WO2000052157A1 - Tumor-associated antigen - Google Patents

Abstract

The invention relates to a tumor-associated antigen, to immunogenic peptides derived therefrom, to DNA molecules coding said tumor-associated antigen and to the use of said DNA molecules in the immunotherapy of cancers.

Description

 Title: Tumor Associated Antigen

The invention relates to the immunotherapy of tumor diseases.

The immune system's task is to protect the organism from a variety of different microorganisms or to actively combat them. The importance of an intact immune system is particularly evident in inherited or acquired immunodeficiencies. In many cases, the use of prophylactic vaccine programs has proven to be an extremely effective and successful immunological intervention in the fight against viral or bacterial infectious diseases. Furthermore, it has been shown that the immune system is also significantly involved in the elimination of tumor cells. The detection of tumor-associated antigens (TAAs) by components of the immune system plays an important role. In the broadest sense, any (peptide or non-peptide) component of a tumor cell that is recognized by an element of the immune system and can be used

Stimulation of an immunological response leads to act as an immunogenic tumor antigen. Of particular importance are those tumor antigens that not only cause an immunological reaction, but also cause rejection of the tumor. The identification of defined antigens that can cause such an immunological reaction is an important step in the development of a molecularly defined tumor vaccine. Although it is not yet clear which elements of the immune system are responsible for rejecting the Tumor are responsible, but there is a consensus that CD8-expressing cytotoxic T-lymphocytes (CTLs) play a major role (Coulie, 1997). Especially for those types of tumors (e.g. melanoma and kidney cancer) that have a relatively high

A spontaneous remission rate showed a correlation between clinical course and the increased occurrence 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 tumor infiltrating lymphocytes (TIL) or peripheral mononuclear blood cells (PBMC) after cocultivation with mostly autologous tumor cells and cytokine stimulation in vitro. In animal models as well as in human cell culture systems cultivated in vitro, the T cell response against tumor cells could be strengthened by transfection of the tumor cells with cytokines (van Elsas et al., 1997;

Gansbacher et al., 1990; Tepper et al., 1989; Fearon et al., 1990; Dranoff et al., 1993).

Because of the correlation between remission and involvement of CD8 ⁺ T cells, the identification of tumor associated antigens (TAA) is made by

CD8-positive CTLs are recognized, a stated main goal on the way to the development of a tumor vaccine (Pardoll, 1998; Robbins and Kawakami, 1996). It is still unclear whether other cell types of the immune system such as CD4 ⁺ T helper cells also play an important role; some studies with MAGE-3 / HLA-A1 peptides in melanoma patients suggest this (Marchand et al., 1995; Boon et al., 1998). A number of TAAs recognized by CTLs have been identified in recent years (Boon et al., 1994; van den Eynde and van der Bruggen, 1997).

T cells recognize antigens as peptide fragments that are presented on cell surfaces of MHC molecules ("major histocompatibility complex", in humans "HLA" = "human leukocyte antigen"). There are two classes of MHC molecules: MHC-I molecules occur on most cells with a nucleus and present peptides (usually 8-10 mers) which are formed by proteolytic degradation of endogenous proteins (so-called antigen processing, "antigen processing"). Peptide: MHC-I complexes are recognized by CD8-positive CTLs. MHC-II molecules only come on so-called

"Professional Antigen Presenting Cells" (APC) and present peptides of exogenous proteins that are absorbed and processed by APC during the endocytosis. Peptide: MHC-II complexes are recognized by CD4 helper T cells. Through an interaction between T-cell receptor and peptide: HC complex, various effector mechanisms can be triggered which lead to apoptosis of the target cell in the case of CTLs, either when the MHC (eg in the case of transplant rejection) or the peptide (eg in the case of intracellular pathogens) ) is recognized as foreign, however not all peptides presented meet the structural and functional requirements for effective interaction with T cells (as described by Rammenee et al., 1995 and below). In principle, several application forms are possible for the use of TAAs in a tumor vaccine: the antigen can either be used as a recombinant protein with suitable adjuvants or carrier systems, or as a cDNA coding for the antigen in plasmid (DNA vaccine; Tighe et al., 1998) , or viral vectors (Restifo, 1997) are applied. Another possibility is the use of recombinant bacteria (eg Listeria, Salmonella), which recombinantly express the human antigen and which have an adjuvative effect due to their additional components (Paterson, 1996; Pardoll, 1998). In all of these cases, processing and presentation of the antigen by so-called "professional antigen-presenting cells" (APC) is necessary. Another possibility is the use of synthetic peptides (Melief et al., 1996), which correspond to the corresponding T cell Epitopes of the antigen correspond and are either loaded onto the APC from the outside (Buschle et al., 1997; Schmidt et al., 1997) or taken up by the APC and transferred intracellularly to the MHC I molecules Antigen is generally determined in clinical studies.

To those recognized by the tumor specific CTLs

Antigens or their epitopes include molecules that can originate from all protein classes (eg transcription factors, receptors, enzymes; for an overview see Rammenee et al., 1995; Robbins and Kawakami, 1996). These proteins do not necessarily have to be located on the cell surface, as is the case with detection by antibodies is required. In order to act as a tumor-specific antigen for detection by CTLs or to be used for therapy, the proteins must meet certain conditions: firstly, the antigen should only be expressed by tumor cells or only in a lower concentration than so-called "critical" normal tissues occur in tumors. Critical normal tissues are essential tissues; an immune reaction against them would have serious, sometimes fatal consequences. Second, the antigen is said to be present not only in the primary tumor, but also in the metastases. Furthermore, in view of a wide clinical use of the antigen, it is desirable if it is present in high concentration in several types of tumor. Another

A prerequisite for the suitability of a TAA as an effective component of a vaccine is the presence of T cell epitopes in the amino acid sequence of the antigen; Peptides derived from TAA are said to lead to an in vitro / in vivo T cell response ("immunogenic" peptide). Another selection criterion for a clinically widely applicable immunogenic peptide is the frequency with which the antigen is found in a given patient population.

The immunogenic tumor-associated antigens (TAAs), most of which have already been shown to have T cell epitopes, can be divided into several categories, including: viral proteins, mutated proteins, overexpressed proteins, fusion proteins formed by chromosomal translocation,

Differentiation antigens, oncofetal antigens (Van den Eynde and Brichard, 1995; van den Eynde and van der Bruggen, 1997).

