CA2365278A1 - Tumour-associated antigen - Google Patents

Abstract

The invention relates to a tumour-associated antigen, to immunogenic peptides derived therefrom, to DNA molecules that code for said peptides and to the use thereof in the immunotherapy of cancer.

Description

Case 12/203 DI Fa/dc 55216 Ingelheim am Rhein (D) Title: Tumour-associated antigen The invention relates to the immunotherapy of tumour diseases.
The immune system has the task of protecting the body from a number of different microorganisms and actively fighting these microorganisms. The importance of an :intact immune system is apparent particularly in the case of inherited or acquired immunodeficiencies. The use of prophylactic vaccine programmes proved in many cases to be an extremely effective and successful immunological intervention in the fight against viral or bacterial infectious diseases. It has also been found that the immune system is also involved to a large extent in eliminating tumour cells.
Recognition of the tumour associated antigens (TAAs) by components of the immune system plays a crucial role. In the broadest sense, any (peptidic or non-peptidic) component of a tumour cell which is recognised by an element of the immune system and leads to stimulation of an immune response, can act as an immunogenic tumour antigen. Those tumour antigens which not only evoke an immunological reaction but also cause rejection of the tumour are of particular importance. The identification of specific antigens which are able to provoke an immunological reaction of this kind constitutes a major step in developing a molecularly defined tumour vaccine.
Although it is not yet clear which elements of the immune system are responsible for rejection of the tumour, there is nevertheless consensus that CD8-expressing cytotoxic , T-lymphocytes (CTLs) play a major part (Coulie, 1997).
Particularly in those types of tumour (such as melanoma r and kidney carcinoma) which have a~relativel.y high spontaneous remission rate, a correlation has been found between the clinical progress 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, 1998). Specific CTL clones were obtained either from tumour-infiltrating lymphocytes (TIL) or peripheral mononuclear blood cells (PBMC) after co-cultivation with generally autologous tumour cells and cytokine stimulation in vitro. Both in animal models and in human cell culture systems cultivated in vitro, the T-cell response against tumour cells was increased by transfection of tumour cells with cytokines (van Elsas et al., 1997; Gansbacher et al., 1990; Tepper et al., 1989; Fearon et al., 1990; Dranoff et al., 1993).
In the light of the correlation between remission and the involvement of CD8+-T cells, the identification of tumour associated antigens (TAA) which are recognised by CD8-positive CTLs is a specific prime objective towards developing a tumour vaccine (Pardoll, 1998; Bobbins and Kawakami, 1996). Whether other cell types of the immune system such as for example CD4+-T-helper cel:Ls play an important part is not yet clear; a number of studies with MAGE-3/HLA-A1 peptides in melanoma patients indicated this (Marchand et al., 1995; Boon et al., 1998). In recent years a number of TAAs which are recognised by CTLs have been identified (Boon et al., 1994; van den Eynde and van der Bruggen, 1997).
T-cells recognise antigens as peptide fragments which are presented on the cell surfaces of MHC molecules ("major histocompatibility complex", in man "HLA" - "human leukocyte antigen"). There are two types of MHC
molecules: MHC-I molecules occur in most cells with a r nucleus and present peptides (usually 8-10-mers) which are produced by proteolytic degradation of endogenous proteins (so-called "antigen processing"). Peptide: MHC-I
complexes are recognised by CD8-positive CTLs. MHC-II
molecules occur only on so-called "professional antigen-presenting cells" (APC) and present peptides of exogenous proteins which are absorbed and processed in the course of endocytosis by APC. Peptide: MHC-II complexes are recognised by CD4-helper-T cells. By interaction between the T-cell receptor and peptide:MHC complex, various effector mechanisms may be triggered which lead to apoptosis of the target cell in the case of CTLs. This occurs if either the MHC (e. g. in the case of transplant rejection) or the peptide (e.g. in the case of intracellular pathogens) is recognised as foreign. In any case, not all the presented peptides meet the structural and functional requirements for effective interaction with T-cells (as described by Rammensee et al., 1995 and hereinafter).
In principle, a number of methods of administration are possible for using TAAs in a tumour vaccine: the antigen can either be administered as a recombinant protein with suitable adjuvants or carrier systems or it may be given as cDNA coding for the antigen in plasmid (DNA vaccine;
Tighe et al., 1998) or viral vectors (Resti:fo, 1997).
Another possibility is to use recombinant bacteria (e. g.
listeria, salmonella) which recombinantly express the human antigen and 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 by so-called "professional antigen presenting cells" (APC). Another possibility is to use synthetic peptides (Melief et al., 1996) which correspond to the equivalent T-cell epitopes of the antigen and are either loaded onto the APC from outside (Buschle et al., ,r 1997; Schmidt et al., 1997) or absorbed by the APC and transferred intracellularly to the MHC I molecules. The most therapeutically efficient method of administration of a specified antigen is generally determined by clinical trials.

w The antigens or epitopes thereof recognised by the tumour-specific CTLs include molecules which can come from any protein classes (e. g. transcription factors, receptors, enzymes; for a survey see Rammensee et al., 1995; Bobbins and Kawakami, 1996). These proteins do not necessarily have to be located on the cell surface, as is necessary for recognition by antibodies. In order to act as a tumour specific antigen for recognition by CTLs or in order to be used for therapy, the proteins must meet certain conditions: first of all, the antigen should be expressed exclusively by tumour cells or should occur in so-called "critical" normal tissues only in smaller concentrations than in tumours. Critical normal tissues are essential tissues; an immune reaction directed against them would have severe, in some cases lethal consequences.
Secondly, the antigen should be present not only in the primary tumour but also in the metastases. Furthermore, with a view to broad clinical use of the antigen, it is desirable for it to be present in high concentrations in several types of tumour. One further precondition for the suitability of a TAA as an effective ingredient of a vaccine is the presence of T-cell epitopes in the amino acid sequence of the antigen; peptides derived from the TAA should lead to an in vitro/in vivo T-ce:l1 response ("immunogenic" peptide). Another criterion for selecting a clinically broadly applicable immunogenic peptide is the frequency with which the antigen is encountered in a given population, of patients.
The immunogenic tumour-associated antigens (TAAs), which ,r have already largely been shown to have T-cell epitopes, can be divided into a number of categories, including viral proteins, mutated proteins, overexpressed proteins, fusion proteins formed by chromosomal translocation, differentiation antigens, oncofoetal antigens (Van den Eynde and Brichard, 1995; van den Eynde and van der Bruggen, 1997).
The methods of identifying and characterising TAAs which form the starting point for the development of a tumour vaccine are based on the one hand on the use of CTLs which have already been induced in patients (cellular immune response) or antibodies (humoral immune response), or are based on drawing up differential transcription profiles between tumours and normal tissues. In the former case, the immunological approach, patient CTLs are used for screening eukaryotic tumour-cDNA expression libraries which present the CTL-epitopes via MHC-I molecules (Boon et al., 1994), whereas by using high affinity patient antisera prokaryotic cDNA expression libraries, the presence of TAAs can be searched directly via immunoblot analysis of the individual plaques (Sahin et. al., 1995).
A combination of CTL reactivity and protein-chemical processes produces the isolation of peptides isolated from MHC-I from tumour cells, which are preselected by reactivity with patient CTLs. The peptides are washed out of the MHC-I complex and identified by mass spectrometry (Falk et al., 1991; Woelfel et al., 1994; Cox et al., 1994). The approaches which use CTLs to characterise antigens involve substantial costs or are not always successful, owing to the need to cultivate and activate CTLs.
Methods of identifying TAAs which are based on comparing the transcription profile of normal and tumour tissue are many and varied; these include differential hybridisation, the establishing of subtraction cDNA banks ("representational difference analysis"; Hubank and Schatz, 1994; Diatchenko et al., 1996) and the use of DNA
chip technology or the SAGE method (Velculescu et al., 1995). In contrast to the above-mentioned immunological method using patient CTLs, when using molecular biological methods it is necessary to show that the potential antigen candidates discovered by this method are tumour-specific (tumour-associated) and do indeed have T-cell epitopes capable of triggering a cytotoxic T-cell response. In at least one case (NY-ESO/LAGE-1) an antigen was identified both by the use of patient sera and by RDA (Chen et al., 1997; Lethe et al., 1998), and moreover CTL-~epitopes of this antigen and a simultaneous spontaneous humoral and T-cell response were described in one patient (lager et al., 1998).
The aim of the present invention was to provide a new tumour-associated antigen (TAA).
This objective was achieved by first establishing a cDNA
subtraction library by RDA (representational difference analysis) between a lung adenocarcinoma cell line (A549) and normal lung tissue. In order to select the antigens which were overexpressed in the tumour, the cDNA clones obtained were then sequenced and compared with sequences available in databanks. Among the genes identified there were 321 unknown genes for which there were mostly EST
entries (expressed sequence tags) in the databank. After further qualitative PCR analysis in cDNA libraries of critical normal tissues and immunoprivileged tissues as well as detailed databank searches, the number of candidate clones was restricted to 56 whose ESTs did not derive from critical normal tissue. It was established by RT-PCR that three of the 56 clones investigated showed expression mainly indifferent tumour tissues and little or no expression in normal tissue. The quantitative comparison (using PCR) of the expression of one of the clones (B99) between tumour tissue and normal tissue showed overexpression of the B99 cDNA in various tumours.
The expression profile analysed by Northern blot also -showed that B99 had no or only poor transcription in the normal tissues investigated.
The human B99 cDNA was cloned; the sequence obtained is shown in SEQ ID N0:1. The sequence analysis of the cloned human B99 cDNA showed that from position 427 to position 1743 there is a continuous open reading frame which, at the nucleotide and protein level, is very similar to the open reading frame of beta-1,3-galactosyl-o-glycosyl-glycoprotein beta-1,6-n-acetylglucosaminyltransferase. The data obtained from Northern Blot experiments lead one to conclude that the B99 transcript has a length of about 3.0 kb. The cloned region of B99 cDNA is 2216 bp, whilst the presence of a PolyA tail at the 3'-end of the sequence is evidence of the completeness of the cDNA in this region. The difference in the size of the cloned B99-cDNA
compared with the size which can be deduced from the Northern Blot analysis can be explained by t:he presence of a PolyA tail of unknown length and an additional sequence in the 5'-untranslated region of B99. In view of the fact that there is no continuous reading frame in the 5' region of the cloned cDNA from position 0 to 427, it can be concluded that the ATG at position 427 is the start codon of B99.
Additional information as to the sequence of B99 located further upstream can be obtained by standard methods of molecular biology, e.g. by 5'-RACE (rapid amplification of cDNA ends). In this method, RNA, preferably mRNA, is reverse transcribed from cells or tissues in which B99 is r transcribed (e. g. colon carcinoma tissue or cell lines derived from lung adenocarcinoma such as A549) and then ligated with an adaptor of known sequence. A PCR with an adaptor primer (binds specifically to the adaptor at the 5'-end of the cDNA) and a B99-specific primer (e.g. SEQ ID
N0:8, 10, 11) allows amplification of corresponding B99 fragments. These PCR products can be cloned by standard methods, as described in Example 1, and characterised particularly by DNA sequencing.
An alternative method of characterising the 5'-end is by screening cDNA libraries by hybridisation with DNA probes or antisera which are specific for B99.
If the screening of cDNA libraries does not achieve the desired outcome, on account of limitations of procedure, e.g. inefficient reverse transcription caused by marked secondary structures of the RNA, genomic libraries can be searched by, for example, isolating clones, as in the screening of cDNA libraries, by hybridising with DNA
probes specific for B99, said clones containing the sequence information located upstream of the 5'-end of the cDNA obtained, e.g. the promoter region of B99.
The isolated cDNA codes for the tumour associated antigen (TAA) designated B99 with the amino acid sequence given in SEQ ID N0:2 (B99-1). This sequence is defined by the start codon at position 427 of the isolated B99-cDNA.
In another attempt at cloning the coding region of B99 in which cDNA from the lung adenocarcinoma cell line A549 was used, a sequence was determined which has an insertion of a nucleotide at position 923, compared with the sequence shown in SEQ ID N0:1 (SEQ ID N0:3 or SEQ ID N0:5). This insertion leads to a change in the open reading frame of B99 with a resulting amended amino acid sequence in the C-terminal region of 'the B99 protein; the sequence of this B99 antigen (B99-2) derived from this reading frame is shown in SEQ ID N0:4. Apart from the insertion, the cDNA
isolated from A549 cells has a nucleotide exchange at position 622 compared with sequence SEQ ID N0:1. This nucleotide exchange causes arginine (SEQ ID N0:2, B99-1) to be replaced by tryptophan (SEQ ID N0:4, B99-2) at position no. 66. Apart from this amino acid exchange the amino acid sequence of B99-2 up to and including position 166 is identical to B99-1.
The insertion of a nucleotide at position 923 produces a second potential reading frame from position 845 to 1744 of the sequence shown in SEQ ID N0:3 (or SEQ ID N0:5). A
protein expressed by a cDNA with this reading frame has the amino acid sequence shown in SEQ ID NO: 6 (B99-3). The sequence of B99-3 is different from B99-1 from position 1 to 27 and identical to B99-1 from position 28 onwards.
Thus, according to a first aspect, the present invention relates to a tumour associated antigen designated B99 selected from the group of polypeptides with the amino acid sequence given in SEQ ID N0:2, SEQ ID rd0:4 or SEQ ID
N0:6.
The amino acid sequences shown in SEQ ID N0:2 (B99-1), SEQ ID N0:4 (B99-2) and SEQ ID N0:6 (B99-3) may have some differences, e.g. those caused by the exchamge of amino acids, if the B99 derivative has the immunogenic properties desired for use in a tumour vaccine. (An example of a B99 polymorphism of this kind is the difference between B99-1 and B99-2 at position 66 caused by point mutation).
Unless otherwise stated, the term "B99" is used~-hereinafter to denote B99-1, B99-2 and B99-3.
r The natural amino acid sequence of B99 (or r_orrespondingly the sequences of the B99-cDNA) can optionally be modified by replacing individual amino acids in a B99 CTL-epitope in order to achieve an increase in the affinity of B99 peptides to MHC-I molecules compared with the natural B99 CTL-epitope, and thus bring about increased immunogenicity and finally greater reactivity to tumours. Modifications in the region of the B99 epitopes may be carried out on the whole B99 protein (this is processed by the APCs to form the corresponding peptides) or on larger B99 protein fragments or on B99 peptides (cf, below).
According to another aspect the present invention relates to immunogenic fragments and peptides derived from B99.
The latter are hereinafter referred to as B99 peptides. A
first group are the B99 peptides which trigger a humoral immune response (induction of antibodies). Such peptides are selected portions of B99 (at least 12 to 15 amino acids) which can be determined by so-called prediction algorithms such as for example the surface probability plot (Emini et al., 1985), the hydrophobicity blot (Kyte and Doolittle, 1982) and the antigenic index. (Jameson and wolf, 1988).
It is known that tumour-associated antigens may have tumour-specific mutations, which contribute to an immunological distinction between tumour and normal tissue (Mandruzzato et al., 1997, Hogan et al., 1998, Gaudi et al., 1999, Wolfel et al., 1995). In order to determine the presence of tumour-specific B99 mutations, the B99 cDNA
may be cloned from one or more different tumours, preferably with probes from the isolated B99 cDNA of the invention, and the obtained sequences are compared with B99 cDNAs from normal tissue. It may be expected that tumour B99 peptides from a sequence section mutated vis a vis normal tissue wild have an increased immunogenicity in comparison with B99 peptides from the corresponding sequence section from normal tissue.
Therefore, the invention relates, in a further aspect, to B99 peptides derived from tumour-expressed B99, which have tumor-specific mutations.

