EP1181312A1

EP1181312A1 - Cytotoxic t-cell epitopes of the papilloma virus l1-protein and use thereof in diagnosis and therapy

Info

Publication number: EP1181312A1
Application number: EP00940295A
Authority: EP
Inventors: Ingrid Jochmus; John Nieland
Original assignee: Medigene AG
Current assignee: Medigene AG
Priority date: 1999-06-01
Filing date: 2000-05-31
Publication date: 2002-02-27
Also published as: CA2375802A1; US6838084B1; US20040258708A1; WO2000073335A1; DE19925199A1; JP2003502027A; AU5527500A

Abstract

The invention relates to a papilloma virus T-cell epitope with an amino acid sequence ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN, SMVTSDAQI, and/or a functionally active variant thereof. The invention also relates to the use thereof in diagnosis and therapy.

Description

Cytotoxic T cell epitopes of the Papillomavirus Ll protein and their use in diagnostics and therapy

The present invention relates to a papillomavirus T cell epitope with an amino acid sequence ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVPVTYP, YL

ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN, SMVTSDAQI, and / or a functionally active variant thereof, and their use in diagnostics and therapy.

The papilloma viruses, also called wart viruses, are double-stranded DNA viruses with a genome size of about 8000 base pairs and an icosahedral capsid with a diameter of approx. 55 nra. To date, more than 100 different human pathogenic papilloma virus (HPV) types are known, some of which, e.g. HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-45, HPV-52 or HPV-58, malignant tumors and others e.g. HPV-6, HPV-1 1 or HPV-42 can cause benign tumors.

The genome of the papillomaviruses can be divided into three areas: The first area concerns a non-coding region that contains regulatory elements for the transcription and replication of the virus. The second region, so-called E (early) region, contains various protein-coding sections E1-E7, of which, for example, the E6 and E7 proteins are responsible for the transformation of epithelial cells and the E1 protein controls the DNA copy number. The E6 and E7 regions are so-called oncogenes that are also malignant degenerate cells are expressed. The third region, also called the L (late) region, contains two protein-coding sections L1 and L2 which code for structural components of the virus capsid. More than 90% of the Ll protein is present in the viral capsid, the ratio of L1: L2 being generally 30: 1. For the purposes of the present invention, the term L1 protein is understood to mean the main capsid protein of the papillomaviruses (Baker T. et al. (1991) Biophys. J. 60, 1445).

In over 50% of cases, HPV-16 has been linked to cervical cancer (cervical cancer). HPV-16 is the main risk factor for the formation of cervical neoplasia. The immune system plays an important role in the progression of the disease. Cellular immune responses and, in particular, antigen-specific T lymphocytes are probably important for the defense mechanism. It was also found that in highly malignant cervical intra-epithelial neoplasia (CIN II / III) and cervical tumors, the E7 gene is constitutively expressed in all layers of the infected epithelium. Therefore, the E7 protein in particular is regarded as a potential tumor antigen and as a target molecule for activated T cells (see e.g. WO 93/20844). However, the E7-induced cellular immune response in the patient does not appear to be strong enough to influence the course of the disease. The immune response may be boosted by appropriate vaccines.

It could be shown that the expression of the Ll gene or the coexpression of the Ll and L2 gene can lead to the formation of capsomers, stable capsomers, capsids or virus-like particles (VLPs for virus-like particles) ( see for example WO 93/02184, WO 94/20137 or WO 94/05792). Capsomers are understood to be an oligomeric configuration that is made up of five Ll proteins. The capsomer is the basic building block from which viral capsids are built. Stable capsomers are capsomers that are unable to form capsids. Capsids are the shell of the papillomavirus, which is composed, for example, of 72 capsomers (Baker T et al (1991) Biophys J 60, 1445) VLP is a capsid which morphologically and in its antigenicity resembles an intact virus. The VLPs were able to trigger a humoral immune response, which is characterized by the formation of neutralizing antibodies, can be used in various deep systems. The formation of virus-neutralizing antibodies against L1 and / or L2 proteins is of less clinical importance, however, if the virus infection has already occurred, since virus is responsible for the infection -fected cells, not antibodies, but a virus-specific cytotoxic T-cell (CTL) response seems to be necessary. And although VLPs are able to trigger a cytotoxic T-cell response, one seems to be exclusively against the capsid proteins L 1 and / or L2 immune response not suitable for combating a tumor caused by papillomaviruses ignet

So-called chimeric papillomavirus-like particles (CVLPs for chimeπc virus-like particles) were therefore developed, which consist of a fusion protein of the capsid protein L1 and the potential tumor antigen E7 (WO 96/1 1272 and Muller, M et al (1997) Virology, 234, 93) The CVLPs triggered a humoral immune response against the E7 protein only to a small extent (Muller, M et al (1997), supra). However, some of the CVLPs tested actually induce the desired E7-specific cytotoxic T cell response Mausen (see also Peng S et al (1998) Virology 240, 147-57) CVLPs are therefore of interest both for the development of a vaccine and for the treatment of already existing infections and resulting tumors, since they are presented via class I MHC molecules E7 peptides of tumor cells were the target molecules of cytotoxic T cells

A vaccine consisting of CVLPs prevents the pseudoinfection of the

Underlying cells by the CVLPs This means that the CVLPs enter the cell like viruses, where they are processed into peptides, the peptides are then processed on MHC Class I and II molecules are loaded and ultimately presented with CD8 or CD4 positive T cells. As a result of this stimulation, CD8 cells can differentiate into cytotoxic T cells and then cause a cellular immune response, while CD4 cells develop to T helper cells and stimulate B cells to a humoral or CD8-positive T cells to a cytotoxic immune response and can even induce the lysis of infected cells

Small peptides can already bind to MHC class I molecules on the cell surface and then stimulate CDS- or CD4-posιtι \ e cells to a cellular immune response without further processing. However, a certain peptide can only be bound by certain MHC molecules Polymorphism of the MHC molecules in natural populations can therefore only bind and present a certain peptide by a small part of a population. Presentation in the sense of the present invention means when a peptide or protein fragment binds to an MHC molecule, whereby this binding can take place, for example, in the endoplasmic reticulum, in the extracellular space, the endosomes, proendosomes, lysosomes or protysosomes, and when this MHC-molecule-peptide complex is then bound to the extracellular side of the cell membrane, so that it is bound by immune cells can be specifically recognized

Since CVLPs trigger both a cellular and humoral immune response and are not MHC-resisting, this technology is generally suitable for the development of vaccines in that the ability to form particles is provided by an Ll component and an additional antigen component in this Ll Share is merged When developing such CVLPs, it is essential to have a functional test system that can be used to directly test the immunogenicity of CVLPs. Such a test system should have the property that CVLPs with different antigen contents can be examined with the same test system. Since the cellular immune response is of crucial importance for immunological therapy methods of tumors or viral diseases, the task was to make the cellular immune response caused by CVLPs measurable.

This problem was solved by identifying T cell epitopes which, in conjunction with MHC molecules, in a special embodiment with HLA A2.01 MHC molecules, trigger a cytotoxic T cell response in vivo and in vitro, for example. These peptides preferably have the sequence ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLWDQDQF, ICDLQDQHQ, YDLWDQDQF. These sequences are part of the Ll and E7 peptides of HPV16. They include the amino acid ranges Ll 86-94 (5104), Ll 123-131 (5106), Ll 285-293 (5107), Ll 275-283 (5108), Ll 238-246 (5109), Ll 426-434 (5112 ), Ll 28-39 (2016). Ll 31 1-320 (2017), Ll 408-417 (2018), Ll 38-47 (2019), Ll 396-404 (2020), Ll 349-357 (2022), Ll 298-306 (27/28) , Ll 90-98 (9), E7 1-9 (43), E7 18-25 (45) and E7 44-53 (47/48). The names of the respective epitopes are given in brackets.

