer. By using the method of the invention which combines morphological parameters, Confocal Laser Scanning Microscopy (CLSM), immuno fluorescence (IF) analysis and digital image analysis with the calculated De or DI index, the ratio of p-Met expression levels in selected regions in the sample are expressed in the form of a 1 5 quantitative prognostic index. Moreover, in accordance with the novel method of the invention, it was found that, based on the distribution of the De or DI index, the breast cancer patients may be divided into three groups: a first group having a large negative De or DI index indicating that pMet in the malignant tissue is of greater diversity or at 0 a higher level than in the adjacent non-malignant tissue (N < T), a second group in which a large positive De or DI index indicates that the pMet is of greater diversity or at a higher level in the non-malignant tissue than in the malignant tissue (N > T) and a third group in which the pMet diversity or levels in both regions are equivalent (N = T). It was found in accordance with the invention that the De or DI 5 index distribution groups correlated with prognosis of the individual from which the sample was obtained. In order to determine to which prognostic group a certain tested individual belongs, two cut-off values are predetermined: a low cut-off value and a high cut-off value. The De or DI index of the tested sample is calculated as mentioned above and described below and its value is then analyzed in comparison 0 to the predetermined cut-off values. The cut-off values are determined for a specific set of parameters (e.g. the type of Abs used for immuno-fluorescence IF analysis) and for these parameters the cut-off values remain constant and can be used for comparing to the calculated De or DI values of tested samples. The poorest prognosis is where the calculated De or DI index is below the low cut-off value. 5 The probability of the tested individual to have or develop a metastatic disease is then the highest with a poor survival expectancy. Where the calculated De or DI index is above the high predetermined cut-off value, the probability of a tested individual to have or develop a metastatic disease is lower ( "medium " prognosis) and wherein the calculated De or DI index is above the low predetermined cut-off I o value but below the high predetermined cut-off value, the probability that the tested individual has or will develop a metastatic disease and the survival expectancy is the lowest ( "better" prognosis). Thus, in accordance with another aspect of the invention, a method is provided for determining in an individual having a primary cancer involving the 15 expression and/or activity of Met the probability of having or developing a metastatic disease and for determining said individual's survival expectancy comprising: (a) obtaining a sample from said individual, the sample comprising at least one malignant region and at least one normal region; 0 (b) identifying in said sample one or more malignant regions and one or more non malignant regions; (c) partitioning said one or more regions of malignant tissue in said sample into a predetermined number N of sub-regions, each sub- region having the same predetermined area a; 5 (d) partitioning said one or more non malignant regions of said sample into a predetermined number M of sub-regions, each sub-region having the same predetermined area a; (e) contacting said sample with anti-pMet mAbs under conditions enabling binding of said anti-pMet mAbs to any pMet present in said 0 N and M regions; (f) determining an extent of binding of the at least one anti-pMet antibody in each of the N regions of malignant tissue in said sample and in each of said M regions of non-malignant tissue in said sample, the extent of binding of said anti-pMet antibodies in each pixel in each one of said N and M regions being an integer between a first integer LI and a second integer L2; (g) for each integer i between L 1 and L2; (ga) defining tpi (in ROI) as the number of pixels in the malignant regions having an extent of binding of the anti-pMet antibody equal to i; (gb) defining by npi the number of non-malignant regions having an extent of binding of the anti-pMet antibody equal to i; (h) determining a first low cut-off value and a second high cut-off value; (i) calculating a prognostic value De according to the algebraic expression De = fe n - fe t wherein and wherein for all i, LI < i < L2 np i tp. npx i = — -,tpx t - npt tpt wherein for all i, LI < i < L2 a De index being below said low cut-off value indicating a high probability that said individual has or will develop metastatic disease and poor survival expectancy; a De index being above said high predetermined cut-off value indicating a medium probability of said tested individual to have or develop a metastatic disease and a better survival expectancy and a De index being above said low cut-off value and below said high cut-off value indicating a low probability of said tested individual to have or develop a metastatic disease and a good survival expectancy. Thus in accordance with another aspect of the invention a method is provided for determining an in individual having a disease involving the level of pMet expression the probability of having or developing a metastatic disease and for determining said individual's survival expectancy comprising: (a) obtaining from a sample from said individual, the sample comprising at least one malignant region and at least one normal region; (b) identifying in said sample one or more malignant regions and one or more non malignant regions; (c) partitioning said one or more regions of malignant tissue in said sample into a predetermined number N of sub-regions, each sub- region having the same predetermined area a; (d) partitioning said one or more non malignant regions of said sample into a predetennined number M or sub-regions, each sub-region having the same predetennined area a; (e) contacting said sample with anti-pMet mAbs of any of Claims 7-10, under conditions enabling binding of said anti-pMet mAbs to any pMet present in said N and M regions; (f) determining an extent of binding of the at least one of said anti-pMet antibodies in each of the N regions of malignant tissue in said sample and in each of said M regions of non-malignant tissue in said sample, the extent of binding of said anti-pMet antibodies in each pixel in each one of said N and M regions being an integer between a first integer LI and a second integer L2; (g) for each integer I between 11 and 12; (ga) defining tpi (in ROI) as the number of pixels in the malignant regions having an extent of binding of the anti-pMet antibody equal to i; (gb) defining by npi the number of non-malignant regions having an extent of binding of the anti-pMet antibody equal to i; (h) determining a first low cut-off value and a second high cut-off value; (i) calculating a prognostic value DI according to the algebraic expression DI = D n - Dt wherein i=X, and wherein for all i, LI < i < L2 wherein for all i, LI < i < L2 a DI index being below said low cut-off value indicating a high probability that said individual has or will develop metastatic disease and poor survival expectancy; a DI index being above said high predetermined cut-off value indicating a medium probability of said tested individual to have or develop a metastatic disease and a better survival expectancy and a DI index being above said low cut-off value and below said high cut-off value indicating a low probability of said tested individual to have or develop a metastatic disease and a good survival expectancy. By a preferred embodiment the above methods are provided for determining the probability of having or developing a metastatic disease and for determining the survival expectancy of an individual having a primary breast