Patent Publication Number: US-2011070253-A1

Title: Tumour-associated peptides binding to mhc molecules

Description:
RELATED APPLICATIONS 
     This application is divisional of U.S. patent application Ser. No. 10/549,718, submitted to the United States Patent and Trademark Office on Sep. 16, 2005 and granted a filing date of Aug. 17, 2007 under 35 U.S.C. §371(c)(1), (c)(2), and (c)(4), which is a National Stage Application of International Application Number PCT/EP04/03077, filed Mar. 23, 2004, which claims priority to German Patent Application Number 103 13 819.6, filed Mar. 24, 2003, the contents of which are hereby incorporated by reference in their entirety. 
    
    
     BACKGROUND 
     The present invention relates to tumour-associated peptides that are able to bind to a molecule of the human major-histocompatibility-complex (MHC), class I. 
     Such peptides are used, for example, in the immunotherapy of tumorous diseases. 
     The recognition of tumour-associated antigens (TAA) by components of the immune system plays a prominent role in the elimination of tumour cells by the immune system. This mechanism is based on the prerequisite that qualitative or quantitative differences exist between tumour cells and normal cells. In order to effect an anti-tumour-response, the tumour cells have to express antigens against which an immunological response takes place that is sufficient for the elimination of the tumour. 
     Involved in the rejection of tumours are in particular CD8-expressing cytotoxic T-lymphocytes (in the following CTLs). For triggering of such an immune reaction by cytotoxic T-cells, foreign proteins/peptides have to be presented to the T-cells. T-cells recognise antigens as peptide fragments only, if these are presented by MHC-molecules on cellular surfaces. These MHC-molecules (“major histocompatibility complex”) are peptide receptors that normally bind peptides within the cell in order to transport them to the cellular surface. This complex of peptide and MHC-molecule can be recognised by the T-cells. The MHC-molecules of the human are also designated as human leukocyte-antigens (HLA). 
     There are two classes of MHC-molecules: MHC-class-1-molecules, that are found on most of the cells with a nucleus, present peptides that are generated by proteolytic degradation of endogenous proteins. MHC-class-II-molecules are only present on professional antigen-presenting cells (APCs), and present peptides of exogenous proteins that are taken up and processed by APCs during the course of endocytosis. Complexes of peptide and MHC-class-I are recognised by CD8-positive cytotoxic T-lymphocytes, complexes of peptide and MHC-class-II are recognised by CD4-helper-T-cells. 
     In order for a peptide to trigger a cellular immune response, it must bind to an MHC-molecule This process is dependent from the allele of the MHC-molecule and the amino acid sequence of the peptides. MHC-class-1-binding peptides are usually 8-10 residues in length, and contain two conserved residues (“anchors”) in their sequence that interact with the corresponding binding groove of the MHC-molecule. 
     In order for the immune system to be able to start an effective CTL-response against tumour-derived peptides, these peptides must not only be able to bind to the particular MHC-class-I-molecules that are expressed by the tumour cells, but they must also be recognised by T-cells having specific T-cell receptors (TCR). 
     The main goal for the development of a tumour vaccine is the identification and characterisation of tumour-associated antigens that are recognised by CD8+ CTLs. 
     The antigens that are recognised by the tumour-specific cytotoxic T-lymphocytes or their epitopes, respectively, can be molecules from all classes of proteins, such as, for example, enzymes, receptors, transcription factors, etc. Another important class of tumour associated antigens are tissue-specific structures, such as, for example, CT (“cancer testis”)-antigens that are expressed in different kinds of tumours, and in healthy tissue of testes. 
     In order for the proteins to be recognised by the cytotoxic T-lymphocytes as tumour-specific antigen, and in order to be able to be used in a therapy, particular prerequisites must be present: The antigen shall mainly be expressed by tumour cells, not by normal tissues or only in lower amounts than in the tumours. It is furthermore desirable that the respective antigen is present not only in one kind of tumour, but also in high concentration in others. In addition, absolutely essential is the presence of epitopes in the amino acid sequence of the antigens, since those of a tumour-associated antigen-derived peptide (“immunogenic peptides”) shall lead to a T-cell-response, whether in vitro or in vivo. 
     Therefore, TAAs provide a starting point for the development of a tumour vaccine. The methods for the identification and characterisation of the TAAs, on the one hand, are based on the use of CTLs that are already induced in patients, or are based on the generation of differential transcription profiles between tumour and normal tissues. 
     The identification of genes that are overexpressed in tumour tissues, or that are selectively expressed in those tissues, nevertheless, did not deliver precise information for a use of the antigens that are transcribed by these genes in immunotherapy. This is due to the fact that in each case only single epitopes of these antigens are suitable for such a use, since only the epitopes of the antigens—and not the whole antigen—trigger a T-cell-response through MHC-presentation. It is therefore important to select those peptides of overexpressed or selectively expressed proteins that are presented with MHC-molecules, whereby starting points for the specific tumour-recognition by cytotoxic T-lymphocytes can be obtained. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In view of this background, it is an object of the present invention to provide at least one novel amino acid sequence for such a peptide that has the ability to bind to a molecule of the human major-histocompatibility-complex (MHC) class-I. 
     According to the invention, this object is solved by the provision of a tumour-associated peptide with an amino acid sequence that is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101 of the attached sequence protocol, wherein the peptide has the ability to bind to a molecule of the human major-histocompatibility-complex (MHC) class-I. 
     Thereby, the object that forms the basis of the invention is completely solved. 
     It shall be understood that the peptides from the tumour as identified can be synthesised or brought to expression in cells in order to obtain larger amounts thereof, and for the use for the purposes as mentioned below. 
     The inventors could isolate and identify the above-mentioned peptides as specific ligands of MHC-class-1-molecules from tumour tissues. Thereby, the term “tumour-associated” designates peptides that were isolated and identified from tumour material. These peptides, that are presented on real (primary) tumours therefore underlie antigen processing in a tumour-cell. 
     The specific ligands can be used in cancer therapy, e.g. in order to induce an immune response against tumour cells that express the corresponding antigens from which the peptides are derived. 
     On the one hand, such an immune response can be achieved in vivo in the form of an induction of CTLs. For this, the peptide, for example in the form of a pharmaceutical composition, is administered to a patient who suffers from a tumorous disease that is associated with the TAA. 
     On the other hand, a CTL-response towards a tumour that expresses the antigens from which the peptides are derived can also be triggered ex vivo. For this, the CTL-precursor cells are incubated together with antigen-presenting cells, and the peptides. Subsequently, the thus stimulated CTL are cultured, and these activated CTL are administered to the patient. 
     Furthermore, the possibility exists to load APC ex vivo with the peptides, and to administer these loaded APCs to the patient who expresses the antigen in the tumorous tissue, from which the peptide is derived from. The APCs, in turn, then are able to present the peptide to the CTLs in vivo, and activate these. 
     Nevertheless, the peptides according to the invention can be used as diagnostic reagents. 
     Thus, using the peptides it can be identified, whether CTLs are present in a CTL-population that are specifically directed against a peptide, or are induced by a therapy. 
     In addition, the increase of precursor T-cells can be tested for with the peptides that exhibit a reactivity against the defined peptide. 
     Furthermore, the peptide can be used as a marker in order to monitor the progression of a disease of a tumours that expresses the antigen from which the peptide is derived. 
     In the attached table 1, the identified peptides are listed. Furthermore, in said table the proteins are given from which the peptides are derived, and the respective positions of the peptides in the respective proteins. Thereby, the English designations of the proteins were maintained in order to avoid mistakable translations. Furthermore, the Acc-numbers are given, respectively that are maintained in the Genbank of the “National Centre for Biotechnology Information” of the National Institute of Health (see http: www.ncbi.nlm.nih.gov). 
     The inventors could isolate the peptides (or ligands) from renal cell tumours of two patients, RCC68, and RCC44. 
     From the tumours of the patients, 101 ligands could be identified, that were bound to the HLA-subtypes HLA-A*02, HLA-A*29, HLA-B*15 or HLA-B*45 (patient RCC68) and to HLA-A*3201, HLA-A*1101, HLA-B*4002, HLA-B*2705 or HLA-Cw*0202 (patient RCC44). 