The methods for identifying and characterizing TAAs, which are the starting point for the development of a tumor vaccine, are based on the one hand on the use of CTLs (cellular immune response) or antibodies (humoral immune response) that have already been induced in patients, or are based on the creation of differential transcription profiles between tumors and normal tissues. In the first case, the immunological approach, patient CTLs are used for screening eukaryotic tumor cDNA expression libraries which present the CTL epitopes via MHC-I molecules (Boon et al., 1994), while using high-affinity patient antisera prokaryotic cDNA expression libraries can be examined directly for the presence of TAAs via an immunoblot analysis of the individual plaques (Sahin et al., 1995). A combination of CTL reactivity and protein chemical methods represents the isolation of peptides isolated from MHC-I from tumor cells which have been preselected for reactivity with patient CTLs. The peptides are washed out of the MHC-I complex and identified using mass spectrometry (Falk et al., 1991; Woelfel et al., 1994; Cox et al., 1994). The approaches that use CTLs to characterize antigens are associated with considerable effort or not always successful due to the required cultivation and activation of CTLs. Methods for the identification of TAAs, which are based on the comparison of the transcription profile of normal with tumor tissue, are diverse; these include differential hybridization, the creation 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 with the help of patient CTLs, the use of molecular biological methods must show that the potential antigen candidates found with them are tumor-specific (tumor-associated) and actually have T-cell epitopes that can trigger a cytotoxic T-cell response . In at least one case

(NY-ESO / LAGE-1), an antigen was identified by both the use of patient sera and RDA (Chen et al., 1997; Lethe et al. 1998), and CTL epitopes of this antigen and a simultaneous spontaneous humoral were and T cell response in a patient (Jager et al., 1998).

The object of the present invention was to provide a new tumor-associated antigen (TAA).

This task was solved by first using RDA ("representational difference analysis") between

Renal cell carcinoma and normal kidney tissue a cDNA subtraction library was prepared and this was hybridized with a testis-specific cDNA library in order to select antigens of the tumor / testis type. The cDNA clones obtained were sequenced and compared with sequences available in databases. Among the genes identified, 29 were unknown, for which ESTs (“expressed sequence tags”) entries existed in the database. Of these genes, 16 were examined in more detail, whose ESTs do not originate from critical normal tissue. RT-PCR was used to determine "Five of the sixteen clones examined showed a clear overexpression in renal cell carcinoma compared to normal kidney tissue. The quantitative comparison (by means of PCR) of the expression of one of the clones (9D7) between tumor and normal tissue showed a clear overexpression of the 9D7 CDNA in the tumor The expression profile analyzed by Northern Blot additionally showed that 9D7 showed no or only weak transcription in the normal tissues examined, while the hybridization of renal cell carcinoma and normal kidney tissue with a 9D7-specific labeled RNA probe confirmed this by RT-PCR, quantitative PCR and Northern Results obtained blot.

The human 9D7 cDNA was completely cloned, the sequence obtained is shown in SEQ ID NO: 1. The sequence of 9D7 shows, at the nucleotide and protein levels, a high homology to SM20, an "immediate early gene" induced by growth factors in rat smooth muscle cells, which has been described as a specific marker for smooth aortic muscle cells (Wax et al. 1994, 1996) Sequence analysis of the human 9D7 cDNA in comparison with SM20 showed that the first 323 bp clearly shows a have lower identity than the rest of the cDNA (40% compared to 96% at the deduced amino acid sequence level). Since the human cDNA has the first ATG at the transition between higher and lower identity with SM20 at position 324, it can be assumed that this is the start codon of the human 9D7 gene. Since the sequence upstream from position 324 also has a continuous reading frame which, although less, shows significant identity with SM20 (40% at the level of the derived amino acid sequence), it cannot be ruled out that a start codon is present further upstream.

The information about the further upstream sequence can be obtained by molecular biological

Standard methods are obtained, for example using 5'-RACE ("rapid amplification of cDNA ends"). In this method, RNA, preferably mRNA, is obtained from cells or tissues in which 9D7 is transcribed (eg RCC tissue or cell lines derived from RCC such as 786 -0, see Example 9) reverse transcribed and then ligated with an adapter of known sequence PCR with an adapter primer (binds specifically to the adapter at the 5 'end of the cDNA) and a 9D7-specific primer (eg SEQ ID NO: 21 , 19, 17, 16, 14, 12, 11, 4) allows the corresponding 9D7 fragments to be amplified.As described in Example 1, these PCR products can be cloned by standard methods and characterized, in particular by DNA sequencing. An alternative method to characterize the

5 'end is the screening of cDNA libraries

Hybridization with DNA probes specific for 9D7 or

Antisera.

Performs the screening of cDNA libraries due to methodological restrictions, e.g. Inefficient reverse transcription due to pronounced secondary structures of the RNA, not to the desired target, genomic libraries can be examined, for example, by cloning, as with the screening of cDNA libraries, by hybridization with DNA probes specific for 9D7 which isolate the upstream of the obtained 5 'end of the cDNA sequence information, for example contain the promoter region of 9D7.

The isolated cDNA codes for the tumor-associated antigen (TAA) of the designation 9D7 with the amino acid sequence given in SEQ ID NO: 2. This sequence is defined by the start codon at position 324 of the isolated 9D7 cDNA.

In a first aspect, the present invention relates to the tumor-associated antigen of the designation 9D7 with the amino acid sequence given in SEQ ID NO: 2.

The amino acid sequence shown in SEQ ID NO: 2 can have deviations, for example those which are caused by conservative exchange of amino acids, provided the 9D7 derivative has the immunogenic properties desired for use in a tumor vaccine. In a further aspect, the invention relates to a polypeptide which contains the 9D7 sequence as a partial sequence. Compared to the 9D7 sequence given in SEQ ID NO: 2, this polypeptide has an extension at the N-terminal end; its sequence is defined by a start codon of the 9D7 cDNA, which may be located upstream from position 324 in the 5 'direction.

The amino acid sequence (or correspondingly the sequence of the 9D7 cDNA) can optionally be modified by exchanging individual amino acids in a 9D7 CTL epitope in order to increase the affinity of 9D7 peptides compared to the natural 9D7 CTL epitope MHC-I molecules and thus an increased immunogenicity and ultimately an increased reactivity to tumors. Modifications in

The region of the 9D7 epitopes can be carried out on the total 9D7 protein (this is processed by the APCs to form the corresponding peptides) or on larger 9D7 protein fragments or on 9D7 peptides (see below).

In another aspect, the present invention relates to immunogenic fragments and peptides derived from 9D7. The latter are referred to below as "9D7 peptides". A first group are 9D7 peptides that trigger a humoral immune response (induction of antibodies). Such peptides are selected sections of 9D7 (at least 12 to 15 amino acids), which are used by means of so-called prediction algorithms such as e.g. the "surface probability blot" (Emini et al., 1985), the

"hydrophobicity blot" (Kyte and Doolittle, 1982) and the "antigenic index" (Jameson and Wolf, 1988) can be determined.