The E7 peptides 43, 45 and 47/48 have already been published as potential epitopes in Käst et al, (1994) Journal of Immunology 152, 3904-3912. However, this publication only shows that these peptides can bind to HLA AI molecules, but not that a cytotoxic T cell response can actually be triggered. Furthermore, no data are listed which document that the peptides are recognized as a component of a protein by T cells. It it has been shown many times that peptides that bind to HLA molecules per se are not necessarily recognized by T cells. Furthermore, it is known that T cells which recognize a peptide, measurably by inducing a T cell response by the peptide, do not necessarily also recognize cells which contain whole proteins - containing the corresponding peptide - were loaded. This can be explained by the fact that peptides often contain protease interfaces, within which the peptides are cut during the processing of the whole proteins in the cell and thus destroyed and thus can no longer be recognized by T cells. This problem is described, for example, in Feltkamp et al. (1993), Eur. J. Immunol. 23: 2242-2249 confirmed.

The present invention therefore relates to a T-cell epitope having an amino acid sequence ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN, SMVTSDAQI, and / or a functionally active variant of it.

Under a functionally active variant of ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL,

SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN or SMVTSDAQizit see for example a T-System-Ep ) one, on the cytotoxicity of ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN or SMVTSDAQI measured cytotoxicity has the minde- least the sum from the mean of the negative controls and three times Standard deviation corresponds, preferably of at least approximately 30%, in particular at least approximately 50% and in a particularly preferred manner of at least approximately 80%.

A preferred variant is for example a T-cell epitope with a sequence homology to ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN or SMVTSDAQI of minde - at least about 65%, preferably at least about 75% and in particular at least about 85% at the amino acid level. Other preferred variants are also T cell epitopes which have a structural homology to ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL,

SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN or SMVTSDAQI. Such epitopes can be found by counteracting the T cell epitopes ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL,

SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN or SMVTSDAQnIl. De (1992) Eur. J. Immunol. 22, 3013-20) and, for example, synthetically produced peptides of choice can be tested for recognition by the peptide-specific T cells (see examples). In particular, T cell epitopes are understood to mean cytotoxic T cell epitopes. However, non-cytotoxic T cells are also known which can also recognize MHC I molecules, so that non-cytotoxic T cell epitopes are also included as a variant of the present invention. Another embodiment of the present invention is a T cell epitope that is part of a compound, the compound being not a naturally occurring Ll protein of a papilloma virus and not an exclusively N-terminal or exclusively C-terminal deletion mutant of a naturally occurring Ll protein of a papilloma virus .

In a particular embodiment, a T-cell epitope with an amino acid sequence ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYIVSKQFQV, YLLGFQVP. and / or a functionally active variant in an Ll protein of another papillomavirus or in a chimeric Ll protein, for example an HPV18L1E7 fusion protein. Such a compound of the invention may have the ability to form CVLPs.

Preferably, the T cell epitope mentioned as part of a compound can be a polypeptide which preferably contains further amino acid sequences, in particular a fusion protein. In particular, the compound can be a polypeptide of at least approximately 50 amino acids, preferably of at least approximately 35 amino acids, in particular of at least approximately 20 amino acids, and in a particularly preferred manner of at least approximately 9-12 amino acids in length.

In order to detect the compound or to modify its binding activity to T cells, it can contain a chemical, radioactive isotope, non-radioactive isotope, and / or fluorescent label of the T cell epitope and / or of the fusion protein mentioned. Examples of chemical substances known to the person skilled in the art which are suitable for a chemical labeling according to the invention are: biotin, FITC (fluorescein isothiocyanate) or streptavidin.

A possible embodiment is that a peptide is modified such that it contains at least one lysine. Biotin or FITC (fluorescein isothiocyanate) is coupled to this lysine in the manner known to the person skilled in the art. A peptide modified in this way is bound to a corresponding MHC molecule or to a cell with corresponding MHC molecules. The peptide can then be detected via labeled avidin or streptavidin or directly via the fluorescence of the FITC.

Examples of isotopes known to the person skilled in the art which are suitable for radioactive isotope labeling according to the invention are: Η, ¹ J, ¹³¹ I, ³² P, ³³ P or

1 C.

Examples of isotopes known to the person skilled in the art which are suitable for an

7 1 ^" . Appropriate non-radioactive isotope labeling are: ^" H or C.

Examples of fluorescent substances known to the person skilled in the art which are suitable for a fluorescent label according to the invention are: Εu, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phaldehyde or fluorescamine.

Those skilled in the art are aware of other markings not listed here which can also be used for marking in the sense of this invention. Examples of chemical modifications according to the invention known to the person skilled in the art are the transfer of acetyl, phosphate and / or monosaccharide groups

Polypeptides according to the invention with an amino acid length of approximately 50 can be produced, for example, by chemical peptide synthesis. Longer polypeptides are preferably genetically engineered. Another object of the present invention is therefore a nucleic acid construct for the expression of said T cell epitope or of the compounds, which contains the following components: (a) at least one regulatory element and (b) at least one nucleic acid which is necessary for an amino acid sequence of encoded connection according to the invention. Said nucleic acid construct is preferably made of DNA or RNA. Suitable regulatory elements allow, for example, the constitutive, regulatable, tissue-specific, cell cycle-specific or metabolically specific expression in eukaryotic cells or the constitutive, metabolically specific or regulatable expression in prokaryotic cells. Regulable elements according to the present invention are promoters, activator sequences, enhancers, silencers, and or repressor sequences.

Examples of suitable regulatable elements which enable constitutive expression in eukaryotes are promoters which are recognized by RNA polymerase III or viral promoters such as CMV enhancers, CMV promoters, SV40 promoters and viral promoter and activator sequences derived from, for example, HBV , HCV, HSV, HPV, EBV HTLV or HIV.

Examples of controllable elements which enable controllable expression in eukaryotes are the tetracycline operator in combination with a corresponding repressor (Gossen M. et al (1994) Curr. Opin. Biotechnol. 5, 516-20). Examples of regulatable elements which enable tissue-specific expression in eukaryotes are promoters or activator sequences from promoters or enhancers of those genes which code for proteins which are only expressed in certain cell types.

Examples of controllable elements which enable cell cycle-specific expression in eukaryotes are the promoters of the following genes: cdc25C, cyclin A, cyclin E, cdc2, E2F, B-myb or DHFR (Zwicker J. and Müller R. (1997) Trends Genet 13, 3-6).

Examples of regulatable elements which enable metabolically specific expression in eukaryotes are promoters which are regulated by hypoxia, by glucose deficiency, by phosphate concentration or by heat shock.

In order to enable the introduction of the nucleic acid mentioned and thus the expression of the polypeptide in a eu or prokaryotic cell by transfection, transformation or infection, the nucleic acid can be present as a plasmid, as part of a viral or non-viral vector. Another object of the present invention is therefore a vector, in particular an expression vector, which contains a nucleic acid coding for a polypeptide according to the invention. The following are particularly suitable as viral vectors: baculoviruses, vaccinia viruses, adenoviruses, adeno-associated viruses and herpes viruses. The following are particularly suitable as non-viral vectors: virosomes, liposomes, cationic lipids, or poly-lysine-conjugated DNA.

