JP2002537808A - Tumor-associated antigen - Google Patents

Abstract

  (57) [Summary] Tumor associated antigens, immunogenic peptides derived therefrom and DNA molecules encoding them and their use in cancer immunotherapy.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to immunotherapy of tumor diseases. The immune system protects the body from various microorganisms and has the function of actively fighting those microorganisms. The utility of the intact immune system is particularly evident in cases of innate or acquired immunodeficiency. The use of prophylactic vaccine programs has proven in many cases to be a very effective and effective immunological intervention in viral or bacterial infectious diseases. It has also been found that the immune system is involved extensively in eliminating tumor cells. Recognition of tumor-associated antigens (TAAs) by components of the immune system plays an important role. In the broadest sense, any (peptidic or non-peptidic) component of a tumor cell will be recognized by a component of the immune system and will stimulate an immune response and may serve as an immunogenic tumor antigen. Of particular interest are tumor antigens that not only cause an immunological reaction but also cause tumor rejection. Identifying specific antigens capable of eliciting this type of immunological response constitutes a major step in the development of molecularly defined tumor vaccines.

[0002] It is not yet clear which elements of the immune system are involved in tumor rejection, however, it is agreed that CD-8 expressing cytotoxic T cells (CTLs) play a major role. (Coulie, 1997). In particular, in tumor types with relatively high spontaneous remission rates (such as melanoma and kidney cancer), there is a correlation between clinical progression and 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 can be obtained from tumor infiltrating lymphocytes (TIL) or peripheral blood monocytic cells (PBMC), generally after co-culture with autologous tumor cells and in vitro cytokine stimulation. In both animal models and in vitro human cell culture systems, T cell responses to tumor cells were enhanced by transfecting tumor cells with cytokines (van Elsas
Gansbacher et al., 1990; Tepper et al., 1989; Fearon et al., 1990; Dranoff et al., 1993).

[0003] In view of the correlation between remission and CD8 ⁺ -T cells, identification of a tumor-associated antigen (TAA) recognized by CD8-positive CTL is a specific first goal toward the development of a tumor vaccine. Yes (Pardoll, 1998; Robbins and Kawakami, 1996). It is not yet clear whether other types of cells of the immune system, such as CD4 ⁺ -T helper cells, play an important role; numerous studies on the MAGE-3 / HLA-A1 peptide in melanoma patients have made this clear. (Marchand et al., 1995; Boon et al., 1998). Recently, a number of TAAs recognized by CTL have been identified (Boon et al., 1994; van den Eyde and van der Bru).
ggen, 1997).

[0004] T cells recognize antigens as cell fragments of MHC molecules (major histocompatibility complex, in humans, HLA = human leukocyte antigen), a peptide fragment presented on human leukocyte antigens. There is: MHC-I molecules are found in most nucleated cells and present peptides (usually 8-10 mer) that are produced by proteolytic degradation of endogenous proteins (so-called antigen processing). : MHC
The -I complex is recognized by CD8-positive CTL. MCH-II molecules are found only on so-called "professional antigen presenting cells (APCs)" and present peptides of exogenous proteins that have been absorbed and processed by APCs during endocytosis. The peptide: MHC-II complex is recognized by CD4-helper T cells. Interactions between the T cell receptor and the peptide: MHC complex may trigger various effector mechanisms, in the case of CTLs, causing apoptosis of target cells. This is M
HC (eg, for transplant rejection) or peptide (eg, for intracellular pathogens)
Occurs when is recognized as foreign. In any case, not all presented peptides fulfill the structural and functional requirements for effective interaction with T cells (as described and described in Ramensee et al., 1995).

[0005] In principle, a number of administration methods are possible for the use of TAA in tumor vaccines: the antigen is administered as a recombinant protein with a suitable adjuvant or carrier system, or a plasmid encoding the antigen ( DNA vaccine; Tighe et al., 199
8) Alternatively, it can be provided as cDNA on a viral vector (Restifo, 1997). Another possibility is to use recombinant bacteria (eg Listeria, Salmonella) that recombinantly express human antigens and have an adjuvant effect as a result of their additional components (Paterson, 1996; Pardoll, 1998). ). In all these cases, the antigen must be processed and presented by so-called "professional antigen presenting cells (APCs)". Another possibility is that the T equivalent to the antigen
Externally loaded on APCs corresponding to cellular epitopes (Buschle et al., 1997; Schmid
t et al., 1997) or a synthetic peptide (Mel) that is intracellularly transferred to MHC I molecules by APC.
ief et al., 1996). The most therapeutically effective method of administering a particular antigen will generally be determined by clinical trial.

[0006] Antigens or epitopes thereof recognized by tumor-specific CTLs include any protein class (eg, transcription factors, receptors, enzymes;
Ensee et al., 1995; see Robbins and Kawakami, 1996). It is not necessary that these proteins be located on the cell surface, as is required for recognition by antibodies. In order to serve as a tumor-specific antigen for recognition by CTLs, or to be used therapeutically, the protein must meet certain conditions; first of all, the antigen is exclusive by tumor cells Or must occur only at lower concentrations in so-called "critical" normal tissues than in tumors. Critical normal tissues are essential tissues; an immune response directed against them will have serious and in some cases lethal consequences.

[0007] Second, this antigen must be expressed not only in primary tumors but also in metastases. Furthermore, in view of the widespread use of antigens, it is desirable to be present at high concentrations in some types of tumors. TA as an effective ingredient in vaccines
A further prerequisite for the suitability of A is the presence of a T cell epitope in the amino acid sequence of the antigen; peptides derived from TAA should elicit in vitro / in vivo T cell responses ("immunogenic" peptide). Another criterion for selecting a clinically broadly applicable immunogenic peptide is the frequency with which the antigen appears in a given patient population. Although immunogenic tumor-associated antigens (TAAs) have been widely shown to already have T cell epitopes, they can be divided into a number of categories, including viral proteins, mutant proteins, overexpressed proteins, chromosomes, Fusion proteins formed by translocation, differentiation antigens, carcinoembryonic antigens (Van den Eynde and Brichard, 1995; va
n den Eynde and van der Bruggen, 1997).

As a starting point for the development of tumor vaccines, methods for identifying and analyzing TAAs include, in part, the use of CTLs that have already been induced in patients (cellular immune response) or the use of antibodies (humoral immune response). Response) or drawing a differential transcription profile between tumor and normal tissue. In the former example, the immunological approach uses patient CTL to screen a eukaryotic tumor cDNA expression library (Boon et al., 1994) that presents CTL epitopes by MHC-I molecules,
By using a high affinity patient antiserum eukaryotic cDNA expression library, the presence of TAA can be probed directly by immunoblot analysis of individual plaques (Sahin et al., 1995). The combination of CTL reactivity and protein-chemical processing results in the isolation of peptides separated from MHC-I of tumor cells, pre-selected by reactivity with patient CTL. This peptide is washed out of the MHC-I complex and identified by mass spectroscopy (Falk et al., 1991; Woelfel et al., 1994; Cox
Et al., 1994). The approach of using CTLs for antigen characterization requires considerable cost or is not always successful due to the need for CTL culture and activation.