cancer. In addition to the above, in accordance with the invention, it has also been found that the ratio between the level of pMet and Met protein in each of the malignant and non-malignant regions of the tested sample obtained from a cancer patient, preferably from a breast cancer patient, may also have a prognostic value in said individual. Thus, in accordance with the invention, the anti-pMet / anti-Met ratio is determined in each malignant and non-malignant region of the sample and a final ratio of the above ratios is determined. A prognostic factor may also be calculated based on the ratio of the measured Met level and the calculated pMet De index. Although the above method of the invention based on calculation of the novel De or DI indices is described mainly in relation with levels and or activation of Met in samples obtained from breast cancer patients, in accordance with the invention it may be used for prognosis of many other malignant diseases involving different expression of a detectable protein in the malignant tissue as compared to adjacent non-malignant tissue. The level of the expression of the relevant protein may be determined in malignant and non-malignant regions of a sample obtained from the tested individual, an index calculated as described above and prognosis provided based on the value of the calculated index. DETAILED DESCRIPTION OF THE INVENTION In accordance with the first aspect of the invention, hybridomas producing monoclonal antibodies which bind phosphorylated Met are provided. The term "phosphorylated Met" relates to any met protein in which at least one of the tyrosines are phosphorylated. Typically, the phosphorylated tyrosines in human Met are the three tyrosines 1349, 1356 and 1365 in the Met docking site. Such hybridomas may be prepared by any of the methods known in the art (e.g. Kohler, G., and Millstein, C, Nature, 256:495-497 (1975)). Typically, in accordance with the invention, a phosphotyrosine peptide comprising at least one Met phosphorylation site, preferably Tyr , linked to a peptide which increases its immunogenecity (e.g. KLH) is injected recurrently into animals, typically mice. Sera collected from the immunized mice is then screened for anti pMet antibodies using any one of the methods known in the art such as ELISA, Western Blotting (WB) radio-immuno assay (RIA), immunoprecipitation (IP) etc. For screening, the peptides used for immunizing the mice may be used as well as any pMet proteins or cells and tissues containing the pMet protein. Hybridoma cell lines are then prepared by fusing spleen cells of the immunized animal, typically a mouse, having a high titer of anti pMet antibodies with myeloma cells and the hybridoma clones are screened again as described above. Generally, immortalized cell lines secreting the mAbs of the invention may be obtained also by additional means known per se, such as by EBV transformation, transfection of appropriate cell lines with DNA constructs encoding the binding site of the antibodies, etc. The present invention also provides antibodies which bind specifically to pMet. Preferably, the mAbs of the invention bind to one of the above mentioned three tyrosine phosphorylation sites in the Met docking site Tyr 9 , Tyr 1336 or Tyr 1365 . The term "antibodies " refers to antibodies of any of the classes IgG, IgM, IgD, IgA and IgE. Although, typically, the antibodies of the invention are produced by hybridoma cell lines as described above, they may also be produced by recombinant genetic methods well known to a person skilled in the art. The antibody may be animal derived, typically a mouse antibody as well as a chimeric antibody, a "humanized antibody ", etc. Wherein the antibodies of the invention are produced by recombinant methods, analogs of the antibodies will also form an aspect of the invention. Analogs of the antibodies which fall under the scope of the present invention are such which substantially retain the Ag binding characteristics of the mAbs of the invention to the peptides of the invention or to cells or tissues expressing pMet. The term "substantially retaining" should be understood to mean a binding of the analogs to pMet as determined by any of the methods mentioned above and below which is statistically non-significantly different than the binding of the whole antibody to the same peptide. The level of binding of the anti-pMet mAbs to each of the regions in the sample may be determined by any one of the methods known in the art such as immunohistochemistry, FACS analysis, ELISA, radio-immuno assay (RIA), Western blotting (WB) etc. However, in accordance with a preferred embodiment of the invention the level of binding of the anti-pMet mAbs to the regions in the sample are determined using confocal laser scanning microscopy (CLSM) and computerized image analysis of samples immunostained with the anti-pMet antibody. The relative levels of binding of the anti-pMet mAbs to the various regions in the sample may be compared using methods known in the art such as, for example, the percentage positive area (PPA) image analysis procedure (Tsarfaty, I. et al, Science, 257: 1258-1261, 1992) Statistical significance of the differences between the binding of the antibodies to the different regions may be calculated by any of the statistical methods known in the art such as Students' t-Test in Microsoft Excel (Microsoft, Redmond, WA). Binding of a mAb of the invention to a malignant region in a sample obtained from a tested individual will be considered to be "substantially different" than its binding to a non-malignant region of the same sample when the difference in binding of the mAb is statistically significantly different as determined by any of the statistical methods known in the art (e.g. Students' t-Test) which are used in connection with results obtained by the experimental methods mentioned herewith. The monoclonal antibodies of the invention may also be labeled with various detectable agents such as a radioactive group, a fluorescent group, an enzyme capable of catalyzing a reaction yielding a detectable product, a biotin group capable of being detected by avidin, etc. The labeling of the antibodies may be carried out by any of the methods known in the art. As will be appreciated by a person versed in the art, the amino acid sequence of the antibodies or peptides of the invention may be altered, for example, by addition, deletion.or conservative or non-conservative substitution of one or more amino acids. The teπn "conservative substitution " refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class if defined by common physiochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as deteπnined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. [Six general classes of amino acid side chains have been characterized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (He, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another Class III residue such as Asn, Gin, or Glu, is a conservative substitution. The term "non-conservative substitution " refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a Class II residue, with a Class III residue such as Asp, Asn, Glu, or Gin. Fragments of the antibodies of the invention which substantially retain the antigen-binding characteristics of the whole antibody are also within the scope of the present invention. Such fragments may be antibodies lacking the Fc portion, single chain antibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, Fab or F(ab) fragments, etc. The antigenic characteristics of the antibodies or fragments thereof may be determined by testing the binding of the antibody or fragment to the pMet peptides or cells or tissues comprising the peptide as described above as compared to the binding of the same Ab or fragment to a non-phosphorylated Met protein or cells or tissues comprising