MXPA02001137A - Tumor-associated antigen (r11). - Google Patents

Abstract

The invention relates to tumor-associated antigens, immunogenic peptides derived therefrom and the DNA molecules coding therefor as well as to their utilization in cancer immunotherapy.

Description

ANTIGENO Rll ASSOCIATED WITH TUMOR DESCRIN OF THE INVENTION The invention relates to the immunotherapy of tumor diseases. The immune system has the task of protecting the body from a number of different microorganisms and actively fighting against these microorganisms. The importance of an intact immune system is evident particularly in the case of inherited or acquired immunodeficiencies. In many cases, the use of prophylactic vaccination programs has proven to be an extremely effective and successful intervention in the immunological field in the fight against infectious diseases of viral or bacterial origin. It has also been found that the immune system is also greatly involved in the elimination of tumor cells. The recognition of tumor-associated antigens (TAA) by the components of the immune system plays a crucial role. In the broadest sense, any component (peic or non-peic) of a tumor cell that is recognized by REF: 135969 an element of the immune system and which causes the stimulation of an immune response, can function as an immunogenic tumor antigen. Those tumor antigens that not only induce an immunological reaction but also cause rejection of the tumor are of particular importance. The identification of specific antigens that can cause an immunological reaction of this type is an important step in the development of a tumor vaccine defined at the molecular level. Although it is not yet clear which elements of the immune system are responsible for the rejection of the tumor, there is nonetheless a consensus that cytotoxic T lymphocytes that express CD8 (CTL) play an important part (Coulie, 1977). Particularly, in those types of tumors (such as melanoma and kidney carcinoma) which have a relatively high rate of spontaneous remission, a correlation has been found between the clinical advance and the increased appearance of CD8 + and CD4 + T cells. (Schendel et al., 1993, Mackensen et al., 1993, Halliday et al., 1995, Kawakami et al., 1995, Kawakami et al., 1996, Wang, 1997, Celluzzi and Falo, 1993). Specific CTL clones were obtained either from lymphocytes that infiltrate the tumor (TIL) or from peripheral blood mononuclear cells (PBMC) after co-culture with generally autologous tumor cells and stimulation of cytokine in vi tro. Both in animal model systems and cell culture systems in humans cultured in vitro, the T cell response against tumor cells was increased by transfection of tumor cells with cytokines (van Elsas et al., 1997; Gansbacher et al., 1990; Tepper et al., 19d9; Fearon et al., 1990; Dranoff et al., 1993). In view of the correlation between remission and the involvement of CDd + T cells, the identification of tumor-associated antigens (TAA) that are recognized by CD8-positive CTLs is a specific primary target for the development of a tumor vaccine ( Pardoll, 1998; Robbins and Kawakami, 1996). It is not yet clear whether other types of cells of the immune system, such as, for example, the CD4 + T helper cells, play an important part or not; a certain number of studies with MAGE-3 / HLA-A1 pees in melanoma patients have indicated this (Marchand et al., 1995, Boon et al., 1998). In recent years a number of TAA have been identified which are recognized by the CTL (Boon et al., 1994, van den Eynde and van der Bruggen, 1997).

The T cells recognize the antigens as pee fragments that occur on the cell surfaces of the MHC molecules (major histocompatibility complex, in human "HLA" = "human leukocyte antigen"). There are two types of MHC molecules: MHC-1 molecules are present in most nucleated cells and present peptides (usually 8-10-méros) which are produced by the proteolytic degradation of endogenous proteins (the so-called antigen processing). Peptide complexes: MHC-I are recognized by CD8-positive CTL. MHC-II molecules are present only in the so-called "professional cells that present antigens" (APC) and present peptides of exogenous proteins which are absorbed and processed in the course of endocytosis by APC. Peptide complexes: MHC-I I are recognized by T helper-CD4 cells. Through the interaction between the T cell receptor and the peptide complex: MHC, various effector mechanisms can be induced which lead to apoptosis of the target cell in the case of CTL. This happens if the MHC (for example in the case of rejection of transplants) or the peptide (for example in the case of intracellular pathogens) are recognized as foreign. In any case, not all the presented peptides meet the structural and functional requirements for an effective interaction with the T cells (as described in Rammensee et al., 1995 and later in the present invention). In principle, a number of administration methods are possible for using TAA in a tumor vaccine: the antigen can be administered as a recombinant protein with appropriate adjuvants or vehicle systems or it can be administered as a cDNA encoding the antigen in a plasmid (DNA vaccine); Tighe et al., 1998) or in viral vectors (Restifo, 1997). Another possibility is to use recombinant bacteria (for example listeria, salmonella) which express recombinantly the human antigen and which have an adjuvant effect as a result of their additional components (Paterson, 1996, Pardoll, 1998). In all these cases, the antigen has to be processed and presented through the so-called "professional antigen presenting cells" (APC). Another possibility is to use synthetic peptides (Melief et al., 1996) that correspond to the T-cell epitopes equivalent to the antigen and that are loaded either in the APC from the outside (Buschle et al., 1997; Schmidt et al., 1997) or that they are absorbed by APC and transferred intracellularly to MHC-I molecules. The therapeutically most efficient method of administration of a specified antigen is usually determined by clinical tests. Antigens or epitopes thereof recognized by tumor-specific CTLs include molecules that can come from any kind of protein (eg, transcription factors, receptors, enzymes, for a research see Rammensee et al., 1995, Robbins and Kawakami, nineteen ninety six) . These proteins do not necessarily have to be located on the cell surface, as is required for recognition by antibodies. To act as a tumor-specific antigen that is recognized by CTL or in order to be used for therapy, the proteins must meet certain conditions: first, the antigen must be expressed exclusively by tumor cells or must be to be present in so-called "critical" normal tissues, not entirely or only in concentrations smaller than that of tumors. Critical normal tissues are essential tissues; an immune reaction directed against these could have serious consequences, in some cases lethal. Secondly. The antigen must be present not only in the primary tumor but also in the metastasis. Furthermore, with a view towards a wider clinical use of the antigen, it is desired that it be present in high concentrations in various types of tumor. An additional precondition for the TAA to be considered as appropriate and as an effective ingredient of a vaccine is the presence of T cell epitopes in the amino acid sequence of the antigen; the peptides derived from TAA must produce a T cell response in vi tro / in vivo ("immunogenic" peptide). Another criterion for selecting a broadly applicable immunogen peptide is the frequency with which the antigen is found in a given population of patients. Immunogenic antigens associated with tumor (TAA), which have already been shown to have T cell epitopes, can be divided into a number of categories, including viral proteins, utate proteins, overexpressed proteins, fusion proteins formed by chromosomal translocation, differentiation antigens, oncofetal antigens ( van den Eynde and Brichard, 1995, van den Eynde and van der Bruggen, 1997). The methods to identify and characterize the TAA, which form the starting point for the development of a tumor vaccine, are based on the one hand on the use of CTL that have already been induced in patients (cellular immune response) or antibodies (humoral immune response), or are based on obtaining differential transcription profiles between tumors and normal tissues. In the first case, the immunological method, the patient's CTL are used to select eukaryotic tumor cDNA expression libraries that display the CTL epitopes by MHC-I molecules (Boon et al., 1994), whereas when using the prokaryotic cDNA expression libraries of high affinity patient antiserum, the presence of the TAA can be investigated directly by immunoblot analysis of the individual plates (Sahin et al., 1995). A combination of the reactivity of the CTLs and the chemical processes of protein produces the isolation of peptides isolated from MHC-I from tumor cells, which are preselected by reactivity with the CTL of the patient. The peptides are cleansed of the MHC-I complex and identified by mass spectrometry (Falk et al., 1991, Woelfel et al., 1994, Cox et al., 1994). Methods that use CTL to characterize antigens involve substantial costs or are not always successful, due to the need to culture and activate CTL. The methods to identify TAA that are based on the comparison of the transcription profile of normal tissue and tumor tissue are many and varied; These include differential hybridization, the establishment of subtraction cDNA libraries ("representative difference analysis", Hubank and Schatz, 1994, Diatchenko et al., 1996) and the use of DNA part technology or the SAGE method (Velculescu et al. al., 1995). In contrast to the aforementioned immunological method using the patient's CTL, when molecular biological methods are used it is necessary to demonstrate that the potential antigen candidates discovered by this method are tumor-specific (tumor-associated) and that they indeed have cell epitopes T that can invoke or induce a cytotoxic T cell response. In at least one case, an antigen was identified (NY-ESO / LAGE-1) both by the use of the patient's serum and by RDA (Chen et al., 1997; Lethe et al., 1998), and were also described. CTL epitopes of this antigen and a spontaneous and simultaneous humoral and T cell response in a patient (Jager et al., 1998). The aim of the present invention is to provide an antigen associated with novel tumor (TAA). This objective is achieved by first establishing a cDNA subtraction library by RDA (representative difference analysis) between a cell line obtained from the patient with pancreatic carcinoma and a combination of 11 different normal tissues. In order to generate the cDNA fragments of the "tester" and "controller" required for the subtractive hybridization, in a deviation from the original method (Diatchenko et al., 1996) a mixture of 6 different restriction enzymes was used. The use of a mixture of different restriction enzymes that require 6 base pairs as the recognition sequence has the following advantages over the original method (Diatchenko et al., 1996): a) selecting two restriction enzymes recognition sequences which are represented by the combinations of six of the bases A / T (for example Ssp I: AATATT) or C / G (for example Nae I: GCCGGC) or A / C / G / T (for example EcoR V: GATATC), both regions rich in GC and AT of a gene are cut in the same way, allowing in this way the homogeneous representation of the region of the complete gene as restriction fragments; b) in addition, this makes it possible to obtain larger cDNA fragments of the candidate gene, in a statistical average (approximately 800 bp), which in turn is quite convenient in the subsequent analysis (sequence determination and annotation) and in the cloning of the "full size" cDNA. In the original method (Diatchenko et al., 1996) a restriction enzyme (Rsa I) is used that recognizes only 4 bases, which leads to an average fragment length of 256 base pairs and can not process in specific form the regions rich in CG or AT. In order to compensate for the kinetics of hybridization changed by the longer insert cDNA fragments, the PCR procedure is modified as described in example 2. In order to select the antigens that are overexpressed in the tumor, they were separated first the cDNA clones obtained and a culture in basic glycerol, a plasmid preparation and a collection representing the insert of the PCR fragments from them in a 96-well plate format was established. First, the sequence of 50 cDNA fragments selected at random from the 3450 clones of the cDNA library subtractive to pancreatic carcinoma was determined in order to select the antigens that are overexpressed in the tumor and compared with the sequences available in the data banks. Among the annotated genes there were 12 unknown, for which EST entries (expressed sequence markers) were made in the database. One clone, Rll, due to its preferential presence in fetal tissue, indicated an EST profile which is appropriate for possible use as a TAA. Further investigations using semiquantitative RT-PCR and Northern Blot analysis confirmed the preferred expression in various tumors (breast tissue carcinoma, kidney and pancreatic cells) and immunoprivileged tissues (testes, placenta and adrenal glands) and little or no expression in normal tissues. In addition, it can be concluded from the data obtained by Northern Blot experiments that the Rll transcript is approximately 7.5 kb in length and that there could be splice variants or homologous genes in the adrenal glands. Human Rll-DNA was cloned from the testes; the sequence obtained is shown in SEQ ID N0: 1 the Rll sequence does not demonstrate identity or homology with any known gene either at the nucleotide or protein level. The -ADNc Rll obtained within the field of the present invention contains two separate open reading frames for a protein of 4001 amino acids long (SEQ ID NO: 2) and for a protein of 357 amino acids long (SEQ ID NO: 3) . The RNI-cloned CND within the field of the present invention has a length of 6582 bp, while the presence of a PoliA residue at the 3 'end of the sequence is an indication of the entire cDNA in this region. Based on the data obtained within the field of the present invention, it can not be established that from the 5 'end of the cDNA to which the sequence was determined, there is another ATG constituting the starting ATG for the first open reading frame (Rll-ORF-1); in this case, the present cDNA contains the region coding for the C-terminal section of Rll-ORF-1 at the 5 'end. The information concerning the 5 'end and a possible coding DNA sequence section also located towards the 5' end can be obtained by standard methods of molecular biology, for example by 5 '-RACE (rapid amplification of cDNA ends). In this method, the RNA, preferably mRNA, is reverse transcribed from cells or tissues into which the Rll is transcribed (eg, tissue from mammary tissue, kidney or pancreas cells) and then it is linked with a known sequence adapter. A PCR with an adapter primer (which binds specifically to the adapter at the 5 'end of the cDNA) and an Rll-specific primer (eg SEQ ID no: 26) allows the corresponding Rll fragments to be amplified. These PCR products can be cloned by standard methods and can be characterized in particular by DNA sequence determination, as described in example 6. An alternative method for characterizing the 5 'end is by selection of cDNA libraries using hybridization with DNA probes that are specific for Rll or by analysis of cDNA expression libraries with antiserum. If the selection of the cDNA libraries does not achieve the desired performance or result, due to the limitations of the method, for example inefficient reverse transcription caused by secondary RNA labeled structures, the genomic libraries can be investigated with respect to, for example, the clone isolation, such as in the selection of cDNA libraries, by hybridization with Rll-specific DNA probes, said clones containing the sequence information located upstream or towards the 5 'end of the 5' end of the cDNA obtained, for example the promoter region of Rll. In the course of the total cloning of the cDNA of Rll it is possible to establish whether the open reading frame of R11-0RF-1 obtained in the region of the cDNA fragment does or does not have a continuation in the 5 'region and / or whether or not alternative reading frames exist. The cDNA isolated in accordance with the field of the present invention has the nucleotide sequence given in SEQ ID NO: 1; it is not considered (see above) that it codes for the C-terminal portion of a tumor-associated antigen (TAA) called Rll-ORF-1 and for another protein that is represented by the second reading frame (Rll-ORF- 2) . The two proteins of the two reading frames expressed by the isolated cDNA have the amino acid sequence shown in SEQ ID NO: 2 and 3, respectively. In a first aspect, the present invention relates to an isolated DNA molecule which has the nucleotide sequence shown in SEQ ID NO: 1; or a polynucleotide that hybridizes with this DNA molecule under astringent conditions. By the phrase "astringent conditions" is meant, for example: incubation overnight at 65 ° C - 68 ° C with 6xSSC (lx SSC = 150 mM NaCl, 15mM trisodium citrate), 5x Denhardt's solution, 0.2% SDS, 50μg / ml salmon sperm DNA, followed by a double wash for 30 min. With 2x SSC, 0.1% SDS at 65 ° C, once for 30 min with 0.2x SSC, 0.1% SDS at 65 ° C and finally optionally a rinse with 0. lx SSC, 0.1% SDS at 65 ° C. In another aspect the present invention relates to an isolated DNA molecule which contains a polynucleotide of the sequence shown in SEQ ID NO: 1; as a partial sequence or containing a polynucleotide that hybridizes with a polynucleotide of this sequence under stringent conditions. DNA molecules or fragments thereof according to the invention encode for (poly) peptides designated R11-ORF1 and R11-0RF2, while R11-ORF2 has the amino acid sequence shown in SEQ ID No: 3 and Rll-ORF2 has the amino acid sequence shown in SEQ ID NO: 2 or contains it; or for protein fragments or peptides obtained from Rll-ORF-1 or R11-ORF2. This therefore includes DNA molecules that comprise deviations from the sequence shown in SEQ ID NO: 1 as a result of the degeneracy of the genetic code.