[0009] Methods of identifying TAAs based on a comparison of the transcriptional profiles of normal and tumor tissues are numerous and diverse; including differential hybridization, establishment of subtraction DNA banks ("representational diff").
erence analysis "); Hubank and Schatz, 1994; Diatchenko et al., 1996) and the use of DNA chip technology or the SAGE method (Velculescu et al. 199). In contrast to the immunological methods described above using patient CTL, When using molecular biology methods, the potential antigen candidates discovered by this method are tumor-specific (tumor-associated) and must be used to identify T-cell epitopes that can trigger cytotoxic T-cell responses. In at least one example (NY-ESO / LAGE-1), the antigen was identified by both patient serum and RDA (Chen et al., 1997; Lethe et al., 1998) CTL epitopes and co-spontaneous humoral and T cell responses have been described for one patient (Jager et al., 1998).

[0010] It is an object of the present invention to provide a novel tumor associated antigen (TAA). The purpose of this was to first establish an RDA between renal cell carcinoma tissue and normal kidney tissue.
By establishing a cDNA subtraction library and hybridizing it with a testis-specific cDNA library to select tumor / testis-type antigens. The resulting cDNA clones were sequenced and compared to sequences available in the databank. Of the genes identified, there were 29 unknown genes with EST (expressed sequence tag) entries in the databank. Of these genes, 16 whose ESTs were not derived from critical normal tissues were further examined. By RT-PCR, 5 of the 16 clones examined showed a clear overexpression in renal cell carcinoma compared to normal kidney tissue. Quantitative comparison (using PCR) of the expression of tumor and normal tissues in one of these clones (9D7) showed a clear overexpression of 9D7 cDNA in the tumor. In normal tissues examined by Northern blot expression profile analysis, 9D7 was completely
Or showed only slight transcription. 9D7 in renal cell carcinoma and normal kidney tissue
In situ hybridization with specific labeled RNA probe is RT-PCR, quantitative
It confirmed the results obtained by PCR and Northern blot.

The human 9D7 cDNA has been completely cloned; the resulting sequence is shown in SEQ ID NO: 1. The 9D7 sequence has high homology at the nucleotide and protein level to SM20, an immediate early gene induced by growth factors in rat smooth muscle cells and described as a marker specific for smooth aortic muscle cells. (Wax et al., 199
4, 1996). Sequence analysis of human 9D7-cDNA compared to SM20 shows the first 323 bp remaining
It was found to have significantly lower homology than cDNA (40% versus 96% at the derived amino acid sequence level). Since human cDNA has the first ATG at transition 324 from high homology to low homology, it can be assumed that this is the start codon for human 9D7. However, since the upstream sequence at position 324 also has a smaller but contiguous reading frame and shows significant homology to SM20 (40% at the derived amino acid sequence level), it is possible that there is an initiation codon further upstream. Cannot be excluded.

Information on sequences located further upstream can be obtained by standard methods of molecular biology, for example, 5′-RACE
(Rapid amplification of DNA ends). In this method, RNA, preferably mRNA, is reverse transcribed from the cell or tissue into which 9D7 is transcribed (eg, RCC tissue or a cell line derived from RCC such as 786-0, see Example 9), and Is ligated with an adapter of known sequence. Adapter primer (for cDNA
The corresponding 8D7 by PCR using a 5D-specific adapter and a 9D7-specific primer (eg, SEQ ID NOs: 21, 19, 17, 16, 14, 12, 11, 4).
The fragment can be amplified. These PCR products can be cloned by standard methods as described in Example 1 and can be particularly characterized by DNA sequencing.

Other methods of characterizing the 5 'end are by hybridization with a DNA probe specific for 9D7 or screening a cDNA library with an antiserum specific for 9D7. If screening of the cDNA library does not achieve the desired result due to limitations of this procedure, such as inefficient reverse transcription due to the significant secondary structure of the RNA, for example, screening of the cDNA library As described in the above, a genomic library is examined by hybridizing with a DNA probe specific to 9D7, and a clone containing information on the sequence located upstream of the 5 ′ end of the obtained cDNA (for example, the promoter region of 9D7) is obtained. It can also be isolated.

[0014] The isolated cDNA encodes a tumor-associated antigen (TAA) having the amino acid sequence set forth in SEQ ID NO: 2, designated 9D7. This sequence is defined by the start codon at position 324 of the isolated 9D7-cDNA. According to a first feature, the present invention provides a 9D7 having the amino acid sequence set forth in SEQ ID NO: 2.
Tumor-associated antigen designated as If the 9D7 derivative has the desired immunogenic properties for use in a tumor vaccine, the amino acid sequence shown in SEQ ID NO: 2 may contain some mutations, for example, by conservative amino acid exchange. According to another aspect, the present invention relates to a polypeptide comprising the 9D7 sequence as a partial sequence. As compared to the 9D7 sequence shown in SEQ ID NO: 2, this polypeptide has an extension at the N-terminus; the sequence is defined by the start codon of the 9D7-cDNA, which is optionally 5 'of position 324. May be located upstream.

The amino acid sequence (or the corresponding 9D7-cDNA sequence) may optionally include 9D7
Can be modified by replacing individual amino acids in the CTL epitope,
Achieving increased affinity of the 9D7 peptide for MHC-I molecules compared to the native 9D7 CTL epitope, thereby resulting in increased immunogenicity and ultimately greater reactivity against tumors it can. Modifications in the 9D7 epitope region may be made in the entire 9D7 protein (which is processed by APC to form the corresponding peptide), or may be made to large 9D7 protein fragments or peptides (see below). Let's do it.

According to another aspect, the invention relates to immunogenic fragments and peptides derived from 9D7. The latter is referred to below as the 9D7 peptide. The first group is the 9D7 peptides that trigger a humoral immune response (induction of antibodies). Such peptides are described, for example, in surface potential blots (Emini et al., 1985), hydrophobic blots (Kyte and Doolittle, 1982).
) And a selected portion of 9D7 (at least 12-15 amino acids) that can be determined by so-called prediction algorithms such as the antigenic index (Jameson and Wolf, 1988). The 9D7 peptide is administered directly or in a modified form (eg, KLH = keyhole limpet hemocyanin) and antibody formation is determined by conventional immunological assays, eg, ELISA.