it- The antibodies and analogs of the invention may also be chemically modified. The term "chemically modified" means a protein where at least one of its amino acid residues is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art. Among the numerous known modifications typical. but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a liquid or lipid derivative, methylation, myristylation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process. The mAbs of the invention may be used as prognostic agents in an individual having a primary breast cancer both in vitro (ex-vivo) as well as in vivo. In vitro use of the mAbs is described above and in the Examples. Generally the mAbs are contacted with a sample obtained from the tested individual being a biopsy specimen, tissue, cells or body fluid comprising cells and the level of binding of the antibodies to the sample is determined using one of the methods known in the art. Wherein the mAbs are used for in situ prognosis, the mAbs are bound to a detectable marker (e.g. radioactive marker) and administered to the individual in a manner which enables to detect them in the body. For this purpose, the antibodies are administered within a composition which comprises at least one of the mAbs of the invention together with a suitable carrier. The carrier is typically a soluble carrier such as any one of the physiological acceptable buffers known in the art (e.g. PBS) or a solid state carrier such as, for example, latex beads. By a further therapeutical aspect of the invention, pharmaceutical compositions comprising, as an active ingredient, one or more of the mAbs of the invention together with a suitable carrier may also be used. The carrier may be any one of the carriers known in the art such as those mentioned above. Treatment of primary breast cancer by administering to an individual in need a therapeutically effective amount of the mAbs of the invention is also within the scope of the present invention. A therapeutically effective amount being an amount capable of alleviating the symptoms of the malignant disease, reducing the symptoms or completely eliminating them. A therapeutically effective amount may, in some cases prevent the development of a metastatic disease in a primary breast cancer patient and in other cases reduce the number of metastatic focuses or their size. The prognostic and therapeutical compositions of the invention are typically administered to the individual in need by means of parenteral administration, e.g. intravenously (i.v.) intraperitoneally (i.p.) or intramuscularly (i.m.). The phosphorylated Met peptides in accordance with the invention may be 5 obtained by enzymatic digestion or chemical digestion of a longer protein. In addition, they may be extracted from cells or tissues containing pMet. In addition, phosphorylated pMet peptides may also be synthesized by methods known in the art such as, for example, solid phase peptide synthesis (F-moc technology) using a peptide synthesizer. The resulting peptides are separated by methods known in the l o art such as by RP-HPLC . The present invention also provides kits, e.g. diagnostic assay kits, for utilizing the mAbs of the invention and carrying out the method disclosed above. In accordance with one embodiment, the diagnostic kit would conventionally include one or more of the above mAbs in one or more containers, a conjugate of a specific 15 binding partner for the mAb, a label capable of producing a detectable signal and directions for its use. The label may be, a priori, bound to the monoclonal antibody or, alternatively, the label may be bound to a carrier molecule which then specifically binds to the mAb. The kit will also comprise low and high De index values to which the calculated De index of the tested sample will be compared. 0 Instructions for use of the kit will also be included. To date, prognosis of breast cancer is typically determined by several factors, the most important being lymph node involvement. However, between 10% and 30% of patients having the same prognosis based on absence of nodal involvement have recurrent breast cancer. Therefore, a clinical test for identifying 5 those patients who are at risk and might benefit from adjuvant therapy is greatly needed. In addition, early determination of the existence of metastatic tumors in an individual having a primary breast cancer disease may be crucial in determining the treatment procedure for the same individual. Currently, the existence of metastatic tumors is very often detected at a late stage. In accordance with the present invention, a novel method was developed for calculating quantitative prognostic De or DI indices. On the basis of these indices, an accurate prognosis can be provided for a primary breast cancer patient, i.e. it is possible to determine relatively accurately the probability of the patient having a metastatic disease or developing one as well as the survival expectancy for the tested individual. In accordance with the method of the invention, a sample is obtained from the tested individual which is typically a biopsy taken from the tumor region of the individual. Typically, the evaluation in accordance with the invention is made on paraffin sections of the biopsy prepared on slides. However, in accordance with the invention, a sample may also be a sample prepared from tissues, cells or body fluids comprising cells expressing the Met protein. The tenn "primary breast cancer patient" is to be understood as an individual in which a breast cancer tumor was detected. The individual may have received a first therapeutic treatment typically being a curative operative, radiological treatment, adjuvant treatment, etc. The patients having primary unilateral breast carcinoma had no other primary cancer. The patients may either be node positive, i.e. having detected malignant cells in nodes distant to the primary location of the tumor or node negative. The tenn "prognosi " relates to a combination of two factors being the probability of having or developing a metastatic disease as well as life expectancy rate. The tenn "metastatic disease " relates to the existence of malignant cells in any location other than the location of the primary breast tumor in the individual. Life expectancy rate relates to expected median time interval between detection of primary tumor in an individual and death. In accordance with the method of the invention, the De or DI indices are calculated on the basis of the binding of anti-pMet mAbs to malignant regions and non-malignant regions of the sample taken from the tested individual. Two cut-off values are predetennined on the basis of different combinations of the De or Di indices as explained above and below. The predetermined values remain constant for a certain set of parameters. As an example only, based on Log-Rank and Wilcoxon P values and further statistical methods described below, and using anti-Met Abs, the low cut-off value was detennined for De and DI to be -60 and - 13 respectively and the high cut-off values for De and DI indices were detennined to be 90 and 10 respectively. In such a case, a patient having a De index of lower 5 than -60 or a DI index lower than -13 will have a poor prognosis, a patient having a De index of above 90 or a DI index greater than 10 will have a medium prognosis and a patient having a De index of between -60 and 90 or a DI index between -13 and 10 will have the best prognosis. These cut-off values calculated when using anti-pMet Abs or when changing any of the other parameters (such as the method l o of detection of binding) may obviously be different. The method of the invention is useful both in patients with no lymph node involvement as well as in patients in which such involvement was diagnosed. Knowing the prognosis of the patient may substantially assist to determine which therapeutic regimen would be most promising in preventing the development of 15 metastatic disease, or on the other hand preventing drastic and harmful chemotherapy from patients with good prognosis. The prognosis may be helpful in detennining, for example, the rate in which the individual should be checked, by which methods, etc. The method of the invention based on calculation of a De or DI prognostic 0 index may also be useful for prognosis of other malignant diseases which involve differential expression of tumor associated proteins in the malignant tissue as compared to non-malignant tissue. In addition, the method may also be used for prognosis of other types of disorders and diseases in which there is a difference in expression of a certain protein in different tissues or tissue parts of the individual. 