EP1183349A1 - Tumor-associated antigen (c42) - Google Patents

Abstract

The invention relates to a tumor-associated antigen, to immunogenic peptides derived therefrom, to coding DNA molecules that code therefor, and to their use in the immune based therapy of cancer diseases.

Description

Tu associated antigen (C42)

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

Showed a spontaneous remission rate, a correlation between clinical course and the increased occurrence of CD8 ⁺ and CD4 ⁺ T cells could be established (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: MHC 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 outside (Buschle et al., 1997; Schmidt et al., 1997) or taken up by the APC and transferred intracellularly to the MHC I molecules - the most therapeutically efficient form of application for a defined 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 function as a tumor-specific antigen for recognition by CTLs or to be used for therapy, the proteins must meet certain conditions: firstly, the antigen should be expressed mainly by tumor cells and not or only to a lesser extent in so-called "critical" normal tissues Concentration than in tumors. Critical normal tissues are essential tissues; directed against them immune response could have serious circumstances, lethal partly consequences. ^"Second, the antigen is not only in the primary tumor, but also in the metastases may be present. Furthermore, it is desirable in view of the widespread clinical use of the antigen when It is present in high concentrations in several types of tumor Another precondition 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 the TAA are said to be 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 to show that the order found potential candidate antigens are tumor-specific (tumor-associated) and actually T-cell epitopes have 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 problem was solved by first using RDA ("representational difference analysis") between a pool of different squamous cell carcinomas of the lungs and a pool of 11 different normal tissues to create a cDNA subtraction library. To generate the cDNA fragments necessary for subtractive hybridization "Tester" and "driver" were different from the original protocol (Diatchenko et al., 1996) used a mixture of 6 different restriction enzymes. The use of a mixture of different restriction enzymes, which require 6 base pairs as a recognition sequence, has the following advantages over the original protocol (Diatchenko et al., 1996): a) by selecting two restriction enzymes each, the recognition sequences of which are combined by a 6-combination of the bases A / T (e.g. Ssp I: AATATT) or C / G (e.g. Nae I: GCCGGC) or A / C / G / T (e.g. EcoR V: GATATC) are shown, both GC and AT-rich regions of a gene are alike cut, which enables a homogeneous representation of the entire gene region as restriction fragments; b) it is also possible to obtain larger cDNA fragments of the candidate gene on average (approx. 800 bp), which in turn is of great advantage in the subsequent analysis (sequencing and annotation) and the cloning of the “full-size” cDNA In the original protocol (Diatchenko et al., 1996), a restriction enzyme (Rsa I) which recognizes only four bases was used, which leads to an average fragment length of 256 bp and cannot specifically process regions rich in CG or AT In order to meet the longer insert cDNA fragments modified hybridization kinetics, the PCR protocol was changed as described in Example 1.

For the selection of the antigens overexpressed in the tumor, the cDNA clones obtained were first isolated and each of them a glycerol stock culture, a plasmid preparation and one representing the insert Collection of PCR fragments established in 96-well plate format. The cDNA fragments of the 4748 clones of the subtractive squamous cell carcinoma cDNA library of the lung were applied to filters in triplicate and each with a testis-specific cDNA library, a mixture of cDNAs from 15 normal tissues or pooled samples from tumor patients (squamous cell carcinoma of the lung) hybridizes to select specific tumor or testis-type antigens. Those 234 clones which gave a signal only with the testis-specific or the tumor-specific or with both hybridization samples, but not with the normal tissue hybridization sample, were selected for further analysis. After sequencing and annotation with in

36 unknown genes, some of which have ESTs (“expressed sequence tags”) in the database, have been obtained from databases available. Of these genes, those 10 were examined in detail whose ESTs did not originate from critical normal tissues.

Using semi-quantitative RT-PCR it was found that a clone (C42) showed a clear overexpression in the tumor and testis, but not in other normal tissues examined. A subsequent Northern blot analysis in different normal and

Tumor tissues confirmed that C42 had no transcription in the normal tissues examined, with the exception of a weak band in the esophagus, but a strong signal was seen in the squamous cell carcinoma of the lungs and esophagus. Furthermore, data obtained from the Northern Blot experiments is too conclude that the C42 transcript is approximately 4.4 kb in length.