Within the scope of the present invention, other preferred 9D7 peptides are those that are presented by MHC molecules and generate a cellular immune response. There are two types of MHC molecules, MHC-I molecules recognized by CD8-positive CTLs and MHC-II molecules recognized by CD4-positive T helper cells. For a peptide to elicit a cellular immune response, the peptide must bind to an MHC molecule, while the molecule in question must be in its repertoire. Thus, determining the MHC-subtype of a patient constitutes one of the most important and essential for the effective use of the peptide in that patient in terms of eliciting a cellular immune response . The sequence of the peptide used therapeutically is determined by the MHC-molecule in question in terms of anchor amino acids and length. The defined anchor position and length ensure that the peptide fits into the peptide binding groove of the MHC-molecule of the patient in question. The result is that if the immune system is stimulated and peptides derived from tumor antigens are used, a cellular immune response directed against the patient's tumor cells will be generated.

[0018] Immunogenic 9D7 peptides may be identified by known methods; one of the basic conditions is a correlation between MHC-binding and CTL-inducibility. Therefore, since the sequence of the immunogenic peptide can be predicted based on the peptide binding motif, the 9D7 peptide constituting the CTL epitope can be identified, and can be synthesized based on the 9D7 protein sequence. A variety of methods are available for doing this, which are the methods used to identify CTL epitopes of known protein antigens; for example, in human papillomavirus
The method described by Stauss et al., 1992 for identifying T cell epitopes. Allele-specific requirements of each MHC-I allele product for peptides that bind to and display MHC molecules have been collected as motifs (eg, Falk et al., 1991). To date, a number of MHC peptide motifs and MHC ligands have both become known. Suitable methods for searching for epitopes of known proteins that fit within a particular MHC-I molecule, within the scope of the present invention, are described by the review of Rammensee et al., 1995. The method includes the following steps: First, the protein sequence is searched for fragments corresponding to the anchor motif, while certain variations in peptide length and anchor occupancy are possible. For example, the motif ends with Ile or Leu
If a 9-mer with a is designated, a 10-mer with the corresponding C-terminus can also be considered, as well as peptides with other aliphatic residues such as Val or Met.

In this way, a large number of peptide candidates are obtained. These are searched for the presence of as many anchor groups as possible in common with known ligands, and / or
Alternatively, it is searched (according to the table of Rammensee et al., 1995) whether they have "preferred" residues for the various MHC molecules. A binding assay is preferably performed to eliminate weakly binding peptides. If the requirements for peptide binding to a particular MHC molecule are known, candidate peptides are searched for non-anchor residues that have a negative or positive effect on binding, or whether they allow binding in the first place. (Ruppert et al., 1993). However,
In this method, it must be kept in mind that the peptide binding motif is not the only determinant when searching for a natural ligand; other aspects, such as enzyme specificity during antigen processing and MHC binding Contributes to the identification of ligands in addition to the specificity of Given these aspects, one suitable method for identifying immunogenic 9D7 peptides within the scope of the present invention is the HL method known in Kawakami et al., 1995, among others.
It was used to identify the gp100 epitope based on the AA ^* 0101 motif.

[0020] The peptides may also be selected for their ability to bind to the MCH-II molecule. MHC-II binding motifs greater than 9 amino acids have a higher degree of degeneracy at the anchor position than MHC-I binding motifs. X-ray structural analysis of MHC-II molecules and methods based on variations in peptide sequences based thereon have been recently developed that allow accurate analysis of MHC-II binding motifs (Rammensee et al., 1995, and cited therein). Original paper). Peptides that bind to MHC-II molecules are typically presented to CD4-T cells by dendritic cells, macrophages or B cells. CD4-T cells then in turn activate CTLs directly, for example by the release of cytokines, increasing the efficiency of antigen presentation by APCs (dendritic cells, macrophages and B cells).

[0021] Recently, prediction algorithms have become available that allow more reliable prediction of peptide epitopes binding to databanks and specific MHC molecules. Within the scope of the present invention, using the algorithm described by Parker et al., 1994, the most important HLA-types, especially HAL-A1, -A ^* 0201, -A3, -B7, -B14 and -B ^* 44
Candidate peptides for 03 were obtained, which are expected to bind to the corresponding HLA molecule and thus are expected to constitute an immunogenic CTL-epitope; the peptides found are listed in Table 3. I have. Similarly, by using other algorithms that take into account the peptide's various characteristics (hydrophobicity, charge, size), or the requirements for the peptide, such as the 3D structure of the HLA molecule, the potential for other HLA types is probably It is possible to discover specific peptide epitopes.

After selecting a candidate 9D7 peptide by the described method, its MHC binding is tested by a peptide binding assay. First, the immunogenicity of peptides with good binding properties is determined (stability of peptide-MHC interactions often correlates with immunogenicity; van der Burg et al., 1996). To determine the immunogenicity of the selected peptide or peptide equivalent, methods as described, e.g., Sette, 1
The method described by 994 et al. Can be used in combination with a quantitative MHC binding assay. Alternatively, the immunogenicity of the selected peptide can be determined in vivo using known methods.
It may be tested by itro CTL induction (as described below for ex vivo CTL induction). The principle of a method of selecting a peptide capable of eliciting a cellular immune response in several steps is described in WO 97/30721. The contents of this document are incorporated herein by reference. A general strategy for obtaining efficient immunogenic peptides within the scope of the present invention is also described by Schweighoffer, 1997.

Instead of using the original peptide that fits into the binding groove of the MHC-I or MHC-II molecule, ie a peptide derived from unmodified 9D7, the anchor position and length specified based on the original peptide sequence Mutations may be made according to the minimum requirements of the present invention. Provided that these mutations not only impair, but preferably enhance, the effective immunogenicity and T cell receptor activating capacity of the peptide formed by the binding affinity to the MHC molecule. In this case, an artificial peptide or peptide equivalent designed to meet the requirements for the ability to bind to the MHC molecule is used.

The peptides modified in this way are called “heteroclitic peptides”. They can be obtained by the following methods: First, for example, use MHC using the principles described by Rammensee et al., 1995.
Obtaining epitopes of -I or MHC-II ligands or variants thereof. The length of this peptide, if it should be compatible with the MHC-I molecule, preferably corresponds to a minimum sequence of 8 to 10 amino acids with the necessary anchor amino acids. If desired, the peptide may be extended at the C-terminus and / or N-terminus, provided that the extension does not affect the ability to bind to the MHC molecule and that the extended peptide may undergo cellular processing to a minimal sequence. It is a condition. The modified peptides are then examined for recognition by TIL (tumor infiltrating lymphocytes), CTL induction and increased MHC binding and immunogenicity as described in Parkhurst et al., 1996 and Becker et al., 1997.

