WO1996034952A1 - Expression of tap and lmp in tumour cells - Google Patents

Abstract

A process for strengthening the immune response to a tumour cell by increasing the intracellular TAP and/or LMP concentration is characterised in that one or several nucleic acids that code for TAP and/or LMP proteins are introduced into the tumour cells and their expression in the tumour cell is activated. Genes that code for TAP-1, TAP-2, LMP 2 and/or LMP-7 are preferred. Another aspect of the invention relates to gene therapies of tumour diseases, in particular processes in which tumour tissues are removed from a patient, one or several nucleic acids that code for TAP protein and/or LMP protein are introduced into the tumour cells, their expression therein is activated, and the tumour cells are reintroduced into the body of the patient. Another aspect of the invention relates to a process for identifying tumour antigens characterised in that (a) the antigen presentation of tumour cells is strenghthened by introducing and expressing one or several nucleic acids that code for TAP-1, TAP-2, LMP-2 and/or LMP-7, (b) tumour-specific cytotoxic T-lymphocytes (CTL) are generated in a mixed cultureof lymphocytes with the tumour cells obtained in (a), (c) cosmide-clones, genomic DNA or complementary DNA (cDNA) obtained or derived from said tumour cells are introduced into and expressed in CTL-resistant cells, (d) transfectants having the property to stimulate the tumour-specific CTL obtained in (b) are selected, and (e) the transfected gene that mediates said property is cloned.

Description

 Expression of TAP and LMP in tumor cells

The invention relates to methods for increasing the immune response to a tumor by transfection of tumor cells with nucleic acids which code for TAP and / or LMP, methods for finding tumor antigens, transfected tumor cells, the use of TAP and / or LMP-specific Nucleic acids for these methods and for the production of agents for the treatment of cancer.

The genes for TAP-1 (= RING 4) and TAP-2 (= RING 11) as well as LMP-2 (= RING 10) and LMP-7 (= RING 12) are known in the prior art (WO 92/11289; Restifo et al, J. Exp. Med 177: 265-272 (1993); Glynne et al, Nature 353: 357 (1991); Bahram et al, Proc. Natl. Acad. Sci. 88: 10094-10098 (1991) ; Powis et al, Nature 354: 528 (1991); Brown et al, Nature 353: 355 (1991); Ortiz-Navarrete et al, Nature 353: 662 (1991); Townsend et al, Cell 62: 285-295 ( 1990); Trowsdale et al, Nature 348: 741-744 (1990)). They play a role in the MHC class I mediated antigen presentation. LMP-2 and - 7 are components of the proteasome, an ATP-dependent cytosolic proteolytic protein complex. TAP-1 and -2 belong to the family of ABC transporters. They form a heterodimeric complex in the membrane of the endoplasmic reticulum (ER) and transport peptides, which are produced by the proteasome through the digestion of proteins, from the cytosol into the interior of the ER lumen (FIG. 1). WO 92/11289, to which reference is hereby made in full, describes the cloning and characterization of the genes mentioned from human cells. It also reports on the transfection of TAP-2 cDNA into a TAP-2-deficient cell line (BM36.1), which has improved the antigen presentation of these cells. Restifo et al, loc. cit., to which reference is also made in full, identified human tumor cell lines of small cell lung carcinoma with low or undetectable expression of TAP-1, -2, LMP-2 and -7, which have a disturbed antigen presentation. Treatment of the cells with interferon-γ increased the expression of these genes and improved the antigen presentation. The authors speculate that reduced antigen processing could be one of the strategies with which tumor cells escape the immune system. The enhancement of antigen processing by increasing the transcription of MHC-encoded proteasome and transporter genes could therefore find therapeutic application.

Different strategies were chosen to determine the nature of antigens recognized by tumor-reactive CTLs. To date, the identity of these antigens largely remained unknown. Only melanoma-associated antigens were defined. Both the genetic and the biochemical approach resulted in the identification of antigens that were recognized by CTL. These belong to the MAGE family, Tyrosinase, gplOO, and Mart-1 / Melan-A. The MAGE-1 and MAGE-3 encoded antigen peptides are presented by the MHC class I molecule HLA-Al, whereas the tyrosinase antigen is presented by HLA-A2. Using the T2 assay, new peptides from MAGE-2 that bind to HLA-A2 were identified. It is very important that a primary CTL response can be induced with antigenic peptides from MAGE-2 and MAGE-3, as previously shown for peptides derived from p53 and human papillomavirus. T. Boon and co-workers developed a method for finding tumor antigens in which tumor-specific CTLs were generated by mixing cultures of peripheral blood lymphocytes with tumor cells (Coulie et al, J. Immunother. 14 (2): 104-9 (1993)). Genomic DNA from these tumor cells was then transfected into a CTL-resistant variant of these tumor cells and transfectants were identified that were able to stimulate the tumor-specific CTL. Then from the selected transfectants the gene was cloned, which mediated this property, ie encoded a tumor antigen.

The object of the present invention was to find a method with which the immune response to a tumor can be increased and to provide means for such methods. Furthermore, a method should be found with which new tumor antigens can be identified more efficiently.

These objects have been achieved with the present invention. One aspect of the invention relates to a method for increasing the immune response to a tumor cell by increasing the intracellular TAP and or LMP concentration, which is characterized in that one or more nucleic acids are introduced into the tumor cell, which are used for TAP Encode protein and / or LMP protein, and the expression of these nucleic acids is effected in the tumor cell. Genes which code for TAP-1, TAP-2, LMP-2 and or LMP-7 are preferred. The nucleic acid to be introduced is advantageously in the form of one or more expression vectors, optionally double or multiple expression vectors. The nucleic acid or nucleic acids can be introduced into the tumor cell, for example, by electroporation or liposome fusion. Transfection methods in which the nucleic acid or the nucleic acids are complexed with a conjugate which contains an endosomolytic agent and a DNA-binding agent are also advantageous. In particular, the endosomolytic agent can be an inactivated adevirus and the DNA-binding agent polylysine (Curiel et al, Human Gene Therapy 3: 147-154 (1992)). Another aspect of the present invention are methods for gene therapy treatment of tumor diseases. These are, in particular, processes in which tumor tissue is removed from a patient and into which tumor cells a nucleic acid or nucleic acids are inserted which code for TAP protein and / or LMP protein and which causes the expression of these nucleic acids in the tumor cell and these tumor cells are reintroduced into the patient's body. Genes which code for TAP-1, TAP-2, LMP-2 and / or LMP-7 are preferred. It is particularly advantageous if the ability of the tumor cells to divide is destroyed before being introduced into the patient's body. Transfectants produced in this way can be used as tumor vaccines. In addition, other genes can be introduced into the tumor cells that increase the immune response to these tumor cells, for example interleukin-2 (IL-2) or GM-CSF.