9D7 peptides are administered directly or in modified form (e.g. coupled to KLH = "keyhole limpet hemocyanine") and the formation of antibodies by means of common immunological assays, e.g. determined by ELISA.

Further 9D7 peptides which are preferred in the context of the present invention are those which are presented by MHC molecules and bring about a cellular immune response. There are two classes of MHC molecules, namely MHC-I molecules that are recognized by CD8-positive CTLs and MHC-II molecules that are recognized by CD4-positive T helper cells.

In order for a peptide to trigger a cellular immune response, it must bind to an MHC molecule, which must have the to molecule in its repertoire. The determination of the patient's MHC subtype thus represents one of the essential prerequisites for the effective application of a peptide to this patient with regard to triggering a cellular immune response.

The sequence of a peptide to be used therapeutically is predetermined by the respective MHC molecule with regard to anchor amino acids and length. Defined anchor positions and lengths ensure that a peptide fits into the peptide binding groove of the patient's respective MHC molecule. As a result, the immune system is stimulated and a cellular immune response is generated, which, in the case of Use of a peptide derived from a tumor antigen against the patient's tumor cells.

Immunogenic 9D7 peptides can be identified by known methods, one of the bases for this is the relationship between MHC binding and CTL induction.

Since the sequence of immunogenic peptides can therefore be predetermined based on their peptide binding motif, 9D7 peptides which represent CTL epitopes can be identified and synthesized on the basis of the 9D7 protein sequence. Various methods are suitable for this, which have been used to identify CTL epitopes of known protein antigens; e.g. the method described by Stauss et al., 1992, for the identification of T cell epitopes in human papillomavirus.

The allele-specific requirements of each MHC-I allele product for a peptide which binds to and is presented by the MHC molecule have been summarized as a motif (eg Falk et al., 1991). A large number of both MHC peptide motifs and MHC ligands are known to date. A method suitable for the purposes of the present invention for searching for epitopes of a known protein which fits into a particular MHC-I molecule has been described in a review article by Rammenee et al., 1995. It comprises the following steps: first, the protein sequence is examined for sections which correspond to the anchor motif, with certain variations in terms of peptide length and anchor occupation being possible. If, for example, a motif 9-mer with Ile or Leu at the end, 10-mer with a corresponding C-terminus can also be considered, as can peptides with other aliphatic residues, such as Val or Met at the C-terminus. In this way a number of peptide candidates are obtained. These are examined for the presence of as many anchor residues as possible, which they have in common with known ligands, and / or for whether they have "preferred" residues for various MHC molecules (according to the table by Rammenee et al., 1995). In order to exclude weakly binding peptides, binding assays are expediently carried out. If the requirements for peptide binding for certain MHC molecules are known, the peptide candidates can also be examined for non-anchor residues which have a negative or positive effect on the binding or which make this possible (Ruppert et al., 1993). With this approach, however, it should be considered that the peptide binding motif is not the only decisive factor in the search for natural ligands; other aspects, such as enzyme specificity during antigen processing, also contribute to the identity of the ligand, in addition to the specificity of the MHC binding. A method which takes these aspects into account and which is suitable in the context of the present invention for the identification of immunogenic 9D7 peptides has been used, inter alia, by Kawakami et al., 1995, to generate gplOO epitopes based on known HLA-A * 0201 motifs to identify.

The peptides can 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 the MHC-I binding motif. Methods have recently been developed, based on the X-ray structure analysis of MHC-II molecules, which allow the precise analysis of the MHC-II binding motifs, and on the basis thereof, variations of the peptide sequence (Rammenee et al., 1995, and the original literature cited therein ). Peptides that bind to MHC-II molecules are typically presented to CD4-T cells by dendritic cells, macrophages or B cells. The CD4-T cells then in turn then directly activate CTLs by, for example, cytokine release and enhance the efficiency of the antigen presentation by APC (dendritic cells, macrophages and B cells).

Databases and prediction algorithms have recently become available which allow the prediction of peptide epitopes which bind to a particular MHC molecule with great reliability.

In the context of the present invention, using the algorithm described by Parker et al., 1994, for the most important HLA types, in particular for HLA-Al, -A * 0201, -A3, -B7, -B14 and -B * 4403, identified candidate peptides that are expected to bind to the corresponding HLA molecules and are therefore immunogenic CTL epitopes; the peptides found are listed in Table 3. In a similar way, if necessary using further algorithms, the different characteristics of the peptides (Hydrophobicity, charge, size) or requirements for the peptides, e.g. B. take into account the 3D structure of the HLA molecule, further potential peptide epitopes for other HLA types can be determined.

After selecting 9D7 peptide candidates using the methods listed, their MHC binding is tested by means of peptide binding assays. The immunogenicity of the peptides with good binding properties is next determined (stability of the peptide-MHC interaction correlates with immunogenicity in most cases; van der Burg et al., 1996). In order to determine the immunogenicity of the selected peptide or peptide equivalent, methods such as e.g. by Sette et al., 1994, can be used in combination with quantitative MHC binding assays. Alternatively, the immunogenicity of the selected peptide can be tested via in vitro CTL induction using known methods (as described below for ex vivo CTL induction). The principle of the method carried out in several steps for the selection of peptides which are capable of triggering a cellular immune response is described in WO 97/30721, the disclosure of which is hereby expressly incorporated by reference. A general strategy for obtaining efficient immunogenic peptides that is useful in the present invention has also been described by Schweighoffer, 1997.

Instead of using the original peptides that fit into the binding groove of MHC-I or MHC-II molecules, that is, peptides that are derived unchanged from 9D7, can be based on the on the basis of Original peptide sequence specified minimum requirements regarding anchor positions and length variations are made, provided by these variations the effective immunogenicity of the peptide, which is composed of its binding affinity to the

MHC molecule and its ability to stimulate T cell receptors is not only not impaired, but is preferably enhanced. In this case, artificial peptides or peptide equivalents are used, which correspond to the requirements of

Binding ability to an MHC molecule are designed.

Peptides modified in this way are referred to as “heteroclitic peptides”. They can be obtained by the following methods:

First, the epitopes of MHC-I or MHC-II ligands or their variation e.g. according to the principle described by Rammenee et al., 1995. The length of the peptide in the case of its adaptation to MHC-I molecules preferably corresponds to a minimum sequence of 8 to 10 amino acids with the required anchor amino acids.

If appropriate, the peptide can also be extended at the C- and / or the N-terminus, provided that this extension does not impair the ability to bind to the MHC molecule or the extended peptide can be processed cellularly for the minimal sequence.

