Title: Prediction of responsiveness to _. anti-estrogen treatment in breast cancer

The invention relates to the field of medical diagnostics, more specifically to the field of cancer diagnostics, especially breast cancer.

Background to the invention Resistance to anti-estrogens is one of the major challenges in the treatment of breat cancer. For more than 25 years, the golden standard for the endocrine treatment of all stages of estrogen receptor-positive breast cancer has been tamoxifen (Jordan, 2003, Nat. Rev. Drug Discov., 2:205-213; Osborne, 1998, N. Eng. J. Med., 339:1609-1618). Approximately 30-50% of ER ⁺ breast tumors do not respond to Tamoxifen treatment (de novo resistance), and those that do respond often eventually progress to a state in which tumor cell proliferation is no longer inhibited, and may even be stimulated, by Tamoxifen treatment (acquired resistance). Also, response rates in patients with ER-α negative primary tumors are very low. The ability to identify tumors that are unlikely to respond to treatment with Tamoxifen or other SERMS, and the development of alternative therapies to treat resistant tumors, are therefore critically needed. Aromatase inhibitors may be more effective than Tamoxifen at treating primary breast cancer, and offer a very promising alternative. In addition, many Tam-resistant tumors retain sensitivity to steroidal antiestrogens such as ICI 182,780 (ICI) (brand names are Fulvestrant or

Faslodex), and this compound is approved as a second line therapy for patients who relapse while undergoing Tamoxifen treatment. However, a significant percentage of patients with advanced breast cancer will likely develop resistance to all endocrine therapies, and additional approaches to treat these patients are needed. Therefore additional biomarkers are needed to identify patients who will not respond and to select patients for various tailored treatments.

On a gene level such a detection has already been described in WO 2005/054510 and Becker, M. et al, 2005, MoI. Cancer Ther. 4:151-168. There, it has been shown that using microarray expression profiling a prediction for the responsiveness or resistance to anti-estrogen therapy is feasible. However, expression-based detection systems suffer from the disadvantages that, in general, not a single biomarker can be used for a proper classification, but that a so-called 'gene signature' needs to be defined consisting of several (up to several hundreds of genes) for which the expression values need to be determined, after which it should be assessed whether or not a patient is in possession of such a gene signature, based on an optimal cut-off in training and validation groups of patients. In WO 2005/054510 two nested signatures have been disclosed consisting of 81 and 44 genes respectively. Another disadvantage is that often classification of a new patient is only possible by pooling the expression data with a standard group of responders and non- responders and then performing statistical analyses (clustering analyses) to evaluate whether the sample of the new patient ends up with pool of responders or with the pool of non-responders.

Therefore, there is still need for more easy to handle biomarkers to identify patients who will and who will not respond to therapy and to select patients for various tailored treatments.

Summary of the invention

The invention comprises a method to predict responsiveness or resistance (non-responsiveness) to anti-estrogen therapy in breast cancer comprising a) isolating a tumor sample from a breast cancer patient; b) assaying said sample for the presence of one or more, preferably both of the proteins selected from the group A essentially consisting of: - nascent polypeptide associated complex alpha subunit (NACA, HSD48) (EMBL Q13765); - splice isoform 1 of epsin 4 (CLINTl, EPN4) (EMBL Q14677-1);

c) assaying said sample for the presence of one or more, preferably two or more, preferably three or more, more preferably four or more, more preferably all of the proteins selected from the group B essentially consisting of:

- adenylate kinase isoenzyme 4, mitochondrial (AK3L1, AK3, AK4) (EMBL P27144);

- annexin A8 (ANXA8, ANX8) (EMBL P13928);

- coronin-lB (COROlB) (EMBL Q9BR76);

- EPH receptor B2 (EPHB2, ERK, EPTH3) (EMBL P29323-3);

- NADH-cytochrome B5 reductase (CYB5R3, DIAl) (EMBL P00387-1); and - splice isoform 2 of basigin precursor (BSG, EMMPRIN, TCSF, CD147)

(EMBL P35613-2); and further d) either:

(i) calculating the ratio between the concentration of the protein(s) of group A with the concentration of the protein(s) of group B; (ii) calculating the normalized ratio of the concentration of the protein of group

A with the concentration of the protein of group A in a control sample from a non-responder;

(iii) calculating the normalized ratio of the concentration of the protein of group B with the concentration of the protein of group A in a control sample from a responder; or

(iv) performing hierarchical clustering and principal components analysis on the obtained data when pooled with corresponding data from groups of known responders and non-responders; and e) classifying the sample as responder or non-responder based upon the result of either (i)-(iv).

Preferably said detection is performed by an immunoassay, comprising one or more antibodies specifically recognizing said protein(s).

In another embodiment, the invention comprises a kit for performing a method as described above, comprising one or more antibodies capable of

binding to one or more proteins of groups A and B, additional reagents, and optionally instructions for performing the assay.