Another aspect of the present invention is a method for finding tumor antigens, characterized in that

(a) the antigen presentation of tumor cells is increased by the introduction and expression of one or more nucleic acids which code for TAP-1 and / or TAP-2 and / or LMP-2 and or LMP-7,

(b) tumor-specific cytotoxic T-lymphocytes (CTL) are generated by mixed culture of lymphocytes with the tumor cells obtained from (a),

(c) cosmid clones, genomic DNA or complementary DNA (cDNA) are introduced into CTL-resistant cells and brought to expression, which is obtained or derived from the tumor cells mentioned,

(d) transfectants are selected which have the property of stimulating the tumor-specific CTL obtained from (b), and

(e) cloning the transfected gene that mediates this property.

The decisive step in comparison with the prior art is that the anti-gene presentation of the tumor cells used to generate the tumor-specific CTL is increased by transfection and expression of nucleic acids which code for TAP and / or LMP proteins. The stimulation of the tumor-specific CTL can be measured, for example, by measuring the TNF-α production of the corresponding CTL clones after incubation with the transfectants. Another aspect of the present invention are tumor cells into which one or more nucleic acids have been introduced which code for one or more TAP proteins and / or LMP proteins, and the use of such tumor cells as tumor vaccines or for the production of tumor-specific CTL.

Another aspect of the present invention is the use of a nucleic acid, which codes for one or more TAP proteins and / or LMP proteins, for increasing the immune response to a tumor cell.

Another aspect of the present invention is the use of a nucleic acid, which codes for one or more TAP proteins and / or LMP proteins, for finding tumor antigens.

Another aspect of the present invention is the use of a nucleic acid, which codes for one or more TAP proteins and / or LMP proteins, for the production of an agent for the treatment of cancer.

Another aspect of the present invention is a method for producing cytotoxic T-lymphocytes (CTL) against autologous tumor cells, characterized in that

(a) one or more nucleic acids which code for TAP protein and / or LMP protein are introduced into tumor cells made of biopsy material or tumor cells derived therefrom and the expression of this nucleic acid or these nucleic acids in the tumor cells is brought about,

(b) the tumor cells treated in this way are brought into contact with lymphocytes, preferably kept in mixed cultures with lymphocytes.

To further improve antigen processing / antigen presentation, the antigen presenting cells mentioned can be stimulated with a cytokine, in particular interferon-γ.

Tumor cells, for example from patient biopsy material, can be obtained by methods known per se. Methods for the isolation of TAP and LMP genes (see the references mentioned above, in particular WO

92/11289), for the construction of eukaryotic expression vectors (Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989, 16.3- 16.73) and the introduction of the constructs into tumor cells can be found in the prior art (Curiel et al, loc. Eil; Sambrook et al, loc. Cit.). It is also possible to use multiple expression vectors, for example double expression vectors, which can control the expression of more than one gene. Methods for electroporation, for liposome fusion or for introducing complexes of DNA, polycations and optionally adenoviral particles are also known from the literature (Curiel et al, loc. Cit .; Sambrook et al, loc. Cit.). The hygromycin gene or the neomycin resistance gene can be used, for example, as a selection marker. After stable transfer of the TAP and / or LMP genes (eg selection of the transfectants with hygromycin or G-418) their integration and expression in the cell lines can be demonstrated by Southern, Northern blot and FACScan analyzes. The success of the transfection can also be checked with the experiments described in the examples, and the efficiency of the TAP-dependent peptide transport, for example with the peptide translocation experiment (see examples). The success of the method according to the invention can also be checked via the CTL-mediated allogeneic lysis of tumor cells, as is described in the examples below. Standard methods are known for destroying the ability of the tumor cells to divide before re-introducing them into the patient's body. Gene therapy strategies and application protocols in which the methods according to the invention can be used are known in the literature (Morgan et Anderson, Ann. Rev. Bio-0 formerly 62: 191-217 (1993), and references therein; Mulligan, Science 260: 926-932 (1993)). Protocols for the production of tumor-specific CTL are also known in the literature. In particular, lymphocytes from peripheral blood can be used for the mixed culture.

5 cytokines, which have an immunomodulatory effect, and their production are known from the literature. For example, the production of interferon-γ is known (Arakawa et al, J. Biol Chem. 260: 14435-9 (1985)).

2 shows a possibility of finding new tumor antigens according to the invention. 0 Tumor cells are transfected with expression vectors that contain TAP and / or LMP genes. The transfectants show an improved antigen presentation and are therefore better suited for the generation of specific CTL. These are generated, for example, by mixing culture with mononuclear cells or lymphocytes from peripheral blood. Genomic DNA from the tumor cells or cDNA, which is derived from mRNA from the tumor cells, is then transfected into CTL-resistant cells, for example a CTL-resistant variant of the tumor cells or COS-7 cells, and expressed. Transfectants that can stimulate tumor-specific CTL express a tumor antigen. Such transfectants are selected and the gene for the tumor antigen is isolated or cloned. there can according to the by T. Boon et al. (Boon, Genetic analysis of tumor rejection antigens, Adv. Cancer Res. 58: 177-210 (1992); Coulie et al, loc. Cit.) Developed procedures.

By introducing TAP and / or LMP genes, the immunogenicity of

Effectively increased tumor cells. This can be used for the therapy of cancer diseases, particularly in gene therapy, in particular in the form of tumor vaccines. Another focus is on methods for the detection of tumor antigens. Appropriate transfectants can be used here for the more efficient production of tumor-specific CTL.

Description of the pictures

1 gives a schematic overview of the MHC class I antigen presentation.

FIG. 2 explains the strategy for identifying tumor antigens that can be recognized by T cells.

Figure 3 shows the alternative nomenclature of the TAP and LMP genes.

4a and b show the lysis of the RCC cell lines MZ1851RC and MZ1851LN by HLA-B7 restricted CTL. It can be seen that the addition of IFN increases the specific lysis. It can also be seen that cells which have been established from a lymph node metastasis (LN) show poorer lysis than those which have been obtained from the corresponding primary tumor (RC).

5 shows the map of the expression vector used for TAP-1.

6 shows the map of the expression vector used for TAP-2.

7 shows the ATP dependence of the peptide translocation and different peptide transport rates between MZ 185 IRC and MZ1851LN. The various peptides were translocated in the presence or absence of ATP in streptolysin-O-permeabilized MZ1851NN, MZ1851RC and MZ1851LN cells. Translocated peptides were isolated with ConA-Sepharose, quantified and as a percentage of the added peptide expressed. The peptides used were lomer: # 63 (RYWANATRSI), # 56 (RYWANATRSR, # 67 (RYWANATRSF), # 600 (TNKTRIDGQY).