In another aspect the present invention relates to tumor-associated antigens referred to as Rll -ORF-1 and Rll-ORF-2, while in the case of R11-0RF-1, there is an extension of an existing open reading frame. in the 5 'direction, the amino acid sequence given in SEQ ID NO: 2 for R11-0RF-1 is a partial sequence. The proteins with the sequences shown in SEQ ID NO: 2 and 3 are products that are translated by a transcript of approximately 7.5 kb in size, or that are translated by transcripts of approximately 3.8 kb and 2.3 kb in size that are derived from variants of splicing of the transcript of 7.5 kb such as those that could be found in the adrenal tissues, or from transcripts of the genes homologous to it. The amino acid sequences shown in SEQ ID NO: 2 and 3 could have deviations, for example those related to the replacement of amino acids, such as those derived from Rll ("R-ll", unless otherwise specified, hereafter indicates R11-ORF1 and / or R11-ORF2) having desirable immunogenic properties for use in a tumor vaccine. The natural amino acid sequence of R11-ORF1 or R11-ORF2 can optionally be modified by replacing individual amino acids in a Rll CTL epitope in order to obtain an increase in the affinity of the Rll peptides towards the MHC-1 molecules in comparison with the CTL epitope of natural Rll, and in this way obtain an increased immunogenic character and finally a greater reactivity towards the tumors. Modifications can be made in the region of the Rll epitopes in the complete Rll protein (this is processed by the APCs to form the corresponding peptides) or in larger Rll protein fragments or Rll peptides (see below). In accordance with another aspect of the present invention this relates to immunogenic polypeptide fragments and peptides obtained from Rll-ORFl or R11-ORF2. The latter are known hereinafter as peptides Rll. A first group are the Rll peptides that induce a humoral immune response (induction of antibodies). Such peptides are selected portions of R11-0RF1 or R11-0RF2 (of at least 12 to 15 amino acids) that can be determined by so-called prediction algorithms such as for example the surface probability graph (Emini et al., 1985), the graph of hydrophobic character (Kyte and Doolittle, 1982) and the antigenic index (Jameson and Wolf, 1988). Also included are all those peptides that are obtained from the N-terminal region of Rll which are obtained optionally in the course of the subsequent cloning. It is known that tumor-associated antigens may have tumor-specific mutations that contribute to an immunological difference between the tumor and normal tissue (Mandruzzato et al., 1997; Hogan et al., 1998; Gaudi et al., 1999; Wolfel et al., 1995). In order to detect the presence of tumor-specific Rll-ORF-1 or Rll-ORF-2 mutations, the Rll cDNA of one or more different tumors is cloned, using appropriately probes of the isolated cDNA from testes of compliance. with the invention, and the sequences obtained are compared to the normal tissue Rll cDNA. It is expected that the tumor Rll peptides from a sequence fragment that is mutated in comparison to the Rll of normal tissue have a higher immunogenic character compared to the Rll peptides of normal tissue from the corresponding fragment. Therefore, according to another aspect, the present invention relates to Rll peptides obtained from regions of an Rll-ORF-1 or R11-0RF-2 expressed in tumor containing tumor-specific mutations. For therapeutic use, the Rll peptides are administered either directly or in a modified form (eg coupled to KLH [lame hemocyanin]) and the production of antibodies is determined by common immunological tests, for example using ELISA. Other Rll peptides that are preferred within the scope of the present invention are those presented by the MHC molecules and that produce a cellular immune response. There are two types of MHC molecules, namely MHC-I molecules that are recognized by the CD8-positive CTL and MHC-II molecules which are recognized by the positive CD4 T helper cells. For a peptide to induce a cellular immune response, it must bind to an MHC molecule, while the patient to be treated must have the molecule of MHC in your repertoire. In this way, the determination of the MHC sub-type of the patient constitutes one of the essential pre-requisites for effective use in this patient, with the intention of inducing a cellular immune response. The sequence of an Rll peptide that will be used in therapeutic form is determined by the MHC molecule in question in terms of anchor amino acids and length. Defined anchor positions and length ensure that a peptide binds to the peptide binding groove of the MHC molecule of the patient in question. The result of this is that the immune system is stimulated and a cellular immune reaction is produced, which is directed against the tumor cells of the patient, if a peptide derived from a tumor antigen is used. Immunogenic Rll peptides can be identified by known methods; one of the basic conditions is the correlation between the binding to the MHC and the induction of CTL. Therefore, because the sequence of the immunogenic peptides can be predicted based on their peptide binding motif, the Rll peptides which constitute the epitopes for CTL can be identified and synthesized based on the Rll protein sequence. . Several methods are available to do this, which are used to identify the CTL epitopes of known protein antigens; for example, the method described by Stauss et al., 1992 to identify T cell epitopes in human papillomavirus. The specific allele requirements of each ______? __ fc_É ^ ______ i__tjAihiájJu «*" * - »• .- - - allelic product of MHC-I with respect to a peptide that binds to the MHC molecule and that is presented with it are assembled as a motif (eg Falk et al., 1991). To date, a large number of both MHC peptide motifs and MHC ligands are known. An appropriate method within the scope of the present invention to search for epitopes of a known protein that conforms to a specific MHC-I molecule is described in an investigation by Rammense et al., 1995. This comprises the following steps: first , the sequence of the protein is sought with respect to fragments corresponding to the anchor motif, while certain variations are possible with respect to the length of the peptide and the occupation of the anchor. If for example a motif prescribes a 9-mer with lie or Leu at the end, 10-mer with a corresponding C-terminus can also be considered, as can peptides with other aliphatic groups such as Val or Met at the end. C-terminal. In this way a number of candidate peptides is obtained. These are investigated with respect to the presence of how many anchor groups as possible have in common with known ligands and / or to investigate whether or not they have groups that are "preferred" by the various molecules of the anchor. tAaséáá * "a" MHC (in accordance with the table prepared by Rammense et al., 1995). In order to exclude weakly binding peptides, binding tests are preferably carried out. If the requirements for the binding of the peptide to specific MHC molecules are known, candidate peptides can also be investigated for non-anchor groups which have a positive or negative effect on binding or which in effect cause this is completely possible (Ruppert et al., 1993). However, it should be kept in mind that the peptide binding motif is not the only decisive factor when investigating natural ligands; other aspects, for example enzyme specificity during antigen processing, also contribute to the identity of the ligand, in addition to the specificity of MHC binding. A method that considers these aspects and that is suitable for identifying immunogenic Rll peptides within the scope of the present invention was used, inter alia, by Kawakami et al., 1995 to identify the epitope gplOO based on the HLA-A * 0201 motifs. known. Peptides can also be selected with respect to their ability to bind to MHC-II molecules. The MHC-II binding motif that extends through nine amino acids has a greater degree of degeneracy at anchor positions than the MHC-I binding motif. Recently methods have been developed, based on X-ray structural analysis of MHC-II molecules, which allow an exact analysis of the MHC-II binding motifs and, based on this, of the variations in the peptide sequence (Rammense et al., 1995, and the original literature cited in the same document). Peptides that bind to MHC-II molecules are typically presented to CD4-T cells by dendritic cells, macrophages or B cells. CD4-T cells in turn directly activate CTLs sequentially by releasing cytokine, for example, and increase the efficiency of antigen presentation by APCs (dendritic cells, macrophages and B cells). Recently, data banks and prediction algorithms have been made available that allow a more reliable prediction of the peptide epitopes that bind to a specific MHC molecule. Within the field of the present invention, using the algorithm described by Parker et al., 1994 and Rammensee et al., 1995, peptides have been identified.

?!! A *? .- MA £ J Í. *. $ -. J > **. * JU: & -,. ~. ... M? IÍ .... > .. .... ~ ... .... ... ^.;. t .., .a »jM, af a» M? »? i« i »* - nnr *» '"? f faith. ^ ifc« ^ a .i ?, .i ..ii. áí? i Ui? / iismi C-terminal fragment candidate for most of the important HLA types, especially for HLA-Al, -A * 0201, -A3, -B7, -B14 and -B * 4403, of which we expect that bind the corresponding HLA molecules and thus constitute the immunogenic CTL epitopes; the discovered peptides are listed in Table 1. Similarly, possibly using other algorithms that take into account the different characteristics of the peptides (hydrophobic character, charge, size) or the requirements made of the peptides, such as the 3D structure of the peptides. the HLA molecule, it is possible to find other potential peptide epitopes; this also applies to the peptide epitopes of other types of HLA. After selecting Rll peptide candidates using the methods described, their binding to MHC is evaluated by peptide binding assays. First, the immunogenic character of the peptides with good binding properties is determined (stability of the peptide-MHC interaction is correlated in most cases with the immunogenic character, van der Burg et al., 1996). In order to determine the immunogenicity of the selected peptide or peptide equivalent, methods could be used as described, for example, by Sette et al., 1994, combined with quantitative MHC binding tests. Alternatively, the immunogenicity of the selected peptide can be assessed by induction of CTL in vi tro using known methods (as described below in the present invention for the induction of CTL ex vivo). The principle of the method, carried out in several steps, to select peptides that can induce a cellular immune response is described in WO 97/30721, whose contents are expressly referred to in the present invention. A general strategy for obtaining efficient immunogenic peptides that are suitable within the scope of the present invention is also described by Schweighoffer, 1997. Rather than using the original peptides that fit the binding groove of the MHC-I or MHC-molecules II ie peptides that are obtained in unaltered form from Rll, variations can be made, adhering to the minimum of requirements with respect to the anchorage and length positions specified based on the original peptide sequence, with the proviso that these variations not only hinder the effective immunogenicity of the peptide being prepared or constituted by this binding affinity to the MHC molecule and its ability to stimulate the T cell receptors, but preferably to intensify it. In this case, artificial peptides or peptide equivalents are therefore used, which are designed to correspond to the requirements regarding the binding capacity of an MHC molecule. Peptides modified in this manner are known as "heteroclite peptides". These can be obtained by the following methods: First of all, ligand epitopes are taken to MHC-I or MHC-II or variations thereof, for example using the principle described by Rammensee et al., 1995. The length of the peptide preferably corresponds to a minimum sequence of 8 to 10 amino acids with the necessary anchor amino acids, if the peptide is being compared to MHC-I molecules. If desired, the peptide may also be extended at the C- and / or N-terminal end provided that this extension does not affect the ability to bind to the molecule of MHC and the extended peptide can be processed in cellular form up to a minimal sequence. The modified peptides are then investigated for their recognition by the TILs (lymphocytes) i.i iJi .i. * ~ *? ua-? q emrt < i .i * i > .j, .i * tumor infiltrators) for the induction of CTL and with respect to increased MHC binding and their immunogenicity or capacity, as described by Parkhurst et al., 1996 and Becker et al., 1997. 5 Another method for finding peptides with a higher immunogenic character than that of natural Rll peptides, which is appropriate for the purposes of the present invention, is to select peptide libraries with CTL that recognize the Rll peptides that occur in 10 natural form in tumors, as described by Blake et al., 1996; in connection with this, the use of combinatorial peptide libraries is proposed in order to design molecules that mimic the tumor epitopes recognized by MHC-I restricted CTL. The Rll polypeptides according to the present invention or the immunogenic fragments or Peptides derived therefrom can be produced in recombinant form or by peptide synthesis, as described in WO 96/10413, which description is 20 referred to in the present invention. For recombinant production, the corresponding DNA molecule is inserted by standard methods into an expression vector, transfected into an appropriate host cell, the cell SÉJJÉÍßltlk Í?, T.A.tí. i? .. Í¡¡áÍ, ij. ? lt f? lM * «m» * > ? rtrtéiW! - *. «« * »«. ^ -....... ....... «A ***. A ~ ... t M¡a? tA ???? t ± íi, ?? k.Í? host is cultured under appropriate expression conditions and the protein is purified, conventional methods for the chemical synthesis of Rll peptides can be used, for example using automated synthesizers of peptides 5 which are commercially available. As an alternative to natural Rll peptides or heteroclite peptides it is also possible to use substances that mimic such peptides, for example "peptide mimics" or "retroinverse peptides". For To evaluate these molecules with respect to their therapeutic use in a tumor vaccine the same method as described above is used for the natural Rll peptides or the Rll peptide equivalents. The two TAAs designated Rll-15 ORF-1 and Rll-ORF-2 according to the present invention can be used and the protein fragments, peptides or peptide equivalents or peptide mimics derived therefrom in cancer therapy, for example to induce an immune response towards the tumor cells expressing the 20 corresponding antigenic determinants. These are preferably used for the treatment of positive Rll-ORF-1 and R11-0RF-2 tumors, particularly in breast, kidney and pancreatic cell carcinoma.

