EP1697408A2 - Antigenes associes aux tumeurs et leur utilisation - Google Patents

Antigenes associes aux tumeurs et leur utilisation

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Publication number
EP1697408A2
EP1697408A2 EP04804228A EP04804228A EP1697408A2 EP 1697408 A2 EP1697408 A2 EP 1697408A2 EP 04804228 A EP04804228 A EP 04804228A EP 04804228 A EP04804228 A EP 04804228A EP 1697408 A2 EP1697408 A2 EP 1697408A2
Authority
EP
European Patent Office
Prior art keywords
cells
cell
protein
antibody
nucleic acid
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.)
Withdrawn
Application number
EP04804228A
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German (de)
English (en)
Inventor
Oliver Gires
Reinhard Zeidler
Markus Muenz
Martina Schaffrik
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vaecgene Biotech GmbH
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Original Assignee
Vaecgene Biotech GmbH
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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Application filed by Vaecgene Biotech GmbH, Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH filed Critical Vaecgene Biotech GmbH
Publication of EP1697408A2 publication Critical patent/EP1697408A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to tumor antigens of squamous cell carcinoma, nucleic acids that encode them, and antibodies that are directed against them.
  • the invention further relates to methods for generating antigen presenting cells and T cells which are specific for such antigens.
  • the invention encompasses diagnostic and therapeutic methods for the detection or treatment of squamous cell carcinoma, in particular squamous cell carcinoma in the ENT area.
  • Carcinoma cells differ from healthy epithelium on both the RNA and protein levels in their expression pattern.
  • the overexpression of certain proteins or the formation of imitated or post-translationally modified proteins in tumor cells can lead to an immune response. This is accompanied by the activation of specific T and B lymphocytes and the formation of specific antibodies.
  • tumor-specific antibodies are suitable in principle as diagnostic markers for the detection of a tumor disease. Since such antibodies can also be present in the serum of patients in early tumor stages, they are also suitable as biomarkers for early cancer detection.
  • tumor-specific antibodies from the serum of cancer patients are a suitable means of identifying immunogenic tumor-associated antigens.
  • AMIDA autoantibody-mediated identification of antigens
  • 2D two-dimensional
  • the protein lysate comes from an established human carcinoma cell line (allogeneic method). This lysate is incubated with the serum of a tumor patient or with pooled immunoglobulins of subclass G (IgGs) from 100 non-tumor patients (commercially available from SIGMA in Deisenhofen).
  • IgGs immunoglobulins of subclass G
  • the protein lysate comes from a tumor biopsy (autologous procedure). The lysate is incubated with serum from the same patient or with pooled sera from 100 non-tumor patients as under 1.
  • each differentially formed and AMIDA-isolated protein is a tumor-associated antigen, which is suitable in principle as a diagnostic marker for the detection of a tumor disease.
  • CK8-specific antibodies have also been described in patients with hepatocellular carcinoma, in esophageal carcinoma and in Nepalese autoimmune diseases. Since the inventors had isolated CK8 from the serum of a tumor patient, they asked about the frequency of such antibodies in other tumor patients and also in non-tumor patients. To quantify these antibodies, the inventors used a detection method in which colored beads were coated with recombinant CK8 protein.
  • the tumor antigens according to the invention can be used in many ways: inter alia, (i) antigen-presenting cells (APCs), such as, for example, dendritic cells or B cells, can be loaded with them and thus used to activate specific T cells, ( ii) Monoclonal and bispecific antibodies can be produced which are of great benefit for the diagnosis of a disease and the therapy of patients, (iii) the antigens can be used as peptides or as DNA vaccines.
  • APCs antigen-presenting cells
  • the antigens can be used as peptides or as DNA vaccines.
  • the present invention is based, inter alia, on the above-mentioned AMIDA method for the identification of antigens which are associated with diseases in which a humoral immune response is formed and thus specific antibodies are formed.
  • This method is based on the precipitation of antigens from cell lysates or bacterial, parasite and / or viral preparations mediated by autologous, allogeneic or xenogeneic antibodies with autologous, allogeneic and / or xenogenic sera, ascites or pleural fluids.
  • the induction of an immune response, which is associated with the production of antibodies, is the only basic requirement for the use of AMIDA.
  • the method is therefore particularly suitable for identifying tumor antigens, but also for those antigens which are associated with autoimmune diseases or bacterial, viral and parasitic infections.
  • antigens as used in this description, is to be understood as structures against which an organism forms antibodies because they are foreign to its immune system. Knowledge of antigens that are as specific as possible is an important prerequisite for diagnosis and immunotherapy of tumor patients and people who suffer from, for example, an autoimmune disease or a chronic infection, such antigens, which are more or less specific to the respective disease, enable the detection and targeting in vivo and in vitro of tumor cells, cells that target one Are autoimmune, as well as infected cells and infectious organisms.
  • the focal points of the invention are thus the diagnostic detection of antigens or of autoantibodies against them using the tumaror antigens according to the invention and the use of the antigens for therapeutic purposes.
  • the invention provides a new and promising tool for the therapy and diagnosis of a large number of diseases and can also make a decisive contribution to the development of new therapies for these diseases.
  • Table 1 27 antigens were isolated by AMLDA from tumor biopsies isolated from patients with head and neck cancer and from cancer cell lines.
  • AMIDA All of the antigens listed in Table 1 were developed using a technology that we developed for the first time (AMIDA; Gires et al (2004) Profile identification of disease-associated humoral antigens using AMIDA, a novel proteomics-based technology. Cell Mol Life Sei 61, 1198-1207 ; Rauch, et al (2004) AUogenic antibody-mediated identification of head and neck cancer antigens.Biochem Biophys Res Commun 323, 156-162) from tumor biopsies.
  • AMIDA is a technology for the identification of tumor antigens based on the presence of specific antibodies in the blood (and other body fluids) of tumor patients. Only those antigens against which antibodies are present in the blood are isolated. This also applies to all antigens listed here.