8 shows the different efficiencies of the peptide transport rate in normal kidney cells (NN), primary (RC) and metastatic (LN) lesions. Peptide # 67 was used to compare the rates of peptide translocation in normal epithelial kidney cells

(MZ1851NN), primary RCC cells (MZ1851RC) and metastatic lesions

(MZ1851LN) used. See also Fig. 7.

9 shows the suppression of all H-2 loci in RMA cells by ras transformation.

Immunofluorescence analyzes were carried out as described. NIH LTRpEJrasC13 cells and parental NIH3T3 cells were stained with the H-2 locus-specific and ras mAb. The results are expressed as the mean specific fluorescence intensity.

Figure 10 shows the decreased lysis of RMAras cells by CTL 10BK.1. Cytotoxicity was measured in a standard chromium release assay as described. The lysis was calculated in%.

20th

Examples

Methods

25 cell lines and cell culture

Various renal carcinoma cell lines, e.g. Cell line MZ 185 IRC (primary tumor cell line) and the cell line MZ1851LN (lymph node metastasis from the same patient), as well as the cell lines MZ1879RC and MZ1940RC were established. HLA typing of 30 patients' peripheral blood mononuclear cells (PBM) was performed.

To establish the tumor cell lines, fresh tumor material was digested enzymatically and single cell suspensions were incubated in CMRL medium (Seromed) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, penicillin and streptomycin 35. Both cell lines could be cultured for more than 25 passages without changing their phenotype. The isolation of short-term cultures from normal corresponding kidney tissue of the same patients was carried out analogously to the establishment of the long-term cultures (MZ1851NN, MZ1879NN, MZ1940NN). Short-term cultures could however, only be maintained for a maximum of 10 passages. The murine T-lymphoma cell lines RMA, the mutant RMA-S derived therefrom, which has a point mutation in the TAP-2 gene (Ljunggren et Karre, J. Exp. Med. 162: 1745 (1985)), the human lymphoblastoid cell line T2 (Hosken et Bevan J. Exp. Med. 175: 719 (1992)) and murine NIH3T3 cells served as controls in temperature shift experiments. Melanoma cells were established and cultivated analogously.

Vectors

The genes for TAP-1, TAP-2, LMP-2 and LMP-7 were cloned (see FIGS. 5 and 6) into the commercially available expression vector pcDNA3 (TAP-lneo, phuTAPl, TAP2neo) Neomycin resistance gene carries.

Electroporation

For electroporation, 1 × 10 ⁶ to 5 × 10 ⁶ cells in 1 ml PBS were carefully mixed with 20 μg plasmid DNA and cooled on ice for 15 minutes. A pulse (Richmond (CA)) with a capacity of 960 μF and a voltage of 250 volts was applied to the suspension in a BIO-RAD gene pulse apparatus. After electroporation, the cells were returned to ice for 10 minutes and then placed in nutrient medium. In another protocol, 2.5 × 10 ⁶ cells with 10 μg linearized TAPl expression plasmid (TAP-lneo, phuTAPl, TAP-2neo) were in an electroporation device (Fischer, Heidelberg, Germany) at 330 V, 1200 mF and 2 msec transfected.

Selection of necfi clones

The frequency of stable transformation was determined by transfection of 20 μg des

Controlled plasmids pAG60, which contains the neomycin resistance gene neo ^) under the control of the He-Simplex-Vims thymidine kinase promoter (Colbere-Garapin et al, J. Mol. Biol 150: 1-14 (1981)). Stable transformants were isolated 48 h after the transfection by selection in a medium which was supplemented with 100-500 μg / ml G-418 (GTBCO, Heidelberg, Germany), depending on the cell line, preferably 200 μg / ml. The number of eo ^ clones was determined 2 weeks later. IFN treatment

Human recombinant IFN-α with a specific activity of 1.8 x 10 ⁸ U / mg protein (Essex-Pharma, Munich, Germany) and IFN-γ with a specific activity of 3 x 10 ⁶ U / mg protein (Thomae , Biberach, Germany). Adherent tumor cells were treated with 50 ng / ml IFN-α or IFN-γ for the specified time. A dose-response analysis showed that the IFN-o / γ concentration did not cause cytotoxic effects in either cell line, but was able to induce MHC class I surface expression.

Norhern blot analysis

Total cellular RNA of 5 × 10 ⁷ cells was extracted with a guanidinium isothiocyanate extraction and a cesium chloride centrifuge according to Chirgwin et al. isolated (J. Biochem. 18: 5294 (1979)). 20 μg of total RNA was subjected to gel electrophoresis in a 1% agarose-formaldehyde gel, size-fractionated and transferred to nylon membranes (Hybond, Amersham Buchler, Braunschweig, Germany). The blots were hybridized successively with specific cDNA sequences which encompass the entire coding regions of TAP-1, TAP-2 (Spies et al. Nature 348: 744 (1990)), β ₂ -m, HLA, LMP -2 and LMP-7 (Ortiz-Navarette et al. Nature 353: 663 (1991)) and were labeled with ³² P according to the random priming procedure (Feinberg et Vogelstein, Anal. Biochem. 132: 6 (1984) ). The hybridizations were carried out at 42 ° C. overnight in a 50% formamide solution which contained 4 mg tRNA, 0.5% SDS and 5 × Denhardt's solution. The membranes were then washed 3 times at 42 ° C. for 10 minutes with decreasing salt concentrations. The blots were used to expose Kodak X-OMAT AR films (Kodak Rochester, NY) overnight. Gene expression was quantified by densitometric analysis and autoradiograms. In order to correct slight deviations in the RNA loading, each lane was normalized to the 28S / 18S signal.

Immune fluorescence analysis

Immunofluorescence analysis was carried out using a monclonal antibody (mAb) MsIgG (negative control; Coulter Clone), the hybridoma supernatants W6 / 32, which

HLA-A, -B, -C locus products complexed with ß ₂ -microglobulin (ß ₂ -; Brodsky et al. Immunol. Rev. 47: 342 (1979)), BB7.2, HLA-A2.1 recognizes 148.3 who

TAP-1 recognizes (Meyer et al, FEBS Lett. 351: 443-447 (1994)), BBM1, (Institut für Hu- mangenetik, Munich, Germany), which recognizes free and complexed ß ₂ -m, 20-8-4S, the H-2K ^b D ^b (American Type Tissue Culture Collection) and 34-1-2S, the H-2K ^d D ^he knows. The second antibody was FITC-labeled goat anti-mouse antibody (Becton Dickinson). Before staining with mAb 148.3 directed against TAP-1, the cells were permeabilized on ice with 70% ethanol for 30 minutes. For cytofluometry, 5 × 10 ⁵ to 1 × 10 ⁶ cells were incubated with an excess of the respective antibody for 30 minutes at 4 ° C. and shaken occasionally, washed twice with ice-cold PBS and then in the dark with FITC-GAM incubated for a further 30 minutes at 4 ° C. The cells were washed twice with PBS and analyzed with a FACScan analyzer (Becton Dickinson). Cytofluometry was performed at least three times. In FACScan histograms, the vertical axis represents the relative cell number and the horizontal axis the log of the fluorescence intensity. The data are presented as fluorescence intensity and represent the mean of at least two independent experiments.