     Some of the ligands were derived from strongly expressed so-called “housekeeping” genes that are uniformly expressed in most tissues, nevertheless, many were characterised by tissue specific and tumour specific expression. 
     Thus, some peptides could be identified that are derived from proteins that are overexpressed, particularly in tumorous tissue. Thus, for example, fragments of vimentin (ALRDVRQQY, position 268-276, SEQ ID NO: 7; EENFAVEA, position 348-355, SEQ ID NO: 15; MEENFAVEA, position 347-355, SEQ ID NO: 45; NYIDKVRFL, position 116-124, SEQ ID NO: 50) could be identified. Young et al., expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers, 2001, Am. J. Pathol., 158:1639-1651) showed that this protein was overexpressed in tissue of renal cell tumours. 
     In addition, the inventors could identify, amongst others, ligands that are derived from alpha-catenin, (LQHPDVAAY, position 229-237, SEQ ID NO: 43), and beta-catenin (AQNAVRLHY, position 481-489, SEQ ID NO: 8). 
     Furthermore, the inventors could show in own experiments that by using of exemplary selected peptides it was possible to generate cytotoxic T-lymphocytes (CTLs) in vitro that were each specific for the selected peptides. Using these CTLs, tumour cells could selectively be killed which expressed the corresponding proteins, and which, in addition, were derived from different tumour cell lines of different patients. Furthermore, said CTLs, for example, also lysed dendritic cells that were “pulsed” (loaded) in advance with the respective peptides. Thus, it could be shown that, with the peptides according to the present invention as epitopes, human T-cells in vitro could be activated in vitro. Accordingly, the inventors could not only show that CTLs that were obtained from peripheral blood-mononuclear-cells (PBMNCs) of a patient, and which were specific for a particular peptide, could kill cells of the same kind of tumour of another patient. In addition, the inventors showed that also cells of other kinds of tumours could be lysed with these CTLs. 
     In a preferred embodiment also peptides could be used for a stimulation of an immune response that exhibited the SEQ ID NO: 1 to 101, and wherein at least one amino acid is replaced by another amino acid having similar chemical properties. 
     With respect to the respective MHC-subtypes, these are, for example, the anchoring amino acids, which can be replaced by amino acids with similar chemical properties. Thus, for example, in case of peptides which are associated with the MHC-subtype HLA-A*02 leucine at position 2 can be replaced by isoleucine, valine or methionine, and vice versa, and at the C-terminus leucine by valine, isoleucine, and alanine, that all have non-polar side chains. 
     It is furthermore possible, to use peptides with the SEQ ID NO: 1 to 101, that N- or/and C-terminally exhibit at least one additional amino acid, or wherein at least one amino acid is deleted. 
     Furthermore, peptides with the SEQ ID NO: 1 to 101 can be used, wherein at least one that amino acid is chemically modified. 
     Thereby, the varying amino acid(s) is(are) chosen in such a manner that the immunogenicity of the peptide is not affected by the variation, i.e. it has a similar binding affinity to the MHC-molecule and the ability for a T-cell-stimulation. 
     According to the invention, the peptide can be used for the treatment of tumorous diseases and/or adenomatous diseases. 
     Thereby, the tumorous diseases to be treated comprise, for example, renal, breast, pancreatic, stomach, testes, and/or skin cancer. In doing so, the listing of the tumorous diseases is only exemplary, and shall not limit the scope of use. The fact that the peptides according to the invention are suitable for such use, could be demonstrated by the inventors in their own experiments. Therein, it was shown that specifically generated CTL that were specific for particular peptides could effectively and selectively kill tumour cells. 
     In general, several application forms are possible for a use of tumour-associated antigens in a tumour vaccine. Tighe et al., 1998, Gene vaccination: plasmid DNA is more than just a blueprint, Immunol. Today 19(2):89-97, described that the antigen can be administered either as recombinant protein together with suitable adjuvants or carrier systems, or as the cDNA encoding for the antigen in plasmid vectors. In these cases, in order to evoke an immune response, the antigen must be processed and presented in the body of the patient by antigen-presenting cells (APCs). 
     Melief et al., 1996, peptides-based cancer vaccines, Curr. Opin. Immunol. 8:651-657, showed an additional possibility, namely the use of synthetic peptides as vaccine. 
     For this, in a preferred embodiment, the peptide can be used with the addition of adjuvants, or else in singular form. 
     The granulocyte-macrophage-colony-stimulating-factor (GM-CSF) can, for example, be used as adjuvant. Further examples for such adjuvants are aluminium hydroxide, emulsions of mineral oils, such as, for example, Freund&#39;s adjuvant, saponines or silicon compounds. 
     The use together with an adjuvant offers the advantage that the immune response that is triggered by the peptide can be enhanced and/or that the peptide is stabilised. 
     In another preferred embodiment, the peptide is used bound to an antigen-presenting cell. 
     These measure has the advantage that the peptides can be presented to the immune system, in particular the cytotoxic T-lymphocytes (CTLs). In doing so, the CTLs can recognise the tumour cells, and specifically kill them. As antigen-presenting cells, for example, dendritic cells, monocytes or B-lymphocytes are suitable for such a use. 
     Thereby, the cells can be loaded, for example ex vivo, with the peptides. On the other hand, the possibility exists to transfect the cells with the DNA encoding for the peptides or the corresponding RNA in order to then bring the peptides to an expression on the cells. 
     The inventors could show in own experiments that it is possible to specifically load dendritic cells (DC) with specific peptides, and that these loaded dendritic cells activate peptide-specific CTLs. This means, that the immune system can be stimulated in order to develop CTLs against the tumours expressing the corresponding peptides. 
     Thereby, the peptide-carrying antigen-presenting cells can either be used directly, or activated before a use with, for example, the heat shock-protein gp96. This heat shock protein induces the expression of MHC-class I-molecules, and of costimulating molecules, such as B7, and additionally stimulates the production of cytokines. Thereby, the overall triggering of an immune response is promoted. 
     In another preferred embodiment, the peptides are used for the labelling of leukocytes, in particular of T-lymphocytes. 
     This use is of advantage if, using the peptides, it shall be elucidated, if CTLs that are specifically directed against a peptide are present in a CTL-population. 
     Furthermore, the peptide can be used as a marker for judging the progression of a therapy in a tumorous disease. 
     The peptide can be used also in other immunisations or therapies for the monitoring of the therapy. Thus, the peptide can not only be used therapeutically, but also diagnostically. 
     In another embodiment, the peptides are used for the production of an antibody. 
     Polyclonal antibodies can be obtained in a common manner by immunisation of animals by means of injection of the peptides, and subsequent purification of the immunoglobulin. 
     Monoclonal antibodies can be produced following standard protocols, such as, for example, described in Methods Enzymol. (1986), 121, Hybridoma technology and monoclonal antibodies. 
     In another aspect, the invention furthermore relates to a pharmaceutical composition that contains one or several of the peptides. 
     This composition, for example, is used for parenteral administration, for example, subcutaneous, intradermal or intramuscular or oral administration. For this, the peptides are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. In addition, the composition can contain auxiliary agents, such as, for example, buffers, binding agents, diluents, etc. 
     The peptides can also be administered together with immune stimulating substances, e.g. cytokines A comprehensive demonstration of auxiliary agents that can be used in such a composition, is, for example, shown in A. Kibbe, Handbook of Pharmaceutical Excipients, 3. Ed., 2000, American Pharmaceutical Association and pharmaceutical press. 
     Thereby, the agent can be used for the prevention, prophylaxis and/or therapy of tumorous diseases and/or adenomatous diseases. 
     The pharmaceutical agent, that at least contains one of the peptides with the SEQ ID NO: 1 to 101, is administered to a patient that suffers from a tumorous disease which is associated with the respective peptide or antigen. By this, a tumour-specific immune response on the basis of tumour-specific CTLs can be triggered. 
     Thereby, the amount of the peptide or the peptides as present in the pharmaceutical composition is a therapeutically effective amount. Thereby, the peptides as contained in the composition can also bind to at least two different HLA-types. 
     In another aspect, the present invention relates to nucleic acid molecules that encode for the peptides having the SEQ ID NO: 1 to 101, as well as the use of at least one of the nucleic acid molecules for producing a medicament for the therapy of tumorous diseases and/or adenomatous diseases. 
     Thereby, the nucleic acid molecules can be DNA- or RNA-molecules, and also be used for the immunotherapy of cancerous diseases. In doing so, the peptide that is induced by the nucleic acid molecule induces an immune response against tumour cells that express the peptide. 
     According to the invention, the nucleic acid molecules can also be present in a vector. 