The human C42 CDNA was cloned, the sequence obtained is shown in SEQ ID NO: 1. The sequence of C42 shows clear homology to a gene family of Ca ²⁺ -activatable Cl ^~ channel proteins, which are expressed in different species (in some cases very tissue-specific) at both the nucleotide and protein levels. 5 representatives of this family have been cloned and partially characterized; the two bovine genes bCLCAl ("bovine Ca ²⁺ -activated Cl ^" channel-1 "; Cunningham et al., 1995) and Lu-ECAM-1 (" bovine lung-endothelial cell adhesion molecule-1 "; Elble et al., 1997), the mouse gene mCLCAl ("murine Ca ²⁺ -activated Cl ^" channel-1; Gandhi, et al.,

1998) and two human genes hCLCAl ("human Ca ²⁺ -activated Cl ^" channel-1; Gruber et al., 1998) and hCLCA3 ("human Ca ²⁺ -activated Cl ^" channel-3; Gruber et al., 1999 ). All representatives of this protein family have typical transmembrane regions and, according to the current state of knowledge, are cleaved post-translationally to form heterodimers, the C-terminal part being glycosylated (Elble et al., 1997) with the exception of hCLCA3, which is a shortened form (Gruber et al., 1999). The bCLCAl gene was exclusively in

Bovine tracheal epithelial cells detected. The closely related Lu-ECAM-1, also isolated from bovine, is expressed in a tissue-specific manner in the vascular endothelial cells of the pulmonary veins. Zhu et al. (1992) demonstrated that Lu-ECAM-1 enables the binding of B16F10 lung metastases from mouse melanoma to endothelial cells. C42 is a new human representative of this protein family.

The C42 cDNA obtained in the context of the present invention has a continuous reading frame coding for 943 amino acids. The C-42 sequence has the five hydrophobic transmembrane regions typical of the protein family (positions nos. 222-252, 416-445, 553-574, 746-766 and 900-926 in SEQ ID: 2), but has a difference to the other representatives a C-terminus, which is built up from highly charged amino acids.

The C42 cDNA cloned in the context of the present invention is 4077 bp, the presence of a polyA tail at the 3 'end of the sequence indicating the completeness of the cDNA in this region.

The cDNA isolated in the context of the present invention has the nucleotide sequence given in SEQ ID NO: 1 and encodes the tumor-associated antigen (TAA) of the designation C42.

One from the isolated cDNA with this towards

The 5 'end of the open, continuous reading frame expressed protein has the amino acid sequence shown in SEQ ID NO: 2.

The invention thus relates in a first aspect to a tumor-associated antigen of the designation C42, which has the amino acid sequence given in SEQ ID NO: 2.

The sequence given in SEQ ID NO: 2 represents a protein which is derived from an approximately 4.4 kb transcript is translated, or is translated from a transcript of approximately 3.5 kb in size, which is derived from a splice variant of the 4.4 kb transcript or from a transcript of a gene homologous thereto.

The amino acid sequence shown in SEQ ID NO: 2 may have deviations, e.g. Those which are caused by the exchange of amino acids, provided that the C42 derivative has the immunogenic properties desired for use in a tumor vaccine.

The natural amino acid sequence of C42 can optionally be modified by exchanging individual amino acids in a C42 CTL epitope in order to increase the affinity of C42 peptides for MHC-I molecules and thus an, compared to the natural C42 CTL epitope cause increased immunogenicity and ultimately increased reactivity to tumors. Modifications in the area of the C42 epitopes can be carried out on the total C42 protein (this is processed by the APCs into the corresponding peptides) or on larger C42 protein fragments or on C42 peptides (see below).

In another aspect, the present invention relates to immunogenic fragments and peptides derived from C42. The latter are referred to below as "C42 peptides". A first group are

C42 peptides that trigger a humoral immune response (induction of antibodies). Such peptides are selected sections of C42 (at least 12 to 15 amino acids) which are used by means of so-called prediction algorithms such as, for example, 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.

Also included are all those peptides that contribute to an immunological differentiation between tumor and normal tissue.

It is known that tumor-associated antigens can have tumor-specific mutations that contribute to an immunological differentiation between tumor and normal tissue (Mandruzzato et al., 1997; Hogan et al., 1998; Gaudi et al., 1999; Wölfel et al., 1995 ). In order to determine the presence of tumor-specific C42 mutations, the C42 cDNA is expediently cloned from one or more different tumors with the aid of probes from the isolated cDNA according to the invention and the sequences obtained are compared with normal tissue C42 cDNA. It is to be expected that tumor C42 peptides from a sequence section mutated compared to normal tissue C42 have an increased immunogenicity compared to normal tissue C42 peptides from the corresponding section. To confirm that any mutations are tumor-specific, antibodies against these areas can be generated and tumor cells can be examined for the expression of possible mutations.

In a further aspect, the present invention thus relates to C42 peptides derived from regions of a tumor-expressed C42 which have tumor-specific mutations. C42 peptides are administered directly or in modified form (for example coupled to KLH = "keyhole limpet hemocyanine") and the formation of antibodies is determined by means of common immunological assays, for example by means of ELISA.