Another object of the present invention is a cell that contains at least one T cell epitope, preferably presented. In a special embodiment, the cell is transfected by one of the vectors mentioned, transformed or infected This cell expresses the polypeptide according to the invention under conditions known to the person skilled in the art which lead to the activation of the regulatable elements used in each case. The polypeptide can then be isolated from this cell and, for example, purified using one of the abovementioned labels for genetic engineering production and subsequent purification of the exposed compounds according to the invention are suitable for prokaryotic and eukaryotic cells, in particular bacterial cells such as E co, yeast cells such as S cerevisiae, insect cells such as Spodoptera frugiperda cells (Sf-9) or Tπchoplusia ni cells or sucker cells such as for example COS cells or HeLa cells

A preferred embodiment is to use the cell itself which expresses the polypeptide according to the invention, in a particularly preferred embodiment the cell presenting parts of the polypeptide according to the invention via MHC-1 molecules on the cell surface. Antigens are used to produce a cell according to the invention -Presenting cells such as B cells, macrophages, dendπtischen cells, fibroblasts or other HLA A2 01 positive cells, in a preferred embodiment JY-, T2, CaSki cells or EBV-transformed B cells, suitable. The cells according to the invention, the one Presenting polypeptide containing a T cell epitope can be used as target cells for restimulating immune cells, in particular T cells and / or for measuring the activation of T cells. Target cell in the sense of the present invention is to be understood as a cell that presents a T cell epitope via MHC molecules and thus specifically one T cell activation causes, especially a cytotoxic T cell response against the cell

Furthermore, the compound containing a T-cell epitope can be part of a complex which is characterized in that the compound is covalent or via hydrophobic interactions, ionic bonding or hydrogen bonding is connected to at least one further species such as peptides, proteins, peptoids, linear or branched oligo- or polysaccharides and nucleic acids.

The present invention therefore relates to a complex containing a T cell epitope or a compound and at least one further compound. A preferred embodiment is that the polypeptide is present in connection with MHC class I molecules, preferably as HLA A2.01 tetramer. Human MHC class I molecules are particularly preferred. Through the technique of Altman J.D. et al. (1996, Science 274, 94-6), for example, HLA A2.01 tetramers can be prepared with the corresponding bound peptides, which are able to bind to the T cell receptors of peptide-specific cytotoxic T cells.

A further embodiment is the immobilization of the compound according to the invention or of the complex mentioned on support materials. Suitable carrier materials are, for example, ceramic, metal, in particular noble metal, glasses, plastics, crystalline materials or thin layers of the carrier, in particular the materials mentioned, or (bio) molecular filaments such as cellulose or framework proteins.

To purify the complex according to the invention, a component of the complex can additionally contain a protein tag. Protein tags according to the invention allow, for example, high affinity absorption to a matrix, stringent washing with suitable buffers without eluting the complex to any appreciable extent, and subsequent targeted elution of the absorbed complex. Examples of protein tags known to the person skilled in the art are a (HIS) ₆ tag, a Myc tag, a FLAG tag, a hemaglutenin tag, glutathione transferase (GST) tag, intein with an affinity chitin binding Tag or maltose binding protein (MBP) Tag The protein tags according to the invention can be N-, C-terminal, and / or internal

Another object of the present invention is a method for in vitro detection of the activation of T cells by at least one compound containing a T cell epitope. Such a method preferably consists of three steps a) In a first step, cells with at least of a compound containing a T-cell epitope stimulated This compound can contain at least one compound according to the invention containing a T-cell epitope, at least one complex according to the invention containing a T-cell epitope, at least one capsomer, at least one stable capsomer, at least one VLP, In a preferred embodiment, immune cells are stimulated by incubation with CVLPs. This stimulation can take place, for example, in the form of a vaccination, or by incubation of immune cells with CVLPs in vitro. Immune cells stimulated in this way are, for example, after a Vaccination or in a tumor patient nten obtained from the blood, from tumors or from lymph nodes, and / or cultured b) In a second step, cells with at least one T cell epitope according to the invention, at least one compound according to the invention containing a T cell epitope, at least one target cell which presents a T cell epitope and / or with at least one according to the invention

Incubated complex c) The activation of T cells is determined in a third step. Methods suitable for this include, for example, the detection of the production or secretion of cytokines by the T cells, the expression of sion of surface molecules on T cells, the lysis of target cells or the proliferation of cells. Suitable methods for this are, for example, a cytokine assay (Chapter 6.2 to 6.24 in Current Protocols in Immunology (1999), edited by Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM and Strober W., John Wiley & Sons), ELISPOT

(Chapter 6.19 in Current Protocols in Immunology, supra), a ⁵¹ Cr release test (Chapter 3.1 1 in Current Protocols in Immunology, supra) or the detection of proliferation (Chapter 3.12 in Cuπent Protocols in Immunology, supra). Depending on the method used, immune cells such as cytotoxic T-cells, T-helper cells, B-

Cells, NK cells and other cells can be distinguished. The use of compounds, complexes and / or cells according to the invention which contain markings according to the invention allows the detection of T cells which recognize the T cell epitope by detecting the binding of labeled compounds, complexes and or cells the T-

Cells. In a preferred embodiment, the binding of MHC-polypeptide complexes according to the invention to the surface of the T cells is detected. This can be carried out in such a way that the MHC complexes themselves are labeled, for example fluorescence-labeled, or that in a further step an MHC-specific, labeled, for example fluorescence-labeled antibody is used in order to in turn detect the MHC complexes. The fluorescence labeling of the T cells can then be measured and evaluated, for example, in a ^' Fluorescens Activated Cell Sorter ^' (FACS). Another possibility for detecting the binding of the complexes to the T cells is again

Measurement of the activation of T cells (cytokine assay, Elispot, ⁵¹ Cr release test, proliferation, see above). However, this requires simultaneous stimulation of coreceptors (e.g. CD28), for example by coreceptor-specific antibodies (anti-CD28) and / or other non-specific activators (IL-2). The present invention also relates to a method which contains an additional step a ') which is introduced after step a). a ') In this additional step a') following step a)), the isolated or cultured cells are loaded with at least one target cell with a compound according to the invention containing a T cell epitope, at least one complex according to the invention containing a T cell epitope, at least one capsomer, at least one stable capsomer, at least one VLP, at least one CVLP and or at least one virus, with at least one complex according to the invention containing a T cell epitope, and / or at least one target cell, the one

T cell epitope presented, co-cultivated for at least about 8 weeks, in particular for at least about 1 week, before step b) follows.

Under cocultivation, the growth of the cells is:

(i) in the presence of at least one target cell loaded with a compound according to the invention containing a T cell epitope, at least one complex according to the invention containing a T cell epitope, at least one capsomer, at least one stable capsomer, at least one VLP, at least a CVLP, and / or at least one virus, (ii) in the presence of at least one complex according to the invention containing a T cell epitope,

(iii) to be understood in the presence of at least one target cell, which presents a T cell epitope, in the same growth medium and the same tissue culture container.

Another object of the present invention is a method for producing a target cell presenting a T cell epitope. It is possible to do this Load target cell with combinations of different T cell epitopes. In a preferred embodiment, the target cell is incubated with at least one compound containing a T cell epitope and / or at least one complex containing a T cell epitope. In a particularly preferred embodiment, the target cell is incubated in growth medium which contains polypeptides according to the invention or with MHC class I complexes with bound polypeptides according to the invention. The MHC class I complexes can be present, for example, as HLA A2.01 tetramers. A tetramer usually binds four peptides. These can either be identical or represent different species of peptides. In a further preferred embodiment, the target cell is transfected, transformed and / or infected with a nucleic acid and or a vector. In a particularly preferred embodiment, the target cell is infected with a vaccinia virus vector. The method according to the invention is carried out with antigen-presenting cells, for example with B cells, macrophages, dendritic cells, embryonic cells or fibroblasts or other HLA A2.01 positive cells, in a preferred embodiment with JY, T2, CaSki cells or EBV cells. transformed B cell lines performed.