Peptide synthesis and peptide binding analysis

Peptides were synthesized on the solid phase with F-moc for temporary NH ^ -terminal protection using an AMS multiple synthesizer (Abimed, Langenfeld, Germany), purified by HPLC and characterized by amino acid analysis.

The peptides used for the peptide binding analysis were HLA-A2 binding HIV IV peptide IV9 (HIVpol 510-518; ILKEPVHGV) and the H2L ^d binding CMV peptide (CMV 168-176, YPHFMPTNL). 5 x 10 ⁵ cells were incubated for 15-18 hours in serum-free medium which contained human β ₂ -microglobulin (Sigma, St. Louis USA) at a concentration at 2.5 μg / ml and 0.5% DMSO, or in the same medium, which contained the respective peptides in a concentration of 100 μM in addition to the β ₂ -microglobulin. Peptides were previously dissolved in DMSO. The final concentration of DMSO was 0.5%. Peptide-labeled cells were stained by indirect immunofluorescence analysis as described.

Peptide translocation assay

The efficiency of the TAP-dependent peptide transport from the cytosol to the ER can be determined on the basis of various model peptides by the so-called peptide translocation experiment. The peptide translocation experiments can be used to determine the peptide transport of primary tumor cell lines, their metastases and cultures from normal painters to compare tissues. This then gives a direct statement about the activity of the TAP heterodimer in the corresponding cells and shows functional TAP deficits.

Peptides were made on the solid phase with F-moc for temporary NH-terminal

Protection synthesized with an AMS multiple synthesizer (Abimed, Langenfeld, Germany), purified by HPLC and characterized by amino acid analysis. 10 μg of the respective peptide was iodinated with the chloramine T method (Neefjes et al, Science 261: 769 (1993)) and had a specific activity of 20-50 μCi / μg. Known protocols were followed (Neefjes et al, loc. Cit .; Momburg et al, J. Exp. Med. 179: 1613 (1994)). 2.5 × 10 ⁶ cells / incubation were harvested and washed once with incubation buffer (130 mM KCI, 10 mM NaCl, ImM CaCl ₂ , 2 mM EGTA, 2 mM MgCl ₂ , 5 mM HEPES, pH 7.3). The kidney carcinoma cells were then permeabilized by treatment with 2 IU streptolysin O / ml (Wellcome Reagent Ltd., Beckenham, UK) in 50 μl incubation buffer for 15 min at 37 ° C. and the cells treated in this way were iodinated with 10 μl ¹²⁵ I , with peptides equipped with a glycosylation site in the presence or absence of 10 μl 10 mM ATP (Boehringer Mannheim, Germany) in a final volume of 100 μl for a further 15 min at 37 ° C. The peptide translocation was inhibited by lysis of the permeabilized cells with 1 ml NP-40 lysis mix. Cores were removed, glycosylated peptides were obtained with ConA-Sepharose (Pharmacia, Uppsala, Sweden) as described (Neefjes et al, loc. Cit.) And quantified in a γ-counter. The data was expressed as a percentage of bound peptide with respect to the added peptide.

Determination of the CTL-mediated cytotoxicity

In order to check the role of TAP and LMP for recognition by the immune system, one can work with allogeneic, on the other hand with autologous MHC class I restricted CTLs.

Two different strategies were chosen for the CTL-mediated allogeneic lysis of the tumor cell lines: firstly, a recombinant vaccinia system was used, and secondly, lysis was detected using peptide-specific MHC-restricted CTLs. First, by infection with recombinant vaccinia virus, which contains the K ^d gene (Vac-K ^d ), the efficiency of the antigen processing was determined by means of a cytotoxicity test in the selected tumor cell lines. The recombinant Vac-K ^d virus and the corresponding K ^d- restricted, Vac-specific CD8-positive LAK cells are from the Literature known (Restifo et al, loc. Cit.). For this experiment, the tumor cells were infected with 10 PFU / cell K ^d expressing vaccinia virus for 2 hours, then labeled with Na ⁵¹ CrO for 1 hour before being incubated with CD8 positive cells for 4 hours. The ⁵¹ Cr released was then measured in the γ-scintillation counter and the specific cell lysis was calculated. The lysis of the tumor cells represents a direct measure of the capacity of the various tumor cell lines to present the K ^d- restricted, Vac-specific CD8-positive T cells Vac antigens, and thus the efficiency of their antigen processing.

Using the same experimental approach, it was tested whether the efficiency of the anti-gene processing when using the recombinant Vac-K ^d system described above in the TAP or LMP transfectants is improved in comparison to the parental tumor cells, which is due to an increased lysis of the tumor cells is detected.

Furthermore, it could be shown in the HLA-A2-expressing tumor cell lines with an HLA-A2-repeated influenza matrix peptide and the use of peptide-specific CTLs that the parental tumor cell lines can be lysed efficiently and that the lysis of TAP and / or LMP transfectants is improved.

CTL clones which were directed against autologous RCC cell lines were produced by mixed lymphocyte tumor cell culture using peripheral blood lymphocytes. The CD8 ⁺ , CD4 "CTL clone MZ1257-CTL5 / 30 with high cytolytic activity for the autologous RCC cell line MZ1257RC was established and used for the determination of its cytolytic activity on allogeneic RCC cell lines and K562 cells. The measurement of the cytotoxicity was measured using a standard ⁵ ^ r release assay.