The immune response in the form of CTL induction can be obtained in vivo or ex vivo. To induce CTL in vivo, a pharmaceutical composition containing the TAA Rll-ORF-1 and Rll-ORF-2 or fragments or a peptide or peptides derived therefrom is administered to a patient suffering from a disease as an active component. tumor associated with the TAA, and the amount of TAA (peptides) must be sufficient to obtain an effective CTL response to the tumor carrying the antigen. Therefore, according to another aspect, the invention relates to a pharmaceutical composition for parenteral, topical oral or local administration. Preferably, the composition is used parenterally for example, for subcutaneous, intradermal or intramuscular application, containing as an active component TAA R11-0RF-1 and R11-0RF-2 or fragments or peptide or peptides derived therefrom. The TAA / Rll peptides are dissolved or suspended in a pharmaceutically acceptable carrier, preferably aqueous. The composition may also contain conventional adjuvants such as buffer solutions etc. The TAA / Rll peptides can be used on their own or together with adjuvants, for example the incomplete Freund's adjuvant, saponins, aluminum salts or, in a ? zA.AÁ.lJ i * UíttdaaHaaaaMélitoh- • «- *" »• - ..1. ^. 1 ^. ^^ J & preferred embodiment, with polycations such as polyarginine or polylysine. The peptides can also be linked to components that help the induction of CTL or the activation of CTL, for example peptides of auxiliary T, 5 lipids or liposomes, or these are administered together with these substances and / or together with immunostimulatory substances, by example cytokines (IL-2, IFN-?). The methods and formulations that are suitable for the preparation and administration of the pharmaceutical composition according to the invention10 are described in documents 95/04542 and WO 97/30721, the descriptions of which are incorporated by reference in the present invention. Rll polypeptide fragments or Rll peptides can also be used to induce a response 15 of CTL ex vivo. An ex vivo CTL response to a tumor expressing the two possible Rll proteins is induced by incubating the CTL precursor cells together with the APCs and Rll peptides or the Rll protein. The active CTLs are then allowed to expand, after which they are returned to 20 administer to the patient. Alternatively, APCs can be loaded with Rll peptides, which leads to efficient activation of cellular immune reactions against Rll-positive tumors (Mayordomo et al., 1995; Zitvogel et al., 1996). An appropriate method for loading the peptides into the cells is described in WO 97/19169. In one embodiment of the invention, a combination of several different Rll peptides or Rll peptide equivalents is used. In another embodiment, the Rll peptides are combined with peptides derived from other TAA. The choice of peptides for such combinations is made in order to detect different types of MHC in order to cover the widest possible population of patients, and / or is directed to the broadest possible spectrum of indications, combining peptides from different antigens of tumor. The number of peptides in a pharmaceutical composition can fluctuate over a broad range, but generally a clinically usable vaccine contains from 1 to 15, preferably 3 to 10, different peptides. The peptides according to the invention can also be used as diagnostic reagents. For example, the peptides can be used to test a patient's response to the cellular or humoral immune response induced by the immunogenic peptide. This offers the possibility of improving a treatment procedure. For example, depending on the form of administration of the TAA ItAA.S A. aJJUii a - * • * "" - »-» • -qfctin > < »^^.» J_? M? * I¿ -. ^ .... t? Í + i? * & i '< t .. ~~ * - & .. > - -, - * - afc, ^?. X. iii * w «B» SJa ^ (peptide, total protein or DNA vaccine), it is possible to investigate the increase of precursor T cells in PBLs that have a reactivity against the defined peptide epitope (Robbins and Kawakami, 1996). references cited in said document). In addition, peptides or total protein or antibodies directed against TAA could be used to characterize the progression of an R11-0RF-1-positive or Rll-ORF-2-positive tumor (e.g. by immunohistochemical analysis of primary tumor and metastasis). A strategy of this type has already proven to be successful in many cases, for example by detecting the estrogen detector as the basis for deciding on endocrine therapy in breast tissue cancer; c-ebrB-2 as the relevant marker in the prognosis and course of therapy in breast cancer (Ravaioli et al, 1998; Revillion et al., 1998); PSMA (prostate-specific membrane antigen) as a marker for epithelial cells of prostate carcinoma in serum or using a monoclonal antibody labeled with 11: LIn against PSMA in an immunological scintillation assay in prostate carcinoma (Murphy et al., 1998 and the references included in said document); CEA (carcinoembryonic antigen) as a serological marker for prognosis and progress in patients suffering from colorectal carcinoma (Jessup and Loda, 1998). Of the DNA molecules according to the invention defined above, there are also included those which lead, by mutation, to an exchange of amino acids in the protein sequence shown in SEQ ID No: 2 or 3, if they encode a derivative or Rll fragments or Rll peptides with the immunogenic properties that are desired to be used as vaccines for tumors. The DNA molecules for Rll of the present invention or the corresponding RNA molecules which are also a subject of the present invention are used, as well as the (poly) peptides encoded by them, for the immunotherapy of cancer diseases. In one embodiment of the invention, DNA molecules encoding natural Rll polypeptides are used. Alternatively to the Rll cDNA or fragments thereof it is therefore also possible to use modified derivatives. These comprise sequences with modifications encoding a protein (fragment) or peptides with a higher immunogenic character, while the same considerations apply to DNA level modifications as applied to the peptides described above. Other The type of modification is the alignment of numerous sequences that code for immunologically relevant peptides such as a string of beads (Toes et al., 1997). The sequences can also be modified by the addition of auxiliary elements, functions, which ensure a more efficient release and processing of the immunogen (Wu et al., 1995). For example, the processing and therefore the presentation and finally the immunogenicity of the antigen can be increased by the addition of a localization sequence in the endoplasmic reticulum ("sequence to select ER target"). In another aspect, the present invention relates to a recombinant DNA molecule containing the Rll DNA in accordance with SEQ ID No: 1 or a partial sequence, in particular the sequence encoding the Rll-ORF-1 or Rll polypeptide. -ORF-2 The Rll DNA molecules of the present invention can preferably be administered in recombinant form as plasmids, directly or as part of a recombinant virus or bacterium. In theory, any method of gene therapy for cancer immunotherapy based on DNA ("DNA vaccine") on Rll DNA could be used, both in vivo and ex vivo. nérifi íiniíntMfíiílriríit MtlHHfctin ri n ii- Ttr * "-" - '-' .Am, Á et.

Examples of in vivo administration are the direct injection of "naked" DNA, either intramuscularly or using a gene gun, which has been shown to lead to the formation of CTL against tumor antigens. Examples of recombinant organisms are vaccinia virus, adenovirus or Listeria monocytogenes a summary is provided by Coulie, 1997). In addition, synthetic vehicles for nucleic acids such as cationic lipids, microspheres, micro-tablets or liposomes could be used for the in vivo administration of nucleic acid molecules encoding the Rll peptide. As with the peptides, different adjuvants can also be administered that enhance the immune response, for example cytokines either in the form of proteins or of plasmids encoding them. The application can optionally be combined with physical methods, for example electroporation. An example of ex vivo administration is the transfection of dendritic cells as described by Tutmg, 1997 or other APCs that are used as vaccines for cellular cancer. Therefore, in accordance with another aspect, the present invention relates to the use of cells expressing Rll, either per se or, optionally, The modified antigen, after transfection with the corresponding coding sequence, in order to produce a vaccine for cancer. In another aspect, the invention relates to antibodies against R11-0RF-1 or R11-0RF-2 (hereinafter referred to as "RII anti-antibodies") or fragments thereof. Polyclonal anti-Rl antibodies are obtained in conventional manner by immunizing animals, in particular rabbits, by injecting the antigen or fragments thereof and subsequently purifying the immunoglobulin. Anti-monoclonal RII antibodies can be obtained by standard procedures following the principle described by Kohler and Milstein, 1975, immunizing animals, in particular mice, then immortalizing the antibody-producing cells from the immunized animals, for example by cell fusion of myeloma, and selecting the supernatant of the hybridomas obtained by standard immunological tests with respect to anti-monoclonal RII antibodies. For therapeutic or diagnostic use in humans, these antibodies of animal origin can optionally be chimerized in the customary manner (Neuberger et al., 1984, Boulianne et al., 1984) or humanized (Riechmann et al., 1988, Graziano et al. ., nineteen ninety five) . Anti-human monoclonal RII antibodies (or fragments thereof) can also be obtained from so-called phage display libraries (Winter et al., 1994, Griffiths et al., 1994, Kruif et al., 1995, Mc Guiness et al., 1996) and by transgenic animals Brüggemann et al., 1996, Jakobovits et al., 1995). The anti-RII antibodies according to the invention can be used in immunohistochemical analysis for diagnostic purposes. In another aspect, the invention relates to the use of antibodies specific for Rll-ORF-1 and Rll-ORF-2 to selectively carry any of the desired substances towards or in the tumor expressing Rll-ORF-1 and / or Rll- ORF-2. Examples of such substances are cytotoxic agents or radioactive nuclides whose activity consists of damaging the tumor in situ. Due to tumor-specific expression of Rll-ORF-1 or Rll-ORF-2, no side effects can be expected or very few side effects can be expected. In accordance with another aspect, substances can be used to exhibit tumors expressing Rll, with the aid of antibodies against Rll-ORF-1 and / or Rll-ORF-2.

