EP1606308A2

EP1606308A2 - Tumour-associated peptides binding to mhc molecules

Info

Publication number: EP1606308A2
Application number: EP04722556A
Authority: EP
Inventors: Hans Georg Rammensee; Stefan Stevanovic; Toni Weinschenk; Claudia Lemmel; Jörn DENGJEL; Oliver Schoor
Original assignee: Immatics Biotechnologies GmbH
Current assignee: Immatics Biotechnologies GmbH
Priority date: 2003-03-24
Filing date: 2004-03-23
Publication date: 2005-12-21
Also published as: AU2004224159A1; US20090221509A1; PL378679A1; WO2004085461A2; HRP20050808A2; EP2295443A1; DE10313819A1; WO2004085461A3; US20110070253A1; CA2520497A1; JP4365405B2; JP2006521093A; AU2004224159B2; CA2731275A1; EP2280023A1

Abstract

The invention relates to a tumour-associated peptide having an amino acid sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 101 from the associated sequence protocol, said peptide being able to bind to a molecule of the human major histocompatibility complex (MHC) class I. The invention also relates to the use of said peptides and the nucleic acids coding therefor, for producing a medicament and for the treatment of tumour diseases and/or adenomatous diseases. The invention further relates to a pharmaceutical composition comprising at least one of said peptides.

Description

Tumor-associated peptides binding to MHC molecules

The present invention relates to tumor-associated peptides which have the ability to bind to a molecule of the major human histocompatibility complex (MHC), Class I.

Such peptides are used, for example, in the immunotherapy of tumor diseases.

The recognition of tumor-associated antigens (TAA) by components of the immune system plays an outstanding role in the elimination of tumor cells by the immune system. This mechanism is based on the premise that see tumor cells and normal cells there are qualitative or quantitative differences. In order to produce an anti-tumor response, the tumor cells must express antigens against which there is an immunological response that is sufficient for the elimination of the tumor.

CD8-expressing cytotoxic T-ymphocytes (hereinafter CTL) are particularly involved in the rejection of tumors. To trigger such an immune reaction by cytotoxic T cells, foreign proteins / peptides must be presented to the T cells. T cells only recognize antigens as peptide fragments if they are presented on the cell surfaces of MHC molecules. These major histocompatibility complex (MHC) molecules are peptide receptors that normally bind peptides within the cell to transport them to the cell surface. This complex of peptide and MHC molecule can be recognized by the T cells. Human MHC molecules are also referred to as human leukocyte antigens (HLA).

There are two classes of MHC molecules: MHC class I molecules, which are found on most cells with a nucleus, present peptides that result from the proteolytic degradation of endogenous proteins. MHC class II molecules occur only on professional antigen-presenting cells (APC) and present peptides of exogenous proteins that are absorbed and processed by APC in the course of endocytosis. Complexes of peptide and MHC class I are recognized by CD8-positive cytotoxic T lymphocytes, complexes of peptide and MHC class II are recognized 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 depends on the allele of the MHC molecule and on the amino acid sequence of the peptide. MHC class I binding peptides are usually 8-10 residues long and contain in their sequence two conserved residues ("anchors") that interact with the corresponding binding groove of the MHC molecule.

In order for the immune system to start an effective CTL response against tumor-derived peptides, not only must these peptides be able to bind to the particular MHC class I molecules that are expressed by the tumor cells, but they must also be from T Cells with specific T cell receptors (TCR, “T cell receptor”) are recognized.

The main goal in developing a tumor vaccine is to identify and characterize tumor-associated antigens that are recognized by CD8 ⁺ CTL.

The antigens that are recognized by the tumor-specific cytotoxic T lymphocytes, or their epitopes, can be molecules from all protein classes, such as e.g. Enzymes, receptors, transcription factors, etc. Another important class of tumor-associated antigens are tissue-specific structures, such as, for example, CT ("cancer testis") antigens, which are expressed in various types of tumor and in healthy testicular tissue.

In order for the proteins to be recognized by the cytotoxic T-lymphocytes as tumor-specific antigen and so that they can be used in therapy, certain conditions must be met: Tumor cells are expressed and not by normal tissues or only in smaller amounts than in the tumors. It is also desirable if the antigen in question is present not only in one tumor type, but also in a high concentration in others. The presence of epitopes in the amino acid sequence of the antigen is also absolutely essential, since such peptides (“immunogenic peptides”) derived from a tumor-associated antigen are said to lead to a T cell response, whether in vitro or in vivo.

TAAs therefore represent a starting point for the development of a tumor vaccine. The methods for identifying and characterizing the TAAs are based on the one hand on the use of CTL that has already been induced in patients or on the creation of differential transcription profiles between tumors and normal tissues.

However, the finding of genes which are overexpressed in tumor tissues or which are selectively expressed in such tissues does not provide precise information for the use of the antigens transcribed by these genes in immunotherapy. The reason for this is that only individual epitopes of these antigens are suitable for such use, since only the epitopes of the antigens - not the entire antigen - cause a T-cell response by means of MHC presentation. It is therefore important to select those peptides from overexpressed or selectively expressed proteins that are presented with MHC molecules, which could provide targets for specific tumor detection by cytotoxic T lymphocytes. Against this background, it is an object of the present invention to provide at least one new amino acid sequence for such a peptide which has the ability to bind to a molecule of the main human histocompatibility complex (MHC) class I.