  • All antigens listed here have a common property. This property is their antigenicity, which felt for the induction of antibodies. In their function, these antigens form a homologous group of proteins, which is a common and characteristic property of these antigens. This property was used to clearly define the antigens with our AMIDA technology. They represent functional homologues in the classic sense. Even if the physiological function of these antigens is not homogeneous (in some cases it is not even known), all of the antigens listed here are so-called tumor antigens, which are principally for diagnosis and / or targeted immunotherapy.
  • the antigens themselves are detected and quantified using suitable methods. Both embodiments are preferably used to detect a tumor disease in a human individual. - Combinations of the antigens listed in Table 1 and / or the autoantibodies recognizing these antigens are used to detect a tumor disease.
  • tumor markers can be known markers (such as PSA, CEA etc.), but they can also be markers that will be discovered in the future and that have diagnostic value or only in combination with one or more of the ones in Table 1 obtained antigens or autoantibodies against these antigens.
  • the present invention relates to an immunogenic squamous cell carcinoma antigen which comprises at least one of the proteins of SEQ ID NO: 1-27 or a variant thereof, the variant comprising one or more additions, insertions, substitutions and / or deletions in the Comparison to the respective protein of SEQ ID NO: 1-27, and wherein the immunogenic activity of the variant of the activity of the respective unmodified protein of SEQ ID NO: 1-27 is essentially the same.
  • immunogenic activity refers to the immunogenic function of the proteins of the invention.
  • the tumor antigens according to the invention are overexpressed in various forms of squamous cell carcinoma and are therefore important targets for immune-based anti-cancer therapies and the detection or early detection of such diseases.
  • the tumor antigens as disclosed hereinbefore are considered to the extent that they are capable of inducing an immune response in mammals, preferably humans, so as to serve as a therapeutic.
  • immunogenic activity relates in particular to the immunogenic function of the proteins according to the invention to produce immune reactions, in particular immune reactions which are mediated by cytotoxic T cells.
  • amino acid sequences of the present invention also encompass all sequences that differ from the sequences disclosed herein by amino acid insertions, deletions and substitutions.
  • Amino acid substitutions are preferably the result of replacing one amino acid with another amino acid with similar structural and / or chemical properties. properties, that is conservative amino acid replacements. Amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues involved.
  • non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine;
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagus and glutamine;
  • positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Inserts” or “deletions” typically range from 1-3 amino acids. The permitted variation can be determined experimentally by systematically inserting, deleting or substituting amino acids in a protein using recombinant DNA techniques and examining the resulting recombinant variants with regard to their immunological activity. For this, the person skilled in the art does not need more than to carry out routine experiments.
  • variant also includes peptide fragments of the tumor antigens according to the invention, such as can be used, for example, for pulsing APC.
  • Such peptide fragments preferably have 5-50, particularly preferably 7-20 and most preferably 8-15 amino acid residues.
  • variants of the proteins according to the invention show at least 50%, particularly preferably at least 70%, most preferably at least 80% sequence identity to the peptides of SEQ ID NO: 1-27.
  • the tumor antigen KIAA1273 / TOB3 (SEQ ID NO: 2) has proven to be a particularly preferred tumor antigen, of function hitherto unknown, which has excellent suitability as a tumor marker.
  • KIAA1273 / TOB3 showed an extremely strong overexpression in head and neck carcinomas, whereas KIAA1273 / TOB3 was only expressed in very low concentrations in cells of the basement membrane in healthy mucous membranes. This makes KIAA1273 / TOB3 a new marker for head and neck cancer.
  • the present invention relates to an isolated nucleic acid encoding one or more of the proteins of claim 1.
  • nucleic acid sequence relates to a heteropolymer of nucleotides or the sequence of these nucleotides.
  • nucleic acid and polynucleotide are used interchangeably herein and refer to a heteropolymer of nucleotides.
  • isolated refers to a naturally occurring nucleic acid having the two sequences of which it (one at the 5 'end and one at the 3' end) in the naturally occurring genome of the organism from which it is derived is obtained, is surrounded, is not immediately adjacent.
  • an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided that the nucleic acid sequences that normally flank this recombinant DNA molecule in a naturally occurring genome are removed or missing.
  • an isolated nucleic acid closes a recombinant DNA without limitation. one that exists as a separate molecule (e.g.
  • an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or a fusion nucleic acid sequence.
  • isolated also includes any non-naturally occurring nucleic acid because non-naturally occurring nucleic acid sequences cannot be found in nature and do not have any immediately adjacent sequences in a naturally-occurring genome.
  • a non-naturally occurring nucleic acid such as for example, a genetically engineered nucleic acid is considered an isolated nucleic acid.
  • a genetically engineered nucleic acid can be made using conventional cloning techniques or chemical nucleic acid synthesis techniques.
  • Isolated non-naturally occurring nucleic acids can be independent of other sequences or into a vector, an autonomously replicating plasmid Virus (for example, a retrovirus, adenovirus or Heipes virus) or built into the genomic DNA of a procaiyte or eukaryote.
  • a non-naturally occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or a fusion nucleic acid sequence.
  • the polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes to the complement of the disclosed nucleotide sequences under moderately stringent or stringent conditions; a polynucleotide that is an allelic variant of any of the polynucleotides described above; a polynucleotide encoding a species homolog of any of the proteins disclosed herein; or a polynucleotide encoding a polypeptide that has an additional specific domain or a truncation or truncation of the disclosed proteins.
  • the stringency of hybridization relates to conditions under which polynucleotide duplexes are stable.
  • the stability of a double strand is a function of sodium ion concentration and temperature (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2n Ed. (Cold Spring Harbor Laboratory, (1989)). those used for hybridization can easily be modified by a person skilled in the art.