Another method for finding peptides having greater immunogenicity than the native 9D7 peptide suitable for the purposes of the present invention is as described in Blake et al., 1996.
Based on screening peptide libraries with CTLs that recognize the 9D7 peptide found naturally on tumors; in this context, designing molecules that mimic tumor epitopes recognized by MHC-I restricted CTLs It is recommended to use a combinatorial peptide library for this. 9D7 peptides or immunogenic fragments or peptides derived therefrom according to the invention may be produced recombinantly as described in WO 96/10413 or may be produced by peptide synthesis. The disclosure of this document is incorporated herein by reference. For recombinant production, the corresponding DNA molecule is inserted into an expression vector by standard methods, transfected into a suitable host cell, the host is cultured under appropriate expression conditions, and the protein is purified. Routine methods may be used for chemical synthesis of the 9D7 peptide, for example, a commercially available automated peptide synthesizer.

In place of the natural 9D7 peptide or heteroclitic peptide, substances that mimic such a peptide, such as “peptide mimetics” or “retroinverse peptides” can also be used. To test these molecules in terms of therapeutic use in tumor vaccines, the same methods described above for native 9D7 peptides or 9D7 peptide equivalents can be used. TAA designated 9D7 of the present invention and protein fragments, peptides or peptide equivalents or peptide mimetics derived therefrom are useful for cancer therapy, e.g., to induce an immune response against tumor cells expressing the corresponding antigenic determinant. Can be used. They are preferably used to treat tumors with strong 9D7 expression, especially tumors with expression in renal cell carcinoma, lung and breast cancer, and Hodgkin's lymphoma. An immune response in the form of CTL induction can be achieved in vivo or ex vivo.

In order to induce CTL in vivo, a pharmaceutical composition comprising TAA 9D7 or a fragment or peptide derived therefrom as an active ingredient is administered to a patient suffering from a cancerous disease associated with the TAA. Peptide) is effective C for antigen-bearing tumors
Must be sufficient to get a TL response. Thus, according to another aspect, the present invention relates to a pharmaceutical composition for parenteral, topical, oral or partial administration. Preferably, the compositions are used parenterally, for example for subcutaneous, intradermal or intramuscular applications. 9D7-TAA / peptide is dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
The composition may include a conventional adjuvant such as a buffer. TAA
The / peptide may be used alone or in combination with an adjuvant, eg, Freund's incomplete adjuvant, saponin, aluminum salt, or in a preferred embodiment a polycation such as polyarginine or polylysine. The peptide may be bound to a component that aids in CTL induction or CTL activation, such as a T helper peptide, lipid or liposome, and may be combined with these substances and / or immunostimulatory substances, such as cytokines (IL-2, IFN-γ). It may be administered. Suitable methods for the preparation and administration of the pharmaceutical compositions of the invention are described in WO 95/04542 and WO
97/30721, the disclosures of which are incorporated herein.

[0028] 9D7 (fragments) or 9D7 peptides may be used to trigger a CTL response ex vivo. Ex vivo CTL responses to tumors expressing 9D7 show that CTL precursors
Induced by incubation with 9D7 peptide or 9D7 protein. The activated CTL are then expanded and subsequently re-administered to the patient.
Alternatively, the APC may be loaded with the 9D7 peptide. This can result in efficient activation of the cellular immune response against 9D7-positive tumors (Mayordomo et al., 1995; Zitvo
gel et al., 1996). One suitable method for loading peptides on cells, for example on dendritic cells, is described in WO 97/19169. In one embodiment of the invention, several different 9D7 peptides or 9D7
A combination of peptide equivalents is used. In another embodiment, the 9D7 peptide is combined with other TAA-derived peptides. The selection of peptides for such combinations is made in terms of detecting different MHC types to cover the broadest patient population by combining peptides from different tumor antigens, and / or Or for the broadest possible spectrum of cases. While the number of peptides in a pharmaceutical composition can vary widely, typically a clinically useful vaccine will contain from 1 to 15, preferably from 3 to 10, different peptides.

[0029] The peptides according to the invention can also be used as diagnostic reagents. For example,
The peptides may be used to test a patient's responsiveness to a humoral or cellular immune response elicited by the immunogenic peptide. This offers the potential to improve treatment procedures. For example, TAA dosage forms (peptides,
Depending on the total protein or DNA vaccine, the increase in T cell precursors in PBLs that are responsive to defined peptide epitopes can be examined (Robbins and K
awakami, 1996 and references cited therein). In addition, antibodies directed against peptides or total protein or TAA can be used to predict the risk of a patient succumbing to a 9D7-associated tumor or to characterize the progression of 9D7-positive tumors (eg,
By immunochemical analysis of secondary tumors and metastases). This type of strategy has proven to be successful in many cases, for example detecting the estrogen receptor as a basis for making an endocrine therapy in breast cancer; c-erbB- as a relevant marker in the prognosis and course of treatment in breast cancer 2 (Raviaioli et al., 1998; Revillion et al.,
1998); PSMA (prostate specific membrane antigen) as serum marker for prostate cancer epithelial cells, or PSMA in immunoscintigraphy for prostate cancer
Of PSMA by Using a ¹¹¹ In-Labeled Monoclonal Antibody Against Murine (Murphy et al., 199
8 and references therein); CEA (carcinoembryonic antigen) as a serological marker for prognosis and progression in colorectal cancer patients (Jessup and Loda, 1998
) Detection.

According to another aspect, the invention relates to an isolated DNA molecule encoding a protein or a fragment thereof having the immunogenic properties of 9D7. Preferably, the present invention provides an isolated DNA molecule comprising a polynucleotide having the sequence set forth in SEQ ID NO: 1 or an isolated DNA comprising a nucleotide that hybridizes under stringent conditions to a polynucleotide having the sequence set forth in SEQ ID NO: 1. Is a molecule. In the present invention, “stringent hybridization conditions” refers to 50
% Formamide, 5 × SSC (1 × SSC = 150 mM NaCl, 15 mM trisodium citrate)
, 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate,
And in a solution containing 20 μg / ml denatured sheared salmon sperm DNA at 42 ° C. overnight, followed by washing the filters with 0.1 × SSC at about 65 ° C., or equivalent conditions.

The DNA molecule of the present invention or a fragment thereof encodes an immunogenic (poly) peptide designated 9D7 having the amino acid sequence shown in SEQ ID NO: 2 or a protein fragment or peptide derived therefrom; And DNA molecules that show mutations from the sequence shown in SEQ ID NO: 1 as a result of the degeneracy of the genetic code.

