JP2003501025A - Tumor-associated antigen (C42) - Google Patents

Abstract

(57) Abstract: 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 has the task of protecting the body from many different microbes and actively fighting these microbes. The importance of the intact immune system is especially apparent in cases of congenital or acquired immunodeficiency. The adoption of vaccine programs for prophylaxis has proven to be a highly effective and successful immune intervention in combating viral or bacterial infections in many cases. It has also been found that the immune system is significantly involved in the elimination of tumor cells. Tumor associated antigen (TAA)
Recognition by components of the immune system plays an important role. In its broadest sense, any (peptidic or non-peptidic) component of a tumor cell that is recognized by elements of the immune system and leads to the stimulation of an immune response can act as an immunogenic tumor antigen. Such tumor antigens, which not only elicit an immune response but also cause tumor rejection, are of particular importance. The identification of specific antigens capable of eliciting this type of immune response constitutes a major step in the development of molecularly defined tumor vaccines. It is not yet clear which elements of the immune system contribute to tumor rejection, but nonetheless cytotoxic T-lymphocytes (CTLs) expressing CD8
It is acknowledged that plays a major role (Coulie, 1997). Especially in the case of tumor types such as those having a relatively high spontaneous remission rate (e.g., malignant melanoma and renal cancer), clinical progression and CD8 ⁺ - and CD4 ⁺ -T- cells appeared correlation between increased (Schendel et al., 1993; Mackensen et al., 1993; Halliday et al., 1995; Kawakami et al., 19).
95; Kawakami et al., 1996; Wang, 1997; Celluzzi and Falo, 1998). Specific CTL clones are generally obtained from either tumor infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) after co-culture with autologous tumor cells and cytokine stimulation in vitro. In both animal models and in vitro cultured human cell culture systems, T-cell responses to tumor cells were enhanced by cytokine transfection of tumor cells (van Elsas et al., 1997; Gansbacher et al., 1990; Te
pper et al., 1989; Fearon et al., 1990; Dranoff et al., 1993).

[0002] In view of the correlation between remission and CD8 ⁺ -T cell involvement, identification of tumor associated antigen (TAA) recognized by CD8-positive CTLs is a particularly important objective for the development of tumor vaccines. (Pardoll, 1998; Robbins and Kawakami, 1996). For example CD4 ⁺ -T
-Whether other cell types of the immune system, such as helper cells, play an important role is still unclear; many studies on MAGE-3 / HLA-A1 peptides in patients with malignant melanoma indicate this (Marchand et al., 1995; Boon et al., 1998). In the last few years, many TAAs recognized by CTL have been identified (Boon et al., 1994; van d.
en Eynde and van der Bruggen, 1997).

[0003] T-cells recognize antigens as cell surface peptide fragments of MHC molecules. (Major histocompatibility complex, in humans "HLA" = "human leukocyte antigen")
). There are two types of MHC molecules: MHC-I molecules appear in most nucleated cells,
It also presents peptides produced by the degradation of endogenous proteins by proteolytic enzymes (usually 8-10-members) (so-called antigen processing). The peptide: MHC-I complex is recognized by CD8-positive CTL. The MHC-II molecule appears only on so-called "professional antigen presenting cells" (APCs) and presents peptides of foreign proteins that are absorbed and processed in the process of phagocytosis by APCs. The peptide: MHC-II complex is recognized by CD4-helper-T cells. The interaction between the T-cell receptor and the peptide: MHC complex may trigger various effector mechanisms leading to target cell apoptosis in the case of CTL. This occurs when either MHC (eg in the case of transplant rejection) or peptides (eg in the case of intracellular pathogens) are recognized as foreign. In all cases, not all of the presented peptides meet the structural and functional requirements for effective interaction with T-cells (Rammensee et al. 1995 and below).

In principle, many administration methods using TAA in tumor vaccines are possible: this antigen is administered as a recombinant protein with a suitable adjuvant or carrier system or as a plasmid (DNA vaccine; Tighe et al., 1998) or can be administered as a cDNA encoding the antigen in a viral vector (Restifo, 1997). Another possibility is that recombinant bacteria which express the human antigen recombinantly and have an adjuvant effect as a result of its additional components (
For example, the use of Listeria, Salmonella) (Paterson, 1996; Pardoll, 199.
8). In all of these cases, this antigen is processed and presented by so-called "professional antigen presenting cells" (APC). Another possibility corresponds to a T-cell epitope equivalent to the antigen and is externally loaded into the APC (Busc
hle et al., 1997; Schmidt et al., 1997), or the use of synthetic peptides that are absorbed by APC and transferred intracellularly to MHC I molecules (Melief et al., 1996). The most therapeutically effective method of administration of the specified antigen will generally be determined by clinical trial.

Those antigens or epitopes recognized by tumor-specific CTL can be either protein class (eg, transcription factor, receptor, enzyme; Rammens for an overview).
ee et al., 1995; see Robbins and Kawakami, 1996). These proteins do not necessarily have to be localized on the cell surface, as required for antibody recognition. In order to act as tumor-specific antigens for recognition by CTLs or to be used therapeutically, these proteins must meet certain conditions: First, the antigens are exclusively associated with tumor cells. It must be expressed or occur in so-called "critical" normal tissues where it is expressed at a lower concentration than the tumor. Critical-onset normal tissues are essential tissues; the immune response to them is severe, with fatal consequences in some cases. Second, the antigen must be present not only in the primary tumor but also in the metastases. Furthermore, in view of the wide clinical use of the antigen, it is desirable that it be present in high concentrations in some types of tumors. One further prerequisite for the suitability of TAA as an active ingredient in vaccines is:
The presence of T-cell epitopes in the amino acid sequence of the antigen; TAA-derived peptides must lead to T-cell responses in vitro / in vivo ("immunogenic" peptides). Another criterion of clinically broadly applicable immunogenic peptide selection is the frequency of encountering the antigen in a given patient population.

Immunogenic tumor-associated antigens that are often shown to already have T-cell epitopes
(TAA) can be divided into many categories, which include viral proteins, mutant proteins, overexpressed proteins, fusion proteins created by chromosomal translocations, differentiation antigens, and embryonal carcinogen antigens (Van den Eynde And Brichard, 1995; van de
n Eynde and van der Bruggen, 1997).

Methods to identify and characterize the TAAs that form the starting point for tumor vaccine development have relied on the use of CTL already induced in patients (cell-mediated immune response) or antibodies (humoral immune response). Or based on the generation of differential transcriptional profiles between tumor and normal tissues. In the former case, which is an immunological method, patient CTL is a tumor of eukaryotic cells that presents CTL-epitope via MHC-I molecule.
-Used to screen a cDNA expression library (Boon et al., 1994), whereas a high affinity patient antiserum prokaryotic cDNA expression library was used to detect the presence of TAA in the immunity of individual plaques. Can be directly examined by blot analysis (S
ahin et al., 1995). The combination of CTL reactivity and protein-chemical processing is shown in Patient C
Reactivity with TL results in the isolation of peptides isolated from MHC-I from tumor cells that have been preselected. These peptides are washed out of the MHC-I complex and identified by mass spectrometry (Falk et al., 1991; Woelfel et al., 1994; Cox et al., 1994).
. Methods that use CTLs to characterize antigens are not always costly and always successful due to the need for CTL culture and activation.