  • weakly stringent hybridization describes conditions that hybridize in 10% formamide, 5 x Denhart's solution, 6 x SSPE, 0.2% SDS at 42 ° C, followed by washing in 1 x SSPE, 0.2% SDS at 50 ° C are equivalent.
  • Denhart's solution and SSPE are well known to those skilled in the art, as are other suitable hybridization buffers.
  • Moderately stringent hybridization means conditions that allow DNA to bind to a complementary nucleic acid that has approximately 60% identity, preferably approximately 75% identity, particularly preferably approximately 85% identity to this DNA; identity of greater than about 90% to this DNA is particularly preferred.
  • Moderately stringent conditions are preferably conditions that hybridize in 50% formamide, 5 x Denhart's solution, 5 x SSPE, 0.2% SDS at 42 ° C followed by washing in 0.2 x SSPE, 0.2% SDS at 65 ° C are equivalent.
  • High stringency hybridization means conditions that allow hybridization only of those nucleic acid sequences that form stable double strands in 0.018 M NaCl at 65 ° C (ie, if a double strand is not stable in 0.018 M NaCl at 65 ° C, it is among those considered here highly stringent conditions not stable).
  • nucleic acid hybridization techniques can be used to identify and recover a nucleic acid that is within the scope of the present invention. Briefly, any nucleic acid with some homology to a sequence or fragment thereof set out in this invention can be used as a probe to identify a similar nucleic acid by hybridization under moderately stringent to highly stringent conditions. Such similar nucleic acids can then be isolated, sequenced and analyzed to determine if they are within the scope of the invention as described herein.
  • the invention also relates to an immunogenic antigen encoded by a nucleic acid as defined above.
  • the invention comprises vectors which comprise one or more of the nucleic acids as defined above.
  • This expression vector preferably comprises one or more regulatory sequences.
  • the term "expression vector” generally refers to a plasmid or a phage or a virus or a vector for expressing a polypeptide from a DNA (RNA) sequence.
  • An expression vector can comprise a transcription unit which has an arrangement of the following: (1) a genetic element or elements with a regulatory role in gene expression, for example promoters and / or enhancers, (2) a structural sequence or coding sequence which transcribes in mRNA and translated into a protein and (3) appropriate transcription start and termination sequences.
  • Structural units that are intended for use in yeasts or higher eukaryotic expression systems preferably include a leader sequence that the extracellular secretion of a translated protein by a host allows.
  • a recombinant protein if expressed without a leader or transport sequence, may include an N-terminal methionine residue. This residue may or may not subsequently be cleaved from the expressed recombinant protein to provide the final product.
  • the present invention further provides hosts, for example host cells, which have been transformed such that they contain the polynucleotides according to the invention.
  • transformation or “transformation” means the introduction of DNA into a suitable host cell so that the DNA can be replicated, either as an extrachromosomal element or by chromosome integration depending on the expression vector used.
  • host cells may contain nucleic acids of the invention introduced into the host cell using known transformation methods in a stable or transient manner.
  • the present invention further provides host cells that have been genetically engineered to express the polynucleotides of the present invention, such polynucleotides being operably linked to a regulatory sequence that is heterologous to the host cell and that express the expression of the polynucleotides in the Cell controls.
  • the host cell may be a higher eukaryotic host cell such as a plant cell, a mammalian cell or a lower eukaryotic host cell such as a yeast cell or may be an insect cell or the host cell may be a prokaryotic cell such as a bacterium.
  • the introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran-mediated transfection, lipofection or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).
  • the most preferred cells are those that do not normally express the particular protein or that express the protein at a low endogenous level or concentration.
  • the proteins according to the invention can be expressed in mammalian cells, yeasts, bacteria or other cells under the control of suitable promoters. Suitable cloning and expression vectors for use with prokaryotic see and eukaryotic hosts and the corresponding procedures are described by Sambrook et al. (1989) A Laboratory Manual, 2 nd Edition, Cold Spring Harbor, New York: Molecular Cloning.
  • the mammalian cell is preferably a CHO, COS, HeLa, 293T, HEH or BHK cell.
  • Bacterial cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells. E. coli and Bacillus subtilis are preferred.
  • Fungal cells can also be used, such as yeast cells (from Saccharomyces, Schizosaccharomyces, Pichia) and Aspergillus cells. Drosophila S2 and Spodoptera frugiperda Sf9 or SF21 cells are preferred as insect cells.
  • N. tabacum or Arabidopsis thaliana cells can be used as plant cells.
  • the present invention provides an antibody or ap- tamer directed against one or more epitopes of a protein according to claim 1.
  • antibody refers to intact antibodies as well as antibody fragments that retain some ability to selectively bind to an epitope. Such fragments include, without limitation, Fab, F (ab ') 2 , recombinant "single chain” Antibodies and Fv Antibody Fragments.
  • epitope determinants usually consist of chemically active surface groups of molecules (for example amino acid or sugar residues) and usually have three-dimensional structural properties as well as special charge properties.
  • the antibodies of the present invention can be made using any known method.
  • the pure protein according to the invention or a fragment thereof can be provided and used as an immunogen in order to elicit an immune response in an animal such that specific antibodies are generated.
  • polyclonal antibodies The preparation of polyclonal antibodies is well known to those skilled in the art. See, for example, Green et al, Production of Polyclonal Antisera, in Immunochemical Protocols (Man- son, editor), pages 1-5 (Humana Press 1992) and Coligan et al, Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in Current Protocols In Immunology, section 2.4.1 (1992). In addition, various techniques of immunology for the purification and concentration of polyclonal antibodies are known to the person skilled in the art, as well as of monoclonal antibodies (Coligan et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).