This object is achieved according to the invention by providing a tumor-associated peptide with an amino acid sequence which is selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 101 from the accompanying sequence listing, wherein the peptide has the ability to bind to a molecule of the major human histocompatibility complex (MHC) class I.

The object underlying the invention is completely achieved in this way.

It goes without saying that the peptides identified by the tumor are synthesized in order to obtain larger amounts and for use for the purposes listed below, or are expressed in cells.

The inventors were able to isolate and identify the above peptides as specific ligands of MHC class I molecules from tumor tissue. Here, the term “tumor-associated” refers to peptides that have been isolated and identified from tumor material. These peptides, which are presented on real (primary) tumors, are therefore subject to antigen processing in a tumor cell. The specific ligands can be used in cancer therapy, for example to induce an immune response against tumor cells that express the corresponding antigens from which the peptides are derived.

Such an immune response in the form of an induction of CTL can be achieved in vivo. For this purpose, the peptide is administered to a patient suffering from a tumor disease associated with the TAA, for example in the form of a pharmaceutical composition.

On the other hand, a CTL response to a tumor 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 the antigen-presenting cells and the peptides. The CTL stimulated thereby is then cultivated and the activated CTL is administered to the patient.

It is also possible to load APC with the peptides ex vivo and to administer these loaded APC to the patient who expresses in the tumor tissue the antigen from which the peptide is derived. The APC can then present the peptide to the CTL in vivo and activate it.

However, the peptides according to the invention can also be used as diagnostic reagents.

The peptides can thus be used to find out whether a CTL population has specifically directed against a peptide or has been induced by therapy. The peptides can also be used to test the increase in precursor T cells which are reactive towards the defined peptide.

Furthermore, the peptide can be used as a marker to monitor the course of the disease of a tumor that expresses the antigen from which the peptide is derived.

The peptides identified are listed in the attached Table 1. The table also shows the proteins from which the peptides are derived and the respective positions of the peptides in the proteins in question. The English names of the proteins have been retained in order to avoid misleading translations. Furthermore, the Acc numbers are given, which are kept in the gene bank of the "National Center for Biotechnology Information" of the National Institute of Health (see http: \\ www.ncbi.nl. Ih. Gov).

The inventors were able to isolate the peptides (or ligands) from renal cell tumors of two patients, RCC68 and RCC44.

101 ligands could be identified from the tumors of the patients, which bind 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 highly expressed so-called "housekeeping" genes, which are uniform in most tissues are expressed, but many were characterized by tissue-specific and tumor-specific expression.

Some peptides have been identified that derive from proteins that are overexpressed, particularly in tumor tissue. 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; NYIDKVRFL, position 116-124) 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 from renal cell tumors.

The inventors were also able to Identify ligands derived from alpha-catenin (LQHPDVAAY, positions 229-237, SEQ ID No. 43) and beta-catenin (AQNAVRLHY, positions 481-489, SEQ ID No. 8).

In addition, the inventors were able to show in their own experiments that it was possible to generate cytotoxic T-lymphocytes (CTL) in vitro, which were specific for the selected peptides, using peptides selected by way of example. With these CTLs it was possible to specifically kill tumor cells which expressed the corresponding proteins and which also came from different tumor cell lines from different patients. Furthermore, the CTL mentioned also lysed, for example, dendritic cells which had previously been “pulsed” (loaded) with the respective peptides. It could thus be shown that human T cells could be activated in vitro with the peptides according to the invention as epitopes. The inventors could not only show that CTL, which were obtained from peripheral blood ononuclear cells (PBMNC) of a patient and which were specific for a certain peptide, could kill cells of the same tumor type of another patient. The inventors also showed that cells of other types of tumor could also be lysed with these CTLs.

In a preferred embodiment, peptides can also be used to stimulate an immune response that have the sequence ID no. 1 to 101 and in which at least one amino acid is replaced by another amino acid with similar chemical properties.

With regard to the respective MHC subtypes, these are, for example, the anchor amino acids, which can be replaced by amino acids with similar chemical properties. For example, in the case of peptides which are associated with the MHC subtype HLA-A * 02, leucine can be exchanged at position 2 with isoleucine, valine or with methionine and vice versa, and at the C-terminus leucine with valine, isoleucine and alanine, all of which have non-polar side chains.

It is also possible to use peptides with the sequence ID no. 1 to 101 to be used which have at the N- or / and C-terminal at least one further amino acid or in which at least one amino acid is deleted.

Furthermore, peptides with the sequence ID no. 1 to 101 can be used in which at least one amino acid is chemically modified. The varying amino acid (s) is (are) chosen so that the variation does not impair the immunogenicity of the peptide, ie has a similar binding affinity to the MHC molecule and the ability to stimulate T cells.

According to the invention, the peptide can be used for the treatment of tumor diseases and / or adenomatous diseases.

