METHODS AND COMPOSITIONS FOR MODULATING TUMOR CELL ACTIVITY

Abstract

Antibodies which target clusterin, a protein involved in the epithelial-to-mesenchymal transition of carcinoma cells, are identified and characterized. The antibodies may be used to modulate tumour cell activity through binding the clusterin.

Description

PRIORITY CLAIM

[0001] This patent application is a divisional of U.S. Ser. No. 11/991,459 filed on Mar. 5, 2008, now U.S. Pat. No. 8,044,179 issued on Oct. 25, 2011 which is a national stage filing under 35 U.S.C. §371 of international application No. PCT/CA2006/001505 filed on Sep. 13, 2006 which claimed priority to U.S. provisional application No. 60/716,086 filed Sep. 13, 2005. The entire contents of each of these priority applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to antibodies, peptides and small molecules which bind clusterin, and their use in modulating tumor cell activity.

BACKGROUND OF THE INVENTION

[0003] Carcinomas, the most common human malignancy, arise from epithelial cells. Progression of epithelial cancers begins with the disruption of cell-cell contacts as well as the acquisition of a migratory (mesenchymal-like) phenotype. This phenomenon, which is called an epithelial-to-mesenchymal transition (EMT), is considered to be a crucial event in late stage tumor progression and metastasis.

[0004] The secreted protein TGF-β suppresses tumor growth initially largely due to its growth inhibitory action on tumor cells of epithelial origin, then at later stages promotes tumor cell progression and metastasis. One mechanism by which TGF-β can promote tumor progression is through the induction of an EMT.

[0005] Due to the dual role that TGF-β plays in carcinogenesis, direct inhibitors of TGF-β may be risky since, while they could benefit late stage tumors, they could also accelerate preneoplastic lesions. A better therapeutic may be one that inhibits the pro-oncogenic EMT-promoting action of TGF-β, while leaving the tumor suppressor growth-inhibitory action of TGF-β unaffected. To develop such an inhibitor it would be necessary to identify the point at which there is a bifurcation of the TGF-β signaling pathway such that the mediators in one branch of the pathway participate in the EMT response, but not the growth inhibitory response to TGF-β. Therapeutics that inhibit mediators that lie exclusively in the EMT-promoting branch of the TGF-β signaling pathway will reduce metastasis while having little or no effect on the acceleration of preneoplastic lesions.

[0006] No TGF-β signal pathway specific components have been generally identified that promote or mediate the EMT-promoting action of TGF-β, yet are not involved in the growth inhibitory action of TGF-β.

[0007] In contrast, an endogenous protein (the YY1 nuclear factor) has been identified that is able to interfere with (as opposed to promote) the protumorigenic EMT action of TGF-β, while leaving the tumor-suppressing action (growth inhibition) intact (Kurisaki et al., 2004).

[0008] Inhibitors that target TGF-β ligands, receptors and the Smad signaling proteins are known. Specifically, soluble receptor ectodomains, antibodies and other binding proteins are able to act as antagonists by interacting with TGF-β ligands and sequestering them away from cell surface receptors. Small molecules are available that inhibit the kinase activity of the Type I TGF-β receptor and endogenous inhibitors of the Smad signaling proteins are also known. Since all of these signaling pathway components are involved in both the pro- and anti-carcinogenic actions of TGF-β, these inhibitors that target them may benefit late stage tumors, however, they could also accelerate preneoplastic lesions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1: TGF-β induces an epithelial to mesenchymal transition (EMT) in JM01 cells.

[0010] (A) This transition is characterized by an elongated morphology, the relocalization of the markers E-cadherin (E-cad), β-catenin (β-Cat) and F-actin and the down-regulation of the marker Zona Occludens-1 (ZO-1). (B) This morphology change is accompanied by an increase in cell motility as shown in a wound healing assay in which the cells' ability to migrate in to a `scratch` area is monitored in the absence or presence of TGF-β. (C) A complementary black ink motility assay was also used to visualize and quantify the motility of individual JM01 cells in the absence or presence of TGF-β. The black ink which is coated on the plastic sticks to the migrating cells, thereby generating the white tracks. Both assays show that the presence of TGF-β increases the motility of the JM01 cells.

[0011] FIG. 2: Analysis of TGF-β-induced gene expression changes using microarray technology. (A) Extensive analysis of microarray data obtained from 7 time-points (0.5, 1, 2, 4, 6, 12, and 24 hrs) during the TGF-β induction of the JM01 cell EMT allowed for the identification of 328 genes that are modulated during the early (0.5, 1 hr), middle (2, 4, 6 hr) or late (12, 24 hr) stages of the transition. (B) Only 5 of these genes are affected over the entire time-course. (C) By comparing our gene list with data on the basal gene expression profiles of the NCI-60 cell line panel (some of these cell lines exhibit a mesenchymal phenotype), and with expression profiling data from clinical samples, we identified 15 genes from our list that are associated with a mesenchymal tumor cell phenotype and with clinical tumor progression.

[0012] FIG. 3: Validation of the TGF-β modulation of selected gene expression and protein levels. (A) Semi-quantitative PCR confirmed the TGF-β-induced clusterin up-regulation and caveolin-1 down-regulation thereby validating the microarray analysis (microarray data shown below PCR results). (B) Western blot analysis of whole cell lysates of JM01 cells treated for 24 hrs with TGF-β demonstrated that these transcriptional changes result in increased clusterin (p-clu=pre-clusterin; s-clu=secreted mature clusterin) and decreased caveolin-1 (Cav-1) protein levels. (C) Immunofluorescent microscopy of JM01 cells treated for 24 hrs with TGF-β further confirmed these changes in clusterin and caveolin-1 protein levels through the visualization of these proteins in the intact cell. Nuclei are stained blue, caveolin-1 and clusterin are stained green and the F-actin fibers are stained red.

[0013] FIG. 4: Identification of secreted clusterin as a mediator of the TGF-β induced EMT. (A) Immunofluorescent microscopy indicated that clusterin is localized to the secretory pathway in JM01 cells and Western blot analysis of conditioned media (CM) indicated that clusterin is secreted (s-clu). (B, C) JM01 cells were treated for 24 hr with TGF-β, or CM taken from TGF-β treated JM01 cells, in the absence or presence of a antibody raised against the C-terminus of the clusterin β chain (anti-clu). Using immunofluorescent microscopy of ZO-1 as a marker of the EMT it was shown that the clusterin antibody blocks the induction of the EMT by both TGF-β and the CM indicating that secreted clusterin is a necessary mediator in the TGF-β EMT pathway. Purified clusterin alone was also shown to promote the EMT indicating that clusterin is not only necessary, but is sufficient for EMT induction. (D) The induction of the EMT by clusterin alone was further confirmed by using FACS analysis of the epithelial marker E-cadherin to monitor the EMT.

[0014] FIG. 5: Clusterin acts as an EMT mediator in cell lines other than the JM01 cells. 4T1 tumor cells (breast) and DU145 tumor cells (prostate) were observed to secrete clusterin and exhibit a motile phenotype in the absence of TGF-β stimulation. Using the wound healing assay to monitor the motility of the 4T1 and DU145 cells, it was observed that a clusterin antibody (anti-clu) inhibits the motility of these cells indicating that clusterin is important for the maintenance of the TGF-β independent mesenchymal phenotype in these cells.

[0015] FIG. 6: Clusterin is a pivotal mediator in the pathway leading to TGF-β induction of EMT but not in the pathway leading to TGF-β growth inhibition. (A) Using the black ink motility assay to monitor the EMT of the JM01 cells, it was confirmed that a clusterin antibody blocks the TGF-β induced EMT and that clusterin alone promotes the EMT. (B) This result was further confirmed by quantifying the motility change as area cleared in the ink per cell. (C) In contrast, as monitored by the incorporation of tritiated thymidine, it was shown that the clusterin antibody does not block TGF-β induced growth inhibition and that clusterin alone does not promote growth inhibition, indicating that clusterin is not a mediator in TGF-β growth inhibitory pathways.