This is useful for the diagnosis and evaluation of the treatment. Therapeutic and diagnostic uses are described in WO 95/33771. The TAAs designated R11-0RF-1 and R11-0RF-2 according to the present invention and the protein fragments, peptides or peptide equivalents or peptide mimics derived therefrom can be used in therapy for cancer, for example to induce an immune response to tumor cells expressing the corresponding antigenic determinants. These are preferably used for the treatment of tumors positive for Rll-ORF-1 and / or R11-0RF-2, in particular in mammary carcinoma of the kidney and pancreas cells. In another application, the Rll can be used as the target molecule of targeted chemotherapy. By the term chemotherapy is meant the therapeutic administration of substances that have either cytostatic or cytotoxic-cytolytic activity interfering with the metabolism of malignant cells, their signal transduction and their cell division processes. In principle, these chemotherapeutic agents develop their activity in all dividing cells; however, the tumor cells demonstrate a greater sensitivity to these substances than that of healthy cells, because they are the cells that proliferate with force those that are affected. The prerequisite for the use of Rll associated with tumor as a target for chemotherapy is - unlike the aforementioned therapeutic immunological methods - knowledge of the function of Rll, Rll-ORF-1 and Rll-ORF-2 proteins or the gene that codes for them. The first step in the so-called functional analysis "downstream" of Rll is conveniently a bioinformatic analysis which points the way for the experimental validation of Rll as a target for chemotherapy. The concepts of bioinformatics based on similarity and modular structure constitute an essential basis for this analysis. The established bioinformatics assistants to determine the similarities are BLAST (http: //www.ncbi. Nlm.mh.gov / BLAST, Altschul et al., 1997) or FASTA (Pearson &Lipman, 1988), data banks such as Pfam (http: // www. sanger .ac. uk / Pfam, Bateman et al., 2000) and SMART (http: // smar. embl-heidelberg .de, Schuitz et al., 2000) which they take domain structures into consideration. fatiá Jiáni H8 - ¿- "** • * < - -. Aifc tt.i. i To refine the analysis, applications such as Clustal can be used (http: // www2.ebi.ac.uk / clustalw, Higgins et al., 1996) HMMer (http: // hmmer.wustl.edu, Durbin et al.), PSI-BLAST (Altschul et al., 1997) and the PROSITE data bank (http: // www. Expasy ch / prosite, Hofmann et al., 1999) Statistical methods of analysis that are not based on homologies make it possible to predict other properties related to structure and function such as secondary structure and the appearance of transmembrane segments and motifs. helix-turn-helix Methods to predict the secondary structure are available, it is worth mentioning Jpred in particular (h p: // barton, ebi.ac.uk / servers / pred.html, Cuff et al., 1998). The prediction of the secondary structure could form the basis for functional hypotheses, for example if the structure of the supposed homologue is known. Subsequently, the Rll is subjected to a biochemical and biological analysis. After the sequence analysis described above has been performed, R11-0RF-1 and Rll-ORF-2 are subjected to biochemical and biological analysis. The choice of the methods used for the subsequent analysis depends on the result of the bioinformatic analysis carried out. ... ^^. ^^^ glia ^ ia ^? uml ^ slíÉklt ^ iiáiain ^ - ^ - - '-V? t? É? á ???. t .t. .i An example of functional analysis is the analysis of proteins theoretically derived partially from chromosome III of the yeast genome. (In such an analysis it is possible to predict more than 70% of the functions of the gene using bioinformation, part of which is confirmed experimentally (Bork, P. et al., 1992; Sharp, P. M. et al. , 1993 and Konnin E. V. et al. , 1994)). In all the studies that are going to be carried out it is important to pre-select those domains of the protein of unknown function that will be analyzed that have an extraordinary structural complexity, because the limited structural information (for example, globular regions) does not contribute to any content of important information. An extensive summary of examples of successful predictions of functions based on protein sequences has been published in Nature Genetics by Bork and Koonin (Bork and Koonin, 1998). In the functional analysis of Rll-ORF-1 carried out within the field of the present invention, it is established that, according to the bioinformatics analysis, this is a protein that belongs to the family of transcription factors that contain zinc fingers. Using appropriate experiments such as, for example, mobility changes, South-Western, UV entanglement, etc., it is possible to demonstrate a direct and / or direct interaction with nucleic acids, particularly in the promoter regions. Appropriate methods for this are known from the literature (e.g. Ausubel et al., 1994). It was possible to demonstrate, for the first 280 amino acids of the protein derived from the Rll-ORF-2 region, a clear homology with a retroviral poly polyprotein. Therefore, R11-0RF-2 could be a possible retrotransposon. Once the function of R11-0RF-1 or Rll-ORF-2 is established, the significance of the gene for Rll and its function or the function of the encoded proteins with respect to the presence of tumors is analyzed. This can be demonstrated, for example, by in vitro proliferation tests or in animal models using tumor cells that overexpress the gene being investigated (constitutively or inducibly) and as a control that expresses it either in suppressed form ( inactive) or negatively regulated by the antisense (see for example Grosveld and Kollias, 1992). The Rll can be used in screening tests to identify substances that modulate, in particular that inhibit, the activity of Rll-ORF-1 or Rll-ORF-2. In one embodiment such a test could consist, for example, of introducing R11-0RF-1 or R11-0RF-2 or an active fragment thereof into cells that react to Rll activity with proliferation or expression of the fragment of Corresponding Rll cDNA in the cell and determine the proliferation of the cells in the presence or absence of a test substance. An example of test cells are cells with a low cleavage rate, for example, primary cells that do not have endogenous Rll. In order to establish that the cells are suitable for a selection test, these are transformed with cDNA-RII, cultured and evaluated with standard tests, for example thymidine incorporation, with respect to their capacity to proliferate. Based on a significant increase in their ability to proliferate after Rll expression, these could be used as test cells, for example in High Throughput Proliferation Screening. Examples of proliferation tests in the High Performance format, for example based on MTS tests, are described in WO 98/00713. Rll inhibitors may be used with an activity that inhibits proliferation to treat tumors | ja__Biá ^ jfcjbáfÍiiÍiÍnJb ^, JB "aa, Í" - '--Íjt-¿< ^ * ».-, at_a« __ i_a < __ ^. afla ^^ ^^^ 11 ^^ ^ ^ that express abundantly Rll, particularly in carcinoma of the breast, kidney or pancreas cells.

BRIEF DESCRIPTION OF THE FIGURES Figures: 1A-1B tra ^ scripci? of Rll in tumor tissues and normal tissues: Semiquantitative RT-PCR. Figure 2: Transcription of Rll in tumor tissues and normal tissues: qualitative PCR. Figure 3: Northern Blot analysis of Rll in normal tissues. Figure 4: Rll transcription: Qualitative RT-PCR of RNA from human tumor cell lines. Figure 5: modified region of the vector pCR3.1 (+).

EXAMPLE 1 Cell culture of the MZ.PC2 m7 # l B7.1 # 3 cell line derived from a human pancreatic carcinoma and poly A * RNA isolation The MZ cell line. PC2 m7 # l B7.1 # 3 is derived from a human pancreatic carcinoma (MZ.PC2; this was obtained * il as follows: first, the tumor cells are passed once through the mouse and a clone is selected for further study (MZ.PC2 m7 # l). This clone is transfected under standard conditions (Ausubel et al., 1994) with a eukaryotic vector (pEF-BOS), the promoter originates from an EF-lalfa gene from human, the selection marker: puromycin, Mizushima and Nagata, 1990), which contains the cDNA of the human B7.1 gene (Selvakumar et al., 1992). An MZ clone is selected. PC2 m7 # l B7.1 # 3 and is grown in T150 cell culture flasks. The nutrient medium used is RPMI 1640 (Gibco plus 4g / l of glucose) containing 10% heat-inactivated fetal bovine serum and 2 mmole of L-glutamine. The cells are cut every 3 or 4 days to propagate them by treating them with trypsin 1: 5. After approximately 80% confluence has been obtained, 4 ml of trypsin solution (containing per liter: 8 g of NaCl) is added., 0.2 g of KCl, 1.13 g of anhydrous Na2HP04, 0.2 g of KH2P04, 100 ml of 2.5% trypsin solution, 1 g of EDTA, sodium salt; pH 7.2-7.4) to each T150 cell culture flask to harvest the cells. In all, 2 x 107 cells were used to isolate the RNA in accordance with the manufacturer's instructions (Rneasy Minikit, QIAgen). Starting with approximately 100 μg of RNA 1 ^ -? R'rtMtbjttt.tu ^ - ^ t?. ^^. ^ »^ - - ^ ••, * ^ -. ^. - ... ^ A --- '" - "«' ^^ total the manufacturer's instructions are followed in order to isolate poly A + RNA using the Oligotex kit (QIAgen). After starting with approximately 0.5 mg of poly A + RNA, cDNA synthesis is carried out in accordance with the instructions of the manufacturer (Clontech Marathon Protokoll).

EXAMPLE 2 Representative difference analysis (RDA) of cell line 10 MZ.PC2 m7 # l B7.1 # 3 of pancreatic carcinoma against a combination of 11 normal tissues Starting from approximately 0.5 μg of poly-A (+) of the MZ cell line. PC2 m7 # l B7.1 # 3 tumor 15 pancreatic and a mixture of 2.5 μg of poly-A (+) RNA from 11 normal tissues (clontech) - bone marrow, heart, kidney, liver, lung, skeletal muscle pancreas, thymus, small intestine and stomach - the RDA is performed (Diatchenko et al., Hubank and Schatz,) using the PCR-20 select ™ kit (Clontech, Palo Alto) in accordance with the manufacturer's instructions: RNA from the pancreatic tumor cell line was used as the "tester" " | iiiTililliiiirlTilliili I I l and RNA from the combination of normal tissue as the "controller" in accordance with the manufacturer's instructions. In contrast to the original procedure, after the synthesis of AD? C using oligo-dT, the AD? C is cut with six restriction enzymes: EcoRV, Nael, NruI, Scal (Promega), SspI, Stul (TakaRa) in Promega regulatory solution A for two hours at 37 ° C and, after increasing the concentration of? aCl up to 150 mM, for another two hours at 37 ° C. The use of this mixture of six different restriction enzymes made it possible to generate AD? C fragments of approximately 800 bp in length, which were used for the representative difference analysis. Equal parts of the AD? C of the tester are ligated with any of the adapters A or B and then hybridized separately with an excess of AD? C from the controller at 68 ° C. Then the two mixtures are combined and subjected to a second hybridization with AD? C of denatured and new controller. Concentrated tester specific AD? C molecules are then amplified exponentially by PCR with the case-specific primers for adapter A or B with a stretch time of two minutes at 72 ° C, through 27 cycles (10 '' at 94 ° C, 30 '' at 66 ° C, 2 'at 72 ° C). For an additional concentration, an aliquot of Itá? & A? .1 .... 1 .1 t ..Mfaa ??? a »*« «j this reaction to a second PCR with specific nested primers from the kit with a stretch time of two minutes at 72 ° C, 10 cycles (10 '' at 94 ° C, 30 '' at 66 ° C, 2 'at 72 ° C). The product resulting from this reaction is ligated into three differently modified, individual pCR3.1 (+) vectors (Invitrogen): vector (l.ORF), vector (2. ORF) and vector (3. ORF) (Fig. 5: CMV cytomegalovirus; BGH is bovine growth hormone; ORF open reading frame) and then transformed into competent E. coli (OneShot ™, Invitrogen). These vectors allow expression in eukaryotic cells in three different reading frames. To construct the three vectors, the vector pCR3.1 (+) (Invitrogen) is cut with Nhel and HindIII (Promega) and ligated with an oligomer of dsDNA that is produced by fixing two ssDNA oligomers (SEQ ID N0: 4 and 5; vector ORFl) or (SEQ ID NO: 6 and 7; vector ORF2) or (SEQ ID NO: 8 and 9; vector 0RF3), using standard methods (for example Ausubel et al., 1994; Smbrook et al., 1989) . The 3 types of vector have a start codon and a cloning site for expression in a reading frame which is different from the other two vectors. The transformation of competent E. coli (OneShot ™ Invitrogen) carried out in the three batches (vector l.ORF, vector 2. ORF and 3-ORF with the cDNA of the subtractive cDNA library produced approximately 9,600 clones) These were examined by PCR analysis for the presence and length of cDNA The following method was used: the 9600 clones were cultured in blocks of 96 cavities in LB-Amp medium for 48 hours at 37 ° C. Then aliquots of 5 μl of the suspension of E. coli were heated to 100 ° C. in 500 μl of the TE buffer for 10 minutes and 1.5 μl thereof is used as the basis for a PCR in which the vector insert with flanking primers (SEQ ID NO: 10 and 11) is amplified through 35 cycles (1 'to 94 ° C, 1 'at 55 ° C, 2' at 72 ° C). The PCR products are revealed using agarose gel electrophoresis and staining with ethidium bromide. The remaining bacterial cultures are stored as reserve cultures in glycerol at -80 ° C. A cDNA subtraction library of 3450 individual clones is obtained in the form of glycerol reserve cultures from E. coli, whose insert length is known through agarose gel electrophoresis. As expected, the inserted cDNA fragments showed an average length of approximately 800 bp.

EXAMPLE 3 Determination of DNA sequence and annotation of clones of the cDNA library subtractive to the cell line MZ.PC2 m7 # l B7.1 # 3 pancreatic tumor DNA-plasmid from 50 clones randomly selected from the cDNA library subtractive in accordance with the manufacturer's instructions (Qiagen) and were determined the sequence using the Sanger method on an ABI-Prism apparatus was isolated. The sequences found in this way were annotated using BLAST-Search (national center for information on biotechnology) and subjected to comparisons with EST data banks. This made it possible to identify 38 known and 12 unknown genes. For the latter there were only EST entries. For the 12 unknown genes, the expression profile was estimated: the starting tissue for the corresponding cDNA library was verified for all ESTs in the data banks having more than 95% identity (BLAST) with the sequence determined in the form experimental. These were subdivided into i) critical normal tissue, ii) fetal, "non-essential" and immunoprivileged tissue and iii) tumors and tumor cell lines. Based on this "virtual mRNA profile", 4 clones (R2, R8, Rll and R12) were selected for further experimental analysis.

EXAMPLE 4 Analysis of transcription of the candidate clones in tumor tissue and in normal tissue They underwent reverse transcription between 2 and 5 μg of total RNA from tumor tissues or normal tissues, using SuperScriptII (GibcoBRL) or AMV-RT (Promega) in accordance with the manufacturer's recommendations. For each individual RNA probe a second test is carried out without reverse transcriptase as a control with respect to contamination by chromosomal DNA. The quality and quantity of cDNA molecules was confirmed by PCR with primers specific for beta-actin (SEQ ID NO: 14 and 15) and with specific primers for GAPDH (SEQ ID NO: 16 and 17) after 30 and 35 cycles (1 'at 95 ° C, 1' at 55 ° C, 1 'at 72 ° C). The four candidate genes were analogously analyzed with specific primers. The PCR products were detected by agarose gel electrophoresis and staining with ethidium bromide. A candidate which is "Rll" has a relatively specific transcription profile for testicular tumor, after 35 cycles with specific Rll primers (SEQ ID NO: 12 and 13); RT-PCR semicuantitiva RNA from breast carcinoma, lung adenocarcinoma, epithelial carcinoma plate lung, carcinoma of kidney, colon carcinoma, heart, lung, liver, kidney, colon, spleen and testis shown in Figure 1) . Another qualitative cDNA from tissue from three human patients with pancreatic tumors using the same Rll-specific primers (SEQ ID NO: 12 and 13) demonstrated expression in human pancreatic tumors (Figure 2). Furthermore, an additional qualitative PCR of cDNA from different tumor cell lines of human lung (LC6, 16) gallbladder (GB 1) and pancreatic tumors (PCI, 2) and two melanomas (Mel 2, 7) it was made the same Rll-specific primers (SEQ ID NO: 12 and 13), which showed clear expression in all tumor cell lines (Figure 4). In this analysis the Perkm Elmer method (GeneAmp RNA PCR kit, # N808-0017) was used (RT reaction: (lx) 15 '/ 42 ° C - 5' / 99 ° C - 5 '/ 4 ° C; of PCR: (35x) 2 '/ 95 ° C - l' / 95 ° C - l '/ 60 ° C and (lx) 7' / 72 ° C -4 ° C (figure 4) As described above, the ^ * ^ a.JM ** ^ A »a * JÉ ° ^ - ^^ - > -j ^ ~ "i -?" J'fe) Tt itr t- í "* J- J" i 1 -i i lÜHünaimiÉi PCR products were developed using agarose gel electrophoresis and ethidium bromide staining. A 1 kb marker prepared by Gibco BRL was used as the size marker.