When selecting B99 peptide candidates the regions of B99-2 and B99-3 which differ from B99-1 deserve special interest. On the understanding that the insertion of the B99 DNA which leads to these differences in the amino acid sequence is a tumour-specific mutation, peptides from this region can be expected to exhibit greater immunogenicity than peptides of B99-1. In order to confirm that the insertion is tumour-specific, antibodies against this region may be generated and tumour cells investigated for the expression of B99-2 and B99-3.
B99 Peptides are administered directly or in modified form (e.g. coupled to~~LH = keyhole limpet hemocyanin) and the formation of antibodies is determined by normal immunological assays, e.g. by ELISA.
Other B99 peptides which are preferred within the scope of the present invention are those which are presented by MHC-molecules and produce a cellular immune response.
There are two types of MHC-molecules, namely MHC-I
molecules which are recognised by CD8-positive CTLs and MHC-II molecules which are recognised by CD4-positive T-helper cells.
In order for a peptide to trigger a cellular immune response, it must bind to an I~-IC-molecule, whilst the patient to be,treated must have the MHC molecule in his or her repertoire. Determining the MHC-subtype of the patient thus constitutes one of the essential prerequisites for effective use of 'a peptide in this patient, with a view to triggering a cellular immune response.
The sequence of a B99 peptide to be used therapeutically is determined by the MHC-molecule in questian in terms of the anchor amino acids and length. Defined anchor positions and length guarantee that a peptide fits 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 tumour cells of the patient, if a peptide derived from a tumour antigen is used.
Immunogenic B99 peptides may be identified by known methods; one of the basic conditions is the correlation between MHC-binding and CTL-induction.
Thus, since the sequence of immunogenic peptides can be predicted on the basis of its peptide binding motif, B99 peptides which constitute CTL-epitopes can be identified and synthesised on the basis of the B99 protein sequence.
Various methods are available for doing this, which are used to identify CTL-epitopes of known protein antigens;
e.g. the method described by Stauss et al., 1992 for identifying T-cell epitopes in human papillama virus.
The allele-specific requirements of each MHC-I allele product with regard to a peptide which binds to the MHC-molecule and is presented thereby have been assembled as a motif (e. g. Falk et al., 1991). Up till now, a large number of both MHC-peptide motifs and MHC-ligands have become known. A suitable method, within the scope of the , present invention, for searching for epitopes of a known protein which fits a specific MHC-I molecule is described in a survey by Rammensee et al., 1995. It comprises the following steps: first, the protein sequence is searched for fragments which correspond to the anchor motif, whilst certain variations are possible with regard to peptide length and anchor occupation. If for example a motif prescribes a 9-mer with Ile or Leu at the end, 10-mers with a corresponding C-terminus can also be considered, as can peptides with other aliphatic groups such as Val or Met at the C-terminus. In this way a number of peptide candidates is obtained. These are searched for the presence of as many anchor groups as possib:Le which they have in common with known ligands and/or to see whether they have groups which are "preferred" for various MHC-molecules (according to the Table by Rammensee et al., 1995). In order to exclude weakly binding peptides, binding assays are preferably carried out. If the requirements for the peptide binding for specific MHC-molecules are known, the peptide candidates can also be searched for non-anchor groups which have a negative or positive effect on the binding or which indeed make it possible at all (Ruppert et al., 1993). However, with this method, it should be borne in mind that the peptide binding motif is not the sole deciding factor when searching for natural ligands; other aspects, e.g. enzyme specificity during antigen processing, also contribute to the identity of the ligand, in addition to 'the specificity of the MHC-binding. One method which takes account of these aspects and which is suitable for identifying immunogenic B99 peptides within the scope of the present invention was used inter alia by Kawakami et al., 1995 for identifying gp100 epitopes on the basis of known HLA-A*0201 motifs.
The peptides may also be selected for their ability to bind to MHC-II molecules. The MHC-II binding motif which extends over nine amino acids has a higher degree of degeneration in the anchor positions than t:he MHC-I
binding motif. Methdds have recently been developed, based on X-ray structural analysis of MHC-II molecules, which allow accurate analysis of the MHC-II binding motifs and, based on that, variations in the peptide sequence (Rammensee et al., 1995, and the original literature cited therein). Peptides which bind to MHC-II molecules are typically presented to the CD4-T cells by dendritic cells, macrophages or B-cells. The CD4-T-cells in turn then activate CTLs directly in sequence by the release of cytokine, for example, and increase the efficiency of antigen presentation by APC (dendritic cells, macrophages and B-cells).
Recently, databanks and prediction algorithms have become available which allow more reliable prediction of peptide epitopes which bind to a specific MHC molecule.
Within the scope of the present invention, using the algorithm described by Parker et al., 1994 and Rammensee et al., 1995, candidate peptides have been identified for the most important HLA-types, especially for HLA-A1, -A*0201, -A3, -B7, -B14 and -B*4403, which c:an be expected to bind to the corresponding HLA molecules and thus constitute immunogenic CTL-epitopes; the peptides discovered are listed in Table 2. Similarly, possibly using other algorithms which take account of the different characteristics of the peptides (hydrophobicity, charge, size) or requirements made of the peptides, such as the 3D structure of the HLA-molecule, it is possible to find potential peptide epitopes; this also applies to peptide epitopes of other HLA types.
After selecting B99-peptide candidates using the methods described, their MHC-binding is tested by peptide binding assays. First, the immunogenicity of the peptides with good binding properties is determined (stability of the peptide-MHC interaction correlates'in most cases with immunogenicity; van der Burg et al., 1996). In order to determine the immunogenicity of the selected peptide or peptide equivalent, methods may be used as described, for example, by Sette et al., 1994, combined with quantitative MHC-binding assays. Alternatively, the immunogenicity of the selected peptide may be tested by in vitro CTL-induction using known methods (as described hereinafter for ex vivo CTL-induction). The principle of the method, carried out in several steps, for selecting peptides which are capable of triggering a cellular immune response is described in WO 97/30721, the contents of which are hereby expressly referred to. A general strategy for obtaining efficient immunogenic peptides which is suitable within the scope of the present invention has also been described by Schweighoffer, 1997.
Instead of using the original peptides which. fit the binding groove of MHC-I or MHC-II molecules, i.e. peptides which are derived unaltered from B99, variations may be carried out, adhering to the minimum requirements regarding anchor positions and length specified on the basis of the original peptide sequence, provided that these variations not only do not impair the effective immunogenicity of the peptide which is made up of its binding affinity to the MHC-molecule and its ability to stimulate T-cell receptors, but preferably enhance it. In this case, artificial peptides or peptide equivalents are thus used which are designed to correspond t.o the requirements regarding binding ability to an MHC-molecule.
Peptides modified in this way are referred to as "heteroclitic peptides". They may be obtained by the following methods:
First of all, the epitopes of MHC-I or MHC-II ligands or variations thereof are undertaken,.e.g. 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 matched to MHC-I
molecules.

If desired, the peptide may also be extended at the C- and/or N-terminus provided that this extension does not affect the ability to bind to the MFiC-molecule and the extended peptide can be cellularly processed down to the minimum sequence.
The modified peptides are then investigated for their recognition by TILs (tumour infiltrating lymphocytes), for CTL-induction and for increased MHC-binding and immunogenicity, as described by Parkhurst et. al., 1996 and Becker et al., 1997. , Another method of finding peptides with greater immunogenicity than that of the natural B99 peptides, which is suitable for the purposes of the present invention, consists in screening peptide lix>raries with CTLs which recognise the B99 peptides naturally occurring on tumours, as described by Blake et al., 1996; in connection with this it is proposed to use combinatorial peptide libraries in order to design molecules which imitate tumour epitopes recognised by MHC-I-restricted CTLs.
The B99 polypeptides according to the present invention or immunogenic fragments or peptides derived therefrom may be produced recombinantly or by peptide synthesis, as described in WO 96/10413,,the disclosure of which is hereby referred to. Fox recombinant production, the corresponding DNA molecule is inserted by standard methods in an expression vector, transfecte'd into a suitable host cell, the host is cultivated under suitable expression conditions and the protein is purified. Conventional methods may be used for the chemical synthesis of B99 peptides, e.g. automatic peptide synthesisers which are commercially available.

Alternatively to natural B99 peptides or heteroclitic peptides it is also possible to use substances which imitate such peptides, e.g. "peptidomimetics" or "retro-inverse peptides". In order to test these molecules with regard to their therapeutic use in a tumour vaccine the same methods are used as described above for the natural B99 peptides or B99 peptide equivalents.
The TAA designated B99 according to the present invention and the protein fragments, peptides or peptide equivalents or peptidomimetics derived therefrom may be used in cancer therapy, e.g. in order to induce an immune response to tumour cells which express the corresponding antigen determinants. They are preferably used for the treatment of B99-positive tumours, particularly in kidney cell-, lung-, colon-, pancreas-, breast- and stomach-carcinoma.
The immune response in the form of induction of CTLs can be achieved in vivo or ex vivo.
In order to induce CTLs in vivo, a pharmaceutical composition containing as active component the TAA B99 or fragments or a peptide or peptides derived therefrom, is administered to a patient suffering from a t,umoral disease associated with the TAA, whilst the quantity of TAA
(peptide) must be sufficient to obtain an effective CTL
response. to the antigen-bearing tumour.
Thus, according to another aspect, the invention relates to a pharmaceutical composition for parenteral, topical, oral or local administration. Preferably, the composition is used parenterally, e.g. for subcutaneous, intradermal or intramuscular application. The B99-TAAs/peptides are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. The composition may also contain conventional adjuvants such as buffers etc. The TAAs/peptides may be used on their own or in conjunction with adjuvants, e.g. incomplete Freund's adjuvant, saponines, aluminium salts or, in a preferred embodiment, polycations such as polyarginine or polylysine. The peptides may also be bound to components which aid CTL
induction or CTL activation, e.g. T-helper peptides, lipids or liposomes, or they are administered together with these substances and/or together with immunostimulant substances, e.g. cytokines (IL-2, IFN-y). Methods and formulations which are suitable for the preparation and administration of the pharmaceutical composition according to the invention are described in WO 95/04542 and WO 97/30721, the disclosure of which is hereby referred to.
B99 (fragments) or B99 peptides may also be used to trigger a CTL response ex vivo. An ex vivo CTL response to a tumour which expresses B99 is induced by incubating the CTL-precursor cells together with APCs and B99 peptides or B99 protein. The activated CTLs are then allowed to expand, whereupon they are re-administered to the patient. Alternatively, APCs may be loaded with B99 peptides, which may lead to efficient activation of cellular immune reactions against B99 positive tumours (Mayordomo et al., 1995; Zitvogel et al., 1996). One suitable method of loading peptides onto cells, e.g.
dendritic cells, is disclosed in WO 97/19169.
In one embodiment of the invention a combination of r several different B99 peptides or B99 peptide equivalents is used. In another embodiment, B99 peptides are combined with peptides derived from other TAAs. The choice of peptides for such combinations is made in the light of detecting different MHC-types in order to cover the broadest possible patient population, and/or it is aimed at the broadest possible spectrum of indications, by combining peptides from several different tumour antigens.
The number of peptides in a pharmaceutical composition can fluctuate over a wide range, but typically a clinically usable vaccine contains 1 to 15, preferably 3 to 10 different peptides.
The peptides according to the invention may also be used as diagnostic reagents. For example, the peptides may be used to test the response of a patient to the humoral or cellular immune response evoked by the immunogenic peptide. This provides a possibility of improving a treatment procedure. For example, depending on the form of administration'(peptide, total protein or DNA vaccine) of the TAA, the increase of precursor T-cells in the PBLs which show reactivity against the defined peptide epitope can be investigated (Bobbins and Kawakami, 1.996 and the references cited therein). Moreover, the peptides or the total protein or antibodies directed against: the TAA may be used to characterise the progression of a B99-positive tumour (e. g. by immunohistochemical analyses of primary tumour and metastases). A strategy of this kind has already proved successful in many cases, e.g. detecting the oestrogen receptor as the basis for deciding on endocrine therapy in breast cancer; c-erbB-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 the serum or by using a 111In-labelled monoclonal antibody .r against PSMA in immunoscintigraphy~on prostate carcinoma (Murphy et al., 1998 and references included therein); CEA
(carcinoembryonic antigen) as a serological marker for the prognosis and progression in patients suffering from colorectal carcinoma (Jessup and Loda, 1998).

According to another aspect the present invention relates to isolated DNA molecules coding for a protein with the immunogenic properties of B99 or fragments thereof.
In one aspect, the present invention relates to an isolated DNA molecule which contains a polynucleotide with the sequence shown in SEQ ID N0:1 or which contains a polynucleotide which hybridises with a polynucleotide of the sequence shown in SEQ ID N0:1, under stringent conditions.
By "stringent hybridization conditions" as used herein is meant overnight incubation at 42°C in a solution comprising: 50~ formamide, 5x SSC (1X SSC = 150 mM NaCl, l5mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10~ dextran sulfate, and 20 ug/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1x SSC at about 65°C, or equivalent conditions.
In another aspect, the present invention relates to an isolated DNA molecule which contains a polynucleotide with the sequence shown in SEQ ID N0:3 (or SEQ II) N0:5) or which contains a polynucleotide which hybridises with a polynucleotide of the sequence shown in SEQ ID N0:3 (or SEQ ID N0:5), under stringent conditions.
The DNA molecules according to the invention or fragments thereof code for (poly)peptides designated B99 (B99-1, B99-2 or B99-3) with the amino acid sequence shown in .r SEQ ID N0:2, SEQ ID N0:4 or SEQ ID N0:6 or for protein fragments or peptides derived therefrom; this includes DNA
molecules which show deviations from the sequence shown in SEQ ID N0:1 or SEQ ID N0:3 or SEQ ID N0:5 as a result of the degeneration of the genetic code.