[0016] FIG. 7: Clusterin is an essential mediator in a TGF-β tumor promoting pathway but not in its tumor suppressing pathway. TGF-β induces secretion of clusterin and antibodies raised against the C-terminus of the clusterin β chain block the TGF-β1 induced EMT, but not the growth inhibitory response of the cells to TGF-β. These results indicate that clusterin is a necessary mediator in the TGF-β EMT pathway but do not address whether other TGF-β-induced mediators act in concert with clusterin to induce the EMT; that is, do not, address the question of whether clusterin alone mediates an EMT. The fact that purified clusterin in the absence of TGF-β also promotes an EMT indicates that clusterin is sufficient to induce this transition.

[0017] FIG. 8: Analysis of the neutralizing activity of anti-clusterin polyclonal antibodies produced at BR1. Sera collected from two rabbits (#9 and #10) immunized with a clusterin peptide (a.a. 421-437) were confirmed to contain antibodies that interact with the peptide using surface plasmon resonance (data not shown), and were tested for their ability to inhibit cell motility in a wound healing assay (1/25 dilution of rabbit serum). The mouse mammary epithelial cell line, 4T1 (top), secretes clusterin and is motile in the absence of TGF-β, whereas the JM01 cell line (bottom) requires stimulation with TGF-β to induce clusterin production and cell motility. The sera of both rabbit #9 and #10 inhibit motility, with #10 serum being more potent. As expected, the pre-immune sera of both rabbits does not affect motility. A commercially available clusterin antibody is shown as a positive control (anti-clu, Santa Cruz).

[0018] FIG. 9: Analysis of the activity of the anti-clusterin monoclonal antibodies produced at BR1. (A) Immunoprecipitations of recombinant human clusterin (500 ng) using either 50 or 100 ng of each of 12 BR1-produced monoclonal antibodies (commercial polyclonal (C18) and monoclonal (B5) antibodies were used as positive controls). Samples were analyzed on a 12% reducing SDS-PAGE. All antibodies were observed to interact with recombinant clusterin by immunoprecipitation. (B) Assessment of the ability of the 12 BR1-produced monoclonal antibodies to inhibit the TGF-b induced motility of JM01 cells using the black ink motility assay (commercial polyclonal (C18) and monoclonal (B5) antibodies were used as positive controls). The bar graph shows the relative values of the motility of the TGF-b treated BR1-JM01 cells in the presence of the various antibodies. Five BR1-produced monoclonal antibodies (21B12, 20E11, 16C11, 16B5 and 11E2) inhibit the TGF-b induced motility of the BR1-JM01 cells. Values are expressed as the clearance/cell/24 hr relative to that of the TGF-b treated (control) cells. The * illustrates the cut-off value that was used when assessing neutralizing ability. When using this cut-off value in the black ink motility assay, there was a good agreement with the evaluation of the neutralizing ability of the monoclonal antibodies when using the wound healing motility assay (data not shown).

[0019] FIG. 10: Two SPR-biosensor (Biacore) approaches to analysing the relationship between the epitopes of antibodies. (A) In the first approach, a rabbit anti-mouse Fc antibody (RAMFc) is covalently immobilized on the sensor chip and one monoclonal (termed Ab 1) is captured on the surface. After binding clusterin to Ab1, the second monoclonal antibody (termed Ab 2) is flowed over the surface. If the epitopes of the two antibodies are overlapping, then Ab2 will not be able to bind to Ab1-bound clusterin. If the two antibodies have unrelated epitopes, then Ab2 will be able to bind to Ab1-bound clusterin. (B) In the second approach, one monoclonal (termed Ab 1) is covalently immobilized on the sensor chip surface. Clusterin is then incubated with a second antibody (monoclonal or polyclonal, termed Ab2) in solution and the complex is then flowed over Ab1. If the epitopes of the two antibodies are overlapping, then Ab2-bound clusterin will not be able to bind to Ab1.

[0020] FIG. 11: Results of the analysis of the relationship of the epitopes of the 5 EMT neutralizing BR1-produced anti-clusterin monoclonals antibodies with each other, and with the peptide epitopes of the C18, pAb#10 and B5 antibodies. This table summarizes all the epitope mapping results obtained using the two SPR-biosensor (Biacore) approaches. A blue + indicates that Ab1 competed with Ab2 for binding to clusterin in the first Biacore approach (i.e. the ratio of RUs of Ab2 to RUs of bound clusterin was 0.1 or less). A red + or +/- indicates that Ab2 competed with Ab1 for binding to clusterin in the second Biacore approach (i.e. the binding of clusterin to Ab1 was inhibited between 30-100% for +, and between 10-30% for +/-, when preincubated with Ab2). It is evident that all of the five neutralizing monolconal antibodies (21B12, 20E11, 16C11, 16B5 and 11 E2) interact with the overlapping peptide epitopes of pAb#10, pAbC18 and mAb B5 since they all compete for each other, and for pAb#10, pAbC18 and mAb B5. *It should be noted that all of the negative results from the first approach (blue -) occurred when Ab 20E11 was used (either as Ab1 or Ab2) indicating that this Ab did not behave well in that experimental set up. Therefore, for A δ 20E11, conclusions are taken primarily from the second experimental approach.

[0021] FIG. 12: Isolation of the Ig variable region cDNAs. Flow diagram indication the steps for the isolation, sequencing, sequence analysis of the monoclonal variable regions.

[0022] FIG. 13: Amino acid sequences of monoclonal antibodies

[0023] FIG. 14: CDR1 and CDR2 alignment of clusterin Ig VH

SUMMARY OF THE INVENTION

[0024] A first object of the invention is to identify a method for inhibiting EMT in tumour cells without inhibiting the tumour-suppressing activity of TGF-β.

[0025] A further object of the invention is to identify molecules or compositions which may inhibit TGF-β-induced EMT in tumour cells without inhibiting the tumour-supressing activity of TGF-β.

[0026] A first aspect of the invention provides for an agent having a binding affinity for clusterin, wherein binding of the agent to clusterin inhibits epithelial-to-mesenchymal transition in carcinoma cells. In particular, the agent may bind to the β-subunit of clusterin, and more specifically, it may bind to the C-terminal portion of the clusterin β-subunit. The agent may, for example, be an antibody, including a monoclonal or polyclonal antibody.

[0027] A second aspect of the invention provides for a method for modulating the activity of carcinoma cells, comprising the steps of exposing the cells to an agent having a binding affinity for clusterin.

[0028] A further aspect of the invention provides for the use of an amino acid sequence in the generation of agents having a binding affinity for clusterin, wherein the sequence comprises SEQ ID NO.: 4 or a portion thereof. In particular, the sequence may comprise shorter portions of SEQ ID NO.: 4, including SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, and SEQ ID NO.: 5.

[0029] A further aspect of the invention provides for a vaccine comprising clusterin or a portion thereof which is involved in epithelial-to-mesenchymal transition in carcinoma cells, and a pharmaceutically suitable carrier. The portion of clusterin may comprise SEQ ID NO.: 4 or a portion thereof.

[0030] A further aspect of the invention provides for the use of an amino acid sequence in the preparation of a vaccine, wherein the sequence comprises SEQ ID NO.: 4 or a portion thereof. In particular, the sequence may comprise shorter portions of SEQ ID NO.: 4, including SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, and SEQ ID NO.: 5.

[0031] A further aspect of the invention provides for a nucleic acid sequence that encodes at least one of SEQ ID NO.: 1 through SEQ ID NO.: 30.

[0032] A further aspect of the invention provides for the use of an agent with a binding affinity for clusterin as a diagnostic tool, wherein binding of the agent to clusterin inhibits epithelial-to-mesenchymal transition in carcinoma cells.

DETAILED DESCRIPTION OF THE INVENTION

[0033] It is disclosed herein that clusterin is a therapeutic target whose inhibition blocks EMT without preventing TGF-β's anti-proliferative tumor suppressor action.