Further, the invention provides for the use of a protein, selected from the group consisting essentially of - nascent polypeptide associated complex alpha subunit (NACA, HSD48) (EMBL Q13765);

- splice isoform 1 of epsin 4 (CLINTl, EPN4) (EMBL Q14677-1);

- adenylate kinase isoenzyme 4, mitochondrial (AK3L1, AK3, AK4) (EMBL P27144); - annexin A8 (ANXA8, ANX8) (EMBL P13928);

- coronin-lB (COEOlB) (EMBL Q9BR76);

- EPH receptor B2 (EPHB2, ERK, EPTH3) (EMBL P29323-3);

- NADH-cytochrome B5 reductase (CYB5R3, DIAl) (EMBL P00387-1); and

- splice isoform 2 of basigin precursor (BSG, EMMPRIN, TCSF, CD 147) (EMBL P35613-2) in an assay for predicting the responsiveness of breast cancer patients to anti- estrogen therapy, more preferably tamoxifen therapy.

Legends to the figures

Fig. 1: Cluster analysis. Hierarchical clustering analysis was performed on the 8-protein profile, calculating z-scores for each protein across the different samples using average protein abundance levels. High protein abundance is shown in red and low abundance in blue. Fig. 2: Principal Components Analysis.

A) PCA on proteins. Proteins highly expressed in either OR (responsive, light blue) or PD (not responsive, dark blue) cluster together.

B) PCA on samples. OR (green) and PD (red) samples cluster together based on their 8-protein expression profile.

Detailed description

In the last couple of years many reports have appeared which have shown that gene expression arrays can be used to discriminate in the total group of patients for patients with good prognosis or poor prognosis, based on metastatic activity and survival rates (see e.g. Van de Vijver, M.J. et al., 2002, N. Eng. J. Med. 347:1999-2009; Bieche, I. et al., 2004, MoI. Cancer 3:37; Bogaerts, J. et al., Nature Clin. Pract. Oncol. 3:540-551; Lin, C-Y. et al., 2007, Breast Cancer Res. 9(2):R25; Frasor, J. et al., 2006, Cancer Res. 66:7334-7340; Hall, P. et al., 2006, BMC Med. 4(1): 16; Yu, K. et al., 2006, Clin. Cancer Res. 12:3288-3296). However, little or no reports have appeared on the identification of protein biomarkers in breast cancer. Of course, since long the relation between estrogen and breast cancer is known, which has led to the use of the estrogen receptor and proteins related to the effect of estrogen on the estrogen receptor as marker for breast tumor cells. Since the general article of Rui, Z. (proteomics, 2003, 3:433-439) only few protein biomarkers for breast cancer have been reported: p27 found by Porter, P. et al., 2006, J. Nat. Cancer. Inst. 98:1723-1731; and the estrogen-responsive finger protein (Efp) reported by Suzuki, T. et al., 2005, Clin. Cancer Res. 11:6148-6151. These two serum- based proteins would appear to be predictive of the prognosis of breast cancer. The group of Li (Li, J. et al., 2005, Clin. Cancer Res. 11:8312-8320; and Li, J. et al., 2005, Clin. Chem. 51:2229-2235) has been able to find differentially expressed proteins in nipple aspirates.

No report however, has been issued on proteins which would be able to discriminate between breast cancer patients that are resistant to anti- estrogen therapy and who would respond to such a therapy. As discussed in the background introduction, two groups have provided for such a distinction on gene expression arrays, but very surprisingly the present inventors have now found a group of 8 proteins, present in breast cancer tissue, that is able to discriminate between responders and non-responders. The surprising aspect of this is that these proteins are not encoded by the genes found by the groups

reporting gene expression array detection methods. This mere fact illustrates that an increase in the expression of a gene does not automatically lead to a corresponding increase in protein levels in tumor cells and vice versa. Apparently other mechanisms, such as excretion of the proteins out of the tumor cells, activity of proteases, turnover time of the proteins, etc. greatly determine the presence and/or level of the proteins in the cells. Further, an important difference between the protein profiling study, that underlies the present invention, and the gene expression profiling studies reported above, is that laser microdissected tumor cells were used for protein biomarker profiling. This means that no interfering signals from surrounding cells were measured in the analysis, in contrast to the gene expression study that was performed on whole tissue sections.

The group of 8 proteins that now has been found to be predictive for responsiveness to anti-estrogen, more specifically tamoxifen therapy is listed in Table 1 below. The amino acid sequences of the proteins of Table 1 are represented herein below.

Table 1.

Surprisingly, although some of the above proteins have been linked with cancer, none of the above proteins has ever been connected to resistance to tamoxifen (or any other resistance), nor is any connection with the estrogen pathway known.

As can be seen in Figure 2, six of the above mentioned proteins, identified as Group B (SEQ ID Nos:3-8) show up as discriminative for non- responders, while two proteins, denominated as group A (SEQ ID Nos: 1 and 2) are predominantly found in responders. Although the present experiments are not conclusive on the absolute levels of these proteins in the tumor, the signature of the 8 proteins as such can be used advantageously to classify a patient as responder or non-responder with respect to anti-estrogen, preferably tamoxifen treatment. Also, in comparison with pre-obtained samples from a

responder and a non-responder, or both, the relative abundance of the proteins can be established and also on basis of this relative level a classification can be obtained. Further, it is believed that also on the absolute level of the protein concentration in the tumor cell a classification can be made. Detection of the presence of any one or more of the proteins of Table

1 starts with obtaining a sample of tumor cells from the primary tumor of the patient. Taking a biopsy of the tumor is a normal procedure for assessing the presence and nature of a tumor in patients suspected of breast cancer. This biopsy can thus further be used to test for the responsiveness to anti-estrogen treatment according to the present invention. In taking the biopsy, or isolating the tumor cells from the biopsy tissue, it should be taken care that, at least for the assay of the present invention, sufficient tumor cells in relation to non- tumor cells are included. It is, however, submitted that even with an underrepresentation of tumor cells, it is believed that the level of one or more of the 8 proteins would be sufficient to perform the assay successfully.