The present invention also provides for deviations from the sequence set forth in SEQ ID NO: 1 resulting from conservative exchange of amino acids, as long as they encode a 9D7 derivative or fragment or peptide having desirable immunogenic properties for use as a tumor vaccine. DNA molecules having. As compared to the molecule shown in SEQ ID NO: 1, the DNA molecule of the present invention may
May be extended at the 5 'end to a start codon that may be located upstream of The 9D7 DNA molecule of the invention, or the corresponding RNA, which is also a subject of the invention, as well as the (poly) peptide encoded thereby, are used for immunotherapy of cancer diseases.

In one embodiment of the invention, a DNA molecule encoding native 9D7 is used. In place of the native 9D7 cDNA or a fragment thereof, a modified derivative may be used. These include sequences with modifications that encode proteins (fragments) or peptides with greater immunogenicity, while the same considerations that apply to peptides described above apply to modifications at the DNA level. . Another type of modification is to align multiple sequences encoding peptides that are immunologically related like a rosary (To
es et al., 1997). The sequence may also be modified by the addition of auxiliary elements to ensure more efficient release and processing of the immunogen, such as functional groups (Wu et al., 1995). For example, antigen processing and, thus, presentation and ultimate immunogenicity are localized to the endoplasmic reticulum ("ER targeting sequences").
Can be increased by adding

[0034] In another aspect, the invention relates to a recombinant DNA molecule comprising 9D7 DNA. The DNA molecules of the present invention may be administered directly in recombinant form, preferably as a plasmid, or as part of a recombinant virus or bacterium. Theoretically, any gene therapy method could be based on DNA (DNA vaccine), 9D7 DNA, in vivo and ex vivo
o Can be used for either cancer immunotherapy. Examples of in vivo administration include "naked" D via the intramuscular route or using a gene gun.
Direct injection of NA, which has been shown to result in the formation of CTL against tumor antigens. Examples of recombinant organisms are vaccinia virus, adenovirus or the Listeria monocyte gene (review is given by Coulie, 1997). In addition, synthetic carriers for nucleic acids such as cationic lipids, microspheres, micropellets or liposomes may be used for in vivo administration of a nucleic acid molecule encoding a 9D7 peptide. Various adjuvants, such as cytokines, either in the form of a protein or in the form of a plasmid encoding it, which enhance the immune response, such as peptides, may be administered. This application is sometimes physical,
For example, it may be combined with electroporation.

Examples of ex vivo administration are the transfection of dendritic cells as described by Tuting, 1997, or of other APCs used as cellular cancer vaccines. Thus, according to another aspect, the invention relates to the use of cells expressing 9D7 itself or optionally modified forms of 9D7 transfected with the corresponding coding sequence for the production of a cancer vaccine. In another aspect, the invention relates to antibodies to 9D7 or a fragment thereof. Polyclonal antibodies are conventionally obtained by immunizing an animal, particularly a rabbit, by injecting the antigen or a fragment thereof followed by purification of the immunoglobulin. The monoclonal anti-9D7 antibody is immunized in an animal, particularly a mouse, and immortalized by fusing the antibody-producing cells of the immunized animal with, for example, myeloma cells, by standard methods according to the principles described by Koler and Milstein, 1975. The obtained hybridoma supernatant can be obtained by screening for anti-9D7 antibody by an immunologically standard assay. For therapeutic or diagnostic use in humans, these animal antibodies may optionally be chimerized in a conventional manner (Neuberger
Et al., 1984) or humanized (Riechamnn et al., 1988; Graziano et al., 1995).

The human monoclonal anti-9D7 antibody (or fragment thereof) is a so-called phage display library (Winter et al., 1994, Griffiths et al., 1994, Kruif et al., 1995, McG
uiness et al., 1996), and transgenic animals (Bruggemann et al., 1996,
Jakobovits et al., 1995). The anti-9D7 antibody of the present invention can be used for immunohistological analysis for diagnostic purposes. In another aspect, the present invention relates to the use of a 9D7-specific antibody for selectively providing a desired substance to or into a tumor expressing 9D7. Examples of such substances are cytotoxic reagents or radioactive nuclei, substances whose activity is to damage the tumor in situ. Due to the tumor-specific expression of 9D7, no or almost no side effects are expected. According to another aspect, a substance for revealing cells expressing 9D7 may be used with the aid of a 9D7 antibody. This is useful for diagnosis and treatment evaluation. The use of therapeutic and diagnostic antibodies applicable to anti-9D7 antibodies is described in WO 95/3377.

Due to the preferential expression of 9D7 in tumor cells, it is hypothesized that this protein has functions important for tumors, such as tumorigenesis, invasion and proliferation. Thus, 9D7 (DNA) can be used in screening assays to identify substances that modulate, especially inhibit, this protein. In one embodiment, such an assay comprises introducing into a cell a 9D7 protein or an active fragment thereof that reacts with 9D7 activity for growth, or expressing the respective 9D7 DNA in a cell, and testing the test substance. Measuring the proliferation of the cells in the presence or absence of the. Substances with an antiproliferative effect can be used for the treatment of tumors with strong expression of 9D7, especially renal cell carcinoma, lung cancer, colon and breast cancer and Hodgkin's lymphoma.

Examples Example 1: RDA (representational analysis) of renal cell carcinoma tissue and normal kidney tissue
Ifference analysis)) human kidney cell carcinoma (RCC of clear cell type from the same patient, the biopsy and normal kidney tissues RCC) immediately after surgical removal and frozen in liquid N _2, -80 ° C. Saved in. For RNA isolation, serial 20 μm sections were placed in a cryomicrotome (Jung CM1800, L
eica), prepared at −20 ° C., and directly dissolved in 4M guanidinium thiocyanate / 1% β-mercaptoethanol. The sample was ultracentrifuged on a CsCl gradient (Samb
rook, 1989).

Starting from 60 μg of total RNA from renal cell carcinoma RCC6 and 350 μg of total RNA from autologous normal kidney N6 and other normal kidney tissue (N3), RDA (Diatchenko et al .; Hubank and Sch
atz) was performed using the PCR-Select ^™ kit (Clontech, Palo Alto) according to the manufacturer's instructions: a polyATtract kit (Promega) was purchased from the manufacturer using RCC (tester) and RNA from normal kidney tissue (driver). Use poly-A (+) RNA as directed
Was isolated. After synthesizing a double-stranded cDNA using oligo-dT, this was digested with RsaI. An equivalent portion of the tester cDNA was ligated with either adapter A or B and each separately hybridized at 65 ° C. with an excess of driver cDNA. The two mixtures were then combined and subjected to a second hybridization with the new denatured driver cDNA. The enriched tester-specific cDNA is then adaptered
Exponentially amplified by PCR using A or B specific primers. An aliquot of this reaction was subjected to a second PCR with specific internal nested primers for further enrichment. The product of this reaction is transferred to the pCRII vector (Invitrogen)
And then transfected into competent E. coli (OneShot ^™ , Invitrogen). 1920 positive transformants (blue-white selection) in a 96-well block
The cells were cultured in LB-Amp medium for 24 to 48 hours at 37 ° C.