The invention also relates to DNA molecules which have deviations from the sequence shown in SEQ ID N0:1 or SEQ ID N0:3 (or SEQ ID N0:5) caused by the conservative exchange of amino acids, if they code for a B99 derivative or fragments or peptides with the immunogenic properties which are desirable for their use as tumour vaccines.
The optionally modified DNA molecules defined above which code for B99-1, B99-2 or B99-3 or for fragments thereof are hereinafter referred to as "B99-DNA molecules", unless stated otherwise.
The B99 DNA molecules of the present invention or the corresponding RNAs which are also a subject of the present invention are used, like the (poly)peptides coded by them, for immunotherapy of cancer diseases.
In one embodiment of the invention, DNA molecules are used which code for natural B99 polypeptides. A:Lternatively to the natural B99 cDNA or fragments thereof it is possible to use modified derivatives. These comprise sequences with modifications which code for a protein (fragment) or peptides with greater immunogenicity, whilst the same considerations apply to modifications at the DNA level as apply to the peptides described above. Another type of modification is the lining up of numerous sequences coding for immunologically relevant peptides like a string of beads (Toes et al., 1997). The sequences may also be modified by the addition of auxiliary elements, e.g.
functions, which ensure more efficient release and processing of the immunogen (Wu et.al., 1995). For example, the processing and hence the presentation and finally the immunogenicity of the antigen can be increased by the addition of a locating sequence in the endoplasmatic reticulum ("ER targeting sequence").

In another aspect, the present invention relates to a recombinant DNA molecule which contains B99-DNA.
The B99 DNA molecules of the present invention may be administered, preferably in recombinant form as plasmids, directly or as part of a recombinant virus or bacterium.
In theory, any method of gene therapy may be used for immunotherapy of cancer based on DNA ("DNA vaccine") on B99-DNA, both in vivo and ex vivo.
Examples of in vivo administration are the direct injection of "naked" DNA, either by intramuscular route or using a gene gun, which has been shown to lead to the formation of CTLs against tumour antigens. Examples of recombinant organisms are vaccinia virus, adenovirus or listeria monocytogenes (a summary was provided by Coulie, 1997). Moreover, synthetic carriers for nucleic acids such as cationic lipids, microspheres, micropellets or liposomes may be used for in vivo administration of nucleic acid molecules coding for B99 peptide. As with peptides, different adjuvants which enhance the immune response may also be administered, e.g. cytokines, either in the form of proteins or plasmids coding :for them. The application may optionally be combined with physical methods, e.g. electroporation.
An example of ex vivo administration is the transfection of dendritic cells as described by Tuting, 1997, or other APCs which are used as cellular cancer vaccine.
r Thus, according to another aspect,~the present invention relates to the use of cells which express B99, either per se or, in optionally modified form, after t:ransfection with the corresponding coding sequence, in order to produce a cancer vaccine.

In another aspect, the invention relates to antibodies against B99 or fragments thereof. Polyclonal antibodies are conventionally obtained by immunising animals, particularly rabbits, by injecting the antigen or fragments thereof and subsequently purifying' the immunogl obul in .
Monoclonal anti-B99-antibodies may be obtained by standard procedures following the principle described by Kohler and Milstein, 1975, by immunising animals, particularly mice, then immortalising antibody-producing cells from the immunised animals, e.g. by fusion with myeloma cells, and screening the supernatant of the hybridomas obtained by immunological standard assays for monoclonal. anti-B99-antibodies. For therapeutic or diagnostic use in humans, these animal antibodies may optionally be chimerised in the conventional way (Neuberger et al., 1984., Boulianne et al., 1984) or humanised (Riechmann et al., 1.988, Graziano et al., 1995).
Human monoclonal anti-B99-antibodies (or fragments thereof) may 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 means of transgenic animals (Bruggemann et al., 1996, Jakobovits et al., 1995).
The anti-B99-antibodies according to the invention may be ,used in immunohistochemical analyses for diagnostic purposes.
r In another aspect, the invention relates to the use of B99-specific antibodies for selectively bringing any desired substances to or into a tumour which expresses B99. Examples of such substances are cytotoxic agents or radioactive nuclides the activity of which consists in damaging the tumour in situ. Because of the tumour-specific expression of B99, no or very few side effects can be expected. According to another aspect, substances for showing up tumours which express B99 may be used, with the aid of B99 antibodies. This is useful f:or the diagnosis and evaluation of the treatment. Therapeutic and diagnostic uses of antibodies which apply to anti-B99-antibodies are described in VETO 95/33771.
The TAA designated B99 according to the present invention and the protein fragments, peptides or peptide equivalents or peptidomimetics derived therefrom may be used in cancer therapy, e.g. to induce an immune response to tumour cells which express the corresponding antigen determinants.
They are preferably used for the treatment of B99-positive tumours, particularly in carcinoma of the kidney cells, lung, colon, pancreas, breast and stomach.
Due to the preferred expression of B99 in tumour cells, it may be assumed that this protein has an important function for the tumour, e.g. for tumour formation, infiltration and growth. B99(DNA) may therefore be employed in screening assays for identifying substances which modulate, in particular inhibit, the activity of this protein. In an embodiment, such an assay may comprise introducing the B99 protein, or an active fragement thereof, into cells or expressing B99 DNA in cells, and determining the proliferation of the cells :in the presence or absence of a test substance. Substances with a proliferation-inhibiting effect can be used for the r treatment of tumors with strong B99 expression, in particular carcinoma of the lung, colon, breast, renal cell carcinoma and for the treatment of Hodgkin lymphoma.
Summary of the drawings Fig. 1: RT-PCR analysis of cDNA pools of different human tumour and normal tissues using B99-specific primers Fig. 2: RT-PCR analysis of individual cDNAs of different human tumour and normal tissues using B99-specific primers Fig. 3: Transcription of B99 in normal tissues: Northern Blot analysis of mRNA from 16 normal tissues Fig. 4: Immunohistochemical analysis of four' different cases of adenocarcinoma with B99 serum Fig. 5: MHC stabilization on T2 cells with different concentrations of B99 peptides Example 1 RDA (Representational Difference Analysis) of the human adenocarcinoma cell line of the lung (A549) and normal lung tissue The human lung adenocarcinoma cell line A549 (CCL 185) obtained from the ATCC was cultured in T150 cell culture bottles. The nutrient medium used was MEM with 10~ heat-inactivated foetal calf serum and 2 mM L-glutamine. Every 3 to 4 days the cells were cleaved by trypsinisation 1:5 to 1:10 in order to propagate them. After about 80~
confluence was achieved, 4 ml of a trypsin solution r (containing per litre: 8 g of NaCl; 0.2 g of KC1, 1.13 g o f anhydrous Na2HP04 , 0 . 2 g o f KHZ P04 , 10 0 rnl o f 2 . 5 ~
trypsin solution and 1 g of EDTA-Na salt; pH 7.2 to 7.4) were added to each T150 cell culture flask in order to harvest the cells. The 4 ml were transferred into a 15 ml Falcon test tube, 8 ml of PBS were added, the mixture was centrifuged at 1200 rpm in a Haereus bench centrifuge (Megafuge 2.OR) for 5 minutes at 4°C, the cell pellet was mixed with 1 ml of lysis buffer (10 mM TrisHCl pH8, 140 mM
NaCl, 1.5 mM MgCl2, 0.5~ NP40), shaken vigorously and centrifuged in a 2 ml Eppendorf vessel at 1:?000 rpm and at 4°C for 5 minutes in a Sigma bench centrifuge (Sigma 202 MK). The supernatant was transferred into another Eppendorf vessel and after the addition of 55 ~1 of 20~
SDS solution it was extracted twice with double the volume of a CHC13/phenol (1:1 v/v) mixture and once with the same volume of CHC13. The aqueous phase containing RNA was mixed with 1/10 volume of 3M NaAc (pH5) and twice the volume of 96~ EtOH and the RNA is precipitated overnight at -20°C. Starting from 1 mg of total RNA, the manufacturer's instructions were followed in order to isolate poly-A(+)RNA using the PolyATtract Kit (Promega).
The storing of the A549 poly-A(+)RNA with a concentration of 1 mg/ml in DEPC-treated H20 was carried out in aliquots at -80°C.
In order to carry out representative differential analysis (RDA; Hubank and Schatz, 1994; Diatchenko et al., 1996) the poly-A(+)RNA of the lung adenocarcinoma cell line A549 was used as the tester while that of normal lung tissue (1 mg/ml; Clontech, Palo Alto; #6524-1) was used as driver. The RDA was carried out using the PCR-selectT'"' kit (Clontech, Palo Alto) in accordance with the manufacturer's instructions, except that a rnodifi,ed primer/adaptor-2-oligonucleotide system having,,the following sequence was used:
r 5'-TGTAGCGTGAAGACGACAGA.AAGGGCGTGGTACCGAGCTCGAG-3' (Adaptor-2-alt-1; SEQ ID N0:19), 5'-AGGGCGTGGTACCGAGCTCGAG-3' (nested-PCR-primer-2-alt;
SEQ ID N0:20) and 5'-GGCTCGAGCTC-3' (Adaptor-2-alt-2;
SEQ ID N0:21). The newly generated primer/adaptor sequences make it possible to excise the relevant cDNA

fragments subsequently by means of the presence of three new restriction enzyme cutting sites (Kpn I, Sac I and Xho I) in the sequence of the nested-PCR-primer-2-alt after cloning of the subtracted cDNA fragments into the pPCRII vector. It was therefore necessary to design a primer/adaptor sequence with a number of available restriction enzyme cutting sites because point mutations were often observed particularly in the primer sequences, caused by the PCR-amplification steps.
After the synthesis of double-stranded cDNA using oligo-dT, the cDNA obtained from the tester and driver was digested with Rsal (RsaI is a 4-base recognising restriction enzyme and yields 256 by long c:DNA fragments as a statistical average). Equal parts of tester cDNA
were ligated with either adaptor 1 or 2 and then separately hybridised with an excess of driver cDNA at 65°C. The two mixtures were then combined and subjected to a second hybridisation with fresh denatured driver cDNA. The concentrated tester-specific cDNAs were then exponentially amplified by PCR with primers specific for the adaptors 1 or 2. For further concentration an aliquot from this reaction was subjected to a second PCR with specific inwardly shifted (nested) primers. The exponentially amplified cDNA fragments resulting from this reaction were ligated directly into the pCRII vector (Invitrogen; "TA cloning vector") and then one third of the ligation mixture was transfected into competent E.coli (OneShot~'", Invitrogen) .
r 712 positive transformants (blue-white selection) were obtained and cultivated in 96-well blocks in LB-Amp medium (1.3 ml per well) at 37°C for 48 hours. 750 ~.1 of the E. coli suspensions were used per well for the preparation of the plasmid DNA (96-well mini preparation method of QIAgen in accordance with the manufacturer's instructions). The bacterial cultures remaining were stored as glycerine stock cultures at -80°C.
A cDNA subtraction library consisting of 712 individual clones was obtained, which was present both in the form of E. coli glycerine stock cultures and also in. the form of purified plasmids.
Example 2 DNA sequencing and annotation of TAA candidates The isolated plasmid-DNA of all 712 clones (see Example 1) was sequenced by the Sanger method using an ABI Prism device. The sequences obtained were annotated using the BioScout Software (LION, Heidelberg) and subjected to databank comparisons (Genbank). Of 712 clones, 678 were able to be sequenced and annotated. The rest (34) had only poly(A) sequences as inserts or corresponded to a relegated vector or could not be sequenced. Of the 678 annotable sequences, 357 proved to be genes with a known function. The remaining 321 represented clones coding for genes of unknown function; 59 of these did not even have entries in the human EST databank. Known genes were not treated further. For those unknown genes for which an EST
entry was available, the expression profile was evaluated:
all those ES'~s with >95~ identity (BLAST), which belonged to the correspondingly experimentally determined sequence of the subtraction libraries, were examined. In the r annotation, a subdivision was made into i) c:ritical normal tissue, ii) foetal, "disposable" and immunoprivileged tissue and iii) tumours and tumour cell lines. On the basis of this "virtual mRNA profile", 200 clones for which no ESTs were found in group i) were selected for further experimental analyses (including the 59 clones for which there was no EST entry). In order to narrow down the candidate clones still further, oligonucleotide primer pairs were designed and synthesised from the sequences determined from the 200 selected clones. First of all, 8 different cDNA libraries derived from human tissue (GibcoBRL SUPERSCRIPTTT') which are directionally cloned in pCMV SPORT, were tested by qualitative PCR for the presence of the candidates in question. The cDNA
libraries used originated from heart tissue (#10419-018), liver (#10422-012), leukocytes (#10421-022), kidney (#10420-016), lung (#10424-018), testis (#10426-013), brain (#10418-010) and foetal brain (#10662-013). The PCR
conditions were as follows: 20 ~l of total volume per PCR
mixture contained 1x TaqPol buffer (50 mM KC1, 10 mM
TrisHCl pH9, 0.1~ Triton X-100), 1.5 mM MgCl2, 0.2 mM
dNTPs (Promega), 0.025 U/~1 Taq-DNA-polymerase (Promega), 5 pM of specific oligonucleotide primer SEQ ID N0:7 and SEQ ID N0:8 and 100 ng of the plasmid DNA to be investigated. As a control, specific primers for GAPDH
(SEQ ID N0:14 and 15) were used. In order t:o check the selective evidence the primer pairs were also tested in parallel on the isolated plasmid. The deter_tability of fragments of the expected length in one of the critical normal tissues (heart, liver, lung, kidney and leukocyte) but not in the cDNA libraries of immunoprivileged tissues (brain, foetal brain and testis) under these PCR
conditions (1 cycle: 3' 94°C; 35 cycles: 1' 94°C - 1' 55°C
- 1' 72°C; 1 cycle: 7' 72°C) was defined as the selection criterion. Using this qualitative PCR analysis the number of candidates could be narrowed down to 56.
.r Example 3 Transcriptional analysis of the candidate clones in various tumour and normal tissues For RT-PCR analysis, cDNA pools were used which had been prepared from 3 ~g of total RNA from 3 different tissues of the same type. The 9 ~g total RNA per tissue pool of tumour or normal tissues was reverse transcribed by means of AMV-RT (Promega) in accordance with the manufacturer's recommendations. To avoid contamination with genomic DNA, the RNA was previously incubated with DNAse I (Boehringer Mannheim). The quality and quantity of the cDNAs were tested by PCR with GAPDH specific primers SEQ ID N0:14 and 15) after 20 cycles (30", 95°C, 90" 60°C). B99-cDNA was amplified by 25, 30 and 35 cycles of the program 1' 95°C, 1' 55°C, 1' 72°C with the B99-specific primers according to SEQ ID N0:7 and 8. The other 55 candidate clones were investigated analogously with specific primers. The PCR
products were detected by agarose gel electrophoresis and ethidium bromide staining. For 3 of the 56 clones investigated, RT-PCR showed increased expression in different tumour tissues compared with normal tissues. An example of candidate B99 is shown in Fig. 1: the RT-PCR
analysis of cDNA pools of different human tumour and normal tissues by means of B99-specific primers produced a strong signal in colon carcinoma and in the lung adenocarcinoma line A549 and a weak signal in breast carcinoma and in kidney cell carcinoma. Of all the normal tissues investigated, a weak signal could be found only in colon tissue. The candidate B99 was evaluated more precisely thereafter.
Example 4 Expression profile of B99 in tumour and normal tissue One of the 56 candidates (designated: B99) showed a signal by means of RT-PCR transcription in 3 out of 5 tumour tissue pools tested. In the majority of normal tissues tested, on the other hand, no transcription could be detected. For a more detailed investigation of the B99 expression, cDNAs of individual tumour and normal tissue samples were analysed by PCR as follows. It was found that the majority of all the colon carcinomas investigated (17/ 23), all the pancreas carcinomas (3/ 3;) and all the stomach carcinomas (4/ 4) express B99. In corresponding normal tissues, expression was detected in :L/ 4, 0/ 3 and 2/ 4 cases, respectively. All the results are shown in Table 1, whilst Fig. 2 shows exemplary resu:Lts for pancreas and colon tissue: by RT-PCR analysis of individual cDNAs of different human tumour <~nd normal tissue by means of B99-specific primers it was possible to detect B99-cDNA in 6 out of 7 tumour samples, whereas only one of the normal tissues investigated (1/ 5) showed a weak expression of B99.