[0034] Clusterin was first identified as a protein possibly involved in EMT using transcriptome analysis, then was analyzed to identify potential binding sites within clusterin. Synthetic peptides were created accordingly, and antibody preparations directed against these peptides were produced or purchased. Additionally, twelve monoclonal antibodies were isolated using full-length recombinant clusterin as the antigen. Both the anti-peptide antibody preparations and the twelve monoclonal antibodies were confirmed to bind to recombinant clusterin. The anti-peptide polyclonal antibody preparations and five of the twelve monoclonal antibodies were shown to inhibit EMT. These five neutralizing monoclonal antibodies were shown to interact with the same peptide epitope as the anti-peptide antibodies.

[0035] Using semi-quantitative RT-PCR, Western blot and immunofluorescent microscopy analysis, it was confirmed that several of the EMT-associated transcriptional changes that were detected by microarray analysis were reflected in changes in message and protein abundance (clusterin and caveolin are shown in FIG. 3). Anti-peptide antibodies were used to demonstrate that clusterin is an essential EMT mediator that is not involved in TGF-β's growth inhibitory pathways (FIGS. 4-6). These results indicate that clusterin is an accessible therapeutic target whose inhibition blocks EMT without preventing TGF-β's anti-proliferative tumor suppressor action.

[0036] The epitope within clusterin that is important for the generation of EMT-inhibiting agents was elucidated using anti-peptide antibody preparations in neutralization assays. Two different commercial polyclonal antibody preparations raised against synthetic peptides corresponding to sections of the C-terminus of the clusterin β sub-unit were used. The first antibody (from RDI Research Diagnostics Inc.) was raised against the synthetic peptide corresponding to amino acids 421-437 of clusterin (VEVSRKNPKF METVAEK, SEQ ID NO 1) (termed RDI) and the second antibody (from Santa Cruz Biotechnology Inc.) was raised against the synthetic peptide corresponding to amino acids 432-443 of clusterin (ETVAEKALQ EYR, SEQ ID NO 2) (termed C-18). An anti-peptide monoclonal antibody against the same peptide (SEQ ID NO 2) was also purchased (termed B5). The overlap between these two epitopes is shown below. The ability of these antibody preparations to block EMT indicates the significance of the C-terminal portion of the clusterin β subunit in inducing EMT (FIG. 4-6, C-18 results shown; similar results obtained with RDI).

TABLE-US-00001 375 (SEQ ID NO.: 4) LTQGED QYYLRVTTVA SHTSDSDVPS GVTEVVVKLF DSDPITVTVP VEVSRKNPKF METVAEKALQ EYRKKHREE 449 Antibody 1 Antibody 2

Prediction of Putative Functional Subdomains in Clusterin Based on Structural Bioinformatics

[0037] Generally, clusterin is thought to be a protein that is only partially structured, containing molten globule fragments. Additionally, it has been classified as an intrinsically disordered protein. Clusterin is postulated to contain several independent classes of binding sites capable of interacting with numerous other binding partners.

[0038] The clusterin sequence was examined using bioinformatics programs, namely: [0039] PredictProtein (Rost, 1996). [0040] GenTHREADER (Jones, 1999). [0041] COILS (Lupas, 1996). [0042] PONDR (Li et al., 1999)

[0043] The C-terminal fragment of the β-subunit was identified as a putative binding region. The fragment (a.a. 375-449, SEQ ID NO.: 4), which starts after the second coiled-coil region, is likely unfolded but has some propensity for β-sheet formation.

[0044] A synthetic peptide was produced corresponding to a.a. 421-437 of clusterin in order to generate polyclonal antibody preparations at BR1 that are similar to the commercial antibody 1 preparation (RDI) (these new polyclonal preparations are termed pAb#9 and #10). Additionally, full-length human clusterin was expressed in 293 cells and purified in order to use as antigen to generate monoclonal antibodies against full-length human clusterin. Twelve monoclonal antibodies were raised against full-length clusterin and were demonstrated to interact with clusterin by ELISA. These twelve antibodies are named 6E12, 7B7, 21B12, 20G3, 20E11, 18F4, 16C11, 16B5, 11E2, 8F6, 7D6, 7C12.

[0045] The polyclonal antibody preparations raised against the a.a. 421-437 epitope (pAb#9 and #10) were confirmed to inhibit the EMT (FIG. 8).

[0046] All twelve monoclonal antibody preparations raised against full-length human clusterin were confirmed to interact with recombinant human clusterin as evidenced by their ability to immunoprecipitate clusterin (FIG. 9A). Five of the twelve monoclonals were shown to be able to neutralize the EMT promoting action of clusterin in the black ink cell motility assay (FIG. 9B) and the wound healing cell motility assay (not shown). The five monoclonal antibodies that neutralize are 11E2, 21B12, 20E11, 16C11, 16B5.

[0047] Two Surface Plasmon Resonance (SPR)-based biosensor epitope mapping assays (FIG. 10) were used to determine whether the five neutralizing monoclonal antibodies generated using full-length clusterin were interacting with the same clusterin peptide epitope as the anti-peptide antibody preparations.

[0048] The two approaches that were used are described below:

1) The monoclonal antibodies were individually captured on a CM5 sensor chip surface on which a Rabbit-anti-Mouse Fc antibody was covalently immobilized (when captured, the mAb is termed mAb1 in this experimental approach). Clusterin was then allowed to bind to mAb1. Then all five monoclonal antibodies were sequentially injected over mAb1-bound clusterin (the injected mAb is termed mAb2 in this experimental approach) in order to determine if both mAb1 and mAb2 are able to interact with clusterin simultaneously (FIG. 11). It was found that all of the five neutralizing mAbs (except 20E11 in some cases) competed with each other for binding to clusterin (when used both as mAb1 or as mAb2). Additionally, they were found to compete with the C18, pAb#10 and B5 anti-peptide antibodies, suggesting that the five neutralizing mAbs interact with the overlapping peptide epitopes of pAb#10, pAbC18 and mAb B5. It should be noted that, although Ab 20E11 appeared to have a distinct epitope in some cases (when used either as mAb1 or mAb2), this conclusion was not supported by the results of the second experimental approach. 2) The monoclonal antibodies were individually covalently immobilized on a CM5 sensor chip surface using amine coupling (when immobilized, the mAb is termed mAb1 in this experimental approach). To demonstrate competition for binding to clusterin, an Ab (termed Ab2 in this approach) was then incubated with clusterin prior to injection of the complex over the mAb1 surface (FIG. 11).

[0049] It was confirmed that all of the five neutralizing mAbs competed with each other for binding to clusterin, and with the C18, pAb#10 and B5 anti-peptide antibodies. This confirms that the five neutralizing mAbs interact with the overlapping peptide epitopes of pAb#10, pAbC18 and mAb B5.

[0050] The hypervariable complementary determining regions (CDRs) of all twelve monoclonal Abs were sequenced. Mammalian light- and heavy-chain Igs contain conserved regions adjacent to the CDRs and the use of appropriately designed oligonucleotide primer sets enabled the CDRs to be specifically amplified using PCR (FIG. 12). These products were then sequenced directly (SEQ ID NO 8-30; see FIG. 13).

[0051] By aligning the CDR sequences of four out of the five neutralizing monoclonal antibodies (11E2, 21B12, 20E11, 16C11), we were able to determine a consensus sequence for VH CDR1 and CDR2 of these anti-clusterin antibodies (see FIG. 14). The following consensus sequences were determined: CDR-1: G-Y-S/T-F-T-X-Y-X (SEQ ID NO.: 6) and CDR-2: I-N/D-P/T-Y/E-X-G-X-P/T (SEQ ID NO.: 7).

[0052] The antibodies or peptides that interact with the epitope of clusterin defined here may be applied as therapeutics, i.e. they may act as a therapeutic in their own right due to their intrinsic ability to neutralize the EMT promoting activity of clusterin. Additionally, these antibodies and peptides may be used as a therapeutic due to their ability to target toxins, suicide genes or other agents with anti-tumor activity to the vicinity of tumor cells through their interaction with secreted clusterin.