  • monoclonal antibodies can be obtained by injecting a composition containing the protein of the invention into mice, then verifying the presence of antibody production by examining a serum sample, removing the spleen for obtaining B-lymphocytes and the B-lymphocytes with myeloma cells for generation hybridomas are fused, the hybridomas are cloned, the positive clones that generate a monoclonal antibody to the protein are selected, and the antibodies are isolated from the hybridoma cultures.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures using a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein A or G Sepharose, size exclusion chromatography and ion exchange chromatography. See, for example, Coligan et al., Sections 2.7.1-2.7.12 and section “Immunoglobulin G (IgG)”, in Methods In Molecular Biology, volume 10, pages 79-104 (Humana Press 1992).
  • antibody in the sense of the present invention means that the respective antibody, due to its antigen specificity, induces or supports a stimulation of the patient's immune system desired in the treatment of the respective disease.
  • immunostimulating antibodies in the sense of the present invention are those which cause T cell activation. Activation of cytotoxic T cells (CTL, "cytotoxic T lymphocytes", so-called T killer cells) is particularly advantageous.
  • CTL cytotoxic T lymphocytes
  • Antikö ⁇ er includes i. S. of the present invention, both polyclonal antibodies and monoclonal antibodies, chimeric antibodies (single chain antibodies), humanized antibodies, which can all be present in bound or soluble form, and also fragments of the aforementioned antibodies. In addition to the fragments of antibodies according to the invention in isolation, antibodies according to the invention can also occur in a recombined form as fusion proteins with other (protein) components.
  • antibodies means both polyclonal, monoclonal, human or humanized or recombinant antibodies or fragments thereof, single chain antibodies or synthetic antibodies.
  • the polyclonal antibodies are heterogeneous mixtures of antibody molecules which are produced from sera from animals which have been immunized with an antigen.
  • the subject of the invention also includes polyclonal monospecific antibodies which are obtained after the antibodies have been purified (for example via a column which is loaded with peptides of a specific epitope).
  • a monoclonal antibody contains an essentially homogeneous population of antibodies which are specifically directed against antigens, the antibodies having essentially the same epitope binding sites.
  • Monoclonal antibodies as mentioned above, can be obtained by the methods known in the art (e.g. Koehler and Milstein, Nature, 256, 495-397, (1975); U.S.
  • Patent 4,376,110 Harlow and Lane, Antibodies: A Laboratory Manual Cold Spring, Harbor Laboratory (1988); Ausubel et al., (Eds), 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York). The description contained in the aforementioned references is incorporated by reference into the disclosure of the present invention.
  • antibodies according to the invention can also be produced by methods as described in the aforementioned publications.
  • antibody-producing cells are grown for this purpose and the mRNA, provided the optical density of the cells is sufficient, is lysed with guanidinium thiocyanate, acidified with sodium acetate, extracted with phenol, chloroform / isoamyl alcohol, precipitated with isopropanol and Wa- see with ethanol isolated from the cells in a known manner. Subsequently, the reverse transcriptase is used to synthesize cDNA from the mRNA.
  • the synthesized cDNA can be inserted directly or after genetic manipulation, for example by "site directed mutagenesis", introduction of insertions, inversions, deletions or base exchanges into suitable animal, fungal, bacterial or viral vectors and expressed in the corresponding host organisms.
  • Bacterial or yeast vectors such as ⁇ BR322, pUC18 / 19, pACYClS4, lambda or yeast mu vectors, are preferred for cloning the genes and for expression in bacteria such as E. coli or in yeast such as Saccharomyces ce-revisiae.
  • Antibodies according to the invention can belong to one of the following immunoglobulin classes: IgG, IgM IgE, IgA, IgD and possibly a subclass of the abovementioned classes, such as the subclasses of the IgG or mixtures thereof.
  • IgG and its subclasses such as IgGl, IgG2, IgG2a, IgG2b, IgG3 or IgGM, are preferred.
  • the IgG subtypes IgGl / k or IgG2b / k are particularly preferred.
  • a hybridoma cell clone that produces monoclonal antibodies according to the invention can be cultured in vitro, in situ or in vivo. Large titans of monoclonal antibodies are preferably produced in vivo or in situ.
  • the chimeric antibodies according to the invention are molecules which contain different constituents, these being derived from different animal species (for example antibodies, which is a variable region which is derived from a mouse monoclonal antibody and a constant region of a human one Have immunoglobulins). Chimeric antibodies are preferably used, on the one hand, to reduce the immunogenicity in use and, on the other hand, to increase the yields in production, e.g. For example, murine monoclonal antibodies give higher yields from Hyforidom cell lines, but also lead to higher immunogenicity in humans, so that human / murine chimeric or fully humanized antibodies are preferably used.
  • a monoclonal antibody that combines the hypervariable complementarity-defining region (CDR) of a murine monoclonal antibody with the other areas of a human antibody.
  • CDR complementarity-defining region
  • Such an antibody is called humanized antibody.
  • Chimeric antibodies and processes for their production are known from the prior art Technique known (Cabilly et al., Proc. Natl. Sci. USA 81: 3273-3277 (1984); Morrison et al. Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984); Boulianne et al.
  • Antikö ⁇ er is intended to include both intact molecules and fragments thereof. Fragments include all shortened or modified antibody fragments with one or two antigen-complementary binding sites, such as antibody parts with a binding site formed by light and heavy chains corresponding to the antibody, such as Fv, Fab or F (ab ') 2 fragments or single-strand fragments. called (see above). Shortened double-strand fragments, such as Fv, Fab or F (ab ') 2 , Fab and F (ab') 2 , fragments are preferably devoid of an Fc fragment, such as present in an intact antibody, so that they are transported faster in the bloodstream can be and have comparatively less non-specific tissue binding than intact Antiköiper.
  • fragments are typically made by proteolytic cleavage using enzymes such as.
  • A. papain (for the production of Fab fragments) or pepsin (for the production of F (ab ') 2 , fragments) can be used or obtained by chemical oxidation or by genetic engineering manipulation of the antibody genes.