Example 1: MHC-x let I and TAP-1 expression is reduced in RCC cell lines

Cell lines from patients with metastatic meridian cell carcinoma (RCC) were identified

Primary tumors and in one case established from a lymph node metastasis. The protein expression of heavy and light chain of the MHC class I and of TAP-1 in these RCC lines was compared with that of normal kidney cell tissue of the same patients corresponding to short-term cultures. Indirect immunofluorescence analyzes were carried out, the monoclonal antibodies W6 / 32, the HLA-A, -B, -C chains, which are associated with ß -m, recognizing, BBM1, which is associated with free and bound ß ₂ -m reacted, and mAbl48.3, which recognizes TAP-1 protein, were used (Table 1). In the normal epithelial cultures MZ1851NN, MZ1879NN and MZ1940NN was a strong positive Staining for TAP-1 and MHC class I was found, whereas a reduced TAP-1 and MHC class I expression pattern was observed in all RCC cells (Table 1). TAP-1 staining was negative in mutant T2 cells and poor staining of these cells was obtained with the mAb W6 / 32. Protein expression was down-regulated by both TAP-1 and MHC class I in primary RCC lines compared to normal kidney cells. In the cell line, which was derived from a lymph node metastasis, the level of expression was reduced even further (Table 1). In order to further analyze the pattern of the MHC class I degradation, the expression of HLA-A2 in the MZ1851 and MZ1879 system was determined. Again, the normal kidney cells MZ1851NN and MZ1879NN had the greatest surface expression in comparison to the malignant counterparts. In addition, HLA-A2 expression was further suppressed in the MZ1851LN cells (Table 1). The extent of down regulation of HLA-A alleles was comparable to that which could be detected with the mAb W6 / 32, which is directed against monomorphic components of the HLA molecule.

Table 1: Expression of TAP, β _j -m, MHC I and HLA-A2 in normal kidney cells and renal carcinoma cells

mean specific fluorescence intensity

Cell line MHCI HLA-A2 ß _r m TAP-1

MZ1851INN 78.5 80.3 82.4 12.5

MZ1851IRC 56 52.5 53.8 9

MZ1851ILN 37.3 31.4 41.7 4.1

MZ1879NN 51.3 48.3 39.7 3.6

MZ1879RC 34.2 31.5 25.4 1.4

MZ1940NN 54.9 n.d. 40.1 5.5

MZ1940RC 28.5 n.d. 16.3 2.3

Example 2: Reduced or missing expression of LMP, TAP,

and HLA not only in renal carcinoma cells but also in melanoma cells

Various melanoma cell lines were examined for the expression of LMP, TAP, β ₂ -microglobulin and HLA using the methods described above. Northern blot so- such as flow cytometry showed heterogeneous expression of TAP, LMP, MHC (heavy and light chain) in the melanoma cell lines tested. A melanoma cell line was completely negative for TAP, LMP and MHC class I expression.

Table 2: Heterogeneous MHC class I membrane expression of melanoma cell lines

Cell line lied to specific fluorescence intensity

MHCI (W6 / 32)

Mel-Juso 14.3

ZKR 30.3

MKrZ 16.8

Mell 7.78

Mell. 5.86 3.44

Mel4 32.4

Mel5 64.1

Mel6 0

Example 3: Effects of reduced temperature on the MHC class I molecules on the cell surface of melanoma cells, RCC and normal renal epithelial cells

Since only the trimeric MHC / β ₂ microglobulin / peptide complex is stably expressed on the cell surface, a TAP or LMP deficiency under normal culture conditions leads to a reduced or no MHC class I membrane expression during incubation of these cells stabilizes the dimeric β ₂ -microglobulin MHC complex at low, unphysiological temperatures and thus causes an increase in the MHC membrane expression. Stabilization of the membrane expression of MHC class I genes can also be brought about by the exogenous addition of MHC class I-specific peptides. Such temperature shift and exogenous peptide loading experiments indicate whether the reduced MHC class I membrane expression is due to deficiency in the genes of the antigen processing machinery. For this reason, these two experimental approaches were carried out with the selected tumor cell lines in comparison to the corresponding normal, non-malignant tissue cultures, the HLA phenotyping of the cells being a prerequisite for the peptide loading experiments. MHC class I membrane expression was determined in melanoma cells after parallel incubation of the cells at 37 ° C. and 26 ° C. for 36 hours.

Table 3: Stabilization of MHC class I by temperature shifi

Cell line% Expression of MHC I at 26 ° C *

Mell 160 Mel4 185 Mel5 125 Mel6 0

Expression expressed in% control (incubation of the cells at 37 ° C.)

MHC class I / β ₂ -m complexes that do not carry a peptide are stably expressed on the cell surface at 26 ° C., but disintegrate at 37 ° C. (Ortiz-Navarette et Hämmerling, Proc. Natl. Acad Sei. USA 88: 3594-1 (1991)). To check the possibility that low MHC class I expression on RCC cells is due to the lack of an adequate amount of peptide, the patient's two RCC lines were MZ1851 and the short-term culture MZ1851NN for 36 hours at 26 ° C or 37 ° C cultivated and then examined by FACScan analysis. As shown in Table 4, MHC class I expression was not increased in normal kidney cells when incubated at low temperature, while MZ1851RC and MZ1851LN cells showed an increase in MHC class I surface expression under these conditions. Interestingly, a 1-fold induction of MHC class I surface expression in MZ1851LN cells at 26 ° C was observed, which was comparable to that of TAP-deficient T2 cells.

Table 4: Stability index of MHC class I molecules at 37 ° C. in normal kidney cells and kidney carcinoma cells

Cell line stability index of MHC class I molecules at 37 ° C

MZ1851NN 1.1 MZ1851IRC 0.63 MZ1851LN 0.48 T2 0.66

MHC class I surface expression of the cell lines of the MZ1851 system, MZ1851NN, MZ 185 IRC and MZ1851LN, and of T2 cells was analyzed after parallel culture of the cells at 37 ° C. and 26 ° C. for 36 hours. The results were presented as the stability index of MHC class I surface at 37 ° C and were obtained by dividing the mean specific fluorescence intensity at 37 ° C by that at 26 ° C. The antibody used was W6 / 32, which reacts with a monomorphic determinant on the MHC class I molecules.

Example 4: The effect of proteasome inhibitors on the stable MHC class I membrane expression of tumor cell lines

In order to clarify the importance of the proteasomes for an effective antigen presentation in various tumor cell lines, we used proteasome inhibitors. These substances inhibit the essential peptidase activities of the 20S and 26S proteasomes and reduce the breakdown of proteins and ubiquitinated protein substrates.

The effect of proteasome inhibitors on MHC class I expression can be demonstrated in the renal carcinoma cell lines using FACScan analyzes. In addition, the effect of these substances on the presentation of an influenza matrix protein, which can be transferred into the kidney carcinoma cells via electroporation, can be checked. This protein introduced into the cytosol is proteolytically cleaved and presented by the MHC class I molecules. The MHC class I molecules containing the peptide derived from the influenza matrix protein can be detected with antigen-specific CTLs. 17

In addition, the proteasome inhibitors can be used to demonstrate whether peptides presented by the MHC class I molecules are generated by the proteasomes. For the investigation of this hypothesis, MHC class I molecules can be precipitated with an antibody, the conformational determinants, which are only present on the intact heterodimer, in the presence or absence of the proteasome inhibitor. If treatment of the kidney carcinoma cells with the proteasome inhibitor prevents the formation of peptides, the exogenous addition of peptide can show whether the MHC class I assembly of proteasome inhibitor-treated cells can be reconstituted.

