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WO1996034952A1 - Expression de tap et de lmp dans des cellules cancereuses - Google Patents

Expression de tap et de lmp dans des cellules cancereuses Download PDF

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Publication number
WO1996034952A1
WO1996034952A1 PCT/EP1996/001666 EP9601666W WO9634952A1 WO 1996034952 A1 WO1996034952 A1 WO 1996034952A1 EP 9601666 W EP9601666 W EP 9601666W WO 9634952 A1 WO9634952 A1 WO 9634952A1
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Prior art keywords
tap
lmp
tumor
cells
expression
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PCT/EP1996/001666
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German (de)
English (en)
Inventor
Barbara Seliger
Christoph Huber
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Boehringer Ingelheim International Gmbh
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Priority claimed from DE1995116040 external-priority patent/DE19516040A1/de
Priority claimed from DE19603668A external-priority patent/DE19603668A1/de
Application filed by Boehringer Ingelheim International Gmbh filed Critical Boehringer Ingelheim International Gmbh
Publication of WO1996034952A1 publication Critical patent/WO1996034952A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • 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.
  • 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.
  • ER endoplasmic reticulum
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • IL-2 interleukin-2
  • GM-CSF GM-CSF
  • Another aspect of the present invention is a method for finding tumor antigens, characterized in that
  • 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,
  • tumor-specific cytotoxic T-lymphocytes are generated by mixed culture of lymphocytes with the tumor cells obtained from (a),
  • 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,
  • transfectants are selected which have the property of stimulating the tumor-specific CTL obtained from (b), and
  • 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- ⁇ 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
  • 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
  • the tumor cells treated in this way are brought into contact with lymphocytes, preferably kept in mixed cultures with lymphocytes.
  • the antigen presenting cells mentioned can be stimulated with a cytokine, in particular interferon- ⁇ .
  • 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
  • the hygromycin gene or the neomycin resistance gene can be used, for example, as a selection marker.
  • 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.
  • lymphocytes from peripheral blood can be used for the mixed culture.
  • 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.
  • tumor cells 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.
  • FIG. 2 explains the strategy for identifying tumor antigens that can be recognized by T cells.
  • FIG. 3 shows the alternative nomenclature of the TAP and LMP genes.
  • 5 shows the map of the expression vector used for TAP-1.
  • FIG 6 shows the map of the expression vector used for TAP-2.
  • FIG. 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).
  • 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%.
  • 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.
  • PBM peripheral blood mononuclear cells
  • CMRL medium Seromed
  • FCS fetal calf serum
  • FCS fetal calf serum
  • Both cell lines could be cultured for more than 25 passages without changing their phenotype.
  • FCS fetal calf serum
  • 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.
  • 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.
  • the frequency of stable transformation was determined by transfection of 20 ⁇ g 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 ⁇ g / ml G-418 (GTBCO, Heidelberg, Germany), depending on the cell line, preferably 200 ⁇ g / ml. The number of eo ⁇ clones was determined 2 weeks later. IFN treatment
  • 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 ⁇ g 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.
  • Immunofluorescence analysis was carried out using a monclonal antibody (mAb) MsIgG (negative control; Coulter Clone), the hybridoma supernatants W6 / 32, which
  • TAP-1 recognizes (Meyer et al, FEBS Lett. 351: 443-447 (1994)), BBM1, (Institut für Hu- mangenetik, Kunststoff, 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.
  • 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.
  • 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.
  • AMS multiple synthesizer Abimed, Langenfeld, Germany
  • 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 ⁇ 2 -microglobulin (Sigma, St. Louis USA) at a concentration at 2.5 ⁇ g / ml and 0.5% DMSO, or in the same medium, which contained the respective peptides in a concentration of 100 ⁇ M in addition to the ⁇ 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.
  • 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.
  • 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 ⁇ -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.
  • 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
  • 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, ⁇ j -m, MHC I and HLA-A2 in normal kidney cells and renal carcinoma cells
  • Example 2 Reduced or missing expression of LMP, TAP, and HLA not only in renal carcinoma cells but also in melanoma cells
  • melanoma cell lines were examined for the expression of LMP, TAP, ⁇ 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.
  • 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
  • MHC class I / ⁇ 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)).
  • 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.
  • 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.
  • MZ1851RC and MZ1851LN cells showed an increase in MHC class I surface expression under these conditions.
  • 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
  • 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
  • proteasome inhibitors 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.
  • proteasome inhibitors on MHC class I expression can be demonstrated in the renal carcinoma cell lines using FACScan analyzes.
  • 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
  • the proteasome inhibitors can be used to demonstrate whether peptides presented by the MHC class I molecules are generated by the proteasomes.
  • 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.
  • the proteasome inhibitors after transfer of a model protein 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.
  • Dose-response analyzes showed a concentration-dependent inhibition of stable MHC class I membrane expression.
  • cytotoxicity of the CTL can be determined either by a 51 Cr release assay or by bioassay (release of TNF- ⁇ ).
  • 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- ⁇ could at least partially reconstitute the MHC class I expression in the proteasome inhibitor-treated cells.
  • FIG. 4a and b shown.
  • 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.
  • 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
  • results are expressed as x-fold induction of MHC class I and TAP-1, based on untransfected control cells (MZ1851RC, MZ1851LN).
  • IFNs Interferons
  • RCC cells were determined by FACScan analysis. Treatment of these cells with either IFN ⁇ and IFN ⁇ resulted in a significant increase in both MHC class I heavy and light chain molecules and TAP-1 protein. Optimal IFN ⁇ doses resulted in an approximately two-fold increase in expression, whereas optimal IFN ⁇ 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 IFN ⁇ -mediated increase in TAP-1 protein was faster and preceded that of MHC class I heavy chain surface protein by about 8 hours.
  • the cells were untreated for 24 h before FACScan analysis or were treated for
  • 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 ⁇ 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).
  • MEM modified Eagle's Medium
  • 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).
  • 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 ⁇ g / 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 ⁇ F 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).
  • 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 ⁇ l hole.
  • FACScan analyzes of EJ ras-transformed NTH3T3 cells, NIH3T3pEJras-Clone3, which constitutively expresses large amounts of the ras protein p21, and of parenta-
  • 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 ⁇ .-2Y & -D d and -L d varied approximately between 10 to 50% of the control cells (Fig. l).
  • 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