The modified peptides are then based on their detection by TILs (tumor-infiltrating lymphocytes, on CTL induction and on enhanced MHC binding and Immunogenicity was tested as described by Parkhurst et al., 1996 and Becker et al., 1997.

Another method suitable for the purposes of the present invention for finding peptides with a greater immunogenicity than that of natural ones

9D7 peptides consist of screening peptide libraries with CTLs that recognize the naturally occurring 9D7 peptides on tumors, as described by Blake et al., 1996; in this context, the use of combinatorial peptide libraries is proposed to design molecules that mimic tumor epitopes recognized by MHC-I restricted CTLs.

The 9D7 polypeptides of the present invention or immunogenic fragments or peptides derived therefrom can be produced recombinantly or by means of peptide synthesis, as described in WO 96/10413, the disclosure of which is hereby incorporated by reference. For the recombinant production, the corresponding DNA molecule is inserted into an expression vector according to standard methods, into a suitable one

Host cell transfected, the host cultivated under suitable expression conditions and the protein purified. Conventional methods can be used for the chemical synthesis of 9D7 peptides, e.g. commercially available automatic peptide synthesizers.

As an alternative to natural 9D7 peptides or heteroclitic peptides, substances which simulate such peptides, for example “peptidomimetics” or “retro-inverse peptides”, can be used. To test these molecules in terms of therapeutic Usability in a tumor vaccine, the same methods are used as above for the natural 9D7 peptides or 9D7 peptide equivalents.

The TAA of the designation 9D7 according to the present invention and the protein fragments, peptides or peptide equivalents or peptidomimetics derived therefrom can be used in cancer therapy, e.g. B. to induce an immune response against tumor cells that express the corresponding antigen determinants. They are preferably used for the therapy of tumors with strong 9D7 expression, in particular for renal cell, lung, colon and breast cancer and for Hodgkin lymphoma.

The immune response in the form of induction of CTLs can be brought about in vivo or ex vivo.

For the in vivo induction of CTLs, a pharmaceutical composition containing as active component the TAA 9D7 or fragments or peptide (s) derived therefrom is administered to a patient who suffers from a tumor disease associated with the TAA, the amount of TAA (peptide) must be sufficient to achieve an effective CTL response to the antigen-bearing tumor.

In a further aspect, the invention thus relates to a pharmaceutical composition for parenteral, topical, oral or local administration. The composition is preferably used for parenteral administration, for example for subcutaneous, intradermal or intramuscular use. The 9D7-TAAs / Peρtide are in a pharmaceutically acceptable, preferred aqueous, carrier dissolved or suspended. The composition can also contain customary auxiliaries, such as buffers, etc. The TAAs / peptides can be used alone or in combination with adjuvants, for example incomplete Freund's adjuvant, saponins, aluminum salts or, in a preferred embodiment, polycations such as polyarginine or polylysine. The peptides can also be bound to components which support CTL induction or activation, for example to T helper peptides, lipids or liposomes, or they are used together with these substances and / or together with immunostimulating substances, for example cytokines (IL -2, IFN-γ) administered. Methods and formulations for the preparation and administration of the pharmaceutical according to the invention

Suitable compositions are described in WO 95/04542 and WO 97/30721, the disclosure of which is hereby incorporated by reference.

9D7 (fragments) or 9D7 peptides can also be used to trigger a CTL response ex vivo. An ex vivo CTL response to a tumor expressing 9D7 is induced by incubating the CTL progenitor cells together with APCs and 9D7 peptides or 9D7 protein. The activated CTLs are then allowed to expand, after which they are re-administered to the patient. Alternatively, APCs can be loaded with 9D7 peptides, which can lead to an efficient activation of cellular immune responses against 9D7 positive tumors (Mayordomo et al., 1995; Zitvogel et al., 1996). A suitable method for loading peptides onto cells, for example dendritic cells, is disclosed in WO 97/19169. In one embodiment of the invention, a combination of several different 9D7 peptides or 9D7 peptide equivalents is used. In a further embodiment, 9D7 peptides are combined with peptides derived from other TAAs. The selection of

Peptides for such combinations are taken with a view to the detection of different MHC types in order to cover the widest possible patient population, and / or they are tailored to the widest possible range of indications by combining peptides from several different tumor antigens. The number of peptides in a pharmaceutical composition can vary over a wide range, typically a clinically applicable vaccine contains 1 to 15, preferably 3 to 10 different peptides.

The peptides according to the invention can also be used as diagnostic reagents. For example, the peptides can be used to test a patient's response to the humoral or cellular immune response elicited by the immunogenic peptide. This makes it possible to improve a treatment protocol. For example, depending on the dosage form (peptide, total protein or DNA vaccine) of the TAA, the increase in precursor T cells in the PBLs that are reactive to the defined peptide epitope can be investigated (Robbins and Kawakami, 1996 and references cited therein) . In addition, the peptides or the total protein or antibodies directed against the TAA can be used to predict a patient's risk of developing a 9D7-associated tumor or to characterize the course of the disease of a 9D7-positive tumor (for example by immunohistochemical analyzes of the primary tumor and metastases). Such a strategy has proven successful several times, for example the detection of the estrogen receptor as a basis for decision-making on endocrine therapy for breast cancer; c-erbB-2 as a relevant marker in the prognosis and course of therapy for breast cancer (Raviaioli et al., 1998; Revillion et al., 1998); PSMA ("prostate specific membrane antigen") as a marker for

Epithelial cells of prostate cancer in serum or by using a ^lu In-labeled monoclonal antibody against PSMA in immunoscintigraphy for prostate cancer (Murphy et al., 1998 and included references); CEA ("carcinoembryonic antigen") as a serological marker for the prognosis and course in patients with colorectal cancer (Jessup and Loda, 1998).

In a further aspect, the present invention relates to isolated DNA molecules coding for a

Protein with the immunogenic properties of 9D7 or for fragments thereof.

The present invention preferably relates to an isolated DNA molecule which contains a polynucleotide with the sequence shown in SEQ ID NO: 1 or which contains a polynucleotide which hybridizes with a polynucleotide of the sequence shown in SEQ ID NO: 1 under stringent conditions.

“Stringent conditions” means, for example: overnight incubation at 65 ° C.-68 ° C. with δxSSC (1 x SSC = 150 mM NaCl, 15mM trisodium citrate), 5xDenhardt's solution, 0.2% SDS, 50 μg / ml salmon sperm DNA, followed by washing twice with 30 min with 2xSSC, 0.1% SDS at 65 ° C, once with 30 min 0.2xSSC, 0.1% SDS at 65 ° C and possibly final

Rinse with O.lxSSC, 0.1% SDS at 65 ° C, or equivalent conditions.