     In addition, the invention relates to cells which have been genetically modified with the aid of the nucleic acid molecule that encodes for the peptides in such a manner that the cell produces a peptide with the SEQ ID NO: 1 to 101. 
     For this, the cells are transfected with the DNA encoding for the peptides or the corresponding RNA, whereby the peptides are brought to an expression on the cells. For such a use as antigen-presenting cells, for example, dendritic cells, monocytes or other human cells are suited, that express suitable molecules for the co-stimulation, such as, for example, B7.1 or B7.2. 
     The invention further relates to a diagnostic method, wherein the presence of one of the novel peptides is used as a diagnostic marker, as well as to a method for the treatment of a pathological condition, wherein an immune response against a protein of interest is triggered, wherein a therapeutically effective amount of at least one of the novel peptides is administered. 
     The inventors have realised that the novel peptides can also be used as markers for a pathological condition, such that a respective diagnostic method, wherein a blood sample of the patient is taken and is examined in a common manner for the presence of lymphocytes that are directed against one of the novel peptides, can be used as an early diagnosis or for the targeted selection of a suitable treatment. 
     Furthermore, the invention relates to an electronic storage medium, which contains the amino acid sequence of at least one of the novel peptides and/or the nucleic acid sequence of nucleic acid molecules that encode for the novel peptides. 
     Starting from this storage medium, then, in case of the presence of a corresponding indication, the information for the peptides that are suitable for the treatment of the pathological condition can be provided quickly. 
     It shall be understood that the above mentioned features and the features to be explained in the following can not only be used in the respectively given combination, but also in a unique positioning without departing from the scope of the present invention. 
     Embodiments of the invention are explained in the following examples. 
     EXAMPLES 
     Example 1 
     1.1. Patient Samples 
     Two samples were obtained from the department for urology, Universität Tübingen, that were derived from patients that suffered from histologically confirmed renal cell tumours. Both patients had received no pre-surgical therapy. Patient No. 1 (in the following designated RCC68) had the following HLA-typing: HLA-A*02 A*29 B*15 B*45; patient No. 2 (in the following designated RCC44) HLA-A*3201 A*1101 B*4002 B*2705 Cw*0202, 
     1.2. Isolation of the MHC-Class-I-Bound Peptides 
     The shock-frozen tumour samples were processed as already described in Schirle, M. et al., Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach, 2000; European Journal of Immunology, 30:2216-2225. The peptides were isolated according to standard protocols, and in particular by using the monoclonal antibody W6/32 that is speCific for HLA-class-I-molecules, or the monoclonal antibody BB7.2 that is specific for HLA-A2. Barnstable, C. J. et al., Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis, 1978, Cell, 14:9-20 and Parham, P. &amp; Brodsky, F. M., Partial purification and some properties of BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28, 1981, Hum. Immunol., 3:277-299, describe the production and uses of these antibodies. 
     1.3. Mass Spectroscopy 
     The peptides were separated by “reversed phase HPLC” (SMART-system, mRPC C2/C18 SC 2.1/19, Amersham Pharmacia Biotech), and the fractions as obtained were analysed by nano-ESI MS. This was done as described in Schirle, M. et al., Identification of tumorassociated MHC class I ligands by a novel T cell-independent approach, 2000, European Journal of Immunology, 30:2216-2225. 
     The peptides that were obtained from tumorous tissue were identified by capillary-LC-MS as just mentioned, nevertheless with slight changes: 100 pl of each of the samples were loaded, desalted, and pre-concentrated on a 300 pm*5 mm C18 p-pre-column (LC Packings). The solvent and the sample were added by means of a syringe pump (PHD 2000, Harvard apparatus, Inc.) with a sealed 100 pl-syringe (1710 RNR, Hamilton) with a speed of 2 pl/min. For the separation of the peptides, the pre-concentration-column was disposed before a 75 tilla*250 mm C-18-column (LC Packings). Subsequently, a binary gradient with 25-60% B was run within 70 min, whereby the flow rate was reduced from 12 pl/min to about 300 nl/min, and in particular by using a TEE-connection (ZT1C, Valco), and a 300 p.m*150 mm C-18-column. 
     In order to ensure that the system was free of residual peptides, in each case a blank sample was measured. Online-fragmentation was performed as described, and the spectra of the fragments were analysed manually. The database searches (NCBInr, EST) were performed using MASCOT (http://www.matrixscience.com). 
     1.4. Identification of the MHC-Class-1-Ligands from Tumorous Tissue of the Patients RCC68 and RCC44 
     In the attached sequence protocol and in the attached table 1 the ligands are listed that were bound to the HLA-molecules of the patients RCC68 and TCC44. The peptides that were associated with HLA-A*02 exhibited the allele-specific peptide motif: Thus, at position 2 leucine, valine, isoleucine, alanine or methionine, and at the C-terminus leucine, valine, isoleucine, or alanine could be found. Most of the ligands were derived from so-called “housekeeping”-proteins, nevertheless, also ligands from proteins could be identified which are associated with tumours. Thus, for example, fragments of vimentin (ALRDVRQQY, position 268-276, SEQ ID NO: 7; EENFAVEA, position 348-355, SEQ ID NO: 15; MEENFAVEA, position 347-355, SEQ ID NO: 45; NYIDKVRFL, position 116-124, SEQ ID NO: 50) could be identified. Young et al. (Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers, 2001, Am. J. Pathol., 158:1639-1651) showed that this protein was overexpressed in tissue of renal cell tumours. 
     1.5. Detection of Peptide-Specific T-Cells in the Normal CD8+-T-Cell-Repertoir 
     For a detection of peptide-specific T-cells, mononuclear cells from peripheral blood of healthy patients were stained with the respective HLA-A*subtype-tetramers that were constituted with the respective peptides: For a production of the tetramers, recombinant HLA-A*subtype-molecules were constituted with the peptides in vitro, purified by gel filtration, biotinylated, and mixed with streptavidin for a linking of the monomers. 
     In general, the results of the double stainings were evaluated by analysis using of FACS, and the specific binding of the peptide-tetramers was detected. 
     Example 2 
     In order to analyse the presentation of the selected peptides by tumour cells, and the recognition of the peptides by CTLs to, CTLs that were specific for the selected peptides were induced in vitro. For this, dendritic cells (DCs) were used that were derived from peripheral blood-mononuclear-cells (PBMNCs) of healthy donors, that had the same respective HLA-(sub)type. 
     2.1. Obtaining of DCs 
     The DCs were isolated by Ficoll/Paque-(Biochrom, Berlin, Germany)-density gradient-centrifugation of PBMNCs from heparinised blood. The heparinised blood was obtained from “buffy coat”-preparations of healthy donors of the blood bank of the Universität Tübingen. The cells were seeded on 6-well-plates (Falcon, Heidelberg, Germany) (1×10 7  cells/3 ml per well) in RP10 medium (RPMI 1640, supplemented with 10% heat-inactivated foetal calf serum and with antibiotics). Following a 2-hour incubation at 37° C. and 5% CO 2 , the non-adhering cells were removed, and the adhering blood monocytes were cultivated in RP10 medium, whereby the following cytokines were added into the medium as supplement: human recombinant GM-CSF (granulocyte macrophage colony stimulating factor; Leukomax, Novartis; 100 ng/ml), interleukin IL-4 (R&amp;D Systems, Wiesbaden, Germany; 1000 IU (ml), and TNF-α (Tumor-Nekrose-Faktor a) (R&amp;D Systems, Wiesbaden, Germany; 10 ng/ml). 
     2.2. Synthesis of the Peptides 
     The exemplary selected peptides were synthesised on a peptide-synthesiser (432A, Applied Biosystems, Weiterstadt, Germany) using F-moc (9-fluoroenylmethyloxycarbonyl)—protective groups, and analysed by “reversed phase” HPLC and mass spectroscopy. By this way, sufficient amounts of the identified peptides could be produced. 
     2.3. Induction of an Antigen-Specific CTL-Response Using Restringed Synthetic Peptides 
     For an induction of CTLs, the DCs (5×10 5 ) as obtained in step 2.1. were pulsed for 2 hours with 50 μg/ml of the peptides obtained from step 2.2., subsequently washed and incubated with 2.5×10 6  autologous PBMC in RP10 medium —  After a 7-day cultivation period, the cells were restimulated with autologous, peptide-pulsed PBMNCs. In doing so, 1 ng/ml human recombinant interleukin 1L-2 (R&amp;D Systems) was added on day 1, 3, and 5. The cytotoxic activity of CTLs that were induced by this way was examined on day 5 following the last restimulation by means of a standardised  51 Cr-release-assay (see below at 2.4.: CTL-assay). 
     2.4. CTL-Assay 
     For the CTL-assays, tumour cells, peptide-pulsed cells of different cell lines, and autologous DCs were used as target-cells. Peptide-pulsed cells were pulsed with 50 μg/ml peptide for 2 hours. All target cells were ( 51 Cr) labelled in RP10 medium (RPMI 1640, supplemented with 10% heat-inactivated foetal calf serum and with antibiotics) for 1 hour at 37° C. with [ 51 Cr] sodium chromate. Subsequently, 10 4  cells/per each well were given on a 96-well-plate with rounded bottoms. Different amounts of CTLs were added in order to reach a final volume of 200 μl, with subsequent incubation for 4 hours at 37° C. Thereafter, the supernatants (50 μl/well) were harvested and counted in a beta-plate-counter. The specific lysis was calculated in percent as follows: 100×(experimental release−spontaneous release/maximal release−spontaneous release). The spontaneous and the maximal release were each determined in the presence of either medium or 2% triton X-100. 