There are a wide variety of methods for identifying TAAs based on comparing the transcriptional profiles of normal and tumor tissues; these include differential hybridization,
Establishment of subtraction cDNA bank ("RDA method: representational difference an
analysis; Hubank and Schatz, 1994; Diatchenko et al., 1996), and the use of DNA chip or SAGE methods (Velculescu et al., 1995). In contrast to the immunological methods using patient CTLs described above, when using molecular biology methods, the potential antigens discovered by this method are tumor-specific (tumor-related). And is actually cytotoxic
It is necessary to show that it has a T-cell epitope that is capable of inducing a T-cell response. In at least one case (NY-ESO / LAGE-1), the antigen was identified both by the use of patient sera and by the RDA method (Chen et al., 1997; Lethe et al., 1998), and also the CTL-epitope of this antigen. And simultaneous spontaneous humoral and T-cell responses
Described by one person (Jager et al., 1998).

The object of the present invention is to provide a novel tumor associated antigen (TAA). This aim was achieved by first establishing a cDNA subtraction library by the RDA method between a pool of various plate epithelial carcinomas of the lung and a pool of 11 different normal tissues. A mixture of six different restriction enzymes, which deviates from the original method (Diatchenko et al., 1996), was used to generate the "test" and "driver" cDNA fragments required for subtraction hybridization. The use of a mixture of different restriction enzymes that requires 6 base pairs as a recognition sequence has the following advantages over the original method (Diatchenko et al., 1996):
a) Combination of 6 bases with 2 restriction enzymes 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) and select the recognition sequence as shown in the following sequence to cleave both the GC- and AT-rich regions of the gene in the same manner, resulting in uniform presentation of the entire gene region as a restriction fragment. B) In addition, this is a statistically average (about 800 bp), with a relatively large c of the candidate gene.
It makes it possible to obtain DNA fragments, which is then a great advantage in the subsequent analysis (sequencing and annotation) and cloning of "full length" cDNAs. In the original method (Diatchenko et al., 1996), a restriction enzyme (Rsa I) that recognizes only 4 bases was used, which results in an average fragment length of 256 bp and specifically processes CG- or AT-rich regions. You cannot do it. The PCR method is modified as described in Example 1 to justify the hybridization kinetics that is altered by the longer cDNA transgene.

To select the antigens overexpressed in the tumor, the resulting cDNA clones were first isolated and cultured in basic glycerol, plasmid preparation and PCR fragment insertion-
From it, the insert-representing collection was established in a 96-well dish format. To select for specific tumor antigens or tumor / testis-type antigens, cDNA fragments of 4748 clones of the lung subtractive plate epithelial tumor cDNA library were placed on filters in triplicate and testis-specific. cDNA library, cDNA from 15 normal tissues or cD from pooled samples of tumor patients (platinum epithelial tumor of the lung)
Hybridized with a mixture of NAs. 234 clones were selected which were found to signal with only one or both testis-specific or tumor-specific hybridization probes, but not with normal tissue hybridization probes and were further analyzed. After sequencing and commenting with the sequences available from the databank, 36 unknown genes with some EST entries (expressed sequence tags) in the databank were obtained. Among these genes, 10 species in which the EST was not derived from the critical tissue for onset criticality were further studied. One clone (C42)
Semi-quantitative RT-PCR confirmed that it showed clear overexpression in tumors and testis but not in other normal tissues examined. Subsequent Northern blot analysis of various normal and tumor tissues revealed that C42 showed no transcription in the normal tissues examined-with the exception of the weak esophageal band-but strong in lung and esophageal flat epithelial tumors. It was confirmed that various signals were detected. In addition, the data obtained from the Northern blot experiment showed that the C42 transcript was approximately 4.4 kb in length.
It was concluded that

[0011]   Human C42 cDNA was cloned; the resulting sequence is shown in SEQ ID NO: 1. C
42 sequences expressed in various species at both the nucleotide and protein level
Ca (in some cases highly tissue-specific) Ca²⁺-Dependency Cl^-Channel protein
It shows clear homology with the gene family. To date, this family
5 representative species have been cloned and partially characterized: 2 cattle
Gene bCLCA1 (bovine Ca²⁺-Dependency Cl^-Channel-1; Cunningham et al., 1995) and Lu-EC.
AM-1 (bovine lung-epithelial cell adhesion molecule-1; Elble et al., 1997), mouse gene mCLCA1 (mouse
Su Ca²⁺-Dependency Cl^-Channel-1; Gandhi et al., 1998) and two human genes hCLCA1 (human
To Ca²⁺-Dependency Cl^-Channel-1; Gruber et al., 1998) and hCLCA3 (human Ca)²⁺-Dependency Cl ^- Channel-3; Gruber et al., 1999). Members of this protein family
All have typical transmembrane regions and, according to current knowledge, are post-translationally cleaved,
Form a heterodimer, the C-terminus of which is glycosylated (Elble et al., 1997)
Except for the shorter form hCLCA3 (Gruber et al., 1999). The bCLCA1 gene is
It is detected only in cattle tracheal epithelial cells. Also isolated from cattle
, A closely related Lu-ECAM-1, is tissue-specific in vascular epithelial cells of the pulmonary vein
Expressed in the form. Zhu et al. (1992) showed that Lu-ECAM-1 is a B16F1O lung translocation in mouse malignant melanoma.
It was shown to bind the translocation to epithelial cells. C42 is this protein family
Is a new human-related member of.

The cDNA obtained within the scope of the present invention has a contiguous open reading frame encoding 943 amino acids which represents the C-terminal part of C42. The C-42 sequence has five hydrophobic transmembrane regions typical of this protein family (locations are 222-252, 416-445, 553-574, 746-766 and 900 of SEQ ID NO: 2). -
926), unlike other members, has a C-terminus formed of strongly charged amino acids.

The cloned C42-cDNA within the scope of the invention has 4077 bp, while the presence of a poly A tail at the 3'end of this sequence is an indication of the integrity of the cDNA in this region. An isolated cDNA within the scope of the invention has the nucleotide sequence set forth in SEQ ID NO: 1 and encodes a tumor associated antigen (TAA) designated C42. Isolated cDN with a continuous open reading frame towards the 5'-end
The protein expressed by A has the amino acid sequence shown in SEQ ID NO: 2. Therefore, according to a first aspect, the invention comprises an amino acid sequence of SEQ ID NO: 2,
It relates to a tumor-associated antigen designated C42.

The amino acid sequence shown in SEQ ID NO: 2 is translated by a transcript of about 4.4 kb in size or is derived from a splicing variant of the 4.4 kb transcript from the transcript of this gene homolog. , A protein translated by a transcript of about 3.5 kb in size. The amino acid sequence shown in SEQ ID NO: 2 has some differences, for example caused by amino acid exchanges when the C42 derivative has the desired immunogenicity for use in tumor vaccines.

The native amino acid sequence of C42 is optionally substituted for individual amino acids of the C42 CTL-epitope in order to increase the affinity of the C42 peptide for MHC-I molecules compared to the native C42 CTL-epitope. Can result in increased immunogenicity and ultimately greater reactivity to the tumor. Modifications of the C42 epitope region can be made in the entire C42 protein, which is processed by APC to form the corresponding peptide, or in a relatively large C42 protein fragment or C42 peptide (compare below).