The DNA molecule according to the invention or fragments thereof code for an immunogenic (poly) peptide of the designation 9D7 with the amino acid sequence shown in SEQ ID NO: 2 or for protein fragments or peptides derived therefrom; this also includes DNA molecules which, due to the degeneration of the genetic code, deviate from the sequence shown in SEQ ID NO: 1.

The invention also relates to DNA molecules which, due to the conservative exchange of amino acids, have deviations from the sequence shown in SEQ ID NO: 1, provided they are for a 9D7 derivative or fragments or peptides with the immunogenic properties desired for use as a tumor vaccine encode.

In comparison to the molecule shown in SEQ ID NO: 1, the DNA molecules according to the invention can be extended at the 5 'end to a start codon which may be located upstream from position 324.

The 9D7 DNA molecules of the present invention or the corresponding RNAs, which are also the subject of the present invention, become like those thereof encoded (poly) peptides, used for the immunotherapy of cancer.

In one embodiment of the invention, DNA molecules coding for the natural 9D7 are used. As an alternative to the natural 9D7 cDNA or

Fragments thereof can be used in modified derivatives. These include sequences with modifications that code for a protein (fragment) or peptides with greater immunogenicity, the same considerations for the modifications at the DNA level as for the peptides described above. Another type of modification is the stringing together of numerous sequences, coding for immunologically relevant peptides, in the manner of a string of pearls ("string-of-beads"; Toes et al., 1997). The sequences can also be modified by adding auxiliary elements, e.g. Functions that ensure more efficient delivery and processing of the immunogen (Wu et al., 1995). For example, by adding a localization sequence into the endoplasmic reticulum ("ER targetting sequence"), the processing and thus the presentation and ultimately the immunogenicity of the antigen can be increased.

In another aspect, the present invention relates to a recombinant DNA molecule which contains 9D7 DNA.

The DNA molecules of the present invention can be administered, preferably in recombinant form as plasmids, directly or as part of a recombinant virus, or bacterium. In principle, everyone can gene therapeutic method for the immunotherapy of cancer based on DNA ("DNA vaccine") on 9D7-DNA can be used, both in vivo and ex vivo.

Examples of in vivo administration are the direct injection of "naked" DNA, either intramuscularly or by means of a gene gun, which has been shown to lead to the formation of CTLs against tumor antigens. Examples of recombinant organisms are vaccinia virus, adenovirus or Listeria monocytogenes (an overview was given by Coulie, 1997). Furthermore, synthetic carriers for nucleic acids, such as cationic lipids, microspheres, microspheres or liposomes, can be used for the in vivo administration of nucleic acid molecules coding for 9D7 peptide. Similar to peptides, various adjuvants that enhance the immune response can be co-administered, e.g. Cytokines, either in the form of proteins or plasmids encoding them. The application can optionally be carried out using 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 that are used as cellular cancer vaccines.

In a further aspect, the present invention thus relates to the use of cells which express 9D7, either on its own or, in optionally modified form, after transfection with the corresponding coding sequence for which

Production of a cancer vaccine.

In a further aspect, the invention relates to antibodies against 9D7 or fragments thereof. Polyclonal antibodies can be obtained in a conventional manner by immunizing animals, in particular rabbits, by injecting the antigen or fragments thereof, and then purifying the immunoglobulin.

Anti-9D7 monoclonal antibodies can be used

Standard protocols are obtained according to the principle described by Köhler and Milstein, 1975, by immunizing animals, especially mice, then immortalizing antibody-producing cells of the immunized animals, e.g. through fusion with

Myeloma cells, and the supernatant of the resulting Hybrido e is screened for monoclonal anti-9D7 antibodies by means of standard immunological assays. For therapeutic or diagnostic use in humans, these animal antibodies can optionally be chimerized in a conventional manner (Neuberger et al., 1984, Boulianne et al., 1984) or humanized (Riechmann et al., 1988, Graziano et al., 1995) .

Human monoclonal anti-9D7 antibodies (fragments) can also be obtained from so-called "phage display libraries" (Winter et al., 1994, Griffiths et al., 1994, Kruif et al., 1995, Mc Guiness et al., 1996) and by means of transgenic animals (Brüggemann et al., 1996, Jakobovits et al., 1995). The anti-9D7 antibodies according to the invention can be used in immunohistochemical analyzes for diagnostic purposes.

In a further aspect, the invention relates to the use of 9D7-specific antibodies in order to selectively bring any substances into or into a tumor which expresses 9D7. Examples of such substances are cytotoxic agents or radioactive nuclides, the effect of which is to damage the tumor on site. Due to the tumor-specific expression of 9D7, no or only minor side effects are to be expected. In another aspect, 9D7 antibodies can be used to visualize tumors that express 9D7. This is useful for the diagnosis and evaluation of the course of therapy. Therapeutic and diagnostic uses of antibodies which are suitable for anti-9D7 antibodies are described in WO 95/33771 for.

Due to the preferred expression of 9D7 in

Tumor cells can be assumed that this protein has an important function for the tumor, for example for its formation, infiltration and growth. 9D7 (DNA) can therefore be used in screening assays to identify substances that modulate, in particular inhibit, the activity of this protein. In one embodiment, such an assay can consist of introducing the 9D7 protein, or an active fragment thereof, into cells which react to the activity of 9D7 with proliferation, or the corresponding 9D7 DNA into the cell for expression bring, and to determine the proliferation of the cells in the presence and in the absence of a test substance. Substances with an anti-proliferative effect can be used for the treatment of tumors with strong 9D7 expression, particularly in kidney cell,

Lung, colon and breast cancer as well as Hodgkin's lymphoma.