[0053] Small molecules that interact with the epitope of clusterin defined here may also act as therapeutics by blocking the EMT promoting activity of clusterin. These antibodies, peptides and small molecules that exert their therapeutic activity by interacting with this clusterin epitope may exhibit less toxicity or side-effects as compared to other agents that remove all activities of clusterin, i.e. antisense or RNAi agents, since, while the EMT activity of clusterin is neutralized when this epitope is blocked, the other activities of clusterin may remain intact.

[0054] Other applications of the antibodies and peptides that interact with the epitope of clusterin defined here may be as 1) non-imaging diagnostics, i,e, they may detect clusterin as a biomarker in accessible body fluids or in tissue/tumor samples for diagnostic and prognostic applications in cancer, and 2) imaging diagnostics, i.e. they may be used to target contrast agents to tumors for imaging in vivo due to their interaction with secreted clusterin.

[0055] Antibodies comprising the heavy and light sequences identified herein, antibodies comprising the CDRs (complementarity determining regions) identified herein (FIG. 13), and antibodies comprising the consensus sequences (FIG. 14) are expected to be useful for the above-mentioned purposes.

[0056] Clusterin itself, or the portions thereof which contain the epitope recognized by the antibodies and peptides discussed above, may be used as a vaccine. Preferably, the clusterin should be combined with a pharmaceutically suitable carrier. Clusterin or epitope-containing portions of clusterin may also be used in the generation of vaccines. Similarly, amino acid sequences having at least 90% identity with SEQ ID NO. 4 or the clusterin epitope identified herein will also be useful, since they are likely to have similar functionality to the specific sequences identified herein.

[0057] Cell culture, antibodies and reagents BR1-JM01 cells were isolated and characterized as described (Lenferink et al., Breast Cancer Res., 6, R514-30 (2004)). Cells were maintained at 37° C. in a humidified, 5% CO₂ atmosphere and cultured in DF/5% FBS (1:1 mixture of Ham's F12 and Dulbecco's modified Eagles Medium (DMEM) with 5% Fetal Bovine Serum (FBS) and antibiotics/antimicotics (both Wisent Inc.)).

[0058] Human recombinant TGF-β1 and pan-TGF-β neutralizing antibody 1D11 were reconstituted according to the manufacturer's instructions (R&D Systems). Purified human serum clusterin was kindly provided by Dr MR Wilson (Wilson and Easterbrook-Smith, 1992). Purified human recombinant clusterin was produced in HEK-293 cells (general expression system described in Durocher et al, 2002). Antibodies against the following proteins were purchased and used in the indicated v/v dilutions: E-cadherin (E-cad, anti-uvomorulin clone Decma-1; Sigma), Zona Occludens-1 (ZO-1; Chemicon), polyclonal antibodies raised against the C-terminus of the human clusterin β chain (cluβ; RDI and Santa Cruz), and Caveolin-1 (cav-1; Santa Cruz). Horseradish peroxidase (HRP) conjugated antibodies were obtained from Jackson ImmunoResearch Laboratories Inc and Alexa-488 labeled antibodies and Texas-red labeled phalloidin were purchased from Molecular Probes. All experiments were carried out with 75-80% confluent monolayers of BR1-JM01 cells in DF/5%. Where indicated, cells were treated for 24 hr or 48 hr with TGF-β1 or purified clusterin at a final concentration of 100 μM or 200 nM, respectively.

RNA Isolation and Labeling

[0059] Monolayers of BR1-JM01 cells were grown in the absence or presence of TGF-β1 for 30 min, 1, 2, 4, 6, 12 or 24 hr. PolyA+ mRNA was extracted (4×150 mm dishes per time point) using the FastTrack® 2.0 kit (Invitrogen) according to the manufacturer's instructions. RNA was isolated and labeled according to Schade et al., 2004.

Hybridization and Data Analysis

[0060] cDNA microarrays (15,264 sequence verified mouse ESTs; http://lgsun.grc.nia.nih.gov/cDNA/15k.html) were obtained from the University Health Network Microarray Center in Toronto (http://www.microarrays.ca/). Slides were hybridized with Cy3 or Cy5 labeled cDNA as described (Enjalbert et al., 2003), scanned using a ScanArray 5000 (Perkin Elmer v2.11) at a 10-micron resolution and 16-bit TIFF files were quantified using QuantArray software (Perkin Elmer, v3.0). Microarray data normalization and analysis was performed as described (Enjalbert et al., 2003).

Northern Blot and Semi-Quantitative RT-PCR (SQ-RT-PCR) Analysis

[0061] For SQ-RT-PCR, 3-5 μg of total RNA was amplified in a 20 μl first-strand RT-PCR reaction using 50 U SuperScript II (Invitrogen) according to the manufacturer's guidelines with modifications. Samples were preincubated (2 min, 42° C.) before adding SuperScript II and the RNaseOUT treatment was omitted. Samples were incubated (90 min, 42° C.) and then cooled on ice. Two μl of first-strand reaction was added to the PCR mix (2.5 U Taq polymerase (New England Biolabs), 10 μM forward/reversed primers) in a final volume of 50 μl, which was heated (2 min, 94° C.) prior to PCR amplification. Primers for the generation of the probes used for northern blot and SQ-RT-PCR are listed in Table 1.

Western Blot Analysis

[0062] BR1-JM01 cells grown in 35 mm dishes were treated with TGF-β1 (24 hr). Cells were lysed in hot 2% SDS. Fifty μg of total protein or 30 μl of conditioned medium was resolved by SDS-PAGE (10%) under reducing conditions. Proteins were transferred to nitrocellulose and membranes incubated with primary antibodies (cluβ, cav-1; 1/500) in TBS-T (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween 20 (v/v)) containing 5% non-fat milk (overnight, 4° C.). Membranes were washed with TBS-T, incubated with secondary HRP-conjugated antibody (1/20,000) in TBS-T+5% milk (1 hr), and washed with TBS-T. Immunoreactive bands were visualized using Enhanced Chemiluminescence (ECL; Perkin Elmer).

Immunofluorescence Microscopy

[0063] BR1-JM01 cells were seeded in glass chamber slides (Lab-Tek) and treated with purified clusterin or TGF-β1 preincubated (30 min) with or without cluβ antibody (8 μg/ml) or 1D11 (100 nM). Conditioned medium, obtained from non-treated and TGF-β1-treated BR1-JM01 cells (24 hr), was preincubated (30 min) with these antibodies prior to incubation with non-treated BR1-JM01 cells. After 24 hr of exposure, cells were fixed with 4% para-formaldehyde (10 min), rinsed twice (PBS), permeabilized (2 min, 0.2% Triton X-100 in PBS), rinsed again, and non-specific sites were blocked with 10% FBS in PBS (40 min). Para-formaldehyde fixed cells were then incubated (1 hr) with primary antibody (E-cad, 1/200; ZO-1, 1/100; cluβ, cav-1; 1/50) in PBS/10% FBS, were rinsed (4× in PBS) and finally were incubated with fluorescently conjugated secondary antibodies (Molecular Probes). Simultaneously, F-actin filaments were labeled with Texas-red labeled phalloidin (1/100) and nuclei were counterstained with 0.4 μg/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma). Slides were rinsed (PBS) and mounted using Prolong anti-fade (Molecular Probes). Fluorescent images were captured using a Princeton Instrument Coolsnap CCD digital camera mounted on Leitz Aristoplan microscope and analyzed using Eclipse (Empix Imaging Inc.) and Photoshop (Adobe) software.

Cell Proliferation Assays

[0064] BR1-JM01 cells (2.5×10⁴ cells/well) were seeded in 24-well plates. The next day the medium was replenished and purified clusterin, TGF-β1, or TGF-β1 pre-incubated for 30 min with 1D11 antibody (100 nM) or cluβ antibody (8 μg/ml), was added to the cells. After 24 hr, cells were pulse-labeled with 0.5 μCi/ml [³H]thymidine (Amersham), rinsed (PBS, 4° C.), trypsinized and [³H]thymidine incorporation was evaluated by liquid scintillation counting.