In another aspect, the invention relates to immunogenic fragments and peptides derived from C42. The latter is hereinafter referred to as C42 peptide. The first group is the C42 peptide that elicits a humoral immune response (antibody induction). Such peptides are for example surface probability blots (Emini et al., 1985), hydrophobicity blots (Kyte and Doolittle, 1982) and antigenic index (James).
on and Wolf, 1988), a selected portion of C42 (at least 12-15 amino acids), as can be determined by so-called predictive algorithms. Further included are all peptides that contribute to the immunological discrimination between tumor and normal tissues.

It is known that tumor-associated antigens can carry tumor-specific mutations, which contribute to the immunological discrimination between tumor and normal tissues (Mandruzzato et al., 1997,
Hogan et al., 1998, Gaudi et al., 1999, Wolfel et al., 1995). To determine the presence of a tumor-specific C42 mutation, the C42 cDNA was cloned from one or more different tumors, preferably by a probe of the isolated C42 cDNA of the invention, and the sequence obtained Is compared to the C42 cDNA of normal tissue. The tumor C42 peptide from the mutated sequence section relative to normal tissue is C42 from the corresponding sequence section from normal tissue.
It is expected that it will have increased immunogenicity as compared to the peptide.

The invention therefore relates, in a further aspect, to a C42 peptide derived from tumor-expressed C42 having a tumor-specific mutation. To confirm that any of the mutations are tumor-specific, antibodies to these regions can be generated and tumor cells can be examined for the expression of potential mutations. The C42 peptide is administered directly or in a modified form (eg in combination with KLH = keyhole limpet hemocyanin) and the formation of this antibody is determined by conventional immunological assays such as ELISA.

Other C42 peptides which are preferably within the scope of the present invention are such that they are presented by MHC-molecules and generate a cellular immune response. There are two types of MHC-molecules, the MHC-I molecule recognized by CD8-positive CTLs and the MHC-II molecule recognized by CD4-positive T-helper cells. In order for the peptide to elicit a cell-mediated immune response, it must bind to an MHC-molecule, while the patient being treated must have the MHC molecule in its repertoire. Therefore, determining the MHC-subtype of a patient is an essential prerequisite for the effective use of the peptide in this patient, in terms of raising a cellular immune response. The sequence of the C42 peptide used in therapy relates to anchor amino acids and length,
Determined by the MHC-molecule in question. The defined position and length of the anchor ensure that the peptide fits into the peptide binding groove of the MHC-molecule of the patient in question. As a result, if peptides derived from tumor antigens are used, the immune system is stimulated and a cellular immune response is generated directed against the tumor cells of the patient.

Immunogenic C42 peptides can be identified by known methods; one of the basic conditions is the correlation between MHC-binding and CTL-induction. Therefore, since the sequence of the immunogenic peptide can be predicted based on its peptide binding motif, the C42 peptide constituting the CTL-epitope can be identified and synthesized based on the C42 protein sequence. To do this, various methods used to identify CTL-epitope of known protein antigens are available; for example, to identify T-cell epitopes in human papillomavirus, Stauss et al. 1992).

The allele-specific requirements of each MHC-I allele product for peptides that bind to and are presented by MHC-molecules have been assembled as motifs (eg, Falk et al., 1991). To date, many MHC-peptide motifs and both MHC-ligands are known. Suitable methods within the scope of the present invention for investigating epitopes of known proteins which match a specific MHC-I molecule are described in the work of Rammensee et al. (1995). This involves the following steps: First, the protein sequence is examined for fragments corresponding to the anchor motif, while certain variations in peptide length and anchor occupancy are possible. For example, the motif is Ile at its end
Or if you specify a 9-member (mer) with Leu, a 10-member with the corresponding C-terminus is C-
It can also be considered as a peptide with another aliphatic group at the terminus, such as Val or Met. Many peptide candidates can be obtained by this method. They can be searched for the presence of as many anchor groups as possible in common with known ligands and / or to see if they can carry "preferred" groups on different MHC-molecules (Rammensee et al. , 1995 table). Binding assays are preferably performed to exclude weakly bound peptides. If the requirements for peptide binding for a specific MHC-molecule are known, the peptide candidate is considered to be a non-anchor group that has a negative or positive effect on binding, or in fact has no possibility at all. Can also be searched for (Ruppert et al., 1993). In this way, however, it should be noted that when searching for natural ligands, the peptide binding motif is not the sole determinant; in addition to MHC-binding specificity, other aspects such as antigens Enzyme specificity during processing also contributes to the identification of this ligand. One method which, considering these aspects and which is suitable within the scope of the invention for the identification of immunogenic C42 peptides is, in particular, the known HLA-A * O2O
The method of Kawakami et al. (1995) for identifying gp100 epitopes based on one motif was used.

These peptides can also be selected for their ability to bind to MHC-II molecules. The MHC-II binding motif spanning 9 amino acids has a higher degree of degeneracy at the anchor position than the MHC-I binding motif. Recently, a method based on the X-ray structure analysis of MHC-II molecules has been developed, which allows the accurate analysis of MHC-II binding motifs and the alteration of peptide sequences based on them. Yes (Rammensee et al., 1995
, And the original documents cited in this document). Peptides that bind to MHC-II molecules are typically presented to CD4-T cells by dendritic cells, macrophages or B-cells. CD4-T-cells then directly activate CTLs in the sequence, for example by releasing cytokines, and increase the efficiency of antigen presentation by APCs (dendritic cells, macrophages and B-cells).

Recently, databanks and prediction algorithms have become available that allow for more reliable prediction of peptide epitopes that bind to specific MHC molecules. Within the scope of the present invention, the algorithm described by Parker et al. (1994) and Rammensee et al. (1995) is used to bind the corresponding HLA molecule and thus contribute to the immunogenic CTL-epitope, a candidate peptide of C42. But the most important HLA-types, especially HLA
Identified for -A1, -A * O2O1, -A3, -B7, -B14 and -B * 44O3; the peptides found are listed in Table 1. Similarly, the use of other algorithms, perhaps taking into account various characteristics of the peptide (hydrophobicity, charge, size) or requirements by the peptide, such as the 3D structure of the HLA-molecule, makes it possible to find potential peptide epitopes. This also applies to other HLA-type peptide epitopes.

After selecting C42-peptide candidates using the method described above, their 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 most often correlates with immunogenicity; van der Burg et al., 1996). To determine the immunogenicity of selected peptides or peptide equivalents, for example, the method described in Sette et al. (1994),
It can be used in combination with a quantitative MHC-binding assay. Alternatively, the immunogenicity of selected peptides can be tested by in vitro CTL-induction (hereinafter referred to as ex vivo CTL-induction) using known methods. The principle of the method for the selection of peptides capable of eliciting a cellular immune response is carried out in several steps,
This is described in WO 97, the content of which is incorporated herein by reference.
/ 30721. A general strategy for obtaining efficacious immunogenic peptides that are also suitable within the scope of the present invention is 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 the peptide derived from the unmodified C42, an anchor specific for the original peptide sequence is used. Variants can be made while adhering to the minimum requirements for position and length, provided that these variants have a binding affinity for the MHC-molecule and its T-
Not only does it impair the effective immunogenicity of the peptide due to its ability to stimulate cell receptors, but preferably it even enhances it. In this case, artificial peptides or peptide equivalents designed to meet the requirements for binding capacity to MHC-molecules are used.