Figure overview

Fig. 1: Transcription of 9D7 in normal tissues and RCC: semiquantitative RT-PCR of RNA from kidneys, heart, leukocytes, liver, lungs, testis and four renal cell carcinomas

2: Transcription of 9D7 in normal tissues: Northern blot analysis of mRNA

16 normal fabrics

Figure 3: In situ hybridization of 9D7 RNA with renal cell carcinoma and normal kidney tissue sections

Fig. 4: Detection of 9D7 expression in renal cell carcinoma

example 1

RDA (Representational Difference Analysis) of renal cell carcinoma and normal kidney tissue

Biopsies of human renal cell carcinomas of the clear cell type (RCC, renal cell carcinoma) as well Normal kidney tissue from the same patient was snap frozen in liquid N ₂ immediately after surgical removal and stored at -80 °. To isolate RNA, serial 20 μm sections were made at -20 ° in a cryomicrotome (Jung CM1800, Leica) and dissolved directly in 4 M guanidinium thiocyanate / 1% ß-mercaptoethanol. This sample was subjected to ultracentrifugation over a CsCl gradient (Sambrook, 1989). Starting with 60 μg total RNA from renal cell carcinoma RCC6 and 350 μg total RNA from the autologous normal kidney N6 as well as another normal kidney tissue (N3), RDA (Diatchenko et al.; Hubank and Schatz,) was used using PCR-select ™ kit (Clontech, Palo Alto) according to the manufacturer's protocol: RNA from RCC ("tester") and normal kidney tissue ("driver") was used to isolate poly-A (+) RNA using the PolyATtract Kit (Promega) used according to the manufacturer's protocol. After the synthesis of double-stranded cDNA using oligo-dT, it was digested with .sal. Equal parts of "tester cDNA" were ligated either with the adapters A or B and then hybridized separately with an excess of "driver cDNA" at 65 ° C. The two approaches were then combined and subjected to a second hybridization with freshly denatured "driver cDNA". The enriched "tester" -specific cDNAs were then amplified exponentially by PCR with primers specific for adapters A and B, respectively. An aliquot of this was used for further enrichment

Reaction subjected to a second PCR with specific inwardly displaced (“nested”) primers product resulting from this reaction was ligated into the pCRII vector (Invitrogen) and then into competent E. coli (OneShot ™, Invitrogen) transfected. 1920 positive transformants (blue-white selection) were cultivated in 96-well blocks in LB-Amp medium for 24-48 h at 37C. 200 μl aliquots of the E. coli suspensions were heated to 100 ° C. for 10 minutes. After briefly centrifuging off the bacterial residues, 140 μl clear supernatant (~ 1 μg plasmid DNA) was taken up in 60 μl 20xSSC and equilibrated

Nylon membranes (Hybond-N, Amersham) are immobilized according to the standard methods for producing DNA dot blots (Sambrook, 1989). The remaining bacterial cultures were stored as gycerin stock cultures at -80 ° C.

A cDNA subtraction library of 1920 single clones was obtained, which can be used both as E. coli glycerol stock cultures as well as immobilized plasmids on DNA dot blots were available.

Example 2

Selection of "Tumor-Testis" antigens by hybridization of the cDNA subtraction library with a Testis-specific cDNA library

"Human Testis-Specific PCR-Select ™ cDNA" (Clontech, Palo Alto) was amplified according to the manufacturer's instructions using 11 cycles of PCR. Aliquots were randomly primed (HiPrime Kit, Boehringer Mannheim) with [α- ³² P] dCTP (NEN, Boston) marked and hybridized according to standard instructions (Sambrook, 1989) under stringent conditions (65 ° C) with the DNA dot blots described in Example 1. Clones which hybridize with the "Human Testis-Specific PCR-Select ™ cDNA" were visualized using standard autoradiography (Xomat DR film, Kodak). The result of Example 2 allowed the selection of 150 clones, which are predominantly in renal cell carcinoma RCC6 and are transcribed into Testis. These clones are candidates for the class of "tumor-Testis" antigens.

Example 3

DNA sequencing and annotation of "tumor testis" antigen candidates

Plasmid DNA from 62 clones which had been selected on the basis of the results obtained in Example 2 was isolated in accordance with the manufacturer's instructions (QIAgen) and sequenced on an ABI-Prism device by the Sanger method. The sequences determined in this way were annotated using the BioWorkBench software (GeneData, Basel) and subjected to database comparisons (Genbank). This allowed 33 known and 29 unknown genes to be identified. Only EST entries existed for the latter. Known genes were not further treated. The expression profile was estimated for the 29 unknown genes: the starting tissue for the corresponding cDNA library was checked for all ESTs with> 95% identity (BLAST) for the experimentally determined sequence in databases. there has been a Subdivision into i) critical normal tissues, ii) fetal, "dispensable" and immune-privileged tissues and iii) tumors and cell lines. Based on this "virtual mRNA profile", 16 clones for which no ESTs were found in group i) were found , selected for further experimental analysis.

Example 4

Transcriptional analysis of the candidate clones in renal cell carcinoma and normal kidney

Between 2 and 5 μg total RNA from tumor or normal tissues were reverse transcribed using SuperscriptII (Gibco) or AMV-RT (Promega) according to the manufacturer's recommendations. For each individual RNA sample, a second approach without reverse transcriptase as a control for contamination by chromosomal DNA was carried out. Alternatively, plasmid cDNA libraries from human heart, leukocytes, kidney, liver, lung and testis (Gibco) were used. The quality and amount of the cDNAs were determined by PCR with β-actin-specific primers (SEQ ID NO: 29 and 30) after 20, 25 and 30 cycles (40 "94 ° C, 45" 55 ° C, 1'30 "72 ° C , from cycle 11 with 30 "55 ° and an extension of 3" 55 ° with every further cycle). 9D7-CDNA was amplified by 30 cycles of the same program with the 9D7-specific primers according to SEQ ID NO: 3 and 4. The other 15 candidate clones were tested analogously, each with specific primers, and the PCR products were detected by agarose gel electrophoresis and ethidium bromide staining The clones examined were able to show a clear overexpression in renal cell carcinoma RCC6 in comparison to the autologous normal kidney tissue N6 by means of RT-PCR. As an additional positive control, the occurrence in Testis cDNA was examined for all clones.

Example 5

Quantitative comparison of 9D7 mRNA expression between tumors and normal tissues

Pools of 9 μg total RNA from 3 tumor or

Normal tissues were reverse transcribed after pretreatment with DNase I (Boehringer Mannheim) and subsequent phenol extraction using oligo-dT and AMV-RT (Promega). The copy number of the cDNA-9D7 or glyceraldehyde-3-phosphate dehydrogenase cDNA (GAPDH) as a reference for quantity and quality of total cDNA was quantitated by 5 'nuclease PCR assay (TaqMan ^® PCR, PE Applied Biosystems). The primers according to SEQ ID NO: 3 and 4 and SEQ ID NO: 31 served as primers or labeled TaqMan probes for cDNA quantification (at the 5 'end with 6-carboxy-flourescein and at the 3' end with 6 -carboxy-tetramethyl-rhodamine) or for GAPDH the primers according to SEQ ID NO: 25 and 26 and the probe according to SEQ ID NO: 32 (at the 5 'end with tetrachloro-6-carboxy-flourescein and at 3' - Marked end with 6-carboxy-tetramethyl-rhodamine). The PCR (40 cycles {15 "95 ° C, 1 '60 ° C} for 9D7 and 40 cycles {15" 95 ° C, 1' 66 ° C} for GAPDH) and the on-line fluorescence measurement were carried out with an ABI PRIΞM 7700 Sequence Detection System (PE Applied Biosystems). Example 5 shows that in pools of renal cell carcinoma and lung carcinoma (SCC) 9D7 is transcribed approximately 1.5 to 6.5 times more strongly than GAPDH. In other tumor types, the transcription rate fluctuates between 0.25-0.6 times the value of GAPDH.