Cell Motility Assays

[0065] Cells (2×10⁴ cells/well) were seeded in ink-coated 12-well plates according to Al-Moustafa et al. (1999) in the absence or presence of TGF-β1, TGF-β1+cluβ antibody, or purified clusterin. Images were captured after 24 hr using a Nikon Coolpix 995 digital camera mounted on Leitz Aristoplan microscope and particle-free tracks were quantified using ImageJ freeware (http://rsb.info.nih.gov/ij/).

Black Ink Motility Assay

[0066] Cells (2×10⁴ cells/well) were seeded in ink-coated 12-well plates according to Al-Moustafa et al. (1999) in the absence or presence of TGF-β1, TGF-β1+cluβ antibody, or purified clusterin. Images were captured after 24 hr using a Nikon Coolpix 995 digital camera mounted on Leitz Aristoplan microscope and particle-free tracks were quantified using ImageJ freeware (http://rsb.info.nih.gov/ij/).

Wound Healing Motility Assay

[0067] Confluent cell monolayers (12-well plates) were "wounded" using a 2 μL pipet tip. The medium was then replenished, to remove cell debris, and the anti-clusterin mAbs were added (final concentration of 4 μg/mL) in the absence or presence of 100 μM TGF-β. Images of the wound were captured prior to and after 24 hr of incubation using a Nikon Coolpix 995 digital camera mounted on Leitz Aristoplan microscope.

Polyclonal Antibody Production

[0068] The peptide (a.a. 421-437 of the clusterin protein) was produced and purified at the University of Calgary (http://peplab.myweb.med.ucalgary.ca/). An extra cysteine was added to the C-terminus of the peptide to facilitate oriented coupling on the surface of the CM-5 sensor chips that were used for screening of the rabbit antisera by surface plasmon resonance (SPR, Biacore® 2000). The peptide was coupled to Keyhole Lympet Hemocyanin (KLH, Imject Mariculture KLH; Pierce) using either glutaraldehyde (Sigma) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCL (Pierce) and dialyzed against PBS (overnight at 4° C.). The peptide preparations that were conjugated by the two methods were mixed (1:1). Pre-immune serum was drawn from two female New Zealand white rabbits (10 ml), which were then injected with the KLH-coupled peptide preparation (1.25 ug peptide per leg/0.5 ml Freund's Incomplete Adjuvant or PBS). Animals were boosted (1.25 ug peptide per leg/0.5 ml Freund's Incomplete Adjuvant or PBS) every third week and serum was drawn (6 ml/kg) every 10 days after each boost until the antibody titer did not increase, at which point the animals were euthanized and exsanguinated.

[0069] Sera were tested for antibody activity using SPR. For this, the peptide was coupled to a CM-5 sensor chip (Biacore Inc.) using the Thiol coupling method (as described by the manufacturer) and dilutions (1/50) of the pre-immune sera, the antibody-containing sera and the commercially available anti-clusterin antibody (Santa Cruz) were run over the peptide surface.

Monoclonal Antibody Production

[0070] Four BALB/c mice were injected subcutaneously (s.c.) and intra-peritoneally (i.p.) with 35 μg of purified human clusterin emulsified in TiterMax adjuvant (Pierce). Animals were re-injected i.p. three weeks later and the serum titer was assessed 10 days later. Ten weeks later, responsive mice was boosted by i.p. injections (50 μg purified clusterin) and sacrificed three days later. Spleen cells harvested, fused with NSO myeloma cells and immediately plated (5×10⁴ cells/well in 96-well microplates; Costar) in Iscove's medium supplemented with 20% FBS, 100 μM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine (HAT medium), murine IL-6 (1 ng/ml), penicillin (50 U/ml) and streptomycin (50 μg/ml). Supernatants (10-20 days post-fusion) were tested for anti-clusterin activity on immobilized purified clusterin by Enzyme-Linked Immunosorbent Assay (ELISA). Antibody producing cells were cloned and retested twice for anti-clusterin activity. Thirteen anti-clusterin antibody producing clones were generated of which frozen stocks were prepared and a large-scale antibody production was initiated.

SPR-Based Biosensor (Biacore) Epitope Mapping

Approach 1:

[0071] Running buffer: [0072] HBS (20 mM Hepes (pH7.4), 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20) [0073] All experiments were run at 5 μL/min [0074] Standard amine coupling of the anti mouse Fc immunoglobulin: [0075] Inject 35 μL of a mixture of 0.05M NHS and 0.2M EDC [0076] Inject antibodies diluted in 10 mM NaAc pH5.0 at concentration of 30 μg/mL until an appropriate amount in captured [0077] Inject 35 μL 1M ethanolamine-HCL pH8.5 [0078] Epitope mapping: [0079] Inject 25 μL of mAb1 at a concentration of 25 or 50 μg/mL. [0080] Inject 25 μL of a mixture of IgG1, IgG2a, IgG2b and IgG3 each one at a concentration of 25 μg/mL. [0081] Inject 25 μL of human recombinant clusterin at a concentration of 30 μg/mL. [0082] Inject 25 μL of mAb2 at a concentration of 25 or 50 μg/mL. [0083] Control: [0084] For each pair of antibodies, the non-specific binding of mab2 was determined by repeating all injections described in the epitope mapping section but injecting running buffer instead of clusterin. [0085] The response (RU) obtained 20 sec after the end of the mab2 injection in the control was subtracted from the response obtained in the presence of clusterin. [0086] Regeneration of the surface: [0087] At the end of each cycle, inject 10 μL of 20 mM glycine pH1.7 followed with 10 μL of 100 mM HCl.

Approach 2:

[0087] [0088] Running buffer: [0089] HBS (20 mM Hepes (pH7.4), 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20) [0090] Standard amine coupling of the antibodies: [0091] Inject 35 μL of a mixture of 0.05M NHS and 0.2M EDC [0092] Inject antibodies diluted in 10 mM NaAc (pH4.5 or 5.0) at concentration raging from 20 to 80 μg/mL until a appropriate amount in captured [0093] Inject 35 μL 1M ethanolamine-HCl pH8.5 [0094] Preparation of control surface [0095] Inject 35 μL of a mixture of 0.05M NHS and 0.2M EDC [0096] Inject 35 μL 1M ethanolamine-HCl pH8.5 [0097] Competition [0098] Mix human recombinant clusterin at 50 nM with 250 nM or 500 nM antibodies in PBS (without Mg++ and Ca++) [0099] Prepare a tube with antibody alone [0100] Inject at a flow of 5 μL/min, 25 μL of clusterin alone, antibody alone or clusterin preincubated with antibodies over the antibody and the control surfaces. [0101] Subtract the response obtained for the antibody alone solution from the response obtained for clusterin preincubated with the same antibody. [0102] Calculate the % binding inhibition by dividing the response obtained for the clusterin preincubated with antibody by the response obtained for clusterin alone. [0103] Regeneration solution [0104] At the end of each cycle, inject 10 μL of 10 mM HCl at a flow rate of 20 μL/min

Immunoprecipitation

[0105] 50 or 100 ng of the various monoclonal antibodies or the polyclonal antibody preparation (C18) was incubated with 204 of protein G slush (1:1 in PBS) overnight at 4° C. Then 500 ng of human recombinant clusterin was added and the mixture was incubated for another 2 hr at 4° C. Immunocomplexes were washed 3 times with 1 mL of buffer (150 nM NaCl, 50 mM Tris pH 8.0, 0.55% NP-40, 50 mM Na fluoride) and 20 μL of reducing sample buffer was added. Samples were boiled for 5 min prior to loading on a 12% SDS-PAGE. Separated proteins were then transferred to nitrocellulose and membranes were probed with anti-clusterin antibodies as described.