     2.5. Results of the CTL-Induction 
     a) CTL-Cytotoxic Activity Versus Peptide-Pulsed DCs 
     In  51 Cr-release-assays (see at 2.4.) the cytotoxic activity of induced CTLs (see at 2.3.) versus T2- or DC-cells was tested. The T2-cell line is HLA-A*02-positive and TAP (transporter associated with antigen processing)—deficient; (TAP-peptide-transporters transport peptide-fragments of a protein antigen from the cytosol into the endoplasmatic reticulum, where they associate with MHC-molecules). 
     The results of these release-assays show that with CTL-cell lines that were obtained after 2-week restimulation, an antigen-specific killing of the cells could be achieved: Only those cells were killed by an increasing amount of CTL that presented each of the selected peptides; the control cells that were loaded with irrelevant peptides were not killed. Thereby, the specificity of the cytolytic activity could be shown. 
     b) CTL-Cytotoxic Activity Versus Tumour Cell Lines 
     In a next step, it was tested again by a  51 Cr-release-assay, whether the CTLs that were specific for the selected peptides recognise and lyse tumour cells that endogenously express the selected peptides. 
     For this, different  51 Cr-labelled cell lines expressing the corresponding HLA-molecules were used: HCT 116 (colon cancer; obtained from Prof G. Pawelec, Tübingen, Germany), A 498, MZ 1257 and MZ 1774 (renal cell carcinoma; obtained from Prof A. Knuth, Frankfurt, Germany), MCF-7 (breast cancer; commercially obtained from the ATCC, American Type Culture Collection), Mel 1479 (melanoma; obtained from Prof. G. Pawelec, Tübingen, Germany), and U 266 (multiple myeloma; obtained from Prof G. Pawelec, Tübingen, Germany). These cell lines express particular proteins as target structures (“targets”). 
     The B-cell line Croft (EBV (Epstein-Barr-Virus)-immortalised; HLA-A*02-positive; obtained from O. J. Finn, Pittsburgh, USA) and the cell line SK-OV-3 (ovarian tumour; HLA-A*03-positive; obtained from O. J. Finn, Pittsburgh, USA) were included in the study as negative controls. K 562 cells (obtainable, for example, at the Deutschen Sammlung von Mikroorganismen and Zellkulturen, DSMZ; ACC 10) were used in order to determine the activity of natural killer cells (NK), since this cell line is highly sensitive against these killer cells. 
     All cell lines were cultivated in RP10 medium (RPMI 1640, supplemented with 10% heat-inactivated foetal calf serum and with antibiotics). 
     With the above tumour cell lines and the CTLs as induced at 2.3.,  51 Cr-release assays (see at 2.4.) were performed. 
     In these tests, the CTLs that were each specific for the selected peptides efficiently lysed tumour cells that expressed both the corresponding HLA-molecule as well as the selected peptides. The specific lysis was—as given above at 2.4.—measured by the  51 Cr-release. In contrast, the control cell line SK-OV-3 (HLA-A-*02-negative) was not lysed by the CTLs that were induced by the peptides that were bound by HLA-A*02. This showed that the peptides must be presented in connection with the corresponding HLA-molecules on the tumour cells in order to efficiently lyse the target-cells. Furthermore, by this the antigen-specificity and the MHC-restriction of the CTLs is confirmed. 
     In addition, the CTL-cells that were induced in vitro by the peptides did not recognise the cell line K562, demonstrating that the cytotoxic activity was not mediated by natural killer cells (NK)-cells. 
     c) Inhibition-Assays 
     In order to further verify the antigen-specificity and the MHC-restriction of the in-vitro induced CTLs, inhibitions-assays were performed with non- 51 Cr-labelled (“cold”) inhibitor-cell lines. 
     Here, the ability of peptide-pulsed cell lines was analysed to inhibit the lysis of tumour cells, or to be competitive. For this, an excess of inhibitor (i.e. of pulsed, non-labelled cells) was used. The ratio of the inhibitor (peptide-pulsed cells) to target (tumour cells) was 20:1. Upon lysis of the inhibitor-cell lines, no  51 Cr could be released since the inhibitor-cell lines were non-labelled. 
     The cell line T2 (HLA-A*02; TAP—deficient; see at 2.5.a)) was used as inhibitor. T his cell line T2 was pulsed before the assays with each of the relevant peptides, or an irrelevant control peptide. 
     In the absence of the inhibitor-cells, a lysis of the tumour cells by CTL was observed. It could furthermore be shown that, in case of an excess of inhibitor-target, no lysis of the tumour cells took place (and thus no  51 Cr-release), as long as the inhibitor-target was pulsed with the corresponding peptides. The activity of the CTLs was directed to the non-labelled T2-cells present in excess, such that these and not the tumour cells were lysed. The T2-cells that were pulsed with an irrelevant peptide could not inhibit the lysis of the tumour cells by the CTLs, such that released  51 Cr could be measured. 
     The MHC-restriction and the antigen-specificity of the cytotoxic activity that was mediated by the HLA-A*02-peptide-induced CTL could be confirmed using a HLA-A*02-specific monoclonal antibody, and in an inhibition-assay with non-labelled (“cold”) inhibitor: The A 498-tumor cells were blocked by the addition of the HLA-A*02-specific antibody (monoclonal antibody BB7.2, IgG2b, obtained from S. Stefanovic, Tubingen), such that they were not lysed by the addition of the CTLs, and no  51 Cr was released. An unspecific antibody served as control that did not block HLA-A*02-molecules (ChromPure mouse IgG, Dianova, Germany). For these inhibition-experiments, the cells were incubated 30 min. with 10 μg/ml antibody before seeding on the 96-well-plates. 
     It could furthermore be found that the T2-competition-cell line that was pulsed with an irrelevant peptide could not inhibit the CTL-mediated lysis of the tumour cell line A 498, but that the T2-inhibitor-cell line pulsed with the corresponding peptide could inhibit the lysis of the tumour-cell line, such that in the latter case no  51 Cr-release could be measured. 
     d) Specific Lysis of Transfected DCs 
     In a next experiment, the cytotoxic activity of the CTLs was analysed in an autologous experimental setting. For this, autologous DCs that were obtained from the same PBMNCs as those that were used for the CTL-induction (see at 2.2.) were used as target cells. Before performing the CTL-assay, the DCs were electroporated with RNA that was isolated earlier either from tumour-cell lines, or that represented control-RNA. The total-RNA was isolated from the tumour cells using the QIAGEN Rneasy mini kit (QIAGEN, Hilden, Germany) in accordance with the manufacturers instructions. Amount and purity of the RNA was determined photometrically, and stored in aliquots at −80° C. 
     Before the electroporation on day 6, immature DCs were washed two times with serumfree X-VIVO 20 medium (BioWhittaker, Walkersville, USA), and resuspended in a final concentration of 2×10 7  cells/ml. Subsequently, 200 μl of the cell suspension were mixed with 10 μg of the total-RNA, and electroporated in a 4 mm cuvette by means of an Easyject™ (Peqlab, Erlangen, Germany) (parameters: 300 V, 150 μF 1540Ω, pulse time: 231 ms). Following the electroporation, the cells were immediately transferred into RP10 medium and again given into the incubator. More than 80% of the cells were viable following the electroporation. 
     After performing the CTL-assays with CTLs that were induced by the selected peptides (see at 2.4.), a specific lysis of DCs could be observed which were electroporated with RNA of peptide-expressing tumour-cell lines. In contrast, DCs that were electroporated with RNA of a non-peptide-expressing tumour-cell line, were not lysed. 
     This shows that—following transfection of the DCs with RNA of peptide-positive tumour-cells—the identified peptides are processed and presented. 
     e) Induction of Peptide-Specific CTLs in a Patient with Chronic Lymphatic Leukaemia 
     In an additional experiment, CTLs that were specific for selected peptides were generated from PBMNCs of a patient with chronic lymphatic leukaemia (CLL). Furthermore, the autologous primary CLL-cells and DCs of this patient were used as  51 Cr-labelled targets in an assay, wherein a  51 Cr-release was mediated by the peptide-induced CTLs. As a result, both the autologous DCs of this patient that were pulsed with the selected peptides, as well as the autologous CLL-cells were lysed by the peptide-induced CTLs. In contrast, DCs that were pulsed with an irrelevant peptide were not lysed. In addition, non-malignant B-cells and the cell line K 562 were not lysed by the CTLs. 
     The specificity of the CTL-response was confirmed in a target-inhibition-assay, whereby the cell line T2 (see above) was used as inhibitor-cells which were pulsed with each of the selected peptides or with an irrelevant peptide. Also in this case, the CTLs that were induced by using the peptides lysed the inhibitor-cell lines present in excess that were pulsed with the relevant peptides, such that in this case the  51 Cr-labelled tumour cells were not lysed. 
     In summary, therefore the inventors could show that the peptides as identified represent promising substances in the context of an immunotherapy in a multitude of (tumorous-) diseases. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                 sequence 
                 Position/Gene type 
                 Acc. No. 
                 SEQ ID-No. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1. 
                 AAFPGASLY 
                 63-7 
                 NM_014764 
                 SEQ ID-No. 1 
               