Tumour tissue Normal tissue Type POSITIVE Type POSITIVE
CASES/CASES CASES/CASES
TESTED TESTED

Colon carcinoma 17/ 23 Colon Liver carcinoma 0/3 Liver 0/4 Lung carcinoma 2/9 Lung 0/4 Breast carcinoma0/10 n.d. , Pancreas- 3/3 Pancreas 0/4 carcinoma r Kidney cell- 1/10 Kidney 0/4 carcinoma Stomach 4/4 Stomach 2/4 carcinoma Lymphocyte 0/1 Small 1/1 intestine Ovary 0/1 Testis 0/4 Prostate 0/1 Thymus 0/1 Spleen 0/4 Muscle 0/1 Brain 0/1 Heart 0/4 For the Northern blot analysis, human multiple tissue Northern blots (Clontech, Palo Alto) were hybridised at 65°C for 16 hours with the [a-32P]dCTP (NEN, Boston) labelled 271 by B99 PCR product. Visualisation was carried out by standard autoradiography (Xomat AR film, Kodak) and exposure on a phosphoirnager (Molecular Dynamics). Fig. 3 shows the results of thi:~ analysis of 16 normal tissues. A transcript of ~3.0 kb in size was found only in the colon and, much weaker, in the duodenum.
The low intensity of the signal leads one to conclude that immunologically relevant expression is improbable.
The results of all the tests regarding the mRNA-profile of B99 (compiled from RT-PCR and Northern blot analysis) are collected in Table 1. Example 4 shows that B99 is clearly transcribed in a high percentage of tumours of various indications, whereas no or only isolated cases of transcription were found in all the normal tissues investigated. , Example 5 Detection of B99 protein expression in human tumours To detect B99 protein expression, B99-specific antibodies were generated in rabbits. For immunisation, the bacterial fusion protein pGEX-ORF-1/1, in which Glutat:hione-S
transferase is fused to a portion of the B99 ORF (position 1278 to 1740 in SEQ ID N0: 1) was used. ThE: obtained serum was affinity-purified with the peptide B99-KML
(SEQ ID NO: 61). To test the specific reactivity of the serum, the complete B99 open reading frame was transiently expressed in COS cells as a GFP fusion protein, and the transfected cells were tested with the serum in a Western Blot. It was shown that the serum clearly reacts with the expressed B99 fusion protein. Subsequently, 56 samples from different tumour types were analysed for B99 expression by immunohistochemistry with the B99 serum (Table 2). In 53 cases, an expression of B99 could be shown in the tumour cells. Examples can be seen in Fig. 4, which shows the immunohistochemical analysis of four different cases of adenocarcinoma (a: colon, b: breast, c: pancreas, d: stomach). In all cases a clear staining of the tumour cells can be seen, while the tumour stroma and the vessels did not show staining. It can also be seen from Fig. 4a that the residual normal colon_~c mucosa does not show reactivity with the antibody. The sections were counterstained with hematoxylin. In a selection of cases, the positive reactivity in the immunohistochemistry was confirmed at RNA level by RT-PCR. In this experiment, also breast tumours yielded a positive PCR signal. The difference of this result from Example 4 can be explained by the use of different PCR primers than in Example 4.
Table 2 Tumour type positive cases/ tested cases breast adenocarcinoma 8/ 8 colon adenocarcinoma 10% 11 lung adenocarcinoma 7/ 8 lung squamous cell 8/ 8 carcinoma _ __ pancreas adenocarcinoma ~ 11/ 12 stomach adenocarcinoma 9/ 9 Example 6 Cloning of B99 Clone B99 has a 271 by long insert of an unknown human gene between the adaptors introduced by the RDA. In order to clone the human sequence fully the following procedure was used: a UniGene Analysis (National Centre for Biotechnology Information) produced the following ESTs homologous to B99: AA315469, AA345780, AA295520. Using these ESTs the B99 sequence could be extended to 439 nucleotides: New primers within this sequence were synthesised (SEQ ID N0:9 to 12). By PCR with various combinations of these primers the theoretical fragment lengths could be amplified from A549 cDNA.
Cloning of the 3' end: 10 ~g of total RNA from the kidney cell carcinoma cell line 786-0 were reverse transcribed using oligo-dT primer and one aliquot of this cDNA was subjected to a PCR the program of which begins with high annealing temperatures so that only the gene-specific primer binds to the cDNA (primer SEQ ID N0:'7 or 9), whereas the second primer (Tm~53°C, SEQ ID N0:13) only binds to the newly synthesised DNA substrate at lower temperatures. This so-called "touch-down PCR"
(Mastercycler Gradient, Eppendorf) was carried out under the following conditions: 20 cycles of {15" 95°C, 30" 75°C
(reduced by 0.7°C per cycle)}, 1 cycle of 7' 72°C and 20 cycles of (15" 95°C, 30" 50°C) , 1 cycle 7' 72°C} .
One aliquot of the above mixture was PCR amplified once more using a second primer combination (SEQ ID N0:7 and 13 or SEQ ID N0:12 and 13) under the same conditions as before. Aliquots of the PCR mixtures were ligated directly into the pGEM-T easy-vector (Promega) and subsequently transformed into competent E. roll J109 (Promega). Positive clones were sequenced after PCR
selection. The sequencing yielded an agreement with the former sequence from the primer used for PCR amplification and in addition 1777 additional nucleotides up to the start of a PolyA sequence in the 3'-region of the sequence. PCR amplification of cDNA from tumour cell lines (786-0, A549) and tissue samples from colon carcinomas with the primers according to SEQ ID N0:16 to 18 yielded, with the original primers, the fragments expected after cloning. In the~cDNA fragment now consisting of 2216 bp, a continuous reading frame could be identified from position 427 to 1743. No additional reading frames could be identified further on in the 5'-region of the sequence, leading one to conclude that the region from 0 to 427 already belongs to the 5'-untranslated region of the B99 mRNA.
Using the primers SEQ ID N0:12 and SEQ ID N0:17 the entire coding region of B99 from A549-cDNA was amplified, cloned and sequenced. In the sequencing, a number of clones were obtained with an insertion of a nucleotide at position 923 compared with SEQ ID N0:1 (codes for B99-1) (SEQ ID N0:3 or SEQ ID N0:5).
This insertion leads to a change in the open reading frame of B99 with a resulting, altered amino acid sequence in r the C-terminal region of the B99 protein; the sequence of the B99 antigen derived from this reading frame (B99-2) is shown in SEQ ID N0:4.
One of the clones isolated from A549 cells exhibited, in addition to the above-mentioned insertion, a nucleotide exchange at position 622 compared with sequence SEQ ID
N0:1. This nucleotide exchange causes, at position No. 66 of the amino acid sequence, arginine (SEQ ID N0:2, B99-1) to be replaced by tryptophan (SEQ ID N0:4, B99-2). Apart from this amino acid exchange, the amino acid sequence of B99-2 is identical to B99-1 as far as position 166.
Moreover, the above-mentioned insertion results in a second potential reading frame from position 845 to 1744 of the sequence shown in SEQ ID N0:3 (or SEQ ID N0:5). A
protein expressed by a cDNA with this reading frame has the amino acid sequence shown in SEQ ID N0:6 (B99-3). The sequence of B99-3 is different from B99-1 from positions 1 to 27 and identical to B99-1 from position 28 onwards.
Example 7:
Potential MHC-binding peptides in the coding region of B99 Potential peptide epitopes within the coding region of B99-1 according to SEQ ID N0:2 (amino acid position:
1-438; Table 3A), of B99-2 according to SEQ ID N0:4 (amino acid position 150-190; Table 3B) and of B99-3 according to SEQ ID N0:6 (amino acid position 1-40; Table 3C) was carried out using the algorithms described by Parker et al., 1994, on the basis of known motifs (Rammensee et al., 1995). For the most important HLA types, particularly for HLA-A1, -A*0201, -A3, -B7, -B14 and -B*4403, 9-mer candidate peptides were identified, which can be expected to bind to the corresponding HLA molecules and therefore constitute immunogenic CTL-epitopes; the peptides found are listed in Table 3. Other potential peptide epitopes for other HLA types or 8- and 10-mer peptides can be determined by proceeding analogously.

Table 3A
Immunogenic B99-peptide candidates (B99-1) Starting HLA

position Sequence in SEQ

ID N0:2 5 Lys Arg Leu Cys Gln Leu B14 His Tyr Leu 12 Tyr Leu Trp Ala Leu Gly A*0201 Cys Tyr Met lg Tyr Met Leu Leu Ala Thr A*0201 Val Ala Leu 21 Leu Leu Ala Thr Val Ala A*0201 Leu Lys Leu 85 Ile Leu Asn Asn Leu Glu A3 Val Lys Lys 90 Glu Val Lys Lys Lys Arg A3 Glu Pro Phe 95 Arg Glu Pro Phe Thr Asp B*4403 Thr His Tyr 96 Glu Pro Phe Thr Asp Thr 87 His Tyr Leu 123 Pro Leu Ser Lys Glu Glu A3 Val Glu Phe 127 Glu Glu Val Glu Phe Pro B*4403 ' Ile Ala Tyr 147 Glu Arg Leu f~euArg Ala B14 Val Tyr Ala 187 Phe Ile Ala Ser Lys Leu A*0201 Val Arg Val 206 Asp Leu AsnCys Met Glu A*0201 Asp Leu Leu 209 Cys Met GluAsp Leu Leu A*0201 Gln Ser SerVal 251 Ser Met GluSer Glu Val A1, A3 Pro Pro Lys 275 Thr Leu HisLeu Thr Asn A3 Lys Lys Lys 305 Phe Val GlnHis Val Leu A3 Lys Asn ProLys 318 Leu Ile GluTrp Val Lys A1 Asp Thr Tyr 400 Trp Met LeuGln Asn His A*0201 His Leu Leu 407 Leu Leu AlaAsn Lys Phe A3 Asp Pro Lys 413 Asp Pro LysVal Asp Asp B7 Asn Ala Leu 422 Gln Cys LeuGlu Glu Tyr A1 Leu Arg Tyr 423 Cys Leu GluGlu Tyr Leu A1, A3 Arg Tyr Lys Table 3B
Immunogenic B99-peptide-candidates (B99-2) Starting - _ position in SEQ

ID N0:4 152 Ala Val Tyr Ala Pro Gln Al, A3 Asn Ile Tyr 154 Tyr Ala Pro Gln Asn Ile A*0201 Tyr Cys Val 160 Tyr Cys Val His Val Asp A*0201 Glu Glu Val 163 His Val Asp Glu Glu Val A1 Pro Arg Asn 164 Val Asp Glu Glu Val Pro A1, B*4403 Arg Asn Phe 172 Phe Gln Arg Gly Gly Gln Al Ser Asn Tyr 177 Gln Ser Asn Tyr Phe Leu A1 Leu Pro Lys 182 Leu Leu Pro Lys Cys Leu A*0201 His Ser Gln r Table 3C
Immunogenic B99-peptide-candidates (B99-3) Starting position in SEQ

ID N0:6 2 Arg Arg Leu Lys Thr Leu A1, B14 Lys Gly Tyr 5 Lys Thr Leu Lys Gly Tyr A*0201 Cys Glu Leu Tyr Cys Glu Leu Cys Met A1 Pro Leu Arg 12 Glu Leu Cys Met Pro Leu A1, A3 Arg Thr Tyr 14 Cys Met Pro Leu Arg Thr A*0201 Tyr Thr Val 16 Pro Leu Arg Thr Tyr Thr A*0201 Val Ser Met 10 Example 8 MHC stabilisation assay with potential B99-specific MHC binding peptides r In a T2 peptide loading assay, potential B99 MHC binding peptides were tested for their ability to stabilise HLA-A2 molecules, which is a hint to their MHC binding capacity.
The assay was conducted as described by Bohm et al., 1998.
The stabilisation was measured by means of FACS analysis with an HLA-A2 specific antibody (BB7.2). Five peptides exhibited a stabilisation effect, which is presented by an increase of the mean fluorescence intensity in comparison with a control containing either no peptide or a MAGE-3 A1 control peptide which does not bind.
Table 4 peptide sequence mean fluorescence intensity no peptide 2,27 MAGE-3 A1 Glu Val Asp Pro Ile Gly His Leu 2,25 negative Tyr control B99-12 Tyr Leu Trp Ala Leu Gly Cys Tyr 7,46 Leu B99-19 Tyr Met Leu Leu Ala Thr Val Ala 7,43 Leu B99-21 Leu Leu Ala Thr Val Ala Leu Lys 3,32 Leu B99-187 Phe Ile Ala Ser Lys Leu Val Arg 3,57 Val B99-209 Cys Met Glu Asp Leu Leu Gln Ser 4,72 Ser Val For further analysis, the peptides were serially diluted and tested in the same test system in order to show whether their binding is concentration-dependent. Fig. 5 shows the MHC stabilisation on T2 cells with various concentrations of B99 peptides. Tyrosinase was used as positive control, a HLA-A1-specific MAGE-3 peptide as negative control. In particular, the peptide's B99-19, B99-187 and B99-209 exhibited a clear dependency of MHC
stabilisation on their concentration, which makes them preferred candidates for immunisation stratE~gies.