The CVLPs used contain a Papillomavirus L1 protein or variants thereof, in particular HPV16 L1 protein and, but not necessarily, a protein heterologous to L1 or variants thereof. The two proteins can be bound directly or indirectly. For the purposes of the invention, directly bound means that there is a covalent bond between the two proteins, for example a peptide bond or a disulfide bond. Bound indirectly means that the proteins are bound via non-covalent bonds, for example hydrophobic interactions, ionic bonds or hydrogen bonds. In a further embodiment, the CVLPs contain a papilloma virus L2 protein in addition to Ll protein or variants thereof. A preferred embodiment of the L1 protein of the present invention are, for example, L1 proteins with one or more deletions, in particular a C-terminal deletion. A C-terminal deletion has the advantage that the efficiency of the formation of virus-like particles can be increased since the nuclear location signal located at the C-terminus is deleted. The C-terminal deletion is therefore preferably up to approximately 35 amino acids, in particular approximately 25 to approximately 35 amino acids, especially approximately 32 to approximately 34 amino acids. For example, a 32-amino acid long C-terminal Deletion of the HPY16 Ll protein sufficient to be able to increase the formation of virus-like particles by at least about ten-fold. Furthermore, the Ll protein can carry one or more mutations or the Ll portion can be composed of Ll proteins from various papillomaviruses his common characteristic of the L1 proteins according to the invention is that they prevent the formation of VLPs or w Allow CVLPs and that they contain at least one T cell epitope according to the invention

In a preferred embodiment, the L1 protein or variants thereof and the protein heterologous to L1 is a fusion protein. Also included are heterologous proteins which are composed of several different proteins or parts thereof. For example, epitopes, in particular cytotoxic T- Cell epitopes of proteins being epitopes in the sense of the invention can also be part of a synthetic polypeptide with a length of approx. 50 amino acids, preferably of at least approx. 35 amino acids, in particular of at least approx. 20 amino acids and in a particularly preferred manner of at least approx. 9 amino acids

Preference is given to proteins which are heterologous to L1 and which are derived from a viral protein, for example derived from HIV, HBV or HCV, preferably from papillomaviruses, in particular from human papillomaviruses In a preferred embodiment, this is an E protein of a papilloma virus, preferably an E6 and / or E7 protein. It is particularly preferred if the E protein is a deleted E protein, preferably a C-terminally deleted, in particular a C-terminally deleted E7 protein, since these constructs in connection with deleted L1 protein preferably virus-like particles can train. Deletions of up to 55 amino acids are particularly preferred, preferably approximately 5 to approximately 55 amino acids, in particular approximately 38 to approximately 55 amino acids.

In a further embodiment, the protein heterologous to L1 can be derived from antigens that are not viral pathogens. They can also be derived from autoimmune antigens such as thyroglobulin, myelin basic protein or zona pellucida glycoprotein 3 (ZP ₃ ), which are associated with certain autoimmune diseases such as thyroiditis, multiple sclerosis, oophoritis or rheumatoid arthritis. In a preferred embodiment, the protein heterologous to L1 is derived from tumor antigens, preferably melanoma antigens such as MART, ovarian carcinoma antigens such as Her2 new (c-erbB2), BCRA-1 or CA125, colon carcinoma antigens such as CA125 or breast carcinoma antigens such as Her2 new (c- erbB2), BCRA-1, BCRA-2.

Another object of this invention is a method for the in vitro detection of the activation of T cells, which are obtained by preparation from samples. This method makes it possible to determine whether papillomavirus L1 protein-specific cytotoxic T cells are present in a sample, for example a blood sample from a patient or in tumors or lymph nodes of a tumor patient. Such a detection method contains the following steps: a ") In a first step, cells are obtained, for example by drawing blood from a patient or by preparing, for example from tumors or lymph nodes. The cells are then taken up in growth medium and cultured. b) In a second step, cells are incubated with at least one target cell which presents a T cell epitope or with at least one complex which comprises as a component a compound containing a T cell epitope.

c) In a third step, the activation of T cells is determined. Suitable methods for this are, for example, the detection of the production or secretion of cytokines by the T cells, the expression of surface molecules on T cells. the lysis of target cells or the proliferation of cells. Methods suitable for this are, for example, a cytokine assay (Chapter 6.2 to 6.24 in Current Protocols in Immunology (1999), edited by Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM and Strober W., John Wiley & Sons), ELISPOT (Chapter 6.19 in Current Protocols in Immunology, supra), a ^{3 i} Cr release test

(Chapter 3.1 1 in Current Protocols in Immunology, supra) or the detection of proliferation (Chapter 3.12 in Cuπent Protocols in Immunology, supra). Depending on the method used, a distinction can also be made between immune cells such as cytotoxic T cells, T helper cells, B cells, NK cells and other cells. The use of compounds, complexes and / or cells according to the invention which contain the labels according to the invention allows the detection of T cells which recognize the T cell epitope by detecting the binding of labeled compounds, complexes and or cells to the T cells . In a preferred embodiment, the binding of MHC polypeptide according to the invention is

Complexes detected on the surface of the T cells. This can be carried out in such a way that the MHC complexes themselves are labeled, for example fluorescence-labeled, or that in a further step an MHC-specific, labeled, for example fluorescence-labeled antibody is used in order to in turn detect the MHC complexes. The fluorescence labeling of the T cells can then be measured and evaluated, for example, in a 'Fluorescens Activated Cell Sorter ^' (FACS). Another way of detecting the binding of the complexes to the T cells is again to measure the activation of T cells (cytokine assay, Elispot, ⁵¹ Cr release test, proliferation, see above). However, this requires simultaneous stimulation of coreceptors (eg CD28), for example by coreceptor-specific antibodies (anti-CD28) and / or other non-specific activators (IL-2).

The present invention also relates to a method which contains an additional step a '), which is introduced after step a "). A') In this additional step a ') following step a"), the isolated or cultured cells are included at least one target cell loaded with a compound according to the invention containing a T cell epitope, at least one complex according to the invention containing a T cell

Cell epitope, at least one capsomer, at least one stable capsomer, at least one VLP, at least one CVLP and / or at least one virus, with at least one complex according to the invention containing a T cell epitope, and / or at least one target cell which contains a T Cell epitope presented, co-cultivated for at least about 8 weeks, in particular for at least about 1 week, before step b) follows.

Under cocultivation, the growth of the cells is:

(i) in the presence of at least one target cell loaded with a compound according to the invention containing a T cell epitope, at least one complex according to the invention containing a T cell

Epitope, at least one capsomer, at least one stable capsomer, at least one VLP, at least one CVLP, and / or at least one virus, (n) in the presence of at least one complex according to the invention containing a T cell epitope,

(in) in the presence of at least one target cell presenting a T cell epitope in the same growth medium and the same tissue culture container

Another object of the invention is a test system (kit) for the in vitro detection of the activation of T cells containing a) at least one T cell epitope according to the invention, at least one compound according to the invention, at least one vector according to the invention, at least one cell according to the invention, and / or at least one complex according to the invention, and b) effector cells of the immune system, preferably T cells, in particular cytotoxic T cells or T helper cells

The test system is used in a special embodiment for determining the L-protein-specific cytotoxic T cells present, for example in a patient's blood sample or in tumors or lymph nodes of a tumor patient. In this case, the cells described in b) are contained in the test system Control cells, the activation of which by the first kit component, the substances mentioned under a), serves as standard. The activation observed in this reaction is compared with the T-cell activation of cells isolated from patients by the kit component a)

In a further special embodiment, the test system is used, for example, to determine the L1 protein-specific antigenicity of a compound containing a T cell epitope, a complex containing a T cell epitope Capsomers, a stable capsomer, a VLP, a CVLP, and / or a virus. In this case, the substances described in a) are control substances whose activating effect on the second kit component, the cells mentioned under b), serves as the standard. The activation observed in this reaction is associated with the activating effect of a compound containing a T cell epitope, a complex containing a T cell epitope, a capsomer, a stable capsomer, a VLP, a CVLP, and / or a virus the kit component b) compared.

Another object of the invention is the use of at least one T cell epitope, at least one compound according to the invention containing a T cell epitope, at least one vector according to the invention containing a nucleic acid coding for a compound containing a T cell epitope, at least one cell according to the invention containing a T cell epitope for, and / or at least one complex according to the invention containing a T cell epitope for triggering or for detecting an immune response.

Cells which present at least one of the molecules according to the invention via their MHC class I molecules are particularly suitable for stimulating immune cells in vitro and in vivo. Suitable cells for antigen presentation are e.g. B. B cells, dendritic cells, macrophages, fibroblasts or other HLA A2.01 positive cells, which can stimulate specific T cells by joint cultivation with immune cells.