The presence of a protein in the sample can be detected using various conventional methods, which will be well known to a person skilled in the art, such as mass spectrometry methods, e.g. MALDI-TOF, nanoLC-MALDI-TOF/TOF and/or MALDI-FTMS. One of the preferred methods for the assay of the invention is a quantitative immunoassay, which can be an enzyme-immunoassay (EIA), and enzyme-linked immunosorbent assay (ELISA), or any other assay using immune reactions. For performing any immunoassay, antibodies to the peptides should be generated.

Once the concentrations of the proteins in the sample have been established, there are several options to classify the sample as responder or non-responder. A first option is formed by pooling the data with data from other breast cancer patients of whom the responsiveness to anti-estrogen treatment is known and performing an analysis as basically described in the Examples. If then a plot is made similar to the plot shown in Fig. 2B it can be easily determined whether the new patient falls in the category of responders

or non-responders. For this option thus a pool of samples from patients with a known history of treatment responsiveness is needed. From these samples, together with one or more samples from patients who need to be classified, the concentration of at least one of the proteins of Group A of Table 1 and at least one of the proteins of Group B of Table 1 should be determined (in any way as described above) and a hierarchical clustering and principal components analysis should be performed on these data, it is submitted that these statistical analysis methods are readily known to a person skilled in the art.

A second option is to regard one or a pool of samples from one known group (responders or non-responders) as a control for a new sample to be tested. Then, using the same method, the concentration of at least one protein of Group A and at least one of the proteins of Group B should be determined in both samples. Using the values obtained in the control sample, the test values of the test samples are corrected. This basically means that - if the control sample would e.g. consist of responders, the determined level of proteins of

Group A, or both, in the test sample are multiplied with (or divided by) a factor to equalize the values obtained in the control sample. This same factor is then used to multiply or divide the data obtained for the at least one protein of Group B, which is then indicated as the normalized concentration. If then the normalized level of said at least one protein of Group B is about similar to the level of said at least one protein of Group B in the control sample, then the test sample can be classified as responder. If the normalized level is much larger than the at least one protein of Group B in the control sample, then the test sample should be classified as non-responder. A third possibility is to calculate the ration between the concentration of at least one of the proteins from Group A and at least one protein of Group B. Preferably, the ratio is calculated for more protein pairs selected from said groups of proteins of Table 1. Preferably, for control purposes also the concentration is measured of a protein which is known to be present at a fairly constant level in tumor cells, whether they origin from

responders or non-responders. Such a protein can for instance be a housekeeping protein such as RNA polymerase or an enzyme from the Krebs cycle. It is then also possible to calculate the ratio of the concentration of at least one of the proteins from Group A and at least one of the proteins of Group B with the concentration of the housekeeping protein. If these ratios are calculated then the ratio of said ratios is determined. If this ratio is below or above a certain predetermined value (based on calculating the same ratio in known responders and non-responders) a classification of the new sample can be established.

By the term "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. As used herein, "enzyme linked immuno- sandwich assay" is an antibody based assay used for the identification of protein and for measurements of protein levels in cell or tissue preparations. "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.

The instruction manual of the kit of the invention may, for example, be affixed to a container which contains the composition or be shipped together with a container which contains the composition. Alternatively, the instruction manual may be shipped separately from the container with the intention that the instructional material and the composition be used cooperatively by the recipient.

The "level" of a nucleic acid or polypeptide, as the term is used herein, refers to a measurable amount of a nucleic acid or polypeptide. By way of a non-limiting example, the level of a protein of Table 1 can be ascertained by measuring the concentration of the protein in weight per unit volume or, as described above, it can be related to the level of a reference protein. As reference protein preferably a protein is used which is available at a nearly constant level in the tumor or surrounding cells, such as is the case for various housekeeping enzymes. By way of another non-limiting example, the complete absence of a protein of Table 1 from an assay sample can be referred to as a "zero level" of said protein in that assay.

The term "peptide" typically refers to short polypeptides. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino- terminus; the right-hand end of a polypeptide sequence is the carboxyl -terminus.

Antibodies against any protein of Table 1 are made by methods known to the skilled artisan, and summarized as follows. The generation of polyclonal antibodies is accomplished by inoculating the desired animal with the antigen and isolating antibodies, which specifically bind the antigen there of.

In general, the proteins listed in Table 1, variants, and fragments thereof may be produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle. Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to

provide the recombinant protein. The precise host cell used is not critical to the invention. A polypeptide of the invention may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., S[pound]21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., supra). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).