Bibliography Becker, D., Kuhn, U., Lempertz, U., Enk, A., Saloga, J., and Knop, J. (1997), J. Immunol. Methods 243, 171-180.
Blake, J., Johnston, J.V., Hellstrom, K.E., Marquardt, H., and, Chen, L. (1996), J. Exp. Med. 1f34, 121-130.
Boon, T, J. C. Cerottini, B. Van den Eynde, P. van der Bruggen, and A. Van Pel (1994), Annu.Rev.Immunol. 12.:

Boon T ,(1998). Tumor antigens recognized by cytolytic T cells. Cancer Vaccine Week - Interantional Symposium, New York, Oct 1998; abstract S01 C. M. Bohm, M. L. Hanski, S. Stefanovic, H. G. Rammensee, H. Stein, J. Taylor-Papadimitriou, E. 0. Riecken, and C. Hanski. Int.J.Cancer 1998.Mar.2. 75:688-693 Boulianne, G. L., et al., (1984), Nature 312: 643-646 Bruggemann, M. and Neuberger, M.S., (1996), Immunol. Today 17: 391-397 Buschle M, Schmidt W, Zauner W, Mechtler K, Trska B, Kirlappos H, Birnstiel M (1997), Proc. :lVatl. Acad. v Sci. U.S.A. ,9:3256-3261 r Celluzzi, CM and Falo, LD, Jr. (1998), J. Immunol. 1~Q.:

Chen, Y.T., Scanlan, M.J., Sahin, U., Tureci, 0., Gure, A.O., Tsang, S., Williamson, B., Stockert, E., Pfreundschuh, M., and Old, L.J. (1997), Proc. Natl.
Acad. Sci. U. S. A. 24, 1914-1918.
Coulie, P.G. (1997) , Mol. Med. Today ~-: 261--268 Cox, AL, Skipper, J, Chen, Y, Henderson, RA, barrow, TL, Shabanowitz, J, Engelhard, VH, Hunt, DF, and Slingluff, CL, Jr. (1994), Science X64: 716-719 Diatchenko, L., Lau, Y.F., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., and Siebert, P.D.
(1996), Proc. Natl. Acad. Sci. U. S. A. 9~., 6025-6030.
Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose, K., Jackson, V., Hamada, H., Pardoll, D., and Mulligan, R. (1993), Proc. Natl.
Acad. Sci. U. S. A 2Q: 3539-3543.
Emini, EA, Hughes, J, Perlow, D, and Boger, J (1985), J.
Virol. ~5.: 836-839 Falk, K., Rotzschke, O., Stevanovi'c, S., Jung, G., and Rammensee, H-G (1991), Nature ~: 290-296.
Fearon, E.R., Pardoll, D.M., Itaya, T., Golumbek, P., Levitsky, H.I., Simons, J.4V., Karasuyama, H., Vogelstein, B., and Frost, P. (1990), Cell f Q:
397-403.
Gansbacher, B., Zier,'K., Daniels, B., Cronin, K., Bannerji, R., and Gilboa, E. (1990), J. Exp. Med. 172:
1217-1224.
Gaudi C, Krerner F, Angevin E, Scott V, Triebel F (1999), J
Immunol 162:1730-1738 Graziano, R.F., et al., (1995), J. Immunol. 155: 4996-5002 Griffiths, A.D., et al., (1994), EMBO J. 13: 3245-3260 Halliday, GM, Patel, A, Hunt, MJ, Tefany, FJ, and Barnetson RS (1995), World J. Surg. 1~: 352-358 Hogan KT, Eisinger DP, Cupp SBC, Lekstrom KJ, Deacon DD, Shabanowitz J, Hunt DF, Engelhard VH, S:Lingluff CL, Ross MM (1998), Cancer Res 58:5144-5150 Hubank, M. and Schatz, D.G., (1994), Nucleic. Acids. Res.
2,,2., 5640-5648.
Japer, E., Chen, Y.T., Drijfhout, J.W., Karbach, J., Ringhoffer, M., Japer, D., Arand, M., W<~da, H., Noguchi, Y., Stockert, E., Old, L.J., and Knuth, A.
(1998), J. Exp. Med. 1$Z, 265-270.
Jakobovits, A., (1995), Curr. Opin. Biotechnol. 6: 561-566 Jameson, BA and Wolf, H (1988) The antigenic index: a novel algorithm for predicting antigenic determinants.
Comput. Appl. Biosci. 4: 181-186 Jessup, JM and Loda, M (1998) Prognostic markers in rectal carcinoma. Semin. Surg: Oncol. 15.: 131-:L40.
Kawakami, Y., Eliyahur, S., Jennings, C., Sakaguchi, K., King, X., Southwood, S., Bobbins, P.F., Sette, A., Appella, E., and Rosenberg, S.A. (1995), J. Immunol.
154: 3961-3968.
Kawakami, Y, Bobbins, PF, and Rosenberg, SA (1996), Keio J. Med. 4~.: 100-108 Kohler, G. and Milstein, C. (1975), Nature 265, 495-497 Kruif, J., et al., (1995), Proc. Natl. Acad. Sci. USA 92:

Kyte, J and Doolittle, RF (1982), J. Mol. Biol. 157:

Lethe, B, Lucas, S, Michaux, L, De Smet, C, Godelaine, D, Serrano, A, De Plaen, E, and Boon, T (1998), Int. J.
Cancer ~: 903-908 Mackensen A, Ferradini L, Carcelain G, Triebel F, Faure F, Viel S, and Hercend T (1993), Cancer Res.

Mandruzzato S, Brasseur F, Andry G, Boon T, van der Bruggen P (1997) , J Exp Med 186:785-79:3 Marchand M, Weynants P, Rankin E et al (1995), Int. J.
Cancer x:883-885 Mayordomo, J.I., Zorina, T., Storkus, W.J., Zitvogel, L., Celluzzi, C., Falo, L.D., Melief, C.J., Ildstad, S.T., Kast, W.M., DeLeo, A.B., and Lotze, M.T. (1995), Nature Medicine 1. 1297-1302.
McGuinnes, B.T., et al., (1996), Nature Biotechnol. 14, r Melief CJ, Offringa R, Toes RE, Kast WM (1996), Curr.
Opin. Immunol. $:651-657 Murphy, G.P., Elgamal, A.A., Su, S.L., Bostwick, D.G., and Holmes, E.H. (1998), Cancer $3., 2259-2269 Neuberger, M.S., et al., (1984), Nature 312: 604-608 Pardoll, D. M.(1998) Nature Medcine 4: 525-531 Parker, K. C., M. A. Bednarek, and J. E. Coligan. (1994), J. Immunol. 15:163.
Parkhurst, M.R., Salgaller, M.L., Southwood, S., Bobbins, P.F., Sette, A., Rosenberg, S.A., and Kawakami, Y.
(1996), J. Immunol. 157, 2539-2548.
Paterson Y, Ikonomidis G (1996), Curr. Opin. Immunol. $:

Rammensee HG, Friede T, Stevanovic S (1995), Immunogenetics 41:178-228 Rapellino, M, Pecchio, F, Baldi, S, Scappaticci, E, and Cavallo, A (1995), Anticancer Res. 1.5.: 1065-1070 Ravaioli, A., Bagli, L., Zucchini, A., and Monti, F.
(1998), Cell. Prolif. 31, 113-126 Revillion, F, Bonneterre, J, and Peyrat, JP (1998), Eur.
J. Cancer ,3~: 791-808 Restifo NP (1996), Curr. Opin. Immunol. 5.: 658-63 Riechmann, L., et al., (1988), Nature 332: 323-327 .r Bobbins, PF and Kawakami, Y (1996), Curr. Opin. Immunol.
$: 628-636 Ruppert, J., Sidney, J., Celis, E., Kubo, R.T., Grey, H.M., and Sette, A. (1993), Cell ,~, 929-937.

Sahin, U., Tiireci, O., Schmitt, H., Cochlovius, B., Johannes, T., Schmits, R., Stenner, F., Luo, G., Schobert, I., and Pfreundschuh, M. (1995), Proc. Natl.
Acad. Sci. U.S.A. ~, 11810-11813 Sambrook, J., Fritsch, E.F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. ColdSpring Harbor Laboratory Press, 1989 Schendel DJ, Gansbacher B, Oberneder R, Kriegmair M, Hofstetter A, Riethmuller G, and Segurado OG (1993), J. Immunol. 151: 4209-4220 Schmidt W, Buschle M, Zauner W, Kirlappos H, Mechtler K, Trska B, Birnstiel M (1997), Proc. Natl. Acad. Sci.
U.S.A 24:3262-3267 Schweighoffer, T. (1997), Onc. Res. ~., 164-:176 Sette, A., Vitiello, A., Reherman, B., Fowler, P., Nayersina, R., Kast, W.M., Melief, C.J.M., Oseroff, C., Yuan, L., Ruppert, J., Sidney, J., del Guercio, M.-F., Southwood, S., Kubo, R.T., Chesmut, R.W., Grey, H.M., and Chisari, F.V. (1994), J. Immunol. 15 5586-5592.
Stauss, H.J., Davies, H., Sadovnikova, E., Chain, B., Horowitz, N., and Sinclair, C. (1992), Proc. Natl.
Acad. Sci. U.S.A $.3, 7871-7875.
r Tepper, R.I., Pattengale, P.K., and Leder, P. (1989), Cell ~Z: 503-512.
Tighe H, Corr, M, Roman M, and Raz E (1998), Immunol.
Today 12:89-97 Toes, R.E., Hoeben, R.C., Van der Voort, E., Ressing, M.E., Van-der-Eb, A.J. Melief, C.J.M., and Offringa, R. (1997), Proc. Natl. Acad. Sci. U.S.A. 2f-(26):

Tuting, T., DeLeo, A.B., Lotze, M.T., and Storkus, W.J., (1997), Eur. J. Immunol. 2~Z, 2702-2707,..
Van den Eynde, B. and Brichard, V.G. (1995),, Curr. Opin.
Immunol. Z, 674-681.
Van den Eynde, BJ, and van der Bruggen, P (1997), Curr.
Opin. Immunol. .x:684-693 van der Bruggen, P., C. Traversari, P. Chomez, C. Lurquin, E. De Plaen, B. Van den Eynde, A. Knuth, and T. Boon.
(1991, Science ,54:1643-1647 van der Burg, S.H., et al., (1996), J. Immunol. 156, van Elsas, A., van der Minne, C.E., Borghi, M., van der Spek, C.W., Braakman, E., Osanto, S., arid Schrier, P.I. (1996), CTL Recognition of an IL-2 Producing Melanoma Vaccine. In: Immunology of human melanoma.
Tumor-host interaction and immunotherapy, edited by M.
Maio, Amsterdam: IOS, 1996, p. 165-173 van Elsas, A., Aarnoudse, C., van der Minne, C.E., van der Spek, C.W., Brouv~enstijn, N., O.santo, S., and Schrier, P.I. (1997), J. Immunother. 2~Q: 343-353.
Vaughan, T.J., et al., (1998), Nature Biotechnol. 16, Case 12/203 SEQUENCE LISTING
<110> Boehringer Ingelheim International GmbH et al.
<120> Tumor associated antigene <130> seqlist12203 <140> ~ .
<141>
<160> 61 <170> PatentIn Ver. 2.1 .
<210> 1 <211> 2216 <212> DNA
<213> Homo sapiens <220>
<221> 5'UTR
<222> (1)..(426) <220>
<221> CDS
<222> (427)..(1743) <220>
<221> 3'UTR
<222> (1744)..(2216) <400> 1 .
G'lCACC~OC~A CZGCCCTIUC '1~~ CTGCCCZTIA C'I~''~IT TI~C~'IT~.'IL3G 60 GA~GCCCTGG GATTCIGCTA ATACC~ C~AGGTGC ~AAOOGAAA C~GATGAACA 120 p~p,~CCIC A~pGAGCTTC CTGmAA7CA GAAGACCAAG C'IGALGCCPG GCAAACATAT 180 4 5 TAA~GGAG CCZGAAACIG TI;CCIT~AC ATCITA' "~' A TG'1'C~rAAAA TACCI'I~f'IGG

AGGGTT~AF~ Gp.TCAGGGGA CA'ICdGTIGIT ~C3CI' GCCAC13GAAC ACCG~C 300 T'I~AC.TIC3GG AF~CAGAAT~' CGCC~1.G~ AGAGATCATC CCTAAGCAOG AC~1C~CI~.1 360 C'IT~AAC~ATT GIGTACTCCT CCACCITCOC T~CI~GT CIC.'C1~C(..'i~T CTCCCATTCT 420 G~ ATG GTT CAA TC~G AAG ~ GTC Tip CAG CTG CAT TAC TTU TGG 468 5 0 Met Val Gln Trp Lys An3 Leu Cys Gln Leu His Tyr Leu Trp GGT CIG GGC ZC,'C TAT ATG CIG CIG GCC ACT GTG GCT CIG AAA CTT TCr 516 Ala Leu Gly Cys Tyr Met Leu Leu Ala 'Thr Val Ala Leu Lys Leu Ser T'IC AGG TIG AAG T~' CSC TCT GAC CAC TIG GGT C'IG GAG TCC FAG GAP. 564 Case 12/203 Phe Arg Leu Lys Cys Asp Ser Asp His Leu Gly Leu Glu Ser An3 Glu TCT CAA PGC CPG TAC Z~'T AOG AAT ATC TIG TAT AAT 612 TTC CTG AAA CIT .