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Abstract

Ce procédé sert à renforcer la réponse immunitaire à une cellule cancéreuse par augmentation de la concentration intracellulaire en TAP et/ou LMP. Ce procédé se caractérise en ce que l'on introduit un ou plusieurs acides nucléiques codant pour des protéines TAP et/ou LMP dans la cellule cancéreuse et en ce que l'on provoque l'expression de ces acides nucléiques dans la cellule cancéreuse. On utilise de préférence les gènes codant pour TAP-1, TAP-2, LMP-2 et/ou LMP-7. Un autre aspect de l'invention concerne des procédés de thérapie génique de cancers, notamment des procédés selon lesquels on prélève des tissus cancéreux d'un patient, on introduit dans les cellules cancéreuses un ou plusieurs acides nucléiques codant pour des protéines TAP et/ou LMP, on provoque l'expression de ces acides nucléiques dans les cellules cancéreuses et on réintroduit ces cellules cancéreuses dans le corps du patient. Un autre aspect de l'invention concerne un procédé d'identification d'antigènes de tumeurs qui se caractérise en ce que (a) on renforce la présentation d'antigènes de cellules cancéreuses par l'introduction et l'expression d'un ou plusieurs acides nucléiques codant pour TAP-1, TAP-2, LMP-2 et/ou LMP-7, (b) on génère des lymphocytes T cytotoxiques (CTL) spécifiques de tumeurs dans une culture mélangée de lymphocytes avec des cellules cancéreuses obtenues pendant l'étape (a), (c) on introduit des clones de cosmide, de l'ADN génomique ou de l'ADN complémentaire (ADNc) extrait ou dérivé des cellules cancéreuses mentionnées dans des cellules résistantes aux CTL et on provoque leur expression, (d) on sélectionne des transfectants qui ont la propriété de stimuler les CTL spécifiques à des tumeurs obtenus pendant l'étape (b), et (e) on clone le gène transfecté médiateur de cette propriété.
PCT/EP1996/001666 1995-05-04 1996-04-20 Expression de tap et de lmp dans des cellules cancereuses WO1996034952A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19516040.1 1995-05-04
DE1995116040 DE19516040A1 (de) 1995-05-04 1995-05-04 Expression TAP und LMP in Tumorzellen
DE19603668.2 1996-02-02
DE19603668A DE19603668A1 (de) 1996-02-02 1996-02-02 Expression von TAP und LMP in Tumorzellen