A variety of expression systems exist for the production of the proteins or polypeptides of table 1. Expression vectors useful for producing such proteins include, without limitation, chromosomal, episomal, and virus- derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof. One particular bacterial expression system for polypeptide production is the E. coli pET expression system (Novagen, Inc., Madison, Wis). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 ENA polymerase in the host cell. This is typically achieved using host strains that express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those

described herein. Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system that is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S- transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX-2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa

Once the recombinant polypeptide of the invention is expressed, it is isolated, e.g., using affinity chromatography. In one example, an antibody (e.g., produced as described herein) raised against a protein of the invention may be attached to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods. Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980). Polypeptides of the invention, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford,

111.). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogues (described herein).

Also included in the invention are proteins, polypeptides, variants, or fragments thereof containing at least one alteration relative to the

sequences of the proteins of Table 1 provided below. Such alterations include certain mutations, deletions, insertions, or post-translational modifications. The invention further includes analogues of any naturally-occurring polypeptide of the invention. Analogues can differ from naturally-occurring polypeptides of the invention by amino acid sequence differences, by post- translational modifications, or by both. Analogues of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring amino acid sequence of the invention. The length of sequence comparison is at least 10, 13, 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably more than 35 amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e- ³ and e ¹⁰ ° indicating a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogues can also differ from the naturally-occurring polypeptides of the invention by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra).

In addition to full-length polypeptides, the invention also can be used with fragments of any one of the polypeptides of the invention. As used herein, the term "a fragment" means at least 5, 10, 13, or 15 amino acids. In other embodiments a fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments at least 60 to 80 or more contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or

may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events). Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY) and in Tuszynsld et al. (1988, Blood, 72:109-115). Human monoclonal antibodies may be prepared by the method described in U.S. patent publication 2003/0224490. Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.

Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4): 125-168) and the references cited therein. Further, the antibody of the invention may be "humanized" using the technology described in Wright et al., (supra) and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77(4): 755-759). To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting PNA is cloned into a suitable bacteriophage vector to

generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Bacteriophages, which encode the desired antibody, may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed. Thus, when bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophages that do not express the antibody will not bind to the cell. Such panning techniques are well known in the art and are described for example, in Wright et al., (supra). Processes such as those described above, have been developed for the production of human antibodies using M13 bacteriophage display (Burton et al., 1994, Adv. Immunol. 57: 191- 280). Essentially, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M 13 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human immunoglobulin rather than cells, which express human immunoglobulin.

The procedures just presented describe the generation of phages ( that encode the Fab portion of an antibody molecule. However, the invention should not be construed to be limited solely to the generation of phage encoding Fab antibodies. Rather, phagesthat encode single chain antibodies

(scFv/phage antibody libraries) are also included in the invention. Fab molecules comprise the entire Ig light chain, that is, they comprise both the variable and constant region of the light chain, but include only the variable region and first constant region domain (CHl) of the heavy chain. Single chain antibody molecules comprise a single chain of protein comprising the Ig Fv fragment. An Ig Fv fragment includes only the variable regions of the heavy and light chains of the antibody, having no constant region contained therein. Phage libraries comprising scFv DNA may be generated following the procedures described in Marks et al., 1991, J. MoI. Biol. 222:581-597. Panning of phage so generated for the isolation of a desired antibody is conducted in a manner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phage display libraries in which the heavy and light chain variable regions may be synthesized such that they include nearly all possible specificities (Barbas, 1995, Nature Medicine 1 :837-839; de Kruif et al., 1995, J. MoI. Biol. 248:97- 105). A preferred method of co-immunoprecipitation is described in the examples herein. See also Harlow et al., (1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Harlow et ah, (1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Various procedures known in the art may be used for the production of polyclonal antibodies to the proteins of Table 1. For the production of antibody, various host animals can be immunized by injection with any of said proteins, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, a protein of Table 1 or a fragment thereof can be conjugated to an immunogenic carrier, e.g. bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin,

pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Galmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a protein of Table 1, or a fragment, analog, or derivative thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to 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; Cote et al., 1983, Proc. Natl.

Acad. Sci. U.S.A., 80:2026-2030), and the EBY-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (WO 89/12690). In fact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, J. Bacteriol. 159:870; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for the protein together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention.

According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 5,476,786 and 5,132,405 to Huston; U.S. Pat. No. 4,946,778) can be adapted to produce specific single chain antibodies for the proteins of Table 1. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the proteins, or its derivative, or analog.

Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays (see Methods in Molecular Biology, Vol. 149; The ELISA

Guidebook by John R. Crowther, Humana Press, Totowa, New Jersey, 2001), immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labelled . Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For selection of an antibody specific to the protein of Table 1 from a particular species of animal, one can select on the basis of positive binding with said protein expressed by or isolated from cells of that species of animal.

The antibodies according to the invention also comprise antibody fragments obtained with the aid of phage libraries as described by Ridder et al., (1995, Biotechnology (NY), 13(3):255-260) or humanized antibodies as described by Reinmann et al. (1997, AIDS Res Hum Retroviruses, 13(ll):933-943) and

Leger et al., (1997, Hum Antibodies, 8(1):3-16).