In a particular embodiment, a compound according to the invention, for example an HPV18 L1E7 fusion protein, which additionally contains a T cell epitope according to the invention, can be used to detect an immune response. Such a compound of the invention may have the ability to form CVLPs. Another object of the invention is a medicament or diagnostic agent containing at least one compound according to the invention containing a T cell epitope, at least one vector containing a nucleic acid coding for a compound containing a T cell epitope, at least one cell according to the invention containing a T cell Epitope, and / or at least one complex according to the invention containing a T cell epitope and optionally a pharmaceutically acceptable carrier.

Examples of supports known to those skilled in the art are glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural or modified cellulose, polyacrylamides, agarose, aluminum hydroxide or magnitide.

A medicament or diagnostic agent according to the invention can be present in solution, bound to a solid matrix, and / or an adjuvant can be added.

The drug or diagnostic agent can be administered in various ways. Examples of administration forms known to the person skilled in the art are parenteral, local and / or systemic administration by, for example. B. oral, intranasal, intravenous, intramuscular, and / or topical application. The preferred form of application is influenced, for example, by the natural route of infection of the respective papillomavirus infection. The amount administered depends on the age, weight, general health of the patient and the type of papillomavirus infection. The medicament or diagnostic agent can be administered in the form of capsules, solution, suspension, elixir (for oral administration) or sterile solutions or suspensions (for parenteral or intranasal administration). For example, saline or phosphate-buffered saline can be used as the inert and immunologically acceptable carrier. The drug is administered in therapeutically effective amounts. This means amounts sufficient to elicit a protective immunological response.

In a particular embodiment, a compound according to the invention, for example an HPV18 L1E7 fusion protein, which additionally contains a T cell epitope according to the invention, can be used as a medicament or diagnostic agent. Such a compound of the invention may have the ability to form CVLPs.

The figures and the following examples are intended to explain the invention in more detail without restricting it.

1 shows the graphical evaluation of the MTT staining of WEHI cell lysates, measured as absorption at 595 nm, which had been incubated with supernatants from T cells, which in turn were stimulated by different antigen-presenting peripheral blood lymphocytes (PBL) were. T cells stimulated by specific antigen-presenting PBL secrete TNF. This induces apoptosis in WEHI cells, so that these cells can no longer process MTT into a brownish dye. Low absorption means low dye production and thus many apoptotic cells, which were thus exposed to a lot of TNFα, so that the associated T cells had been stimulated. Thus, the lower the absorption for a particular antigen at 595 nm, the better the stimulation of the T cells.

2 shows the graphical evaluation of the fluorescence of T2 cells - measured in a FACS analysis - whose MHC-1 molecules on the cell surface had been labeled by a FITC-labeled antibody. Cell len, whose MHC-1 molecules can specifically bind the peptides plotted on the X axis, have increased MHC-1 molecules, since the specific binding stabilizes the MHC complexes so that they can accumulate on the cell surface.

3 shows the evaluation of three FACScan experiments after restimulation of CVLP-specific human T cells with peripheral blood mononuclear cells (PBMC), which present different antigens. The content of CD3, which is specific for T cells, and the content of human interferon γ, which is specific for activated cells, are plotted from left to right.

4 shows the evaluation of FACScan experiments after restimulation of specific human T cells from an HLA AI positive donor with peripheral blood mononuclear cells (PBMC) which present different antigens. The name of the respective peptide with which the PBMC was loaded is plotted from left to right. "None" stands for PBMC, which were only incubated with buffer. The proportion of CD8-positive T cells which were classified as reactive on the basis of interferon γ expression in the FACScan experiment is plotted on the Y axis.

5 shows the evaluation of FACScan experiments after restimulation of specific human T cells from a non-HLA-typed donor with peripheral blood mononuclear cells (PBMC), which present different antigens. The name of the respective peptide with which the PBMC was loaded is plotted from left to right. "None" stands for PBMC, which were only incubated with buffer. The proportion of CD4-positive T cells which were classified as reactive on the basis of interferon γ expression in the FACScan experiment is plotted on the Y axis. FIG. 6 shows the evaluation of FACScan experiments after restimulation of specific human T cells from an HLA AI positive donor with peripheral blood mononuclear cells (PBMC) which present different antigens. The name of the respective peptide pool with which the PBMC was loaded is plotted from left to right. "None" stands for PBMC, which were only incubated with buffer. The proportion of CD8-positive T cells which were classified as reactive on the basis of interferon γ expression in the FACScan experiment is plotted on the Y axis.

FIG. 7 shows the evaluation of FACScan experiments after restimulation of specific human T cells from an HLA A24 positive donor with peripheral blood mononuclear cells (BLCL), which present different antigens. The name of the respective peptide pool with which the BLCL were loaded is plotted from left to right. "None" stands for BLCL which were only incubated with buffer. The proportion of CD8-positive T cells which were classified as reactive on the basis of interferon γ expression in the FACScan experiment is plotted on the Y axis.

8 shows the evaluation of a ⁵¹ Cr release experiment after loading BLCL cells of an HLA A24 positive donor (= target cells) with the peptide 9. The target cells were lysed by T cells (= effector cells) stimulated with peptides 1-43 . The ratio of the effector cells used to the target cells used is plotted on the X axis, and the% of the specifically lysed target cells, determined by the release of ⁵¹ Cr from the target cells, is plotted on the Y axis. The% values were calculated using the formula given in Example 7. Examples

1. Description of the raw materials

• HPV16 L1 _{ΛC *} E7 _{I -55} CVLPs were produced in accordance with German patent application DE 198 12 941, see also Müller M. et al. (1997) Virology

234, 93-1 1 1.

• The production of Ll ^VLP's was performed according to Müller M. et al. (1997) Virology 234, 93-11 1.

• T2 cells, which can be obtained under the ATCC number: CRL-1992, have a defect in the antigen processing-associated transport which leads to

Prevents the loading of MHC-1 molecules in the endoplasmic reticulum. The unloaded MHC-1 molecules still present on the cell surface can be loaded, for example, by incubating the cells in peptide-containing media, so that these cells are very well suited for the presentation of an antigen.

• WEHI cells are available under ATCC number CRL-2148.

• PMBC means peripheral blood mononuclear cells, the isolation of which is described, for example, in Rudolf MP. et al. (1999), Biol. Chem. 380, 335-40. • BLCL means a B cell line transformed with the help of the Epstein-Barr virus (obtained from Dr. Andreas Kaufmann, Friedrich-Schiller-University, Jena, Germany).

• BB7.2 means an α-HLA A2.01-specific monoclonal mouse antibody (ATCC HB-82). Α-hum CD28 means a monoclonal mouse antibody which is directed against the extracellular part of human CD28. • α-hum CD3 / PE means a monoclonal mouse antibody that is directed against the extracellular part of human CD3 (ε) and contains the fluorescent marker phycoerithrin (Medac, Hamburg, Germany).

• α-hum CD4 / Cychrome means a monoclonal mouse antibody which is directed against the extracellular part of human CD4 and which contains the fluorescence marker cychrome (DAKO; Glostrup, Denmark).

Α-hum interferon γ / FITC means a monoclonal antibody which is directed against human interferon γ and contains the fluorescent marker FITC (Medac, Hamburg, Germany). • α-hum CD8 / PE means a monoclonal mouse antibody which is directed against the extracellular part of human CD8 and contains the fluorescence marker phycoerithrin (Pharmingen, Heidelberg, Germany).

• InfluenzaMP means amino acids 58-66 GILGFVFTL of the influenza matrix protein (see Dunbar P.R. et al. (1998) Curr. Biol. 26, 413-6). • HPV16E7 peptide means amino acids 11-20 YMLDLQPETT of the human papillomavirus E7 protein.