A 200 μl aliquot of the E. coli suspension was heated to 100 ° C. for 10 minutes. After brief centrifugation of the bacterial residue, 140 μl of the clear supernatant (（1 μg of plasmid DNA)
Transfer to μl 20xSSC and use standard method for making DNA dot blots (Sambro
ok, 1989) and immobilized on a nylon membrane (Hybond-N, Amersham) equilibrated. The remaining bacterial culture was stored at -80 ° C as the glycerin parent culture. A cDNA subtraction library was obtained from 1920 individual clones. They exist both as E. coli glycerin parent cultures and as plasmids immobilized on DNA dot blots.

Example 2 Selection of Tumor-Testis Antigen by Hybridizing a cDNA Subtraction Library with a Testis-Specific cDNA Library “Human Testis-Specific PCR-Select ^™ cDNA” (Human Testis-Specific PCR-Select) (Clontech, Palo, Alto) were amplified by 11 cycles of PCR according to the manufacturer's instructions. Aliquots are random primed (HiPrime Kit, Boehring
er Mannheim) with [α- ³² P] dCTP (NEN, Boston) and the DNA dot blot described in Example 1 under stringent conditions (65 ° C.) by standard methods (Sambrook, 1989). Hybridized. Human Testis-Specific PCR-S
Clones hybridizing to the elect ^™ cDNA were visualized by standard autoradiography (Xomat DR film, Kodak). According to the results of Example 2, 150 clones predominantly transcribed in renal cell carcinoma RCC6 and testis could be selected. These clones are candidates for a class of tumor-testis antigens.

Example 3 DNA Sequencing and Annotation of Tumor Testis Antigen Plasmids from 62 Clones Selected Based on the Results Obtained in Example 2
DNA was isolated according to the manufacturer's instructions (QIAgen) and sequenced by the Sanger method on an ABI-prism instrument. The sequence determined in this way is transferred to BioWorkBench software (GeneData, Basl
Analysis was performed using e) and the data bank (Genbank) was compared. This allowed the identification of 33 known genes and 29 unknown genes. For the latter,
There was only EST entry. Known genes were not processed further. Expression profiles were evaluated for 29 unknown genes: experimentally determined sequences and 95
For all ESTs in the data bank with greater than% homology (BALST), the starting tissue was checked against the corresponding cDNA library. These were subdivided into i) critical normal tissues, ii) fetal, "disposable" and immunodominant tissues and iii) tumors and cell lines. ES based on this “virtual mRNA profile”
The 16 clones in which T was not found in group i) were selected for further experimental analysis.

Example 4 Transcriptional Analysis of Candidate Clones in Renal Cell Carcinoma and Normal Kidney 2-5 μg of total RNA from tumor tissue or normal tissue was analyzed by Superscript II (Gibco) or AMV-RT (Promega) according to the manufacturer's recommendations Reverse transcribed. For each individual RNA probe, a second batch was run without reverse transcription as a control for chromosomal DNA contamination. Or human heart, leukocytes,
Kidney, liver, lung and testis plasmid cDNA libraries (Gibco) were used. C
20, 25 and 30 cycles of PCR (40 sec at 94 ° C., 45 sec at 55 ° C., 72 ° C.) using β-actin specific primers (SEQ ID NOs: 29 and 30) 1 minute 30 seconds, from cycle 11 on each additional cycle for 30 seconds at 55 ° C and
(3 seconds elongation at 55 ° C.). The 9D7 cDNA was replaced with the 9D7
Amplified by 30 cycles of the same program with specific primers. The other 15 candidate clones were also tested with specific primers. PCR products were detected by agarose electrophoresis and ethidium bromide staining. Five of the 16 clones examined were compared with their normal kidney tissue N6 by RT-PCR for renal cell carcinoma RCC.
In Figure 6, clear overexpression could be detected. Appearance in testis cDNA was examined as an additional positive control for all clones.

Example 5: Quantitative Comparison of 9D7 mRNA Expression between Tumor and Normal Tissues A 9 μg pool of total RNA from three tumor and normal tissues was prepared using DNaseI (Boehringer
Mannheim), followed by phenol extraction, followed by oligo-dT and AMV-RT (P
reverse transcripts using the following method. 9D7 cDNA of glyceraldehyde-3-phosphate as a control of copy number or total cDNA quantity and quality - dehydrogenase cDNA and (GAPDH) 5'-nuclease PCR assay ^{(TaqMan R PCR, PE Applied Biosystems} ) quantitated using a did.
Primers or labeled TaqMan probes for cDNA quantification are SEQ ID NOs: 3 and 4,
And SEQ ID NO: 31 (labeled at the 5 'end with 6-carboxy-fluorescein and labeled at the 3' end with 6-carboxy-tetramethyl-rhodamine),
For GAPDH, the primers described in SEQ ID NOS: 25 and 26, and SEQ ID NO: 32 (the 5 ′ end is labeled with tetrachloro-6-carboxy-fluorescein, and the 3 ′ end is labeled with 6-carboxy-tetramethyl-rhodamine) Labeled). ABI
Using the PRISM 7700 sequence detection system (PE Applied Biosystems), PCR (9D
For 7 cycles, 40 cycles {95 seconds at 95 ° C, 1 minute at 60 ° C} or for GAPDH, 40 cycles {15 seconds at 95 ° C, 1 minute at 66 ° C}) and online fluorescence measurements .

Example 5 showed that 9D7 was transcribed approximately 1.5 to 6.5 times more strongly than GAPDH in a pool of renal cell carcinoma and lung cancer (SCC). In other types of tumors, the rate of transcription varies between 0.25 and 0.6 times the value of GAPDH. Normal liver and testis show about 10% value of GAPDH transcript, while other normal tissues examined show virtually no 9D7 transcript (Table 1).

Example 6: Expression profile of 9D7 in tumor and normal tissues One of the 15 candidates (designated 9D7) was identified by RT-PCR in all renal cell carcinomas and some other It was shown to be strongly transcribed in tumor types. However, no transcript was found in most normal tissues. FIG. 1 shows a comparison between four normal kidneys (N1, 2, 4, 5) and four renal cell carcinomas (RCC1, 2, 3, 6); and in this test, the myocardium (he), Leukocyte (leu), kidney (ki), liver (li), lung (lu) and testis (tes) cDNAs were also tested; controls were performed with β-actin primers. These results were confirmed by independent experiments using three different primer pairs (SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 10, and SEQ ID NOs: 11 and 15).