Peptides modified in this way are hereinafter referred to as "heteroclitic peptides". These can be obtained by the following method: First, the epitopes of the MHC-I or MHC-II ligands or variants thereof are harvested, for example, using the principles described in Rammensee et al. (1995). It The length of this peptide preferably corresponds to a minimal sequence of 8-10 amino acids with the required anchor amino acids, if the peptide corresponds to an MHC-I molecule.

If desired, the peptide can also be extended at the C- and / or N-termini, provided that this extension does not affect the ability to bind MHC-molecules and the extended peptide has a minimal sequence. And that it can be processed on cells. The modified peptides were subsequently analyzed by Parkhurst et al. (19) for their recognition by TILs (tumor infiltrating lymphocytes), CTL-induction, and increased MHC-binding and immunogenicity.
96) and Becker et al. (1997).

Another way of finding peptides with greater immunogenicity than that of the native C42 peptide suitable for the purposes of the invention is as described in the article by Blake et al. (1996): It consisted of screening a peptide library with CTLs that recognize the naturally occurring C42 peptide on tumors; in this context MHC-I-restricted (restric
The use of combinatorial peptide libraries to design molecules that mimic tumor epitopes recognized by (ted) CTL has been proposed.

International Publication No. WO 96/104, the content of which is the C42 polypeptide of the present invention or an immunogenic fragment or peptide derived therefrom, which is incorporated herein by reference.
It can be produced recombinantly or by peptide synthesis as disclosed in No. 13. For recombinant production, the corresponding DNA molecule is inserted into an expression vector by standard techniques, transfected into a suitable host cell, the host cultured under suitable expression conditions and the protein purified. Conventional methods, such as commercially available automated peptide synthesizers, can be used for the chemical synthesis of C42 peptides.

Instead of the natural C42 peptide or the irregularly-altering peptide, substances that mimic such peptides, such as "peptidomimetics" or "reverse inverse peptides"
It is also possible to use (retro inverse peptide) ”. To test these molecules for their therapeutic use in tumor vaccines, the same methods as described above can be used with the native C42 peptide or C42 peptide equivalents.

The TAA-designated C42 and protein fragments, peptides or peptide equivalents or peptidomimetics thereof of the present invention may be used, for example, to elicit an immune response against tumor cells expressing the corresponding antigenic determinant. , Can be used in cancer treatment. Preferably they are used for the treatment of C42-positive tumors, in particular lung, breast and esophageal cancers.

The 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 containing TAA C42 or a fragment or peptide or a peptide derived therefrom as an active ingredient is administered to a patient suffering from a TAA-associated tumor disease, but at the same time TAA ( The amount of peptide) should be sufficient to obtain CTL that respond efficiently to the tumor that gives rise to the antigen.

Accordingly, in another aspect, the invention relates to a pharmaceutical composition for parenteral, topical, oral or topical administration. Preferably, the composition is applied parenterally, eg subcutaneously, intracutaneously or intramuscularly. The C42-TAA / peptide is dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier. The composition may also contain conventional adjuvants such as buffers. The C42-TAA / peptide may be used by itself or in combination with an adjuvant, such as Freund's incomplete adjuvant, saponin, aluminum salt, or, in a preferred embodiment, a polycation such as polyarginine or polylysine. it can. These peptides may also be bound to components that assist CTL induction or CTL activation, such as T-helper peptides, lipids or liposomes, or the peptides may be bound to these substances and / or cytokines (IL-2, It can also be co-administered with an immunostimulatory substance such as IFN-γ). Suitable methods and formulations for the preparation and administration of the pharmaceutical compositions of the present invention are disclosed in WO 95/04542 and WO 97/30721, the contents of which are referred to herein. Is incorporated as.

The C42 fragment or C42 peptide can also be used to trigger the CTL reaction ex vivo. The ex vivo response of CTL to C42-expressing tumors is
-Induced by incubating progenitor cells with APC and C42 peptide or C42 protein. The subsequently activated CTLs are able to proliferate whenever they are re-administered to the patient. Alternatively, APCs can be loaded with the C42 peptide, which leads to effective activation of the cellular immune response against C42-positive tumors (Mayordomo et al. 1995; Zitvogel et al. 1996). One suitable method of loading peptides into cells, eg 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 embodiment, the C42 peptide is another T
Combined with peptides derived from AA. The selection of peptides for such a combination is performed in terms of detection of various MHC-types to cover the widest possible population of patients, and / or this may be done in several different ways. By combining peptides derived from tumor antigens, we aim for the broadest possible indication spectrum. The number of peptides in the pharmaceutical composition can vary widely, but typically clinically useful vaccines contain 1 to 15, preferably 3 to 10 different peptides.

The peptides of the present invention can also be used as diagnostic agents. For example, these peptides can be used to test a patient's response to a humoral or cellular immune response elicited by an immunogenic peptide. This offers the potential for improved treatment. For example, PB showing reactivity to defined peptide epitopes, depending on the TAA dosage form (peptide, total protein or DNA vaccine)
An increase in T-cell precursors in L can be investigated (Robbins and Kawakami, 1996 and references cited therein). Furthermore, peptides or total proteins or antibodies shown against TAA can be used to predict patient risk of developing C42-related tumors and to characterize the progression of C42-positive tumors (e.g., primary tumors). And immunohistochemical analysis of metastases). This kind of strategy has already proven successful in many cases, for example the detection of the estrogen receptor underlying the decision of endocrine therapy in breast cancer; c- as a relevant marker in the prognosis and course of treatment of breast cancer. erbB
-2 (Ravaioli et al., 1998; Revillion et al., 1998); ¹¹¹ I against PSMA as an epithelial cell marker for prostate cancer in serum and in immunoscintigraphy of prostate cancer
As a marker by using n-labeled monoclonal antibody, PSMA (
Prostate-specific membrane antigen (Murphy et al., 1998, and references cited therein); CEA (carcinoembryonic antigen) as a serological marker of prognosis and progression in patients with colorectal cancer (Jessup and Loda, 1998).

In another aspect, the invention relates to an isolated DNA molecule encoding a protein with immunogenicity of C42 or a fragment thereof. In another aspect, the invention comprises a polynucleotide having the sequence set forth in SEQ ID NO: 1, or a polynucleotide that hybridizes under stringent conditions to a polynucleotide of the sequence set forth in SEQ ID NO: 1. It relates to an isolated DNA molecule comprising nucleotides.

As used herein, “stringent hybridization conditions” means 50% formamide, 5 × SSC (1 × SSC = 150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6). 42 in a solution containing 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured and sheared salmon sperm DNA.
C. overnight incubation, followed by filter wash in 0.1 × SSC at about 65 ° C., or equivalent conditions.

The DNA molecule of the present invention or a fragment thereof encodes a (poly) peptide designated C42 having the amino acid sequence shown in SEQ ID NO: 2 or a protein fragment or peptide derived therefrom; SEQ ID NO: as a result of the degeneracy of the genetic code:
Includes DNA molecules that show derivatives from the sequence shown in 1. The present invention provides for mutations in the protein sequence shown in SEQ ID NO: 2 if they encode C42 derivatives or fragments or peptides with desirable immunogenicity for their use as tumor vaccines. It also relates to DNA molecules that lead to the exchange of amino acids. The C42 DNA molecules of the invention or the corresponding RNAs which are also the subject of the invention, as well as the (poly) peptides encoded by them, can be used for immunotherapy of cancer diseases.