Sequencing of the Monoclonal Antibody Variable Region

[0106] Total RNA was isolated from the 12 hybridomas and first strand cDNA was prepared with reverse transcriptase and the Ig-3 constant region primer followed by amplification with the appropriate Ig-5' primer. These primer sets used in conjunction with KOD Hot Start DNA Polymerase specifically amplify the variable regions of light- and heavy-chain cDNAs. PCR products can be directly cloned with Novagen's pSTBlue-1 Perfectly Blunt® Cloning Kit or treated with the Single dA® Tailing Kit and cloned into the pSTBlue-1 AccepTor® Vector. For details see FIG. 13.

TABLE-US-00002 TABLE 1 Primer sets used for the validation of some of the 328 TGF-β modulated genes in the BRI-JM01 cells. Gene Gene Bank # Reverse Forward size(bp) Eefla1 AW556381 CTGGCTTCACTGCTCAGGT TGGCCAATTGAGACAAACAG 457 Clusterin AU041878 TGGTGAAAGCTGTTTGACTCTG AAGGCGGCTTTTATTGGATT 355 Integrin α6 AW556992 ATGTGCCATTGTTGCTTTGA CAAGCGATGAGCACTTTTGT 517 Caveolin-1 AU016590 GTGCAGGAAGGAGAGAATGG GCACACCAAGGAGATTGACC 247 Ptpn13 AW548343 CCTGCAATGGTTCTTGGTTT GGGAAAATCGATGTTGGAGA 300 14-3-3σ AA410123 GGGCTGTTGGCTATCTCGTA AGAGACCGAGCTCAGAGGTG 297

[0107] Inclusion of a reference is neither an admission nor a suggestion that it is relevant to the patentability of anything disclosed herein [0108] Bailey et al., Biochemistry. 2001; 40:11828-40 [0109] Dunker et al., J Mol Graph Model. 2001; 19 (1):26-59 [0110] Li et al., Genome Inform. Ser. Workshop Genome Inform. 1999; 10: 30- [0111] Jones, J. Mol. Biol. 1999; 287: 797-815 [0112] Lupas, Meth. in Enzym. 1996; 266: 513-525 [0113] Rost, Meth. in Enzym. 1996; 266: 525-539 [0114] Singh et al., Curr Opin Drug Discov Devel. 2004: 437-445 [0115] Al-Moustafa et al., Biotechniques. 1999: 60-62 [0116] Durocher et at Nucleic Acids Res 2002: E9 [0117] Enjalbert et al., Mol Biol Cell. 2003: 1460-1467 [0118] Schade et al., Mol Blot Cell 2004: 5492-5502 [0119] Wilson and Easterbrook-Smith, Biochim Biophys Acta 1992: 319-326

Sequence CWU 1

203117PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 1Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu1 5 10 15Lys212PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 2Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg1 5 1036PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 3Glu Thr Val Ala Glu Lys1 5475PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 4Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala1 5 10 15Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val 20 25 30Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu 35 40 45Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala 50 55 60Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu65 70 75523PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 5Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu1 5 10 15Lys Ala Leu Gln Glu Tyr Arg 2068PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 6Gly Tyr Xaa Phe Thr Xaa Tyr Xaa1 578PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 7Ile Xaa Xaa Xaa Xaa Gly Xaa Xaa1 58106PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 8Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu65 70 75 80Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 1059107PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile 35 40 45His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10510111PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 10Asp Ile Val Leu Thr Leu Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Asn Ser Ser 20 25 30Asn Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95Glu Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 11011113PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 11Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ile Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110Lys12112PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 12Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Phe Lys 100 105 11013112PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 13Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Pro Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 11014112PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 14Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 11015113PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 15Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly1 5 10 15Gln Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 85 90 95His Tyr Asn Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110Lys16112PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 16Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asp Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 11017112PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 17Asp Val Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 11018107PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 18Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10 15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro65 70 75 80Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10519108PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 19Glu Ile Val Leu Thr Gln Ser Pro Thr Thr Met Thr Ala Ser Pro Gly1 5 10 15Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn 20 25 30Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Phe Ser Pro Lys Leu Leu 35 40 45Ile Tyr Arg Thr Ser Asn Leu Pro Ser Gly Val Pro Pro Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu65 70 75 80Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser Leu Pro 85 90 95Arg Thr Phe Gly Ala Gly Thr Lys Leu Ala Leu Lys 100 10520120PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 20Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Asn Met Asn Trp Val Lys Gln Asn Asn Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Thr Pro Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Leu Asn Ser Leu Leu Arg Leu Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Ser Val Thr Val Ser Ser 115 12021118PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 21Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Thr Gly Ser Ser Gly Tyr Phe Asp Cys Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val Ser Ser 115 22118PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 22Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Asp Gly Phe Leu Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val Ser Ser 11523117PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 23Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Arg Leu Ser Cys Thr Thr Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala Tyr Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Tyr Asp Thr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110Val Thr Val Ser Ser 11524118PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 24Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Gly Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Asn Met Tyr Trp Val Lys Gln Ser His Arg Lys Ser Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Ser 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr65 70 75 80Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Gly Ile Tyr Tyr Cys 85 90 95Ala Arg Gly Ala Tyr Gly Ser Ser Tyr Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Ala Val Ser Ala 11525122PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Glu Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65

70 75 80Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Thr Arg Ile Tyr Tyr Asp Tyr Gly Ser Trp Asp Gly Phe Ala Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 12026120PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 26Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Asp Tyr Ser Phe Thr Thr Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ser Arg Asp Gly Asn Tyr Arg Tyr Tyr Thr Leu Asp Phe Trp Gly Gln 100 105 110Gly Thr Ser Val Thr Val Ser Ser 115 12027119PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 27Thr Cys Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ser Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Thr Ile Gly Ser Tyr Thr Asp Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Cys Cys 85 90 95Thr Arg Glu Asp Tyr Arg Tyr Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ala 11528120PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 28Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Asp Tyr Ser Phe Thr Thr Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ser Arg Asp Gly Asn Tyr Arg Ser Tyr Thr Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Ser Val Thr Val Ser Ser 115 12029115PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 29Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Phe Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Asn Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Ile 35 40 45Ala Glu Ile Ser Ser Gly Gly Ser Asp Thr Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Leu65 70 75 80Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95Arg Asp Gly Asn Trp Asp Gly Gly Ser Leu Thr Thr Gly Ala Lys Ala 100 105 110Pro Leu Ser 11530118PRTArtificial SequenceDescription of Artificial Sequence Synthetic protein construct 30Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Asp Tyr 20 25 30Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Val Asp Asp Phe 50 55 60Lys Arg Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Ala Ala Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Thr Arg Asp Gly Ser Ser Thr Trp Phe Ser Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala 1153119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 31ctggcttcac tgctcaggt 193220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 32tggccaattg agacaaacag 203322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 33tggtgaaagc tgtttgactc tg 223420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 34aaggcggctt ttattggatt 203520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 35atgtgccatt gttgctttga 203620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 36caagcgatga gcacttttgt 203720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 37gtgcaggaag gagagaatgg 203820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 38gcacaccaag gagattgacc 203920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 39cctgcaatgg ttcttggttt 204020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 40gggaaaatcg atgttggaga 204120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 41gggctgttgg ctatctcgta 204220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 42agagaccgag ctcagaggtg 20438PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 43Gly Tyr Ser Phe Thr Gly Tyr Asn1 5448PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 44Gly Tyr Ser Phe Thr Gly Tyr Asn1 5458PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 45Gly Tyr Thr Phe Thr Asn Tyr Gly1 5468PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 46Gly Tyr Thr Phe Thr Asp Tyr Ser1 5478PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 47Ile Asp Pro Tyr Asn Gly Asp Thr1 5488PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 48Ile Asp Pro Tyr Tyr Gly Thr Pro1 5498PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 49Ile Asn Thr Tyr Thr Gly Glu Pro1 5508PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 50Ile Asn Thr Glu Thr Gly Glu Pro1 55111PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 51Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser1 5 105211PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 52Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala1 5 105326PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 53Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Ser Ala Ser 20 255426PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 54Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Gly Lys Val Thr Ile Thr Cys Lys Ala Ser 20 255526PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 55Asp Ile Val Leu Thr Leu Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser 20 25 5626PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 56Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Lys Ser Ser 20 255726PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 57Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Pro Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 20 255826PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 58Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 20 255926PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 59Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly1 5 10 15Gln Arg Val Thr Met Ser Cys Lys Ser Ser 20 256026PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 60Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 20 25615PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 61Ser Ser Val Ser Tyr1 5626PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 62Gln Asp Ile Asn Lys Tyr1 56310PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 63Gln Ser Val Asn Ser Ser Asn Tyr Ser Tyr1 5 106412PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 64Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr1 5 106512PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 65Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr1 5 106611PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 66Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr1 5 106711PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 67Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr1 5 106812PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 68Gln Ser Leu Leu Asn Ser Asn Asn Gln Lys Asn Tyr1 5 106911PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 69Gln Ser Leu Val His Ser Asn Gly Asp Thr Tyr1 5 107026PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 70Asp Val Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 20 257126PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 71Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10 15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser 20 25 7226PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 72Glu Ile Val Leu Thr Gln Ser Pro Thr Thr Met Thr Ala Ser Pro Gly1 5 10 15Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser 20 257311PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 73Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr1 5 10746PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 74Gln Ser Ile Ser Asp Tyr1 5757PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 75Ser Ser Ile Ser Ser Asn Phe1 57625PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 76Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser 20 257725PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 77Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser 20 257825PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 78Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser 20 257925PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 79Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Arg Leu Ser Cys Thr Thr Ser 20 258025PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 80Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Gly Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser 20 258125PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 81Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser 20 258225PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 82Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser 20 258325PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 83Thr Cys Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser 20 258425PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 84Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser 20 25858PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 85Gly Tyr Ser Phe Thr Gly Tyr Asn1 5868PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 86Gly Tyr Thr Phe Thr Asp Tyr Ser1 5878PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 87Gly Tyr Thr Phe Thr Asn Tyr Gly1 5888PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 88Gly Phe Asn Ile Lys Asp Ile Tyr1 5898PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 89Gly Tyr Ser Phe Thr Gly Tyr Asn1 5908PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 90Gly Phe Thr Phe Ser Ser Tyr Ala1 5918PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 91Asp Tyr Ser Phe Thr Thr Tyr Trp1 5928PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 92Gly Phe Thr Phe Ser Ser Tyr Ser1 5938PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 93Asp Tyr Ser Phe Thr Thr Tyr Trp1 59425PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 94Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Phe Ser Cys Ala Ala Ser 20 259525PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 95Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser 20 25968PRTArtificial