Normal liver and testis show a value of about 10% of the GAPDH transcription, while the other normal tissues examined show practically no transcription of 9D7 (Tab. 1).

Example 6

Expression profile of 9D7 in tumor and normal tissues

One of the 15 candidates (designation: 9D7) showed strong transcription in all tested renal cell carcinomas and several other tumor types by means of RT-PCR. However, no transcription could be found in most normal tissues. Fig. 1 shows a comparison between four normal kidneys (Nl, 2, 4, 5) and four renal cell carcinomas (RCC1, 2, 3, 6); In addition, cDNA from cardiac muscle (he),

Leukocytes (leu), kidney (ki), liver (left), lung (lu) and testis (tes) tested and a control performed with ß-actin primers. These results were confirmed in independent experiments with three different primer pairs (SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 10 and SEQ ID NO: 11 and 15). For the Northern blot analysis, human multiple tissue Northern blots (Clontech, Palo Alto) were hybridized with the [α- ³² P] dCTP (NEN, Boston) labeled 170 bp 9D7 PCR product at 65 ° for 16 h. The visualization was done by Standard autoradiography (Xomat AR film, Kodak). 2 shows the result of this analysis: of 16 normal tissues (1: leukocytes, 2: colon, 3: small intestine, 4: ovaries, 5: testes, 6: prostate, 7: thymus, 8: spleen, 9: pancreas, 10 : Kidney, 11: skeletal muscle, 12: liver, 13: lung, 14: placenta, 15: brain, 16: heart muscle), only a transcript of ~ 2.2 kb in size was shown in the heart muscle. However, the low intensity of the signal makes an immunologically relevant expression seem unlikely.

The results of all experiments with regard to the mRNA profile of 9D7 (compiled from RT-PCR, quantitative RT-PCR and Northern blot analysis) are summarized in Tab. 2. Example 6 shows that 9D7 is strongly transcribed in a high percentage of tumors of various indications, whereas no or only weak transcription was found in all examined normal tissues.

Example 7

hybridization of a 9D7-specific RNA probe with renal cell carcinoma and normal kidney tissue

A 280 bp Notl fragment with 220 bp sequence information from 9D7 was subcloned into pBluescript and then E competent for the transformation. coli

DH5-α used. The RNA probes were synthesized by in vitro transcription with T3 or T7 RNA polymerase (sense: Sac I, T3; antisense: BamH I, T7) with the addition of digoxigenin UTP in accordance with Manufacturer recommendations (DIG RNA labeling kit, Boehringer Mannheim). After cleaning the transcripts (Sephadex G50 quick spin columns, Boehringer Mannheim), the labeling efficiency was determined using dot blot. The in situ hybridization was made after

Standard procedures carried out (Boehringer Mannheim, Nonradioactive In Situ Hybridzation Application Manual, 1996): 5 μm frozen sections were denatured after paraformaldehyde fixation, acetylation and dehydration and rehydration and hybridized overnight at 52 ° C in a moist chamber. After the usual washing steps, unspecific protein binding sites were saturated with 10% FCS and 3% goat serum (DAKO) for 15 minutes. The visualization of the probe hybridized to 9D7 mRNA was carried out by

Incubation with an α-DIG-AP conjugate and NBT / BCIP (Boehringer Mannheim) development at 4 ° C in the dark.

3 shows the result of an in situ hybridization of renal cell carcinoma or normal kidney tissue labeled with a 9D7-specific digoxigenin UTP

RNA probe ("antisense"): the tumor tissue shows strong and homogeneous staining, while the normal tissue does not react; the specificity of the staining is shown by the negative control ("sense").

Example 7 shows that 9D7 in tumor cells from

Renal cell carcinoma tissues, but not in any cell type is transcribed from normal kidney tissue and thus confirms the results described in Examples 4-6. Example 8

Detection of protein expression of 9D7 in renal cell carcinoma

To detect the protein expression of 9D7, 9D7-specific antibodies were generated in rabbits. The peptide 9D7-3aa of the sequence: DAEERAEAKKKFRNLTRKTES (SEQ ID NO: 59) was used for immunization, and the serum obtained was affinity-purified using peptide 9D7-3aa. To check the specific reactivity of serum 9D7-3aa, the entire

Reading frame of 9D7 as GFP fusion protein transiently expressed in COS cells and the transfected cells tested in a Western blot with serum 9D7-3aa. It was shown that the serum clearly reacts with the eukaryotic expressed 9D7 fusion protein. Subsequently, 18 different renal cell carcinomas were analyzed immunohistochemically with the serum 9D7-3aa for 9D7 expression. In 15 cases, expression of 9D7 was found in tumor cells, whereas no expression of 9D7 was detectable in the tumor stroma. Examples can be seen in Fig. 4.

Example 9

Cloning 9D7

Clone 9D7 has a 221 bp insert of an unknown human gene between the adapters introduced by the RDA. Database comparisons showed a homology of 9D7 to rat SM20, 69 bp of the C-terminal coding region (corresponding to 23 amino acids), the stop codon and 149 bp of the 3 'untranslated region of SM20 corresponding to the 221 bp of 9D7 in an "antisense" orientation. The

23 / amino acids show only 2 exchanges between the rat and human sequences. SM20 has been described as a "immedia te early gene" induced by growth factors and a specific marker for cells of the smooth aorta muscles (Wax et al.,; Wax et al.,).

The complete cloning of the human sequence was carried out as follows: a UniGene analysis (National Center for Biotechnology Information) revealed the following ESTs homologous to 9D7: AA234127, AA233899, AA917421, AA878927. Total RNA from tumor cell lines 786-0 (ATCC # CRL-1932) or A549 (ATCC # CCL 185) was isolated using TRIzol (Gibco) and using Superscript II MMLV-RT (Gibco) and an adapter oligo-dT primer (SEQ ID NO: 27) reverse transcribed according to the manufacturer's instructions. PCR amplification of this cDNA with the primers according to SEQ ID NO: 5, 6, 7 and 10 gave the fragments expected according to the homologous sequences with the original primers (SEQ ID NO: 3 and 4) and with one another. The longest fragment of 804 bp was obtained by combining the primers according to SEQ ID NO: 10 and 5. The primers of the sequence SEQ ID NO: 8 and 9 are in a sequence region of EST-AA234127 which have no homology to SM-20 and consequently did not result in an amplification product with any of the other primers.