The antibodies according to the invention are useful in immunological detection tests intended for the identification of the presence and/or of the quantity of antigens present in a sample. Therefore, an antibody according to the invention may comprise, in addition, a detectable marker that is isotopic or non-isotopic, for example fluorescent, or may be coupled to a molecule such as biotin, according to techniques well known to persons skilled in the art. In addition, a protein that specifically binds with a protein of Table

1 can be identified using, for example, a yeast two-hybrid assay. Yeast two hybrid assay methods are well-known in the art and can be performed using well documented techniques, for example those described in Bartel and Fields, (The Yeast Two-Hybrid System, Oxford University Press, Gary, N.C.). Therefore, once armed with the teachings provided herein, e.g., the full amino and derivable nucleic acid sequences of the protein(s) of Table 1, one skilled in the art can easily identify a protein that specifically binds with said protein(s). By "detectable amino acid sequence" or "detectable moiety" is meant a composition that when linked with the nucleic acid or protein molecule of interest renders the latter detectable, via any means, including spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens. "Microarray" means a collection of nucleic acid molecules or polypeptides from one or more organisms arranged on a solid support (for example, a chip, plate, or bead).

The diagnostic methods of the invention are used to assay the concentration of any protein of Table 1 in a biological sample relative to a reference (e.g., the level of said protein present in a corresponding control

tissue). In one embodiment, the level of a protein is detected using an antibody that specifically binds said protein. Methods for measuring an antibody- protein complex include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index. Optical methods include microscopy (both confocal and non- confocal), imaging methods and non-imaging methods. Methods for performing these assays are readily known in the art. Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay. These methods are also described in, e.g., Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991); and Harlow & Lane, supra. Immunoassays can be used to determine the quantity of a protein of Table 1 in a sample, where is the above described calculation methods are diagnostic of a patient being prone to anti-estrogen therapy.

The invention also provides kits for performing the diagnostic method of the invention in a biological sample obtained from a subject. In one embodiment, the kit detects an increase in the concentration of any protein of Table 1 relative to a reference level. In preferred embodiments, the kit includes an antibody that binds to a protein of Table 1, more specifically the kit comprises a set of antibodies, wherein each antibody is able to bind to one of the proteins of Table 1. More preferably said kit comprises a set of 8 antibodies, wherein each antibody is capable to bind to a different protein of Table 1. In another embodiment, the kit also comprises an antibody which is able to specifically bind to a housekeeping protein.

Optionally, the kit includes directions for monitoring the levels of the protein(s) of table 1 in a biological sample derived from a subject. In other embodiments, the kit comprises a sterile container, which contains the antibody or antibodies, or other detection regents; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other

suitable container form known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding nucleic acids. The instructions will generally include information about the use of diagnostic reagents described herein and their use in performing the diagnostic method of the invention. In other embodiments, the instructions include at least one of the following: description of the antibody or antibodies; methods for using the enclosed materials for the diagnostic method according to the invention; precautions; warnings; indications; clinical or research studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The diagnostic methods of the invention may also be used in microarray-based assays that provide for the high-throughput analysis of tissue samples. The proteins of Table 1 or fragments or variants thereof are useful as hybridizable array elements in such a microarray. The array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate. Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res. 28:e3.i-e3.vii, 2000), MacBeath et al., (Science 289:1760-1763, 2000), Zhu et al. (Nature Genet. 26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by reference.

Typically, protein microarrays feature a protein, or fragment thereof, bound to a solid support. Suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer- based films (e.g., polystyrene), beads, or glass slides. For some applications, the proteins of Table 1, or fragments or variants thereof, or,

alternatively, antibodies capable of binding said proteins, fragments or variants, are spotted on a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer). Preferably, such methods retain the biological activity or function of the protein bound to the substrate (e.g., antibody binding). The protein microarray is hybridized with a detectable probe. Such probes can be polypeptide (e.g., an antibody) or small molecules. Binding conditions (e.g., temperature, pH, protein concentration, and ionic strength) are optimized to promote specific interactions. Such conditions are known to the skilled artisan and are described, for example, in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual. 1998, New York: Cold

Spring Harbor Laboratories. After removal of non-specific probes, specifically bound probes are detected, for example, by fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric assay), direct immunoassay, radiometric assay, or any other suitable detectable method known to the skilled artisan. Optionally, detection of one or more of the proteins of Table 1 may be combined with the detection of other biomarkers, where the presence or level of the biomarker is correlated with the responsiveness to anti-estrogen treatment.

Contacting the biological sample with the (detecting) reagent (protein, peptide or antibody) is generally a matter of simply adding the composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with the antigen. Washing of the sample (i.e., tissue section, ELISA plate, dot blot or Western blot, microarray) is generally required to remove any non-specifically bound antibody species. The antigen-antibody complex (immunocomplex) is then detected using specific reagents.

When, for example, the antigen detecting reagent is an antibody (a specific antibody), this antibody may be (directly) labeled with a marker (fluorophore, chromophore, dye, enzyme, radioisotope, etc.) for enabling the detection of the complex. In other instances, it may be advantageous to use a

secondary binding ligand such as a secondary antibody or a biotin/avidin (streptavidin) (binding/ligand complex) arrangement, as is known in the art. Again, secondary antibodies may be labeled with a marker as described above or with an arrangement of biotin/avidin (i.e. avidin peroxidase) or biotin/streptavidin (i.e. streptavidin coupled with a reporter molecule (e .g., peroxidase)), which allow the detection of the immunocomplex. United States Patents concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Usually, the secondary antibody will be an antibody directed to the specific antibody (primary antibody) of a defined isotype and species such as, for example, an anti-mouse IgG.