SequenceDescription of Artificial Sequence Synthetic peptide 96Gly Phe Thr Phe Ile Asn Tyr Ala1 5978PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 97Gly Tyr Thr Leu Thr Asp Tyr Ser1 59817PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 98Met His Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile1 5 10 15Tyr9917PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 99Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile1 5 10 15His10017PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 100Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile1 5 10 15Lys10117PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 101Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile1 5 10 15Tyr10217PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 102Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile1 5 10 15Tyr10317PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 103Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile1 5 10 15Tyr10417PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 104Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile1 5 10 15Tyr10517PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 105Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Val1 5 10 15Tyr10617PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 106Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile1 5 10 15Tyr10717PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 107Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile1 5 10 15Tyr10817PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 108Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile1 5 10 15Lys10917PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 109Leu His Trp Tyr Gln Gln Lys Pro Gly Phe Ser Pro Lys Leu Leu Ile1 5 10 15Tyr11017PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 110Met Asn Trp Val Lys Gln Asn Asn Gly Lys Ser Leu Glu Trp Ile Gly1 5 10 15Asn11117PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 111Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly1 5 10 15Trp11217PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 112Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly1 5 10 15Trp11317PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 113Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly1 5 10 15Arg11417PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 114Met Tyr Trp Val Lys Gln Ser His Arg Lys Ser Leu Glu Trp Ile Gly1 5 10 15Tyr11517PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 115Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val Ala1 5 10 15Glu11617PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 116Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly1 5 10 15Met11717PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 117Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala1 5 10 15Thr11817PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 118Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly1 5 10 15Met1198PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 119Ile Asp Pro Tyr Tyr Gly Thr Pro1 51208PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 120Ile Asn Thr Glu Thr Gly Glu Pro1 51218PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 121Ile Asn Thr Tyr Thr Gly Glu Pro1 51228PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 122Ile Asp Pro Ala Tyr Gly Asn Thr1 51238PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 123Ile Asp Pro Tyr Asn Gly Asp Thr1 51248PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 124Ile Ser Ser Gly Gly Thr Tyr Thr1 51258PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 125Ile Asp Pro Ser Asp Ser Glu Thr1 51268PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 126Ile Ser Thr Ile Gly Ser Tyr Thr1 51278PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 127Ile Asp Pro Ser Asp Ser Glu Thr1 512817PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 128Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Ile Ala1 5 10 15Glu12917PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 129Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly1 5 10 15Trp1308PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 130Ile Ser Ser Gly Gly Ser Asp Thr1 51318PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 131Ile Asn Thr Glu Thr Gly Glu Pro1 513236PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 132Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Val Ala 20 25 30Thr Tyr Tyr Cys 3513336PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 133Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala 20 25 30Thr Tyr Tyr Cys 3513436PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 134Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Thr His Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Thr Ala 20 25 30Thr Tyr Tyr Cys 3513536PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 135Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly1 5 10 15Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala 20 25 30Val Tyr Tyr Cys 3513636PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 136Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly1 5 10 15Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala 20 25 30Val Tyr Tyr Cys 3513736PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 137Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 20 25 30Val Tyr Tyr Cys 3513836PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 138Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 20 25 30Val Tyr Phe Cys 3513936PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 139Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly1 5 10 15Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala 20 25 30Asp Tyr Phe Cys 3514036PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 140Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 20 25 30Val Tyr Phe Cys 351419PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 141Phe Gln Gly Ser Gly Tyr Pro Phe Thr1 51429PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 142Leu Gln Tyr Asp Asn Leu Leu Arg Thr1 51439PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 143Gln His Ser Trp Glu Ile Pro Trp Thr1 51449PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 144Gln Gln Tyr Tyr Ile Tyr Pro Arg Thr1 51458PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 145Lys Gln Ser Tyr Asn Leu Trp Thr1 51469PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 146Phe Gln Gly Ser His Val Pro Tyr Thr1 51479PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 147Ser Gln Ser Thr His Ile Pro Arg Thr1 51489PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 148Gln Gln His Tyr Asn Thr Pro Leu Thr1 51499PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 149Ser Gln Ser Thr His Val Pro Arg Thr1 515036PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 150Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Thr Asp Phe Thr Leu Asn Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 20 25 30Val Tyr Phe Cys 3515136PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 151Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly 20 25 30Val Tyr Tyr Cys 3515236PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 152Asn Leu Pro Ser Gly Val Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu Ala Glu Asp Val Ala 20 25 30Thr Tyr Tyr Cys 351539PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 153Ser Gln Ser Thr His Val Pro Arg Thr1 51549PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 154Gln Asn Gly His Ser Phe Pro Tyr Thr1 51559PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 155Gln Gln Gly Ser Ser Leu Pro Arg Thr1 515638PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 156Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys1 5 10 15Ser Ser Ser Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp 20 25 30Ser Ala Val Tyr Tyr Cys 3515738PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 157Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr1 5 10 15Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp 20 25 30Thr Ala Thr Tyr Phe Cys 3515838PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 158Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr1 5 10 15Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp 20 25 30Thr Ala Thr Tyr Phe Cys 3515938PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 159Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr1 5 10 15Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp 20 25 30Thr Ala Val Tyr Tyr Cys 3516038PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 160Ser Tyr Asn Gln Lys Ser Lys Gly Lys Ala Thr Leu Thr Ala Asp Arg1 5 10 15Ser Ser Ser Thr Ala Tyr Met His Leu Asn Ser Leu Thr Ser Glu Asp 20 25 30Ser Gly Ile Tyr Tyr Cys 3516138PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 161Tyr Tyr Pro Asp Thr Val Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn1 5 10 15Ala Lys Asn Thr Leu Tyr Leu Glu Met Ser Ser Leu Arg Ser Glu Asp 20 25 30Thr Ala Met Tyr Tyr Cys 3516238PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 162Arg Leu Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys1 5 10 15Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp 20 25 30Ser Ala Val Tyr Tyr Cys 3516338PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 163Asp Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn1 5 10 15Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp 20 25 30Thr Ala Met Tyr Cys Cys 3516438PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 164Arg Leu Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys1 5 10 15Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp 20 25 30Ser Ala Val Tyr Tyr Cys 3516513PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 165Ala Leu Asn Ser Leu Leu Arg Leu Asn Ala Met Asp Tyr1 5 1016611PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 166Ala Arg Thr Gly Ser Ser Gly Tyr Phe Asp Cys1 5 1016711PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 167Ala Arg Asp Gly Phe Leu Tyr Phe Phe Asp Tyr1 5 1016810PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 168Ala Arg Arg Tyr Asp Thr Ala Met Asp Tyr1 5 1016911PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 169Ala Arg Gly Ala Tyr Gly Ser Ser Tyr Ala Tyr1 5 1017015PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 170Thr Arg Ile Tyr Tyr Asp Tyr Gly Ser Trp Asp Gly Phe Ala Tyr1 5 10 1517113PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 171Ser Arg Asp Gly Asn Tyr Arg Tyr Tyr Thr Leu Asp Phe1 5 1017212PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 172Thr Arg Glu Asp Tyr Arg Tyr Ala Trp Phe Ala Tyr1 5 1017313PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 173Ser Arg Asp Gly Asn Tyr Arg Ser Tyr Thr Met Asp Tyr1 5 1017437PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 174Tyr Tyr Pro Asp Thr Val Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn1 5 10 15Ala Lys Asn Thr Leu Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr 20 25 30Ala Met Tyr Tyr Cys