5 EXAMPLES The various aspects of the invention will now be illustrated by the following non-limiting examples with occasional reference to the attached figures. 0 BRIEF DESCRIPTION OF THE FIGURES: Fig. 1 is a schematic representation showing binding of mAbs of several hybridomas including the hybridoma of the lf8 (ECACC Deposit No. 00041122), with pMet (grey) or Met (crossed lines) in an ELISA assay. Fig. 2A is a photograph showing an immunoblot prepared from cell lysates of untreated DA3 cells (time 0) or from cell lysates of DA3 cells treated for ten minutes with HGS/SF (time 10 mins.). The cell lysates were resolved by 7.5% SDS-PAGE and immunoblotted with the following tested mAbs: lf8 (lane 3), lf6 (lane 4), 3g4 (lane 5), 7cl l (lane 6). Fig. 2B is a photograph showing an immunoblot of the cell lysates prepared from the same cells as those in Fig. 2A which were immunoprecipitated with C260 anit-mouse Met peptide antibody, resolved by 7.5% SDS-PAGE and immunoblotted with the following: anti-Met polyclonal Ab (lane 1), anti-P-Tyr Ab (lane 2), lf8 (lane 3), lfό (lane 4), 3g4 (lane 5), 7c 11 (lane 6). Fig. 3 A is a photograph showing an immunoblot of NIH/3T3 (Fig 3 A, lane 1) and HMH (Fig. 3 A, lane 2) cell lysates which were immunoprecipitated with lf8 pMet mAb and immunoblotted with anti-human Met antibody C28. NIH/3T3 cells were used as negative control (Fig. 3A1) and HMH cell immunoprecipitated with anti- human Met 19S and immunoblotted with anti-p-Tyr mAb (4G10) were used as positive control (Fig. 3 A3). Fig. 3B is a photograph showing immunofluorescence staining of NIH/3T3 cells and HMH cells. The cells were subjected to indirect immunofluorescence staining with 3g4 mAb supernatant (top row, green) and with anti-human Met C28 antibody (bottom row, red). Staining was analyzed by CLSM. For co-localization analysis the green and red staining were overlaid (middle row), yellow indicates co-localization of the red and green staining. Fig. 3C is a photograph showing indirect immunofluorescence staining of 3T3 cells transfected with HGF/SF or with different combinations of tyrosine mutations in Met docking site co-stained for Met and pMet. Columns 1, 3 and 5 show staining of the wild type Y1354 transfectants and columns 2, 4 and 6 show the staining of mutated Y1354 transfectants. Additional Tyr mutations in Met mu docking site in combination with Y1354 wild type/mutated are shown in rows: (a) substitution of Y1347; (b) substitution of Y1363; (c) substitution of both Y1347 and docking site in combination with Y1354 wild type/mutated are shown in rows: 5 (a) substitution of Y1347; (b) substitution of Y1363; (c) substitution of both Y1347 and Y1363; (d) wild type Tyr. All substitutions were to Phe. Cells were subjected to indirect immunofluorescence staining with 1F8 Mab, (columns 1 and 2, green) and anti-mouse Met C260 (columns 3 and 4, red), staining was analyzed by CLSM. For co-localization analysis the red and green staining were overlaid. Yellow indicates l o co-localization of the red and green staining (columns 5 and 6). Fig. 4 is a photograph showing the time course of Met phosphorylation in DA3 mammary cells. Fig. 4A shows lysates from DA3 cells (0 min.) or DA3 cells treated for 5, 10 and 20 mins. with HGF/SF, resolved by 7.5% SDS-PAGE, immunoprecipitated 15 with C260 anti-mouse Met antibody and followed by western blot analysis using the C260 antibody. Fig. 4B is a photograph showing the lysates of Fig. 4A in which the antibody used for the western blot analysis was an anti-pMet antibody (lfδ). Fig. 4C is a photograph showing indirect immunofluorescence of DA3 cells 0 which were treated with HGF/SF for 0, 5, 10 and 20 mins. The staining (seen as red staining) was with mAb lf8. Fig. 5 are photographs showing the level of pMet in different breast tissue biopsies. Paraffin-embedded sections of breast adenocarcinoma were subjected to indirect immunofluorescence staining with anti-human Met C28 antibody (red) and 5 lf8 mAb (green), the staining was analyzed by CLSM. For co- localization analysis the green and red staining were overlaid. Yellow indicates co-localization of the red and green staining. Fig. 5A shows the expression of Met (c) and pMet (d) in normal breast ducts. Co-localization analysis (a), computed co-localization (middle square) is 0 depicted in blue (b). Fig. 5B shows comparison of met/pMet expression levels in nonnal (c-d) and tumor (a-b) human breast sections immunostained with 1 f8 (a, c) or co-stained with anti-human Met C28 (red) and lf8 (green) antibodies (b, d). Fig. 5C shows different breast tumor sections co-stained with Met-pMet. Upper row shows samples with low Met and pMet; middle row shows samples with high Met and high pMet; bottom row shows samples with high Met and low pMet. Fig. 6 is a photograph showing immunohistchemical CLSM of Met protein expression in breast cancer biopsies. Images (a, c, e, g, i, k, m and o) are of normal breast cancer biopsies and images (b, d, f, h, j, 1, n and p) are of tumor breast cancer biopsies. The intensity is color coded based on the Zeiss "Glow Scale " look-up table, which labels intensity in a graded series of colors from black (lowest) through red, orange, yellow, and white (highest). Panels a-d are cases in which the level of Met is greater in the normal tissue relative to the tumor (N>T). Panels e-1 are representative of N-T cases. Panels e-h are representative of overall higher levels than are shown in panels 1-1. Panels m-p are cases in which the level of Met is greater in the tumor N < T. Fig. 7A is a photograph showing a western blot analysis of normal and tumor breast tissues. HT29 cell extract was stained with the C-28 Met antibody (1) or with C-28 antibody in the presence of competing antigen (2). Normal breast tissue (3,5,7,9) and tumor breast tissue (4,6,8,10) were stained with the C-28 Met antibody (3,4,7,8) or with C-28 antibody in the presence of competing antigen (5,6,9,10). Reactivity with anti-actin antibody was added to the samples in lanes 3,4,7, and 8 to detennine levels of protein loaded from tumor and normal tissues. Paired lanes 3/4 show N>T, whereas lanes 7/8 show T<N. Fig. 7B shows an immunofluorescence and CLSM analyses of breast tumor section with C-28 antibody alone (1) or in the presence of competing antigen (2). Loss of intensity is evident in panel 2 relative to panel 1. Fig. 7C shows CLSM analysis of costaining using anti-Met DI monoclonal antibody (1, red) and C-28 (2, green). In the overlay of the DI and C-28 images in panel 3, yellow indicates colocalization of the two signals. Panel 4 shows the X-Y graphic representation of fluorescence intensities in panel 3. Fig. 8 is a schematic representation showing the distribution and cumulative percentages of the De index using anti-Met antibodies. The distribution of De frequencies for 182 patients (bars) is shown with the cumulative percentages (line). Fig. 9 is a schematic representation showing distribution of statistical significance with different De cut-off points in survival