In one embodiment of the invention, a DNA molecule encoding the native C42 polypeptide is used. Modified derivatives can be used in place of the native C42 cDNA or fragments thereof. While these include sequences with modifications encoding proteins (fragments) or peptides with greater immunogenicity, the same considerations that apply to the peptides described above apply to modifications at the DNA level. can do. Another type of modification is the alignment of many sequences that encode immunologically related peptides, such as bead strings (Toes et al., 1997). This array is also
It is modified by the addition of auxiliary elements, such as functions that ensure more efficient release and processing of the immunogen (Wu et al., 1995). For example, the localization sequence in the endoplasmic reticulum (locat
ing sequence) (“ER targeting sequence”) can increase the processing of this antigen and the resulting antigen presentation and ultimately immunogenicity.

In another aspect, the invention features a recombinant DNA molecule that comprises C42-DNA. The C42 DNA molecule of the present invention can be administered directly in recombinant form, preferably as a plasmid, or as part of a recombinant virus or bacterium. Theoretically, any method of gene therapy for DNA-based cancer immunotherapy (“DNA vaccines”) against C42-DNA both in vivo and ex vivo can be used.

An example of in vivo administration is the direct injection of “naked” DNA, either by the intramuscular route or the use of a gene gun, which has been shown to lead to the formation of CTLs against tumor antigens. . Examples of recombinant organisms are vaccinia virus, adenovirus or Listeria monocytogenes (outline provided by Coulie, 1997). In addition, synthetic carriers for nucleic acids such as cationic lipids, microspheres, micropellets or liposomes can be used for in vivo administration of nucleic acid molecules encoding C42 peptides. Various adjuvants, such as peptides, which enhance the immune response, such as cytokines, can also be administered, either in the form of proteins or plasmids encoding them. This application is optional by physical method,
For example, it can be combined with electroporation.

An example of ex vivo administration is the transfection of dendritic cells as described in the article by Tuting (1997), or of other APCs such as used as a cellular cancer vaccine. Therefore, in another embodiment, the present invention provides for C4, after transfection of the corresponding coding sequence, either by itself or in optionally modified form.
Using cells expressing 2 to make a cancer vaccine.

In another aspect, the invention relates to an antibody against C42 or a fragment thereof.
Polyclonal antibodies are usually obtained by immunization of animals, especially rabbits, injection of antigen or fragments thereof, and subsequent purification of immunoglobulins. Monoclonal anti-C42-antibodies are used in standard procedures according to the principles described by Kohler and Milstein (1975) to immunize animals, especially mice, followed by antibody-producing cells from the immunized animals, such as myeloma cells. Immortalization by fusion and screening of the resulting hybridoma supernatants by immunological standard assays for monoclonal anti-C42 antibodies. For therapeutic or diagnostic use in humans, the antibodies of these animals are optionally chimerized by standard methods (Neuberger et al., 19
84, Boulianne et al., 1984) or humanized (Riechmann et al., 1988, Graziano et al., 19).
95).

Human monoclonal anti-C42-antibodies (or fragments thereof) have been derived from so-called protein-displaying phage libraries (Winter et al., 1994, Griffiths et al., 1994, Kruif et al., 1995, Mc Guiness et al., 1996), and trans. Using transgenic animals (Bruggema
nn et al., 1996, Jakobovits et al., 1995). The anti-C42-antibodies of the invention can also be used in immunohistochemical analysis for diagnostic purposes.

In another embodiment, the invention relates to the use of C42-specific antibodies for the selective production of any desired substance against or against a C42-expressing tumor.
Examples of such substances are cytotoxic substances or radionuclides whose activity damages tumors in situ. Side effects are either unexpected or minimal due to the tumor-specific expression of C42. In another embodiment, agents that reveal C42-expressing tumors can be used with the help of C42 antibodies. This is useful in diagnosis and treatment evaluation. The therapeutic and diagnostic uses of antibodies applied to anti-C42 antibodies are disclosed in WO 95/33771.

The C42 designated TAA in the present invention and protein fragments, peptides or peptide equivalents or peptidomimetics derived therefrom, for example, induce an immune response against tumor cells expressing the corresponding antigenic determinant. It can be used to treat cancer, such as. They are preferably used for the treatment of C42-positive tumors, in particular lung, breast and esophageal cancers.

Example 1 Biopsies of Various Human Lung Cancers of RDA Method SCC Type (Squamous Cell Carcinoma, Squamous Cell Tumor) Compared to 11 Normal Tissue Pools of Various Pulmonary Platen Tumor Tumor Pools Immediately after surgical removal of the specimen, it was flash frozen in liquid nitrogen,
Stored at -80 °. To isolate RNA, a series of 20 μm sections were prepared on a -20 ° cryomicrotome (Jung CM1800, Leica) and directly lysed in 4M guanidine thiocyanate / 1% β-mercaptoethanol. Isolated. These samples were ultracentrifuged on a CsCl gradient (Sambrook, 1989).

Several representative tissue pieces (5 m) were fixed on slides and stained with Harris hematoxylin (Sigma) and eosin for histological examination. This was utilized to provide the most homogeneous tumor tissue possible as a starting material for obtaining RNA. Only samples classified as SCC were further processed.

From 110 μg of total RNA from 5 different lung slab epithelial tumors, PolyAtract Ki
Poly-A (+) RNA was isolated (SCC pool) using t (Promega) according to the manufacturer's instructions. This SCC pool and 11 kinds of normal tissues-bone marrow, heart, kidney, liver, lung, pancreas, skeletal muscle, spleen, thymus, small intestine and stomach-derived poly-A (-4-) RNA (Clontech) 2.5. μg
Starting with the PCR-se1ect® kit (Clontech, Palo Alto) according to the manufacturer's instructions and performing the RDA method (Diatchenko et al; Hubank and Schatz): S
RNA from the CC pool (tester) and RNA from normal tissue (driver) were used according to the manufacturer's instructions. Unlike the original method, after double-stranded cDNA was synthesized using oligo-dT, this cDNA was digested with 6 restriction enzymes: ECoRV, NaeI, NruI, ScaI (Prome
ga), SspI, and StuI (TaKaRa) were used for cleavage in Promega's buffer A at 37 ° C. for 2 hours, and the NaCl concentration was increased to 150 mM, followed by further cleavage at 37 ° C. for 2 hours. The use of a mixture of these 6 different restriction enzymes allows the production of cDNA fragments of approximately 800 bp,
This fragment was used in the RDA method.

Equal parts of the tester cDNA were ligated with either adapter A or B and then individually hybridized with excess driver-cDNA at 68 ° C. The two mixtures were then combined and subjected to a second hybridization with the newly denatured driver cDNA. The concentrated tester-cDNA was then subjected to an extension time of 72 ° C. for 2 minutes, 27 cycles or more (94 ° C. for 10 seconds, 66 seconds, 66 times, using the primer of the kit specific for adapter A or B).
Amplification was performed exponentially by PCR at 30 ° C. for 30 seconds and 72 ° C. for 2 minutes. To further concentrate, in one aliquot of this reaction, use the specific nested primers of the kit,
A second PCR was performed with an extension time of 72 ° C for 2 minutes and 27 cycles or more (94 ° C for 10 seconds, 66 ° C for 30 seconds, 72 ° C for 2 minutes). The product obtained from this reaction was used as the pCR2.1 vector (Invitrogen).
) And then competent E. coil (OneShot®, Invitrogen
). 4748 transformants were obtained, 96- in LB-Amp medium
Incubated at 37 ° C. for 48 hours in a well block. Then, a 5 μl aliquot of the E. coil suspension was heated in 500 μ1 TE buffer for 10 minutes at 100 ° C.
Used as the basis for the vector, the insert fragment of the vector was amplified with flanking primers (SEQ ID NOs: 9 and 10) for 35 cycles or more (94 ° C. 1 minute, 55 ° C. 1 minute, 72 ° C. 2 minutes).
The PCR product was revealed by agarose gel electrophoresis and ethidium bromide staining. 17 μl of the PCR product was collected in 6 × SSC so that the final volume was 252 μl, and equilibrated on a nylon membrane (Hybond-N, Amersham), DNA-Dot-Blots (Sambrook, 1989).
Was fixed at 3 copies according to standard techniques for making. The remaining bacterial culture was stored at -80 ° C as a glycerol stock culture.