               
                   
                   
                 DAZ associated protein 2 
                   
                   
               
               
                   
               
               
                 2. 
                 AELATRALP 
                 137-145 
                 NM_002230 
                 SEQ ID-No. 2 
               
               
                   
                   
                 junction placoglobin 
                   
                   
               
               
                   
               
               
                 3. 
                 AFFAERLYY 
                 397-405 
                 NM_001156 
                 SEQ ID-No. 3 
               
               
                   
                   
                 annexin A7 
                   
                   
               
               
                   
               
               
                 4. 
                 ALATLIHQV 
                 26-34 
                 NM_016319 
                 SEQ ID-No. 4 
               
               
                   
                   
                 COP9 constitutive 
                   
                   
               
               
                   
                   
                 photomorphogenic homolog 
                   
                   
               
               
                   
                   
                 subunit 7A ( Arabidopsis ) 
                   
                   
               
               
                   
               
               
                 5. 
                 ALAVIITSY 
                 318-326 
                 NM_005765 
                 SEQ ID-No. 5 
               
               
                   
                   
                 ATPase, H+ transporting, 
                   
                   
               
               
                   
                   
                 lysosomal (vacuolar proton pump) 
                   
                   
               
               
                   
                   
                 membrane sector associated protein 
                   
                   
               
               
                   
                   
                 M8-9 
                   
                   
               
               
                   
               
               
                 6. 
                 ALQEMVHQV 
                 806-814 
                 NM_006403 
                 SEQ ID-No. 6 
               
               
                   
                   
                 enhancer of filamentation 1 
                   
                   
               
               
                   
               
               
                 7. 
                 ALRDVRQQY 
                 268-276 
                 NM_003380 
                 SEQ ID-No. 7 
               
               
                   
                   
                 vimentin 
                   
                   
               
               
                   
               
               
                 8. 
                 AQNAVRLHY 
                 481-489 
                 NM_001904 
                 SEQ ID-No. 8 
               
               
                   
                   
                 catenin (cadherin-associated 
                   
                   
               
               
                   
                   
                 protein), beta 1, 88 kDa 
                   
                   
               
               
                   
               
               
                 9. 
                 AQPGFFDRF 
                 1006-1014 
                 NM_001849 
                 SEQ ID-No. 9 
               
               
                   
                   
                 collagen, type VI, alpha 2 
                   
                   
               
               
                   
                   
                 (COL6A2), transcript variant 2C2 
                   
                   
               
               
                   
               
               
                 10. 
                 AVCEVALDY 
                 2260-2268 
                 NM_003128 
                 SEQ ID-No. 10 
               
               
                   
                   
                 spectrin, beta, non-erythrocytic 1 
                   
                   
               
               
                   
               
               
                 11. 
                 AVLGAVVAV 
                 161-169 
                 M12679 
                 SEQ ID-No. 11 
               
               
                   
                   
                 Cw1 antigen 
                   
                   
               
               
                   
               
               
                 12. 
                 DAILEELSA 
                 154-162 
                 NM_024591 
                 SEQ ID-No. 12 
               
               
                   
                   
                 hypothetical protein FLJ11749 
                   
                   
               
               
                   
               
               
                 13. 
                 EEHPTLLTEA 
                 101-110 
                 NM_002388 
                 SEQ ID-No. 13 
               
               
                   
                   
                 actin, alpha 2, smooth muscle, aorta 
                   
                   
               
               
                   
               
               