Ser Gln Ser Gln Tyr Cys Arg Asn Ile Leu Tyr Asn Phe Leu Lys Leu CCA GCA AAG AGG TCT ATC AAC zGT TCA GOG GTC ACC 660 CGA GOG GAC QUA

Ptro Ala Lys And Ser Ile Asn Cys Ser Gly Val Thr Arg Gly Asp Gln ~ ~ ~ ~T (~G GCT ATT CIG AAT AzIC CIG GAG GTC 708 AAG AAG AAG

Glu Ala Val Leu Gln Ala Ile Leu Asn Asn Leu Glu Val Lys Lys Lys 80 85 ~ 90 CU~A GAG CCT!' TI'C ACF1 CAC ACC C~.C TAC ChC 756 TCC ChC ACC PGA G'AC 'IGT

An3 Glu Pro Phe Zhr Asp Thr His Tyr Leu Ser Leu Thr Arg Asp Gds 95 100 105 11.0 2 G~ cAC Trc A~ ccr c~ ~ AAG Trc ATA c~ Trc ccA ao4 0 cIG ~C A~

Glu His Phe Lys Ala Glu A~ Lys Phe Ile Gln Phe Pro Leu Ser Lys GAA GAG GIG GAG TrC CCT ATT GCA TAC TCT AZG GIG 852 ATT CAT GAG AAG

2 Glu Glu Val Glu Phe Piro Ile Ala Tyr Ser lit Val 5 Ile His Glu Lys ATT GAA AAC TIT G~1A AC~G CTA CIG 03A GCT GIs 900 TAT GCC CCT CAG AAC

Ile Glu Asn Phz Glu Arg Leu Leu Azg Ala Val Tyr Ala Piro Gln Asn ATA TAC TuT GTC CAT GIG GAT GAG AAG TCC CC~1 GAA 948 ACT TIC AAA G~1G

Ile Tyr Cys Val His Val Asp Glu Lys Ser Pno Glu 'Thr Phe Lys Glu GCG GTC AAA GCA ATT ATT TC.'r TC~C TI'C CC'A AAT 996 GTC TTC ATA GCC AGT

Ala Val Lys Ala Ile Ile Ser Cys Phe Pro Asn Val Phe Ile Ala Ser 4 A~ CIG GIT Ct3G GIG GTT TAT GCC TG'C TC~G TCC 1044 Lys Leu Val Azg Val Val Tyr Ala Ser Trp Ser Azg Val Gln Ala Asp .

CTC AAC TGC A'I~ GAA GAC TIG GTC CI~G pCC TCA 1092 GIG CCG 'Ir3G AAA TAC

4 Leu Asn Cys I~t Glu Asp Leu Leu Gln Ser Ser Val 5 Piro Trp Lys Tyr TIC CIG AAT AC3~ TGT GG1G ACS GAC TIT CCT ATA 1140 AAG PGC AAT GCA GAG

Phe Leu Asn Thr Cys Gly Thr Asp Phe Pro Ile Lys Ser Asn Ala Glu AZG GTC CpG GGT CTC A'9G ATG TIG AAT GGG PGG AAT 1188 1~GC AZG GAG TCA

Met Val Gln Ala Leu Lys Met Leu Asn Gly Arg Asn Ser Met Glu Ser GAG GTA CCT CCT AAG CAC AAA GAA ACC CGC 'IC~G 1236 AAA TAT C~ TTT GAG

Glu Val Pro Pro Lys His Lys Glu Thr AnJ Trp Lys Tyr His Phe Glu ' . CA 02365278 2001-10-26 Case 12/203 GTP. GZG ACS GAC ACS TTA CAC CIA ACC AAC APB 1284 AAG AAG GAT CCT CCC

Val Val Arg Asp Thr Leu His Leu Thr Asn Lys Lys Lys Asp Pro Faro CCT TAT AAT TIA ACT A'IG TIT A~'A GGG AAT GCG 1332 TAC ATT GIG C,~C,T TCC

pro Tyr Asn Leu Thr Met Phe Thr Gly Asn Ala Tyr Ile Val Ala Ser CuA GAT TTC GhC CAA CAT GTT TIG APG AP.C CCT 1380 AAA TCC CAA CAA CIG

An3 Asp Phe Val Gln H:is Val Leu Lys Asn Pro Lys Ser Gln Gln Leu 1 ATT GAA TGG GTA AAA G~ ACr TAT AGC CC~ GAT GAA 1428 Ile Glu Tzp Val Lys Asp Thr Tyr Ser Pro Asp Glu His Leu Txp A7:a ACC CIT CF1G CGT GCA ~G 'IGG AZG CCT GGC TCT 1476 GTr CCC AAC C~ CCC

2 Thr Leu Gln Arg Ala Arg Trp Met Pm Gly Ser Val 0 Pro Asn His Pro 335 340 ' 345 350 AAG TAC GAC ATC Tt~ CSC ATG ACT TCT ATT GCC ~ 1524 CIG GTC A~G 'IC3G

Lys Tyr Asp Ile Ser Asp Met Thr Ser Ile Ala Arg Leu Val Lys Trp CAG GGT CAT GaG GGA GAC ATC GAT ApG GGT GGT C~.T1572 TAT GCT CCC 2~'.C .

Gln Gly His Glu Gly Asp Ile Asp Lys Gly Ala Pro Tyr Ala Pro Cys TCT GGA ATC CAC CAG COG GCT ATC Tv~C GIT TAT 1620 GGG GG'I' GGG GAC T1G

Ser Gly Ile His Gln And Ala Ile Cys Val Tyr Gly Ala Gly Asp Leu 5 AAG TIT CAC CC~

Asn Trp Met Leu Gln Asn His His Leu Leu Ala Asn Lys Phe Asp Pro 400 405 . 410 AAG G'IP. GAT GAT AAT C'CT CTT CAG TGC TTA GAA 1716 GAA TAC C'IP. CST TAT

4 Lys Val Asp Asp Asn Ala Leu Gln Cys Leu Glu Glu 0 Tyr Leu Arg Tyr AAG GCC ATC TAT OGG ACT CAA CIT TC~. GACp~AC1'AT1763 C~1~~IZG

Lys Ala Ile Tyr Gly Thr Glu Leu CTACCIGIC3G GGC~l~GAGC'A TGT~ TGCI~GAAC TIGCICY3GAC1823 AGIGIGGGTG

GG~?GACCI~GG GCITIGCAAT ~GCATC CrTTAGGGATA AG~~(30GCIGC1883 TATTAG~TIG

ZGC3GTAP~GIT3 GATG'1TTIGC CTIGCAAATT C~GCCIC~OG 1943 ~AA~ TTGI''CCTCCC

ACCCC1~1A~CC CTAC~TAGrPC CTCC~C1T~AC TIZC'T'CAC'IP~2003 AGIG<~1AZG ~1GAACIGCIG

5 ~,G ~~; ~,TGIGG T1~C~CIT GATTTCAGIT GAATC~C1GC 2063 Case 12/203 AA~tT ZGAZC3GAAAG AC~A~CTTGC CTTC'!G'~' GI~1A~A AAA'I~AATAG 2183 ChGC'IGATTC AAAGTAAAAA AAAAAAAAAA AAA 2216 <210> 2 <211> 439 <212> PRT
<213> Homo sapiens <400> 2 1 5 Met Val Gln Trp Lys Azg Leu Cys Gln.Leu His Tyr Leu Trp Ala Leu G1Y Cps Tyr Met L~eu Leu Ala Tt~r Val Ala Leu Lys I~eu Ser Phe Arg Izu Lys Cys Asp Ser Asp His~Leu Gly I~eu Glu Ser Arg Glu 5er Gln Ser Gln Tyr Cps Azg Asn Ile Leu Tyr Asn Phe Leu Lys Leu Pzro Al.a Lys Arg Ser Ile Asn Cys Ser Gly Val Thr Azg Gly Asp Gln Glu Al.a .

3 0 Val Leu Gln Ala Ile Leu Asn Asn Leu Glu Val Lys Lys Lys Azg Glu Pzro Phe Thr Asp Thr His Tyr Leu Ser 1xu Thr Arg Asp Cys Glu His Phe Lys Ala Glu Arg Lys Phe Ile Gln Phe Piro Leu Ser Lys Glu Glu Val Glu Phe Pro Ile Ala Tyr Ser Met Val Ile His Glu Lys Ile Glu Asn Phe Glu Arg Leu Leu Azg Ala Val Tyr Ala Pro Gln Asn Ile Tyr 4 5 Cps Val His Val Asp Glu Lys Ser Pn~ Glu Thr Phe Lys Glu Ala Val Lys Ala Ile Ile Ser Cys Phe Pro Asn Val Phe Ile Ala Ser Lys Leu Val Arg Val Val Tyr Ala Ser Tzp Ser Azg Val G1n Ala Asp Leu Asn 5 5 Cys Met Glu Asp Leu Leu Gln Ser Ser Val Piro Trp Lys Tyr Phe Leu ' , ~ CA 02365278 2001-10-26 Case 12/203 Asn Thr Cys Gly 'Il~r Asp Phe Pro Ile Lys Ser Asn Ala Glu Met Val Gln Ala Leu Lys Met Leu,Asn Gly Arg Asn Ser Met Glu Ser Glu Val Pro Pro Lys His Lys Glu Thr Axg Trp Lys Tyr His Phe Glu Val Val 1 0 Azg Asp Thr Leu His Leu T'hr Asn Lys Lys Lys Asp Pro Pro Pro 'I~' Asn Leu Thr Met Phe Tnr Gly Asn Ala Tyr Ile Val Ala Ser Axg Asp Phe Val Gln His Val Leu Lys~Asn Pro Lys Ser Gln Gln Leu Ile Glu ' Trp Val Lys Asp Zhr 'Tyr Ser Pro Asp Glu His Leu Trp Ala Thr Leu , Gln An3 Ala Axg Trp Met Pro Gly Ser Val Pro Asn His Piro Lys 'Iyr 2 5 Asp Ile Ser Asp Met 'rhr Ser Ile Ala Azg Leu Val Lys Tip Gln Gly 355 360 ' 365 His Glu Gly Asp Ile Asp Lys Gly Ala Ptro Tyr Ala Pro Cys Ser Gly Ile His Gln Azg Ala Ile Cys Val Tyr Gly Ala Gly Asp Leu Asn Tip Met Lsu Gln Asn His His Leu Leu Ala Asn Lys Phe Asp Pro Lys Val 3 5 ~ 405 410 415 Asp Asp Asn Ala Leu Gln Cys Leu Glu Glu Tyr Leu Axg Tyr Lys Ala 4 0 Ile Zyr Gly I'hr Glu Leu 45 <210> 3 <211> 2217 <212> DNA
<213> Homo sapiens 50 <220>
<221> 5'UTR
<222> (1)..(426) <220>
55 <221> CDS
<222> (427)..(999) Case 12/203 <220> .
<221> 3'UTR
<222> (1000)..(2217) <220>
<221> 5'UTR
<222> (1)..(844) <220>
<221> CDS
<222> (845)..(1744) <220>
<221> 3'UTR
<222> (1745)..(2217) <400> 3 GTCA~JOCGAA CIGCCC.t~CyC TAC.'ITGIGAC CIGOC~TTT'A C't'C~CCAGIT TTTGThCIGG 60 C~1GCCC~G GATIATACCTATCA CIGI~G'IGC ~AAOGCd~AA CAGA~ 120 2 5 AO'A~Ci'C AAGGAGCITC CMG~C~A'n'aP. C~AG~CAAG CIGA~CCIG GCAAF~GATAT 180 TAAAC~F~C~~G CCI~AAACIG TTCC.TInC'~C AAA ~GAAA1~ TAOCITT'Ir3G 240 AODGTTAGAA GA'ICPGGC3C~GA CA'IC~GTI~IT CACATTIGCT GCCALC'~AAC AGTC 300 TIC~C,'ILC3CG AAC~P.TrA CCCCTIGZ'Gp. AGAGA~C~~'C CGTAAGCAGG AGAGAAC~TA 360 ~CIP~AAC3GATT GTGT~~CCT CCACCTI'CCC TGIGCT03Gr G'TCCACCICGT ~'IT'CT 420 3 5 G~ A'I~ GTT CAA TC~G AFG Ate. CIC 'IGC CAG CIG CAT TAC TIG 'IC~',~ 468 Met Val Gln Trp Lys Arg Leu Cys Gln Leu His Tyr Leu Trp GCr CIG GGC TGC TAT A'I~ CIA CIG GCC ACT GIG GCT CIG AAA CIT TCT 516 4 0 Ala Leu Gly Cys Tyr Met Leu Leu Ala Thr Val Ala Leu Lys Leu Sex TIC AC~G TIG AAG ~T GAC TCT GAC CAC TIG GGT CIG GAG TCC ~1C~G GF1A 564 Phe Arg Leu Lys Cys Asp Ser Asp His Leu Gly Leu Glu Ser Azg Glu '1'CI' CAA AGC C~1G TAC SGT hC~G AAT A'I~ dT'IG T'AT AAT TIC CIG AAA CIT 612 Ser Gln Ser Gln Tyr Cys Azg Asn Ile Leu Tyr Asn Phe Leu Lys Leu 50 v CCA GCA AAG TGG TCI' ATC .'4AC 'IC~T TCA GOG GTC ACC f~A GGG GAC CAA 660 Pro Ala Lys Trp Ser Ile Asn Cys Ser Gly Val Thr Azg Gly Asp G:In 5 5 GF1G GCA G'IG CIT CAG GCT ATT CIG AAT AAC CIG GAG GTC AAG AAG AAG 708 Glu Ala Val Leu Gln Ala Ile Leu Asn Asn Leu Glu Val Lys Lys Lys Case 12/203 CGA GAG OGT T1L~ ACA GAC ACC C3~C TAC CTC TCC CTC ACC AGA GAC 'IGT 756 Azg Glu Pro Phe Thr Asp Thr His Tyr Leu Ser Leu Thr Axg Asp Cys 95 100 105 11.0 GAG CAC TTC AAG GCT GAA AGG AAG TTC ATA C~G TTC CC~. CIG AGC AAA 804 Glu His Phe Lys Ala Glu Azg Lys Phe Ile Gln Phe Pro Leu Ser Lys 1 0 GAA GAG GTG GPG TIC CC.T ATr GCA TAC TCP A'IG GIG ATT CAT GAG AAG 852 Glu Glu Val Glu Phe Pzn Ile Ala Tyr Ser Met Val Ile His Glu Lys ATT GAA AAC TTT GAA A(,3G CTA CIG CGA.GGT GIG TAT GCC CCT CAG AAC 900 1 5 Ile Glu Asn Phe Glu Arg Leu Leu Arg Ala Val Tyr Ala Pro Gln Asn ATA TAC ~T GTC CAT GIG GAT GAA G~AA GhC CCC ~1GA AAC TIT CAA AC3P. 948 Ile Tyr Cys Val His Val Asp Glu Glu Val Pro Axg Asn Phe Gln Axg GGC GGT CAA PGC AAT 'IP.T TIC TIG CIT CCC AAA TGT CTT CAT AGC C~G 996 Gly Gly Gln Ser Asn Tyr Phe I~eu Leu Pro Lys C'ys Leu His Ser Gln TAA GCIGGITJOG GIC3C~TTI'ATG CCrCC~'C CAOOGTGCAA GCIGACCTC~. 1049 * , 3 0 ACIGCATOGA AG~'A~.'~IGChC CAGAGCIC'AG ZGCLGInC~FIA ATACI'PCCIG
AATAC'A'IGIG 1109 GCCTT TCCTATAAAG AGC~AZGC~ AGA'I~Y'A GGChCIC~AG A'IV'!'IGAA'IG 1169 pCApG~,ATAG CAIr3G~ GA~~'ACC:TC CTA~CAC~A AGAAACCGGC TGC~AAT~.TC 1229 ~~~~ pCC Tpp,~,ACAF. G~GAA~3GAT CCICCCCCTT 1289 A~,ATITAAC Tp,~CA C~AATGC~T ACATIGIGGC ThCCCGAGAT ZTCGTC'CAAC 1349 4 0 CAF. G~1ACCCTAAA T'~P~CAAC ZGATIC~ATG GGTAAAAGAC P~~CC 1409 C~,TG~1ACA CC~'IGGGCC ACCCrTCAGC GTGCACX3GZG GFiIGCCIC3GC TCIGI'hCCCA 1469 ACCACCCCAA GTACGACATC TC~1GACAIGA CIT4'rATIGC CACGCIC3GI'C AAG'rGGCAG3G 1529 GTC~1TGAC~~ l~CACp.TCGAT a~ACIC~CzIGCI'C CITACIGCTC'lGGA ATCCACX,'AGC 1589 G~CTKTCIG Q~GG GCIC3GOGACT ~AA2'IC~GAT GCTICA~AAC CA~~ 1649 5 0 TG~C~ACAP. GIfiiG'9CCCA P~AC~GI~A'IG ATAAZGCTCr TQ1G:IGCITA GAAC~1ATAGC