Cloning of the 3 'end; 10 μg total RNA from the 786-0 renal cell carcinoma cell line were reversed was transcribed and an 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 (T _rn ~ 93 ° C, SEQ ID NO: 22), while the second primer (T _m ~ 53 ° C, SEQ ID NO: 28) only binds to the newly synthesized DNA template at a lower temperature. This so-called "touch-down PCR" (Mastercycler Gradient, Eppendorf) was carried out under the following conditions: 20 cycles {15 "95 ° C, 30" 80 ° C (reduced by 1 ° C per cycle), 2'72 ° C } as well as 20 cycles {15 "95 ° C, 30" 50 ° C, 2 '72 ° C}.

Cloning of the 5 'end: 1 μg of total RNA from the renal cell carcinoma cell line 786-0 was carried out according to a modified SMART ™ protocol (SMART ™ PCR cDNA

Synthesis Kit, Clontech) using the primers SEQ ID NO: 4 and Smart-II (SEQ ID NO: 23) reverse transcribed: the cDNA with the 9d7-specific primer according to SEQ ID NO: 4 and with the addition of RNasin ( Promega) synthesized. The

9D7-specific primers according to SEQ ID NO: 11 were used in a final concentration of 0.4 μM, the SMART-PCR primer (SEQ ID NO: 24) with 0.1 μM. The PCR amplification was carried out with 40 cycles each (15 "95 ° C, 30" 65 ° C, 2 '72 ° C).

Aliquots of the PCR batches were ligated directly into the pCR2.1 vector (Invitrogen) and then transformed into competent E. coli (OneShot ™, Invitrogen). Positive clones were sequenced after blue and white selection. Sequencing revealed 50 additional nucleotides using EST-AA878927 matched to the 3 'end confirmed until then and 870 additional nucleotides to the 5' end confirmed until then. PCR amplification of cDNA from tumor cell lines (786-0, A549) and tissue biopsies of renal cell carcinomas with the primers according to SEQ ID NO: 14 to 21 gave the fragments expected after cloning with the original primers and among themselves. The identity of all PCR products was verified by sequencing. During the sequencing, several clones with or without an exon of 282 bp

(Pos. 399-680) identified. The presence of both splice variants was verified by RT-PCR with the primers according to SEQ ID NO: 11 and 15 or 13 in several tumor samples (renal cell carcinoma, lung carcinoma / SCC, colon carcinoma / AC).

Example 10

Potential MHC binding peptides in the coding region of 9D7

Potential peptide epitopes within the coding region of 9D7 according to SEQ ID NO: 2 were carried out using the algorithms described by Parker et.al., 1994, based on known motifs (Rammenee et al. 1995). For the most important HLA types, in particular for HLA-Al, -A * 0201, -A3, -B7, -B14 and - B * 4403, candidate peptides were identified which are expected to bind to the corresponding HLA Bind molecules and therefore represent immunogenic CTL epitopes; the peptides found are listed in Table 3. By analogous procedure, others can potential peptide epitopes for other HLA types can be determined.

Table 1:

Transcription of 9D7 in tumor and normal tissues relative to GAPDH

Tumor tissue 9D7 / normal tissue 9D7 / GAPDH GAPDH

Colon cancer 0.48 Colon 0.02

Lung cancer 6.54 lung 0.04 (SCC)

Lung cancer 0.26 spleen 0.03 (AC)

Hodgkin's 0.35 skeleton = 0.03 lymphoma muscle

Breast cancer 0.63 liver 0.10

Renal cell = 1.55 testis 0.08 carcinoma

Table 2: mRNA expression profile of 9D7

Normal = mRNA tumor mRNA common = tissue

Kidney kidney cell = ++++ 4/4 carcinoma

Lungs Lungs = +++ 2/3 carcinoma / SCC

Lungs = ++ 3/3 carcinoma / AC

Colon cancer 3/3

Breast cancer ++ 2/3

Hodgkin's lymphoma

pancreas

stomach

liver

Small intestine

Skeleton = muscle

Heart muscle: +) spleen

Thymus

Leukocytes

placenta

Ovaries

Testes

prostate

brain

Table 3

Immunogenic 9D7 peptide candidates

205 Arg Tyr Ala Met Thr Val A24, Cw * 0401 Trp Tyr Phe

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Claims

215758Patent claims

1. tumor-associated antigen of the designation 9D7, characterized in that it has the amino acid sequence defined in SEQ ID NO: 2.

2. Polypeptide, characterized in that it contains the sequence defined in SEQ ID NO: 2 as a partial sequence.

3. Immunogenic protein fragment or peptide, characterized in that it is derived from the tumor-associated antigen defined in claim 1.

4. Immunogenic (poly) peptide according to one of claims 1 to 3, which triggers a humoral immune response.

5. Immunogenic (poly) peptide according to one of claims 1 to 3, or its degradation products are presented by MHC molecules and trigger a cellular immune response.

6. Immunogenic peptide according to claim 5, selected from the group of peptides according to SEQ ID NO: 34 to 58.

7. Immunogenic (poly) peptide according to one of claims 1 to 6 for the immunotherapy of

Cancers in vivo or ex vivo, wherein the (poly) peptide induces an immune response against patient tumor cells that express 9D7.

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

9. Pharmaceutical composition according to claim 8, characterized in that it contains various immunogenic peptides derived from 9D7.

10. Pharmaceutical composition according to claim 9, characterized in that it contains one or more peptides derived from 9D7 in a mixture with peptides derived from other tumor-associated antigens.

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

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

13. DNA molecule according to claim 12, coding for an immunogenic polypeptide of the designation 9D7 with the amino acid sequence shown in SEQ ID NO: 2 or for protein fragments or peptides derived therefrom.

14. DNA molecule according to claim 13, characterized in that it is a polynucleotide with the sequence shown in SEQ ID NO: 1 or contains or that it is or contains a polynucleotide that with a polynucleotide of SEQ ID NO: l sequence shown hybridized under stringent conditions.

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

16. DNA molecule according to one of claims 12 to 15, for the immunotherapy of cancer, wherein the 9D7- (poly) peptide expressed by the DNA molecule induces an immune response against tumor cells of the patient, which express 9D7.

17. Use of cells which express the tumor-associated antigen defined in claim 1 or 2 for the production of a cancer vaccine.

18. Antibodies against a (poly) peptide defined in one of claims 1 to 6.

19. Antibody according to claim 18, characterized in that it is monoclonal.

20. Antibody according to claim 18 or 19 for the

Treatment and diagnosis of cancer associated with the expression of 9D7.