                 14. 
                 EEMPQVHTP 
                 715-723 
                 NM_002388 
                 SEQ ID-No. 14 
               
               
                   
                   
                 MCM3 minichromosome 
                   
                   
               
               
                   
                   
                 maintenance deficient 3 ( S. cerevisiae ) 
                   
                   
               
               
                   
               
               
                 15. 
                 EENFAVEA 
                 348-355 
                 NM_00380 
                 SEQ ID-No. 15 
               
               
                   
                   
                 vimentin 
                   
                   
               
               
                   
               
               
                 16. 
                 EENKLIYTP 
                 56-64 
                 NM_012106 
                 SEQ ID-No. 16 
               
               
                   
                   
                 binder of Arl Two 
                   
                   
               
               
                   
               
               
                 17. 
                 FAEGFRAL 
                 110-118 
                 NM-018834 
                 SEQ ID-No. 17 
               
               
                   
                   
                 v-jun sarcoma virus 17 oncogen 
                   
                   
               
               
                   
                   
                 homolog (avian 
                   
                   
               
               
                   
               
               
                 18. 
                 FFGETSHNY 
                 235-243 
                 NM_108834 
                 SEQ ID-No. 18 
               
               
                   
                   
                 matrin 3 
                   
                   
               
               
                   
               
               
                 19. 
                 FLPHMAYTY 
                 931-939 
                 NM_014795 
                 SEQ ID-No. 19 
               
               
                   
                   
                 zinc finger homeobox 1 b 
                   
                   
               
               
                   
               
               
                 20. 
                 GEPRFISVGY 
                 42-51 
                 Z46810 
                 SEQ ID-No. 20 
               
               
                   
                   
                 major histocompatibility complex, 
                   
                   
               
               
                   
                   
                 class 1, C 
                   
                   
               
               
                   
               
               
                 21. 
                 GLATDVQTV 
                 55-63 
                 NM_002795 
                 SEQ ID-No. 21 
               
               
                   
                   
                 proteasome (prosome, macropain) 
                   
                   
               
               
                   
                   
                 subunit, beta type, 3 
                   
                   
               
               
                   
               
               
                 22. 
                 GLNDETYGY 
                 161-169 
                 NM_001677 
                 SEQ ID-No. 22 
               
               
                   
                   
                 ATPase, Na+/K+ transporting, beta 
                   
                   
               
               
                   
                   
                 1 polypeptide 
                   
                   
               
               
                   
               
               
                 23. 
                 GQEFIRVGY 
                 103-111 
                 NM_018154 
                 SEQ ID-No. 23 
               
               
                   
                   
                 anti-silencing function 1B 
                   
                   
               
               
                   
               
               
                 24. 
                 GQFPGHNEF 
                 76-84 
                 NM_006449 
                 SEQ ID-No. 24 
               
               
                   
                   
                 CDC42 effector protein (Rho 
                   
                   
               
               
                   
                   
                 GTPase binding) 3 
                   
                   
               
               
                   
               
               
                 25. 
                 GQPWVSVTV 
                 121-129 
                 AC005912 
                 SEQ ID-No. 25 
               
               
                   
                   
                 FLJ00063 
                   
                   
               
               
                   
               
               
                 26. 
                 GYLHDFLKY 
                 254-262 
                 NM_012286 
                 SEQ ID-No. 26 
               
               
                   
                   
                 mortality factor 4 like 2 
                   
                   
               
               
                   
               
               
                 27. 
                 HQITVLHVY 
                 137-145 
                 NM_021814 
                 SEQ ID-No. 27 
               
               
                   
                   
                 homolog of yeast long chain 
                   
                   
               
               
                   
                   
                 polyunsaturated fatty acid 
                   
                   
               
               
                   
                   
                 elongation enzyme 2 
                   
                   
               
               
                   
               
               
                 28. 
                 HVIDVKFLY 
                 163-171 
                 NM_001923 
                 SEQ ID-No. 28 
               
               
                   
                   
                 damage-specific DNA binding 
                   
                   
               
               
                   
                   
                 protein 1, 127 kDa 
                   
                   
               
               
                   
               
               
                 29. 
                 HVNDLFLQY 
                 484-492 
                 AB023222 
                 SEQ ID-No. 29 
               
               
                   
                   
                 KIAA1005 
                   
                   
               
               
                   
               
               
                 30. 
                 IAMATVTAL 
                 249-257 
                 NM_000034 
                 SEQ ID-No. 30 
               
               
                   
                   
                 aldolase A, fructose-bisphosphate 
                   
                   
               
               
                   
               
               
                 31. 
                 IGIDLGTTY 
                 7-15 
                 NM_005345 
                 SEQ ID-No. 31 
               
               
                   
                   
                 heat shock 70 kDa protein 1A 
                   
                   
               
               
                   
               
               
                 32. 
                 ILHDDEVTV 
                 15-23 
                 NM_001003 
                 SEQ ID-No. 32 
               
               
                   
                   
                 ribosomal protein, large P1 
                   
                   
               
               
                   
               
               
                 33. 
                 IQKESTLHL 
                 61-69 
                   
                 SEQ ID-No. 33 
               
               
                   
                   
                 ubiquitin A-52 residue ribosomal 
                   
                   
               
               
                   
                   
                 protein fusion product 1 
                   
                   
               
               
                   
               
               
                 34. 
                 ISRELYEY 
                 70-77 
                 BC022821 
                 SEQ ID-No. 34 
               
               
                   
                   
                 clone MCG: 39264 
                   
                   
               
               
                   
                   
                 IMAGE: 5087938 
                   
                   
               
               
                   
               
               
                 35. 
                 KLHGVNINV 
                 59-67 
                 NM_002896 
                 SEQ ID-No. 35 
               
               
                   
                   
                 RNA binding motif protein 4 
                   
                   
               
               
                   
               
               
                 36. 
                 KQMEQVAQF 
                 89-97 
                 NM_003186 
                 SEQ ID-No. 36 
               
               
                   
                   
                 transgelin 
                   
                   
               
               
                   
               
               
                 37. 
                 KVADMALHY 
                 296-304 
                 NM_006585 
                 SEQ ID-No. 37 
               
               
                   
                   
                 chaperonin containing TCPI, 
                   
                   
               
               
                   
                   
                 subunit 8 (theta) 
                   
                   
               
               
                   
               
               
                 38. 
                 LEEDSAREI 
                 68-76 
                 XM_119113 
                 SEQ ID-No. 38 
               
               
                   
                   
                 LOC204689 
                   
                   
               
               
                   
               
               
                 39. 
                 LLAERDLYL 
                 576-584 
                 NM_004613 
                 SEQ ID-No. 39 
               
               
                   
                   
                 transglutaminase 2 (C polypeptide, 
                   
                   
               
               
                   
                   
                 protein factor 4 like 2 
                   
                   
               
               
                   
               
               
                 40. 
                 LLDEEISRV 
                 44-52 
                 AB067800 
                 SEQ ID-No. 40 
               
               
                   
                   
                 RNA binding protein HQK-7 
                   
                   
               
               
                   
               
               
                 41. 
                 LLYPTEITV 
                 830-838 
                 NM_002204 
                 SEQ ID-No. 41 
               
               
                   
                   
                 integrin, alpha 3 (antigen CD49C, 
                   
                   
               
               
                   
                   
                 alpha 3 subunit of VLA-3 receptor) 
                   
                   
               
               
                   
               
               
                 42. 
                 LMDHTIPEV 
                 290-298 
                 NM_005625 
                 SEQ ID-No. 42 
               
               
                   
                   
                 syndecan binding protein 
                   
                   
               
               
                   
               
               
                 43. 
                 LQHPDVAAY 
                 229-237 
                 NM_001903 
                 SEQ ID-No. 43 
               
               
                   
                   
                 catenin (cadherin-associated 
                   
                   
               
               
                   