• Using the algorithm for potential HLA A2.01-binding peptides (Parker, KC et al. (1994) J. Immunol. 152: 163), carried out in the Parker peptide prediction program at http: // www-bimas. dcrt.nih.gov/molbio/- hla bind index.html were the following peptides as candidates for HPV16

Ll identified and synthetically manufactured. The indicated amino acid positions of the respective peptide are given relative to the Met (+1) of the sequence for Ll stored under Genbank accession number k02718 (see Table 1 below). Table 1: Potential HLA A2.01 binding peptides from HPN16 Ll

Peptide ame sequence rel. Ll position

5104 ILVPKVSGL (86-94)

5105 SMDYKQTQL (174-182)

5106 RLVWACVGV (123-131)

5107 HLFNRAGTV (285-293)

5108 YLRREQMFV (275-283)

5109 TLQANKSEV (238-246)

51 12 ILEDWNFGL (426-434)

5113 TLEDTYRFV (441-449)

2016 SLWLPSEATVYL (28-39)

2017 NLASSNYFPT (31 1-320)

2018 TLTADVMTYI (408-417)

2019 YLPPVPVSKV (38-47)

2020 YDLQFIFQL (396-404)

2021 FQLCKITLT (402-410)

2022 ICWGNQLFV (349-357)

2023 KVVSTDEYV (46-54)

2024 QLFVTVVDT (354-362)

2025 GLQYRVFRI (93-101)

• Furthermore, 20mer peptides overlapping by 9 amino acids each were synthesized, which comprise the sequence of the L1 and the E7 protein of HPV16. The peptides were numbered from 1 to 52. Her name and hers Sequences are summarized in the following table, in which "restr." restricted means

Table 2: Synthetic overlapping 20mer peptides from HPN16 Ll and E7

Peptide sequence relative epitope

Name Position Information Ll peptides

1 MSL LPSEATVY PPVPVSK (1-20)

2 YLPPVPVSKWSTDEYVART (12-31)

3 STDEYVARTNIYYHAGTSRL (23-42)

4 YYHAGTSRLLAVGHPYFPIK (34-53)

5 VGHPYFPIKKPNNNKILVPK (45-64)

6 NN KILVPKVSGLQYRVFRI (56-75)

7 GLQYRVFRIHLPDPNKFGFP (67-86)

8 PDPNKFGFPDTSFYNPDTQR (78-97)

9 SFYNPDTQRLVWACVGVEVG (89-108) zvtotox. Epitope, HLA A24 restrict.

10 WACVGVEVGRGQPLGVGISG (100-119)

11 QPLGVGISGHPLLNKLDDTE (111-130)

12 LLNKLDDTENASAYAANAGV (122-141)

13 SAYAANAGVDNRECISMDYK (133-152)

14 RECISMDYKQTQLCLIGCKP (144-163)

15 Q CLIGCKPPIGEH GKGSP (155-174)

16 GO GKGSPCTNVAVNPGDC (166-185)

17 NVAVNPGDCPPLELINTVIQ (177-196)

18 LELINTVIQDGDMVDTGFGA (188-207)

19 DMNDTGFGA DFTTLQANKS (199-218)

20 FTTLQANKSEVPLDICTSIC (210-229)

21 PLDICTSICKYPDYIKMNSE (221-240)

22 PDYIKMVSEPYGDSLFFYLR (232-251) 23 GDSLFFYLRREQMFVRHLFN (243-262) 24 QMFVRHLFNRAGAVGENVPD (254-273) 25 GAVGENVPDDLYIKGSGSTA (265-284) 26 YIKGSGSTANLASSNYFPQQ (276-295) 27 ASSNYF7PSQN (286-295) 27 ASSNYF7PSQQ epitope 29 KPYWLQRAQGHNNGIC GNQ (309-328) 30 NNGICWGNQLFVTWDTTRS (320-339) 31 VTWDTTRSTNMSLCAAIST (331-350) 32 MSLCAAISTSETTYKNTNFK (342-361) 33 TTYKNTNFKEYLRHGEEYDL (353-372) 34 LRHGEEYDLQFIFQLCKITL (364-383) 35 IFQLCKITLTADVMTYIHSM (375-394 ) DVMTYIHSMNSTILEDWNFG 36 (386-405) 37 TILEDWNFGLQPPPGGTLED (397-416) 38 PPPGGTLEDTYRFVTSQAIA (408-427) 39 RFVTSQAIACQKHTPPAPKE (419-438) 40 KHTPPAPKEDPLKKYTFWEV (430-449) 41 LKKYTFWEVNLKEKFSADLD (441-460) 42 KEKFSADLDQFPLGRKFLLQ (452-471) 43 PLGRKFLLQAGMHGDTPTLH (463-482) cytotox. Epitope HLA AI restrict.

E7 peptides

44 MHGDTPTLHEYMLDLQPETT (1-20)

45 MLDLQPETTDLYCYEQL DS (12-31) cytotox. Epitope HLA AI restrict.

46 YCYEQLNDSSEEEDEIDGPA (23-42) 47 EEDEIDGPAGQAEPDRAHYN (34-53) cytotox. Epitope HLA AI restrict.

48 AEPDRAHYNIVTFCCKCDST (45-64) cytotox. Epitope HLA AI restrict.

49 TFCCKCDSTLRLCVQSTHVD (56-75) 50 LCVQSTHVDIRTLEDLLMGT (67-86) 51 TLEDLLMGTLGIVCPICSQKP (78-97) Influenza control peptide

52 KEYLRHGEEGILGFVFTLCK

• Golgi Plug is available from Pharmingen (Hamburg, Germany).

• Monensin is available from Sigma (Deisenhofen, Germany). • IL-2 was obtained from Becton Dickinson (Hamburg, Germany).

• MTT solution in PBS means 2.5 mg / ml of 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide in PBS (Sigma, Deisenhofen, Germany).

• PBS means “phosphate buffered saline” and consists of 16.6 mM Na ₂ HPO 8.4 mM NaH ₂ PO ₄ , 150 mM NaCl pH 7.4. • Cells were each in 37 ° C. and 5% CO ₂ in RPMI -Medium (Gibco BRL, Eggenstein; Germany) cultivated with 10% fetal calf serum, kanamycin and ampicillin.

• Luma plates and the Canberra-Packerd B-Plate Counter were purchased from Canberra-Packerd, Dreieich, Germany. • FACScan calibur means 'fluorescens activated cell sorter'. The equipment was purchased from Becton Dickenson (Hamburg, Germany).

• Cellquest software was obtained from Becton Dickinson (Hamburg, Germany).

2. Peptide-specific TNFα secretion by CVLP-stimulated T cells

a) Production of CVLP-specific T cells

Human T cells (4χ l0 ⁵ ) from an HLA A2.01 positive donor were cured for 8 weeks with HPV 16 Ll _ΛC * E7ι_ ₅ 5 CVLPs at 37 ° C with weekly addition of 1 μg / ml CVLPs, 10 irradiated peripheral blood mononuclear cells ( PMBC) and 10 IU / ml IL-2 stimulated and harvested.

b) Stimulation with antigens The cells were overnight in 100 ul medium at 37 ° C with different antigens (PMBC + E7 peptide; PMBC + HPV16 Ll _{ΔC *} E7ι. ₅₅ (CVLP); PMBC + 5104, 5105, 5106, 5107 , 5108, 5109, 51 12, 5113 each 10 μg / ml) in the presence of 10 IU / ml IL-2 stimulated. During this time stimulated cells produce TNFα.

c) TNFα detection

The next day, 50 μl of supernatant were removed, frozen, thawed again (in order to kill any cells that had been taken along) and added to 50 μl of a cell suspension which contained 0.9 × 10 ⁶ WEHI cells, 2 μg / ml actinomycin D and 400 mM LiCl . The cells were incubated for 24 h at 37 ° C. During this time, TNFα (if contained in the supernatant) induces apoptosis of the WEHI cells. By adding 50 μl of a 2.5 mg / ml MTT solution in PBS, non-apoptotic cells were stained brown within 3 hours, whereas apoptotic cells remained yellow. All cells were lysed by adding 100 μl lysis buffer (34% N, N dimethylformamide, 20% sodium dodecyl sulfate) and incubating for at least 6 hours at 37 °, so that the dyes were released. Finally, the absorption of the solution was measured at 595 nm. Fig. 1 shows the different antigens plotted against the measured absorption at 595 nm. Low absorption means low dye production and thus many apoptotic cells, which were thus exposed to a lot of TNFα, so that the associated T cells had been stimulated. Thus, the lower the absorption at 595 nm, the better the stimulation of the T cells.

