EP1214407A2 - Procede d'identification d'antigenes a restriction cmh - Google Patents

Procede d'identification d'antigenes a restriction cmh

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Publication number
EP1214407A2
EP1214407A2 EP00960681A EP00960681A EP1214407A2 EP 1214407 A2 EP1214407 A2 EP 1214407A2 EP 00960681 A EP00960681 A EP 00960681A EP 00960681 A EP00960681 A EP 00960681A EP 1214407 A2 EP1214407 A2 EP 1214407A2
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EP
European Patent Office
Prior art keywords
cells
cell
antigen
cdna
autologous
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00960681A
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German (de)
English (en)
Inventor
Georg W. Bornkamm
Gerd Hobom
Josef Mautner
Falk Nimmerjahn
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Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Artemis Pharmaceuticals GmbH
Original Assignee
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Artemis Pharmaceuticals GmbH
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Priority claimed from DE19962508A external-priority patent/DE19962508C2/de
Application filed by Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH, Artemis Pharmaceuticals GmbH filed Critical Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Publication of EP1214407A2 publication Critical patent/EP1214407A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16141—Use of virus, viral particle or viral elements as a vector
    • C12N2760/16143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • the present invention relates to a method for the identification of MHC-restricted T-cell antigens.
  • Tumor formation is the result of genetic changes in the cell that lead to the expression of aberrant gene products.
  • T cells are able to recognize such changes in the protein pattern of degenerate cells. Activation of antigen-specific T cells is a prerequisite for the induction of an antitumor immune response.
  • the molecular identification of T-cell tumor antigens therefore creates the conditions for the development of antigen-specific vaccines and other forms of T-cell-mediated immunotherapy (Rosenberg, 1996, 1999).
  • antigens In recent years, in particular in the case of malignant melanoma, several antigens have been identified that are recognized by the patient's autologous T cells. A possible therapeutic benefit of these antigens is currently being investigated in clinical studies. For a broad clinical application, however, it is necessary to identify as many tumor antigens as possible, because: 1. The previously known antigens are usually only expressed in a small percentage of all malignant melanomas and can therefore only be used in a small patient population. 2. Because antigens are peptides of HLA molecules are presented and HLA molecules in the human population are highly polymorphic, one must identify as many antigens as possible in order to have antigens available for vaccination for each HLA constellation. 3.
  • Vaccinations with only one antigen often lead to the tumor being switched off by this tumor and thus to the development of resistance.
  • Such resistance development should be prevented by simultaneous vaccination with as many antigens as possible.
  • the identification of other tumor antigens in melanoma as well as in other tumors is therefore an essential prerequisite for successful immunotherapy.
  • the identification of tumor antigens consists of two steps. First, the isolation of the patient's tumor-specific T cells by repeated in vitro stimulation with autologous tumor cells, and second, the molecular identification of the antigens recognized by the T cells. A simple and generally applicable method is desirable for this.
  • Some methods for identifying MHC-restricted T cell antigens are already known from the prior art.
  • the known approaches include in particular the following methods (Rosenberg, 1999): transient transfection of allogeneic or xenogeneic cell lines; Elution and HPLC fractionation of MHC-bound peptides; Retroviral transduction of autologous fibroblasts.
  • MHC class I-restricted tumor antigens can also be identified biochemically (Cox et al., 1994). For this purpose, tumor cells are lysed and the restricting MHC molecules are immunoprecipitated using monoclonal antibodies. The peptides bound to them are eluted from these MHC molecules and separated by reverse phase HPLC. Target cells are then loaded with the individual peptide fractions. These target cells are characterized by the fact that they only express the respective restriction element in peptide-unbound form. The exogenous addition of peptides therefore quickly leads to binding by the "empty" MHC molecules.
  • fibroblasts can only be cultured in vitro for a limited number of passages. Since fibroblasts also do not express MHC class II, this method is also limited to the identification of MHC class I-restricted antigens. In comparison to the transient expression cloning, as described under 1, the retroviral transduction of the target cells is associated with a considerably greater amount of work. However, the main disadvantage of this method is the comparatively low expression of the genes introduced retrovirally. This low level of expression requires about 10 times less sensitivity compared to transient transfection and thus 10 times more screening effort.
  • Figure 1 LCL infection with influenza
  • the cell line LCL 1.26 was incubated with recombinant influenza viruses (FPV-1 104 as vector), which carry the gene for the green fluorescence protein under the control of a promoter, which is characterized by an increased activity against the Wild-type promoter distinguished (promoter-up variant). After 24 hours, the cells glowing green under UV light were counted.
  • FPV-1 104 recombinant influenza viruses
  • Figure 2 Presentation of the model antigen in the context of MHC class II molecules after infection with recombinant influenza virus.
  • LCL 1.11 were infected with wild-type (wt) or recombinant FPV influenza viruses that express the model antigen under the control of the promoter-up variant.
  • MA model antigen
  • GM-CSF is released by the model antigen-specific T cell clone, but not after wild-type virus (wt) infection or after infection with GFP gene-bearing influenza viruses.
  • Figure 4 Presentation of the model antigen in the context of MHC class II molecules after infection with recombinant retroviruses.
  • the present invention thus provides a method for identifying MHC-restricted antigens, specifically both MHC class I and / or class II-restricted antigens. The following steps are included in the method according to the invention:
  • step (c) infecting immortalized autologous cells expressing MHC class I and / or MHC class U molecules on their surface with the recombinant virus particles obtained in step (b);
  • the invention is based on the fact that the mRNAs are isolated from a cell, for example an animal or human tumor cell, and a cDNA library is created therefrom. Analogously, it can also be cells that are infected with a microorganism are, for example a bacterium, a virus, a fungus or a protozoan. Of course, mixed infected cells are also possible. As an alternative to producing a cDNA bank from the infected cell, it is also possible to start from a gene bank that would be produced directly from the microorganism to be examined.
  • the cDNA or the DNA of the gene bank is introduced into the genome of retroviruses or as additional vRNA in modified influenza viruses, whereby recombinant retroviruses or influenza viruses arise.
  • the retroviruses used are preferably amphotropic or pseudotyped retroviruses.
  • the production of recombinant retroviruses and examples of amphotropic retroviruses are described in Kinsella et al. (1996), examples of pseudotyped retroviruses in Miletic et al. (1999).
  • Another example of retroviruses is the group of lentiviruses, with HTV and SIV in particular being mentioned here.
  • FPV Bratislava promoter-up variants are preferably used as modified influenza viruses.
  • the production of such influenza viruses with promoter up variants is described in Neumann and Hobom (1995), Flick and Hobom (1999) and WO-A-96/10641.
  • influenza promoter up variants mentioned have an increased transcription rate (both with the vRNA promoter and in the cRNA promoter of the complementary sequence) and an increased replication and / or expression rate relative to the wild type and differ from the wild type in that that they have at least one segment (one of the naturally present or an additional one) in which a total of up to 5 nucleotides are exchanged in the 5 'and 3' conserved region of the wild type.
  • the nucleotides in positions 3 and 8 are preferably replaced by other nucleotides, the nucleotides now inserted forming a base pair (item 3: G, then item 8: C; Pos. 3: G, then Pos. 8: G; etc.).
  • the nucleotide in position 5 of the 3 'conserved region can also be replaced.
  • the 3 'conserved regions of the wild-type influenza viruses have the following sequences
  • exchanges can also be made in the 13 nucleotide long 5 'conserved region of the wild type, e.g. B. in positions 3 and 8, again provided that the nucleotides now inserted form a base pair.
  • the 5 'conserved regions of the wild-type influenza viruses have the following sequences:
  • Influenza A 5'-AGUAGAAACAAGG-3 'Influenza B: 5'-AGUAG (A / U) AACA (A / G) NN-3' Influenza C 5'-AGCAGUAGCAAG (G / A) -3 '
  • Influenza virus mutants in which the exchanges G3A and C8U are carried out in the 3 'conserved region are preferably used in the present invention.
  • Most preferred influenza mutants are influenza A mutants and especially those that still have the U5C exchange (the above mutations are numbered from the 3 'end; such counting from the 3' end is also indicated by a line on the number, for example G 3A).
  • Further preferred influenza mutants include the mutations G3C, U5C and C8G (counted from the 3 'end) in the 3'-terminal nucleotide sequence, which gives the following 3'-terminal nucleotide sequence: (5') - CCUGGUUCUCCU-3 ⁇
  • influenza viruses defined above, those are particularly preferred which have the following 3'-terminal nucleotide sequence: (5 ') - CCUGUUUCUACU-3'
  • the modified segment preferably still has the modifications U3A and A8U in its 5'-terminal sequence, in the case of influenza C viruses it can still have the modifications C3U and G8A in its 5'- have terminal sequence.
  • influenza promoter up variants of the present invention have the following general structures:
  • Influenza A promoter-up variant "1 104": 5'-AGUAGA ⁇ CAAGGNN U5.6 ... (880-2300 ntd) ... N'N ⁇ 'CCUGUUUOJACU-3 ,
  • Influenza A promoter-up variant "1920" ⁇ : 5'-AGAAGAAUCAAGGNNNU 5. 6 ... (880-2300 ntd) ... N'N , N'CCUGUUUCuACU-3 '
  • Influenza A (Promoter-up version "1948"): 5 ⁇ -AGUAGAAACAAGGNNNU fifth 6. ⁇ (880-2300 ntd) ... N • N'N , CCUGGUUCuCcU-3 ,
  • N and N ' indefinite but base-paired positions, complementary between 5' and 3 'end, different for different of the eight segments, but always constant across all isolates;
  • the recombinant viruses are grown on suitable host cells and, if necessary, isolated by methods known per se.
  • Immortalized autologous cells preferably B cells or dendritic cells that express MHC class I and / or MHC class II molecules, are then infected with these recombinant retroviruses or influenza viruses. This is followed by steps d) to g), as described above.
  • autologous means the origin of the cells, i.e. the cells are from the same individual as the T cells used to screen for antigen expression.
  • T cell clones are used which recognize an antigen in certain cells. Since the identity of the antigen recognized by the T cells is unknown, the aim is to find out which antigen is recognized by the T cells. The availability of the T cell clones thus says nothing about the nature of the antigen. For example, one assumes T cell clones that recognize a prostate carcinoma line, but not EBV-immortalized cells and fibroblasts from the same individual. The aim now is to use a cDNA bank to identify the still unknown antigen from the prostate cancer cells, the autologous EBV-immortalized cells as recipient cells for the cDNA bank and the specific T cell clones.
  • the invention was preceded by the establishment of tumor-specific T cell clones. Since T cells only recognize antigen in connection with MHC molecules, in some clones However, an identification of the restricting MHC molecule was not possible, already established methods for the identification of T cell antigens could not be used for these clones. Since cells from different tissues of an individual express identical MHC molecules, possibilities were sought to find autologous B cells of the To make patients usable as recipient cells for the expression of cDNA banks from the tumor. B cells from any individual can be infected with Epstein-Barr virus, a human representative of the lymphocryptovirus group or related primate viruses from this group, immortalized and made available in practically unlimited quantities as so-called lymphoblastoid cell lines (LCL).
  • LCL lymphoblastoid cell lines
  • the use of the patient's autologous LCL as target cells requires efficient gene transfer to these cells, which has so far not been achieved by chemical or physical transfection methods. Comparatively little is known about the infection rate of LCL with viruses.
  • modified influenza A viruses firstly results in a uniquely efficient infection of LCL, secondly it occurs through the use of the transcriptionally activated viral regulatory elements in the FPV virus variants to an unmatched high level of gene expression of the genetic information that has been introduced, which results in high sensitivity and thus simplicity of detection.
  • influenza A virus-derived vectors for gene transfer in LCL described here required a determination of the infection efficiency. As shown in Figure 1, up to 80% of the lymphoblastoid cell lines LCL1.26 are infected with these viruses. In addition to a high infection rate, a number of other prerequisites had to be met for the intended application so that the influenza virus system could be used for the stated purpose.
  • a high expression rate of the foreign sequence introduced into the cells is essential for a high sensitivity of the detection method.
  • influenza viral transcription / translation machinery as demonstrated in Western analyzes for a model antigen, an approximately 5 times higher expression rate than after transient transfection and an approximately 10 times higher expression level than after retroviral transduction could be achieved.
  • influenza virus does not interfere with the presentation of the antigen.
  • influenza virus infection does not result in non-specific T cell activation or in the cytokine release by the infected LCL.
  • T cell activation requires at least 20 hours of co-cultivation of target cells and T cells.
  • a possible infection of T cells by the influenza viruses used from the supernatant or released from LCL) and a lysis of the cells after 8 hours would make the detection of a specific T cell activation impossible.
  • the influenza viruses used do not infect the T cells or at least not productively, ie viral genes are not expressed.
  • the LCL 1.26 cell line was incubated with recombinant influenza viruses that carry the gene for the green fluorescence protein. After 24 hours, the cells glowing green under UV light were counted.
  • Retroviruses were also considered as a possible alternative to gene transfer by recombinant influenza viruses and were therefore included in our investigations. As shown in Figure 3, very good efficiency of gene transfer could also be achieved with recombinant retroviruses. With the help of model antigens, an antigen-specific T cell stimulation could also be demonstrated with recombinant retroviruses (Figure 4). However, retroviruses do not reach the high level of expression of influenza viruses. The invention is generally illustrated below.
  • the starting point of the method of the invention is the isolation of mRNA or of DNA from cells whose antigens are to be examined.
  • cells of human origin are used in particular, although cells of animal origin, for example from rodents such as mice or rats, can also be used. It is preferably cells from a patient suffering from a tumor, e.g. B. a tumor of the hematopoietic system such as a B Zeil tumor, e.g. B. a leukemia.
  • the method can also be used for the identification of autoantigens or foreign antigens, e.g. from microbially infected tissue (bacteria, viruses, fungi, protozoa and any mixed infection). If this is a known microorganism, its genetic information (in the form of RNA or genomic DNA) can also be used directly.
  • the mRNA or the DNA is isolated by known molecular biological methods. For example, see Sambrook et al., (1989). The mRNA is then rewritten into its cDNA by techniques which are also known to produce a 14
  • the genetic information contained in the mixture of the cDNA or DNA fragments is now introduced into the genome of influenza viruses.
  • the procedure is generally shown as follows:
  • the genetic information for the proteins encoded by influenza A viruses is located on 8 negative strand RNAs, the coding regions being flanked in each case by the virus-specific promoter and termination sequences, which encompass both the transcription and the replication and the packaging of the Control vRNAs in the virus particles.
  • RNA and RNA carries the viral promoter or packaging signals presupposes that this gene is firstly present as a negative strand RNA and secondly carries the viral promoter or packaging signals.
  • This can be achieved, for example, by using a plasmid vector which, in addition to a resistance gene, for example for ampicillin, and a bacterial origin of replication, additionally has a polylinker sequence which is flanked directly on both sides by the non-translated (cDNA) promoter sequences of influenza A virus is, but in a modified form, which leads to increased activity of the promoter.
  • the viral 5 ' and 3' promoter sequences are in turn surrounded by the sequences of the human RNA polymerase I promoter and terminator, which were isolated from the human rDNA.
  • the cDNAs from the tissue to be examined are cloned into the polylinker sequence of this plasmid in the inverse orientation with respect to the RNA polymerase I promoter and amplified in E. coli. After transient transfection of these plasmids into suitable target cells, the transcriptional activity of RNA polymerase I results in pseudoviral negative-strand RNAs from the cloned-in cDNAs, which carry the viral transcription and packaging signals at their ends.
  • autologous B cells are infected with the recombinant virus particles, which contain one or more copies of cDNAs as negative strand vRNAs.
  • the B cells have to be immortalized before the infection, but EBV genes are preferably used other immorization systems are also known, for example oncogenes. The procedure is as follows
  • Lymphocytes are purified from the donor's peripheral blood using a Ficoll gradient and incubated with cell culture containing EBV.
  • the B95-8 cell line was incubated and immortalized
  • the infection of the immortalized autologous B cells leads to an expression of the pseudoviral gene segments originating from the original cell, which, like the genetic information of the virus's negative strand RNAs, are expressed as proteins, and cleavage products of these proteins are expressed by the cell's own antigen presentation machinery Compound with MHC molecules on the cell surface of the B cells where they can be recognized by antigen-specific T cells
  • APC antigen presenting cells
  • a stable transfection of the APCs with the cDNAs is forbidden for the method according to the invention for two reasons firstly, the stable transfection with the help of resistance genes always requires selection for growth-promoting and against growth-inhibiting genes. This leads to a shift in the representation of the cDNA bank due to the selection-related long culture period of the cells. Secondly, the expression of toxic, eg apoptosis, fördemden genes for the death of the transfected cells and thus for the loss of these genes In order to avoid these selection mechanisms, which would prevent identification of potential antigens, the shortest possible experimental conditions must be sought after transient transfect tion of cells with plasmids, the maximum level of expression is reached after 48-72 hours.
  • influenza system is superior to the normal transient transfection with plasmids. Due to the virus' own transcription machinery, maximum expression of the introduced foreign gene occurs as early as 6-12 hours after infection. This shortens the test times after introducing the foreign gene from around 72 hours to just 24 hours
  • the B cells infected with recombinant influenza viruses are therefore co-cultivated with autologous T cell clones which have a specificity for the antigen to be identified. If the influenza viruses were used to insert a cDNA as a vRNA into the B- If cells are inserted and expressed, which codes for the antigen recognized by the T cells, the antigen-specific T cells are stimulated.
  • the stimulation of the T cells via their T cell receptor is associated with a release of cytokines which are associated with Known methods, for example, ELISA-V, can be detected Of course, other methods can also be used with which the antigen-specific T cell stimulation can be detected.
  • this also includes the detection of T cell-mediated cytotoxicity and others measurable parameters of T cell activation If virus particles were contained in the influenza virus population which code for the antigen in question and have caused T cells to be released from the cytokine, the virus population is divided into smaller pools and with them the infection of the B cells and the procedure for antigen recognition are repeated after the viruses have been separated , which code for the antigen, the isolation and identification of the antigen recognized by the T cells can be carried out in a subsequent step
  • retroviruses can also be used for the infection of autologous LCL and for the expression of the antigens to be identified. With retroviruses, a similarly good transduction efficiency as with modified influenza viruses can be achieved, but retroviruses do not achieve the high expression level of the modified influenza viruses.
  • the production of recombinant retroviruses is described in detail in the prior art and reference is made here only to the publication by Kinsella et al (1996) as an example.
  • the open reading frame of the neomycin phosphotransferase U gene was cloned into the polylinker sequence of the influenza vector plasmid described above in inverse orientation with respect to the polymerase I promoter, and E. coli was transformed therewith.
  • Antigen-specific T cells are available in the laboratory for the detection of the neomycin phosphotransferase ⁇ gene product.
  • 5 ⁇ g plasmid DNA were mixed with 185 ⁇ l medium and 15 ⁇ l Lipofectamine TM (Gibco BRL) and incubated for 30 min at room temperature.
  • influenza A vuus 1
  • lxl 0 7 MDCK cells were incubated with 1 ml virus supernatant With lO ⁇ l of the culture supernatant harvested again after 15 hours, 1 ⁇ 10 5 LCL were infected and then co-cultivated with the same number of antigen-specific T cells. After 20 hours, the GM-CSF concentration in the culture supernatant was determined using an ELISA
  • the neomycin phosphotransferase II gene was again used as the model antigen for MHC class II restricted T line detection after retroviral transduction, and the green fluorescence protem (GFP) was used for the determination of the infection efficiency.
  • the open reading frames of both genes were retrovirally between the 5 and 3 Long Terminal Repeats (LTR) from Moloney murme leukemia virus (M-MuLV) was cloned into the plasmid PDMCO (Gngnani et al, 1998), and amplified as described above with E. coli.
  • lxlO 5 cells were taken up in 2 ml of virus supernatant and polybrene was added at a concentration of 2 ⁇ g / ml. The cells were then centrifuged at 1800 ⁇ m in a Va ⁇ fuge 3 2RS (Heraeus) for 30 min at room temperature. After adding new virus supernatant and again The cells were used for centrifugation Incubated for 12 hours at 37 ° C. The virus supernatant was then replaced by culture medium. 48 hours after infection, lxl 0 3 infected LCL were co-cultivated with the same number of antigen-specific T cells. After 20 hours, the GM-CSF concentration in the culture supernatant was determined using an ELIS As.

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  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Mycology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé d'identification d'antigènes à restriction CMH.
EP00960681A 1999-09-21 2000-09-20 Procede d'identification d'antigenes a restriction cmh Withdrawn EP1214407A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
DE19945171 1999-09-21
DE19945171 1999-09-21
DE19951543 1999-10-26
DE19951543 1999-10-26
DE19962508 1999-12-23
DE19962508A DE19962508C2 (de) 1999-09-21 1999-12-23 Verfahren zur Identifizierung von MHC-Restringierten Antigenen
PCT/EP2000/009217 WO2001022083A2 (fr) 1999-09-21 2000-09-20 Procede d'identification d'antigenes a restriction cmh

Publications (1)

Publication Number Publication Date
EP1214407A2 true EP1214407A2 (fr) 2002-06-19

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP00960681A Withdrawn EP1214407A2 (fr) 1999-09-21 2000-09-20 Procede d'identification d'antigenes a restriction cmh

Country Status (3)

Country Link
EP (1) EP1214407A2 (fr)
AU (1) AU7288600A (fr)
WO (1) WO2001022083A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5792604A (en) * 1996-03-12 1998-08-11 University Of British Columbia Method of identifying MHC-class I restricted antigens endogenously processed by cellular secretory pathway
WO1999041383A1 (fr) * 1998-02-11 1999-08-19 Maxygen, Inc. Immunisation par bibliotheque d'antigenes
CA2350207C (fr) * 1998-11-10 2011-02-08 University Of Rochester Lymphocytes t specifiques d'antigenes cible, vaccins prepares a partir desdits lymphocytes, et methodes associees

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0122083A2 *

Also Published As

Publication number Publication date
WO2001022083A3 (fr) 2001-10-11
AU7288600A (en) 2001-04-24
WO2001022083A2 (fr) 2001-03-29

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