For example, it could be shown that the proteasome inhibitors after transfer of a model protein (ovalbumin) prevent the presentation of ovalbumin-specific peptides by the MHC class I complex, which is based on an inhibition of the proteolytic activity of the proteasome complex.

We investigated the effect of protease inhibitors on the MHC class I presentation in melanoma and kidney carcinoma cell lines:

Dose-response analyzes showed a concentration-dependent inhibition of stable MHC class I membrane expression.

Table 5: Dois effect analysis of proetasome inhibitor on MHC I expression using the example of the melanoma cell line Mel4

Concentration proteasome inhibitor log specific fluorescence intensity

untreated 31.7

0.1 μM 32.5

0.5 μM 30.8

1.0 μM 28.4

5.0 μM 22.3

10 μM 19.1

50 μM 13.7

100 μM 14.1

200 μM 13.9 Depending on the cell lines, the use of 50 μM inhibitor led to an approximately 30 to 70-fold reduction in MHC class I membrane expression.

j Table 6: Effect of the proi asome inhibitor on the MHC class I membrane expression of different tumor cell lines

Cell line tumor% MHC class I membrane expression from untreated control

MZ1851RC renal cancer 28.5

MZ1851LN LK metastasis / renal carcinoma. 62.6

MZ1257RC renal carcinoma 59.4

Mel4 Melanoma 43.2

Mel5 melanoma 38.0

Some patients can generate CTL against the autologous tumor relatively easily, while others find this impossible. The reason for this discrepancy can be a loss, a reduced or altered peptide presentation of tumor cells, which then leads to a reduced recognition by CTL. It was determined in the various tumor cell lines and TAP and LMP transfectants in the presence and absence of is cytokines whether (1) whether the transfer of TAP or LMP genes into correspondingly deficient cells and (2) whether cytokine treatment and thus the induction of the TAP / LMP expression and MHC genes not only improves the antigen processing (Vac-K ^d system), but also the generation of MHC-restricted CTLs and the CTL-mediated lysis in the autologous tumor cell / CTL system become.

20th

First, using the Vac-K ^d system described in detail above, it was demonstrated that, in addition to gene transfer, induction of LMP and / or TAP expression by cytokine treatment increases the lysis of the K ^d -specific CTLs. In systems in which pairs of tumor cells and CTLs are already established, it can be shown that improved TAP and / or LMP expression after cytokine treatment leads to more efficient CTL-mediated elimination of the tumor cells. For these examinations, the cytotoxicity of the CTL can be determined either by a ⁵¹ Cr release assay or by bioassay (release of TNF-α). In addition, kidney carcinoma cells, which have a reduced capacity for antigen processing, can then be used Treatment with cytolcins MHC-restricted autologous CTLs against the corresponding tumor cell lines can be established using mixed lymphocyte tumor cell cultures.

Exogenous addition of peptide or IFN-γ could at least partially reconstitute the MHC class I expression in the proteasome inhibitor-treated cells.

Table 7: Effect of peptide and IFN on MHC I of proteasome inhibitor-treated cells using the example of the cell line MZl 85 IRC

Treatment% MHC class I expression of untreated control

Proteasome inhibitor 28.5 IFN-γ 143.5

Proteasome inhibitor + IFN 92.7 Proteasome inhibitor + HLA A2 peptide 87.6

Example 5: Reduced MHC class I membrane expression correlates with reduced CTL lysis

It was functionally tested with the cell lines MZ1851RC (primary tumor) and MZ1851LN (metastasis) whether the greatly reduced MHC class I expression of the MZ1851LN

Cells results in reduced CTL-mediated lysis. Allogeneic HLA-B7 restricted CTL were used for this experimental approach. The results of these experiments are in

Fig. 4a and b shown.

Example 6: Reconstitution of MHC class I expression in TAP transfectants

A TAP-1 expression vector was introduced into MZl 85 IRC cells by electroporation.

were selected and the TAP-1 gene expression was measured with the TAP-1-specific antibody mAb 148.3. The staining of the three wec clones showed an increase in TAP-1 expression compared to the parental MZl 85 IRC cells. Furthermore, RCC clones expressing the recombinant TAP-1 gene had higher densities of the MHC class I antigen on the surface. The poor MHC class I expression of the parental RCC lines could be reconstituted by TAP-1 gene transfer (measurement with mAb W6 / 32; Table 8). The transfectants had an approximately 1.5-fold increase in MHC class I membrane expression. Peptide translocation assays also resulted in an approximately 1.6-fold more efficient peptide transport from the cytoplasm to the ER.

Table 8: MHC and TAP-1 expression in TAP-1 transfected cells, based on untransfected control cells

X-fold induction of the FI of the untransfected control

Transfectant MHC class I TAP-1

MZ1851RC-A1 1.4 1.6 MZ1851LN-B1 2.3 1.8 MZ1851LN-X 1.5 2.7

The results are expressed as x-fold induction of MHC class I and TAP-1, based on untransfected control cells (MZ1851RC, MZ1851LN).

Example 7: Interferons (IFNs) induce coordinated expression of TAP and MHC class I genes in RCC cell lines

The TAP and MHC class I protein levels in untreated and IFN-treated

RCC cells were determined by FACScan analysis. Treatment of these cells with either IFNα and IFNγ resulted in a significant increase in both MHC class I heavy and light chain molecules and TAP-1 protein. Optimal IFNγ doses resulted in an approximately two-fold increase in expression, whereas optimal IFNα doses only caused a 1.3-1.6-fold increase. Some cell lines such as MZl 846RC showed almost no increase. No differences were observed between cell lines with a constitutively temperature-stable or temperature-labile MHC class I membrane phenotype.

The kinetics of IFN-mediated induction of TAP-1 and MHC class I mRNA and protein were investigated in more detail on the MZ1257RC cell line. Both Northem blot and FACScan analyzes showed coordinated regulation of mRNA and protein Level of the genes of the antigen processing apparatus. The IFNγ-mediated increase in TAP-1 protein was faster and preceded that of MHC class I heavy chain surface protein by about 8 hours.

Table 9: IFN-mediated modulation of MHC class I and TAP-1 protein expression in RCC cells

The cells were untreated for 24 h before FACScan analysis or were treated for

Treated with IFN 24h.

¹ The results are expressed as ratios by dividing the mean specific fluorescence intensity of the IFN-treated RCC lines by the untreated ones.