                   
                 protein), alpha 1, 102 kDa 
                   
                   
               
               
                   
               
               
                 44. 
                 MEDIKILIA 
                 632-640 
                 NM_001530 
                 SEQ ID-No. 44 
               
               
                   
                   
                 hypoxia-inducible factor 1, alpha 
                   
                   
               
               
                   
                   
                 subunit (basic helix-loop-helix 
                   
                   
               
               
                   
                   
                 transcription factor) 
                   
                   
               
               
                   
               
               
                 45. 
                 MEENFAVEA 
                 347-355 
                 NM_003380 
                 SEQ ID-No. 45 
               
               
                   
                   
                 vimentin 
                   
                   
               
               
                   
               
               
                 46. 
                 MQKEITAL 
                 313-320 
                 NM_001101 
                 SEQ ID-No. 46 
               
               
                   
                   
                 actin, beta 
                   
                   
               
               
                   
               
               
                 47. 
                 NEDLRSWTA 
                 151-159 
                 NM_002127 
                 SEQ ID-No. 47 
               
               
                   
                   
                 HLA-G histocompatibility antigen, 
                   
                   
               
               
                   
                   
                 class I, G 
                   
                   
               
               
                   
               
               
                 48. 
                 NEIKDSVVA 
                 673-681 
                 NM_001961 
                 SEQ ID-No. 48 
               
               
                   
                   
                 eukaryotic translation elongation 
                   
                   
               
               
                   
                   
                 factor 2 
                   
                   
               
               
                   
               
               
                 49. 
                 NVTQVRAFY 
                 439-447 
                 NM_001752 
                 SEQ ID-No. 49 
               
               
                   
                   
                 catalase 
                   
                   
               
               
                   
               
               
                 50. 
                 NYIDKVRFL 
                 116-124 
                 NM_003380 
                 SEQ ID-No. 50 
               
               
                   
                   
                 vimentin 
                   
                   
               
               
                   
               
               
                 51. 
                 PTQELGLPAY 
                 392-401 
                 NM_017827 
                 SEQ ID-No. 51 
               
               
                   
                   
                 seryl-tRNA synthetase 2 
                   
                   
               
               
                   
               
               
                 52. 
                 QEQSFVIRA 
                 422-430 
                 NM-000211 
                 SEQ ID-No. 52 
               
               
                   
                   
                 integrin, beta 2 (antigen CD18 
                   
                   
               
               
                   
                   
                 (p95), lymphocyte function- 
                   
                   
               
               
                   
                   
                 assocaited antigen 1; macrophage 
                   
                   
               
               
                   
                   
                 antigen 1 (mac-1) beta subunit) 
                   
                   
               
               
                   
               
               
                 53. 
                 QQKLSRLQY 
                 636-644 
                 NM_002204 
                 SEQ ID-No. 53 
               
               
                   
                   
                 integrin, alpha 3 (antigen CD49C, 
                   
                   
               
               
                   
                   
                 alpha 3 subunit of VLA-3 receptor) 
                   
                   
               
               
                   
               
               
                 54. 
                 QVAEIVSKY 
                 217-225 
                 NM_002210 
                 SEQ ID-No. 54 
               
               
                   
                   
                 integin, alpha V (vitronectin 
                   
                   
               
               
                   
                   
                 receptor, alpha polypeptide, antigen 
                   
                   
               
               
                   
                   
                 CD51) 
                   
                   
               
               
                   
               
               
                 55. 
                 REHAPFLVA 
                 30-38 
                 XM_208570 
                 SEQ ID-No. 55 
               
               
                   
                   
                 transport-secretion protein 2.2 
                   
                   
               
               
                   
               
               
                 56. 
                 RLAAAAAQSVY 
                 5-15 
                 NM_000581 
                 SEQ ID-No. 56 
               
               
                   
                   
                 glutathione peroxidase 1 
                   
                   
               
               
                   
               
               
                 57. 
                 RLASYLDKV 
                 90-98 
                 Y00503 
                 SEQ ID-No. 57 
               
               
                   
                   
                 keratin 19 
                   
                   
               
               
                   
               
               
                 58. 
                 RNADVFLYKY 
                 1020-1028 
                 NM_007118 
                 SEQ ID-No. 58 
               
               
                   
                   
                 triple function domain (PTPRF 
                   
                   
               
               
                   
                   
                 interacting) 
                   
                   
               
               
                   
               
               
                 59. 
                 RQGFVPAAY 
                 1012-1020 
                 NM_003127 
                 SEQ ID-No. 59 
               
               
                   
                   
                 spectrin, alpha, non-erythrocytic 1 
                   
                   
               
               
                   
                   
                 (alpha-fodrin) 
                   
                   
               
               
                   
               
               
                 60. 
                 RVIEEAKTAF 
                 198-207 
                 NM_002133 
                 SEQ ID-No. 60 
               
               
                   
                   
                 heme oxygenase (decyclingZ) 1 
                   
                   
               
               
                   
               
               
                 61. 
                 RVQPKVTVY 
                 89-97 
                 AF450316 
                 SEQ ID-No. 61 
               
               
                   
                   
                 MHC class II antigen 
                   
                   
               
               
                   
               
               
                 62. 
                 RVYPEVTVY 
                 123-131 
                 L42143 
                 SEQ ID-No. 62 
               
               
                   
                   
                 MHC HLA-DRB1*0411 
                   
                   
               
               
                   
               
               
                 63. 
                 SDHHIYL 
                 218-224 
                 NM_000034 
                 SEQ ID-No. 63 
               
               
                   
                   
                 aldolase A, fructose-bisphosphate 
                   
                   
               
               
                   
               
               
                 64. 
                 SHAILEALA 
                 204-212 
                 NM_018378 
                 SEQ ID-No. 64 
               
               
                   
                   
                 F-box and leucine-rich repeat 
                   
                   
               
               
                   
                   
                 protein 8 
                   
                   
               
               
                   
               
               
                 65. 
                 SISGVTAAY 
                 728-736 
                 NM_003870 
                 SEQ ID-No. 65 
               
               
                   
                   
                 IQ motif containing GTPase 
                   
                   
               
               
                   
                   
                 activating protein 1 
                   
                   
               
               
                   
               
               
                 66. 
                 SPVYVGRV 
                 216-223 
                 NM_004613 
                 SEQ ID-No. 66 
               
               
                   
                   
                 transglutaminase 2 (C polypeptide, 
                   
                   
               
               
                   
                   
                 protein-glutamine-gamma- 
                   
                   
               
               
                   
                   
                 glutamyltransferase) 
                   
                   
               
               
                   
               
               
                 67. 
                 SQFGTVTRF 
                 66-74 
                 NM_032390 
                 SEQ ID-No. 67 
               
               
                   
                   
                 MK167 (FHA domain) interacting 
                   
                   
               
               
                   
                   
                 necleolar phosphoprotein 
                   
                   
               
               
                   
               
               
                 68. 
                 SWNNHSYLY 
                 156-164 
                 NM_000821 
                 SEQ ID-No. 68 
               
               
                   
                   
                 gamma-glutamyl carboxylase 
                   
                   
               
               
                   
               
               
                 69. 
                 TFMDHVLRY 
                 700-708 
                 NM_001096 
                 SEQ ID-No. 69 
               
               
                   
                   
                 ATP citrate lyase 
                   
                   
               
               
                   
               
               
                 70. 
                 TLADLVHHV 
                 378-386 
                 NM_003496 
                 SEQ ID-No. 70 
               
               
                   
                   
                 transformation/transcription 
                   
                   
               
               
                   
                   
                 domain-associated protein 
                   
                   
               
               
                   
               
               
                 71. 
                 TLGALTVIDV 
                 1336-1345 
                 NM_017539 
                 SEQ ID-No. 71 
               
               
                   
                   
                 hypothetical protein 
                   
                   
               
               
                   