  • Antibodies of this type can be used, for example, for the treatment of cancer cells or cells infected with a pathogenic pathogen, in that they are directed against a surface determinant of a tumor cell or a pathogen and, according to a preferred embodiment, a toxin, for example ricin or Pseudomonas toxins, is coupled to the antibody become; summarized z. B. Valera (1994) Blood 83: 309-317; Vitetta et al. (1993) Immunol. Today 14: 252 to 259.
  • Immunostimulating antibodies in the sense of the present invention are preferably produced by recombinant DNA techniques. Particularly preferred immunostimulatory antibodies are multiply specific, in particular bispecific, and / or multiply, especially trifunctional.
  • bispecific antibodies in particular, recombinant antibody molecules which are produced by recombinant techniques are to be mentioned, for example scFv molecules (so-called “single chain antibodies”), diabodies etc.
  • scFv molecules single chain antibodies
  • the basic structure of bispecific antibodies and immune conjugates is described, for example, in van Spriel et al. (2000) Immunol. Today 21: 391-397.
  • Bispecific antibodies can of course also be produced by known hybridoma techniques. Processes for producing multivalent and bispecific Antikö ⁇ er fragments are known to a person skilled in the art and are described, for example, in Tomlinson and Holliger (2000) Meth. Enzymol. 326: 461 ff.
  • bispecific antibody is a trifunctional bispecific antibody, to whose Fc part, that is to say the part of the antibody which is not directly involved in antigen binding, can bind accessory immune cells (see Zeidler et al .: British Journal of Cancer 2000, Journal of Immunology 1999).
  • the antibody can therefore be administered intraperitoneally, systemically (intravenously or intraarterially, inframuscularly, intradermally, subcutaneously, intratumorally) or else selectively in or via a defined organ.
  • administration via the bone marrow (as an immunological organ) or via a superselective catheter into a vessel supplying the respective organ (artery) or the direct intratumoral application can be mentioned.
  • a specific example of such an application by means of a catheter can be given in the A. hepatica for selective application in the liver or for systemic administration after passage through the organ.
  • organ-specific application are those in the liver via the portal vein, in the kidney via the renal artery, intrathecal application in cerebral tumors, in the colon area via mesentarial vessels, in the pancreas via the celiac trunk and the superior mesenteria and in Limb tumors via the corresponding arteries. Furthermore, it can also be applied directly to a tumor.
  • the invention also provides a pharmaceutical composition which comprises at least one antibody as defined above, as defined above.
  • the pharmaceutical composition of the present invention is suitable especially for the treatment of the diseases listed above. If appropriate, the pharmacologically active constituents of the pharmaceutical composition according to the invention are present in conjunction with one or more carriers and / or auxiliaries, as is explained in more detail below.
  • the antibody is advantageously provided in suitable formulations.
  • suitable formulations are known to a person skilled in the art and contain, in addition to the substances having a therapeutic or immunostimulatory effect, one or more pharmaceutically acceptable carriers and / or pharmaceutically acceptable vehicles.
  • Corresponding ways of appropriately formulating and producing such formulations are disclosed, for example, in "Remington's Pharmaceutical Sciences” (Mack Pub. Co., Easton, PA, 1980), which forms part of the disclosure of the present invention.
  • Suitable carriers for parenteral administration are, for example, sterile water, sterile saline, polyalkylene glycols, hydrogenated naphthalenes and in particular biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers.
  • Pharmaceutical compositions according to the invention can contain fillers or substances, such as lactose, manitol, substances for covalently attaching polymers, such as, for. B.
  • polyethylene glycol to immunostimulatory antibodies according to the invention complexation with metal ions or inclusion of materials in or on special preparations of polymer compounds, such as polylactate, polyglycolic acid, hydrogel or on liposomes, microemulsions, micelles, unilameral or multilamelar vesicles, erythrocyte fragments or spheroblasts.
  • polymer compounds such as polylactate, polyglycolic acid, hydrogel or on liposomes, microemulsions, micelles, unilameral or multilamelar vesicles, erythrocyte fragments or spheroblasts.
  • the respective embodiments of the pharmaceutical compositions are selected depending on the physical behavior, for example with regard to solubility, stability, bioavailability or degradability.
  • Controlled or constant release of the active ingredient components according to the invention includes the formulation based on lipophilic depots (e.g. fatty acids, waxes or oils).
  • coatings of pharmaceutical compositions or medicaments according to the invention containing the therapeutically active substances are also disclosed (for example polyoxamers or polyoxamines).
  • therapeutically active substances or compositions according to the invention can be protective coatings, for. B. protease inhibitors or permeability enhancers, point.
  • Preferred carriers are typically aqueous carrier materials, using water for injection (WFI) or water buffered with phosphate, citrate, HEPES or acetate, etc. and the pH typically being 5.0 to 8.0 (preferably 6.5 to 7, 5) is set.
  • the carrier or vehicle will additionally preferably contain salt components, e.g.
  • the carrier or vehicle can furthermore contain additional components, such as human serum albumin (HSA), polysorbate 80, sugar or amino acids, etc.
  • HSA human serum albumin
  • polysorbate 80 polysorbate 80
  • sugar or amino acids etc.
  • the manner of administration and the dosage of the medicament according to the invention or of the pharmaceutical compositions depend on the type of disease to be controlled, possibly its stage, the antigen to be controlled as well as the body weight, age and gender of the patient.
  • concentration of the active components in the formulations according to the invention can be varied within a wide range. Doses of the antibody according to the invention vary in the range from about 1 ⁇ g to about 10 mg.
  • the pharmaceutical composition of the present invention can comprise one or more of the proteins according to the invention, one or more of the nucleic acids according to the invention, a vector as defined above, an APC or T cell as defined below and a pharmaceutically acceptable carrier.
  • the present invention relates to a hybridoma which produces a monoclonal antibody which has a binding specificity for a protein according to claim 1.
  • the invention comprises an ex vtvo method for generating a population of autologous or allogeneic antigen presenting cells (APCs) which are capable of inducing an effective immune response against a protein according to the invention, which comprises the following steps:
  • an ex v / vo process for the production of genetically engineered APCs which are capable of inducing an effective immune reaction against a protein according to the invention comprises the following steps:
  • the nucleic acid in step a) is provided in an expression vector.
  • the present invention relates to an antigen presenting cell (APC) which can be obtained by one of the methods mentioned above.
  • APC antigen presenting cell
  • this APC is a dendritic cell or a B cell.
  • An eighth aspect of the invention relates to an ex vivo method for the detection and production of T cells which are specific for a protein according to the invention, which comprises the following step:
  • step c) determining the presence of a specific activity of the T cells against such an APC, and d) optional selection and cultivation / expansion of those T cells which in step c) showed specificity for such an APC as defined above.
  • the term activation as used herein is defined as stimulation of T cells.
  • the cocultivation step which is carried out in b), is necessary in order to activate and thus select specific T cells. This can be done, for example, by applying the proteins of the present invention directly to a sample, preferably a blood sample, obtained from the patient.
  • PBMC mononuclear peripheral blood mononuclear cells
  • APCs for example B cells
  • the pulsed APCs can be prepared separately before they are used in step b), simply by pulsing APCs, preferably autologous APCs, with the proteins according to the invention in accordance with methods known in the art and then co-cultivating the sample with the APCs.
  • APCs are preferably selected to express the appropriate MHC class I molecule.
  • the T cells are preferably cytotoxic T cells, particularly preferably CD8 + T cells.
  • the activity of the T cells in step c) above can be determined by methods known per se in the art. Further information can be found, for example, in Immunology, 5th edition, 2001.
  • this determination can be carried out using a 51 Cr release assay.
  • T cell function can be determined by a 51 Cr release assay using a T cell bioassay based on the killing of a target cell by a cytotoxic T cell. This assay is based on the uptake of radioactively labeled sodium chromate, Na 2 51 CrO, by living cells that do not spontaneously release this sodium chromate. When these labeled cells are killed, the radioactive chromate is released and its presence in the supernatant of the mixtures of target cells and cytotoxic T cells can be measured.
  • target cells are T2 cells which have been pulsed with one or more of the proteins according to the invention.
  • the determination in step c) is carried out by measuring the amount of cytokines in the T cells.
  • the following procedures can be used for this purpose:
  • the cytokines can be measured by intracellular cytokine staining or staining.
  • the intracellular cytokine staining approach is based on the use of metabolic poisons that inhibit protein export from the cell. Then the cytokine accumulates within the endoplasmic reticulum and the vesicular network of the cell. If the cells are then fixed and made permeable using mild surfactants, antibodies can gain access to these intracellular compartments and are able to detect the cytokine.
  • the cytokines are measured extracellularly using an ELISPOT assay.
  • the ELISPOT assay is a modification of an ELISA antigen capture assay. It is an excellent approach for measuring the frequency of T-cell reactions. Populations of T-cells are stimulated with the antigen of interest and are then allowed to settle on plastic plates coated with antibodies and against the one to be examined Cytokine are targeted. All cytokines secreted by the T cell are captured by the antibody on this plastic plate.
  • the cells are removed and a second antibody for the cytokine is added to the plate, so that a circle of bound cytokine is shown which surrounds the position of each activated T cell, counting each spot and knowing the number of the T cells originally added to the plate allows easy calculation of the frequency of T cells that secrete the particular cytokine.
  • the invention relates to T cells which can be obtained by the method explained above.
  • the present invention relates to a diagnostic composition
  • a diagnostic composition comprising one or more of the proteins according to the invention, an antibody as defined above or a T cell according to the invention.
  • composition can be offered, for example, in the form of a test kit, with which e.g. the corresponding tumor antigens are detected in a sample (e.g. biopsy) via the antibody according to the invention, or with which the autoantibodies of a patient to be examined are detected via the tumor antigens according to the invention, or the T cells directed against an antigen according to the invention e.g. be determined with the help of specific tetramers.
  • a test kit with which e.g. the corresponding tumor antigens are detected in a sample (e.g. biopsy) via the antibody according to the invention, or with which the autoantibodies of a patient to be examined are detected via the tumor antigens according to the invention, or the T cells directed against an antigen according to the invention e.g. be determined with the help of specific tetramers.
  • the invention relates to the use of the above-mentioned compositions in the diagnosis or therapy of squamous cell carcinoma, in particular squamous cell carcinoma in the ENT area, e.g. of the head and neck area.
  • Figure 1 Principle of the autologous version of AMIDA.
  • the step of immunoprecipitation isolates only those proteins against which autoantibodies are present in sera from tumor patients and non-tumor patients.
  • Autoantikö ⁇ er which are only present in patient sera, can be used to detect a tumor disease.
  • the ZelUysat is obtained from an established tumor cell line. Allogenic sera from tumor patients are then compared with those from non-tumor patients.
  • Figure 2 CK8-specific antibodies are significantly increased in the blood of most tumor patients (orange) compared to non-tumor patients (green). Quantified in a modified ELISA system (and calculated as 'relative light units'), the sensitivity of the test is 83.3% and the specificity is 89.5%).
  • KLAA 1937 is expressed in all examined carcinomas of the head and neck area (T232-T246). In contrast, expression in almost all healthy control tissues (Nl, N2, N4-9) is not detectable.
  • GHD-1 is the carcinoma cell line that was used to identify KIAA1937 as a tumor antigen. It originated from hypopharyngeal carcinoma and was established in the inventors' laboratory.
  • FIG. 4 KIAA1273 / TOB3 mRNA expression in healthy mucosa and in carcinomas. Cryosections of healthy mucosa (a and b) and carcinoma samples (ch) were performed by in situ hybridization using a labeled KIAA1273 / TOB3 / AAA-ATPase KIAA1273 / TOB3 complementary anti-sense RNA (AAA-ATPase KIAA1273 / TOB3 AS) and as a control labeled KIAA1273 / TOB3 / AAA-ATPase KIAA1273 / TOB3 Sense RNA (AAA-ATPase KIAA1273 / TOB3 S) performed. Healthy mucosa stained weakly with the complementary RNA in cells of the basement membrane layer, whereas carcinoma cells showed a strong and specific staining.
  • FIG. 5 Immunohistochemis rather detection of e-FABP (ac), Grb2 (df) and hnRNP H (gi) in healthy oral mucosa (a, d, g), primary carcinomas of the head and neck region (b, e, h) and derived from them Metastases (c, f, i).
  • FIG. 6 Detection of CK8 expression on the cell surface. FaDu cells were treated with sucrose / EGS and the plasma membrane was imperialized. Subsequently Antibodies were used specifically for CK8 or actin in combination with FITC or Texas Red conjugated secondary antibodies for the detection of both molecules. (B) Fa- Du cells were permeabilized with Triton X-100 and actin was detected with specific antibodies. Confocal laser scanning microscopy images of the colorations are shown.
  • Figure 7 CK8 detection in autologous tissues. Frozen sections of healthy (a, d), hypeiplastic (b) and tumor tissue (c, e) or metastases (f) of a patient were made and stained with CK8-specific antibodies. The expression of CK8 increased with increasing tissue transformation.
  • Sera from patients suffering from squamous cell carcinoma of the upper air and food passages were collected together with samples of the autologous tumors and subjected to an AMIDA screening process, as shown schematically in Figure 1.
  • Cancer cell biopsies were processed into single cell suspensions and a defined number of cells was lysed to obtain crude protein preparations or membrane-associated protein fractions.
  • Potential tumor-associated antigens (TAAs) were obtained overnight by immunoprecipitation (IP) with immobilized autologous serum antibodies and separated by 2DE, as described in the experimental methods of the PCT application mentioned above (WO 03/025568).
  • IP immunoprecipitation
  • 2DE immunoprecipitation
  • As a control autologous leukocytes were processed as a protein reference in parallel with the tumor material.
  • Serum antibodies coupled to Sepharose A beads were also processed and served as a second negative control.
  • an AMIDA "screen” was carried out allogenously.
  • Two established ENT carcinoma cell lines served as the protein source.
  • the proteins were immunoprecipitated with immobilized antibodies according to the allogeneic AMIDA variant (see above) and separated in a 2DE.
  • the proteins were also immunoprecipitated with pooled IgGs from 100 healthy donors.
  • An analysis of 2DE protein patterns and the differentiation of different protein spots was carried out with the Imagemaster 2DE software (FIGS. 1 and 2). Tumor IP-specific protein spots were excised, subjected to in-gel tryptic digestion and the resulting peptides were examined by MALDI-TOF mass spectrometry.
  • a total of six squamous cell carcinomas of the upper airways and food passages were examined independently of one another in an autologous AMIDA procedure, and the IgGs of 8 carcinoma patients were tested against two tumor cell lines in an allogeneic AMIDA procedure.
  • the combination of both variations of the AMIDA method allowed the identification of 27 potential tumor-associated antigens (see Table 1).
  • In situ hybridization- Frozen tissue sections (5 ⁇ m) were fixed in 4% paraformaldehyde and washed in PBS, dehydrated or dehydrated in ethanol and stored at -70 ° C. After thawing, inactivation of endogenous alkaline phosphatase with HC1 (0.2 N), and digestion with Proteinase K (10 ⁇ g / mL), the slides were treated with 0.1 M glycine / 0.05 M PBS and 4% paraformaldehyde at RT. PBS washes were performed between each treatment.
  • Sections were then permeabilized with 0.1 M triethanolamine / 0.25% acetic anhydride, washed with 2X SSC and in prehybridization buffer (5 h, RT, 50% deionized formamide, 4X SSC, 5X Denhardf s, 25 ⁇ g / ml salmon seed DNA, 0.1% SDS, 50 ⁇ g / ml t-RNA, 5% dextran sulfate) incubated.
  • prehybridization buffer 5 h, RT, 50% deionized formamide, 4X SSC, 5X Denhardf s, 25 ⁇ g / ml salmon seed DNA, 0.1% SDS, 50 ⁇ g / ml t-RNA, 5% dextran sulfate
  • KIAA1273 / TOB3 cDNA was amplified by PCR using the forward primer 5'-CGATGGTACCGATCCTGGGTGCAGATGCAGCTGGAAG-3 'and the reverse primer 5'-ATCGCTCGAGCTACAACAGGGGGTGCCCTGGG11ZP9D with the R3 as the IR3.
  • the PCR product was cloned into the pDrive vector (Qiagen, Hilden, Germany).
  • pDrive-KIAA1273 / TOB3 was linearized by BamHI or, alternatively, HindIII digestion for in vitro transmission to generate sense and antisense DIG-labeled RNA probes using the DIG RNA labeling kit (Röche, Mannheim, Germany) , including the T7 and SP6 RNA polymerases.
  • the resulting probes were diluted in pre-hybridization buffer to hybridize the sections overnight at 70 ° C. After stringent washing (2 x SSC and 0.2 x SSC), the sections were washed further with TI buffer (0.5 M maleic acid, 750 mM NaCl, pH 7.5), and incubated in T2 buffer (TI buffer + 1% blocking agent, 1 h, RT).
  • KIAA1273 / TOB3 is specifically expressed in carcinoma cells
  • the AMIDA screening enabled the isolation and identification of KIAA1273 / TOB3 (KIAA1273 / TOB3, SEQ ID NO: 2) and the examination of its tumor specificity.
  • Weak KIAA1273 / TOB3 mRNA expression was observed in the basal membrane layer of healthy mucosa, whereas highly differentiated epithelia did not express KIAA1273 / TOB3 mRNA (Fig. 4a).
  • carcinoma cells from neck and head samples expressed large amounts of the mRNA for KIAA1273 / TOB3 (Fig. 4c, e, g).
  • KIAA1273 / TOB3 de novo was thus expressed in carcinoma cells of the upper airways and food passages.
  • KIAA1273 / TOB3 is a newly identified member of the TlS21 / PC3 / BTGl / -TOB family, which contains a putative AAA-ATPase motif, with unknown function (Parng, C. et al.) And through the proto-oncogene c-Myc its expression is regulated.
  • the KIAA1273 / TOB3 overexpression may result from a direct influence on c-myc regulation in cancer cells, since c-myc is also often overexpressed in tumors.
  • GRB2 GRB2, hnRNP H and E-FABP were isolated as potential tumor-associated antigens using AMLDA technology.
  • Grb2, hnRNP H and E-FABP were strongly overexpressed in primary carcinomas as well as in locoregional metastases compared to healthy tissue (Figure 5).
  • the overexpress sion of E-FABP and hnRNP H has been studied in a number of healthy tissues and carcinomas. Both AMIDA antigens showed significant overexpression in transformed tissue (Tables 2 and 3).
  • Oropharynx 0/5 (0%) 0/5 (0%) 2/5 (40%) 3/5 (60%)
  • Cytokeratin 8 is an intermediate filament protein that causes a humoral response in vivo in HNSCC patients. In contrast to healthy epithelia, in cancer, CK8 is also expressed on the outside of the plasma membrane, as was shown by flow cytometry. Although several references describe such a phenomenon, i.e. the presence of keratins on the cell surface (21-26), this remains controversial (27). For this reason, FaDu carcinoma cells were treated with a sucrose / EGS mixture (ethylene glycol bis-succinimidyl succinate) and the plasma membrane was made impermeable. Then CK8 and, as a control, actin were detected immunohistochemically and visualized in a confocal laser scanning microscope.
  • sucrose / EGS mixture ethylene glycol bis-succinimidyl succinate
  • actin could not be detected in treated cells. be shown, although the antibody used allowed staining of actin in permeabilized cells. CK8 in turn could be detected in SukiOse / EGS treated cells on the outer side of the plasma membrane ( Figure 6).
  • E-FABP was identified in an allogeneic AMIDA approach with serum antibodies from three of eight patients. Serum reactivity to E-FABP was determined in a modified Bio-Plex approach for 48 healthy blood donors and 59 HNSCC patients. With a calculated specificity of 95%, the E-FABP-specific serum reactivity reached a sensitivity of 22%, i.e. 22% of the cancer patients were diagnosed retrospectively specifically (Figure 8).
  • Elongin A2 1 maagsttlha veklqvrlat ktepkkleky lqklsalpmt adilaetgir ktvkrlrkhq 61 hvgdfardla ar kklvlvd rntrpgpqdp eesasrqrfg ealqdqekaw gfpenatapr 121 spshspehrr tarrtppgqq rphprshsre praerkcpri apadsgryra sptrtaplrm 181 pegpepaapg kqpgrghtha aqggpllcpg cqgqpqgkav vshskghkss rqekrplcaq 241 gdwhsptlir ekscgaclre etprmpswas ardrq
  • Paraspekle protein 1 alpha isofor MMLRGNLKQV RIEKNPARLR ALESAVG ⁇ SE PAAAAAMAL ⁇ LAGEPAPPAP APPEDHPDEE MGFTIDIKSF LKPGEKTYTQ RCRLFVGNLP TDITEEDFKR LFERYGEPSE VFINRDRGFG FIRLESRTLA EIAKAELDGT ILKSRPLRIR FATHGAALTV KNLSPWSNE LLEQAFSQFG PVEKAVWVD DRGRATGKGF VEFAAKPPAR KALERCGDGA FLLTTTPRPV IVEPMEQFDD EDGLPEKLMQ KTQQYHKERE QPPRFAQPGT FEFEYASR K ALDEMEKQQR EQVDRNIREA KEKLEAEMEA ARHEHQLMLM RQDLMRRQEE LRRLEELRNQ ELQKRKQIQL RHEEEHRRRE EEMIRHREQE ELRRQQEGFK PNYMENREQ

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Abstract

L'invention concerne des antigènes associés aux tumeurs du carcinome de l'épithélium pavimenteux, l'acide nucléique codant ces antigènes, ainsi que des anticorps qui sont dirigés contre ceux-ci. L'invention concerne en outre un procédé de production de cellules présentant des antigènes et des cellules T qui sont spécifiques pour de tels antigènes. Enfin, l'invention concerne des procédés de diagnostic et thérapeutiques permettant de déceler et de traiter un carcinome de l'épithélium pavimenteux, en particulier un carcinome de l'épithélium pavimenteux dans le domaine HNO.
EP04804228A 2003-12-22 2004-12-22 Antigenes associes aux tumeurs et leur utilisation Withdrawn EP1697408A2 (fr)

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DE10360456A DE10360456A1 (de) 2003-12-22 2003-12-22 Tumorantigene und deren Verwendung
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WO2009055820A2 (fr) 2007-10-26 2009-04-30 The Regents Of The University Of California Biomarqueurs de protéines salivaires pour le cancer de la bouche de l'homme
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MX2011013452A (es) 2009-07-02 2012-04-30 Ith Immune Therapy Holdings Ab Tratamiento de cancer basado en exosomas.
EP2404618A1 (fr) 2010-07-07 2012-01-11 Stichting Katholieke Universiteit meer in het bijzonder Radboud Universiteit Nijmegen Configuration de protéine immunomodulatoire avec un polymer à squelette hélicale
GB201511191D0 (en) * 2015-06-25 2015-08-12 Immatics Biotechnologies Gmbh T-cell epitopes for the immunotherapy of myeloma
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