Publications (1)

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WO1996034952A1 true WO1996034952A1 (fr) 1996-11-07

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2114442A4 (fr) * 2007-01-19 2013-08-21 Univ British Columbia Promoteurs de l'acétylation de hat et utilisations de compositions à base de ceux-ci pour améliorer l'immunogénicité

Citations (3)

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WO1992011289A1 (fr) * 1990-12-19 1992-07-09 Imperial Cancer Research Technology Ltd. Formation d'antigene
EP0600591A2 (fr) * 1992-10-02 1994-06-08 Bristol-Myers Squibb Company Inhibition de la croissance de cellules tumorales par l'administration de cellules transformées pour l'expression de la B7 protéine
WO1996009380A1 (fr) * 1994-09-23 1996-03-28 The University Of British Columbia Procede d'accentuation de l'expression de molecules de classe i du complexe majeur d'histocompatibilite portant des peptides endogenes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992011289A1 (fr) * 1990-12-19 1992-07-09 Imperial Cancer Research Technology Ltd. Formation d'antigene
EP0600591A2 (fr) * 1992-10-02 1994-06-08 Bristol-Myers Squibb Company Inhibition de la croissance de cellules tumorales par l'administration de cellules transformées pour l'expression de la B7 protéine
WO1996009380A1 (fr) * 1994-09-23 1996-03-28 The University Of British Columbia Procede d'accentuation de l'expression de molecules de classe i du complexe majeur d'histocompatibilite portant des peptides endogenes

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Title
CURIEL ET AL: "HIGH-EFFICIENCY GENE TRANSFER MEDIATED BY ADENOVIRUS COUPLED TO DNA-POLYLYSINE COMPLEXES", HUMAN GENE THERAPY, vol. 3, 1992, pages 147 - 154, XP000579759 *
GABATHULER ET AL: "COMPARISON OF CELL LINES DEFICIENT IN ANTIGEN PRESENTATION REVEALS A FUNCTIONAL ROLE FOR TAP-1 ALONE IN ANTIGEN PROCESSING", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 180, October 1994 (1994-10-01), pages 1415 - 1425, XP000579776 *
MONACO: "A MOLECULAR MODEL OF MHC CLASS-I-RESTRICTED ANTIGEN PROCESSING", IMMUNOLOGY TODAY, vol. 13, no. 5, 1992, pages 173 - 179, XP002011887 *
SELIGER ET AL: "ANALYSIS OF THE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I ANTIGEN PRESENTATION MACHINERY IN NORMAL AND MALIGNANT RENAL CELLS: EVIDENCE FOR DEFICIENCIES ASSOCIATED WITH TRANSFORMATION AND PROGRESSION", CANCER RESEARCH, vol. 56, 15 April 1996 (1996-04-15), pages 1756 - 1760, XP002011888 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2114442A4 (fr) * 2007-01-19 2013-08-21 Univ British Columbia Promoteurs de l'acétylation de hat et utilisations de compositions à base de ceux-ci pour améliorer l'immunogénicité

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