For Northern blot analysis, multiple human tissue Northern blots (Clonte
ch, Palo Alto) and a 170bp 9D7 PCR product labeled with [α- ³² P] dCTP (NEN, Boston).
Hybridization was performed at 65 ° C. for 16 hours. Visualization was obtained by standard autoradiography (Xomat AR film, Kodak). Figure 2 shows the results of this analysis: 1
6. Normal tissues (1. white blood cells, 2. large intestine, 3. small intestine, 4. ovary, 5. testis, 6. prostate, 7. thymus, 8. spleen, 9. pancreas, 10. kidney, 11. skeletal muscle, (12 liver, 13. lung, 14. placenta, 15. brain, 16. myocardium), transcripts of ~ 2.2 kb were present only in myocardium. However, immunologically relevant expression is unlikely due to the low signal intensity. All experimental results for the 9D7 mRNA profile (collected from RT-PCR, quantitative RT-PCR and Northern blot analysis) are shown in Table 2. Example 6 showed that 9D7 was strongly transcribed in a high percentage of tumors of various indices, while no or little transcript was found in any of the normal tissues examined.

Example 7: In situ hybridization of 9D7-specific RNA probe to renal cell carcinoma tissue and normal kidney tissue A 280 bp NotI fragment having 220 bp sequence information of 9D7 was subcloned into pBluescript and then used. To transform competent E. coli DH5-α. Using T3- and T7-RNA polymerases (sense; SacI, T3; antisense: BamHI
, T7), add digoxigenin-UTP and add to the manufacturer's recommendations (DIG RNA labeling kit, Boeh
RNA probes were synthesized by in vitro transcription according to Ringer Mannheim). After transcript purification (Sephadex G50 Quick Spin Column, Boehringer Mannheim)
Labeling efficiency was measured by dot blot. In situ hybridization was performed using standard methods (Boehringer Mannheim, Non-radioactive In situ hybridization Applica- tion Manual).
Replication Manual), 1996): 5 μm frozen sections were denatured after fixation with paraformaldehyde, acetylated and dehydrated or rehydrated and hybridized overnight at 52 ° C. in a moist chamber. After a normal washing step, 10 non-specific protein binding sites
Saturated with% FCS and 3% goat serum (DAKO) for 15 minutes. Visualization of the probe hybridized to 9D7 mRNA was performed by incubation with α-DIG-AP conjugate with NBT / BCIP (Boehringer Mannheim) and development at 4 ° C. in the dark.

FIG. 3 shows that renal cell carcinoma tissue or normal kidney tissue and 9D7-specific digoxigenin-UTP
Shown is the result of in situ hybridization with a labeled RNA probe (antisense): tumor tissue shows strong homogeneous staining, but normal tissue is unreactive; staining specificity is negative control ( Sense). Example 7 shows that 9D7 is transcribed in tumor cells of renal cell carcinoma tissue, but not in any cell type of normal kidney tissue, and is therefore described in Examples 4-6. The result is confirmed.

Example 8: To detect 9D7 protein expression, 9D7-specific antibodies were raised in rabbits.
A peptide named 9D7-3aa having the sequence of DAEERAEAKKKFRNLTRKTES (SEQ ID NO: 59) was used for immunization, and the resulting serum was affinity-purified with peptide 9D7-3aa. To determine the specific reactivity of 9D7-3aa sera, the complete 9D7 reading frame was transiently expressed in COS cells as a GFP fusion protein, and transfected cells were tested by western blot with 9D7-3aa sera . This serum clearly reacted with the eukaryotically expressed fusion protein. Subsequently, 18 different renal cell carcinomas were analyzed immunohistochemically for 9D7 expression using 9D7-3aa serum.
Expression in tumor cells was detected in 15 cases, whereas 9D7 expression was not detectable in gastric cancer. These examples are shown in FIG.

Example 9: Cloning of 9D7 Clone 9D7 has an insertion of an unknown human gene of 221 bp in length between the adapters introduced by RDA. Comparison with the data bank shows the homology of 9D7 to rat SM20, with a 69 bp C-terminal coding region (corresponding to 23 amino acids), a stop codon for SM20 and a 149 bp 3 ′ untranslated region with 9D7 in the antisense orientation. It corresponds to 221 bp. The 23 amino acids show only two exchanges between rat and human. SM20 has been described as a growth factor-induced immediate early gene and as a specific marker of smooth aortic muscle (Wax et al., 1994; Wax et al., 1996). To fully clone the human sequence, the following procedure was performed: UniGene Analysis (National Center
for Biotechnology Information) generated the following EST homologous to 9D7: AA23412
7, AA233899, AA917421, AA878927. Tumor cell line 786-0 (ATCC # CRL-1932) or
Total RNA was isolated from A549 (ATCC # CCL 185) using TRIzol (Gibco).
Reverse transcription was performed with II MMLV-RT (Gibco) and adapter-oligo dT-primer (SEQ ID NO: 27) according to the manufacturer's instructions. The primers of SEQ ID NOs: 5, 6, 7 and 10 of this cDNA and the original primers (SEQ ID NOs: 3 and 4), and PC
R amplification produced the expected fragment according to the homologous sequence. The longest 804 bp fragment was obtained by combining the primers of SEQ ID NOs: 10 and 5. The primers of SEQ ID NOs: 8 and 9 are located within the sequence range of EST-AA234127, which sequence sequence has no homology to SM-20 and thus did not generate an amplification product with any other primer.

Cloning of 3 ′ end: reverse transcription of 10 μg of total RNA from renal cell carcinoma cell line 786-0
However, one aliquot of the cDNA was lacking in PCR. PCR programs are gene-specific
High annealing temperature (Tm-93 ° C, sequence
No. 22), while a second primer (Tm-53 ° C., SEQ ID NO: 28)
) Binds only to newly synthesized DNA at low temperatures. This so-called
Touchdown PCR (Mastercycler Gradient, Eppendorf) was performed under the following conditions
: 20 cycles of {95 ° C for 15 seconds, 80 ° C for 30 seconds (1 ° C decrease for each cycle),
72 ° C for 2 minutes} and 20 cycles of {95 ° C for 15 seconds, 50 ° C for 30 seconds, 72 ° C for 2 seconds
Minutes}. 5 'end cloning: 1 μg of total RNA from renal cell carcinoma cell line 786-0 modified SMART ^TM Instructions (SMART^TM PCR cDNA Synthethic Kit, Clontech)
And Smart-II (SEQ ID NO: 23) were reverse transcribed: cDNA was SEQ ID NO: 4
RNasin (Promega) was added using the 9D7-specific primer described in (1). Array
No. 11 9D7-specific primer at a final concentration of 0.4 μM and SMART-PCR
A primer (SEQ ID NO: 24) was used at 0.1 μM. PCR amplification is performed at 95 ° C for 15 seconds, 65 ° C
For 30 seconds and 72 ° C. for 2 minutes) for 40 cycles.