3517538PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 175Thr Tyr Val Asp Asp Phe Lys Arg Arg Phe Ala Phe Ser Leu Glu Thr1 5 10 15Ser Ala Ser Ala Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp 20 25 30Thr Ala Thr Tyr Phe Cys 3517612PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 176Ala Arg Asp Gly Asn Trp Asp Gly Gly Ser Leu Thr1 5 1017711PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 177Thr Arg Asp Gly Ser Ser Thr Trp Phe Ser Tyr1 5 101789PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 178Gly Ser Gly Thr Lys Leu Glu Ile Lys1 517910PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 179Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys1 5 1018010PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 180Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys1 5 1018110PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 181Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys1 5 1018210PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 182Phe Gly Gly Gly Thr Lys Leu Glu Phe Lys1 5 1018310PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 183Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys1 5 1018410PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 184Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys1 5 1018510PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 185Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys1 5 1018610PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 186Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys1 5 1018710PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 187Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys1 5 1018810PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 188Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys1 5 1018910PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 189Phe Gly Ala Gly Thr Lys Leu Ala Leu Lys1 5 1019011PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 190Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser1 5 1019111PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 191Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser1 5 1019211PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 192Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser1 5 1019311PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 193Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser1 5 1019411PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 194Trp Gly Gln Gly Thr Leu Val Ala Val Ser Ala1 5 1019511PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 195Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala1 5 1019611PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 196Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser1 5 1019711PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 197Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala1 5 1019811PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 198Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser1 5 1019911PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 199Gly Ala Lys Ala Pro Leu Ser Gln Ser Pro Gln1 5 1020015PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 200Leu Trp Thr Thr Gly Val Lys Glu Pro Gln Ser Pro Ser Pro Gln1 5 10 1520112PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 201Leu Leu Gly Pro Arg Asp Ser Gly Arg Cys Leu Cys1 5 1020216PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 202Ile Leu Trp Thr Thr Gly Val Lys Glu Pro Gln Ser Pro Ser Pro Gln1 5 10 1520314PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 203Leu Thr Thr Gly Ala Lys Ala Pro Leu Ser Gln Ser Pro Gln1 5 10

Claims

1-30. (canceled)

31. A monoclonal antibody or antigen binding fragment capable of specific binding to a β-subunit of human clusterin, wherein said monoclonal antibody binds to an epitope including amino acids 421-443 of the β-subunit of human clusterin more efficiently than a second monoclonal antibody raised against a synthetic peptide including or comprised within said epitope.

32. The monoclonal antibody or antigen binding fragment of claim 31, wherein said monoclonal antibody does not bind significantly to a synthetic peptide including or comprised within said epitope.

33. The monoclonal antibody or antigen binding fragment of claim 31, wherein the monoclonal antibody inhibits migration of cells in an ink clearance assay better than the second antibody.

34. The monoclonal antibody or antigen binding fragment of claim 31, wherein the monoclonal antibody or antigen binding fragment is capable of inhibiting epithelial-to-mesenchymal transitions in carcinoma cells.

35. A monoclonal antibody or an antigen binding fragment capable of specific binding to a β-subunit of human clusterin, wherein said monoclonal antibody binds to an epitope including amino acids 421-443 of the β-subunit of human clusterin and comprises a) a light chain variable region; and b) a heavy chain variable region comprising a CDR1 as set forth in SEQ ID NO.:6 and a CDR2 as set forth in SEQ ID NO.:7.

36. The monoclonal antibody or antigen binding fragment of claim 35, wherein the antibody or antigen binding fragment comprises a light chain variable region having complementary determining regions identical to those of SEQ ID NO.:8 or SEQ ID NO.:9 and a heavy chain variable having a CDR1 as set forth in SEQ ID NO.:6, a CDR2 as set forth in SEQ ID NO.:7 and a CDR3 as set forth in SEQ ID NO.165.

37. The monoclonal antibody or antigen binding fragment of claim 36, wherein the CDR1 is as set forth in SEQ ID NO.: 85.

38. The monoclonal antibody or antigen binding fragment of claim 36, wherein the CDR2 is as set forth in SEQ ID NO.:119.

39. The monoclonal antibody or antigen binding fragment of claim 35, wherein the antibody or antigen binding fragment comprises a light chain variable region having complementary determining regions identical to those of SEQ ID NO.:10 and a heavy chain variable having a CDR1 as set forth in SEQ ID NO.:6, a CDR2 as set forth in SEQ ID NO.:7 and a CDR3 as set forth in SEQ ID NO.166.

40. The monoclonal antibody or antigen binding fragment of claim 39, wherein the CDR1 is as set forth in SEQ ID NO.:86.

41. The monoclonal antibody or antigen binding fragment of claim 39, wherein the CDR2 is as set forth in SEQ ID NO.:120.

42. The monoclonal antibody or antigen binding fragment of claim 35, wherein the antibody or antigen binding fragment comprises a light chain variable region having complementary determining regions identical to those of SEQ ID NO.:11 and a heavy chain variable having a CDR1 as set forth in SEQ ID NO.:6, a CDR2 as set forth in SEQ ID NO.:7 and a CDR3 as set forth in SEQ ID NO.167.

43. The monoclonal antibody or antigen binding fragment of claim 42, wherein the CDR1 is as set forth in SEQ ID NO.:87.

44. The monoclonal antibody or antigen binding fragment of claim 42, wherein the CDR2 is as set forth in SEQ NO.:121.

45. A conjugate comprising a) an antibody or antigen binding fragment capable of specific binding to a β-subunit of human clusterin, wherein said monoclonal antibody binds to an epitope including amino acids 421-443 of the β-subunit of human clusterin and b) an agent having anti-tumor activity or a contrast agent.

46. The conjugate of claim 45, wherein the agent having anti-tumor activity is a toxin.

47. A pharmaceutical composition comprising the antibody or antigen binding fragment of claim 31.

48. A pharmaceutical composition comprising the antibody or antigen binding fragment of claim 35.

49. A pharmaceutical composition comprising the conjugate of claim 45.

50. The pharmaceutical composition, wherein the agent having anti-tumor activity is a toxin.

Images