analyses using anti-Met Abs. Distribution of p values in survival analyses are shown using different combinations of lower and upper cutoff points. Comparisons of survival curves were evaluated with log-rank (grey bars) and Wilcoxon (crossed bars) statistics. Fig. 10 is a schematic representation showing the alteration of Met expression in the tumor as a prognostic indicator of breast cancer. The overall survival curve of 182 patients is shown. Fig. 11 is a schematic representation showing the alteration of Met expression in node-negative breast cancer patients. Fig. 11A shows the overall survival curves for node-negative patients: n > t, n = t and n < t; Fig. 11B shows the survival curves for node-negative patients: n = t, and n < or > t; Fig. 12A is a histogram of values obtained for the DI index (bars) and cumulative percentages (line); Fig. 12B shows the statistical significance of survival analysis using different combinations of lower and upper cut-off points (bars with horizontal lines - logrank, gray bars - Wicoxon; and Fig. 12C shows Kaplan-Meier analysis for overall survival of patients grouped by lower and upper cut-offs of 13 and 10 respectively respectively ( A - N > T, ■ - N « T, . N< T) . Specificity of the anti-pMet mAbs to pMet 1.1. Generation of anti pMet monoclonal antibodies. Tyr J: " is a major tyrosine autophosphorylation site of the Met hu receptor. A phosphotyrosine peptide corresponding to the Met autophosphorylation site (Val His Val Asn Ala Thr Tyr (P0 3 H) Val Asn Val Lys Cys) was synthesized and analyzed by mass spectrometry. The phosphorylated peptide was linked to KLH (keyhole limpet hemocyanin) to increase its immunogenicity. Seven mice were immunized with this antigen. After five boosters with the same antigen, blood was collected from the mice. These sera were screened for anti pMet antibodies by ELISA and western blot (WB) analysis using the immunizing antigen. Four of the seven mice immunized with the phosphorylated peptide demonstrated high titers of anti pMet antibodies. The spleen of the mouse with the highest titer was harvested and fused with myeloma cells to generate hybridoma lines. The hybridoma clones were screened by ELISA using the phosphorylated Met peptide. Analysis of non-specific reaction was carried out using the same peptide not phosphorylated and KLH. Twenty-five clones showing specific positive results were expanded and re-analyzed with the same antigens by ELISA. As seen in Fig. 1, clones lf8, the hybridoma having the ECCC Deposit No. 00041 122, 7c 1 1 and lf6 showed high reactivity with low non- specific reaction, while clone 3g4 showed relatively low yet significant reactivity with the phosphorylated Met peptide. 1.2 Binding of the anti-pMet mAbs to DA3 cell extracts Supernatants from clones lf8, lf6, 3g4 and 7c 11 were also analyzed by WB analysis using blots containing both treated and untreated DA3 cells lysates (Figs. 2A, lanes 3, 4, 5, 6 respectively). DA3 is a mammary adenocarcinoma cell line, cloned from a tumor induced in BALB/c mice by dimethyl benzanthracene, and is a metastatic cell line. DA3 cells express high levels of Met protein pl40 met that is rapidly phosphorylated upon HGF/SF treatment. To determine whether the mAbs react with pMet, DA3 cells were treated with HGF/SF for 10 min at room temp. Treated and untreated DA3 cell lysates were immunoprecipitated using anti-mouse Met polyclonal antibody C260; Immunoprecipitates were subjected to SDS -poly aery lamide gel electrophoresis, followed by immunoblot analysis using anti-pTyr (4G10- UBI) (Fig. 2 B2) or supernatants of mAbs lfδ (lane 3), lf6 (lane 4), 3g4 (lane 5) and 7cl 1 (lane 6). As seen in Fig. 2, the supernatants of the four tested hybridomas showed binding (to different extents) to the extracts obtained from the DA3 cells activated with HGF/SF. 1.3 Reactivity of the mAbs with pMet in met + hgf/sf transfected cell lines HMH cells, NIH/3T3 mouse fibroblasts co-expressing human Met and human HGF/SF, express high levels of Met which is constitutively activated due to an autocrine loop. NIH/3T3 and HMH cell lysates were immunoprecipitated with lf8 pMet mAb and subsequently immunoblotted with anti human Met Ab C28 (Fig. 3 A, 1 and 2, respectively). The negative control NIH 3T3 cells did not show any reactivity (Fig. 3 A 1) while the positive control, HMH immunoprecipited with anti human Met 19S and immunoblotted with anti-P-Tyr mAb (4G10), exhibited strong reactivity (Fig. 3 A 3). These results indicate that lf8 specifically recognizes phosphorylated Met. To further establish the observation that the mAb react with phosphorylated Met (red) the same cells were co-stained with C28 rabbit polyclonal Ab against Met along with one of each of the five mAb candidate supernatants (green). NIH/3T3 cells, which do not express detectable levels of Met, served as control. Examples of results with 3g4 are shown in fig 3. High levels of tluorescein staining was observed with clone 3g4 in HMH cells, while in the 3T3 control cells, the staining was only at background levels (Fig. 3 B green staining). The staining with C28 was of greater intensity and appeared in both cell cytoplasm and membrane regions (Fig 3 B- red). The 3g4 and C28 staining results were overlaid allowing co-localization analysis. Co-localization (Fig 3 B yellow) was observed mostly on the cell membrane. These results suggest that indeed the 3g4 mAb recognize pMet, which is localized mainly on the cell surface. Some of the mAb staining was in the cytoplasm. 1.4 Specificity of mAbs of the hybridoma lf8 to Y Met mu To confirm the specificity of the lf8 Ab to Y 1354 Met™ , NIH/3T3 cells were transfected with HGF/SF and seven different expression vectors were constructed by introducing point mutations to the three major tyrosines in Met 1 ™ docking site turning them into phenylalanine (phe). The constructs were co-transfected into NIH3T3 cells along with a plasmid coding for human HGF/SF as previously described (Rong et al, Mol Cell. Biol, 12:5152-8, 1992). The different clones were designated according to the mutation introduced: wild type clone was designated YYY and the mutated Met clones: FFY, FYY, FYF, YFY, YFF, YYF, and FFF. Although similar levels of Met were observed in all the NIH/3T3 transfectants (Fig. 3C column 3 and 4 - red), staining of the pMet was relatively low in the Y1354-mutated transfectants (Fig. 3C column 2 - green). There was co-localization between the pMet (green) and Met staining (red) (Fig. 3C column 5) as depicted by the yellow staining in the CLSM co-staining analysis. Mutation in the other tyrosines in the docking site did not affect staining (Fig. 3 C a-d in all columns). These results show that indeed the lf8 mAb recognizes pMet and is specific to + tyrosi ■ne 1354 1.5 Binding of anti-pMet mAbs to DA3 cells activated with HGF/SF Further analysis of the mAbs was carried out using DA3 cells. Unlike HMH cells where Met is constitutively phosphorylated at high levels, in DA3 cells Met is activated (phosphorylated) after treatment with HGF/SF (Fig 4 A, B). The mAb staining in treated DA3 cells was at least ten-fold higher than the staining levels in untreated cells (Fig 4 C comparing time 0 to 10). A similar increase in staining was observed with the anti-pTyr Ab (data not shown). 