Example 8: Reduced peptide transport rate in RCC lines

Transporter activity is necessary for an effective peptide loading of MHC class I molecules and stable surface expression. The efficiency of peptide transport in the MZl 851 cell system was examined. ¹²⁵ I-labeled peptides containing the consensus sequence for N-linked glycosylation were inserted into the ER (endoplasmic reticulum). lum) streptolysin-O-permeabilized MZl 851 cells and the glycosylated fraction isolated with ConA-Sepharose. The amount of translocated peptides was determined in a γ counter and compared with the amount of peptide entered. As a control, permeabilized cells were incubated with peptides in the absence of ATP. Figure 8 shows that the peptide RYWANATRSF (peptide # 67) was transported with different efficiency in MZl 851 cells. In comparison with the transport rate of corresponding normal kidney cells, the peptide translocation was reduced to about 57% in MZl 85 IRC and to 25% in MZ1851LN cells. The different transport rates of RCC from primary and metastatic lesion were consistent when three other peptides were used (# 56: RYWANATRSR, # 63: RYWANATRSI, # 600: TNKTRIDGQY) (Fig. 7).

The reduced cell surface expression of both RCC cells was therefore at least partially caused by the lack of peptides in the ER. This led to an inadequate peptide loading and stabilization of the β ₂ -m / MHC class I complex.

Example 9:

Cell lines and cell culture

The parental murine fibroblast cell line NIH3T3 and the constitutive c-Ha-ras expressing cell line NIH EJras cl3, which had been established after transfection of NTH3T3 cells with the mutated human c-Ha-ras, were used. These are described in the literature (Seliger et al, J. Virol. 65: 6307 (1991)). Both cell lines were cultivated in modified Eagle's Medium (MEM), which was supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, penicillin (100 U / ml), and streptomycin (100 μg / ml). The mouse T-lymphoma cell line RMA transformed with the Rauscher virus (Ljunggren et al, J. Exp. Med 162: 1745 (1985)) was kept in CRPMI medium containing 10% FCS, glutamine and the corresponding antibiotics was supplemented. The H-2K ^b restricted CTL 10BK.1 with specificity for ovalbumin (OVA) (Dick et al, Proc. Natl. Acad Sei. USA 86: 2316 (1989)) was cultivated in Iscove's modified Dulbecco's medium (IDMEM), the was supplemented with 5% FCS and 3 U / ml recombinant IL-2 (Boehringer Mannheim, Germany). This clone has recently been shown to recognize the peptide OVA 257-264, which is a common by-product in OVA preparations (Staege et al, Immunol. 81: 333 (1994)). Immunofluorescence analysis

The following antibodies were used for flow cytometry: mAb 9C5 with specificity for H-2D ^b (Jankovic et al, J.Exp. Med. 163: 1459 (1986)), mAb B8-24-3 (American Type Tissue Culture Collection, ATCC, USA) with specificity for H-2K ^b , P, P ^a , ^r (Köhler et al, In: Steinberg CM, Lefkovits I (ed.) The immune System. Vol.2, p.202, Karger, Basel ( 1981)), mAb 34-1-2S with specificity for H-2K (ATCC, USA), 31-3-4S with specificity for H-2K ^d , 34-5-8S with specificity for H-2D ^d , 30- 5-7S with specificity for H-2L ^d (Cedarlane, Hornby, Ontario, Canada), and mAb-1, which recognizes Ha-, Ki- and N-ras (Dianova, Hamburg; Germany). Immunofluorescence analysis was carried out as described in the other examples. For intracellular staining with the mAb anti-ras, the cells were permeabilized on ice with 70% ethanol for 30 min. Then 5 × 10 ⁵ cells were incubated with a suitable concentration of the first antibody for 25 min at 4 ° C., washed (PBS, 1% [w / v] FCS) and then for a further 30 min at 4 ° C. in Incubated in the dark with fluorescent anti-mouse Ig antibodies (Coulter, USA). The cells were washed twice with PBS before the fluorescence intensity was analyzed with a FACScan flow cytometer (Coulter, USA). Cytofluometry analyzes were carried out at least three times.

Electroporation:

Electroporation was carried out as described elsewhere (Oellig et al, J. Neurosci. Res. 26: 390 (1990)). 1 x 10 ⁶ - 5 x 10 ⁶ RMA cells were suspended in 1 ml PBS containing 10 μg / ml linearized c-Ha-ras plasmid DNA (Seliger et al, J. Virol. 61: 2567 ( 1988)), and chilled on ice for 15 min. The suspension was pulsed once, maintaining 960 μF and 250 V and using a BIORAD Gene Pulser Apparatus (BIORAD, Richmond, USA). The cells were immediately placed back on ice, cooled for a further 10 minutes, and sown in the respective culture medium. 36 hours after transfection, the cells were aliquoted in 24-well plates and selected in a CRPMI medium which was supplemented with 1.1 mg / ml G418 (Gibco, Gaithersburg).

Cell mediated cytotoxicity in vitro

Cytotoxicity was determined using a ⁵¹ Cr release assay as described (Dick et al, J. Immunol 150: 2575 (1993)). Target cells were pulsed with 1.5 mg / ml OVA for 2 hours at 37 ° C, with 3.75 MBq Na ₂ ⁵¹ CrO ₄ (Amersham Buchler, Braunschweig, Germany) marked for 1 hour at 37 ° C., washed twice, resuspended in IMDM with 10% FCS, and sown on 96-well microtiter plates with a V-shaped bottom at a cell number of 5000 cells per hole. 10BK.1 cells in varying concentrations were added to a final volume of 150 μl hole. After 4 hours, the supernatant of the samples (100 μl) was harvested and counted in a γ counter. The percentage of specific lysis was calculated as follows:% lysis = (experimental cpm - spontaneous cpm) / (maximum cpm - spontaneous cpm) x 100.

i o Oncogene-mediated reduction in MHC class I surface expression affects all loci:

FACScan analyzes of EJ ras-transformed NTH3T3 cells, NIH3T3pEJras-Clone3, which constitutively expresses large amounts of the ras protein p21, and of parenta-

15 len untransformed NTH3T3 cells were performed with a set of H-2 locus-specific mAbs. N_H3T3pEJrasC13 cells showed a reduced level of MHC class I surface expression compared to the parental control. The ras-mediated suppression of MHC class I expression affected all H-2 loci, but the extent of the decrease in Η.-2Y & -D ^d and -L ^d varied approximately between 10 to 50% of the control cells (Fig. l).