On the other hand, the detecting reagent may also be a polypeptide having affinity for a protein of Table 1, which forms a complex (i.e., polypeptide-polypeptide complex complex). In that case, the polypeptide itself may be labeled using the markers described above, allowing direct detection. Again, the complex may be detected indirectly by adding a secondary (labeled) antibody or polypeptide.

Immunodetection methods, such as enzyme-linked immunosorbent assays (ELISA), Western blots, etc. have utility in the diagnostic method of the present invention. However, these methods also have applications to nonclinical samples, such as in the titering of antibody samples, in the selection of hybridomas, and the like.

Immunoassays that may be performed using reagents of the present invention includes, for example, enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays(RIA), which are known in the art lmmunohistochemical detection using tissue sections is also particularly useful However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like also may be used.

Examples of ELISA assays include the following, one or more antibodies binding to one or more proteins of Table 1 are immobilized onto a selected surface (i.e., a suitable substrate) exhibiting protein affinity, such as a well in a polystyrene microtiter plate (E.LISA plate) Then, a sample suspected of containing the polypeptide is added to the wells of the plate After binding and washing to remove non-specifically bound immunocomplexes, the bound protein may be detected Detection may be achieved by the addition of a second antibody specific for the target polypeptide, which is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA " Detection also may be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label (marker). Another example of ELISA assay is the following, the samples suspected of containing the protein of interest are immobilized onto the surface of a suitable substrate and then contacted with the antibodies capable of binding said protein. After binding and washing to remove non-specifically bound immunocomplexes, the bound protein is detected The immunocomplexes may be detected directly or indirectly as described herein. An additional example of an ELISA assay is the following, again, proteins are immobilized to a substrate, however, in that case the assay involves a competition step In this ELISA, a known amount of the protein of interest is adsorbed to the plate The amount of protein in an unknown sample is then determined by mixing the sample with a specific antibody before or during incubation with wells containing the immobilized protein. A detection reagent is added (e g , 2 ^nd antibody, labeled) to quantify the antibody that is able to bind to the immobilized polypeptide. The presence of the protein in the sample acts to reduce the amount of antibody available for binding to the protein contained in the well (immobilized polypeptide) and thus reduces the signal.

In order to get a correlation between the signal and the amount (concentration) of polypeptide in an unknown sample, a control sample may be

included during the assay. For example, known (predetermined) quantities of a polypeptide (usually in a substantially pure form) may be measured (detected) at the same time as the unknown sample. The signal obtained for the unknown sample is then compared with the signal obtained for the control. The intensity (level) of the signal is usually proportional to the amount of polypeptide (antibody bound to the polypeptide) in a sample. However, the amount of control polypeptide and antibodies required to generate a quantitative assay needs to be evaluated first.

In coating a plate with either an antigen (polypeptide) or antibody, one will generally incubate the wells of the plate with a solution of the protein or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a non-specific protein that is antigenically neutral with regard to the test samples. These include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of non-specific adsorption sites on the immobilizing surface and thus reduces the background caused by non-specific binding of antisera onto the surface. Conditions that may allow immunocomplex (antigen/antibody) formation include diluting the proteins and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of non-specific background. Suitable conditions involves that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 h, at temperatures preferably on the order of 20 ⁰ C to 27°C, or may be overnight at about 4°C or so.

Often, the detection of the immunocomplex is performed with a reagent that is linked to an enzyme. Detection usually requires the addition of the enzyme's substrate. Enzymes such as, for example, a phosphatase (e.g., alkaline phosphatase), a peroxidase, etc. when given an appropriate substrate will generate a reaction that may be quantified by measuring the intensity

(degree) of color (radioactivity, fluorescence, etc.) produced. The reaction is usually linear over a wide range of concentrations and may be quantified using a visible spectra spectrophotometer.

The present invention also relates to immunodetection kits and reagents for use with the immunodetection methods described above. As the polypeptide of the present invention may be employed to detect antibodies and the corresponding antibodies may be employed to detect the polypeptide, either or both of such components may be provided in the kit. The immunodetection kits may thus comprise, in suitable container means, one or more proteins selected from the proteins listed in Table 1, or one or more of a first antibody that binds to said one or more protein and/or an immunodetection reagent. The kit may comprise also a suitable matrix, to which the antibody or protein of choice may already be bound. Suitable matrices include an ELISA plate. The plate provided with the kit may already be coated with the antibody or protein of choice. The coated ELISA plate may also have been blocked using reagents described herein to prevent unspecific binding. Detection reagents may also be provided and may include, for example, a secondary antibody or a ligand, which may carry the label or marker and/or an enzyme substrate. Kits may further comprise an antibody or protein (usually of known titer or concentration) that may be used for control. Reagents may be provided, for example, lyophilized or in liquid form (of a defined concentration) and are provided in suitable containers (ensuring stability of reagents, safety etc.). It is to be understood herein, that if a "range", "group of substances" or particular characteristic (e.g., temperature, concentration, time and the like) is mentioned, the present invention relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges or sub-groups therein whatsoever. Thus, any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub- ranges or sub-

groups therein. Thus, for example, with respect to reaction time, a time of 1 minute or more is to be understood as specifically incorporating herein each and every individual time, as well as sub-range, above 1 minute, such as for example 1 minute, 3 to 15 minutes, 1 minute to 20 hours, 1 to 3 hours, 16 hours, 3 hours to 20 hours etc.; and similarly with respect to other parameters such as concentrations, temperature, etc...