An aliquot of the PCR batch was ligated directly to PCR 2.1 (Invitrogen) and transformed into competent E. coli (OneShot ^™ , Invitrogen). Positive clones were sequenced by blue-white selection. Sequencing revealed 50 additional nucleotides at the 3 'end, previously identified in EST-AA878927,
And so far identified 870 additional nucleotides at the 5 'end. PCR amplification of cDNA with the primers of SEQ ID NOs: 14-21 from tumor cell line (786-0, A549) and renal cell carcinoma biopsies is expected after cloning using the original primers and each other A fragment was generated. The identity of all PCR products was confirmed by sequencing. 282 during sequencing
Numerous clones with or without exons of bp (positions 399-680) were revealed. The presence of two splice variants was shown in several tumor probes (renal cell carcinoma, lung cancer / SCC, colon cancer / AC) by RT-PCR using primers as set forth in SEQ ID NOs: 11 and 15 or 13.

Example 10: Potential MHC-binding peptide in the coding region of 9D7 A potential peptide epitope in the coding region of 9D7 of SEQ ID NO: 2 is described by Parker et al., 1
Using the algorithm described by 994, known motifs (Rammensee et al.
1995). The most important HLA types, especially HLA-A1, A * 0201, A3, B7,
For B14 and B * 4403, candidate peptides expected to be able to bind to the corresponding HLA molecule and thus expected to be immunogenic CTL-epitope were identified; these peptides found are listed in Table 3. Raised. Similar methods can be used to find other potential peptide epitopes for other HLA types.

[0055]

[Table 1] Table 1. Relative transcription of 9D7 to GAPDH in tumor and normal tissues

[0056]

Table 2. mRNA expression profile of 9D7

[0057]

[Table 3] Table 3. Immunogenic 9D7 peptide candidate

[0058]

[Table 4] Table 3. Continued

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[Sequence list]

[Brief description of the drawings]

FIG. 1. Transcription of 9D7 in normal tissues and RCC: semi-quantitative RT-PCR for RNA from kidney, heart, leukocytes, liver, lung, testis and four renal cell carcinomas.

FIG. 2. Transcription of 9D7 in normal tissues: Northern blot analysis of mRNA of 16 normal tissues.

FIG. 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.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61K 48/00 A61P 37/04 A61P 35/00 C07K 14/47 37/04 16/28 C07K 14/47 C12P 21/08 16/28 C12N 15/00 ZNAA // C12P 21/08 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AU, BG, BR, CA, CN, CZ, EE , HU, ID, IL, IN, JP, KR, LT, LV, MX, NO, NZ, PL, RO, SG, SI, SK, TR, UA, US, UZ, VN, YU ZA (72) Inventor Adolf Günther Austria Aer-1070 Vin Schuttgasse 15-17 / 10 (72) Inventor Sommergrober Wolfgang Austria Aer 3002 Purkasd Ruff Linzer-Strasse 19 Haus 4 (72) Inventor Haider Carl- Heinz Austria A-2000 Stockerau Johann-Strauss Promenade 4/11 F-term (reference) 4B024 AA01 AA11 AA12 BA36 BA54 CA04 DA06 EA04 4B064 AG26 AG27 AG31 DA03 DA05 DA14 4C084 AA02 AA07 AA13 BA02 CA53 MA52 A55 NA14 BB11 BB31 CC01 DD62 GG08 GG10 4H045 AA10 AA11 BA09 BA54 CA41 DA76 DA86 EA22 EA51 FA74

Claims (20)

[Claims] 1. A tumor-associated antigen 9D7 having the amino acid sequence of SEQ ID NO: 2. 2. A polypeptide comprising the amino acid sequence of SEQ ID NO: 2 as a partial sequence. 3. An immunogenic protein fragment or peptide derived from the tumor-associated antigen according to claim 1. 4. The immunogenic (poly) peptide according to claim 1, which elicits a humoral immune response. 5. The immunogenic (poly) peptide or a degradation product thereof according to claim 1, which is presented by an MHC molecule and activates a cellular immune response. 6. The immunogenic peptide according to claim 5, which is selected from the group of peptides set forth in SEQ ID NOs: 34 to 58. 7. The method of claim 1 for in vivo or ex vivo immunotherapy of a cancer disease.
An immunogenic (poly) peptide according to any one of claims 6 to 9 wherein the patient has 9D7
(Poly) peptides that induce an immune response against tumor cells expressing 8. Parenteral administration, topical administration, oral or partial administration, characterized in that it comprises the immunogenic (poly) peptide according to any one of claims 1 to 6 as an active ingredient. Pharmaceutical composition. 9. The pharmaceutical composition according to claim 8, comprising various peptides derived from 9D7. 10. The pharmaceutical composition according to claim 9, comprising one or more peptides derived from 9D7 mixed with peptides derived from other tumor-associated antigens. 11. The pharmaceutical composition according to claim 9, wherein the peptide binds to at least two different HLA types. 12. An isolated DNA molecule encoding a protein having immunogenic properties of the tumor-associated antigen according to claim 1 or a fragment thereof. 13. The DNA molecule according to claim 12, which encodes the immunogenic polypeptide 9D7 having the amino acid sequence of SEQ ID NO: 2, or a protein fragment or peptide derived from 9D7. 14. A polynucleotide having the sequence of SEQ ID NO: 1.
A DNA molecule comprising a DNA molecule or a polynucleotide having the sequence of SEQ ID NO: 1, or a DNA molecule or sequence that is a polynucleotide that hybridizes under stringent conditions to a polynucleotide having the sequence of SEQ ID NO: 1 A DNA molecule comprising a polynucleotide that hybridizes under stringent conditions to a polynucleotide having the sequence of No. 1. 15. A recombinant DNA molecule comprising the DNA molecule according to any one of claims 12 to 14. 16. The DNA molecule according to any one of claims 12 to 15 for immunotherapy of cancer, wherein the 9D7 (poly) peptide expressed by the DNA molecule expresses 9D7 in a patient. The DNA molecule that induces an immune response against the tumor cell in question. 17. Use of a cell expressing the tumor-associated antigen according to claim 1 or 2 for the preparation of a cancer vaccine. 18. An antibody against the (poly) peptide of any one of claims 1 to 6. 19. The antibody according to claim 18, which is a monoclonal antibody. 20. The antibody according to claim 18 or 19, for treating and diagnosing cancer associated with 9D7 expression.