                   
                 DKFZp434N074 
                   
                   
               
               
                   
               
               
                 72. 
                 TQMPDPKTF 
                 46-54 
                 NM_016096 
                 SEQ ID-No. 72 
               
               
                   
                   
                 HSPC038 protein 
                   
                   
               
               
                   
               
               
                 73. 
                 VEHPSLTSP 
                 170-178 
                 M15374 
                 SEQ ID-No. 73 
               
               
                   
                   
                 HLA-DR beta gene, exon 2 
                   
                   
               
               
                   
               
               
                 74. 
                 VEPDHFKVA 
                 204-212 
                 NM_002306 
                 SEQ ID-No. 74 
               
               
                   
                   
                 lectin, galactoside-binding, soluble, 
                   
                   
               
               
                   
                   
                 3 (galectin 3) 
                   
                   
               
               
                   
               
               
                 75. 
                 VEREVEQV 
                 64-71 
                 AI278671 
                 SEQ ID-No. 75 
               
               
                   
                   
                 EST reading frame +2 
                   
                   
               
               
                   
               
               
                 76. 
                 VFIGTGATGATLY 
                 20-32 
                 NM_002489 
                 SEQ ID-No. 76 
               
               
                   
                   
                 NADH dehydrogenase 
                   
                   
               
               
                   
                   
                 (ubiquinone) 1 alpha subcomplex, 
                   
                   
               
               
                   
                   
                 4, 9 kDa 
                   
                   
               
               
                   
               
               
                 77. 
                 VLREIAEEY 
                 822-830 
                 NM_005336 
                 SEQ ID-No. 77 
               
               
                   
                   
                 high density lipoprotein binding 
                   
                   
               
               
                   
                   
                 protein (vigilin) 
                   
                   
               
               
                   
               
               
                 78. 
                 VLSLLSSVAL 
                 27-36 
                 XM_098362 
                 SEQ ID-No. 78 
               
               
                   
                   
                 LOC153339 
                   
                   
               
               
                   
               
               
                 79. 
                 VLYDRVLKY 
                 484-492 
                 NM_014230 
                 SEQ ID-No. 79 
               
               
                   
                   
                 signal recognition particle 68 kDa 
                   
                   
               
               
                   
               
               
                 80. 
                 VMDSKIVQV 
                 432-440 
                 NM_012316 
                 SEQ ID-No. 80 
               
               
                   
                   
                 karyopherin alpha 6 (importin alpha 
                   
                   
               
               
                   
                   
                 7) 
                   
                   
               
               
                   
               
               
                 81. 
                 VQRTLMAL 
                 126-133 
                 NM_003186 
                 SEQ ID-No. 81 
               
               
                   
                   
                 transgelin 
                   
                   
               
               
                   
               
               
                 82. 
                 YFEYIEENKY 
                 238-247 
                 NM_004501 
                 SEQ ID-No. 82 
               
               
                   
                   
                 heterogeneous nuclear 
                   
                   
               
               
                   
                   
                 ribonucleoprotein U (scaffold 
                   
                   
               
               
                   
                   
                 attachment factor A) 
                   
                   
               
               
                   
               
               
                 83. 
                 YIFKERESF 
                 303-311 
                 NM_015947 
                 SEQ ID-No. 83 
               
               
                   
                   
                 CGI-18 protein 
                   
                   
               
               
                   
               
               
                 84. 
                 YVYEYPSRY 
                 164-172 
                 NM_006403 
                 SEQ ID-No. 84 
               
               
                   
                   
                 enhancer of filamentation 1 
                   
                   
               
               
                   
               
               
                 85. 
                 YYRYPTGESY 
                 354-363 
                 NM_004566 
                 SEQ ID-No. 85 
               
               
                   
                   
                 6-phosphofructo-2-kinase/fructose- 
                   
                   
               
               
                   
                   
                 2, 6-biphosphatase 3 
                   
                   
               
               
                   
               
               
                 86. 
                 YYSNKAYQY 
                 230-238 
                 NM_024711 
                 SEQ ID-No. 86 
               
               
                   
                   
                 human immune associated 
                   
                   
               
               
                   
                   
                 nucleotide 2 
                   
                   
               
               
                   
               
               
                 87. 
                 SSLPTQLFK 
                 5-13 
                 NM_000618 
                 SEQ ID-No. 87 
               
               
                   
                   
                 insulin-like growth factor 1 
                   
                   
               
               
                   
               
               
                 88. 
                 ATFPDTLTY 
                 702-710 
                 NM_000210 
                 SEQ ID-No. 88 
               
               
                   
                   
                 integrin, alpha 6 
                   
                   
               
               
                   
               
               
                 89. 
                 SIFDGRVVAK 
                 107-116 
                 NM_019026 
                 SEQ ID-No. 89 
               
               
                   
                   
                 putative membrane protein 
                   
                   
               
               
                   
               
               
                 90. 
                 FRFENVNGY 
                 32-40 
                 NM_001673 
                 SEQ ID-No. 90 
               
               
                   
                   
                 asparagine synthetase 
                   
                   
               
               
                   
               
               
                 91. 
                 QRYGFSAVGF 
                 82-91 
                 NM_016321 
                 SEQ ID-No. 91 
               
               
                   
                   
                 Rh type C glycoprotein 
                   
                   
               
               
                   
               
               
                 92. 
                 AARLSLTYERL 
                 307-316 
                 NM_001183 
                 SEQ ID-No. 92 
               
               
                   
                   
                 ATPase, H+ transporting, 
                   
                   
               
               
                   
                   
                 lysosomal interacting protecin 1 
                   
                   
               
               
                   
               
               
                 93. 
                 GRYQVSWSL 
                 84-92 
                 NM_006280 
                 SEQ ID-No. 93 
               
               
                   
                   
                 signal sequence receptor, delta 
                   
                   
               
               
                   
               
               
                 94. 
                 KRFDDKYTL 
                 61-69 
                 NM_014752 
                 SEQ ID-No. 94 
               
               
                   
                   
                 KIAA0102 
                   
                   
               
               
                   
               
               
                 95. 
                 TRWNKIVLK 
                 37-45 
                 NM_024292 
                 SEQ ID-No. 95 
               
               
                   
                   
                 ubiquitin-like 6 
                   
                   
               
               
                   
               
               
                 96. 
                 LRFDGALNV 
                 242-250 
                 NM_006001 
                 SEQ ID-No. 96 
               
               
                   
                   
                 tubulin, alpha 2 
                   
                   
               
               
                   
               
               
                 97. 
                 ARFSGNLLV 
                 310-318 
                 NM_013336 
                 SEQ ID-No. 97 
               
               
                   
                   
                 protein transport protein SEC61 
                   
                   
               
               
                   
                   
                 alpha subunit isoform 1 
                   
                   
               
               
                   
               
               
                 98. 
                 NRIKFVIKR 
                 491-499 
                 NM_001518 
                 SEQ ID-No. 98 
               
               
                   
                   
                 general transcription factor II, I 
                   
                   
               
               
                   
               
               
                 99. 
                 GRVFIIKSY 
                 410-418 
                 NM_016258 
                 SEQ ID-No. 99 
               
               
                   
                   
                 high-glucose-regulated protein 8 
                   
                   
               
               
                   
               
               
                 100. 
                 SRFGNAFHL 
                 538-546 
                 NM_006445 
                 SEQ ID-No. 100 
               
               
                   
                   
                 PRP8 pre-mRNA processing factor 
                   
                   
               
               
                   
                   
                 8 homolog (yeast) 
                   
                   
               
               
                   
               
               
                 101. 
                 GRTGGSWFK 
                 26-34 
                 NM_001677 
                 SEQ ID-No. 101 
               
               
                   
                   
                 ATPase, Na+K+ transporting, beta 
                   
                   
               
               
                   
                   
                 1 polypeptide