A cDNA subtraction library of 4748 individual clones was obtained in the form of an E. coil glycerol stock culture, the fixed PCR product of which was applied to a nylon membrane and the length of its insert was determined. It was examined by agarose electrophoresis. As expected, the inserted cDNA fragment was found to have an average length of approximately 800 bp.

[0053] Example 2: cDNA subtraction library oncogenes and tumor by differential hybridization of - testis genes selected human testis - specific cDNA library (Human Testis Specific PCR-Select (TM) cDNA; Clontech, Inc., Palo Alto) according to the manufacturer's instructions (94 ° C for 10 seconds,
It was amplified by 11 cycles of PCR at 6 ° C for 30 seconds and 72 ° C for 1.5 minutes). Aliquot the RTS Ra
Labeled with [α- ³² P] dCTP (NEN, Boston) using the dPrime DNA Labeling System (GibcoBRL) according to the manufacturer's instructions, and using the standard method (Sambrook, 1989). The resulting DNA-Dot-Blots were hybridized under stringent conditions (68 ° C). Human Testis Specific PCR-Select (registered trademark) cD
Clones hybridized with NA were visualized by autoradiography (Xomat DR film, Kodak). The second set of DNA-Dot-Blots hybridized with a labeled probe of the cDNA SCC pool as described above. Third DNA-
Dot-Blots set is a cDNA of 15 normal tissues (bone marrow, heart, kidney, liver, lung, pancreas, skeletal muscle, spleen, thymus, small intestine, stomach, lymph node, mammary gland, prostate and trachea) in the same manner. Hybridized with a probe from the normal tissue pool.

By comparing the images and the net number of differentially hybridized spots of the autoradiography or set, it is possible to select clones in which the mRNA is overexpressed only in the tumor compared to the normal tissue. Alternatively, it was transcribed both in tumor and testis (as an example of immunoprivileged tissue). The former are candidates for the tumor antigen class and the latter are candidates for the tumor-testis antigen class.

Example 3 Tumor and Tumor-Testicular Antigen Candidate DNA Sequencing and Remarks Plasmid-DNA from 234 clones selected based on the results obtained in Example 2.
Was isolated according to the manufacturer's instructions (QIAgen), and by the Sanger method, ABI-Pris
Sequenced on m instrument. The sequence recognized in this way is BLAST-Search (Natio
nal Center for Biotechnology Information) and compared with the EST databank. This made it possible to identify 198 known genes and 36 unknown genes. For the latter, only EST input is performed. The expression profile was estimated for 36 unknown genes: the starting tissues for the corresponding cDNA libraries were all ESTs in the databank with> 95% identity (BLAST) to the experimentally determined sequence. Checked. These were subdivided into i) criticality normal tissues, ii) fetal "disposal" and immunoprivileged tissues, and iii) tumors and tumor cell lines. This "virtual mRNA profile"
On the basis of 10 clones were selected for further experimental analysis.

Example 4 Transcriptional Analysis of Candidate Clones in Tumor and Normal Tissues 2-5 μg of total RNA from tumor or normal tissues was transferred to SuperScriptII (GibcoBRL).
Alternatively, AMV-RT (Promega) was used as recommended by the manufacturer for reverse transcription. A second test was performed on each individual RNA probe without reverse transcriptase and served as a control for chromosomal DNA contamination. The properties and amounts of these cDNAs were determined using β-actin-specific primers (SEQ ID NOs: 3 and 4) and GAPDH specific primers (SEQ ID NOs: 5 and 6) for 30 and 35 cycles (1 min at 95 ° C). , 1 min at 55 ° C, 1 min at 72 ° C). Ten candidate genes were analyzed for similarity with specific primers.
These PCR products were detected by agarose gel electrophoresis and ethidium bromide staining. The candidate designated "C42" exhibits a relatively specific tumor / testis transcript profile after 30 cycles with C42-specific primers (SEQ ID NOs: 7 and 8); breast cancer,
Semi-quantitative RT-PCR of RNA from lung adenomas, slab epithelial tumors of the lung, kidney cancer, colon cancer, heart, lung, liver, kidney, colon, spleen and testis is shown in FIG. This clone C42 contains an insert of 549 bp, which was subsequently subjected to further analysis.

Example 5: Transcriptional profile of C42 in tumor and normal tissues Human Multiple Tissue Northern Blots (MT) for Northern blot analysis.
N; Clontech, Palo Alto) and (Invitrogen) were hybridized with [α- ³² P] dCTP (NEN, Boston) labeled C42 PCR product of length 549 bp at 68 ° for 2 hours.
Visualization was performed by standard autoradiography (Hyperfilm, Amersham). Figure 2
The results of this analysis are shown: 20 normal tissues (pancreas, adrenal medulla, thyroid, adrenal cortex, testis, thymus, small intestine, stomach, brain, heart, skeletal muscle, colon, spleen, kidney, liver, placenta, Lungs, white blood cells, gallbladder, esophagus) and four tumor tissues (gallbladder and gastric adenomas, and esophageal and lung slab epithelial tumors). For C42, a prominent transcript of 4.4 kb in length was found in the tumor tissue of esophageal and lung-derived plate epithelial tumors. In normal tissues, a weak transcript with a length of 4.4 kb was found only in the esophagus. The low intensity signal results in immunologically relevant expression that seems unlikely. Another 3.5 kb transcript is probably a splicing variant of the 4.4 kb transcript, or derived from a homologous gene, which was identified in both tumor tissues but not in normal tissues ( Figure 2). TAA in plate and epithelial tumors of the lung and esophagus
The presence of'C42 'highly corresponds to the starting material (pool from 5 individual patients with pulmonary plate epithelial tumors) originally used in the RDA method, and the emerging TAAs are specific to this type of cancer. Is shown.

Example 6: Cloning of C42 cDNA The human C42 cDNA was cloned using the following procedure: 549 bp long C42 cDNA insert obtained in Example 4 from the BLAST search ("original fragment"). The following ES overlapping with
Obtained T: AA429919; AA430055; AA446075; AA430264; AA160879. Array AA430055
Starting with the clone C42 overlapping contigs, TigemNet (http: //gcg.ti
It was recognized by Est Extractor of gem.it/cgi-bin/uniestass.pl). This contig and the overlapping part of the sequence of the original fragment obtained in Example 4 and having a length of 549 bp (SEQ ID NO: 19) are the contigs of the C42-specific primer and lung tumor cDNA (SEQ ID NO: 11 and 1).
It was verified by PCR amplification with the primer located at the 3'end of 2). Extensions in the 5'direction were obtained with the sequences AA430246 and AA446075 overlapping the original fragment of C42.
Primer pairs (SEQ ID NOS: 13 and 14) for the first PCR, and "nested" P
Using the primer pair (SEQ ID NOs: 15 and 16) for CR, two other fragments belonging to C42 were analyzed by the Advantage cDNA PCR Kit (Clontech) and consecutively described there by two consecutive PCRs. Using the SuperScript® Testis cDNA Library (Gibco
BRL). Starting from this new sequence, we found three more EST inputs belonging to C42: A1493356; AA443218; AA443258. Knowledge of these new sequences is then followed by the primer pair (SEQ ID NOs: 13 and 17) for the first PCR and the primer pair for the internally offset "nested" PCR. (SEQ ID NOS: 15 and 18) were used, and it was possible to perform two types of continuous PCR further upstream. The other C42 fragment was cloned in this way.