TACGTTAATAA GGCCATC'IAT ~~AC~AC TI'IC~GA~CAC ACS .'TACC 1769 ~G~, ~p~ ~GCI'C ~CITGCT GOGACAGIGT GGGZGGGAGP. 1829 C~~GGGCLZT GC~ATIGGIG GCA'IGCITIA GGATAAGAC~G GCIGGTATTA GATIG'.~' 1889 Case 12/203 p,~T TPIGCCIT~ AAATIGCICC CIC3GGG'IC~~AT GCIGCITGrT CTCTC'~!CC.C 1949 Th,ACaTAGT ~GIT~~CA CI~1A~.TTTCr CF1,G'TAAGIGA GAATG~AC 1GCIGIC'~ATA 2009 GC~GAC'PDGAG3GATA T<'~3GIp1'~G CAC1IGATIT CAG'I7GAAZG CGIG2069 GCITTIGCAT TCIGIGC~AGC TGC'~I'tL''CT AA'I~1A'~I~C~ C3GI'IT3GTAG ~'IC~G~AG 2129 AACIrl'IGATG GAAAG~C~ CI'I'CCCrI'C'T GAP. CTTA,AAAP.TA AATAGCI'C<.'r 2189 ~ p~ p~,~A 2217 <210> 4 <211> 190 <212> PRT
<213> Homo sapiens <400> 4 ' Met Val Gln Trp Lys Azg Leu Cys Gln Leu His Tyr Leu Trp Ala Leu Gly Cys Tyr Met I~eu Leu Ala Thr Val Ala Leu Lys Lzu Ser Phe A~eg Leu Lys Cys Asp Ser Asp His Leu Gly Leu Glu Ser Axg Glu Ser Gln Ser Gln Tyr Cys Azg Asn Ile Leu Tyr Asn Phe Leu Lys Leu Pro Ala 3 5 Lys Tzp Ser Ile Asn Cys Ser Gly Val Thr Arg Gly Asp G1n Glu Ala Val Leu Gln Ala Ile Leu Asn Asn Leu Glu Val Lys Lys Lys Azg Glu Pro Phe Thr Asp Thr His Tyr Leu Ser Leu T'hr Axg Asp Cps Glu His Phe Lys Ala Glu Arg Lys Phe Ile Gln Phe Pro Leu Ser Lys Glu Glu Val Glu Phe Pro Ile Ala Tyr Ser Met Val Ile His Glu Lys Ile Glu 5 0 Asn Phe Glu Azg Leu Leu Arg l~la Val Tyr Ala Pro Gln Asn Ile Tyr Cys Val His Val Asp Glu Glu Val Pro Azg Asn Phe Gln Arg Gly Gly Gln Ser Asn Tyr Phe Leu Leu Piro Lys Cys Leu His Ser Gln g Case 12/203 <210> 5 <211> 2217 <212> DNA

<213> Homo sapiens <220>

1 <221> 5'UTR

<222> (1) . . (844) <220>

<221> CDS

<222> (845)..(1744) <220>

<221> 3'UTR ' <222> (1745)..(.2217) w <400> 5 GI'CA030GAA CIGCCCTIGC Zi,GAC CIGCCCITlA CTCAGCAGIT60 'I~G

GAE~GC~G GATI~'IGC'm ATACCI~.TCA CIGI~OGIGC TGF~C~OGAAA120 CAGA'IGAAGA

A~'ATG'P~CTC AA~GCTrC CIGrCAA~ C-AAGACC~AG G'IGA~'7GCCrG180 C'CAAAGAT~1T ~

3 TAAAGAGGAG CCl~~AAAC'.I~ TTCCITaGAC AZ'CITATGAA 240 0 ~G~AAA TACCTTT1GG

AGGG'T17!GAA C~TC~GGGp. CA'IC3GI~Tr CAC~T'IT~.T 300 GCC~GOGAAC ACC~CC~1GI'C

TTC~CIZ~~OG 19AC~P.1CA CGCCr'~IG~ AG~TCATC CCTAAGCAGG360 AGF~GAAC~C'.IT~

CTAA~GP.TI' GTC'zTAG!'CGT CCACCTI~CC ~Cr00CT CTC'CACCIGT420 CTCCCAITGT

GI~~.~G TmAAT3GAA G~PL~I~'~C CF~GCIC~~.TT AGTIGIC30CC480 TC'IC~GC.'ICdC

4 TATA'IGCZGC 'IC3GCCACIGT GGCI~AAA CTI'I'C'ITTCPr 540 0 OG~'l~AAGIG ~.'rCl.~AC

C~ICrIC3GGTC 'I~C'.~IC.''C~1G GGAAT~a AGCCAGTACr 600 GIAC~AATP.T CTIG'lATAAT

TI~CCIGAAAC T'ICCA~~AA GIGGrCTA'rC AACIC~'~TCpG 660 GGGII~CCCG Af3GGGP.C:C~A

C~AGIGC TTCAGG<.'rAT TCIGAA'rAAC C~CaGAf3G'TC'A 720 AGAAG~1AGCG PG~1GC~TrC

ACI~C~CCC ACIP,~JCrCTC CCrCA~ G~P~C AGC ~AAALaC'~780 5 cr Tcec~~crG~ C~~ GrGc crATyc~TA cz~~~Gr~ a4o ATIC ATG PC,~ AGA TIG AAA ACT TIG AAA GGC TAC 889 'IGC C~ CIG ~T A~

Met Arg Arg Leu Lys Thr Leu Lys Gly Tyr Cys Glu Leu Cys Met CCC CTC AGA ACA TAT ACT GIG TCC A'I~ ZC3G A~ AAG 937 AAG Ti C GC'A GAA

Ptro Leu Azg Thr Tyr Thr Val Ser Met Trp Met Lys Lys Ser Ftro G1u Case 12/203 20 . 25 30 AL'r TTC AAA GAG GCS GIC AAA GCA ATT ATT TCT IGC 985 TTC CCA AAT GTC

Thr Phe Lys Glu Ala Val Lys Ala Ile Ile Ser Cys Phe Pro Asn Val TIC ATA GCC AGT .'9AG CIG GTT 0f3G GIG GTT TAT 1033 GCC TCC Tu~G TCC AGG

Phe Ile Ala Ser Lys Leu Val Azg Val Val Tyr Ala Sex Trp Ser Azg 50 55 . 60 Grc cAA ccT c-~c cTC AAC TcC A~ c~ c~.c TIC c-rc foal c~c Arc TCA cIC

Val G1n Ala Asp Leu Asn Cys Met Glu Asp Leu Leu Gln Ser Ser Val C~ 'IC3G AAA TAC TTC C1G AAT ACA TC~T GGG ACJ 1129 S GAC TIT CCT ATA AFG

Pn~ Trp Lys Tyr Phe Leu Asn Thr Cys Gly Thr Asp Phe Pro Ile Lys P~GC AAT GCA GAG A'IG GTC CAG GCT CTC AAG ATG 1177 TIG AAT GGG PDG AAT

2 Ser Asn Ala Glu Met Val Gln Ala Leu Lys Met Leu 0 Asn Gly Arg Asn ~

~ 105 110 AGC ATG GAG Tv~ GPG GTP. GCT CGT AAG C~.C AAA 1225 GAA ACC CSC 'n.3G AAA

Ser Met Glu Ser Glu Val Pro Pro Lys His Lys Glu Zhr Arg Trp Lys TAT CAC TIT GAG GTA GIG AGA CAC AO'A Tg1 CAC CI'A1273 ACC AAC AAG AFG .

Tyr His Phe Glu Val Val Ar3 Asp 'I~r Leu His Leu Thr Asn Lys Lys AAG GAT CCT C'CC C'C'T TAT AAT TTA ACT ATG TTT 1321 ACA GOG AAT GCG TAC

Lys Asp Pro Pro Pro Tyr Asn Leu Thr Met Phe Thr Gly Asn Ala Tyr Ile Val Ala Ser Arg Asp Phe Val Gln His Val Leu Lys Asn Pro Lys TCC CAA CAA CIG ATT GAA ~ G'IA AAA GAC ACT TAT 1417 AGC CC~1 GAT GAF.

4 Ser Gln Gln Leu Ile Glu Trp Val Lys Asp Thr Tyr 0 Ser Piro Asp Glu CAC CIC 'IC~G GCC ACC CTT CFG CGT GCA 03G ~G AZG 1465 CCT GGC TCT GIZ' His Leu.Trp Ala Thr Leu Gln Axg Ala An3 Txp Met Pzro Gly Ser Va1 CCC AAC C~ OCC AAG T~1C GAC ATC TCA GAC AZG ACT 1513 TCT ATT GCC PLUG

Pro Asn His Pm Lys Tyr Asp Ile Ser Asp Met Thr Ser I1~ Ala Arg 50 v CIG G1'C AFG n3G CAG GGT CAT GAG GGA GAC ATC GAT 1561 AAG C3GT GCT C'G'I' Letz Val Lys Trp Gln Gly His Glu Gly Asp Ile Asp Lys Gly Ala Pro 5 TAT GCT . CCC IGC TCT GGA ATC CAC C~SG CMG GCT 1609 5 ATC 'IC'C GTr TAT GOG

Tyr Ala Pn~ Cys Ser Gly Ile His Gln Azg Ala Ile Cys Val Tyr Gly Case 12/203 Gcr c~oG GG~cc Tlc AAT TGG ~ crr c~A AAC cAT c~c cIC TIC c~ AAC 1657 Ala Gly Asp Leu Asn Trp Met Leu Gln Asn His His Leu Leu Ala Asn AAG TIT GAC CC~1 AAG GTA GAT GAT AP.T GCT CTT CAG Tu"C TTA GAA GAA 1705 Lys Phe Asp Pro Lys Val Asp Asp Asn Ala Leu Gln Cys Leu Glu Glu TAC CTA QiT TAT AAG GCC ATC TAT GGG ACT GAA CTT ~ C~ACACAO.'~AT 1754 Tyr Leu Arg Tyr Lys Ala Ile Tyr Gly Thr Glu Txu G~AG~G CTAGCIGIC~G GC'~CAAG~GGA ZG. ~CZG~AC TIGCI~~C 1814 .~C~IGZ~IG GGAGACCAGG GCITIGCAAT T~1~~GC'P.'I~ C'1'ITI~CACCIL;C 1874 Tp~T~,~ TuG~ ~C C?'IGG~AATT GCIGG'C'tC~3G TuAAZGC'1~"'.~C 1934 2 0 'I~TCPCPC ACCCGI'AACC CT~GI~'ITC CTCCACTAAC TTTC'11CACTA PA'IG 1994 ~~ ~CGPG FLIT C'~ C~TGIC3G TPGA(sCAC.TT C3ATTIC~IT 2054 GAATGCCIG~C C.'CIT ~CAT'tGTG'I' GGAGCIGCCG TICCTAA'I~A TTCCAGGTIT 2114 GGTAC'~DG AGGAGAAC'TT WC~AAG P~ACCTI'CC C'II!' GTT~ACZTAA 2174 AAATAAP.TAG CTCCIGATl'C AAAGI'AAAAA AAAAAAAAAA AAA ~ ~ 2217 <210> 6 <211> 300 <212> PRT
3 5 <213> Homo sapiena , <400> 6 4 4 Met Arg Azg Leu Lys Thr Leu Lys Gly Tyr Cps Glu Leu Cys Met Pro Leu Azg Thr Tyr Thr Val Ser Met Tzp Met Lys Lys Ser Pzro Glu Thzv Phe Lys Glu Ala Val Lys Ala Ile Ile Ser Cys Phe Pro Asn Val Phe Ile Ala Ser Lys Leu Val An3 Val Val Tyr Ala Ser Trp Ser An3 Val 5 0 50 55 v 60 Gln Ala Asp Leu Asn Cys Met Glu Asp Leu Leu Gln Ser Ser Val Pra 5 5 Trp Lys Tyr Phe Leu Asn Thr Cys Gly Thr Asp Phe Pro Ile Lys Ser Case 12/203 Asn Ala Glu Met Val Gln Ala Leu Lys Met Leu Asn Gly Azg Asn Ser Met Glu Ser Glu Val Pro Pro Lys His Lys Glu Thr An3 Trp Lys Tyr 115 120 ~5 His Phe Glu Val Val Arg Asp Thr Leu His Leu 'Il~r Asn Lys Lys Lys Asp Pro Pro Pro Tyr Asn Leu 'iii' Met Phe Thr Gly Asn Ala Tyr Ile Val Ala Ser Axg Asp Phe Val G7n His Val Leu Lys Asn Pro Lys Ser Gln Gln Leu Ile Glu Txp Val Lys Asp Thr Tyr Ser Pro Asp Glu His Leu Tzp Ala Thr Leu Gln Axg Ala Azg Trp Met Pro Gly Ser Val Pro Asn His Pro Lys Tyr Asp Ile,Ser Asp Met~I'hr Ser Ile Ala An3 Leu 2 5 Val Lys Trp Gln Gly His Glu Gly Asp Ile Asp Lys Gly Ala Pro Tyr Ala Pro Cys Ser Gly Ile His Gln Azg Ala Ile Cys Val Tyr Gly Ala Gly Asp Leu Asn Trp Met Isu Gln Asn His His 7xu Ixu Ala Asn Lys Phe Asp Pro Lys. Val Asp Asp Asn Ala Leu Gln Cys Leu Glu Glu Tyr Lieu Azg Tyr Lys Ala Ile Tyr Gly Thr Glu Leu <210> 7 <211> 25 <212> PRT
4 5 <213> Homo Sapiens <400> 7 °