EXAMPLE 5 Transcription profile of Rll in normal tissues For the Northern Blot analysis, multiple human tissue Northern Blots were hybridized (Clontech, Palo Alto and Invitrogen) for 2 hours at 68 ° with the product of almost a thousand base pairs long Rll labeled with [α- 32P] dCTP (NEN, Boston). The visualization is carried out by standard autoradiography (Hyperfilm, Amersham). Figure 3 shows the results of this analysis: of 19 normal tissues (pancreas, adrenal medulla, thyroid, adrenal cortex, testes, thymus, small intestine, stomach, brain, heart, skeletal muscle, colon, spleen, kidney, liver, placenta , lung, leukocyte). For Rll, there is a transcript of 7.5 kb long prominent in the placenta, adrenal medulla, adrenal cortex and in the testes. As well »* • a very weak band of 7.5 kb can be detected in the brain, because all these normal tissues have an immunoprivileged state (Streilein, 1995) can not be considered an attack by CTL in any immunotherapy based on this antigen Other transcripts of 3.8 kb and 2.3 kb were identified, which could possibly be splice variants of the 7.5 kb transcript or could be obtained from a homologous gene, in the adrenal medulla and in the adrenal cortex (figure 3).

EXAMPLE 6 Cloning of Rll cDNA The following procedure was used to clone the human Rll cDNA: a BLAST search identified a fragment AF038197 and a plurality of ESTs, such as for example N42343, W69539, H82474, H51766, N28313, which overlap with the "original sequence" of Rll (796 bp) obtained in example 3 by sequence analysis. Starting from the sequence AF038197, an overlapping contiguous with the clone Rll was found using the EstExtractor in TigemNet (http://gcg.tigem.it/cgi-bin/uniestass.pl). The overlap of the contiguous sequence and the "original Rll sequence of 796 base pairs long was confirmed by PCR amplification" an original sequence "of 5 Rll-specific primer and an initiator located in the contiguous overlap (SEQ ID NO. : 18 and 19) of a Superscript ™ human testis cDNA library (GibcoBRL) and subsequent sequence determination, by means of a PCR with a specific Rll primer (SEQ ID NO: 20) and a specific primer of vector (SEQ ID NO: 21), other fragments belonging to Rll from the SuperScript ™ human testis cDNA library were amplified using the Advantage cDNA PCR kit (Clontech) and the standard procedure 15 described therein is followed. The knowledge of these new sequences, in turn, makes it possible to carry out additional PCR reactions with Rll-specific primers (SEQ ID NO: 22 and 23) and the vector-specific primer (SEQ ID NO: 21) 20 To extend the Rll cDNA additionally, a panel from the human testis rapid selection cDNA library (OriGene Technologies, Inc.) is selected with specific primers for Rll (SEQ ID NO: 24). and 25) ? wiH btmW t BmWt ^ * í ^ & ^ * ~? i ^ ii? d * ^ it ** ?? *? i ^ ***. «" ^ Tlj.jBaJtt.j ». ^ T. < l__. ? i.í. ^ Ji ^ t? ^ UÜUJ ^^ t ?? a?,. ú? e aa? ^^^^? ^ l? í? .. ^ ¡^ a. ^ .A- í i1 '' | i__ | t_rir! JMÜJM under the standard PCR conditions specified by the manufacturer. From the positive cavities, an aliquot is amplified as a template for a PCR with a specific primer for Rll and a primer specific for the vector (SEQ ID NO: 26 and 27) using the Advantage cDNA PCR kit (Clontech) and following the standard procedure described therein. For sequence analysis, aliquots of the PCR preparations were ligated directly into the pCR2.1 vector (Invitrogen) and then transformed into competent E. coli (OneShot ™, Invitrogen) and their sequence determined as described in the example 3. Starting from these newly identified sequences, regions of 5 'sequence located upstream towards the 5' end from a human SuperScript ™ testis cDNA library could be cloned.

(GibcoBRL) with the following additional oligonucleotide primers specific for Rll (SEQ ID NO: 28 to 43). The primers were used with a specific primer for plasmid (SEQ ID NO: 21) described above in the present invention or combined with one another for PCR cloning using the Advantage cDNA PCR kit (Clontech).

The cloned region of the Rll cDNA is 6582 bp, whereas the presence of a poly A trace at the 3 'end of the sequence is an indication of the entire cDNA in this region. Two separate continuous reading frames were identified. The first reading frame at the 5 'end (Rll-ORF-1; SEQ ID NO: 2) is represented by the start codon at position 218 and by the stop codon (TAG) at position 1421 in SEQ ID N0: 1 Because there are no entries in the gene data banks known for this gene, no comments can be made regarding its function.

In profile analysis of the protein (http: // www. expasy. ch / prosite /) gave a reference for three possible N-glycosylation sites (position number 62-65, 76-79 and 117-120 in SEQ ID NO: SEQ ID NO: 2) , a cAMP-dependent protein phosphorylation site of cAMP and cGMP (position number 11-14 in SEQ ID NO.-2), as well as seven possible phosphorylation sites of PKC (position number 9-11, 14-16, 78-80, 119-121, 183-185, 202-204 and 210-212 in SEQ ID NO: 2) and six possible casein kinase II phosphorylation sites (position number 119-122, 127-130, 183-186, 256-259, 295-298 and 358-361 in SEQ ID NO: 2). The zinc finger motif (ZF-CCHC; E = 0, 11, Pfam-A HMM) from position number J? A &???? á? ?? AH-% ¡-t¡gtátiki. *%. • «-« * "- - ~ -A < fa« ... »> rf-« «.«. A »., ^ Mtu¿'-» ** - »- ~ t? ___ a ^ í ? fe1? fc? n_É »371 to position 384 in SEQ ID NO: 2 (CLYCGTGGHYADNC) should be of particular interest to predict a possible function of R11-0RF-1. It is known that members of the protein family that have these motifs have no insertion or deletion in the motif itself, this is also true for the Rll -ORF-1 protein. Although a typical SH3 binding sequence can not be found, it is conceivable that the P-rich region ( position number 36-56 in SEQ ID NO: 2) could interact with an SH3 domain Using the COILS algorithm, we can predict a coiled helical structure with more than 99% probability for the amino acid groups in the region of position number up to approximately 125. Based on these two domains, the zinc finger motif and the coiled helical domain, it can be concluded that Rll-ORF-1 is possibly a factor of transcription whose oligomerization is controlled by these two domains. In the second open reading frame, Rl-ORF-2, which is defined by a start codon at position number 1498 and by a stop codon (TAA) at position 2569, in addition to the two sections rich in proline evident (position number 128-141 and 330-351 in SEQ ID NO: 2), motifs i potentials for two N-glycosylation sites (104-107 and 251-254), one protein kinase C phosphorylation site (108-110), five casein kinase II phosphorylation sites (99-102, 165-168, 198-201, 200-203 and 274-277) and a region that resembles the active center of eukaryotic and viral aspartate proteinases (16-17). The clear homology of the first 280 amino acids of Rll-ORF-2 with the retroviral polyprotein is particularly evident. In contrast, at the C-terminal end, no homologies were discovered. The amino acids from position 9 through 277 are clearly aligned in blastp with the Fugu pol polyprotein (position number 104-365; 2e ~ 22). The aforementioned aspartate protease pattern number 16-27 comprises the nucleophile active aspartate (number 19) of the active center of the protease of the pol region; position number 215 to number 277 corresponding to the domain part of the reverse transcriptase. Therefore, the protein derived from Rll-ORF-2 is possibly a retrotransposon. lAAA I i iiBüim f JL? A, 1. i. Love - *** EXAMPLE 7 Potential MHC-binding peptides in the regions encoding the two Rll reading frames, R11-0RF-1 and R11-0RF-2 Potential peptide epitopes were performed within the two Rll reading frames in accordance with SEQ ID NO: 2 or 3 using the algorithms by Parker et al., 1994 on the basis of known motifs (Rammense et al., 1995) . 9-mer candidate peptides have been identified for most of the important types of HLA, especially for HLA-A1, -A * 0201, -A3, -B7, -B14 and -B * 4403, of which one can expect that bind to the corresponding HLA molecules and thus constitute the immunogenic CTL epitopes; the discovered peptides are listed in Table 1 (Rll-ORF-1) and Table 2 (Rll-ORF-2). By analogous methods, other potential peptide epitopes can be discovered for other types of HLA or for 8-meric or 10-mer peptides. ^ _iaaáaj .. ^ ___ a_a «. ££ .... ¿LLM A. jH» * ~ fc ~ > .. »?. t..l3teJÍ? & .. '-« att ,. J- ~ * < fc > ^ '. ^ ^ Ii ^ AJ Aww i .. * »• **' lM 'tt' 'to TABLE 1 Candidate R11-0RF immunogen peptide-1 (401 amino acids t jA = &t ^ -tá ^ fc TABLE 2 Candidates of immunogenic peptide of R11-0RF-2 (357 amino acids 10 15 twenty J A. a .A.1. i * ¿- -? JBt * ej * .... «Idfc.i ^ t.fc.á Literature Altschul, S.F., Madden, T.L., Scháffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. Gapped BLAST and PSI-BLAST: a new generation of protein datbase search programs. Nucleic Acids Res. 25, 3389-3402 (1997). Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Smith, JA, Seidman, JG, Struhl, K. (Eds), (1994), Vol. 1 + 2"Current Protocols in Molecular Biology", John Wiley & Sons. Inc. Bateman, A., Birney, E., Durbin, R., Eddy, S.R., Howe, K.L. and Sonnhammer, E.L. The Pfam Protein Families Datábase. Nucleic Acids Res. 28, 263-266 (2000). Becker, D., Kuhn, U., Lempertz, U., Enk, A., Saloga, J., and Knop, J. (1997), J. Immunol. Methods 203, 171-ldO. 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It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