Further C42 peptides 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, with the patient to be treated having the MHC molecule in his

Must have a repertoire. The determination of the MHC subtype of the patient 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 C42 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 using a peptide derived from a tumor antigen, is directed against the patient's tumor cells. Immunogenic C42 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,

C42 peptides, which are CTL epitopes, are identified and synthesized based on the C42 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. For example, if a motif prescribes a 9-mer with Ile or Leu at the end, 10-mer with a corresponding C-terminus can also be considered, as can peptides with others 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 the 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, e.g. the enzyme specificity during antigen processing, in addition to the specificity of the MHC binding, contribute to the identity of the ligand. A method which takes these aspects into account and which is suitable within the scope of the present invention for the identification of immunogenic C42 peptides has been, inter alia, by Kawakami et al., 1995, to identify gplOO epitopes based on known HLA-A * 0201 motifs.

The peptides can also be selected for their ability to bind to MHC-II molecules. The MHC-II binding motif spanning nine

Amino acids, has a higher degree of degeneration in the anchor positions than the MHC-I Tie 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 in turn then directly activate CTLs by, for example

Cytokine release and enhance the efficiency of 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 and Rammenee et al., 1995, the most important types of HLA, in particular HLA-Al, -A * 0201, -A3, -B7 , -B14 and -B * 4403, candidate peptides of C42 identified which are expected to bind to the corresponding HLA molecules and are therefore immunogenic CTL epitopes; the peptides found are listed in Table 1. In a similar manner, further potentials may be used, possibly using further algorithms that take into account the different characteristics of the peptides (hydrophobicity, charge, size) or requirements on the peptides, for example the 3D structure of the HLA molecule Peptide epitopes can be determined; this also applies to peptide epitopes of other HLA types.

After selecting C42 peptide candidates using the methods listed, their MHC binding is tested using peptide binding assays. Next, the immunogenicity of the peptides is good

Binding properties determined (stability of the peptide-MHC interaction correlates in most cases with immunogenicity; van der Burg et al., 1996). To determine the immunogenicity of the selected peptide or peptide

To determine equivalents, methods such as 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 multi-step method for the selection of peptides capable of triggering a cellular immune response is in the

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, i.e. peptides that are derived unchanged from C42, variations can be made based on the minimum requirements regarding anchor positions and length based on the original peptide sequence are carried out, provided that the effective immunogenicity of the peptide, which is composed of its binding affinity for the MHC molecule and its ability to stimulate T cell receptors, is not only not impaired by these variations, but is preferably enhanced. In this case, artificial peptides or peptide equivalents are used, which are designed according to the requirements of the binding ability to an MHC molecule.

Such modified peptides are called

“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 method by Rammenee et al. , 1995, principle described. 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 tested for recognition by TILs ("tu or infiltrating lymphocytes"), for CTL induction, and for enhanced MHC binding and immunogenicity, as described by Parkhurst et al., 1996 and Becker et al., 1997 , described. Another method suitable for the purposes of the present invention for finding peptides with greater immunogenicity than that of the natural C42 peptides is to screen peptide libraries with CTLs which recognize the naturally occurring C42 peptides on tumors, as described by Blake et al. , 1996, described; 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 C42 polypeptide 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, transfected into a suitable host cell, the host is cultivated under suitable expression conditions and the protein is purified. Conventional methods can be used for the chemical synthesis of C42 peptides, e.g. commercially available automatic peptide synthesizers.

As an alternative to natural C42 peptides or heteroclitic peptides, substances which simulate such peptides, for example “peptidomimetics” or “retro-inverse peptides”, can be used. To test these molecules with regard to their therapeutic utility in a tumor vaccine, the same methods are used as above for the natural C42 peptides or C42 peptide equivalents. The TAA of the designation C42 according to the present invention and the protein fragments, peptides or peptide equivalents or peptidomimetics derived therefrom can be used in cancer therapy, for example to induce an immune response against tumor cells which express the corresponding antigen determinants. They are preferably used for the therapy of C42-positive tumors, in particular for lung, breast and esophageal carcinoma.

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 the active component TAA C42 or fragments or peptide (s) derived therefrom is administered to a patient suffering 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 C42-TAA / peptides are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. The composition can also contain customary auxiliaries, such as buffers, etc. The C42-TAA / peptides can be used alone or in combination with adjuvants, e.g. 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 which are suitable for producing and administering the pharmaceutical composition according to the invention are described in WO 95/04542 and WO 97/30721, the disclosure of which is hereby incorporated by reference.