For sequence analysis, an aliquot of the PCR preparation was added to the pCR2.1 vector (Invitrogen
Direct connection to Competent E. coil (OneShot®, Invitroge
(Company n) and sequenced as above. Using two consecutive PCRs, primer pair SEQ ID NO: 13 and SEQ ID NO: 3 for the first PCR and primer pair SEQ ID NO: 15 for the “nested” PCR, SuperScript® ) Another C42 fragment from a human testis cDNA library (Gibco BRL) was used for Advantage cDNA PCR.
It was amplified using a kit (Clontech). New upstream C42 with additional upstream primer
No fragment was identified. Therefore, C42 (SEQ ID NO: 1 of 4077 nucleotides in length is
It is presumed that the full-length mRNA (of the cDNA sequence) was identified. This result is shown in M
This is consistent with the recognition of the C42 band of approximately 4.4 kb on TN. (The difference between the sequenced length of the polynucleotide chain and the C42 sequence recognized by MTN was due to the poly A tail and mRN
This may be due to the inaccuracy of the A fraction). This mRNA has an initiation codon at nucleotides 564-566 indicating the start of a continuous open reading frame up to nucleotide 3392 encoding a 943 amino acid long polypeptide (SEQ ID NO: 2).
.

[0060] C42 sequences of which are (in some cases, a high degree of tissue specificity) expression in various species Ca ²⁺ - dependent Cl ^- - and gene family channel proteins, clearly at both levels of nucleotide and protein Shows homology. Ever 5 of this family
Cloned species of members, and are partially characterized: two bovine genetic BCLCAl (bovine Ca ²⁺ - dependent Cl ^- - channel -1; Cunningham et al., 1995) and Lu
-ECAM-1 (bovine lung epithelial cell adhesion molecule -1; Elble et al., 1997), the mouse gene MCLCA1 (murine Ca ²⁺ - dependent Cl ^- - channel -1; Gandhi et al, 1998) and two human genes hCLCA1
(Human Ca ²⁺ - dependent Cl ^- - channel; Gruber et al., 1998) and HCLCA3 (human Ca ²⁺ - dependent
Cl ⁻ −Channel-3; Gruber et al., 1999). All members of this protein family have a typical transmembrane region and what is presently known is that, except for hCLCA3, it is post-translationally cleaved to form a heterodimer, the C-terminal portion of which is glycosyl. (Elble et al., 1997). For example, bovine Lu-ECAM-1 causes B16F1O lung metastases in mouse malignant melanoma and binds to epithelial cells (Zhu et al., 1992).

To check the accuracy of the nucleotide sequence, overlapping portions of the C42 fragment from lung squamous cell tumor and testis were analyzed using primer pair SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8.
, SEQ ID NOS: 9 and 10, SEQ ID NOS: 11 and 12, SEQ ID NOS: 13 and 14, SEQ ID NO: 1
Amplified and sequenced with 5 and 16, SEQ ID NOS: 17 and 18, SEQ ID NOS: 19 and 20. Alignment of both sequences showed no difference except for the silent mutation at position 2399 (G → A).

This C42 sequence has five hydrophobic transmembrane regions typical of the protein family (positions 222-252, 416-445, 553-574, 746-766 and 900-92 in SEQ ID NO: 2). However, unlike other members, it had a C-terminus formed by a strongly charged amino acid.

Example 7: Possible MHC-Binding Peptide of the Region Coding the C-Terminal Portion of C42 Peptide within the Coding Region of the C-Terminal Fragment of C42 (SEQ ID NO: 2) Epitopes were based on known motifs (Rammnensee et al., 1995) using the algorithm described by Parker et al., 1994. The 9-member candidate peptides that are predicted to bind the corresponding HLA molecule and thus constitute an immunogenic CTL-epitope are the most important HLA-types, especially HLA-A1, -A * 0201, -A3. , -B7, -B14 and -B * 4403 were identified; the peptides found were listed in Table 1. By similar methods, other potential peptide epitopes can be identified for other HLA types or 8- or 10-membered peptides.

Table 1: Candidate immunogenic peptides of the C-terminal fragment of C42 (742 amino acids)

Example 8: T2-A2 Peptide Loading Assay This assay is utilized to identify peptides that may bind to HLA-A2 molecules and thus present T-cells. Potential C42 epitopes were identified by the prediction algorithm shown in Example 7 and these peptides were synthesized (SEQ ID NO: 9
0-101). The HLA-A2 peptide epitope of tyrosinase (SEQ ID NO: 102) was used as a positive control, and the HLA-A1 epitope of MAGE3 (SEQ ID NO: 103) was used as a negative control. These peptides were dissolved in DMSO (Sigma) at a concentration of 40 mg / ml, and
Diluted with PBS. T2-cells (ATCC Deposit No .: CRL-1992) were added to RPMI 1640 at a concentration of 2.5 x.
The cells were resuspended at 10 ⁶ and 10 μg / ml β ₂ -microglobulin (ICN) was added. These cells were seeded at 200 μl / well on 96-well microtiter plates,
And loaded with 0-320 μg of serial peptide dilutions. After incubation at 37 ° C. for 16 hours, the amount of stable peptide-HLA-β ₂ -microglobulin was measured using a fluorescence-activated cell sorter (anti-HLA antibody recognized by the second antibody R0480 (DAKO)). FACS) (Becton Dickinson) (FIG. 3). The fluorescence shift reflects the amount of stabilized peptide HLA-β ₂ -microglobulin complex on the cell surface.
The reaction of 5 C42-CTL peptides selected by 12 C42-CTL according to the above test is shown in FIG. Peptides C42-5, C42-6, C42-8 and C42-11 (SEQ ID NOs: 94, 95
, 97 and 100) show binding to HLA-A 0201. Therefore, these are T-cells-
It is a good candidate for a specific peptide epitope.