LIST OF SEQUENCES < 110 > Boehringer Ingelheim International GmbH < 120 > Rll antigen associated with Tumor < 130 > 12211aa < 140 > < 141 > < 160 > 102 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 6582 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > 5'UTR < 222 > (1) .. (217) < 220 > < 221 > CDS < 222 > (218) .. (1423) < 220 > < 221 > uncertain < 222 > (1424) .. (1497) < 220 > < 221 > CDS < 222 > (1498) .. (2571) < 220 > < 221 > 3'ÜTR < 222 > (2572) .. (6582) < 400 > 1 acacgcgctt caacttcgg tggtgtgtgt cgaagaaacc tgactgcgcc ctgaggagaa 60 ggtccaccga cagcggagaa gcctggcgaa aggtccgctg agcgggctgt cgtccggagc 120 cactccgggc tgcggagcac ccagtggaga ccgcgcctgg ctcaggtgtg ggaccccatc 180 cttcctgtct tcgcagagga gtcctcgcgt ggtgagt atg cga aat aag cgg gtt 235 Met Arg Asn Lys Arg Val 1 May ttg aaa here aaa aaa aga agg agt gga aga ggg ggc cag gat cea ggc 283 Leu Lys Tnr Lys Lys Arg Arg Ser Gly Arg Gly Gly Gln Asp Pro Gly 10 15 20 ctc cat ccc cac aga agt gaa gct here ggg agg tet cct ccc acc 331 Leu His Pro His Arg Ser Glu Aia Thr Wing Gly Arg Ser Pro Pro Thr 25 30 35 cea acc gtc acc ctg ggt ccc gac tgc cea cct cct cct cct ccc cct 379 Pro Thr Val Thr Leu Gly Pro Asp Cys Pro Pro Pro Pro Pro Pro Pro 40 45 50 ccc ccc aac aac aac aac aac aac c aac c aac c ac g gc cat aag 427 Pro Pro Asn Asn Asn Asn Asn Asn Asn Ser Lys His Thr Gly His Lys 55 60 65 70 agt gcg tgt gtc ccc aac atg acc gaa cga aga agg gac gac ctc tet 475 Ser Ala Cys Val Pro Asn Met Thr Glu Arg Arg Arg Asp Glu Leu Ser 75 80 85 gaa gag atc aac aac tta aga gag aag gtc atg aag cag teg gag gag 523 Glu Glu lie Asn Asn Leu Arg Glu Lys Val Met Lys Gln Ser Glu Glu 90 95 100 aac aac aac ctg cag age cag gtg cag aag ctc here gag gag aac acc 571 Asn Asn Asn Leu Gln Ser Gln Val Gln Lys Leu Thr Glu Glu Asn Thr 105 110 115 acc ett cga gag caa gtg gaa ccc acc cct gag gat gag gat gat gac 619 Thr Leu Arg Glu Gln Val Glu Pro Thr Pro Glu Asp Glu Asp Asp Asp 120 125 130 atc gag ctc cgc ggt gct gca gca gct gct gcc cea ccc cct cea ata 667 He Glu Leu Arg Gly Ala Ala Ala Ala Ala Ala Ala Pro Pro Pro Pro H e 135 140 145 150 gag gaa gag tgc cea gaa gac ctc cea gag aag ttc gat ggc aac cea 715 Glu Glu Glu Cys Pro Glu Asp Leu Pro Glu Lys Phe Asp Gly Asn Pro 155 160 165 gac atg ctg gct cct ttc atg gcc cag tgc cag atc ttc atg gaa aag 763 Asp Met Leu Wing Pro Phe Met Wing Gln Cys Gln He Phe Met Glu Lys 170 175 180 age acc agg gat ttc tea gtt gat cgt gtc cgt gtc tgc ttc gtg here 811 Ser Thr Arg Asp Phe Ser Val Asp Arg Val Arg Val Cys Phe Val Thr 185 190 195 age atg atg acc ggc cgt gct gcc cgt tgg gcc tea gca aag ctg gag 859 Met Met Met Thr Gly Arg Ala Ala Arg Trp Ala Ser Ala Lys Leu Glu 200 205 210 cgc tcc cac tac ctg atg cac aac tac cea gct ttc atg atg gaa atg 907 Arg Ser His Tyr Leu Met Kis Asn Tyr Pro Wing Phe Met Met Glu Met 215 220 225 230 aag cat gtc ttt gaa gac cct cag agg cga gag gtt gcc aaa cgc aag 955 Lys His Val Phe Glu Asp Pro Gln Arg Arg Glu Val Wing Lys Arg Lys 235 240 245 atc aga cgc ctg cgc ca ggc atg ggg tet gtc atc gac tac tcc aat 1003 He Arg Arg Leu Arg Gln Gly Met Gly Ser Val He Asp Tyr Ser Asn 250 255 260 gct ttc cag atg att gcc cag gac ctg gat tgg aac gag cct gcg ctg 1051 Wing Phe Gln Met Wing Gln Asp Leu Asp Trp Asn Glu Pro Wing Leu 265 270 275 t ^ ^. «Jü.i? .. loves ,? - - ^ jter, ,, l f ,, | .. ^ _ fc * .s *. ^ ._ t, ¿feA ^^ tfc4¡ < , .Aj 9 att gac cag tac cac gag ggc ctc age gac cac att cag gag gag ctc 1099 He Asp Gln Tyr His Glu Gly Leu Ser Asp His He Gln Glu Glu Leu 280 285 290 tcc cac ctc gag gtc gcc aag teg ctg tet gct ctg att ggg cag tgc 1147 Ser His Leu Glu Val Ala Lys Ser Leu Ser Ala Leu He Gly Gln Cys 295 300 305 310 att cac att gag aga agg ctg gcc agg gct gct gca gct cgc aag cea 1195 He His He Glu Arg Arg Leu Wing Arg Wing Wing Wing Wing Arg Lys Pro 315 320"325 cgc teg cea ccc cgg gcg ctg gtg ttg cct cac att gca age cac cac 1243 Arg Ser Pro Pro Arg Ala Leu Val Leu Pro His He Wing Ser His His 330 335 340 cag gta gat cea acc gag gag gtg gga ggt gcc cgc atg cgc ctg acg 1291 Gln Val Asp Pro Thr Glu Pro Val Gly Gly Ala Arg Met Arg Leu Thr 345 350 355 cag gaa gaa aaa gaa aga cgc aga aag ctg aac ctg aac ctc tac tc t 1339 Gln Glu Glu Lys Glu Arg Arg Arg Lys Leu Asn Leu Cys Leu Tyr Cys 360 365 370 gga here gga ggt cac tac gct gac aat tgt cct gcc aag gcc tea aag 1387 Gly Thr Gly Gly His Tyr Wing Asp Asn Cys Pro Wing Lys Wing Ser Lys 375 380 385 390 tet teg ceg gcg gga aac tcc ceg gcc ceg ctg tag agggaccttc 1433 Ser Ser Pro Ala Gly Asn Ser Pro Ala Pro Leu 395 400 agegaceggg ccagaaataa taaggtcccc acaagatgat gcctcatctc cacacttgca 1493 agtg atg ctc cag att cat ett ceg ggc aga cac acc ctg ttc gtc cga 1542 Me t Leu Gln He His Leu Pro Gly Arg His Thr Leu Phe Val Arg 405 410 415 gcc atg atc gat tet ggt gct tet ggc aac ttc att gat cac gaa tat 1590 Wing Met He Asp Ser Gly Wing Ser Gly Asn Phe He Asp His Glu Tyr 420 425 430 gtt gct caat aat gga att cct cta aga atc aag gac tgg cea ata ett 1638 Val Ala Gln Asn Gly He Pro Leu Arg He Lys Asp Trp Pro He Leu 435 440 445 gtg gaa gca att gat ggg cgc ccc ata gca teg ggc cea gtt gtc cac 1686 Val Glu Ala He Asp Gly Arg Pro He Wing Ser Gly Pro Val Val His 450 455 460 465 gaa act cac gac ctg ata gtt gac ctg gga gat cac cga gag gtg ctg 1734 Glu Thr His Asp Leu He Val Asp Leu Gly Asp His Arg Glu Val Leu 470 475 480 tea ttt gat gtg act cag tet cea ttc ttc cct gtc gtc cta ggg gtt 1782 Ser Pne Asp Val Thr Gln Ser Pro Phe Phe Pro Val Val Leu Gly Val 485 490 495 cgc tgg ctg age here cat gat ccc aat atc here tgg age act cga tet 1830 Arg Trp Leu Ser Thr His Asp Pro Asn He Thr Trp Ser Thr Arg Ser 500 505 510 atc gtc ttt gat tet gaa tac TGC CGC tac cac TGC CGG atg tat tet 1878 I Val Phe Asp Ser Glu Tyr Cys Arg Tyr His Cys Arg Met Tyr Ser 515,520,525 cea ata cea cea TEG ctc cea cea cea cea GCA caá ceg cea ctc tat 1926 Pro He Pro Pro Pro Pro Le Pro Pro Pro Pro Pro Pro Leu Tyr 530 535 540 545 tat cea gta gat gga tac aga gtt tac caa cea gtg agg tat tac tat 1974 Tyr Pro Val Asp Gly Tyr Arg Val Tyr Gln Pro Val Arg Tyr Tyr Tyr 550 555 560 gtc cag aat gtg tac act cea gta gat gag cac gtc tac cea gat cac 2022 Val Gln Asn Val Tyr Thr Pro Val Asp Glu His Val Tyr Pro Asp His 565 570 575 cgc ctg gtt gac cct cac ata gaa atg ata cct gga gca cac agt att 2070 Arg Leu Val Asp Pro His He Glu Met He Pro Gly Wing His Ser He 580 585 590 ccc agt gga cat gtg tat tea ctg tcc gaa cct gaa atg gca gct ett 2118 Pro Ser Gly His Val Tyr Ser Leu Ser Glu Pro Glu Met Ala Ala Leu 595 600 605 cga gat ttt gtg gca aga aat gta aaa gat ggg cta att act cea acg 2166 Arg Asp Phe Val Wing Arg Asn Val Lys Asp G ly Leu He Thr Pro Thr 610 615 620 625 att gca cct aat gga gcc caa gtt ctc cag gtg aag agg ggg tgg aaa 2214 He Ala Pro Asn Gly Ala Gln Val Leu Gln Val Lys Arg Gly Trp Lys 630 635 640 ctg caa gtt tet tat gat TGC CGA GCT cea aac aat ttt act atc cag 2262 Leu Gln Val Ser Tyr Asp Cys Arg Ala Pro Asn Asn Phe Thr I Gln 645 650 655 aat cag tat cct CGC cta tet att cea AAT TTA GAA GAC GCA caá cac 2310 Asn Gln Tyr Pro Arg Leu Ser He Pro Asn Leu Glu Asp Gln Ala His 660 665 670 ctg gca acg tac act gaa ttc cct gta caa ata cct gga tac caa here 2358 Leu Ala Thr Tyr Thr Glu Phe Val Pro Gln He Pro Gly Tyr Gln Thr 675 680 685 tac ccc here tat gcc GCG tac ceg acc tac gta cea gga ttc gcc TGG 2406 Tyr Pro Thr Tyr Ala Ala Tyr Pro Thr Tyr Pro Val Gly Phe Ala Trp 690695700705 tac cea GTG CGA GGA GGA GAC caá gga tea cta tat aga gta cct gtg 2454 Tyr Pro Val Gly Arg Asp Gly Gln Gly Arg Ser Leu Tyr Val Pro Val 710 715 720 atg atc act tgg aat cea cac tgg tac cgc cag cct ceg gta cea cag 2502 Met He Thr Trp Asn Pro His Trp Tyr Arg Gln Pro Pro Val Pro Gln 725 730 • 735 tac ceg ceg cea cag ceg cc ce ce ce ce ce ce ce ce ce cec ceg cec cct 2550 Tyr Pro Pro Pro Pro Gln Pro Pro Pro Pro Pro Pro Pro pro Pro 740 745 750 cea tet tac CTG AGT ACC taa atacctgtca tgtccttcag gatctctgcc 2601 Pro Ser Leu Tyr Ser Th.r 755,760 ctcaaaattt attcctgttc agcttctcaa tcagtgactg tgtgctaaat tttaggctac 2661 ggccacctga tgtatcttca ggcacatcct ctctgaaacg gctatggaag gttagggeca 2721 ctctggactg gcacacatcc taaagcacca aaagacette gagageaaca aacattttct 2781 caataaatga gagtatttgc tetetcattt ttccaccttg actgccaatc taactaaaat 2841 aattaataag tttactttcc agccagtcct ggaagtctgg gttttacctg ccaaaacctc 2901. catcaccatc taaattatag gctgccaaat ttgctgttta acatttacag agaagctgat 2961 gaaatgctga acaaacgcag gagggggaga tttctttatg gaggacatga cgaggaggag 3021 cttttcttgc ggtttcggta ccctcttttt aaatcactgg aggactgagg cettattaag 3081 gaagccaaaa ttatcggtgc agtgtggaaa ggcttccgtg atcctctcgc tgcaccctta 3141 gaaacttcac cgtcttcaaa ctccatttcc atggttctgt taattetcaa ggagcagcaa 3201 tctcccagga ctcgactggt gcaggaaaaa cccttgtgac atgaaacatc tcaggcctga 3261 aaagaaagtg ctctctcaga tggactcttg catgttaaga ctatgtcttc acatcatggt 3321 10 gcaaatcaca tgtacccaat gactccggct ttgacacaac accttaccat catcatgcca 3381 tgatggcttc cacaaagcat taaacctggt aaccagagat tactggtggc tccagcgttg 3441 ttagatgttc atgaaatgtg accacctctc aatcaccttt gagggctaaa gagtageaca 3501 tcaaaaggac tccaaaatcc catacccaac tettaagaga tttgtcctgg tacttcagaa 3561 agaattttca tgagtgttct taattggctg gaaaagcacc agctgacgtt ttggaagaat 3621 etatecatgt gtctscctcc atatgeatet gggcatttca tcttcagtcc cetcattaga 3681 ctgtagcatt aggatgtgtg gagagaggag aaatgattta gcacccagat tcacactcct 3741 15 atgcctggaa gggggacatc tttgaagaag aggaattagg gctgtggaca ctgtcttgag 3801 gatgtggact tccttagtga gctccacatt acttgatggt aaccacttca aaaggatcag 3861 aatccacgta atgaaaaagg tccctctaga ggatggagct gatgtgaagc tgccaatgga 3921 tgaaaagcct cagaaagcaa ctcaaaggac tcaaagcaac ggacaacaca agagttgtct 3981 tcagcccagt gacacctctg atgtcccctg gaagctttgt gctaacctgg gactgcctga 4041 cttcctttag cctggtccct tgctactacc ttgaactgtt ttatetaace tctctttttc 4101 tgtttaattc tttgctactg ccattgaccc tgctgcagga tttgtgtcat tttcctgcct 4161 20 ggttgctgag actccatttt gctgccacac acagagatgt aagaggcagg ctttaattgc 4221 caaagcacag tttgagcagt agaaaacaac atggtgtata tctcaaattg cctgacatga 4281 ili I l ¥ l MílllffiÉíiÉÉ 1?. ** *?. *? * l **. * Mi? .. > , *, - .. agaggagtet aaeggtgaag tttcactttt catcagcatc atettteaca tgtteattat 4341 catccgctct tattcttgca tgtttaaaca cttaaaattt ttagtataat ttttagtgtg 4401 ttttgaagtg gtgactaggc tttcaaaaac ttccattgaa ttacaaagea etatecagtt 4461 aactaagtaa cttattgtta aaatgataag taacatagtg taaaatattc ctttactgtg 4521 aaettettac aatgctgtga atgagaggct cctcagaact ggagcatttg tataataatt 4581 catcctgttc atettcaatt ttaacatcat atataattte aattetatca attgggccCC 4641 taaaaatcat ataaaaggat ataaaatttg aaaagagaaa cctaattggc tatttaatec 4701 aaaacaactt ttttttttcc ttcaatggaa teagaaaget tgtcaatcac tcatgtgttt 4761 tagagtaatt acttttaaaa tggtgcattt gtgcttctga actattttga agagtcactt 4821 caagtatcaa ctgtttacct atacatttga ttcatcctcc ttttttgtca attcaagttg 4881 aatttacagt tgtcaattga tettcaaget gcagggtgcc tagaaatggg ccgttgtctg 4941 tagccctggc atgtgcacac ggacatttgc caccactgca agcaaaagtc tggagaagtt 5001 caccaacgac aagaaegatt agggaaaata tgctgctgtg ggttaacaac tcagaaagtc 5061 cctgatccac atttggctgt ttactaaagc ttgtgattaa ctttttggca gtgtgtact to 5121 10 tgctctattg etatatatge tatetataaa tgtagatgtt aaggataagt aattctaaat 5181 ttattattct atagttttga agtttggtta agtttccttt caetcaattg atttattttg 5241 ttgttaatca aatttatgtt aattggatcc tttaaatttt ttttggcatt ttccaacaaa 5301 ttcataagaa aatggcttta aggaaaaaaa tcaatggaat ttgatatcta aagaagttag 5361 aaataaaaaa aaagggagca cataaaggag atagatgaat tagtaagcaa atcagtagtc 5421 aactggcaaa gagtttttca attaattaat tgacttttag cccaaattta cattgttaat 5481 aggaagaaga taaatcaaga tetaagaget cccattgata gagagaacta ggcaagccta 5541 - [- gctaaattta teatgetagg atattgaaac acagaaagtt tacatacatt tatgaagggt 5601 tggacagtga caatttagtt ggtatttgtc ttagtggaaa aaaggagaat tagtctgatc 5661 aaatcgtgaa gtaatacagt gaacttgcag gtgcacaaaa taagagggcc acatetatat 5721 ggtgcagtct ggaattctgt ttaagtttgt aggtacctct tggacttctg aattgatcca 5781 accacagaca gttgtcatcc tctcacatca gatacagaca tgacaacaga gttccaagat 5841 gaacaacctg ctggaaagac ctgggcagaa atggagagcc ctgcgggaac catgetacat 5901 tttcatctaa agagagaatg cacatctgat gagactgaaa gttctttgtt gttttagatt 5961 0 gtagaatggt attgaattgg tctgtggaaa attgcattgc ttttatttct ttgtgtaatc 6021 aagtttaagt aataggggat atataatcat aagcatttta gggtgggagg gactattaag 6081 taattttaag tgggtggggt tatttagaat gttagaataa tattatgtat tagatatege 6141 tataagtgga catgcgtact tacttgtaac cctttaccct teettaaaga ataattgeta 6201 tttcaaataa actcggaggg aactgcaggg agaccaactt atttagagcg aattggacat 6261 cccagtggga ggataaaaac gaaagttcaa aggtgattag attaataatt taatagagga 6321 tgagtgacct ctgataaatt actgctagaa tgaacttgtc aatgatggat ggtaaatttt 6381 catggaagtt ataaaagtga taaataaaaa cccttgcttt tacccctgtc agtagccctc 6441 ctcctaccac tgaaccccat tgcccctacc cctccttcta actttattgc tgtattctct 6501 tttctctcta tcactctata tttgctaata ttgcattgct gttacaataa aaattcaata 6561 aagatttagt ggttaagtgc t 6582 < 210 > 2 < 211 > 401 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Arg Asn Lys Arg Val Leu Lys Thr Lys Lys Arg Arg Ser Gly Arg 1 5 10 15 Gly Gly Gln Asp Pro Gly Leu His Pro His Arg Ser Glu Wing Thr Wing 20 25 30 Gly Arg Ser Pro Pro Thr Pro Thr Val Thr Leu Gly Pro Asp Cys Pro 35 40 45 Pro Pro Pro Pro Pro Pro As Asn Asn Asn Asn Asn Asn Ser 50 55 60 Lys Kis Thr Gly His Lys Ser Wing Cys Val Pro Asn Met Thr Glu Arg 65 70 75 80 Arg Arg Asp Glu Leu Ser Glu Glu He Asn Asn Leu Arg Glu Lys Val 85 90 95 Met Lys Gln Ser Glu Glu Asn Asn Asn Leu Gln Ser Gln Val Gln Lys 100 105 110 Leu Thr Glu Glu Asn Thr Thr Leu Arg Glu Gln Val Glu Pro Thr Pro 115 120 125 Glu Asp Glu Asp Asp Asp He Glu Leu Arg Gly Wing Wing Wing Wing 130 135 140 Wing Pro Pro Pro Pro He Glu Glu Glu Cys Pro Glu Asp Leu Pro Glu 145 150 155 160 Lys Phe Asp Gly Asn Pro Asp Met Leu Ala Pro Phe Met Ala Gln Cys 165 170 175 Gln He Phe Met Glu Lys Ser Tnr Arg Asp Phe Ser Val Asp Arg Val 180 185 190 Axg Val Cys Phe Val Thr Ser Met Met Thr Gly Arg Ala Ala Arg Trp 195. 