C42 fragments or C42 peptides can also be used to trigger a CTL response ex vivo. An ex vivo CTL response to a tumor expressing C42 is induced by incubating the CTL progenitor cells together with APCs and C42 peptides or C42 protein. The activated CTLs are then allowed to expand, after which they are re-administered to the patient. Alternatively, APCs can be loaded with C42 peptides, which can lead to an efficient activation of cellular immune responses against C42 positive tumors (Mayordomo et al., 1995; Zitvogel et al., 1996). A suitable method for peptides on cells, e.g. Loading dendritic cells is disclosed in WO 97/19169.

In one embodiment of the invention, a combination of several different C42 peptides or C42 peptide equivalents is used. In another In one embodiment, C42 peptides are combined with peptides derived from other TAAs. The selection of peptides for such combinations is made with a view to the detection of different MHC types in order to cover the widest possible patient population and / or it is based on 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 a C42-associated tumor or to predict the course of the disease of a C42-positive tumor (for example by immunohistochemical analyzes of Primary tumor and metastases). Such 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 (Ravaioli 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 an ^nl 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 C42 or for fragments thereof.

In a further aspect, the present invention 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 contains a polynucleotide with the sequence shown in SEQ ID NO: 1 under stringent conditions hybridizes.

“Stringent conditions” mean, for example: overnight incubation at 65 ° C.-68 ° C. with 6xSSC (1 × SSC = 150 mM NaCl, 15 mM Tri-sodium citrate), 5 × Denhardt's solution, 0.2% SDS, 50 μg / ml salmon sperm DNA, then washing twice for 30 min with 2xSSC, 0.1% SDS at 65 ° C, once for 30 min with 0.2xSSC, 0.1% SDS at 65 ° C and, if necessary, finally rinsing with O.lxSSC, 0.1% SDS at 65 ° C, or equivalent conditions.

The DNA molecule according to the invention or fragments thereof code for (poly) peptides of the designation C42, containing 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, by mutation, lead to an exchange of amino acids in the protein sequence shown in SEQ ID NO: 2, provided that they are for a C42 derivative or fragments or peptides with the immunogenic properties desired for use as tumor vaccines encode.

The C42 DNA molecules of the present invention or the corresponding RNAs, which are also the subject of the present invention, are used, like the (poly) peptides encoded therein, for the immunotherapy of cancerous diseases.

In one embodiment of the invention

DNA molecules encoding natural C42 polypeptides used. As an alternative to the natural C42 cDNA or fragments thereof, modified derivatives can be used. These include sequences with modifications for a protein (fragment) or peptides encode stronger immunogenicity, with 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, for example functions which 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 a further aspect, the present invention relates to a recombinant DNA molecule which contains the C42 DNA.

The C42 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, any gene therapy method for cancer immunotherapy based on DNA ("DNA vaccine") on C42-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 form 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 for the in vivo administration of nucleic acid molecules coding for C42 peptide can be used. 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 C42, either on their own or, in optionally modified form, after transfection with the corresponding coding sequence, for the production of a cancer vaccine.

The invention relates in a further aspect

Antibodies against C42 or fragments thereof. Polyclonal antibodies can be obtained in a conventional manner by immunizing animals, in particular rabbits, by injection of the antigen or fragments thereof, and subsequent purification of the immunoglobulin can be obtained.

Monoclonal anti-C42 antibodies can be obtained according to standard protocols according to the principle described by Köhler and Milstein, 1975, by immunizing animals, in particular mice, then immortalizing antibody-producing cells of the immunized animals, e.g. by fusion with myeloma cells, and the supernatant of the hybridomas obtained is screened for monoclonal anti-C42 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-C42 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-C42 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 C42-specific antibodies in order to selectively bring any substances into or into a tumor which expresses C42. Examples of such substances are cytotoxic agents or radioactive ones Nuclides, the effect of which is to damage the tumor on site. Due to the tumor-specific expression of C42, no or only minor side effects are expected. In a further aspect, substances can be used to visualize tumors that express C42 with the help of C42 antibodies. This is useful for the diagnosis and evaluation of the course of therapy. Therapeutic and diagnostic applications of antibodies which are suitable for anti-C42 antibodies are described in WO 95/33771.

The TAA of the designation C42 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 C42-positive tumors, in particular for lung, breast and esophageal carcinoma.

Based on the preferred expression of C42 in tumor cells, it can be assumed that this protein has an important function for the tumor, for example for its formation, infiltration and growth. C42 (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 may consist of introducing the C42 protein, or an active fragment thereof, into cells that respond to the activity of C42 with proliferation to express the corresponding C42 DNA in the cell, 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 C42 expression, particularly in particular in the case of lung, breast and esophageal carcinoma.

Figure overview

Fig. 1: Transcription of C42 in tumor tissues and normal tissues: semiquantitative RT-PCR of RNA from different tissues

Fig. 2: Northern blot analysis of C42 in tumor and normal tissues

3: Binding test of five selected C42-CTL peptides on HLA-A * 0201

Example 1:

Representative Difference Analysis (RDA) of a pool of various squamous cell carcinomas of the lungs against a pool of 11 normal tissues

Biopsies of various human SCC (squamous cell carcinoma) lung carcinomas were shock-frozen in liquid nitrogen immediately after surgical removal and stored at -80 °

Isolation of RNA, serial 20 μm sections were made at -20 ° in a cryomicrotome (Jung CM1800, Leica) and directly in 4 M Guanidinium thiocyanate / 1% ß-mercaptoethanol dissolved to isolate the RNA. These samples were subjected to ultracentrifugation over a CsCl gradient (Sambrook, 1989).

Some representative tissue sections (5 μm) were fixed on a slide for histological diagnosis and stained with Harris' Hematoxyline (Sigma) and eosin. This served to provide tumor tissue as homogeneous as possible as a starting material for the extraction of the RNA. Only samples that were classified as SCC were processed further.

The poly-A (+) RNA was isolated from 110 μg total RNA from 5 different squamous cell carcinomas of the lung using the PolyAtract Kit (Promega) according to the manufacturer's instructions (SCC pool).

Starting from this SCC pool and a pool of 2.5 μg poly-A (+) RNA from 11 normal tissues (Clontech) bone marrow, heart, kidney, liver, lung, pancreas, skeletal muscle, spleen, thymus, small intestine and stomach became RDA (Diatchenko et al.,; Hubank and Schatz,) using the PCR-select ™ kit (Clontech, Palo Alto) according to the manufacturer's protocol: RNA from the SCC pool ("tester") and from normal tissue (" driver ") according to the manufacturer's protocol. In contrast to the original

The protocol was based on the synthesis of double-stranded cDNA using oligo-dT using the cDNA

6 restriction enzymes: EcoRV, Nael, Nrul -

Scal (Promega), Sspl, StuI (TaKaRa) in Promega buffer A for 2 hours at 37 ° C and after increasing the NaCl

Concentration on 150mM for a further 2 hours at 37 ° C cut. The use of this mixture of 6 different restriction enzymes allowed the generation of approx. 800 bp long cDNA fragments, which were used for the representative difference analysis.

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 68 ° C. The two batches were then combined and subjected to a second hybridization with freshly denatured “driver cDNA”. The enriched “tester” -specific cDNAs were then PCR-treated with primers of the kit specific for the adapters A and B, with an elongation time of 2 minutes at 72 ° C amplified exponentially, 27 cycles (10 "94 ° C, 30" 66 ° C, 2 '72 ° C). For further enrichment, an aliquot of this reaction was subjected to a second PCR with specific nested primers of the kit with an elongation time of 2 minutes at 72 ° C, 10 cycles (10 "94 ° C, 30" 66 ° C, The product resulting from this reaction was ligated into the pCR2.1 vector (Invitrogen) and then transfected into competent E. coli (OneShot ™, Invitrogen). 4748 transformants were obtained and in 96-well blocks cultivated in LB-Amp medium for 48 h at 37 ° C. Subsequently, 5 μl aliquots of the E. coli suspensions in 500 μl TE buffer were heated at 100 ° C. for 10 minutes and 1.5 μl of it was used as a template for a PCR in which the insert of the vector was amplified with flanking primers (SEQ ID NO: 9 and 10), 35 cycles (1'94 ° C., 1'55 ° C., 2'72 ° C.) The PCR products were analyzed using agarose -Gel- Electrophoresis and ethidium bromide staining demonstrated. 17 μl of the respective PCR product was taken up in a final volume of 252 μl 6xSSC and immobilized in 3 replicas on equilibrated nylon membranes (Hybond-N, Amersham) 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 was obtained from 4748 individual clones, which were present as E. coli glycerol stock cultures, whose immobilized PCR products were applied to nylon membranes and whose insert length was known from agarose gel electrophoresis. As expected, an average length of the inserted cDNA fragments of approximately 800 bp could be detected.

Example 2:

Selection of "tumor" and "tumor testis" genes by differential hybridization of the cDNA subtraction library

A human Testis-specific cDNA library ("Human Testis-Specific PCR-Select ™ cDNA"; Clontech, Palo Alto) was amplified according to the manufacturer's instructions by means of 11 cycles by PCR (10 "94 ° C, 30" 66 ° C, 1, 5'72 ° C.) Aliquots were labeled with “RTS RadPrime DNA Labeling System” (GibcoBRL) with [α- ³ P] dCTP (NEN, Boston) according to the manufacturer's instructions and according to standard instructions (Sambrook, 1989) for hybridization used under stringent conditions (68 ° C) with the DNA dot blots described in Example 1. Clones which hybridize with the "Human Testis-Specific PCR-Select ™ cDNA" were visualized by means of autoradiography (Xomat DR film, Kodak). The second set of DNA dot blots was carried out as described above with a labeled probe of a cDNA SCC Pools hybridized The third set of DNA dot blots was similarly probed with a cDNA normal tissue pool of 15 normal tissues (bone marrow, heart, kidney, liver,

Lungs, pancreas, skeletal muscle, spleen, thymus, small intestine, stomach, lymph nodes, mammary gland, prostate and trachea) hybridized.

The comparison of the image images and the autoradiographs or the net accounts of the differentially hybridized spots of a set allowed the selection of clones whose mRNA was overexpressed only in the tumor compared to normal tissue or transcribed both in the tumor and in testis (as a representative of an immune-privileged tissue) become. The former are candidates of the "tumor" class, the latter of the "tumor testis" antigen class.

Example 3:

DNA sequencing and annotation of "tumor" and "tumor testis" antigen candidates

The plasmid DNA of 234 clones selected on the basis of the results obtained in Example 2 was selected according to the manufacturer's instructions (QIAgen) isolated and sequenced according to the Sanger method on an ABI-Prism device. The sequences determined in this way were annotated by BLAST search (National Center for Biotechnology Information) and subjected to EST database comparisons. That allowed the

Identification of 198 known and 36 unknown genes. Only EST entries existed for the latter. The expression profile was estimated for the 36 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. A division was made into i) critical normal tissues, ii) fetal, "dispensable" and immune-privileged tissues and iii) tumors and tumor cell lines. On the basis of this "virtual mRNA profile", 10 clones were selected for further experimental analysis.

Example 4:

Transcriptional analysis of the candidate clones in tumor and normal tissue

Between 2 and 5 μg total RNA from tumor or normal tissues were reverse transcribed using SuperScriptll (GibcoBRL) 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. The quality and amount of the cDNAs was determined by PCR with β-actin Primers (SEQ ID NO: 3 and 4) and GAPDH specific primers (SEQ ID NO: 5 and 6) checked after 30 and 35 cycles (1 '95 ° C, 1' 55 ° C, 1 '72 ° C). The 10 candidate genes were analyzed analogously with specific primers. The PCR products were detected by agarose gel electrophoresis and ethidium bromide staining. A candidate named "C42" showed a relatively specific tumor / testis after 30 cycles with C42-specific primers (SEQ ID NO: 7 and 8).

Transcription profile; the semiquantitative RT-PCR of RNA from breast carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, kidney carcinoma, colon carcinoma, heart, lung, liver, kidney, colon, spleen and testis is shown in FIG. 1). This clone C42, which contains an insert of 549 bp, was subsequently subjected to a more detailed analysis.

Example 5:

C42 transcription profile in tumor and normal tissues

For the Northern blot analysis, human multiple tissue Northern blots (MTN; Clontech, Palo Alto) and (Invitrogen) were labeled with the [α- ³² P] dCTP (NEN, Boston) 549 bp C42 PCR product at 68 ° for 2 h hybridizes. The visualization was carried out by standard autoradiography (Hyperfilm, Amersham). 2 shows the result of this analysis: of 20 normal tissues (pancreas, adrenal medulla, thyroid, adrenal cortex, testes, thymus, small intestine, stomach, brain, heart, Skeletal muscle, colon, spleen, kidney, liver, placenta, lungs, leukocytes, bile, esophagus) and 4 tumor tissues (adenocarcinoma of the bile and stomach as well as squamous cell carcinoma of the esophagus and lungs). For C42 the tumor tissue shows the

Squamous cell carcinoma of the esophagus and lungs is a prominent transcript 4.4 kb in length. In normal tissues, there is only a weak 4.4 kb transcript in the esophagus. The low intensity of the signal makes an immunologically relevant expression seem unlikely. Another 3.5 kb transcript, which may represent a splice variant of the 4.4 kb transcript or is derived from a homologous gene, was also identified in both tumors, but not in normal tissues (FIG. 2). The presence of the TAA "C42" in squamous cell carcinoma of the lungs and esophagus is in good agreement with the original material used for the RDA (pool of 5 different patients with squamous cell carcinoma of the lung) and indicates a TAA that is specific for this type of Carcinomas.

Example 6:

Cloning of the C42 cDNA

The cloning of the human C42 cDNA was carried out as follows: A BLAST search gave the following ESTs overlapping with the C42 cDNA insert of 549 bp obtained in Example 4 (“original fragment”): AA429919; AA430055; AA446075; AA430264; AA160879. Starting from the sequence AA430055, a contig overlapping with the clone C42 was found with the EstExtractor on TigemNet (http://gcg.tigem.it/cgi-bin/uniestass.pl). The overlap of the contig and the sequence of the in

Original fragments of 549 bp (SEQ ID NO: 19) obtained in Example 4 could be verified by PCR aplification with a C42-specific primer and a primer located at the 3 'end of the contig (SEQ ID NO: 11 and 12) in lung tumor cDNA. With the help of the sequences AA430246 and AA446075 overlapping with the original fragment of C42, an extension in the 5 'direction was obtained. By two successive PCRs with the primer pair (SEQ ID NO: 13 and 14) for the first PCR and with the primer pair

(SEQ ID NO: 15 and 16) for the “nested” PCR, additional fragments belonging to C42 were amplified from the SuperScript ™ Human Testis cDNA Library (GibcoBRL) using the Advantage cDNA PCR Kit (Clontech) and the standard protocol described there. Based on this new sequence, three further EST entries belonging to C42 could be found: AI493356; AA443218; AA443258. Knowledge of these new sequences in turn enabled two PCRs with the primer pair (SEQ ID NO: 13 and 17) for the first PCR and with the primer pair (SEQ ID NO: 15 and 18) for the inwardly displaced "nested" PCR. This enabled further C42 fragments to be cloned.

For the sequence analysis, aliquots of the PCR batches were ligated directly into the pCR2.1 vector (Invitrogen) and then transformed into competent E. coli (OneShot ™, Invitrogen) and sequenced as described above.

The SuperScript ™ was obtained from two successive PCRs with the primer pair SEQ ID NO: 13 and SEQ ID NO: 20 for the first PCR and with the primer pair SEQ ID NO: 15 and SEQ ID NO: 21 for the "nested" PCR Human Testis cDNA Library (GibcoBRL) amplified further fragments belonging to C42 using the Advantage cDNA PCR Kit (Clontech) and the standard protocol described there.No new fragments belonging to C42 could be identified with other upstream primers, so it can be assumed that the "full-size" mRNA sequence of C42 (SEQ ID NO: 1) with a length of 4077nt was identified, which corresponds to the detection of a band of C42 on the MTN (FIG. 2) at about 4.4 kb The addition of a poly A tail and an inaccuracy in the separation of the RNA at the MTN can lead to deviations in the length of the sequenced polynucleotide chain from C42 to that recognized on the MTN.

The mRNA has a start codon at nucleotide 564-566, which forms the beginning of a continuous ORF to nucleotide 3392, which codes for a 943 AS long protein (SEQ ID NO: 2). The sequence of C42 shows a clear homology to a gene family of Ca ²⁺ -activatable Cl ^" channel proteins, which are expressed in different species (sometimes very tissue-specific) at both the nucleotide and protein levels.

5 representatives of this family have been cloned and partially characterized; the two bovine genes bCLCAl ("bovine Ca ²⁺ -activated Cl ^" channel-1 "; Cunningham et al., 1995) and Lu-ECAM-1 (" bovine lung-endothelial cell adhesion molecule-1 "; Elble et al., 1997), the mouse gene mCLCAl ("murine Ca ²⁺ -activated Cl ^" channel-1; Gandhi, et al., 1998) and the two human genes hCLCAl ("human Ca ²⁺ -activated Cl ^" channel; Gruber et al. , 1998) and hCLCA3 ("human Ca ⁺ -activated Cl ^" channel-3; Gruber et al., 1999). All representatives of this protein family have typical transmembrane regions and, according to the current state of knowledge, with the exception of hCLCA3, are cleaved post-translationally to form heterodimers, the C-terminal part being glycosylated (Elble et al., 1997). The bovine Lu-ECAM-1 enables, for example, the binding of the B16F10 lung metastases of mouse melanoma to endothelial cells (Zhu et al., 1992).

To check the correctness of the nucleotide sequence, overlapping fragments of C42 from lung squamous cell carcinoma and from Testis with the primer pairs (SEQ ID NO: 22 and 23) (SEQ ID NO: 24 and 25) (SEQ ID NO: 26 + 27) (SEQ ID NO: 28 + 29) (SEQ ID NO: 30 + 31) (SEQ ID NO: 32 + 33) (SEQ ID NO: 34 + 35) (SEQ ID NO: 36 + 37) and (SEQ ID NO : 38 and 39) amplified and sequenced. The alignment of both sequences shows - with the exception of a silent mutation in position 2399 G -> A- no differences.

The sequence of C42 has the five hydrophobic transmembrane regions typical of the protein family (positions Nos. 222-252, 416-445, 553-574, 746-766 and 900-926 in SEQ ID: 2), but has the difference to the other representatives have a C-terminus that is built up from highly charged amino acids.

Example 7:

Potential MHC binding peptides in the region coding for the C-terminal part of C42

Potential peptide epitopes within the coding region of the C-terminal fragment of C42 according to (SEQ ID NO: 2) were determined using the method described by Parker et. al., 1994 described algorithms performed on the basis of 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, 9-mer candidate peptides were identified which are expected to be present bind the corresponding HLA molecules and therefore represent immunogenic CTL epitopes; the peptides found are listed in Table 1. Using an analogous procedure, further potential peptide epitopes for other HLA types or 8- and 10-mer peptides can be determined.

Table 1

Immunogenic peptide candidates of the C-terminal fragment of C42 (742 amino acids)

Example 8:

T2-A2 peptide loading assay

This test is used to identify peptides that bind to HLA-A2 molecules and thus represent potential T cell epitopes. As described in Example 7, potential C42 epitopes were identified using prediction algorithms and the peptides synthesized (SEQ ID NO: 90-101). The HLA-A2 peptide epitope of tyrosinase was used as a positive control (SEQ ID NO: 102), and the HLA-Al as negative control

Epitope from MAGE3 (SEQ ID NO: 103). The peptides were dissolved in a concentration of 0 mg / ml in DMSO (Sigma) and diluted in PBS. T2 cells (ATCC number: CRL-1992) were resuspended in a concentration of 2.5 × 10 6 / ml in RPMI 1640 and provided with 10 μg / ml β ₂ microglobulin (ICN). Of these, 200 μl / well were sown in a 96-well plate and loaded in a dilution series with 0-320 μg / ml peptide. After an incubation of 16 h at 37 ° C, the amount of stable peptide HLA-ß ₂ microglobulin complex was measured in an FACS (Becton Dickinson) using an anti HLA antibody, which in turn is recognized by a second antibody R0480 (DAKO) ( Fig. 3). The amount of the fluorescence shift provides information about the amount of stabilized peptide-HLA-ß ₂ microglobulin complex on the cell surface. The behavior of five C42-CTL peptides selected from 12 C42-CTLs according to the test described above is shown in FIG. 3. The peptides show C42-5, C42-6, C42-8 and C42-11 (SEQ ID NO: 94, 95, 97 and 100)

CORRECTED SHEET (RULE 91) ISA / EP Binding to HLA-A 0201. They are therefore good candidates for T cell-specific peptide epitopes.

CORRECTED SHEET (RULE 91) ISA / EP literature

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Claims

claims

1. Tumor-associated antigen of the designation C42, characterized in that it contains the amino acid sequence defined in SEQ ID NO: 2.

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

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

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

5. Immunogenic peptide according to claim 4, selected from the group of peptides according to SEQ ID NO: 0 to 89.

6. Immunogenic (poly) peptide according to one of claims 1 to 5 for the immunotherapy of cancer in vivo or ex vivo, wherein the (poly) peptide induces an immune response against tumor cells of the patient, which express C42.

7. Immunogenic (poly) peptide, derived from areas of a tumor-expressed tumor-associated antigen C42, which has one or more tumor-specific mutations.

CORRECTED SHEET (RULE 91), ISA / EP

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 7 as an effective component.

9. Pharmaceutical composition according to claim 8, characterized in that it contains various immunogenic peptides derived from C42.

10. Pharmaceutical composition according to claim 9, characterized in that it contains one or more peptides derived from C42 in a mixture with peptides derived from other tumor-associated antigens.

11. Pharmaceutical composition according to one of the

Claims 8 to 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 C42, which contains the amino acid sequence contained 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 is or contains the sequence shown in SEQ ID NO: 1 or is or contains a polynucleotide which hybridizes with a polynucleotide of the sequence shown in SEQ ID NO: 1 under stringent conditions.

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

16. DNA molecule according to one of claims 12 to 15, for the immunotherapy of cancer, wherein the C42 expressed by the DNA molecule

(Poly) peptide induces an immune response against patient's tumor cells that express C42.

17. Use of cells which express the antigen defined in claim 1 for the production of a cancer vaccine.

18. Antibodies against a (poly) peptide defined in one of claims 1 to 5.

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 C42.