Result: The peptides 5104, 5106, 5107, 5108, 5109 and 51 12 were able to stimulate CVLP-specific T cells.

3. Binding of peptides to T2 cells

a) Loading of the T2 cells 2.5 × 10 ⁶ T2 cells from an HLA A2.01 positive donor were carried out overnight in medium containing 2% human serum in the presence of 0, 10 or 100 μg / ml 5105, 5106, 5107, 5109 , 5112, 5113, InfluenzaMP, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025 peptide incubated at 37 ° C. During this time, suitable peptides can bind to the unphysiologically empty MHC-1 molecules and thus stabilize them, whereas MHC-1 molecules without suitable peptides are absorbed back into the cell relatively quickly. Thus, specifically binding peptides increase the number of MHC-1 molecules on the cell surface.

b) Detection of the peptide binding to MHC-1 complexes of the T2 cells

The next morning the cells were harvested, washed with PBS containing 0.5% bovine serum albumin (BSA) and the MHC-1 molecules demonstrated. This is done by incubating for 30 min on ice with the BB7.2 antibody, washing and staining with an α-mouse FITC antibody for a further 30 min on ice. The cells were washed again, measured in a FACScan calibur and analyzed with Cellquest software.

2 shows the different peptides plotted against the measured fluorescence.

Result: T2 cells after incubation with the Ll peptides 5106, 5107, 5109, 51 12, 2016, 2017, 2018, 2019, 2020 and 2022 show, as after incubation with the known influenza peptide MP, significantly more MHC molecules on the Cell surface, which indicates a binding of the corresponding peptides to the MHC molecules.

Restimulation of CVLP-stimulated T cells with different antigen presenting cells

Human T cells (4χl0 ⁵ ) from an HLA A2.01 positive donor were irradiated for 8 weeks with HPV16 L1 _{ΛC *} E7 _{I -55} CVLPS at 37 ° C with weekly addition of 1 μg ml CVLPs and 10 ⁵ antigen-presenting cells (irradiated PMBC) stimulated and harvested. The cells were then restimulated in 100 μl medium at 37 ° C. with 10 μg / ml different antigens in the presence of 10 IU / ml IL2 and 0.5 μg / ml α-human CD28: a) overnight with PBMC-incubated with CVLP b ) PBMC incubated overnight with Ll 2022 peptide c) incubated overnight with Ll 2025 peptide (control peptide)

PBMC

After one hour, 1 μl of Golgi plug was added. The cells were incubated for a further 5 hours at 37 ° C. The cells were then fixed and permeabilized, stained with α-hum CD3 / PE, with α-hum CD4 / cychrome and with α-hum interferon γ / FITC. The cells were examined for their labeling in a FACScan calibur and the measurement results were analyzed using Cellquest software.

Result: FIG. 3 shows that the CVLP-incubated PBMC, like the PBMC incubated with the L1 peptide 2022, caused restimulation of CVLP-stimulated T cells, but not the PBMC incubated with the control peptide.

5. Restimulation of CVLP-stimulated T cells with different antigen-presenting cells

Human T cells (4x10 ^{"^} ) of an HLA AI positive donor were for 5 weeks with HPV 16L 1 _{ΛC *} E7 _I _ ₅₅ CVLPs at 37 ° C with weekly addition of 1 μg / ml CVLPs and 10 ⁵ antigen-presenting cells ( irradiated PMBC) and the cells were then restimulated in 100 μl medium at 37 ° C. with 10 μg / ml of the peptides indicated on the X axis of FIG. 4 in the presence of 10 IU / ml IL2 as a negative control served only cells incubated with buffer.

After one hour, 1 µl monensin (300 µM) was added. The cells were incubated for a further 5 hours at 37 ° C. The cells were then fixed and permeabilized, with α-hum CD8 / PE, with α-hum CD4 / Cychrome and with α- hum interferon γ / FITC stained The cells were examined for their labeling in a FACScan calibur and the measurement results were analyzed using Cellquest software

Result FIG. 4 shows that the PBMC incubated with peptides 43, 47 and 48 caused restimulation of CVLP-stimulated T cells, but not the PBMC incubated with the native peptides. Peptide 43 contains the 9mer peptide of the sequence MHGDTPTLH, the two overlapping peptides 47 and 48 contain the lOmer peptide of the sequence QAEPDRAHYN, which were described as HLA AI-binding peptides in Käst et al (supra), but for which no functional detection has yet been carried out

6 Restimulation of CVLP-stimulated T cells with different antigen-presenting cells

Human T cells (4x10) from a non-HLA-typed donor were stimulated for 6 weeks with HPV16Llj, c * E7ι _5i CVLPs at 37 ° C with weekly addition of 1 μg / ml CVLPs and 10 antigen-presenting cells (irradiated PMBC) and harvested Then the cells were restimulated in 100 μl medium at 37 ° C. with 10 μg / ml of the peptides indicated on the X-axis of FIG. 5 in the presence of 10 IU / ml IL2. Only cells incubated with buffer served as a negative control

After one hour, 1 μl of monensin (300 μM) was added. The cells were incubated for a further 5 hours at 37 ° C. The cells were then fixed and permeabilized, with α-hum CD8 / PE, with α-hum CD4 / Cychrome and with α - hum interferon γ / FITC stained The cells were marked for their labeling examined in a FACScan calibur and the measurement results analyzed using Cellquest software.

Result: FIG. 5 shows that the PBMC incubated with peptides 27 and 28 caused restimulation of CVLP-stimulated T cells, but not the PBMC incubated with the other peptides. The two overlapping peptides 27 and 28 contain the peptide of the sequence SMVTSDAQI, so that the peptide actually recognized must essentially comprise this sequence.

Human T cells (4x10) from an HLA AI positive donor were irradiated for 6 weeks with HPV16L1 _{ΛC *} E7 _I _ ₅₅ CVLPs at 37 ° C with weekly addition of 1 μg / ml CVLPs and 10 ⁵ antigen-presenting cells (irradiated PMBC) stimulated and harvested.

The 20mer peptides 1 to 51 were according to the matrix

summarized in peptide pools A to H or 1 to 7.

The T cells of an HLA AI positive donor were then restimulated in 100 μl medium at 37 ° C. with the peptide pools in the presence of 10 IU / ml IL2. The amounts of the peptide pools used were such that 1 μg / ml was added for each individual peptide. Only cells incubated with buffer served as a negative control.

After one hour, 1 μl of Golgi plug was added. The cells were incubated for a further 5 hours at 37 ° C. The cells were then fixed and permeabilized, stained with α-hum CD8 / PE, with α-hum CD4 / cychrome and with α-hum interferon γ / FITC. The cells were examined for their labeling in a FACScan calibur and the measurement results were analyzed using Cellquest software.

Result: FIG. 6 shows that the PBMC incubated with the peptide pools E and 6 brought about a restimulation of CVLP-stimulated T cells, but not the PBMC incubated with the other peptide pools. The peptide pools E and 6 together contain the peptide 45, which is therefore likely to be responsible for the restimulation of the CVLP-stimulated T cells. The peptide 45 in turn contains the peptide ETTDLYCY, which Käst et al. (supra) has described as a HLA AI binding peptide, but for which no functional detection has yet been possible. Furthermore, the T cells of an HLA A24 positive donor with the peptide pools were restimulated and analyzed as above.