Documents Becker, D., Kuhn, U., Lempertz, U., Enk, A., Saloga, J., and Knop, J. (1
997), J. Immunol. Methods 203 , 171-180. Blake, J., Johnston, JV, Hellstrom, KE, Marquardt, H., and, Chen, L.
(1996), J. Exp. Med. 184 , 121-130. Boon, T, JC Cerottini, B. Van den Eynde, P. van der Bruggen, and A. V.
an Pel (1994), Annu.Rev.Immunol. 12 : 337-365 Boon T, (1998), Tumor antigens recognized by cytolytic Tcells. Cancer Va
ccine Week − International Symposium, New York, Oct 1998; abstract S01
Boulianne, GL, et al., (1984), Nature 312 , 643-646 Bruggemann, M. and Neuberger, MS, (1996), Immunol. Today 17 ,, 391-397 Buschle M, Schmidt W, Zauner W, Mechtler K , Trska B, Kirlappos H, Birnst
iel M (1997), Proc. Natl. Acad. Sci. USA 94 , 3256-3261 Celluzzi, CM and Falo, LD, Jr. (1998), J. Immunol. 160 , 3081-3085 Chen, YT, Scanlan, MJ , Sahin, U., Tureci, O., Gure, AO, Tsang, S.,
Williamson, B., Stockert, E., Pfreundschuh, M., and Old, LJ (1997), P
roc. Natl. Acad. Sci. USA 94 , 1914-1918. Coulie, PG (1997), Mol. Med. Today 3 , 261-268 Cox, AL, Skipper, J, Chen, Y, Henderson, RA, Darrow, TL, Shabanowitz, J,
Engelhard, VH, Hunt, DF, and Slingluff, CL, Jr. (1994), Science 264 , 71
6-719

Cunningham SA, Awayda MS, Bubien JK, Ismailov II, Arrate MP, Berdiev BK,
Benos DJ, Fuller CM (1995), J. Biol. Chem. 270 , 31016-31026 Diatchenko, L., Lau, YF, Campbell, AP, Chenchik, A., Moqadam, F., Hu
ang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, ED, and S
iebert, PD (1996), Proc. Natl. Acad. Sci. USA 93 , 6025-6030.Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose,
K., Jackson, V., Hamada, H., Pardoll, D., and Mulligan, R. (1993), Proc
Natl. Acad. Sci. US A 90 , 3539-3543. Elble, R., Widom, J., Gruber, AD, Abdel-Ghany, M., Levine, R., Goodwin
, A., Cheng, H.-C., and Pauli, BU (1997), J. Biol. Chem. 272 , 27853-27
861. Emini, EA, Hughes, J, Perlow, D, and Boger, J (1985), J. Virol. 55 , 836-
839 Falk, K., Rotzschke, O., Stevanovi′c, S., Jung, G., and Rammensee, HG
(1991), Nature 351 , 290-296. Fearon, ER, Pardoll, DM, Itaya, T., Golumbek, P., Levitsky, HI, Si
mons, JW, Karasuyama, H., Vogelstein, B., and Frost, P. (1990), Cell 6 0, 397-403. Gandhi, R., Elble, RC, Gruber, AD, Ji, H.-J ., Copeland, SM, Fuller
, CM, and Pauli, BU (1998), J. Biol. Chem. 273 , 32096-32101. Gansbacher, B., Zier, K., Daniels, B., Cronin, K., Bannerji, R., and Gil
boa, E. (1990), J. Exp. Med. 172 , 1217-1224.

Graziano, RF, et al., (1995), J. Immunol. 155 , 4996-5002 Griffiths, AD, et al., (1994), EMBO J. 13 , 3245-3260 Gruber, AD, Elble, RC, Ji, H.-J,., Schreur, KD, Fuller, CM, and
Pauli, BU (1998), Genomics 54 , 200-214. Gruber, AD and Pauli, BU (1999), Biochim. Biophys. Acta 1444 , 418-42
3. Halliday, GM, Patel, A, Hunt, MJ, Tefany, FJ, and Barnetson RS (1995), W
orld J. Surg. 19 , 352-358 Hubank, M. and Schatz, DG, (1994), Nucleic. Acids. Res. 22 , 5640-5648. Jager, E., Chen, YT, Drijfhout, JW, Karbach, J., Ringhoffer, M., Jag
er, D., Arand, M., Wada, H., Noguchi, Y., Stockert, E., Old, LJ, and K
nuth, A. (1998), J. Exp. Med. 187 , 26-270. Jakobovits, A., (1995), Curr. Opin. Biotechnol. 6 , 561-566 Jameson, BA and Wolf, H (1988) , The antigenic index: a novel algorithm f
or predicting antigenic determinants. Comput. Appl. Biosci. 4 , 181-186

Jessup, JM and Loda, M (1998), Prognostic markers in rectal carcinoma. S
emin. Surg. Oncol. 15 , 131-140. Kawakami, Y., Eliyahu, S., Jennings, C., Sakaguchi, K., King, X., Southw
ood, S., Robbins, PF, Sette, A., Appella, E., and Rosenberg, SA (199
5), J. Immunol. 154 , 3961-3968. Kawakami, Y, Robbins, PF, and Rosenberg, SA (1996), Keio J. Med. 45 , 100.
-108 Kohler, G. and Milstein, C. (1975), Nature 265 , 495-497 Kruif, J., et al., (1995), Proc. Natl. Acad. Sci. USA 92 , 3938-3942 Kyte, J and Doolittle, RF (1982), J. Mol. Biol. 157 , 105-132 Lethe, B, Lucas, S, Michaux, L, De Smet, C, Godelaine, D, Serrano, A, De
Plaen, E, and Boon, T (1998), Int. J. Cancer 76 , 903-908 Mackensen A, Ferradini L, Carcelain G, Triebel F, Faure F, Viel S, and H
ercend T (1993), Cancer Res. 53 , 3569-3573 Marchand M, Weynants P, Rankin E et al (1995), Int. J. Cancer 63 : 883-885

Mayordomo, JI, Zorina, T., Storkus, WJ, Zitvogel, L., Celluzzi, C.,
Falo, LD, Melief, CJ, Ildstad, ST, Kast, WM, DeLeo, AB, and Lo
tze, MT (1995), Nature Medicine 1 , 1297-1302. McGuinnes, BT, et al., (1996), Nature Biotechnol. 14 , 1149 Melief CJ, Offringa R, Toes RE, Kast WM (1996), Curr. Opin. Immunol. 8 ,
651-657 Murphy, GP, Elgamal, AA, Su, SL, Bostwick, DG, and Holmes, EH
(1998), Cancer 83 , 2259-2269 Neuberger, MS, et al., (1984), Nature 312 , 604-608 Pardoll, DM (1998), Nature Medcine 4 , 525-531 Parker, KC, MA Bednarek, and JE Coligan. (1994), J. Immunol. 15 2 , 163. Parkhurst, MR, Salgaller, ML, Southwood, S., Robbins, PF, Sette, A
., Rosenberg, SA, and Kawakami, Y. (1996), J. Immunol. 157 , 2539-2548.

Paterson Y, Ikonomidis G (1996), Curr. Opin. Immunol. 5 , 664-669 Rammensee HG, Friede T, Stevanovic S (1995), Immunogenetics 41 , 178-228 Rapellino, M, Pecchio, F, Baldi , S, Scappaticci, E, and Cavallo, A (1995
), Anticancer Res. 15 , 1065-1070 Ravaioli, A., Bagli, L., Zucchini, A., and Monti, F. (1998), Cell. Proli
f. 31 , 113-126 Revillion, F, Bonneterre, J, and Peyrat, JP (1998), Eur. J. Cancer 34 , 7
91-808 Restifo NP (1996), Curr. Opin. Immunol. 5 , 658-63 Riechmann, L., et al., (1988), Nature 332: 323-327 Robbins, PF and Kawakami, Y (1996), Curr. Opin. Immunol. 8 , 628-636 Ruppert, J., Sidney, J., Celis, E., Kubo, RT, Gray, HM, and Sette, A
(1993), Cell 74 , 929-937. Sahin, U., Tureci, O., Schmitt, H., Cochlovius, B., