EP1697408A2 - Tumor antigens, and use thereof - Google Patents

Abstract

The invention relates to squamous cell carcinoma antigens, nucleic acids coding the same, and antibodies targeted thereagainst. The invention further relates to methods for producing antigen-presenting cells and T cells that are specific for such antigens. Also disclosed are diagnostic and therapeutic methods for detecting/treating squamous cell carcinomas, especially squamous cell carcinomas in the ENT region.

Description

 Tumor antigens and their use

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. Finally, 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.

Such 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.

Since a human cell may produce up to 100,000 and more different proteins, tumor-specific antibodies from the serum of cancer patients are a suitable means of identifying immunogenic tumor-associated antigens.

A technology has been developed (AMIDA for autoantibody-mediated identification of antigens) that uses such antibodies to isolate only those proteins against which specific antibodies exist from the lysate of tumor cells by immunoprecipitation and subsequent two-dimensional (2D) gel electrophoresis. This technology is described in PCT application No. WO 03/025568, the content of which is incorporated by reference. We know that there is an individual set of autoantibodies in every person, the origin and meaning of which are unknown. Usually these antibodies do not cause any symptoms. In order to be able to differentiate proteins which are recognized by these "natural" autoanticöipem from tumor-specific proteins, the inventors carried out AMIDA for control purposes also with immunoglobulins from non-tumor patients. There are two different methods:

1. 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).

2. 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.

Both methods have in common that after the lysate has been incubated with a serum, an immunoprecipitation routine and a separation of the precipitated proteins are carried out in 2D gel electrophoresis. In the comparison of the protein patterns of tumor patients and non-tumor patients resulting on the 2D gels, only those proteins can be identified exclusively against which antibodies are only present in sera from tumor patients. By definition, 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.

Using this principle, the inventors tested AMIDA with various tumor samples obtained from patients with tumors of the head, neck, intestine and lungs. One of the proteins isolated by the inventors is cytokeratin 8 (CK8), which has already been described as a tumor antigen. 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. These beads were made with Sera from tumor and non-tumor patients incubated. The antibodies bound to the CK8 beads were detected with a fluorescein-coupled second antibody (mouse anti-human IgG) and quantified in a Bio-Plex measuring device. Figure 2 shows that tumor patients have significantly more CK8 antibodies in their serum than non-tumor patients.

The targeting of cells in vivo and in vitro using immunological tools such as T cells and antibodies is therefore very strongly dependent on the knowledge of specific proteins on target cells.

The identification of molecular diagnostics for the early detection of tumors has become a particular challenge due to the high mortality caused by cancer. Accordingly, protein approaches have experienced a strong resuscitation and are of recognized importance with regard to target protein identification (HANASH, 2003). Targets, d. H. disease-specific markers are useful for antibody-based approaches, DNA or protein vaccination and for the generation of antigen-specific cytotoxic T lymphocytes. In vivo, the humoral immune response is one of the weapons of our immune system to fight pathogenic and transformed cells.

It is therefore an object of the present invention to provide new tumor antigens which can be used as improved diagnostic markers in tumor detection. It is a further object of the invention to provide new and improved therapies for combating tumor diseases, but in particular squamous cell carcinoma, based on these tumor antigens.

These tasks are solved by the subject matter of the independent claims. Preferred embodiments result from the dependent claims.

Once isolated and characterized, 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. 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.

The term “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.

Among the antigens identified by the inventors are 27 proteins not previously known as tumor antigens. These proteins are listed in Table 1. SEQ Acc. N ° Protein ID score Reference

ID NO:

1 Q9p2V9 Elongin A2 66 (?)

2 BAA86587 KLAA 1273 / TOB3 = KIAA 1273 / TOB3 100 (14) (AAH18701, NP 114127, AAH09938, AAH02542)

3 Q13347 TGF-beta receptor interacting protein 1 168 (17)

4 P29354 growth factor receptor-bound protein 2 122 (16)

55 Q8WXF1 paraspeckle protein 1 alpha isoform 186 (18)

6 P31943 heterogenous nuclear ribonucleoprotein H 107 (19)

7 1KAX Heat shock cognate protein 70 99 a-Ag (2)

8 P08670 Vimentin 298 a-Ag (3)

9 O60588 c-NAPl 61 a-Ag (4)

10 O00109 mutant keratin 9 110 (5)

11 D42825 Kruppel-type zinc finger protein ZNF-70 76 (6)

12 Q9UJD9 Similar to RPS2 69 TREMBL

13 AAA69899 AHNAK nucleoprotein 61 a-Ag (l)

14 P06753 Tropomyosin alpha 72 (8)

15 1MABB ATP synthase beta chain 129 (9)

16 O63042 Small ribosomal protein 4 68 (10)

17 S55024 Nebulin 87 (11)

18 Q96ES1 Similar to serine proteinase 107 (12)

19 C1HURB Complement subcomponent Clr 106 (13)

20 Q96PV1 KIAA1937 99 (14)

21 Q9Y2K3 KLAA 1000 63 (14)

22 Q9ULK3 KIAA1217 65 (14)

23 Q96LL7 CDNA FLJ25393 64 TREMBL

24 Q96I66 Unknown protein 72 TREMBL

25 Q9B5S71 Hyp. 41.3 kD protein 96 TREMBL

26 P32320 cytidine deaminase 86 (15)

27 P35080 profilin II 108 (20) Table 1: 27 antigens were isolated by AMLDA from tumor biopsies isolated from patients with head and neck cancer and from cancer cell lines.

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 complete sequence information for SEQ ID NO: 1-27 can be found in the sequence overview at the end of the description.

According to the invention, the following methods were used:

- Appropriate methods are used to detect antibodies which recognize one or more of the antigens listed in Table 1. Whole blood or derivatives thereof, such as serum or plasma from a human individual, serve as the source of these autoantibodies.

- 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.

- Combination of one or more of the antigens listed in Table 1 and / or autoantibodies against these antigens are used with other tumor arteries. These 'other 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.

According to a first aspect, 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.

The term "immunogenic activity" as used herein refers to the immunogenic function of the proteins of the invention. As mentioned above, 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. Thus, 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. The term 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.

The 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. For example, 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.

The term “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.

It is fundamentally preferred in this application that the 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. In a second aspect, the present invention relates to an isolated nucleic acid encoding one or more of the proteins of claim 1.

The term "nucleic acid sequence" relates to a heteropolymer of nucleotides or the sequence of these nucleotides. The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to a heteropolymer of nucleotides.

The term "isolated" as used herein in relation to nucleic acids 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.

For example, 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. Thus, an isolated nucleic acid closes a recombinant DNA without limitation. one that exists as a separate molecule (e.g. a cDNA or a genomic DNA fragment generated by PCR or treatment with a restriction endonuclease) independent of other sequences, as well as recombinant DNA which is converted into a vector, an autonomously replicating plasmid, a virus ( for example, a retrovirus, adenovirus or Heφesvims) or built into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or a fusion nucleic acid sequence.

The term "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. For example, 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. In addition, 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, as used herein, relates to conditions under which polynucleotide duplexes are stable. As is known to those skilled in the art, 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.

The term 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).

Furthermore, 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.

According to a third aspect, the invention comprises vectors which comprise one or more of the nucleic acids as defined above.

This is preferably an expression vector which comprises one or more of the nucleic acid sequences as defined above and comprises one or more regulatory sequences. 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. Alternatively, 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. The term “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.

For example, such 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.

According to a fourth aspect, the present invention provides an antibody or ap- tamer directed against one or more epitopes of a protein according to claim 1.

The term "antibody" as used herein 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. The term “epitope” relates to any antigenic determinant on an antigen to which the paratope of an antibody binds. 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. For example, 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.

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).

The production of monoclonal antibodies is also familiar to the person skilled in the art. See, for example, Koehler & Milstein, Nature 256: 495 (1975); Coligan et al. Sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988). Briefly, 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).

The term “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.

In particular, 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. The term "Antiköφer" 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. Fragments as such or fragments of antibodies according to the invention as constituents of fusion proteins are typically produced by the methods of enzymatic cleavage, protein synthesis or the recombination methods familiar to the person skilled in the art. According to the present invention, the term “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. However, 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.

Genetically manipulated antibodies according to the invention can also be produced by methods as described in the aforementioned publications. In short, 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 ρBR322, 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. Even more preferred is a monoclonal antibody that combines the hypervariable complementarity-defining region (CDR) of a murine monoclonal antibody with the other areas of a human antibody. 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. Nature 312: 643-646 (1984); Cabilly et al., EP-A-125023; Neuberger et al., Nature 314: 268-270 (1985); Taniguchi et al., EP-A-171496; Moirion et al., EP-A-173494; Neuberger et al., WO 86/01533; Kudo et al, EP-A-184187; Sahagan et al., J. Immunol. 137: 1066-1074 (1986); Robinson et al., WO 87/02671; Liu et al., Proc. Natl. Acad. Sei USA, 84: 3439-3443 (1987); Sun et al., Proc. Natl. Acad. Be UA 84: 214218 (1987 ); Better et al., Science 240: 1041-1043 (1988) and Harlow and Line, Antibodies: A Laboratory Manual, supra. These references are incorporated herein by reference.

The term "Antiköφer" 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. Such fragments are typically made by proteolytic cleavage using enzymes such as. B. 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. In the case of 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. 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 Antiköφer 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. A particularly preferred example of a 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).

There are no restrictions with regard to the manner in which the antibody according to the invention is administered. The antibody can therefore be administered intraperitoneally, systemically (intravenously or intraarterially, inframuscularly, intradermally, subcutaneously, intratumorally) or else selectively in or via a defined organ. As an example of a selective application into or via an 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. Other examples of 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.

Accordingly, 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.

In the pharmaceutical composition according to the invention or in the use according to the invention, the antibody is advantageously provided in suitable formulations. Such 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. 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). Within the scope of the present invention, coatings of pharmaceutical compositions or medicaments according to the invention containing the therapeutically active substances, namely coatings with polymers, are also disclosed (for example polyoxamers or polyoxamines). Furthermore, 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. As sodium chloride, potassium chloride or other components that make the solution isotonic, for example. In addition to the constituents mentioned above, the carrier or vehicle can furthermore contain additional components, such as human serum albumin (HSA), 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.

The 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 μg to about 10 mg.

As an alternative to the above-mentioned antibody, 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.

According to a fifth aspect, the present invention relates to a hybridoma which produces a monoclonal antibody which has a binding specificity for a protein according to claim 1.

According to a sixth aspect, 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:

a) providing autologous or allogeneic APCs; b) contacting the APCs with an effective amount of a peptide fragment of a protein according to the invention under conditions which enable endocytosis, processing and presentation of the peptide fragments by these APCs, and c) isolating the APCs presenting the corresponding peptides.

Alternatively, 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:

a) providing a nucleic acid which codes for a protein or peptide fragment thereof, b) transfecting the APCs with the nucleic acid and c) selecting APCs which present the peptide fragments.

According to a preferred embodiment, the nucleic acid in step a) is provided in an expression vector.

According to a seventh aspect, the present invention relates to an antigen presenting cell (APC) which can be obtained by one of the methods mentioned above.

In one embodiment, 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:

a) providing mammalian, in particular human, T cells or PBMCs,

b) co-culturing the cells with an APC according to the seventh aspect of the present invention under conditions which enable activation of T cells, and

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. Thus, mononuclear peripheral blood mononuclear cells (PBMC), which contain the T cells and which can be easily produced from patient blood samples, can provide APCs (for example B cells) which are pulsed by the peptides according to the invention.

Alternatively, 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. These APCs are preferably selected to express the appropriate MHC class I molecule.

The T cells, the activity of which can be determined by the method according to the invention, 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.

According to one embodiment, this determination can be carried out using a ⁵¹ Cr release assay. In particular, 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. An example of target cells are T2 cells which have been pulsed with one or more of the proteins according to the invention.

According to a preferred embodiment, 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:

First, 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.

Alternatively, 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. After a defined period of time, 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 procedure for generating specific T cell lines is described in detail in Moosmann et al., B cells immortalized by a mini-Epstein-Barr virus encoding a foreign antigen efficiently reactivate specific cytotoxic T cells, Blood, September 1, 2002, Vol. 100, No. 5.

According to a ninth aspect, the invention relates to T cells which can be obtained by the method explained above.

In a tenth aspect, the present invention relates to 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.

This 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.

According to an eleventh aspect, 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.

Unless otherwise specified, all technical and scientific terms used herein have the meaning commonly assigned to them by those skilled in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present description including the definitions will decide. In addition, the materials and methods and examples are illustrative only and are not intended to be limiting.

The invention will now be illustrated by examples and the accompanying drawings, which illustrate: 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. Autoantiköφer, which are only present in patient sera, can be used to detect a tumor disease. In the allogeneic version, 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%).

Figure 3: 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.

Figure 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.

Figure 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).

Figure 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.

Figure 8: E-FABP serum reactivity was determined for normal human sera (NHS, n = 48) and HNSCC patients (n = 59) with recombinant E-FABP in a modified Bio-Plex approach. The mean fluorescence values from three independent experiments are shown.

Examples:

1. Identification of tumor associated antigens using AMIDA

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). 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. In addition, 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. As a control, 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 μm) 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 μg / 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 μg / ml salmon seed DNA, 0.1% SDS, 50 μg / ml t-RNA, 5% dextran sulfate) incubated. 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). The sections were then incubated (1 h, RT) with an alkaline-phosphatase-conjugated anti-DIG antibody (Röche, Mannheim, Germany). The signals were visualized with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) as substrates (Röche, Mannheim, Germany). The reaction was stopped by washing the slides in PBS buffer. Control experiments were carried out in parallel for each test using DIG-labeled sense RNA probes.

2. 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). In sharp contrast to this, 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.

3. Immunohistochemical detection of Grb2, hnRNP H and E-FABP overexpression in head and neck carcinomas

GRB2, hnRNP H and E-FABP were isolated as potential tumor-associated antigens using AMLDA technology. The expression of these three antigens in healthy tissue, in carcinomas of the head and neck region and metastases derived therefrom was examined below. Ultrathin sections of tissue samples of the respective tissue were made and immunohistochemically stained with specific antibodies. 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).

Table 2: Expression patterns of E-FABP in HNSCC. Localization expression pattern - + ++ +++

Pharynx 0/11 (0%) 4/11 (36%) 2/11 (18%) 5/11 (46%)

Larynx 0/2 (0%) 0/2 (0%) 1/2 (50%) 1/2 (50%)

Metastases 0/5 (0%) 0/5 (0%) 1/5 (20%) 4/5 (80%)

Healthy mucosa 0/3 (0%) 3/3 (100%) 0/3 (0%) 0/3 (0%)

+++ strong expression; ++ moderate expression; + weak expression; - no expression.

Table 3 Expression patterns of hnRNP H in HNSCC

Localization Expression Patterns Q-25% * 26-50% * 51-75% * 76-100% *

Oropharynx 0/5 (0%) 0/5 (0%) 2/5 (40%) 3/5 (60%)

Hypopharynx 1/5 (20%) 0/5 (0%) 0/5 (0%) 4/5 (80%)

Larynx 0/3 (0%) 0/3 (0%) 0/3 (0%) 3/3 (100%)

Metastases 0/3 (0%) 1/3 (33%) 0/3 (0%) 2/3 (67%)

Healthy mucosa 8/8 (100%) ** 0/8 (0%) 0/8 (0%) 0/8 (0%)

* Percentage of hnRNPH-positive cells in tissue samples.

** with the exception of the basal cell layer in which hnRNPH was always expressed.

4. Examination of CK8 overexpression in healthy and transformed epithelia

Cytokeratin 8 (CK8) 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. As expected, 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).

Furthermore, the expression of in healthy, hypoplastic and tumor tissue of a patient was examined immunohistochemically. Frozen sections were generated and stained with specific antibodies. CK8 was weakly detectable in basal cell cells in healthy tissue. Already with the onset of hypothyroidism there was a clear overexpression of CK8 in the tissue, which increased in the course of tissue transformation (Figure 7).

5. Determination of the E-FABP-specific serum reactivity in healthy and cancer patients

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).

Sequence overview for SEQ ID NO: 1-27 (assignment see table 1):

AHNA nucleoprotein

1 mpgikvggsg vnvnakgldl ggrggvqvpa vdissslggr pvevqgpsle sgdhakikfp 61 tmkvpkfgvs tgregqtpka glrvsapevs vghkggkpgl tiqapqlevs vpsaniegle 121 gklkgpqitg pslegdlglk gakpqghigv dasapqiggs itgpsvevqa pdidvqgpgs 181 klnvpkmkvp kfsvsgakge etgidvtlpt gevtvpgvsg dvslpeiatg glegkmkgtk 241 vktpemiiqk pkismqdvdl slgspklkgd ikvsapgvqg dvkgpqvalk gsrvdietpn 301 legtltgprl gspsgktgtc rismsevdln vaapkvkggv dvtlprvegk vkvpevdvrg 361 pkvdvsapdv eahgpewnlk mpkmkmptfs tpgakgegpd vhmtlpkgdi sisgpkvnve 421 apdvnleglg gklkgpdvkl pd svktpki smpdvdlhvk gtkvkgeydv tvpklegelk 481 gpkvdidapd vdvhgpdwhl kmpkmkmpkf svpgfkaegp evdvnlpkad vdisgpkidv 541 tapdvsieep egklkgpkfk mpemnikvpk ismpdvdlhl kgpnvkgeyd vtmpkvesei 601 kvpdvelksa kmdidvpdve vqgpd hlk pkmkmpkfsm pgfkaegpev dvnlpkadvd 661 isgpkvgvev pdvniegpeg klkgpkfkmp emnikapkis mpdvdlhmkg pkvkgeydmt 721 vpklegdlkg pkvdvsapdv emqgpd nlk mpkikmpkfs mpslkgegpe fdvnlskanv 781 disapkvdtn apdlslegpe gklkgpkfkm pemhfrapkm slpdvdldlk gpkmkgnvdi 841 sapkiegemq vpdvdirgp k vdikapdveg qgld slkip kmkmpkfsmp slkgegpevd 901 vnlpkadvdv sgpkvdieap dvslegpegk lkgpkfkmpe mhfktpkism pdvdlhlkgp 961 kvkgdvdvsv pkvegemkvp dveikgpkmd idapdvevqg pd hlkmpkm kmpkfsmpgf 1021 kgegrevdvn Ipkadidvsg pkvdvevpdv slegpegklk gpkfkmpemh fkapkismpd 1081 vdlnlkgpkl kgdvdvslpe vegemkvpdv dikgpkvdis apdvdvhgpd whlkmpkvkm 1141 pkfsmpgfkg egpevdvklp kadvdvsgpk mdaevpdvni egpdaklkgp kfkmpemsik 1201 pqkisipdvg Ihlkgpkmkg dydvtvpkve geikapdvdi kgpkvdinap dvevhgpd h 1261 lkmpkvkmpk fsmpgfkgeg pevdmnlpka dlgvsgpkvd idvpdvnlea pegklkgpkf 1321 kmpsmniqth kismpdvgln lkapklktdv dvslpkvegd lkgpeidvka pkmdvnvgdi 1381 diegpegklk gpkfkmpemh fkapkismpd vdlhlkgpkv kgdmdvsvpk vegemkvpdv 1441 dikgpkvdid apdvevhdpd whlkmpkmkm pkfsmpgfka egpevdvnlr kadidvsgps 1501 vdtdapdldi egpegklkgs kfkmpklnik apkvsmpdvd lnlkgpklkg eidasvpele 1561 gdlrgpqvdv kgplveaevp dvdlecpdak lkgpkfkmpe mhfkapkism pdvdlhlkgp 1621 kvkgdadvsv pklegdltgp svgvevpdve lecpdaklkg pkfkmpdmhf kapkismpdv 1681 DLH

Heat shock cognate protein 70

1 mskgpavgid lgttyscvgv fqhgkveiia ndqgnrttps yvaftdterl igdaaknqva

61 mnptntvfda mrligrrfdd avvqsdmkhw pfmvvndagr pkvqveykge tksfypeevs

121 smvltkmkei aeaylgktvt navvtvpayf ndsqrqatkd agtiaglnvl riineptaaa

181 iaygldkkvg aernvlifdl gggtfdvsil tiedgifevk stagdthlgg edfdnrmvnh

241 fiaefkrkhk kdisenkrav rrlrtacera krtlssstqa sieidslyeg idfytsitra

301 rfeelnadlf rgtldpveka lrdakldksq ihdivlvggs tripkiqkll qdffngkeln

361 ksinpdeava ygaavqaail s

Vimei- in

1 mstrsvssss yrrmfggpgt asrpsssrsy vttstrtysl gsalrpstsr slyasspggv

61 yatrssavrl rssvpgvrll qdsvdfslad aintefkntr tnekvelqel ndrfanyidk

121 vrfleqqnki llaeleqlkg qgksrlgdly eeemrelrrq vdqltndkar veverdnlae

181 dimrlreklq eemlqreeae ntlqsfrqdv dnaslarldl erkveslqee iaflkklhee

241 eiqelqaqiq eqhvqidvdv skpdltaalr dvrqqyesva aknlqeaeew ykskfadlse

301 aanrnndalr qakqesteyr rqvqsltcev dalkgtnesl erqmremeen faveaanyqd

361 tigrlqdeiq nmkeemarhl reyqdllnvk maldieiaty rkllegeesr islplpnfss

421 lnlretnlds lplvdthskr tfliktvetr dgqvinetsq hhddle

c-nAPL

1 metrspglnn mkpqslqlvl eeqvlalqqq maenqaaswr klknsqeaqq rqatlvrklq

61 akvlqyrswc qelekrleat ggpipqrwen veepnldell vrleeeqqrc eslaevntql

121 rlhmekadvv nkalredvek ltvd srard elmrkesqwq meqeffkgyl kgehgrllsl

181 wrevvtfrrh flemksatdr dlmelkaehv rlsgslltcc lrltvgaqsr epngsgrmdg

241 repaqlllll aktqelekea hersqeliql ksqgdlekae Iqdrvtelsa lltqsqkqne

301 dyekmikalr etveiletnh telmeheasl srnaqeekls lqqvikditq vmveegdnia

361 qgsghensle ldssifsqfd yqdadkaltl vrsvltrrrq avqdlrqqla gcqeavnllq

421 qqhdqweeeg kalrqrlqkl tgerdtlagq tvdlqgevds lskerellqk areelrqqle

481 vleqeawrlr rvnvelqlqg dsaqgqkeeq qeelhlavre rerlqemlmg leakqsesls

541 elitlreale sshlegellr qeqtevtaal araeqsiael sssentlkte vadlraaavk

601 lsalnealal dkvglnqqll qleeenqsvc srmeaaeqar nalqvdlaea ekrrealwek

661 nthleaqlqk aeeagaelqa dlrdiqeeke eiqkklsesr hqqeaattql eqlhqeakrq

721 eevlaravqe kealvrekaa levrlqaver drqdlaeqlq glssakelle sslfeaqqqn

781 svievtkgql evqiqtvtqa keviqgevrc lkleldters qaeqerdaaa rqlaqaeqeg

841 ktaleqqkaa hekevnqlre k ekerswhq qelakalesl erekmelemr lkeqqtemea

901 iqaqreeert qaesalcqmq letekervsl letllqtqke ladasqqler lrqdmkvqkl

961 keqettgilq tqlqeaqrel keaarqhrdd laalqeesss llqdkmdlqk qvedlksqlv 1021 aqddsqrlve qevqeklret qeynriqkel erekasltls Imekeqrllv lqeadsirqq

1081 elsalrqdmq eaqgeqkels aqmellrqev kekeadflaq eaqlleelea shiteqqlra

1141 slwaqeakaa qlqlrlrste sqlealaaeq qpgnqaqaqa qlaslysalq qalgsvcesr

1201 pelsgggdsa psv glepdq ngarslfkrg plltalsaea vasalhklhq dlwktqqtrd

1261 vlrdqvqkle erltdteaek sqvhtelqdl qrqlsqnqee kskwegkqns leselmelhe

1321 tmaslqsrlr raelqrmeaq gerellqaak enltaqvehl qaavvearaq asaagileed

1381 lrtarsalkl kneevesere raqalqeqge lkvaqgkalq enlalltqtl aereeevetl

1441 rgqiqelekq remqkaalel Isldlkkrnq evdlqqeqiq elekcrsvle hlpmavqere

1501 qkltvqreqi relekdretq rnvlehqlle lekkdqmies qrgqvqdlkk qlvtleclal

1561 eleenhhkme cqqklikele gqretqrval thltldleer sqelqaqssq ihdleshstv

1621 larelqerdq evksqreqie elqrqkehl qdlerrdqel mlqkeriqvl edqrtrqtki

1681 leedleqikl slrergrelt tqrqlmqera eegkgpskaq rgslehmkli lrdkekevec

1741 qqehihelqe lkdqleqqlq glhrkvgets lllsqreqei vvlqqqlqea reqgelkeqs

1801 lqsqldeaqr alaqrdqele alqqeqqqaq gqeervkeka dalqgaleqa hmtlkerhge

1861 lqdhkeqarr leeelavegr rvqaleevlg dlraesreqe kallalqqqc aeqaqeheve

1921 tralqdswlq aqavlkerdq elealraesq ssrhqeeaar araealqeal gkahaalqgk

1981 eqhlleqael srsleastat lqasldacqa hsrqleealr iqegeiqdqd lryqedvqql

2041 qqalaqrdee Irhqqereql lekslaqrvq enmiqekqnl gqereeeeir glhqsvrelq

2101 ltlaqkeqei lelretqqrn nlealphshk tspmeeqslk ldsleprlqr elerlqaalr

2161 qteareie r ekaqdlalsl aqtkasvssl qevamflqas vlerdseqqr Iqdeleltrr

2221 alekerlhsp gatstaelgs rgeqgvqlge vsgveaepsp dgmekqswrq rlehlqqava

2281 rleidrsrlq rhnvqlrstl eqvererrkl kreamraaqa gsleiskata ssptqqdgrg

2341 qknsdakcva elqkevvllq aqltlerkqk qdyitrsaqt srelaglhhs Ishsllavaq

2401 apeatvleae trrldesltq sltspgpvll hpspsttqaa sr

Mutant keratin 9

1 mscrqfsssy ltsggggggg lgsggsirss ysrfsssggr ggggrfssss gygggssrvc 61 grggggsfgy sygggsgggf sasslgggfg ggsrgfggas gggysssggf gggfgggsgg 121 gfgggygsgf gglggfggga gggdggilta nekstmqeln srlasyldkv qaleeanndl 181 enkiqdwydk kgpaaiqkny spyyntiddl kdqivdltvg nnktlldidn trmtlddfri 241 kfemeqnlrq gvdadinglr qvldnltmek sdlemqyetl qeelmalkkn hkeemsqltg 301 qnsgdvnvei nvapgkdltk tlndmrqeye qliaknrkdi enqyetqitq iehevsssgq 361 evqssakevt qlrhgvqele ielqsqlskk aaleksledt knrycgqlqm iqeqisnlea 421 qitdvrqeie cqnqeyslll sikmrlekei etyhnllegg qedfessgag kiglggrggs 481 ggsygrgsrg gsggsygggg sgggygggsg srggsggsyg ggsgsgggsg ggygggsggg 541 hsggsggghs ggsggnyggg sgsgggsggg ygggsgsrgg sggshgggsg fggesggsyg 601 ggeeasgsgg gygggsgkss hs

Kruppel-type zinc finger protein ZNF-70

1 ihtgerpyqc kecgksfnqs sglsqhrkih tlkkphecdl cgkafchrsh lirhqrihtg 61 kkpykcdecg kafsqssnli Ehrkthtgek p

Similar to RPS2

1 maddagaagr pggpggpgmg nrggfrggfg sgirgrgrgr grgrgqgrga regkpedkew

61 mpvtklgrlv kdmkikslee iylfslpike seiidfflgt slkdevlkim pvqkqtragq

121 rtrfkvfvai gdynghvglg vkcskevata irgaiilakl sivpvcrgyw gnkigkphtv

181 pckvtgrcss vlvrlipapr gtgivsapvp kkllmmagid dcytsargct atlgnfakat

241 fdaisktysy lppdlwketv ftkslyeeft dhlikthtrv saqrtqapav att

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 ardrqpsdfk tdkeggqags gqrvpaleea 301 pdshqkrpqh shsnkkrpsl dgrdpgngth glspeekeql sndretqegk pptahldrts 361 vsslseveev dmaeefeqpt lscekyltyd qlrkqkkktg ksattalgdk qrkaneskgt 421 reswdsakkl ppvqesqser lqaagadsag pktvpnhvfs elwdlseawm qanydplsds 481 dsmtsqakpe alsspkfree aafpgrrvna kmpvysgsrp acqlqvptlr qqcaqvlrnn 541 pdalsdvgev pywvlepvle gwrpdqlyrr kkdnhalvre tdelrrnhcf qdfkeekpqe 601 nktwreqylr Ipdapeqrlr vmttnirsar gnnpngreak micfksvakt pydtsrrqek 661 sagdadpeng eikpaskpag sshtpssqss sgggrdssss ilrwlpekra npclsssneh 721 aapaaktrkq aakkvaplma kairdykrrf srr

Tropo yosin alpha

1 meaikkkmqm lkldkenald raeqaeaeqk qaeerskqle delaamqkkl kgtedeldky

61 sealkdaqek lelaekkaad aeaevaslnr riqlveeeld raqerlatal qkleeaekaa

121 desergmkvi enralkdeek melqeiqlke akhiaeeadr kyeevarklv iiegdlerte

181 eraelaeskc seleeelknv tnnlksleaq aekysqkedk yeeeikiltd klkeaetrae

241 faersvakle ktiddledel yaqklkykai seeldhalnd mtsi

ATP synthase beta chain (NP 001677)

1 mlgfvgrvaa apasgalrrl tpsaslppaq lllraaptav hpvrdyaaqt spspkagaat

61 grivavigav vdvqfdeglp pilnalevqg retrlvleva qhlgestvrt iamdgteglv

121 rgqkvldsga pikipvgpet lgrimnvige pidergpikt kqfapihaea pefmemsveq

181 eilvtgikvv dllapyakgg kiglfggagv gktvlimeli nnvakahggy svfagvgert

241 regndlyhem iesgvinlkd atskvalvyg qmneppgara rvaltgltva eyfrdqegqd

301 vllfidnifr ftqagsevsa llgripsavg yqptlatdmg tmqeritttk kgsitsvqai

361 yvpaddltdp apattfahld attvlsraia elgiypavdp Idstsrimdp nivgsehydv

421 argvqkilqd ykslqdiiai Igmdelseed kltvsrarki qrflsqpfqv aevftghmgk

481 lvplketikg fqqilageyd hlpeqafymv gpieeavaka dklaeehss

Small ribosomal protein 4

1 PGLTNKTSQ KSSSINQSTS NKKISQYRIR EEKQK RFH YGIIERQLLN YVRIARKAKG

61 STGEVLLQLL ΞMRSDNVIFR LGMAPTIPGA RQLVNHRHIL VNDRIVDIPS YRCKPQDFIT

121 IK RQKSQAI INKNIDFYQK YKIPNHLTYS SLEKKGFVNQ I DRESIGLK INELLWEYY 181 SRQA

Nebulin

1 maddedyeev veyyteevvy eevpgetitk iyettttrts dyeqsetskp alaqpalaqp

61 asakpverrk virkkvdpsk fmtpyiahsq kmqdlfspnk ykekfektkg qpyasttdtp

121 elrrikkvqd qlsevkyrmd gdvaktichv dekakdieha kkvsqqvskv lykqnwedtk

181 dkyllppdap elvqavknta mfskklyted eadkslfyp yndspelrrv aqaqkalsdv

241 aykkglaeqq aqftpladpp diefakkvtn qvskqkyked yenkikgkws etpcfevana

301 rmnadnistr kyqedfenmk dqiyfmqtet peykmnkkag vaaskvkyke dyeknkgkad

361 ynvlpasenp qlrqlkaagd alsdklyken yektkaksin ycetpkfkld tvlqnfssdk

421 kykdsylkdi lghyvgsfed pyhshcmkvt aqnsdknyka eyeedrgkgf fpqtitqeye

481 aikkldqckd htykvhpdkt kftqvtdspv llqaqvnskq lsdlnykakh esekfkchip

541 pdtpafiqhk vnaynlsdnl ykqdwekska kkfdikvdai pllaakantk ntsdvmykkd

601 yeknkgkmig vlsinddpkm lhslkvaknq sdrlykenye ktkaksmnyc etpkyqldtq 661 lknfsearyk dlyvkdvlgh yvgsmedpyh thcmkvaaqn sdksykaeye edkgkcyfpq 721 titqeydaik kldqckdhty kvhpdktkft avtdspvllq aqlntkqlsd lnykakhege 781 rfkchipada pqfiqhrvna ynlsdnvykq dwekskakkf dikvdaipll aakantknts 841 dvmykkdyek skgkmigals inddpkmlhs lktaknqsdr eyrkdyeksk tiytapldml 901 qvtqakksqa iasdvdykhi lhsysyppds invdlakkay alqsdveyka dynswmkgcg 961 wvpfgsleme kakrasdiln ekkyrqhpdt lkftsiedap itvqskinqa qrsdiaykak 1021 geeiihnynl ppdlpqfiqa kvnaynisen mykadlkdls kkgydlrtda ipiraakaar 1081 qaasdvqykk dyekakgkmv gfqslqddpk lvhymnvaki qsdreykkdy ektkskyntp 1141 hdmfnvvaak kaqdvvsnvn ykhslhhyty lpdamdlels knmmqiqsdn vykedynn m 1201 kgigwipigs ldvekvkkag dalnekkyrq hpdtlkftsi vdspvmvqak qntkqvsdil 1261 ykakgedvkh kytmspdlpq flqakcnays isdvcykrd hdlirkgnnv lgdaipitaa 1321 kasrniasdy kykeayeksk gkhvgfrslq ddpklvhymn vaklqsdrey kknyentkts 1381 yhtpgdmvti taakmaqdva tnvnykqplh hytylpdams lehtrnvnqi qsdnvykdey 1441 nsflkgigwi pigslevekv kkagdalner kyrqhpdtvk ftsvpdsmgm mlaqhntkql 1501 sdlnykve ge klkhkytidp elpqfiqakv nalnmsdahy kadwkktirk gydlrpdaip 1561 ivaakssrni asdckykeay ekakgkqvgf lslqddpklv hymnvakiqs dreykkgyea 1621 sktkyhtpld mvsvtaakks qevatnanyr qsyhhytllp dalnvehsrn amqiqsdnly 1681 ksdftnwmkg igwvpiesle vekakkagei lsekkyrqhp eklkftyamd tmeqalnksn 1741 klnmdkrlyt ekwnkdktti hvmpdtpdil lsrvnqitms dklykag ee ekkkgydlrp 1801 daiaikaara srdiasdyky kkayeqakgk higfrsledd pklvhfmqva kmqsdreykk 1861 gyeksktsfh tpvdmls va akksqevatn anyrnvihty nmlpdamsfe laknmmqiqs 1921 dnqykadyad fmkgigwlpl gsleaeknkk ameiisekky rqhpdtlkys tlmdsmn vl 1981 aqnnakimne hlykqawead ktkvhimpdi pqiilakana inisdklykl sleeskkkgy

2041 dlrpdaipik aakasrdias dykykynyek gkgkmvgfrs leddpklvhs mqvakmqsdr 2101 eykknyentk tsyhtpadml svtaakdaqa nitntnykhl ihkyillpda mnieltrnmn 2161 riqsdneykq dynewykglg wspagsleve kakkateyas dqkyrqhpsn fqfkkltdsm 2221 dmvlakqnah tmnkhlytid wnkdktkihv mpdtpdilqa kqnqtlysqk lyklgweeal 2281 kkgydlpvda isvqlakasr diasdykykq gyrkqlghhv gfrslqddpk Ivlsmnvakm 2341 qsereykkdf ekwktkfssp vd lgvvlak kcqelvsdvd yknylhqwtc lpdqndvvqa 2401 kkvyelqsen lyksdlewlr gigwsplgsl eaeknkrase iisekkyrqp pdrnkftsip 2461 damdivlakt naknrsdrly reawdkdktq ihimpdtpdi vlakanlint sdklyrmgye 2521 elkrkgydlp vdaipikaak asreiaseyk ykegfrkqlg hhigarnied dpkmmwsmhv 2581 akiqsdreyk kdfekwktkf sspvdmlgvv laykcqtlvs dvdyknylhq wtclpdqsdv 2641 iharqaydlq sdnlyksdlq wlkgigwmts gsledeknkr atqilsdhvy rqhpdqfkfs 2701 slmdsipmvl aknnaitmnh rlyteawdkd kttvhimpdt pevllakqnk vnyseklykl 2761 gleeakrkgy dmrvdaipik aakasrdias efkykegyrk qlghhigara irddpkmmws 2821 mhvakiqsdr eykkdfekwk tkfsspvdml gvvlakkcqt lvsdvdykny lhqwtclpdq 2881 s dviharqay dlqsdnmyks dlqwmrgigw vsigsldvek ckrateilsd kiyrqppdrf 2941 kftsvtdsle qvlaknnaln mnkrlyteaw dkdktqihim pdtpeimlar qnkinysetl

3001 yklaneeakk kgydlrsdai pivaakasrd visdykykdg yrkqlghhig arnieddpkm 3061 mwsmhvakiq sdreykkdfe kwktkfsspv dmlgvvlakk cqtlvsdvdy knylhewtcl 3121 pdqndvihar qaydlqsdni yksdlqwlrg igwvpigsmd vvkckraaei lsdniyrqpp 3181 dklkftsvtd sleqvlaknn alnmnkrlyt eawdkdktqv himpdtpeim larqnkinys 3241 eslyrqamee akkegydlrs daipivaaka srdiasdyky keayrkqlgh higaravhdd 3301 pkimwslhia kvqsdreykk dfekyktrys spvdmlgivl akkcqtlvsd vdykhplhec 3361 iclpdqndii harkaydlqs dnlyksdlew mkgigwvpid slevvrakra gellsdtiyr 3421 qrpetlkfts itdtpeqvla knnalnmnkr lyteawdndk ktihv PDTP eimlaklnri 3481 nysdklykla leeskkegyd lrldaipiqa akasrdiasd ykykegyrkq lghhigarni 3541 kddpkmmwsi hvakiqsdre ykkefekwkt kfsspvdmlg vlakkcqil vsdidykhpl 3601 hewtclpdqn dviqarkayd lqsdaiyksd lewlrgigwv pigsvevekv krageilsdr 3661 kyrqpadqlk ftcitdtpei vlaknnaltm skhlyteawd adktsihvmp dtpdillaks 3721 nsanisqkly tkgwdeskmk dydlradais iksakasrdi asdykykeay ekqkghhiga 3781 qsieddpkim caihaekiqs ereykkefqk wktkfsspvd mlsillakkc qtlvtdiyyr 3841 ny lhewtcmp dqndiiqakk aydlqsdaly kadlewlrgi gwmpqgspev lrvknaqnif 3901 cdsvyrtpvv nlkytsivdt pe vlaksna enisipkyre vwdkdktsih impdtpeinl 3961 aranalnvsn klyregwdem kagcdvrlda ipiqaakasr eiasdykykl dhekqkghyv 4021 gtltarddnk irwaliadkl qnereyrldw akwkakiqsp vdmlsilhsk nsqalvsdmd 4081 yrnylhqwtc mpdqndviqa kkayelqsdn vykadlewlr gigwmpndsv svnhakhaad 4141 ifsekkyrtk ietlnftpvd drvdyvtakq sgeilddiky rkdwnatksk ytltetpllh

4201 taqeaarild qylykegwer qkatgyilpp davpfvhahh cndvqselky kaehvkqkgh

4261 yvgvptmrdd pklvwfehag qiqnerlyke dyhktkakin ipadmvsvla akqgqtlvsd

4321 idyrnylhqw mchpdqndvi qarkaydlqs dnvyradlew lrgigwipld svdhvrvtkn 4381 qemmsqikyk knalenypnf tsvvdppeiv lakinsvnqs dvkyketfnk akgkytfspd 4441 tphishskdm gklystilyk gawegtkayg ytlderyipi vgakhadlvn selkyketye 4501 kqkghylagk vigefpgvvh cldfqkmrsa lnyrkhyedt kanv ipnd mnhvlakrcq 4561 yilsdleyrh yfhqwtslle epnvirvrna qeilsdnvyk ddlnwlkgig cyvwdtpqil 4621 hakksydlqs qlqytaagke nlqnynlvtd tplyvtavqs ginasevkyk enyhqikdky 4681 ttvletvdyd rtrnlknlys snlykeawdr vkatsyilps stlslthakn qkhlashiky 4741 reeyekfkal ytlprsvddd pntarclrvg klnidrlyrs vyeknkmkih ivpdmvemvt 4801 akdsqkkvse idyrlrlhew ichpdlqvnd hvrkvtdqis divykddlnw lkgigcyvwd 4861 tpeilhakha ydlrddikyk ahmlktrndy klvtdtpvyv qavksgkqls davyhydyvh 4921 svrgkvaptt ktvdldralh ayklqssnly ktslrtlptg yrlpgdtphf khikdtryms 4981 syfkykeaye htkaygytlg pkdvpfvhvr rvnnvtserl yrelyhklkd kihttpdppe 5041 irqvkktqea vseliyksdf fkmqghmisl pytpqvihcr yvgditsdik ykedlqvlkg 5101 fgcflydtpd mvrsrhlrkl wsnylytdka remrdkykvv ldtpeyrkvq elkthlselv 5161 yr aagkkqks iftsvpdtpd llrakrgqkl qsqylyvela tkerphhhag nqttalkhak 5221 dvkdmvsekk ykiqyekmkd kytpvpdtpi lirakraywn asdlryketf qktkgkyhtv 5281 kdaldivydvdqdqdqdqfdqdqfddkyvvqdqqdqdqddkvdqddqdddkvdqdqdqddkvdqdqdqdddvdddkvdqddkdddvdkvdqdddkyvdkdddkydkdddkhdddddkvdkdddkydkdddkhddkvddkhddkdddkdddkddddddddd

5341 t lkgigcya ydtpdftlae knktlyskyk ykevfertks dfkyvadspi nrhfkyatql

5401 mnekkyrady eqrkdkyhlv vdeprhllak trsdqisqik yrknyekskd kftsivdtpe

5461 hlrttkvnkq isdilykley nkakprgytt ihdtpmllhv rkvkdevsdl kykevyqrnk

5521 snctiepdav hikaakdayk vntnldykkq yeankahwkw tpdrpdflqa aksslqqsdf

5581 eykldreflk gcklsvtddk ntvlalrntl iesdlkykek hvkergtcha vpdtpqilla

5641 ktvsnlvsen kykdhvkkhl aqgsyttlpe trdtvhvkev tkhvsdtnyk kkfvkekgks

5701 nysimleppe vkhamevakk qsdvayrkda kenlhyttva drpdikkatq aakqasevey

5761 rakhrkegsh glsmlgrpdi emakkaakls sqvkyrenfd kekgktpkyn pkdsqlykvm

5821 kdannlasev kykadlkklh kpvtdmkesl imnhvlntsq lassyqykkk yekskghyht

5881 ipdnleqlhl keatelqsiv kykekyeker gkpmldfetp tyitakesqq mqsgkeyrkd

5941 yeesikgrnl tglevtpall hvkyatkias ekeyrkdlee sirgkgltem edtpdmlrak

6001 natqilneke ykrdlelevk grglnamane tpdfmrarna tdiasqikyk qsaemekanf

6061 tsvvdtpeii haqqvknlss qkkykedaek smsyyetvld tpeiqrvren qknfsllqyq

6121 cdlknskgki tvvqdtpeil rvkenqknfs svlykedvsp gtaigktpem mrvkqtqdhi

6181 ssvkykeaig qgtpipdlpe vkrvketqkh issvmykenl gtgipttvtp eiervkrnqe

6241 nfssvlyken Igkgiptpit pemervkrnq enfssvlyke nmgkgtplpv tpemervkhn

6301 qenissvlyk envgkatatp vtpemqrvkr nqenissvly kenlgkatpt pftpemervk

6361 rnqenfssvl ykenmrkatp tpvtpemera krnqenissv lysdsfrkqi qgkaayvldt

6421 pemrrvretq rhistvkyhe dfekhkgcft p vtdpiter vkknmqdfsd inyrgiqrkv

6481 vemeqkrndq dqetitglrv wrtnpgsvfd ydpaedniqs rslhminvqa qrrsreqsrs

6541 asalsvsgge eksehseapd hhlstysdgg vfavstaykh akttelpqqr sssvatqqtt

6601 vssipshpst agkiframyd ymaadadevs fkdgdaiinv qaidegwmyg tvqrtgrtgm 6661 lpanyveai

Similar to serine protease

1 mpssvswgil llaglcclvp vslaedpqgd aaqktdtshh dqdhptfnki tpnlaefafs

61 lyrqlahqsn stniffspvs iatafamlsl gtkadthdei leglnfnlte ipeaqihegf

121 qellrtlnqp dsqlqlttgn glflseglkl vdkfledvkk lyhseaftvn fgdteeakkq

181 indyvekgtq gkivdlvkel drdtvfalvn yiffkgkwer pfevkdteee dfhvdqvttv

241 kvpmmkrlgm fniqhckkls swvllmkylg nataifflpd egklqhlene Ithdiitkfl

301 enedrrsasl hlpklsitgt ydlksvlgql gitkvfsnga dlsgvteeap lklskavhka

361 vltidekgte aagamfleai pmsippevkf nkpfvflmie qntksplfmg kvvnptqk Complerne t component Clr

1 mwllyllvpa lfcraggsip ipqklfgevt splfpkpypn nfetttvitv ptgyrvklvf

61 qqfdlepseg cfydyvkisa dkkslgrfcg qlgsplgnpp gkkefmsqgn kmlltfhtdf

121 sneengtimf ykgflayyqa vdldecasrs ksgeedpqpq cqhlchnyvg gyfcscrpgy

181 elqedrhscq aecsselyte asgyissley prsyppdlrc nysirvergl tlhlkflepf

241 diddhqqvhc pydqlqiyan gknigefcgk qrppdldtss navdllfftd esgdsrgwkl

301 rytteiikcp qpktldefti iqnlqpqyqf rdyfiatckq gyqliegnqv Ihsftavcqd

361 dgtwhrampr ckikdcgqpr nlpngdfryt ttmgvntyka riqyychepy ykmqtragr

421 eseqgvytct aqgiwkneqk gekiprclpv cgkpvnpveq rqriiggqka kmgnfpwqvf

481 tnihgrggga llgdrwilta ahtlypkehe aqsnasldvf Ightnveelm klgnhpirrv

541 svhpdyrqde synfegdial lelensvtlg pnllpiclpd ndtfydlglm gyvsgfgvme

601 ekiahdlrfv rlpvanpqac enwlrgknrm dvfsqnmfca ghpslkqdac qgdsggvfav

661 rdpntdrwva tgivswgigc srgygfytkv lnyvdwikke meeed

KIAA1273 / TOB3 - KIAA1273 / TOB3

1 swvqmqleal nllhtlvwar slcragavqt qerlsgsasp eqvpagecca lqeyeaaveq

61 lkseqiraqa eerrktlsee trqhqaraqy qdklarqrye dqlkqqqlln eenlrkqees

121 vqkqeamrra tveremelrh knemlrvete ararakaere nadiireqir lkasehrqtv

181 lesirtagtl fgegfrafvt drdkvtatva gltllavgvy saknatavtg rfiearlgkp

241 slvretsrit vlealrhpiq vsrrllsrpq dvlegvvlsp slearvrdia iatrntkknr

301 glyrhillyg ppgtgktlfa kklalhsgmd yaimtggdva pmgregvtam hklfdwants

361 rrglllfmde adaflrkrat eeiskdlrat lnaflyhmgq hsnkfmlvla snlpeqfdca

421 insridvmvh fdlpqqeere rlvrlhfdnc vlkpategkr rlklaqfdyg rkcsevarlt

481 egmsgreiaq lavswqatay askdgvltea mmdacvqdav qqyrqkmrwl kaegpgrgve

541 hplsgvqget ltswslatdp sypclagpct fricswmgtg lcpgplsprm scgggrpfcp 601 pghpll

KIAA1937

VLGG L LGV AQLGLFLEEK LREHLAEKEK NEER EQEE KLKAKIRQ T EEKAALEEYI TQERNRAKET EEERKRMQE ESLIAQQKK A AKSITQEK NRVKEALEEE QTRVQELEER LARQKEISES NIAYEKRKAK EAMEKEKKKV QDLENRLTKQ KEELELKEQK EDVLNNKLSD A AMVEETQK TKÄTESLKAE SLALKLNETL AELETTKTKM IMVEERLILQ QKMVKALQDE QESQRHGFGE EI EYKEQIK QHAQTIVSLE EKLQKVTQHH KKIEGEIATL KDNDPAPKEE RPQDP VAPM TESSAKDMAY EHLIDDL AA QKEILSQQEV IMKLRKDLTE AHSRMSDLRG ELNEKQKMEL EQ WLVQQQ SKE SV KEK MAQMSSLVEK KDRELKALEE A RASQEKHR LQLNTEKEQK PRKKTQTCDT SVQIEPVHTΞ AFSSSQEQQS FSDLGVRCKG SRHEEVIQRQ KKA SELRAR IKE EKARSP DHKDHQNESF LDLKNLRMEN NVQKILLDAK PDLPTLSRIE I APQNG CN ARFGSAMΞKS GKMDVAEALE LSEKLYLDMS KTLGS MNIK SGHVS KY LSRQEREKVN Q RQRDLDLV FDKITQLKNQ LGRKEE LRG YΞKDVEQLRR SKVSIEMYQS QVAK EDDIY KEAEEKALLK EALERMEHQ CQEKRINRAI RQQK

KIAA1000 Y PHLTKKI MVESCLLTFR AFFWWIALIK MDLSDLGEAA AF RRSEAE LLLQATALDG KKKC IPDGE NAYIEAEVKG SEDDGTVIVE TADGES SIK EDKIQQMNPP EFEMIEDMAM LTH NEASVL HT KRRYGQ MIYTYSGLFC VTINPYK LP VYQKEV AAY KGKRRSEAPP HIFAVAN AF QDMLHNRENQ SILFTGESGA GKTVNSKHII QYFATIAAMI ESRKKQGA E DQIMQANTI EAFGNAKTLR NDNSSRFGKF IRMHFGARGM LSSVDIDIY EKSRVIFQQ AGERNYHIFY QILSGQKELH DLL VSANPS DFHFCSCGAV TVESLDDAEE LLATEQAMDI LGF PDEKYG CYKLTGAIMH FGNMKFKQKP REEQLEADGT ENADKAAFLM GINSSELVKC LIHPRIKVGN EYVTRGQTIE QVTCAVGA S KSMYERMFKW VARINRALD AK SRQFFIG ILDITGFEI EYNSLEQLCI NFTNEKLQQF FNWY FVLEQ EEYKKESIEW VSIGFG DQ ACIDLIEKPM GILSILEEEC MFPKATDLTF KTKLFDNHFG KSVHLQKPKP DKKKFEAHFE LVHYAGWPY NI SGWLEKNK DLLNETWAV FQKSSNRLLA SLFENYMSTD SAI PFGEKKR KKGASFQTVA SLHKENLNKL MTNLKSTAPH FVRCINPNVN KI PGI LDPYL VLQQLRCNGV LEGTRI CREG FPNRLQYADF KQRYCILNPR TFPKSKFVSS RKAAEΞLLGS LEIDHTQYRF GITKVFFKAG FLGQLEAIRD ERLSKVFTLF QARAQGKLMR I KFQKILEER DALILIQ NI RAFMAVKN P MRLFFKI KP LVKSSEVGEE VAGLKEECAQ LQKALEKSEF QREELKAKQV SLTQEKNDLI LQLQAEQETL ANVEEQCE L IKSKIQLEAR VKELSERVEE EEΞINSELTA RGRKLEDECF ELKKEIDDLE TMLVKSEKEK RTTEHKVKNL TEEVEFLNED ISKLNRAAKV VQEAHQQTLD DLHMEEEKLS SLSKANLKLE QQVDELEGAL EQERKARMNC ERELHKLEGN LKLNRESMEN LESSQRHLAE ELRKKELELS QMNSKVENEK GLVAQLQKTV KELQTQI KDL KEKLEAERTT RAKMERERAD LTQDLADLNE RLEEVGGSSL AQLEITKKQE TKIQKLHRDM EEATLHFETT SASLKKRHAD SLAELEGQVE NLQQVKQKLE KDKSDLQLEV DDLLTRVEQM TRAKANAEKL CTLYEERLHE ATAKLDKVTQ LANDLAAQKT KLWSESGEFL RRLEEKEALI NQLSREKSNF TRQIEDLRGQ LEKETKSQSA LAHALQKAQR DCDLLREQYE EEQEVKAELH RTLSK VNAEM VQ RMKYENN VI QRTEDLED AKKELAIRLQ EAAEAMGVAN ARNASLERAR HQLQLELGDA LSDLGKVRSA AARLDQKQLQ SGKALAD KQ KHEESQALLD ASQKEVQALS TELLKLKNTY EESIVGQETL RRENKNLQEE ISNLTNQVRE GTKNLTEMEK VKKLIEEEKT EVQVTLEETE GALERNESKI LHFQLELLEA KAELERKLSE KDEEIENFRR KQQCTIDSLQ SSLDSEAKSR IEVTRLKKKM EEDLNEMELQ LSCANRQVSE ATKSLGQLQI QI KDLQMQLD DSTQLNSDLK EQVAVAERRN SLLQSELEDL RSLQEQTERG RRLSEEELLE ATERINLFYT QNTSLLSQKK KLEADVARMQ KEAEEWQEC QNAEEKAKKA AIEAANLSEE LKKKQDTIAH LERTRENMEQ TITDLQKRLA EAEQMALMGS RKQIQKLESR VRELEGELEG EIRRSAEAQR GARRLERCI K ELTYQAEEDK KNLSRMQTQM DKLQLKVQNY KQQVEVAETQ ANQYLSKYKK QQHELNEVKE RAEVAESQVN KLKI KAREFG KKVQEE

K AA1217

KTALLLHGVF LLLLI FFLEQ GKGNLHVTSP EDAECRRTKE RLSNGNSRGS VSKSSRNI PR RHTLGGPRSS KEILGMQTSE MDRKREAFLE HLKQKYPHHA SAIMGHQERL RDQTRSPKLS HSPQPPSLGD PVEHLSETSA DSLEAMSEGD APTPFSRGSR TRASLPWRS TNQTKERSLG VLYLQYGDΞT KQLRMPNEIT SADTIRALFV SAFPQQLTMK MLESPSVAIY IKDESRNVYY ELNDVRNIQD RSLLKVYNKD PAHAFNHTPK TMNGDMRMQR ELVYARGDGP GAPRPGSTAH PPHAI PNSPP STPVPHSMPP SPSRI PYGGT RSMWPGNAT I PRDRISSLP VSRPI SPSPS AILERRDVKP DEDMSGKNIA MYRNEGFYAD PYLYHEGRMS lASSHGGHPL DVPDHI IAYH RTAI RSASAY CNPSMQAEMH MEQSLYRQKS RKYPDSHLPT LGSKTPPASP HRVSDLRMID MHAHYNAHGP PHTMQPDRAS PSRQAFKKEP GTLVYIEKPR SAAGLSSLVD LGPPLMEKQV FAYSTATI PK DRETRERMQA MEKQIASLTG LVQSALFKGP ITSYSKDASS EKMMKTTANR NHTDSAGTPH VSGGKMLSAL ESTVPPSQPP PVGTSAIHMS LLEMRRSVAE LRLQLQQMRQ LQLQNQELLR AMMKKAELEI SGKVMETMKR LEDPVQRQRV LVEQERQKYL HEEEKIVKKL CELEDFVEDL KKDSTAASRL VTLKDVEDGA FLLRQVGEAV ATLKGEFPTL QNKMRAILRI EVEAVRFLKE EPHKLDSLLK RVRSMTDVLT MLRRHVTDGL LKGTDAAQAA QYMAMEKATA AEVLKSQEEA AHTSGQPFHS TGAPGDAKSE WPLSGMMVR HAQSSPWIQ PSQHSVALLN pAQN LPHVAS SPAVPQEATS TLQMSQAPQS PQI PMNGSAM QSLF IEEIHS VSAKNRAVSI EKAEKK EEK RQNLDHYNGK EFEKLLEEAQ ANIMKSI PNL EMPPATGPLP RGDAPVDKVE LSEDSPNSEQ DLEKLGGKSP PPPPPPPRRS YLPGSGLTTT RSGDWYTGR KENITAKASS EDAGPSPQTR ATKYPAEEPA SA TPSPPPV TTSSSKDEEE EEEEGDKIMA ELQGSSGAPQ TSRMPVPMSA KNRPGTLDKP GKQSKLQDPR QYRQANGSAK KSGGDFKPTS PSLPASKI PA LSPSSGKSSS LPSSSGDSSN LPNPPATKPS IASNPLSPQT GPPAHSASLI PSVSNGSLKF QSLTHTGKGH HLSFSPQSQN GRAPPPLSFS SSPPSPASSV SLNQGAKGTR TIHTPSLTSY KAQNGSSSKA TPSTAKETS

cDNA FLJ25393

MTQNYEGQLQ SLKAQFSKLT NNFEKLRLHQ MKQNKVPRKE LPHLKEEI PF ELSNLNQKLE EFRAKSRE D KQEILYQTHL I SLDAQQKLL SEKCNQFQKQ AQSYQTQLNG KKQCLEDSSS EIPRLICDPD PNCEINERDE FIIEKLKSAV NEIALSRNKL QDENQKLLQE LKMYQRQCQA

MEAGLSEVKS ELQSRDDLLR IIEMERLQLH RELLKIGECQ NAQGNKTRLE SSYLPSIKEP

ERKIKELFSV MQDQPNHEKE LNKIRSQLQQ VEEYHNSEQE RMRNEISDLT EELHQkeitI

ATVTKKAALL EKQLKMELEI KEKMLAKQKV SDMKYKAVRT ENTHLKGMMG DLDPGRYMSM

DFTNREQSRH TSINKLQYEN ERLRNDLAKL HVNGKSTWTN QNTYEETGRY AYQSQIKVEK

NEERLSHDCE PNRSTMPPLP PSTFQAKEMT SPLVSDDDVF PLSPPDMSFP ASLAAQHFLL

EEΞKRAKELE KLLNTHIDEL QRHTEFTLNK YSKLKQNRHI

Unknown protein

HEGPRRRGRL WDSSGVPQRQ KRPGP RTQT QEQMSRDVCI HTWPCTYYLE PKRR VTGQL SLTSLSLRFM TDSTGEILVS FPLSSIVEIK KEASHFIFSS ITILEKGHAK H FSSLRPSR NWFSIIEHF WRELLLSQPG AMADASVPRT RGEELTGLMA GSQKRLEDTA RVLHHQGQQL DSVMRGLDKM ESDLEVADRL LTELESPAW PFSSKL KTP PETKPREDVS MTSCEPFGKE GILIKIPAVI SHRTESHVKP GRLTVLVSGL EIHDSSSLLM HRFEREDVDD IKVHSPYEIS IRQRFIGKPD MAYRLISAKM PEVIPILEVQ FSKKMELLED ALVLRSARTS SPAEKSCSV HAASGLMGRT LHREPPAGDQ EGTALHLQTS LPALSEADTQ ELTQILRRMK GLALEAESEL ERQDEALDGV AAAVDRATLT IDKHNRRMKR LT

Hyp.41.3kD protein

MVLGLTEMLR SCLLYALRPH CCHCHRMPFS IRGHL Cytidine deaminase

1 maqkrpactl kpecvqqllv csqeakksay cpyshfpvga alltqegrif kgcnienacy 61 plgicaerta iqkavsegyk dfraiaiasd mqddfispcg acrqvmrefg tnwpvymtkp 121 dgtyivmtvq ellpssfgpe dl

Growth factor receptor-bound protein 2

1 meaiakydfk ataddelsfk rgdilkvlne ecdqnwykae lngkdgfipk nyiemkphpw

61 ffgkipraka eemlskqrhd gaflireses apgdfslsvk fgndvqhfkv lrdgagkyfl

121 wvvkfnslne lvdyhrstsv srnqqiflrd ieqvpqqpty vqalfdfdpq edgelgfrrg

181 dfihvmdnsd pnwwkgachg qtgmfprnyv tpvnrnv

TGF-beta receptor interacting protein 1

1 mkpillqghe rsitqikynr egdllftvak dpivnvwysv ngerlgtymg htgavwcvda

61 dwdtkhvltg sadnscrlwd cetgkqlall ktnsavrtcg fdfggniimf stdkqmgyqc

121 fvsffdlrdp sqidnnepym kipcndskit savwgplgec iiaghesgel nqysaksgev

181 Ivnvkehsrq indiqlsrdm tmfvtaskdn taklfdsttl ehqktfrter pvnsaalspn

241 ydhvvlgggq eamdvtttst rigkfearff hlafeeefgr vkghfgpins vafhpdgksy

301 ssggedgyvr ihyfdpqyfe fefea

Paraspekle protein 1 alpha isofor MMLRGNLKQV RIEKNPARLR ALESAVGΞSE PAAAAAMALΑ 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 PNYMENREQE MRMGDMGPRG AINMGDAFSP APAGNQGPPP MMGMNMNNRA TIPGPPMGPG PAMGPEGAAN MGTPMMPDNG AVHNDRFPQG PPSQMGSPMG SRTGSETPGFGPSGGGPGQGPS

Heterogenous nuclear ribonucleoprotein H

1 mmlgteggeg fvvkvrglpw scsadevqrf fsdckiqnga qgirfiytre grpsgeafve

61 lesedevkla lkkdretmgh ryvevfksnn vemdwvlkht gpnspdtand gfvrlrglpf

121 gcskeeivqf fsgleivpng itlpvdfqgr stgeafvqfa sqeiaekalk khkerighry

181 ieifkssrae vrthydpprk lmamqrpgpy drpgagrgyn sigrgagfer mrrgaygggy

241 ggyddyngyn dgygfgsdrf grdlnycfsg msdhrygdgg stfqsttghc vhmrglpyra

301 tendiynffs plnpvrvhie igpdgrvtge advefathed avaamskdka nmqhryvelf

361 Instagasgg ayehryvelf Instagasgg aygsqmmggm glsnqssygg pasqqlsggy

421 gggyggqssm sgydqvlqen ssdfqsnia

Profilin II

1 magwqsyvdn lmcdgccqea aivgycdaky vwaataggvf qsitpieidm ivgkdregff 61 tngltlgakk csvirdslyv dgdctmdirt ksqggeptyn vavgragrvl vfvmgkegvh 121 ggglnkfays makylrds

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Claims

Tumor antigens and their use

1. Immunogenic squamous cell carcinoma antigen which comprises at least one of the proteins of SEQ ID NO: 1-27 or variants thereof, the variants one or more additions, insertions, substitutions and / or deletions compared 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 substantially the same.

2. An isolated nucleic acid encoding one or more of the proteins of claim 1.

3. An isolated nucleic acid which is a transcription product of the nucleic acid of claim 2.

4. Isolated nucleic acid which hybridizes selectively to the nucleic acids according to claim 2 or 3 under moderately stringent conditions.

5. Immunogenic antigen which is encoded by a nucleic acid according to any one of claims 2 to 4.

6. Vector comprising one or more of the nucleic acids according to any one of claims 2-4.

7. Expression vector comprising one or more of the nucleic acid sequences according to one of claims 2-4 and one or more regulatory sequences.

8. The vector of claim 6 or 7, which is a plasmid.

9. host cell transformed with the vector of any of claims 6-8.

10. Host cell according to spoke 9, which is a eukaryotic cell.

11. Host cell according to spoke 10, which is a mammalian cell, plant cell, yeast cell or an insect cell.

12. A mammalian cell according to claim 11 which is a CHO, COS, HeLa, 293T, HEH or BHK cell

13. Host cell according to spoke 9, which is a prokaryotic cell.

14. The host cell of claim 13 which is E. coli or Bacillus subtilis.

15. Antiköφer or aptamer, which is directed against one or more epitopes of a protein according to claim 1.

16. Antibody according to spoke 15, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a chimeric antibody and a synthetic antibody.

17. Antibody according to spoke 15 or 16, which is bound to a toxic and / or detectable agent.

18. Hybridoma which produces a monoclonal antibody which has a binding specificity for a protein according to claim 1.

19. An ex v / vσ method for generating a population of autologous or allogeneic antigen presenting cells (APCs) capable of inducing an effective immune response against a protein according to claim 1, comprising the following steps:

a) providing autologous or allogeneic APCs; b) contacting the APCs with an effective amount of a peptide fragment of a protein according to claim 1 under conditions which enable endocytosis, processing and presentation of the peptide fragments by these APCs, and c) isolating the APCs presenting the corresponding peptides ,

20. An ex vivo method for producing genetically engineered APCs capable of inducing an effective immune response against a protein according to claim 1, comprising the following steps:

a) providing a nucleic acid which codes for a protein according to claim 1 or peptide fragment thereof, b) transfecting the APCs with the nucleic acid and c) selecting APCs which present the peptide fragments.

21. The method according to claim 20, wherein the nucleic acid in step a) is provided in an expression vector.

22. APC, which can be obtained by the method according to one or more of claims 19 to 21.

23. The APC of claim 22 which is a dendritic cell or a B cell.

24. Ex vivo method for the detection and production of T cells which are specific for a protein according to spoke 1, which comprises the following steps:

a) providing mammalian, in particular human, T cells or PBMCs,

b) co-culturing the T cells with an APC according to spoke 22 or 23 under conditions which enable activation of the T cells, and

c) determining the presence of a specific activity of the T cells against an APC according to claim 22 or 23, d) optional selection and cultivation / expansion of those T cells which in step c) showed specificity for an APC according to claim 22 or 23.

25. T cells which can be obtained by the method according to claim 24.

26. A diagnostic composition comprising one or more of the proteins according to claim 1, an antibody according to one or more of claims 15-17 or a T cell according to claim 25.

27. A pharmaceutical composition comprising a therapeutically effective amount of one or more of the proteins according to claim 1, one or more of the nucleic acids according to 2-4, a vector according to claims 6-8, a monoclonal antibody according to one or more of claims 15-17 , an APC according to claim 22 or 23 or a T cell according to claim 25 and a pharmaceutically acceptable carrier.

28. A pharmaceutical composition according to claim 27 which is a vaccine.

29. Use of the pharmaceutical composition according to spoke 27 or 28 in the diagnosis and therapy of a squamous cell carcinoma, in particular a squamous cell carcinoma in the ENT area.