200 205 Wing Wing Wing Lys Leu Glu Arg Ser His Tyr Leu Met His Asn Tyr Pro 210 215 220 Wing Phe Met Met Glu Met Lys His Val Phe Glu Asp Pro Gln Arg Arg 225 230 235 240 Glu Val- Ala Lys Arg Lys He Arg Arg Leu Arg Gln Gly Met Gly Ser 245 250 255 Val He Asp Tyr Ser Asn Wing Phe Gln Met Wing Gln Asp Leu Asp 260 265 270 Trp Asn Glu Pro Wing Leu He Asp Gln Tyr His Glu Gly Leu Ser Asp 275 280 285 His He Gln Glu Glu Leu Ser His Leu Glu Val Wing Lys Ser Leu Ser 290 295 300 Wing Leu He Gly Gln Cys He His Glu Arg Arg Leu Wing Arg Wing 305 310 315 320 Wing Wing Arg Lys Pro Arg Ser Pro Pro Arg Wing Leu Val Leu Pro 325 330 335 - His He Wing His His Gln Val Asp Pro Thr Glu Pro Val Gly Gly 340 345 350 Wing Arg Met Arg Leu Thr Gln Glu Glu Lys Glu Arg Arg Arg Lys Leu 355 360 365 Asn Leu Cys Leu Tyr Cys Gly Thr Gly Gly His Tyr Wing Asp Asn Cys 370 375 380 Pro Wing Lys Wing Ser Lys Ser Ser Pro Wing Gly Asn Ser Pro Wing Pro 385 390 395 400 Leu < 210 > 3 < 211 > 357 < 212 > PRT < 213 > Homo sapiens < 400 > 3 Met Leu Gln He His Leu Pro Gly Arg His Thr Leu Phe Val Arg Ala 1 5 10 15 Met He Asp Ser Gly Ala Ser Gly Asn Phe He Asp His Glu Tyr Val 20 25 30 Wing Gln Asn Gly He Pro Leu Arg He Lys Asp Trp Pro He Leu Val 35 40 45 Glu Wing He Asp Gly Arg Pro He Wing Ser Gly Pro Val Val His Glu 50 55 60 Thr His Asp Leu He Val Asp Leu Gly Asp His Arg Glu. Val Leu Ser 65 70 75 80 Phe Asp Val Thr Gln Ser Pro Phe Phe Pro Val Val Leu Gly Val Arg 85 90 95 Trp Leu Ser Thr His Asp Pro Asn He Thi Trp Ser Thr Arg Ser He 100 105 110 Val Phe Asp Ser Glu Tyr Cys Arg Tyr His Cys Arg Met Tyr Ser Pro 115 120 125 Pro Pro Pro Le Pro Pro Pro Pro Pro Pro Pro Leu Tyr 130 135 140 Val Pro Asp Gly Tyr Arg Val Tyr Gln Pro Val Arg Tyr Tyr Val Tyr 150 155 160 Gln Asn Val Tyr Thr Pro Val Asp Glu His Val Tyr Pro Asp His Arg 165 170 175 Leu Val Asp Pro His He Glu Met He Pro Gly Ala His Ser He Pro 180 185 190 Ser Gly His Val Tyr Ser Leu Ser Glu Pro Glu Met Ala Ala Ala Leu Arg 195 200 205 ASD Phe Val Ala Arg Asn Val Lys Asp Gly Leu He Thr Pro Thr He 210 215 220 Wing Pro Asn Gly Wing Gln Val Leu Gln Val Lys Arg Gly Trp Lys Leu 225 230 235 240 Gln Val Ser Tyr Asp Cys Arg Ala Pro Asn Asn Phe Thr He Gln Asn 245 250 255 Gln Tyr Pro Arg Leu Ser He Pro Asn Leu Glu Asp Gln Wing His Leu 260 265 270 Wing Thr Tyr Thr Glu Phe Val Pro Gln He Pro Gl and Tyr Gln Thr Tyr 275 280 285 Pro Thr Tyr Wing Wing Tyr Pro Thr Tyr Pro Val Gly Phe Wing Trp Tyr 290 295 300 Pro Val Gly Arg Asp Gly Gln Gly Arg Ser Leu Tyr Val Pro Val Met 305 310 315 320 He Thr Trp Asn Pro His Trp Tyr Arg Gln Pro Pro Val Pro Gln Tyr 325 330 335 Pro Pro Pro Gln Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 340 345 350 Ser Tyr Ser Thr Leu 355 * lAíkt & A * t? A¿ktAAt! ká ¿, ___ já_to_ »¿., __ ,. Já.fci > ? - < 210 > 4 < 211 > 31 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 4 ctagcccacc atggcatctg cagccacgtg at 31 < 210 > 5 < 211 > 30 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 5 agcttcacgt ggctgcagat gccatggtgg 30 < 210 > 6 < 211 > 30 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 6 ctagcccacc atggcatctg cagcacgtga 30 < 210 > 7 < 211 > 29 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 7 agcttcacgt ggtgcagatg ccatggtgg 29 < 210 > 8 < 211 > 29 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator _ .. fc. ^ _ ia_a¡ ____ Í ___ aÍ__a¡l_ __, aiMMÍ S lE firr < 400 > 8 ctagcccacc atggcatctg cacacgtga 29 < 210 > 9 < 211 > 28 < 212 > DNA < 213 > synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 9 agcttcacgt gtgcagatgc catggtgg 28 < 210 > 10 < 211 > 23 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 10 gggcggtagg cgtgtacggt ggg 23 < 210 > 11 < 211 > 26 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 11 gcaactagaa ggcacagtcg aggctg 26 < 210 > 12 < 211 > 27 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 12 gtttggacag tgaggtattt gtcttag 27 < 210 > 13 < 211 > 27 < 212 > DNA < 213 > Synthetic sequence fciá ? i iíí ^ ^, ¿I iButi ri i fi i j ^ 8"" ^ < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 13 ctttccagca ggttgttctc tgttgtc 27 < 210 > 14 < 211 > 30 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of Synthetic Sequence: Synthetic Initiator < 400 > 14 tgacggggtc acccacactg tgcccatcta 30 < 210 > 15 < 211 > 29 < 212 > DNA < 213 > synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 15 ctagaagcat tgcggtggac gatggaggg 29 < 210 > 16 < 211 > 22 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 16 aaggtgaagg tcggagtcaa cg 22 < 210 > 17 < 211 > 24 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 17 ggcagagatg atgacccttt tggc 24 < 210 > 18 < 211 > 26 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 18 tattttgctc cctttctaac ttcttt 26 < 210 > 19 < 211 > 30 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 19 tttcactttt catcagcatc atettteaca 30 < 210 > 20 < 211 > 27 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 20 cgttagactc etetteatgt caggcaa 27 < 210 > 21 < 211 > 22 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 21 ggtgacacta tagaaggtac ge 22 < 210 > 22 < 211 > 28 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 22 caggcctgag atgtttcatg tcacaagg 28 < 210 > 23 < 211 > 29 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 23 gcatttcctg cgtttgtatc agcttctct 29 < 210 > 24 < 211 > 30 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 24] _Q accagcacca caaccgccac tctattatcc 30 < 210 > 25 < 211 > 30 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 25 catatagtga tcttccttgt ccgtctcgtc 30 5 < 210 > 26 < 211 > 26 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 26 gcgcccatca attgcttcca caagta 26 0 < 210 > 27 < 211 > twenty-one * iA »My? mWbH? MiJkS * ~ fUt ± * * íz-jucy *. *? Hn. «AaM tSÉftiflHÍ? L? F?» < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 27 gcagagctcg tttagtgaac c 21 < 210 > 28 < 211 > 30 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 28 ggccagaaat aataaggtcc ccacaagatg 30 < 210 > 29 < 211 > 29 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 29 agctttctgc gtctttcttt ttcttcctg 29 < 210 > 30 < 211 > 26 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 30 aggtcgccaa gtcgctgtct gctctg 26 < 210 > 31 < 211 > 37 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 31 tgggtagttg tgcatcaggt agtgggagcg ctccagc 37 < 210 > 32 < 211 > 27 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 32 ctcgaagggt ggtgttctcc tctgtga 27 < 210 > 33 < 211 > 22 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 33 gagctcgtcc cttcttcgtt cg 22 < 210 > 34 < 211 > 40 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 34 cataagagtg cgtgtgtccc caacatgacc gaacgaagaa 40 < 210 > 35 < 211 > 47 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 35 tcgtcccttc ttcgttcggt catgttgggg acacacgcac tcttatg 47 < 210 > 36 < 211 > 40 < 212 > DNA < 213 > Synthetic sequence ^^^^^ g ifa? A t »< 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 36 ttcttcgttc ggtcatgttg gggacacacg cactcttatg 40 < 210 > 37 < 211 > 28 < 212 > AD < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 37 cagggtgacg gttggggtgg gaggagac 28 < 210 > 38 < 211 > 29 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 38 gcttcacttc tgtggggatg gaggcctgg 29 < 210 > 39 < 211 > 22 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 39 atgcgaaata agcgggtttt ga 22 < 210 > 40 < 211 > 29 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 40 cgcagaggag tcctcgcgtg gtgagtatg 29 . -? í &uAi,? ia. ... ^ ia? í? u ^ .. U í ...... _a. ^ 4 < 210 > 41 < 211 > 29 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 41 ggctcaggtg tgggacccca tccttcctg 29 < 210 > 42 < 211 > 29 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator- < 400 > 42 gctccggacg acagcccgct cagcggacc 29 < 210 > 43 < 211 > 24 < 212 > DNA < 213 > Synthetic sequence < 220 > < 223 > Description of the synthetic sequence: Initiator < 400 > 43 gaagaaacct gactgcgccc tgag 24 < 210 > 44 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 44 Met Leu Gln He His Leu Pro Gly Arg 1 5 < 210 > 45 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 45 He His Leu Pro Gly Arg His Thr Leu 1 5 < 210 > 46 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 46 His Leu Pro Gly Arg His Thr Leu Phe 1 5 < 210 > 47 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 47 Tyr Val Ala Gln Asn Gly He Pro Leu 1 5 < 210 > 48 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 48 Tyr Pro Arg Leu Ser He Pro Asn Leu 1 5 < 210 > 49 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 49 He Leu Val Glu Ala He Asp Gly Arg 1 5 < 210 > 50 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 50 Gly Arg Pro He Wing Ser Gly Pro Val 1 5 < 210 > 51 < 211 > 9 < 212 > PRT < 213 > Homo saDiens < 400 > 51 Glu Thr His Asp Leu He Val Asp Leu lii il'f 'iiliiitiíllí íkj ^ afa < 210 > 52 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 52 Asp Leu Gly Asp His Arg Glu Val Leu 1 5 < 210 > 53 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 53 Gly Asp His Arg Glu Val Leu Ser Phe 1 5 < 210 > 54 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 54 Gln Ser Pro Phe Phe Pro Val Val Leu 1 5 < 210 > 55 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 55 Val Leu Gly Pro Arg TrD Leu Ser Wing 1 5 < 210 > 56 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 56 Trp Leu Ser Ala His Asp Pro Asn He 1 5 < 210 > 57 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 57 Arg Ser He Val Phe Asp Ser Glu Tyr 1 5 < 210 > 58 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 58 He Val Phe Asp Ser Glu Tyr Cys Arg 1 5 < 210 > 59 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 59 Pro Pro Pro Wing Pro Gln Pro Pro Leu 1 5 < 210 > 60 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 60 Pro Leu Tyr Tyr Pro Val Asp Gly Tyr 1 5 < 210 > 61 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 61 Arg Val Tyr Gln Pro Val Arg Tyr Tyi 1 5 < 210 > 62 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 62 Tyr Gln Pro Val Arg Tyr Tyr Tyr Val 1 5 < 210 > 63 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 63 Val Arg Tyr Tyr Tyr Val Gln Asn Val 1 5 < 210 > 64 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 64 Tyr Val Gln Asn Val Tyr Thr Pro Val 1 5 < 210 > 65 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 65 Glu His Val Tyr Pro Asp His Arg Leu 1 5 < 210 > 66 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 66 Leu Val Asp Pro His He Glu Met He He 1 5 < 210 > 67 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 67 Glu Met He Pro Gly Ala His Ser He 1 5 < 210 > 68 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 68 His Ser He Pro Ser Gly His Val Tyr 1 5 < 210 > 69 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 69 He Pro Ser Gly His Val Tyr Ser Leu 1 5 < 210 > 70 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 70 Ser Leu Ser Glu Pro Glu Met Ala Ala 1 5 < 210 > 71 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 71 Leu Ser Glu Pro Glu Met Ala Ala Leu 1 5 < 210 > 72 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 72 Pro Glu Met Ala Ala Leu Arg Asp Phe 1 5 < 210 > 73 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 73 Glu Met Ala Ala Ala Leu Arg Asp Phe Val 1 5 < 210 > 74 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 74 Ala Leu Arg Asp Phe Val Ala Arg Asn -, & & &. * »** F - * -" i ímiíi i .Í .Í .... M < 210 > 75 < 211 > 9 < 212 > PRT < 213> Homo sapiens <400> 75 Val Ala Arg Asn Val Lys ASD Gly Leu 1 5 < 210 > 76 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 76 Thr He Ala Pro Asn Gly Ala Gln Val 1 5 < 210 > 77 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 77 He Ala Pro Asn Gly Ala Gln Val Leu 1 5 < 210 > 78 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 78 Val Leu Gln Val Lys Arg Gly Trp Lys 1 5 < 210 > 79 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 79 Leu Gln Val Lys Arg Gly Trp Lys Leu 1 5 < 210 > 80 < 211 > 9 < 212 > PRT < 213 > Homo sapiens 1 .. i? Í, í..í, Í. -l < 400 > 80 Tyr Pro Arg Leu Ser He Pro Asn Leu 1 5 < 210 > 81 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 81 Glu Asp Gln Ala His Leu Ala Thr Tyr 1 5 < 210 > 82 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 82 His Leu Wing Thr Tyr Thr Glu Phe Val 1 5 < 210 > 83 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 83 Gly Arg Asp Gly Gln Gly Arg Ser Leu 1 5 < 210 > 84 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 84 Arg Asp Gly Gln Gly Arg Ser Leu Tyr < 210 > 85 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 85 Asp Gly Gln Gly Arg Ser Leu Tyr Val 1 5 < 210 > 86 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 86 Ser Leu Tyr Val Pro Val Met He Thr 1 5 < 210 > 87 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 87 He Thr Trp Asn Pro His Trp Tyr Arg 1 5 < 210 > 88 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 88 Ser Pro Pro Thr Pro Thr Val Thr Leu 1 5 < 210 > 89 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 89 Leu Ser Glu Glu He Asn Asn Leu Arg 1 5 < 210 > 90 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 90 Lys Leu Thr Glu Glu Asn Thr Thr Leu 1 5 < 210 > 91 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 91 Leu Thr Glu Glu Asn Thr Thr Leu Arg 1 5 ^ s ^? ^? Ésik ^ iM ^^ & < 210 > 92 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 92 He Glu Leu Arg Gly Ala Ala Ala Ala 1 5 < 210 > 93 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 93 Phe Met Wing Gln Cys Gln He Phe Met 1 5 < 210 > 94 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 94 Met Met Met Thr Gly Arg Ala Ala Arg 1 5 < 210 > 95 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 95 Ala Ala Arg Trp Ala Ser Ala Lys Leu 1 5 < 210 > 96 < 211 > 8 < 212 > PRT < 213 > Homo sapiens < 400 > 96 Ala Lys Leu Glu Arg Ser His Tyr 1 5 < 210 > 97 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 97 Gln Gly Met Gly Ser Val He Asp Tyr 1 5 < 210 > 98 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 98 Asn Glu Pro Wing Leu He Asp Gln Tyr 1 5 < 210 > 99 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 99 Arg Arg Leu Ala Arg Ala Ala Ala Ala 1 5 < 210 > 100 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 100 Lys Pro Arg Ser Pro Pro Arg Ala Leu 1 5 < 210 > 101 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 101 Arg Met Arg Leu Thr Gln Glu Glu Lys 1 5 < 210 > 102 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 400 > 102 Pro Thr Glu Pro Val Gly Gly Ala Arg 1 5 32 , .t., .. m. ^, ^ * ..? . , ft < * > , A ^ MO, ??? ^