Examples

To identify protein biomarkers indicative of tamoxifen-resistance in breast cancer, a comparative proteomics analysis was performed using nanoscale liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (nanoLC-FTICR MS). Two independently processed datasets (n=27 and n=28) of both OR (objective responsive to tamoxifen treatment) and PD (progressive disease despite tamoxifen treatment) tumors were subjected to laser capture microdissection (LCM), ~ 4000 tumor cells were collected and subsequently pooled into groups of seven. Tryptic digests were prepared and analyzed in triplicate; ~550 ng of peptides per analysis were separated on a 50 μm x 80 cm reversed phase nanoLC column prior to FTICR MS analysis. Peptide mass and elution time features were matched to information in previously generated accurate mass and time (AMT) tag reference databases to identify peptide sequences and proteins, and the MS peak intensities were used to determine relative peptide abundances. More than 20,000 unique peptides were identified that corresponded to a total of 2309 non-redundant proteins identified with two or more peptides. From this total, 1713 (74%) proteins that overlapped between the two datasets were used for further statistical analysis. The two datasets were separated into a 'training' and Validation' set and analyzed for differential protein abundance between OR and PD groups, using a univariate t-test from BRB array-tools software package, followed by a Wilcoxon rank-sum test. In both datasets, 100 differentially abundant proteins were identified (p<0.05), of which 8 were

present in both sets (p<0.015). These 8 proteins were subjected to hierarchical clustering and principal components analysis. Based upon this 8-protein profile, PD and OR samples clustered into two separate groups that correctly predicted therapy-response. The sensitivity and specificity of this protein profile will be determined in an independent validation set.

In summary, an 8-protein profile observed to predict tamoxifen-resistance in breast cancer was revealed by ultra-sensitive nanoLC-FTICE, technology for comprehensive proteome analyses of LCM cells. The 8 proteins are listed in Table 1 and their amino acid sequences are provided below.

Sequence listing

NACA (SEQ ID NO: 1) 1 mpgeatetvp ateqelpqpq aetgsgtesd sdesvpelee qdstqattqq aqlaaaaeid

61 eepvskakqs rsekkarkam sklglrqvtg vtrvtirksk nilfvitkpd vykspasdty 121 ivfgeakied lsqqaqlaaa ekfkvqgeav sniqentqtp tvqeeseeee vdetgvevkd 181 ielvmsqanv srakavralk nnsndivnai meltin CLINTl (SEQ ID NO:2)

1 mlnmwkvrel vdkatnvvmn yseieskvre atnddpwgps gqlmgeiaka tfmyeqfpel

61 mnmlwsrmlk dnkknwrrvy ksllllayli rngserwts arehiydlrs lenyhfvdeh.

121 gkdqginirq kvkelvefaq dddrlreerk kakknkdkyv gvssdsvggf ryserydpep

181 kskwdeewdk nksafpfsdk lgelsdkigs tiddtiskfr rkdredsper csdsdeekka 241 rrgrspkgef kdeeetvttk hihitqatet tttrhkrtan psktidlgaa ahytgdkasp 301 dqnasthtpq ssvktsvpss kssgdlvdlf dgtsqstggs adlfggfadf gsaaasgsfp 361 sqvtatsgng dfgdwsafnq apsgpvassg effgsasqpa velvsgsqsa lgpppaasns 421 sdlfdlmgss qatmtssqsm nfsmmstntv glglpmsrsq ntdmvqksvs ktlpstwsdp 481 svnisldnll pgmqpskpqq pslntmiqqq nmqqpmnvmt qsfgavnlss psnmlpvrpq 541 tnaliggpmp msmpnvmtgt mgmaplgntp mmnqsmmgmn mnigmsaagm gltgtmgmgm

601 pniamtsgtv qpkqdafanf anfsk

AK3L1 (SEQ ID NO:3) 1 maskllravi lgppgsgkgt vcqriaqnfg lqhlssghfl renikastev gemakqyiek

61 sllvpdhvit rlmmselenr rgqhwlldgf prtlgqaeal dkicevdlvi slnipfetlk

121 drlsrrwihp psgrvynldf npphvhgidd vtgeplvqqe ddkpeavaar lrqykdvakp

181 vielyksrgv lhqfsgtetn kiwpyvytlf snkitpiqsk eay ANXA8 (SEQ ID NO:4)

1 mawwkawieq egvtvksssh fnpdpdaetl ykamkgigtn eqaiidvltk rsntqrqqia

61 ksfkaqfgkd ltetlksels gkferlival myppyryeak elhdamkglg tkegviieil

121 asrtknqlre imkayeedyg ssleediqad tsgylerilv cllqgsrddv ssfvdpglal

181 qdaqdlyaag ekirgtdemk fitilctrsa thllrvfeey ekianksied siksethgsl 241 eeamltwkc tqnlhsyfae rlyyamkgag trdgtlirni vsrseidlnl ikchfkkmyg

301 ktlssmimed tsgdyknall slvgsdp

COROlB (SEQ ID NO:5)

1 msfrkvvrqs kfrhvfgqpv kndqcyedir vsrvtwdstf cavnpkflav iveasgggaf 61 lvlplsktgr idkayptvcg htgpvldidw cphndevias gsedctvmvw qipengltsp

121 ltepvvvleg htkrvgiiaw hptarnvlls agcdnvvliw nvgtaeelyr ldslhpdliy

181 nvswnhngsl fcsackdksv riidprrgtl vaerekaheg arpmraifla dgkvfttgfs

241 rmserqlalw dpenleepma Iqeldssnga llpfydpdts vvyvcgkgds siryfeitee

301 ppyihflntf tskepqrgmg smpkrglevs kceiarfykl herkcepivm tvprksdlfq 361 ddlypdtagp eaaleaeewv sgrdadpili slreayvpsk qrdlkisrrn vlsdsrpama

421 pgsshlgapa stttaadatp sgslaragea gkleevmqel ralralvkeq gdricrleeq

481 lgrinengda EPHB2 (SEQ ID NO:6)

1 malrrlgaal lllpllaave etlmdsttat aelgwmvhpp sgweevsgyd enmntirtyq

61 vcnvfessqn nwlrtkfirr rgahrihvem kfsvrdcssi psvpgscket fnlyyyeadf

121 dsatktfpnw menpwvkvdt iaadesfsqv dlggrvmkin tevrsfgpvs rsgfylafqd

181 yggcmsliav rvfyrkcpri iqngaifqet lsgaestslv aargsciana eevdvpikly

241 cngdgewlvp igrcmckagf eavengtvcr gcpsgtfkan qgdeacthcp insrttsega

301 tncvcrngyy radldpldmp cttipsapqa vissvnetsl mlewtpprds ggredlvyni 361 ickscgsgrg actrcgdnvq yaprqlglte priyisdlla htqytfeiqa vngvtdqspf

421 spqfasvnit tnqaapsavs irahqvsrtvd sitlswsqpd qpngvildye lqyyekelse

481 ynataikspt ntvtvqglka gaiyvfqvra rtvagygrys gkmyfqtrαte aeyqtsiqek

541 lpliigssaa glvfliavvv iaivcnrrgf eradseytdk lqhytsghmt pgmkiyidpf

601 tyedpneavr efakeidisc vkieqvigag efgevcsghl klpgkreifv aiktlksgyt 661 ekqrrdflse asimgqfdhp nvihlegvvt kstpvmiite fmengsldsf lrqndgqftv

721 iqlvgmlrgi aagmkyladm nyvhrdlaar nilvnsnlvc kvsdfglsrf leddtsdpty

781 tsalggkipi rwtapeaiqy rkftsasdvw sygivmwevm sygerpywdm tnqdvinaie

841 qdyrlpppmd cpsalhqlml dcwqkdrnhr pkfgqivntl dkmirnpnsl kamaplssgi

901 nlplldrtip dytsfntvde wleaikmgqy kesfanagft sfdwsqmmm edilrvgvtl 961 aghqkkilns iqvmraqmnq iqsvegqpla rrpratgrtk rcqprdvtkk tcnsndgkkk

1021 gmgkkktdpg rgreiqgiff kedshkesnd cscgg

CYB5R3 (SEQ ID NO:7)

1 mgaqlstlgh mvlfpvwfly sllmklfqrs tpaitlespd ikyplrlidr eiishdtrrf 61 rfalpspqhi lglpvgqhiy lsaridgnlv vrpytpissd ddkgfvdlvi kvyfkdthpk

121 fpaggkmsqy lesmqigdti efrgpsgllv yqgkgkfair pdkksnpiir tvksvgmiag

181 gtgitpmlqv iraimkdpdd htvchllfan qtekdillrp eleelrnkhs arfklwytld

241 rapeawdygq gfvneemird hlpppeeepl vlmcgpppmi qyaclpnldh vghptercfv

301 f

BSG (SEQ ID NO:8)

1 maaalfvllg fallgthgas gaagfvqapl sqqrwvggsv elhceavgsp vpeiqwwfeg

61 qgpndtcsql wdgarldrvh ihatyhqhaa stisidtlve edtgtyecra sndpdrnhlt

121 raprvkwvra qavvlvlepg tvfttvedlg skilltcsln dsatevtghr wlkggvvlke

181 dalpgqktef kvdsddqwge yscvflpepm gtaniqlhgp prvkavksse hinegetaml 241 vcksesvppv tdwawykitd aedkalmngs esrffvsssq grselhienl nmeadpgqyr 301 cngtsskgsd qaiitlrvrs hlaalwpflg ivaevlvlvt iifiyekrrk pedvldddda 361 gsaplkssgq hqndkgknvr qrnss