1.6 Colocalization of Met and pMet Colocalization analysis was performed on samples of simultaneously detected Met (Fig. 5A d - green) and pMet (Fig. 5A c - red) using the Zeiss co-localization procedure. This procedure results in a graphic representation of the distribution of green (Y axis) and red (X axis) fluorescence of each pixel. A range of fluorescence intensities is selected from graphic representation of pixel distribution. These selected intensities of co-localization are then overlaid as a blue image on the red/green image (Fig. 5A b). The small graph in Fig 5A demonstrates such analysis. The co-localization between Met and pMet staining further demonstrates the possibility to use the pMet Ab in immunostaining and shows the specificity of the pMet Ab. The above results show that the anti-pMet mAbs of the invention indeed react specifically with pMet. 2. Binding of anti-Met and the anti-pMet mAbs to breast tumor biopsy specimens The work described in the Example below was carried out with anti-Met Abs. Anti-pMet mAbs are used in the identical experiment. Materials and Methods 2.1 Patients and Histological Material. Excised primary breast tumors were collected from patients treated in two locations: 1) Centre Rene Huguenin in Saint-Cloud, France, from 1978 to 1993 (86 tumors, of which 79 were ductal carcinomas) and 2) Israeli hospitals from 1975 to 1990 (96 tumors, of which 90 were ductal carcinomas). Median age of patients was 57.5 years (range 27 - 89 years). The patients underwent curative operation (radical mastectomy, 50 (21.5%) patient; modified mastectomy, 105 (57.7%) patients and lumpectomy 27 (14.8%)). Of the total 182 patients 116 were determined to be node negative. From the 182 patients 1 14 were examined for estrogen and progesterone receptor's, from which 50.9 % were estrogen receptor positive and 46.0 were progesterone receptor positive. Disease recurrence was documented by physical examination, radiological and laboratory tests and/or other relevant diagnostic procedures. Median follow-up period was 9 years (range, 1 - 18 years). Metastatic free survival was defined as the time interval between surgery and identification of distal metastasis. Overall free survival was defined as the time interval between surgery and death or latest physical check-up. For all cases death was due to breast cancer. The patients in this retrospective study met the following criteria: primary unilateral breast carcinoma; no other primary cancer at the time of diagnosis; and clinical follow-up. The evaluation was made on the initial presentation biopsy sample (paraffin sections). In those few cases where the original tissue sample on the slide did not contain adjacent, uninvolved normal terminal ductal epithelium as well as tumor tissue, additional sections were prepared from the same block or adjacent blocks, which contained both nonnal and tumor tissue. 2.2 Tissue Preparation and Staining Methods Paraffin blocks from human primary breast carcinomas were serially sectioned at 5-6 μm. Tissue samples were stained with hematoxylin and eosin (HE) to identify "normal" and tumor areas. 2.3 Evaluation of Met and pMet expression in primary breast carcinoma biopsies Breast cancer tumor sections were deparaffinized, washed in phosphate buffered saline (PBS), incubated with anti-pMet (lf8) and anti Met polyclonal primary antibodies (C28), washed in PBS and incubated with a FITC-labeled anti mouse antibody and Rhodamine labeled anti-rabbit Ab. The sections were imaged by Nomarski optics (differential interference contrast microscopy) and fluorescence staining was visualized using an inverted, Zeiss LSM 4 confocal microscope. For each sample, Met and pMet expression in non-malignant and malignant regions of the biopsy was evaluated. Non-malignant regions are defined as the histologic areas adjacent to the neoplastic tissue that appeared to be uninvolved. We defined "malignant" as areas in which the malignant cells had clearly invaded muscle or surrounding connective tissue, but were still within the primary tumor parenchyma. 2.4 Western Analysis of Met in Breast Tumor Biopsies Breast tumor and adjacent normal tissue were surgically dissected from patient biopsies and frozen in liquid nitrogen. Tissues were homogenized in 1 ml of lysis buffer (20 mM Tris-HCl, pH 7.8, 100 mM NaCl, 50 mM NaF, 1% NP-40, 0.1% SDS, 2 mM EDTA, 10% glycerol) with protease inhibitor cocktail (Boehringer Mannheim, Germany). One hundred micrograms of protein was resolved on 7.5% SDS-PAGE and subjected to Western analysis using the C-28 antibody (1 :500). For competition experiments, the C-28 -immunizing peptide was incubated with the antibody for 2 h at RT prior to incubation with the blot. For actin analysis, the Western blot was cut below the 68-kd marker and reacted with anti-actin antibody (Boehringer Mannheim). HT29 colon cells served as a control for Met expression (31). Visualization was achieved using HRP-conjugated Protein A (1 :5000) (Amersham, Arlington Heights, IL), ECL reaction, and exposure to X-ray film (Fuji, Japan). Results 2.5 Differential expression of pMet in malignant and non malignant tissues of human breast adenocarcinoma biopsy specimen 2.5.1 Immunostaining Representative images of various levels of Met expression in nonnal versus malignant tissues of human breast adenocarcinoma biopsies are shown in Fig. 6. Normal histologic areas contained ducts and appeared to be uninvolved with the tumor, whereas the "tumor" contained malignant cells, often with overt invasion of muscle or connective tissue. Based on subjective evaluation, Met expression was higher in the normal tissue (Fig. 6a, c) than in the adjacent breast tumor tissue (Fig. 6b, d) in approximately 40% of the cases. In contrast, we found no significant difference between Met expression in the normal tissue and that in the tumor tissue in approximately 40% of the cases. Examples of the N=T cases are shown in Fig. 6 (high e-h and low i-1). We found lower Met expression in the normal tissue (Fig. lm, o) relative to the adjacent breast tumor (Fig. 6n, p) in about 20% of the cases (N<T). 2.5.2 Western blotting analysis To determine whether the ratio of Met expression in normal and tumor tissue extracts also varied between patients, we performed Western analysis. Samples from two patients are shown in Fig. 7A. In samples from one patient, higher Met levels were observed in the normal tissue relative to the tumor tissue (Fig. 7A, lanes 3 and 4, respectively), whereas in samples from second patients, higher Met levels were observed in the tumor relative to the normal tissue (Fig. 7A, lanes 7 and 8, respectively). We also observed reduced immunostaining of C-28 in the presence of competing C-28 peptide (cf. Fig. 7B, panels 1 and 2). The immunofluorescence specificity was further demonstrated by staining the same section with both the C-28 anti-Met polyclonal antibody and a monoclonal antibody (Di) to the human Met receptor extracellular domain. This analysis shows that both antibodies (Fig. 7C, panels 1 and 2) display the same reactivity pattern, depicted by yellow in the overlay images (Fig. 7C, panel 3), and a positive correlation in fluorescence intensity (Fig. 7C, panel 4). Furthermore, multiple sections from several patients, containing both normal and tumor regions, displayed equivalent fluorescence intensity between sections and exhibited no significant differences in fluorescence signal between tubular duct lobular units and large ducts cells (data not shown). 