Claims (30)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An isolated DNA molecule characterized in that it has the nucleotide sequence shown in SEQ ID. NO: 1 or a polynucleotide that hybridizes with this DNA molecule under stringent conditions or a fragment thereof. 2. - The isolated DNA molecule according to claim 1, further characterized in that it contains a DNA sequence encoding a tumor-associated antigen with the immunogenic properties of the polypeptide of the amino acid sequence according to SEQ ID NO: 2 or an antigen associated with tumor, of which the polypeptide shown in SEQ ID NO: 2 constitutes a partial sequence, or for fragments thereof. 3. The isolated DNA molecule according to claim 1, further characterized in that it contains a DNA sequence encoding a tumor-associated antigen that has the immunogenic properties of the polypeptide with the amino acid sequence in accordance with the invention. with SEQ ID NO: 3, or for fragments thereof. 4. The tumor associated antigen called Rll-ORF-1, characterized in that it has the amino acid sequence defined in SEQ ID NO: 2 or contains it as a partial sequence. 5. The protein fragment or immunogenic peptide, characterized in that it is obtained from the antigen associated with tumor defined in the claim. 6. The immunogenic (poly) peptide according to claim 5, further characterized in that it induces a humoral immune response. 7. The (poly i) peptide that is immunogenic, according to claim 5, or the degradation products thereof, further characterized because it is or is presented by MHC molecules and induces a cellular immune response. 8. The immunogenic peptide according to claim 1, further characterized in that it is selected from the group of peptides according to SEQ ID NO: 88 to 102. 9. The immunogenic (poly) peptide according to any of the claims 5. to 8 for the Hü.fflafai, .- ^? ^. T,! L, ^ "A41.I i immunotherapy of cancers in vivo or ex vivo, characterized in that the (poly) peptide induces an immune response against the tumor cells in the patient Express Rll. 10. The pharmaceutical composition for parenteral, topical, oral or local administration, characterized in that it contains as an active component one or more immunogenic (poly) peptides according to any of claims 5 to 9. 11.- The pharmaceutical composition according to claim 10, further characterized in that it contains various immunogenic peptides derived from Rll-ORF-1. 12. - The pharmaceutical composition according to claim 10 or 11, further characterized in that it contains one or more peptides derived from Rll-ORF-1 intimately mixed with peptides derived from other antigens associated with tumor. 13. The pharmaceutical composition according to claim 11 or 12, further characterized in that the peptides bind to at least two different types of HLA. 14. - The antigen associated with tumor called Rll-ORF-2, characterized in that it has the sequence of amino acids defined in SEQ ID No: 3. 15.- The immunogenic peptide or protein fragment, characterized in that it is derived from the tumor associated antigen defined in claim 14. 16.- The immunogenic (poly) peptide according to the claim 15, further characterized in that it induces a humoral immune response. 17. The immunogenic (poly) peptide according to claim 5, or the degradation products thereof, characterized in that it is or is presented by MHC molecules and induces a cellular immune response. 18. The immunogenic peptide according to claim 17, further characterized in that it is selected from the group of peptides according to SEQ ID No: 44 to 87. 19. The immunogenic (poly) peptide according to any of the claims 15 to 18 for the unpaired api of cancers in vi v or ex vi ve, further characterized because the (pol i) peptide induces an immune response against the tumor cells in the patient expressing Rll. 20.- The pharmaceutical composition for parenteral, topical, oral or local administration, . ¿.. characterized in that it contains as an active component one or more (poly) immunogenic peptides according to any of claims 15 to 19. 21. The pharmaceutical composition according to claim 20, further characterized in that it contains various Immunogenic peptides derived from Rll-ORF-2. 22. The pharmaceutical composition according to claim 21, further characterized in that it contains one or more peptides derived from Rll-ORF-2 intimately mixed with peptides derived from other antigens associated with tumor. 23. The pharmaceutical composition according to claim 21 or 22, further characterized in that the peptides bind to at least two different types of HLA. 24. The recombinant DNA molecule characterized in that it contains a DNA molecule according to any of claims 1 to 4. The DNA molecule according to one of claims 1 to 4 or 24, for the immunotherapy of cancers, further characterized in that the (poly) peptide of Rll-ORF-1 or Rll-ORF-2 expressed by the DNA molecule or a fragment thereof induces an immune response against the tumor cells of the patient expressing R11-0RF -1 and / or R11-0RF-2. 26. The use of cells that express the antigen defined in claim 5 and / or 15, to prepare a vaccine against cancer. 27. The antibody against a (poly) peptide defined in any of claims 5 to 9. 28.- The antibody against a (poly) peptide defined in any of claims 15 to 19. 29.- The antibody according to Claim 27 or 28, further characterized in that it is monoclonal. 30. The antibody according to any of claims 27 to 29, characterized in that it is for the treatment and diagnosis of cancers associated with the expression of Rll. r? tri¡fttt iíiifT 'J --- • -fc- * i- í .AtAu-Á ^