<210> 8 <211> 22 <212> DNA
<213> Artificial sequence Case 12/203 <220>
<223> Primer <400> 8 <210> 9 <211> 26 <212> DNA
<213> Artificial sequence <220>
<223> Primer <400> 9 _ <210> 10 <211> 27 <212> DNA
<213> Artificial sequence <220>
<223> Primer <400> 10 <210> 11 <211> 21 <212> DNA
<213> Artificial sequence <220>
4 5 <223> Primer <400> 11 <210> 12 <211> 21 5 5 <212> DNA
<213> Artificial sequence Case 12/203 <220> .
<223> Primer <400> 12 <210> 13 <211> 20 <212> DNA
<213> Artificial sequence <a2o>
<223> Primer <400> 13 <210> 14 <211> 22 <212> DNA
<213> Artificial sequence .
<220>
<223> Primer <400> 14 <210> 15 <211> 24 4 0 <212> DNA
<213> Artificial sequence <220>
<223> Primer <400> 15 ~
<210> 16 <211> 27 <212> DNA
5 5 <213> Artificial sequence <220>
1~

Case 12/203 <223> Primer <400> 16 <210> 17 <211> 28 <212> DNA
<213> Artificial sequence <220>
<223> Primer <400> 17 TTCCATCAAA GTTCTCCTCC ACGCTACC ~~ ~ 28 <210> 18 <211> 28 <212> DNA
<213> Artificial sequence <220>
<223> Primer <400> 18 <210> 19 <211> 43 <212> DNA

4 <213> Artificial sequence <220>

<223> Primer <400> 19 TGTAGCGTGA
AGACGACAGA
AAGGGCGTGG
TACCGAGCTC
GAG

v <210> 20 <211> 22 <212> DNA

<213> Artificial sequence <220>

<223> Primer Case 12/203 <400> 20 <210> 21 <211> 11 <212> DNA
<213> Artificial sequence <220>
<223> Primer <400> 21 <210> 22 <211> 9 <212> PRT
<213> Homo Sapiens <400> 22 Lys Arg Leu Cys Gln Leu His Tyr Leu <210> 23 ' <211> 9 <212> PRT
<213>.Homo Sapiens <400> 23 Tyr Leu Trp Ala Leu Gly Cys Tyr Met <210> 24 .
<211> 9 5 0 <212> PRT t <213> Homo Sapiens <400> 24 Ile Leu Asn Asn Leu Glu Val Lys Lys 't6 Case 12/203 <210> 25 <211> 9 <212> PRT
<213> Homo Sapiens <400> 25 Glu Val Lys Lys Lys Arg Glu Pro Phe <210> 26 <211> 9 <212> PRT
2 0 <213> Homo Sapiens <400> 26 2 5 Arg Glu Pro Phe Thr Asp.Thr His Tyr 30 <210> 27 <211> 9 <212> PRT
<213> Homo sapiens <400> 27 Glu Pro Phe Thr Asp Thr His Tyr Leu <210> 28 <211> 9 4 5 <212> PRT
<213> Homo sapiens <400> 28 Pro Leu Ser Lys Glu Glu Val Glu Phe <210> 29 <211> 9 Case 12/203 <212> PRT
<213> Homo sapiens <400> 29 Glu Glu Val Glu Phe Pro Ile Ala Tyr <210> 30 <211> 9 <212> PRT
<213> Homo sapiens <400> 30 Glu Arg Leu Leu Arg Alal'Val Tyr Ala <210> 31 <211> 9 .
<212> PRT
<213> Homo Sapiens <400> 31 Phe Ile Ala Ser Lys Leu Val Arg Val <210> 32 <211> 9 <212> PRT
<213> Homo sapiens <400> 32 Ash Leu Asn Cys Met Glu Asp Leu Leu <210> 33 <211> 9 <212> PRT
<213> Homo sapiens Case 12/203 <400> 33 Ser Met Glu Ser Glu Val Pro Pro Lys <210> 34 <211> 9 <212> PRT
<213> Homo sapiens <400> 34 Thr Leu His Leu Thr Asn Lys Lys Lys <210> 35 <211> 10 ' <212> PRT
<213> Homo sapiens <400> 35 ' Phe Val Gln His~Val Leu Lys Asn Pro Lys <210> 36 <211> 9 <212> PRT
<213> Homo sapiens <400> 36 Leu Ile Glu Trp Val Lys Asp Thr Tyr <210> 37 <211> 9 <212> PRT
5 0 <213> Homo sapiens r <400> 37 5 5 Trp Met Leu Gln Asn His His Leu Leu Case 12/203 <210> 3a <211> 9 <212> PRT
<213> Homo Sapiens <400> 38 Leu Leu Ala Asn Lys Phe Asp Pro Lys <210> 39' <211> 9 <212> PRT
<213> Homo Sapiens <400> 39 Asp Pro Lys Val Asp Asp Asn Ala Leu <210> 40 <211> 9 <212 > PRT
<213> Homo sapiens <400> 40 Gln Cys Leu Glu Glu Tyr Leu Arg Tyr <210> 41 <211> 9 <212> PRT
4 5 <213> Homo sapiens <400> 41 Cys Leu Glu Glu Tyr Leu Arg Tyr Lys <210> 42 <211> 9 <212> PRT

Case 12/203 <213> Homo sapiens~
<400> 42 Ala Val Tyr Ala Pro Gln Asn Ile Tyr <210> 43 <211> 9 <212> PRT
<213> Homo sapiens <400> 43 Tyr Ala Pro Gln Asn Ile Tyr Cys Val <210> 44 <211> 9 <212> PRT
<213> Homo sapiens <400> 44 Tyr Cys Val His Val Asp Glu Glu Val <210> 45 <211> 9 <212> PRT

4 <213> Homo sapiens <400> 45 4 His 5 Val Asp Glu Glu Val Pro Arg Asn 50 <210> 46 <211> 9 <212> PRT

<213> Homo sapiens <400> 46 z~

Case 12/203 Val Asp Glu Glu Val Pro Arg Asn Phe <210> 47 <211> 9 <212> PRT
<213> Homo sapiens <400> 47 Phe Gln Arg Gly Gly Gln Ser Asn Tyr <210> 4a <211> 9 <212> PRT
<213> Homo sapiens <400> 48 Gln Ser Asn Tyr Phe Leu Leu Pro Lys <210> 49 <211> 9 <212> PRT
<213> Homo sapiens <400> 49 4 0 Leu Leu Pro Lys Cys Leu His Ser Gln <210> 50 <211> 9 <212> PRT
<213> Homo sapiens <400> 50 Arg Arg Leu Lys Thr Leu Lys Gly Tyr ZZ

Case 12/203 <210> 51 <211> 9 <212> PRT
<213> Homo sapiens <400> 51 Lys Thr Leu Lys Gly Tyr Cys Glu Leu <210> 52 <211> 9 <212> PRT
<213> Homo sapiens <400> 52 Tyr Cys Glu Leu Cys Met Pro Leu Arg <210> 53 <211> 9 <212> PRT
3 0 <213> Homo Sapiens <400> 53 Glu Leu Cys Met Pro Leu Arg Thr Tyr <210> 54 <211> 9 <212> PRT
<213> Homo Sapiens <400> 54 "
Cys Met Pro Leu Arg Thr Tyr Thr Val _ <210 > 55 <211> 9 5 5 <212> PRT
<213> Homo sapiens ' , CA 02365278 2001-10-26 Case 12/203 <400> 55 Pro Leu Arg Thr Tyr Thr Val Ser Met <210> 56 <211> 10 <212> PRT
<213> Homo sapiens ' <400> 56 Tyr Leu Trp Ala 2eu Gly Cys Tyr Met Leu <210> 57 <211> 9 <212> PRT
<213> Homo sapiens <400> 57 Tyr Met Leu Leu Ala Thr Val Ala Leu <210> 58 <211> 9 <212> PRT
4 0 <213> Homo sapiens <400> 58 4 5 Leu Leu Ala Thr Val Ala Leu Lys Leu ° 1 5 r 50 <210> 59 <211> 10 <212> PRT
<213> Homo sapiens Z~

Case 12/203 <400> 59 Cys Met Glu Asp Leu Leu Gln Ser Ser Val <210> 60 <211> 9 1 0 <212> PRT
<213> Homo sapiens <400> 60 Glu Val Asp Pro Ile Gly His Leu Tyr <210> 61 <211> 218 <212> PRT
<213> Homo sapiens <400> 61 3 0 Asn Lys Phe Asp Pro Lys Val Asp Asp Asn Ala Leu Gln. Cys Leu Glu Glu Tyr .r zs Velculescu, VE, Zhang, L, Vogelstein, B, and Kinzler, KW
(1995), Science 270: 484-487 Wang, L., et al., (1997), Mol. Immunol. 34: 609-618 Wang, RF (1997), Mol. Med. 3.: 716-731 Wax, S.D., Rosenfield, C.L., and Taubman, M.B., (1994), J.
Biol. Chem. 269, 13041-13047.
Wax, S.D., Tsao, L., Lieb, M.E., Fallon, J.'f., and Taubman, M.B. (1996), Lab. Invest. 24, ',797-808.
Winter, G., et al., (1994), Annu. Rev. Immunol. 12, Woelfel, T, Schneider, J, Zum Buschenfelde, Meyer, KH, Rammensee, HG, Rotzschke, 0, and Falk, K (1994), Int.
J. Cancer 52: 413-418 Woelfel T, Hauer M, Schneider J, Serrano M, Wolfel C, Klehmann-Hieb E, De Plaen E, Hankeln T, Meyer zum Biischenfelde KH, Beach D (1995), Science 269:

Wu, T.C., Guarnieri, F.G., Staveley-O'Carrol.l, K.F., Viscidi, R.P., Levitsky, H.I., Hedrick, L., Cho, K.R., August, J.T., and Pardoll, D.M. (1995), Proc. Natl.
Acad. Sci. U.S.A. x(25): 11671-11675.
Zitvogel, L., Mayordomo, J.I., Tjandrawan, T., DeLeo, A.B., Clarke, M.R., Lotze, M.T., and Storkus, W.J.
(1996), J. Exp. Med. 1~3, 87-97.

Claims (21)

Claims

1. Tumour-associated antigen designated B99, selected from the group of polypeptides having the amino acid sequence given in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

2. Immunogenic protein fragment or peptide, characterised in that it is derived from a tumour-associated antigen as defined in claim 1.

3. Immunogenic (poly)peptide according to claim 1 or 2, which triggers a humoral immune response.

4. Immunogenic (poly)peptide according to claim 1 or 2, which, or the breakdown products of which, are presented by MHC molecules and trigger a cellular immune response.

5. Immunogenic peptide according to claim 4, selected from the group of peptides according to SEQ ID NO:22 to 55.

6. Immunogenic peptide according to claim 5, selected from the group of peptides according to SEQ ID NO:31, 57 and 59.

7. Immunogenic (poly)peptide according to one of claims 1 to 6 for the immunotherapy of cancer diseases in vivo or ex vivo, wherein the (poly)peptide induces an immune response against the patient's tumour cells which express B99.

8. Pharmaceutical composition for parenteral, topical, oral or local administration, characterised in that it contains as active component one or more immunogenic (poly)peptides according to one of claims 1 to 6.

9. Pharmaceutical composition according to claim 8, characterised in that it contains various immunogenic peptides derived from B99.

10. Pharmaceutical composition according to claim 9, characterised in that it contains one or more peptides derived from B99 in admixture with peptides derived from other tumour-associated antigens.

11. Pharmaceutical composition according to claim 9 or 10, characterised in that the peptides bind to at least two different HLA types.

12. Isolated DNA molecule, coding for a protein having the immunogenic properties of a tumour-associated antigen as defined in claim 1 or for fragments thereof.

13. DNA molecule according to claim 12, coding for an immunogenic polypeptide designated B99 having the amino acid sequence shown in SEQ ID NO:2, 4 or 6 or for protein fragments or peptides derived therefrom.

14. DNA molecule according to claim 13, characterised in that it is a polynucleotide having the sequence shown in SEQ ID NO:1, 3 or 5 or hybridises with a polynucleotide having the sequence shown in SEQ ID
NO:1, 3 or 5 under stringent conditions.

15. Recombinant DNA molecule, containing a DNA molecule according to one of claims 12 to 14.

16. DNA molecule according to one of claims 12 to 15, for the immunotherapy of cancer diseases, wherein the B99 (poly)peptide expressed by the DNA molecule induces an immune response against the patients tumour cells which express B99.

17. Pharmaceutical composition, containing as active ingredient one of more of the DNA molecules defined in one of claims 12 to 16.

18. Use of cells which express the tumour-associated antigen defined in claim 1, for the production of a cancer vaccine.

19. Antibody against a (poly)peptide as defined in one of claims 1 to 6.

20. Antibody according to claim 19, characterised in that it is monoclonal.

21. Antibody according to claim 19 or 20 for the therapy and diagnosis of cancer diseases associated with the expression of B99.