2.5.3 Quantification of Relative pMet Expression Levels by Image Analysis and use of the De and/or DI Indices of the invention a. Calculation of indices for a tested sample Normal and tumor areas in breast biopsy sections were identified on HE stained slides by a certified pathologist. The pathologies marked the identified normal and tumor region in un-stained serial sections corresponding to HE. Immunostaining was subsequently performed on marked un-stained slides. The immunostained sections were then imaged by Nomarski differential interference contrast (N/DIC) microscopy and fluorescence, using a Zeiss LSM 310 confocal microscope (Oberkochen, Germany). Regions of interest (ROIs) for both nonnal and tumor areas were outlined by a cursor on the Nomarski images according to morphological characteristics, using the Optimas image analysis software (Seattle, WA). This method eliminates any observer bias toward "bright" fluorescent areas during the selection of the ROI. The defined areas were overlaid onto the fluorescent images and the digital informatin for reach ROI collected (Klineberg, E., et al, Cell Vision, 5:402-406, 1996). The digital information of the confocal image consists of the gray values (0-255) describing the brightness of every point within the image (pixel). The pixel intensity histogram of the fluorescence image was then determined for each ROIs. Two indices were developed to reveal differences between Met expression in the same biopsy. The indices provide a quantitative measurement of variations Met expression between normal and tumor ROIs in a single patient's breast biopsy section. The De index is defined according to the algebraic expression: wherein A = C-iXwpx. iogzføpx,-) i=L, and wherein for all i, LI < i < L2 np. tp i npx t = — -, tpx x = npt tpt wherein for all i, LI < i < L2 npt The DI index is defined according to the algorithmic expression: DI = D n = D t wherein and The immunostaining, CLSM analysis, and quantification were perfonned blind to all clinical information. Statistical Methods The distributions of the De and DI indices were examined and divided into three distinct biological groups as described below. Analyses of metastasis-free and overall survival were performed with the Kaplan-Meier analyses (Kaplan, E.L. and Meier, P., J. Am. Stat. Soc, 53:457-481, 1958) A series of Kaplan-Meier analyses was performed to determine the optimal separation of the three groups (Tandon, A.K. et al, N. Engl J. Med., 322:297-302, 1990). Subgroup analyses were also performed on those node-negative patients who showed no evidence of nodal involvement at the time of diagnosis. Computations were performed with SAS (SAS Institute, Inc., SAS/STAT Software: Changes and Enhancements through Release 6.1 1, Cary, NC, 1996) and S-Plus (Statistical Sciences, S-PLUS Guide to Statistical and Mathematical Analysis, Version 3.3, StatSci, Mathsoft, Inc., Seattle, WA, 1995) software. 2.5.4 Grouping of patients into 3 groups on the basis of the De and DI indices and cut-off values Met expression was evaluated in 182 primary breast carcinoma specimens. For each sample, we evaluated Met expression in both normal and tumor regions of the biopsy using the De or DI index to process the digital CLSM output. These results were used to evaluate the clinical significance of alteration in Met expression in breast cancer. Visual inspection of frequencies for the De index (Fig. 9 d c e g respectively) and DI index (Figs. 12, a, b) show suggested that there are three groups, with the middle group in the "normal" range (N≡T). A large negative De or DI index indicates that Met expression in the tumor is greater than in the adjacent normal tissue (N<T), whereas a large positive De or DI index indicates that Met expression is greater in the normal tissue than in tumor tissue (N>T). Establishing cutoff values among the different groups is one requirement in assessing the significance of a marker. We therefore used different combinations for both the De and DI indices to define the cutoff values of the three groups for each index. Figure 9 shows both log-rank and Wilcoxon P values for analysis of the De index. The log-rank statistic attained its maximum at De = -60 and De = 90. A histogram of the DI value frequencies is shown in Fig. 12a. Determination of cut-off values, by non-parametric analysis, revealed a large range of values in which the DI calculator inhibited statistically significant results. Fig. 12b shows both log-rank and Wilcoxon P values for this analysis. An example of Kaplan-Meier analysis, shown in Fig. 5c for the cut-off values -13 and 10, produced results for log rank of 0.014 and Wilcoxon 0.0095. 3. Prognosis of primary breast cancer patients on the basis of the method of the invention 3.1 Met expression in breast cancer patients Log-rank and discrete Cox proportional hazards regression analyses was performed to determine whether the designated Met expression groups (N<T, N≡T, and N>T) are a prognostic indicator for overall and metastasis-free survival. Kaplan-Meier plots for overall survival (Fig. 10) for the three groups were significantly different {p = 0.0023 (log- rank), p=0.0104 (Wilcoxon)} . Thus, for overall survival the N<T group was significant for predicting poor prognosis. 3.2 Met expression in node negative breast cancer patients The relative levels of Met expression (N<T, N≡T, and N>T) in tumors of the node-negative patients were compared to the level of normal tissue in the same patient. Overall survival among these three groups was significantly different {p=0.0041 (log-rank) and p=0.0102 (Wilcoxon)} . The overall survival data showed that 11% of the N≡T group died, whereas 37.5% and 25% of the N<T and N>T groups died, respectively. When the latter two groups (N<T and N>T) were combined, then in a period of greater than 10 years, only 10 out of 88 (11%) in the N=T group died, whereas 19 out of 28 (37%) in the N<T + N>T group died. The difference in survival when the data were grouped in this manner was also statistically significant {p=0.0012 (log-rank) p=0.0027 (Wilcoxon)}, but there was no significant difference in the relative levels of Met expression with respect to metastasis-free survival. Thus, the method of the invention is especially useful for determining prognosis of a node negative (Stage I) breast cancer patient.

Claims: CLAIMS: 1. Hybridoma cell lines which produce monoclonal antibodies (mAbs) that bind to phosphorylated Met (pMet) and do not bind to non-phosphorylated Met. 2. Hybridoma cell lines according to Claim 1, wherein said anti-pMet mAbs bind to one or more phosphorylated tyrosines present in the phosphorylated form of the Met docking site. 3. Hybridoma cell lines according to Claim 2, wherein said phosphorylated . . . „ 1356 tyrosine is Tyr 4. A hybrodima cell line, which was deposited on April 12, 2000, in the Centre for Applied Microbiology and Research (CAMR), European Collection of Cell Cultures (ECACC) depository under Accession No. 00041122. 5. Monoclonal antibodies (mAbs) produced by hybridoma cell lines of any of Claims 1 to 4 or fragments of said antibodies which substantially retain the antigen binding characteristics of the whole antibody. 6. Antibodies which bind to an antigenic epitope bound by any one of the antibodies of Claim 5. 7. Antibodies which bind to any antigenic epitope bound by the antibodies of Claim 4. 8. Monoclonal Abs according to Claims 5 or 6, wherein said mAbs bind to the pMet docking site. 9. Monoclonal Abs according to Claim 8, wherein said mAbs bind to one or more of the phosphorylated tyrosines present in the phosphorylated form of the Met docking site. 10. Monoclonal Abs according to Claim 9, which bind to the major tyrosine phosphorylated tyrosine Tyr 11. A prognostic composition comprising one or more of the mAbs of any of Claims 5-10, together with a suitable carrier. 12. A phannaceutical composition comprising, as an active ingredient, one or more of the mAbs of any of Claims 5-10, together with a pharmaceutically acceptable carrier. 13. A kit for use in the prognosis of a disorder or disease involving pMet expression comprising one ore more anti-pMet mAbs of any of claims 5-10, reagents required for detennining the level of binding of said mAbs to said sample, low and high cut-off values and instructions for use. 14. A kit according to Claim 13, wherein said disease is cancer. 15. A kit according to Claim 14, wherein said cancer is breast cancer. 16. A peptide being the antigen of the mAbs of any of Claims 5-10 selected from the group consisting of: (a) a peptide having the amino acid (a.a) sequence: Val His Val Asn Ala Thr Tyr Val Asn Val Lys Cys of Seq. ID NO. 1 ; (b) a peptide having at least 85% identity to the peptide of (a) above; and (d) a protein or peptide comprising the peptide of (a) or (b) above. 