20 The strongest suppression was observed for the H-2L ^d -Loci.

Heterogeneous but reduced levels of MHC class I expression in RMA-ras transformants:

25

The T-lymphoma cell line RMA, which expresses large amounts of H-2K ^b , was stably transfected with a plasmid containing Ha-ras. Necfi mass cultures of ras transformants contain a mixed population of tumor clones with a wide range of ras and H-2K ^b expression. Therefore, the mass cultures were cloned to

Obtain 30 RMA ras clones. The clones were then screened for expression of p21ras, H-2K ^b and H-2D ^b with flow cytometry. Intracellular ras and H-2 surface expressions of a number of clones are shown in Table 10. Although a broad distribution was observed in the pattern of ras and H-2K ^b / H-2D ^b expression, the H-2 surface expression was reduced in all RMAras clones. There was also an inverse

35 Correlation between levels of ras and MHC class I expression in the majority of RMAras clones. Table 10: Expression of MHC class I and ras in ras transformants

Specific fluorescence (% RMA)

Cell line H-2K ^b / H-2D ^b ras

RMAras5SKl 44.5 290 RMAras5SK2 54.5 260 RMAras5SK4 65.7 129 RMAras7SK4 74.3 186 RMAras7SK6 83.5 126

Immunofluorescence analysis of RMAras clones was carried out with the corresponding antibodies. Parental RMA cells served as controls. The results are expressed as% of the mean specific fluorescence intensity of the RMA cells.

The role of MHC class I and ras expression for lytic susceptibility

The question arises as to whether the extent of MHC class I antigen expression can influence tumor detection by CTL and offers a selective advantage in vivo to tumor cells that express less MHC antigens. Selected RMAras clones were tested for lysis by CTL 10BK.1. This is an H-2K-restricted CTL clone with specificity for OVA, which has been shown to recognize a specific OVA peptide. The lysis of two independent representative RMAras clones, which were pulsed with OVA peptide and lysed by CTL 10BK.1 at an effector-target cell ratio (E: T) of 0.063: 1 to 8: 1, is shown in Fig. 10 . Compared to parent RMA cells, the CTL-mediated lysis of RMAras5SKl and RMAras5SK2 was significantly reduced. Interestingly, the two clones have a different sensitivity to lysis by CTL 10BK.1, which is inversely linked to lower levels of H-2K ^b antigens, which in turn are due to high levels of p21ras expression. S EQU ENZ PROTOCOL

( 1. GENERAL INFORMATION :

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(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Arg Tyr Trp Ala Asn Ala Thr Arg Ser Ile 1 5 10

(2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE LABEL:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Arg Tyr Trp Ala Asn Ala Thr Arg Ser Arg 1 5 10

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE LABEL:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Arg Tyr Trp Ala Asn Ala Thr Arg Ser Phe 1 5 10

(2) INFORMATION ON SEQ ID NO: 4:

(i) SEQUENCE LABEL:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Thr Asn Lys Thr Arg Ile Asp Gly Gin Tyr 1 5 10

(2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE LABEL:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Ile Leu Lys Glu Pro Val His Gly Val 1 5

(2) INFORMATION ON SEQ ID NO: 6: (i) SEQUENCE LABEL:

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(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Tyr Pro His Phe Met Pro Thr Asn Leu 1 5

Claims

claims

1. A method for increasing the immune response to a tumor cell by increasing the intracellular TAP and / or LMP concentration, characterized in that one or more nucleic acids are introduced into the tumor cell, which code for TAP protein and / or LMP protein , and the expression of these nucleic acids in the tumor cell is effected.

2. The method according to claim 1, characterized in that it is TAP-1 w and / or TAP-2.

3. The method according to claim 1, characterized in that it is LMP-2 and / or LMP-7.

is 4. The method according to any one of claims 1 to 3, characterized in that the nucleic acid or nucleic acids in question are one or more expression vectors, optionally double or multiple expression vectors.

5. The method according to any one of claims 1 to 4, characterized in that the 20 nucleic acid or the nucleic acids by electroporation or liposome fusion in the

Tumor cell are introduced.

6. The method according to any one of claims 1 to 5, characterized in that the nucleic acid or the nucleic acids are complexed with a conjugate which is an endo-

Contains 25 somolytic agent and a DNA binding agent.

7. The method according to claim 6, characterized in that the endosomolytic agent is an inactivated adenovirus.

30 8. The method according to any one of claims 5 or 6, characterized in that

DNA binding agent is polylysine.

9. The method according to any one of claims 1 to 8, characterized in that tumor tissue is removed from a patient, the nucleic acid or the nucleic acids are introduced into tumor cells of this tumor tissue, and these tumor cells are reintroduced into the patient's body.

10. The method according to claim 9, characterized in that the ability to divide the tumor cells is destroyed before insertion into the patient's body.

11. A method for finding tumor antigens, characterized in that

(a) the antigen presentation of tumor cells is increased by the introduction and expression of one or more nucleic acids which code for TAP-1 and / or TAP-2 and / or LMP-2 and / or LMP-7,

(b) tumor-specific cytotoxic T-lymphocytes (CTL) by mixed culture of

Lymphocytes are generated with the tumor cells obtained from (a)

(c) cosmid clones, genomic DNA or complementary DNA (cDNA) are introduced and expressed in CTL-resistant cells, which is obtained or derived from the tumor cells mentioned,

(d) transfectants are selected which have the property of stimulating the tumor-specific CTL obtained from (b), and

(e) cloning the transfected gene that mediates this property.

12. The method according to claim 11, characterized in that the stimulation of the tumor-specific CTL is measured in that the TNF-α production of the CTL is measured after incubation with the tumor cells.

13. Tumor cells into which one or more nucleic acids have been introduced which code for TAP protein and / or LMP protein.

14. Use of tumor cells according to claim 13 as a tumor vaccine.

15. Use of a nucleic acid which codes for TAP protein and / or LMP protein to increase the immune response to a tumor cell.

16. Use of a nucleic acid coding for TAP protein and / or LMP protein for the detection of tumor antigens.

17. Use of a nucleic acid coding for TAP protein and / or LMP protein for the production of an agent for the treatment of cancer.

18. A process for the production of cytotoxic T lymphocytes (CTL) against autologous tumor cells, characterized in that

(a) introducing one or more nucleic acids into tumor cells made of biopsy material or tumor cells derived therefrom which code for TAP protein and / or LMP protein, and which expression of this nucleic acid or these nucleic acids in the tumor cells is effected,

(b) the tumor cells treated in this way are brought into contact with lymphocytes, preferably in mixed cultures with lymphocytes.

19. The method according to any one of claims 1 to 12 or 18, characterized in that the TAP and / or LMP transfected cells are stimulated with a cytokine.

is 20. The method according to claim 19, characterized in that the cytokine is interferon-γ.

21. The method according to any one of claims 1 to 12 or 18 to 20, characterized gekenn¬ characterized in that the tumor cells are additionally transfected with a nucleic acid necessary for B7

20 coded.

22. Use of tumor cells according to claim 13 for the production of a tumor vaccine.