The tumor diseases to be treated include, for example, kidney, breast, pancreas, stomach, testicular and / or skin cancer. The list of tumor diseases is only an example and is not intended to limit the area of use. The inventors were able to show in their own experiments that the peptides according to the invention are suitable for such a use. It proved that specially generated CTLs, which were specific for certain peptides, could effectively and selectively kill tumor cells.

In principle, several application forms are possible for the use of tumor-associated antigens in a tumor vaccine. Tighe et al. , 1998, Gene vaccination: plasmid DNA is more than just a blueprint, Immunol. Today 19 (2): 89-97 that the antigen can either be administered as a recombinant protein with suitable adjuvants or carrier systems or as the cDNA coding for the antigen in plasmid vectors. In these cases, the antigen in the patient's body must be processed and presented by antigen-presenting cells (APC) in order for an immune response to be triggered. Melief et al., 1996, Peptide-based cancer vaccines, Curr. Opin. I munol. 8: 651-657, show a further possibility, namely the use of synthetic peptides as vaccines.

In a preferred embodiment, the peptide can be used with the addition of adjuvants, or else on its own.

For example, the granulocyte macrophage colony stimulating factor (GM-CSF) can be used as an adjuvant. Further examples of such adjuvants are aluminum hydroxide, emulsions of mineral oils, such as Freund's adjuvant, saponins or silicon compounds.

The use with adjuvants offers the advantage that the immune response triggered by the peptide can be strengthened and / or that the peptide is stabilized.

In another preferred embodiment, the peptide is used bound on an antigen-presenting cell.

This measure has the advantage that the peptides can be presented to the immune system, in particular the cytotoxic T-lymphocytes (CTL). This enables the CTL to recognize the tumor cells and specifically kill them. As antigen-presenting cells, for example, dendritic cells, monocytes or B-lyitiphoocytes are suitable for such an application.

The cells will be loaded with the peptides ex vivo, for example. On the other hand, there is also the possibility of the cells with the DNA coding for the peptides or the corresponding Transfect RNA to then express the peptides on the cells.

The inventors were able to show in their own experiments that it is possible to load dendritic cells (DC) with specific peptides and that these loaded dendritic cells activate peptide-specific CTL. This means that the immune system can be stimulated to develop CTL against the tumors that express the corresponding peptides.

The antigen-presenting cells carrying the peptide can either be used directly, or they can be activated, for example, with the heat shock protein gp96 before use. This heat shock protein induces the expression of MHC class I molecules and of costimulatory molecules such as B7 and also stimulates the production of cytokines. Overall, this triggers the triggering of immune responses.

In another preferred embodiment, the peptides are used for labeling leukocytes, in particular T-lymphocytes.

This use is advantageous if the peptides are to be used to find out whether there is a CTL population specifically directed against a peptide.

Furthermore, the peptide can be used as a marker for assessing a course of therapy for a tumor disease. The peptide can also be used for monitoring the therapy in other immunizations or therapies. The peptide can therefore not only be used therapeutically but also diagnostically.

In a further embodiment, the peptides are used to produce an antibody.

Polyclonal antibodies can be obtained in a conventional manner by immunizing animals by injection of the peptides and subsequent purification of the immunoglobulin.

Monoclonal antibodies can be produced according to standard protocols, for example in Methods Enzymol. (1986), 121, Hybridoma technology and monoclonal antibodies.

In another aspect, the invention also relates to a pharmaceutical composition which contains one or more of the peptides.

This composition is used, for example, for parenteral administration, for example subcutaneously, intradermally or intramuscularly, or for oral administration. The peptides are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. In addition, the composition can contain auxiliary substances such as buffers, binders, diluents, etc. contain.

The peptides can also be administered together with immunostimulating substances, for example cytokines. A comprehensive description of auxiliaries, such as can be used in such a composition, is presented, for example, in A. Kibbe, Handbook of Pharmaceutical Excipients, 3rd Ed., 2000, American Pharmaceutical Association and pharmaceutical press. The agent can be used for the prevention, prophylaxis and / or therapy of tumor diseases and / or adenomatous diseases.

The pharmaceutical agent which contains at least one of the peptides with the sequence ID no. 1 to 101 is administered to a patient suffering from a tumor disease with which the peptide or antigen in question is associated. This can trigger a tumor-specific immune response based on tumor-specific CTL.

The amount of the peptide or peptides present in the pharmaceutical composition is present in a therapeutically effective amount. The peptides contained in the composition can also bind to at least two different HLA types.

In a further aspect, the present invention relates to nucleic acid molecules which code for the peptides with the sequence ID numbers 1 to 101, and to the use of at least one of the nucleic acid molecules for the production of a medicament for the treatment of tumor diseases and / or adenomatous diseases.

The nucleic acid molecules can be DNA or RNA molecules and can also be used for the immunotherapy of cancer. The peptide expressed by the nucleic acid molecule induces an immune response against tumor 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 are generated with the aid of the nucleic acid molecule which codes for the peptides. was changed in such a way that it contains a peptide with the sequence ID no. 1 to 101 produced.

For this purpose, the cells are transfected with the DNA encoding the peptides or the corresponding RNA, as a result of which the peptides are expressed on the cells. For such use, suitable antigen-presenting cells are, for example, dendritic cells, monocytes or other human cells which express molecules which are suitable for co-stimulation, such as, for example, B7.1 or B7.2.

The invention further relates to a diagnostic method in which the presence of one of the new peptides is used as a diagnostic marker, and to a method for treating a pathological condition in which an immune response against a protein of interest is triggered, a therapeutically effective amount of at least one of the new peptides is administered.

The inventors have recognized that the new peptides can also be used as markers for a pathological condition, so that a corresponding diagnostic method in which a blood sample is taken from the patient and examined in the usual way for the presence of lymphocytes directed against one of the new peptides is used as early detection or for the targeted selection of a suitable treatment.

The invention further relates to an electronic storage medium which contains the amino acid sequence of at least one of the new peptides and / or the nucleic acid sequence of nucleic acid molecules coding for the new peptides.

Based on this storage medium, the information for the pep- tide are quickly provided, which are suitable for the treatment of the pathological condition.

It goes without saying that the features mentioned above and those yet to be explained below can be used not only in the combination indicated in each case, but also on their own without departing from the scope of the present invention.

Exemplary embodiments of the invention are explained in the following examples.

example 1

1.1. patient samples

From the Department of Urology, University of Tübingen, two samples were obtained from patients who had histologically confirmed renal cell tumors. Neither patient had received preoperative therapy. Patient No. 1 (hereinafter referred to as RCC68) had the following HLA typing: HLA-A * 02 A * 29 B * 15 B * 45; patient No. 2 (hereinafter referred to as RCC44) HLA-A * 3201 A * 1101 B * 4002 B * 2705 Cw * 0202.

1.2. Isolation of MHC class I bound peptides

The shock-frozen tumor 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 using the W6 / 32 monoclonal antibody specific for HLA class I molecules or the BB7.2 monoclonal antibody specific for HLA-A2. Barnstable, CJ 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. & Brodsky, FM, 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, described the production and use of these antibodies.

1.3. mass spectroscopy

The peptides were separated by "reversed phase HPLC" (SMART system, µRPC C2 / C18 SC 2.1 / 19, Amersham Pharmacia Biotech) and the fractions obtained were analyzed by nanoESI MS. The procedure was as 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 Immunolgy, 30: 2216-2225.

The peptides obtained from tumor tissue were identified by capillary LC-MS as just mentioned, but with minor changes: 100 μl of the samples were each loaded, desalted and on a 300 μm * 5 mm C18 μ precolumn (LC packings ) pre-concentrated. The solvent and sample were delivered using a syringe pump (PHD 2000, Harvard Apparatur, Inc.) with a sealed 100 ul syringe (1710 RNR, Hamilton) at a rate of 2 ul / min. To separate the peptides, the preconcentration column was placed in front of a 75 μm * 250 mm C-18 column (LC Packings). A binary gradient with 25-60% B was then run within 70 min, the flow rate being reduced from 12 μl / min to approximately 300 nl / min, using a TEE connection (ZT1C, Valco) and a 300 μm * 150 mm C-18 column.

To ensure that the system was free of remaining peptides, an empty sample was measured. On-line- Fragmentation was performed as described and the spectra of the fragments were analyzed manually. The database searches (NCBInr, EST) were carried out using MASCOT (http://www.matrixscience.com).

1.4. Identification of the MHC class I ligands from tumor tissue of patients RCC68 and RCC44

In the attached sequence listing and in attached Table 1 the ligands are listed which were bound to the HLA molecules of the patients RCC68 and TCC44. The peptides associated with HLA-A * 02 had the allele-specific peptide motif: Leucine, valine, isoleucine, alanine or methionine were found at position 2 and leucine, valine, isoleucine, or alanine was found at the C-terminus , Most of the ligands were derived from so-called "housekeeping" proteins, but ligands of proteins associated with tumors could also be identified. For example, fragments of Vi entin (ALRDVRQQY, position 268-276, SEQ ID No. 7; EENFAVEA, position 348-355, SEQ ID No. 15; MEENFAVEA, position 347-355; NYIDKVRFL, position 116-124 ) 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 from renal cell tumors.

1.5. Detection of peptide-specific T cells in the normal CD8 ⁺ T cell repertoire

To detect peptide-specific T cells, mononuclear cells from peripheral blood of healthy patients were treated with the HLA-A * subtype tetramers in question, which were constituted with peptides in question: To produce the tetramers, recombinant HLA-A * subtype molecules were constituted in vitro with the peptides, purified by gel filtration, biotinylated and mixed with streptavidin to link the monomers ,

Basically, the results of the double staining are evaluated by analysis using FACS and the specific binding of the peptide tetramers is verified.

Example 2

In order to analyze the presentation of the selected peptides by tumor cells and the recognition of the peptides by CTL, CTLs were induced in vitro which were specific for the selected peptides. For this purpose, dendritic cells (DC) were used, which came from peripheral blood mononuclear cells (PBMNC) from healthy donors, which had the HLA (sub) type in question.

2.1. Obtaining the DC

The DC were isolated from heparinized blood by Ficoll / Paque (Biochrom, Berlin, Germany) density gradient centrifugation from PBMNC. The heparinized blood was obtained from "buffy coat" preparations from healthy donors of the blood bank of the University of Tübingen. The cells were placed on 6-well plates (Falcon, Heidelberg, Germany) (1 x 10 ⁷ cells / 3 ml per well) in RP10 Medium (RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum and with antibiotics) sows. After a 2-hour incubation at 37 ° C and 5% CO ₂ , the non-adherent cells were removed and the adherent blood monocytes were cultured in RP10 medium, the following cytokines being added to the medium: human recombinant GM-CSF ( granulocyte macrophage colony stimulating factor; Leukomax, Novartis; lOOng / ml), Interleukin IL-4 (R&D Systems, Wiesbaden, Germany; 1000 IU (ml) and TNF-α (tumor necrosis factor α) (R&D Systems, Wiesbaden, Germany; 10 ng / ml).

2.2. Synthesis of the peptides

The peptides selected by way of example were synthesized using F-moc (9-fluorenylmethyloxycarbonyl) protective groups on a peptide synthesizer (432A, Applied Biosystems, Weiterstadt, Germany) and analyzed by “reversed phase” HPLC and mass spectroscopy Amounts of the identified peptides are made.

2.3. Induction of an antigen-specific CTL response using restricted synthetic peptides

For the induction of CTL the steps in 2.1. obtained DC (5 x 10 ⁵ ) with the from step 2.2. The peptides obtained were pulsed at 50 μg / ml for 2 hours, then washed and incubated with 2.5 × 10 ⁶ autologous PBMNC in RP10 medium. After a 7-day cultivation period, the cells were restimulated with autologous, peptide-pulsed PBMNC. 1 ng / ml human recombinant interleukin IL-2 (R&D Systems) was added on days 1, 3 and 5. The cytotoxic activity of on CTL induced in this way was examined on day 5 after the last re-stimulation by means of a standardized ⁵¹ Cr release assay (see below, under 2.4 .: CTL assay).

2.4. CTL assay

Tumor cells, peptide-pulsed cells from different cell lines and autologous DC were used as target cells for the CTL assays. Peptide-pulsed cells were pulsed with 50 ug / ml peptide for 2 hours. All target cells were labeled in RPIO medium (RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum and with antibiotics) for 1 hour at 37 ° C. with [ ⁵¹ Cr] sodium chromate ( ⁵¹ Cr). Then 10 ⁴ cells / per well were placed on a 96-well plate with a rounded bottom. Different amounts of CTL were added to reach a final volume of 200 ul, followed by incubation for 4 hours at 37 ° C. The supernatants (50 μl / well) were then harvested and counted in a beta plate counter. The specific lysis was calculated in percent as follows: 100 x (experimental release - spontaneous release / maximum release - spontaneous release). The spontaneous and maximum release were determined in the presence of either medium or 2% Triton X-100.

2nd 5. Results of CTL induction

a) CTL cytotoxic activity against peptide-pulsed DC

In ⁵¹ Cr release assays (see under 2.4.) The cytotoxic activity of induced CTL (see under 2.3.) Was tested against T2 or DC cells. 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 endoplasmic reticulum, where they associate with MHC molecules.)

The results of this release assasy showed that antigen-specific cell killing could be achieved with CTL cell lines obtained after 2 weeks of restimulation: only those cells that killed the selected ones were killed by an increasing amount of CTL Presented peptides; the control cells loaded with irrelevant peptides were not killed. This showed the specificity of the cytolytic activity.

b) CTL cytotoxic activity against tumor cell lines

In a next step, a ⁵¹ Cr release assay was used to test whether the CTL, which were specific for the selected peptides, recognize and lyse tumor cells which endogenously express the selected peptides.

Various ⁵¹ Cr-labeled cell lines expressing the corresponding HLA molecules were used for this purpose: HCT 116 (colorectal 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; purchased from the ATCC, American Type Culture Collection), Mel 1479 (melanoma; received from Prof. G. Pawelec, Tübingen, Germany) and U 266 (multiple myeloma; obtained from Prof. G. Pawelec, Tübingen, Germany). These cell lines express certain proteins as target structures. The B cell line Croft (EBV (Epstein-Barr virus) immortalized; HLA-A * 02 positive; obtained from OJ Finn, Pittsburgh, USA) and the cell line SK-OV-3 (ovarian tumor; HLA-A * 03 -positive; obtained from OJ Finn, Pittsburgh, USA) were included in the studies as negative controls. K 562 cells (for example available from the German Collection of Microorganisms and Cell Cultures, DSMZ; ACC 10) were used to determine the activity of natural killer cells (NK), since this cell line is highly sensitive to these killer cells.

All cell lines were cultivated in RP10 medium (RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum and with antibiotics).

With the above tumor cell lines and the same as in 2.3. induced CTL, ⁵¹ Cr release assays were performed (see 2.4.).

In these tests, the CTLs, which are specific for the selected peptides, efficiently lysed tumor cells that express both the corresponding HLA molecule and the selected peptides. The specific lysis was - as in 2.4 above. indicated - measured by the ⁵¹ Cr release. The control cell line SK-OV-3 (HLA-A- * 02 negative), however, was not lysed by the CTL induced by the peptides bound by HLA-A * 02. This showed that the peptides in connection with the corresponding HLA molecules had to be presented on the tumor cells in order to efficiently lyse the target cells. This also confirms the antigen specificity and the MHC restriction of the CTL. The CTL cells induced in vitro by the peptides also did not recognize the cell line K562, which shows 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 CTL, inhibition assays were carried out with non- ⁵¹ Cr-labeled (“cold”) inhibitor cell lines.

The ability of peptide-pulsed cell lines to inhibit or compete for the lysis of tumor cells was analyzed. An excess of inhibitor (ie pulsed, unlabeled cells) was used for this. The ratio of the inhibitor (peptide-pulsed cells) to the target (tumor cells) was 20: 1. When the inhibitor cell lines were lysed, ⁵¹ Cr could not be released because the inhibitor cell lines were unlabeled.

Cell line T2 (HLA-A * 02; TAP deficient; see under 2.5.a)) was used as an inhibitor. This cell line T2 was pulsed with the relevant peptides or an irrelevant control peptide before the assays.

Without the inhibitor cells, lysis of the tumor cells by CTL was observed. It was also possible to show that if the inhibitor target was excess, there was no lysis of the tumor cells (and thus no ⁵¹ Cr release), provided the inhibitor target was pulsed with the corresponding peptides. The activity of the CTL was directed towards the unlabeled T2 cells present in excess, so that these and not the tumor cells were lysed. The T2 cells, which were pulsed with an irrelevant peptide, could not inhibit the lysis of the tumor cells by the CTL, so that released ⁵¹ Cr could be measured.

The MHC restriction and the antigen specificity of the cytotoxic activity mediated by the HLA-A * 02 peptide-induced CTL could be determined using an HLA-A * 02-specific monoclonal antibody and in an inhibition assay with unlabelled ("cold") inhibitor: The A 498 tumor cells were blocked by adding the HLA-A * 02-specific antibody (monoclonal antibody BB7.2, IgG2b, obtained from S. Stefanovic, Tübingen), so that they were not lysed by adding the CTL and no ⁵¹ Cr was released. A non-specific antibody which did not block the HLA-A * 02 molecule (ChromPure mouse IgG, Dianova, Germany) was used as a control. The cells were used for these inhibition experiments Sowing on the 96-well plates incubated for 30 min with 10 μg / ml antibody.

Furthermore, it was found that the T2 competition cell line, which was pulsed with an irrelevant peptide, could not inhibit the CTL-mediated lysis of the tumor cell line A 498, but that the T2 inhibitor cell line pulsed with the corresponding peptide could inhibit the lysis of the tumor Cell line could inhibit, so that in the latter case no ⁵¹ Cr release could be measured. d) Specific lysis of transfected DC

In a next experiment, the cytotoxic activity of the CTL was analyzed using an autologous approach. For this purpose, autologous DC, which were obtained from the same PBMNC as those used for CTL induction (see under 2.2.), Were used as target cells. Before the CTL assay was carried out, the DCs were electroporated with RNA which had either been isolated from tumor cell lines beforehand or which was control RNA. The total RNA was isolated from the tumor cells using the QIAGEN Rneasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The amount and purity of the RNA was determined photometrically and stored in aliquots at -80 ° C.

Prior to electroporation on day 6, immature DCs were washed twice with serum-free X-VIVO 20 medium (BioWhittaker, Walkersville, USA) and resuspended at a final concentration of 2 x 10 ⁷ cells / ml. Then 200 μl of the cell suspension were mixed with 10 μg of the total RNA and electroporated in a 4 mm cuvette using an Easyject Plus ™ (Peqlab, Erlangen Germany) (parameters: 300 V, 150 μF, 1540 Ω, pulse time: 231 ms). After electroporation, the cells were immediately transferred to RP10 medium and placed back in the incubator. More than 80% of the cells were viable after electroporation.

After performing the CTL assay with CTL induced by the selected peptides (see under 2.4.), A specific lysis of DC was observed, which was electroporated with RNA from peptide-expressing tumor cell lines. DC that were electroporated with RNA from a non-peptide-expressing tumor cell line, however, were not lysed.

This shows that - after transfection of the DC with RNA from peptide-positive tumor cells - the identified peptides are processed and presented.

e) Induction of peptide-specific CTL in a patient with chronic lymphoblastic leukemia

In a further experiment, CTL was generated from the PBMNC of a patient with chronic lymphatic leukemia (CLL), which were specific for selected peptides. Furthermore, autologous primary CLL cells and DC of this patient were used as ⁵¹ Cr-labeled targets in an assay in which ^sl Cr release was mediated by the peptide-induced CTL. As a result, both the autologous DC of this patient, which was pulsed with the selected peptides, and the autologous CLL cells were lysed by the peptide-induced CTL. DC, which were pulsed with an irrelevant peptide, however, were not lysed. Non-malignant B cells and the cell line K 562 were also not lysed by the CTL.

The specificity of the CTL response was confirmed in a target inhibition assay, the cell line T2 (see above) being used as the inhibitor cells, each of which was pulsed with the selected peptides or with an irrelevant peptide. The CTL induced by the use of the peptides also lysed the excess inhibitor cell lines which were pulsed with the relevant peptides that the ⁵¹ Cr-labeled tumor cells were not lysed in this case.

In summary, the inventors were able to show that the identified peptides are promising substances in the context of immunotherapy for a large number of (tumor) diseases.

Table 1

Sequence position / gene type Acc. No. SEQ ID No. •

1. AAFPGASLY 63-71 N JD14764 SEQ ID no. 1

DAZ associated protein 2

2. AELATRA P 137-145 NM_002230 SEQ ID no. 2 junction placoglobin

3. AFFAER YY 397-405 NM_00U56 SEQ ID no. 3 annexin A7

4.ALAT IHQV 26-34 NM_016319 SEQ ID no. 4

C0P9 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. A QEMVHQV 806-814 N _006403 SEQ ID no. 6 enhancer of filamentation 1

7. A RDVRQQY 268-276 N _003380 SEQ ID no. 7 vimentin

8. AQNAVR HY 481-489 NM_001904 SEQ ID no. 8 σatenin (cadherin-associated protein), beta 1, 88kDa

9. AQPGFFDRF 1006-1014 N _001849 SEQ ID no. 9 collages, type VI, alpha 2

(CO 6A2), transcript variant

2C2

10. AVCEVALDY 2260-2268 NM_003128 SEQ ID no. 10 spectrin, beta, non-erythrocytic 1

11. AVLGAWAV 161-169 12679 SEQ ID no. 11 Cwl antigen

12. DAILEELSA 154-162 N _024591 SEQ ID no. 12 hypothetical protein F J11749

13. EEHPT TEA 101-110 NM_001613 SEQ ID no. 13 actin, alpha 2, smooth muscle, aorta 14. EEMPQVHTP NM 002388 SEQ ID no. 14

15.EENFAVEA NM 003380 SEQ ID no. 15

16. EENKLIYTP NM 012106 SEQ ID no. 16

17.FAEGFVRAL NM 002228 SEQ ID no. 17

18. FFGETSHNY NM 018834 SEQ ID no. 18

19. F PH AYTY NM 014795 SEQ ID no. 19

20.GEPRFISVGY Z46810 SEQ ID no. 20

21.GLATDVQTV NM 002795 SEQ ID no. 21

22. G NDETYGY NM 001677 SEQ ID no. 22

23. GQEFIRVGY NM 018154 SEQ ID no. 23

24. GQFPGHNEF NM 006449 SEQ ID no. 24

25. GQPWVSVTV AC005912 SEQ ID no. 25

26. GYLHDF KY NM 012286 SEQ ID no. 26

27. HQITV HVY NM 021814 SEQ ID no. 27

28. HVIDVKF Y NM 001923 SEQ ID no. 28

29. HVND FLQY 484-492 AB023222 SEQ ID no. 29 KIAA1005

30. IAMATVTAL 249-257 NM 000034 SEQ ID no. 30 aldolase A, fructose bisphosphates

31. IGID GTTY 7-15 NM 005345 SEQ ID no. 31 heat shock 70kDa protein 1A

32. ILHDDEVTV 15-23 NM 001003 SEQ ID no. 32 ribosomal protein, large, pl

33. IQKEST H 61-69 NM 003333 SEQ ID no. 33 ubiquitin A-52 residue ribosomal protein fusion product 1

34. ISRΞLYEY 70-77 BC022821 SEQ ID no. 34 σlone MGC: 39264 IMAGE: 5087938

35. KLHGVNINV 59-67 NM 002896 SEQ ID no. 35

RNA binding motif protein 4

36. KQMΞQVAQF 89-97 NM 003186 SEQ ID no. 36 transgeline

37. VADMA HY 296-304 NM 006585 SEQ ID no. 37 σhaperonin containing TCPl, subunit 8 (theta)

38. LEΞDSAREI 68-76 XM 119113 SEQ ID no. 38 OC204689

39. AERDLYL 576-584 NM 004613 SEQ ID no. 39 transglutaminase 2 (C polypeptides, protein-glutamine-gamma-gluta yl transferase)

40. LDEEISRV 44-52 AB067800 SEQ ID no. 40

RNA binding protein HQK-7

41. L YPTEITV 830-838 NM 002204 SEQ ID no. 41 integrin, alpha 3 (antigen

CD49C, alpha 3 subunit of VLA-3 reσeptor)

42. LMDHTIPEV 290-298 NM 005625 SEQ ID no. 42 syndecan binding protein

43. LQHPDVAAY 229-237 NM 001903 SEQ ID no. 43 σatenin (σadherin-associated protein), alpha 1, 102kDa 44. MEDIKI IA NM 001530 SEQ ID no. 44

45. MEENFAVEA NM 003380 SEQ ID no. 45

46. MQKΞITAL NM 001101 SEQ ID no. 46

47. NED RS TA NM 002127 SEQ ID no. 47

48. NEIKDSWA NM 001961 SEQ ID no. 48

49. NVTQVRAFY NM 001752 SEQ ID no. 49

50. NYIDKVRFL NM 003380 SEQ ID no. 50

51. PTQELGLPAY NM 017827 SEQ ID no. 51

52.QEQSFVIRA NM 000211 SEQ ID no. 52

53.QQKLSRLQY NM 002204 SEQ ID no. 53

54. QVAEIVSKY NM 002210 SEQ ID no. 54

55. RΞHAPFLVA XM 208570 SEQ ID no. 55

56. RLAAAAAQΞVY NM 000581 SEQ ID no. 56

57. RLASYLDKV Y00503 SEQ ID no. 57 58. RNADVFLKY 1020-1028 NM 007118 SEQ ID no. 58 triple functional 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 (decycling) 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-DRBl * 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-riσh 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 polypeptides, protein-glutamine-gamma-glut amy lt rans f eras e)

67.SQFGTVTRF 66-74 NM 032390 SEQ ID no. 67

MKI67 (FHA domain) interacting nucleolar phosphoprotein

68.SWNNHSYLY 156-164 NM 000821 SEQ ID no. 68 ga ma-glutamyl carboxylase

69. TFMDHVLRY 700-708 NM 001096 SEQ ID no. 69

ATP nitrate 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 leαtin, galaαtoside-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, 4,9kDa

77. VLREIAEEY 822-830 NM 005336 SEQ ID no. 77 high density lipoprotein binding protein (vigilin)

78.VLSLLΞSVAL 27-36 XM 098362 SEQ ID no. 78 LOC153339

79. VLYDRVLKY 484-492 NM 014230 SEQ ID no. 79 signal recognition particle

68kDa

80. VMDSKIVQV 432-440 NM 012316 SEQ ID no. 80 karyopherin alpha 6 (importin alpha 7)

81st VQRTLMAL 126-133 NM 003186 SEQ ID no. 81 transgeline

82. YFΞYIEENKY 238-247 NM 004501 SEQ ID no. 82 heterogeneous nuclear ribonu- σleoprotein U (scaffold attach- ment faσtor A)

83. YIFKERESF 303-311 NM 015947 SEQ ID no. 83 CGI-18 protein

84. YVYEYPSRY 164-172 NM 006403 SEQ ID no. 84 enhanσer of filamentation 1

85. YYRYPTGΞSY 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 size th factor 1 88.ATFPDTLTY 702-710 NM_000210 SEQ ID no. 88 integrin, alpha 6

89.SIFDGRWAK 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. ARLSLTYERL 307-316 NM_001183 SEQ ID no. 92

ATPase, H + transporting, lysosomal interacting protein 1

93. GRYQVS SL 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 5 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 SΞC61 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 homologous (yeast)

101. GRTGGSWFK 26-34 NM 001677 SEQ ID No. 101

ATPase, Na + / K + transporting, beta 1 polypeptides

Claims

claims

1. Tumor-associated peptide with an amino acid sequence which is selected from the group consisting of SEQ ID no. 1 to SEQ ID no. 101 from the attached sequence listing, wherein the peptide has the ability to bind to a molecule of the major human histocompatibility complex (MHC) class I.

2. Peptide according to claim 1, characterized in that at least one amino acid is replaced by another amino acid with similar chemical properties.

3. Peptide according to claim 1 or 2, characterized in that N- or / and C-terminal at least one further amino acid is present.

4. Peptide according to one of claims 1 to 3, characterized in that at least one amino acid is deleted.

5. Peptide according to one of claims 1 to 4, characterized in that at least one amino acid is chemically modified.

6. Use of one or more peptides according to one of claims 1 to 5 for the manufacture of a medicament for the treatment of tumor diseases and / or adenomatous diseases.

7. Use of the peptide according to one of claims 1 to 5 for the treatment of tumor diseases and / or adenomatous diseases.

8. Use according to claim 6 or 7, characterized in that the disease is kidney, breast, pancreatic, stomach, bladder and / or testicular cancer.

9. Use according to claim 7 or 8, characterized in that the peptide is used together with an adjuvant.

10. Use according to claim 7 or 8, characterized in that the peptide is used bound on an antigen-presenting cell.

11. Use of the peptide according to one of claims 1 to 5 for labeling leukocytes, in particular T-lymphocytes.

12. Use according to claim 11 for assessing a course of therapy in a tumor disease.

13. Use of the peptide according to one of claims 1 to 5 for the production of an antibody.

14. Pharmaceutical composition containing one or more peptides according to one of claims 1 to 5.

15. A nucleic acid molecule coding for the peptide according to one of claims 1 to 5.

16. Use of at least one nucleic acid molecule coding for the peptide according to one of claims 1 to 5, for the manufacture of a medicament for the treatment of tumor diseases and / or adenomatous diseases.

17. Vector comprising the nucleic acid molecule according to claim 15.

18. Cell which has been genetically modified with the aid of the nucleic acid molecule according to claim 15 or with the aid of the vector according to claim 17 in such a way that it produces a peptide according to one of claims 1 to 5.

19. Diagnostic method in which the presence of a peptide according to one of claims 1 to 5 is used as a diagnostic marker.

20. A method for treating a pathological condition in which an immune response is triggered against a protein of interest, characterized in that a therapeutically effective amount of at least one of the peptides according to one of claims 1 to 5 is administered.

21. Electronic storage medium which contains the amino acid sequence of at least one of the peptides according to one of claims 1 to 5 and / or the nucleic acid sequence of the nucleic acid molecule according to claim 15.