17. A DNA sequence encoding the amino acids of any of the peptides of Claim 16. 18. Cells comprising one or more DNA sequences of Claim 17. 19. A hybridoma cell line producing monoclonal antibodies that bind to a peptide of Claim 16. 20. Antibodies which bind to the peptide of Claim 16. 21. A pharmaceutical composition comprising as an active material an effective amount of a peptide in accordance with Claim 16. 22. A method for determining in an individual having a malignant disease involving the expression of pMet the probability of having or developing a metastatic disease and for determining said individual's survival expectancy comprising: (a) obtaining a sample from the tested individual, said sample comprising a malignant region and a non-malignant region; (b) identifying in the sample one or more malignant regions and one or more non-malignant regions; (c) contacting said one or more malignant regions of the sample with one or more of the anti-pMet mAbs of any of Claims 5-10, under 5 conditions enabling binding of the mAbs to the sample; (d) contacting said one or more non-malignant regions of the sample with said one or more anti-pMet mAbs, under conditions enabling binding of the mAbs to the sample; (e) comparing the extent of binding of said mAbs in (c) and (d) above, a 10 substantially higher level of binding in (c) above as compared to (d) above indicating a high risk of having or developing a metastatic disease and a poor survival rate in said tested individual. 23. A method according to Claim 22, wherein said malignant disease is breast cancer. 15 24. A method for determining in an individual having a disease involving the expression of pMet the probability of having or developing a metastatic disease and for determining said individual's survival expectancy comprising: (a) obtaining a sample from said individual, the sample comprising at least one malignant region and at least one nonnal region; 0 (b) identifying in said sample one or more malignant regions and one or more non malignant regions; (c) partitioning said one or more regions, of malignant tissue in said sample into a predetermined number N of sub-regions, each sub- region having the same predetermined area a; 5 (d) partitioning said one or more non malignant regions of said sample into a predetermined number M of sub-regions, each sub-region having the same predetermined area a; (e) contacting said sample with anti-pMet mAbs of any of Claims 5-10, under conditions enabling binding of said anti-pMet mAbs to any 0 pMet present in said N and M regions; (f) detennining an extent of binding of the at least one of said anti-pMet antibodies in each of the N regions of malignant tissue in said sample and in each of said M regions of non-malignant tissue in said sample, the extent of binding of said anti-pMet antibodies in each pixel in each one of said N and M regions being an integer between a first integer LI and a second integer L2; (g) for each integer i between 11 and 12; (ga) defining tpi (in ROI) as the number of pixels in the malignant regions having an extent of binding of the anti-pMet antibody equal to i; (gb) defining by npi the number of non-malignant regions having an extent of binding of the anti-pMet antibody equal to i; (h) determining a first low cut-off value and a second high cut-off value; (i) calculating a prognostic value De according to the algebraic expression De = fe n - fe t wherein L 2 fe n = ∑(-l)tø?x ( .)log 2 (πpx,.) i=L and wherein for all i, LI < i < L2 wherein for all i, LI < i < L2 i=L \ i-L \ a De index being below said low cut-off value indicating a high probability that said individual has or will develop metastatic disease and poor survival expectancy; a De index being above said high predetermined cut-off value indicating a medium probability of said tested individual to have or develop a metastatic disease and a better survival expectancy and a De index being above said low cut-off value and below said high cut-off value indicating a low probability of said tested individual to have or develop a metastatic disease and a good survival expectancy. 25. A method according to Claim 24, wherein said disease is breast cancer. 26. The method of Claim 23 or Claim 25, wherein said individual has node negative breast cancer. 27. The method of Claim 23 or Claim 25, wherein said individual has node positive breast cancer. 28. A method according to any one of Claims 24-27, wherein said De index is pMetT calculated on the basis of the ratio , ( , . pMetN MetN 29. A method for determining in an individual having a malignant disease involving the expression of pMet the probability of having or developing a metastatic disease and for detennining said individual's survival expectancy comprising: (a) obtaining a sample from said individual, the sample comprising at least one malignant region and at least one nonnal region; (b) identifying in said sample one or more malignant regions and one or more non malignant regions; (c) partitioning said one or more regions of malignant tissue in said sample into a predetermined number N of sub-regions, each sub- region having the same predetermined area a; (d) partitioning said one or more non malignant regions of said sample into a predetermined number M of sub-regions, each sub-region having the same predetennined area a; (e) contacting said sample with anti-pMet mAbs of any of Claims 5-10, under conditions enabling binding of said anti-pMet mAbs to any pMet present in said N and M regions; (f) determining an extent of binding of the at least one of said anti-pMet antibodies in each of the N regions of malignant tissue in said sample and in each of said M regions of non-malignant tissue in said sample, the extent of binding of said anti-pMet antibodies in each pixel in each one of said N and M regions being an integer between a first integer LI and a second integer L2; (g) for each integer i between 11 and 12; (ga) defining tpi (in ROI) as the number of pixels in the malignant regions having an extent of binding of the anti-pMet antibody equal to i; (gb) defining by npi the number of non-malignant regions having an extent of binding of the anti-pMet antibody equal to i; (h) detennining a first low cut-off value and a second high cut-off value; (i) calculating a prognostic value De according to the algebraic expression DI = D„ = D t wherein and wherein for all i, LI < i < L2 npx i = — n Pι - , tpx i = — tpi - npt tpt wherein for all i, LI < i < L2 i=L \ =£, a DI index being below said low cut-off value indicating a high probability that said individual has or will develop metastatic disease and poor survival expectancy; a DI index being above said high predetermined cut-off value indicating a medium probability of said tested individual to have or develop a metastatic disease and a better survival expectancy and a DI index being above said low cut-off value and below said high cut-off value indicating a low probability of said tested individual to have or develop a metastatic disease and a good survival expectancy. 30. A method according to Claim 29, wherein said disease is breast cancer. 31. The method of Claim 30, wherein said individual has node negative breast cancer. 32. The method of Claim 30, wherein said individual has node positive breast cancer. 33. A method according to any one of Claims 29-32, wherein said DI index is pMetT calculated on the basis of the ratio , , ^ τ . pMetN MetN

Inventors: ALTSTOCK ROM, STIEN GIDEON Y, TSARFATY ILAN, ALTSTOCK, ROM, STIEN, GIDEON, Y., TSARFATY, ILAN
Applicants: ALTSTOCK ROM, FBIT LTD, STIEN GIDEON Y, TSARFATY ILAN, ALTSTOCK, ROM, FBIT LTD., STIEN, GIDEON, Y., TSARFATY, ILAN
Application year: 2000
Main classification: C07K16
IPC codes: A61K38, A61K39, A61P15, A61P35, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1, G01N33