Result: FIG. 7 shows that the PBMC incubated with the peptide pools A and 2 brought about a restimulation of CVLP-stimulated T cells, but not the PBMC incubated with the other peptide pools. The peptide pools A and 2 together contain the peptide 9, which is therefore likely to be responsible for the restimulation of the CVLP-stimulated T cells. The Parker et al. (supra) results in a potential peptide for HLA A24 with the sequence FYNPDTQRL, which is therefore likely to be responsible for the activity of peptide 9.

8. Lvse of BLCL cells loaded with peptide 9

BLCL cells from an HLA A24-positive donor were incubated with ^{5 l} Cr for one hour at 37 ° C., washed three times with medium and divided into 2 aliquots. On the one hand, 10 μg / ml of peptide 9 were added to the aliquot of the cells, the other aliquot served as a negative control in the absence of a peptide. Then 2000 of the cells (= target cells) were added to increasing amounts of T cells (= effector cells) in a total volume of 150 μl. The T cells had previously been stimulated for 5 weeks with a mixture of 43 peptides (peptides 1-43, 1 μg / ml each). In parallel, approaches for spontaneous and maximum lysis of the cells were set up. Target cells which were incubated in medium were used for the spontaneous lysis, target cells which were incubated with 0.5% Triton were used for the maximum lysis. The batches were incubated at 37 ° C. for 5 h. 50 μl of the supernatant of the batches were placed on luma plates and added overnight Room temperature dried. The next morning, the amount of radioactive ⁵¹ Cr was determined using a Canbeπa-Packerd B-plate counter (counts) and related to the maximally lysed cells of the Triton approach. The% specific lysis was determined according to the formula: x = 100 • (counts - spontaneous counts) / (maximum counts - spontaneous counts).

FIG. 8 shows that the BLCL cells loaded with peptide 9 could be effectively lysed by the T cells, but the non-loaded BLCL cells could not. Peptide 9 is thus an HLA A24 restricted cytotoxic T cell epitope.

Claims

claims

1. T cell epitope with an amino acid sequence ILVPKVSGL,

RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNGDYDDLQV, FYNGDDQTL, FYNGDDQTLQ, FYNGDDTLQV, FYNGDDTLQV, FYNGDDTLQV, FYNGDDQV

2. T cell epitope according to claim 1, characterized in that said

Variant of a sequence homology to ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YLLQQDQFQQVQQQQQQQQQQQQQQQQQQ

65%, preferably at least about 75% and in particular at least about 85% at the amino acid level.

3. T cell epitope according to claim 1, characterized in that said variant has a structural homology to ILVPKVSGL, RLVWACVGV,

HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV FYNPDTQRL, MHGTTPDLYHQ, MHGDTPAEDHY

T-cell epitope according to one of claims 1-3, characterized in that the T-cell epitope is a cytotoxic T-cell epitope.

5. A compound containing a T cell epitope according to one of claims 1 to

4, wherein the compound is not a naturally occurring Ll protein of one

Papillomavirus and none exclusively N-terminal or exclusively

C-terminal deletion mutant of a naturally occurring Ll protein of a papillomavirus.

Compound according to claim 5, characterized in that the compound is a polypeptide, in particular a fusion protein.

7. A compound according to claim 5 or 6, characterized in that the

Compound is a polypeptide of at least about 50 amino acids, preferably of at least about 35 amino acids, in particular of at least about 20 amino acids and in a particularly preferred manner of at least about 9-13 amino acids in length.

8. A compound according to any one of claims 5-7, characterized in that the compound is a chemical, radioactive, non-radioactive isotope and / or fluorescent label of the T cell epitope and / or said fusion protein, and or a chemical modification of the T- Contains cell epitops and / or fusion protein.

9. nucleic acid, characterized in that it is for a T cell epitope or a compound containing a T cell epitope according to one of the claims

5-8 coded.

10. Vector, in particular an expression vector, characterized in that it contains a nucleic acid according to claim 9.

1 1. cell, characterized in that it contains at least one T cell epitope according to any one of claims 5-8, preferably presented.

12. Cell according to claim 1 1, characterized in that the cell is transfected, transformed, and / or infected with a nucleic acid according to claim 9 and / or a vector according to claim 10.

13. Cell according to claim 1 1, characterized in that the cell with at least one compound according to one of claims 5-8 and / or at least one complex according to one of claims 15-17 containing a T-cell epitope according to one of the Claims 5-8 was incubated.

14. Cell according to claim 1 1 or 12, characterized in that the cell is a B cell, a macrophage, a dendritic cell, a fibroblast, in particular a JY, T2, CaSki cell or EBV-transformed cell.

15. Complex containing a T cell epitope according to one of claims 1-4 or a compound according to one of claims 5-8 and at least one further compound.

16. Complex according to claim 15, characterized in that the complex contains at least one MHC class I molecule, preferably as HLA A2.01 tetamer.

17. A complex according to claim 16, characterized in that said MHC class I molecule is a human MHC class I molecule, in particular an HLA A2.01 molecule.

18. A method for the in vitro detection of the activation of T cells by at least one compound containing a T cell epitope according to any one of claims 1-4, which contains the following steps: a) stimulation of cells with at least one said compound; b) adding at least one target cell presenting a T cell epitope according to any one of claims 1-4 or a complex according to any one of claims 15-17, and c) determining the activation of T cells.

19. The method according to claim 18, characterized in that it contains the following additional step a ') after step a):

a ') Co-cultivation of the cells for at least about 1 week, in particular at least about 8 weeks with:

(i) at least one target cell loaded with a compound according to one of claims 5-8, at least one complex according to one of claims 15-17, at least one capsomer, at least one stable capsomer, at least one VLP, at least one CVLP, and or at least one virus,

(ii) at least one complex according to any one of claims 15-17,

(iii) and / or at least one target cell, which presents a T cell epitope according to one of claims 1-4, before step b) follows.

20. A method for producing a target cell according to one of claims 1 1, 13, 14, 18 or 19, characterized in that the target cell with at least one compound according to one of claims 5-8 and / or at least one complex according to one of claims 15 -17 containing a T cell epitope according to any one of claims 5-8, is incubated.

21. A method for producing a target cell according to one of claims 1 1, 12, 14, 18 or 19, characterized in that the target cells are transfected, transformed and / or infected with a nucleic acid according to claim 9 and / or a vector according to claim 10 .

22. A method for producing a target cell according to claim 20 or 21, characterized in that the target cell is a B cell, a macrophage, a dendritic cell, a fibroblast, in particular a JY, T2, CaSki cell or EBV-transformed cell is.

23. The method according to claim 18 or 19, characterized in that instead of step a) the following step a ") is carried out: a") Obtaining and preparing samples containing T cells and subsequent cultivation.

24. Test system for the in vitro detection of the activation of T cells comprising: a) at least one T cell epitope according to one of claims 1-4, at least one compound according to one of claims 5-8, at least one vector according to claim 10, at least one cell according to any one of claims 1-14, and / or at least one complex according to any one of claims 15-17, and b) Effector cells of the immune system, preferably T cells, in particular cytotoxic T cells or T helper cells.

25. Use of at least one T cell epitope according to one of claims 1-4, at least one compound according to one of claims 5-8, at least one vector according to claim 10, at least one cell according to one of claims 1 1-14, and / or at least one complex according to any one of claims 15-17 for triggering or for detecting an immune response.

26. Medicament or diagnostic agent containing at least one compound according to one of claims 5-8, at least one vector according to claim 10, at least one cell according to one of claims 1 1-14, and / or at least one complex according to one of claims 15-17 and optionally a pharmaceutically acceptable carrier.

27. Medicament or diagnostic agent according to claim 26, characterized in that at least one compound according to one of claims 5-8, at least one vector according to claim 10, at least one cell according to one of claims 1 1-14, and / or at least one complex according one of claims 15-17 is in solution, is bound to a solid matrix, and / or has an adjuvant added.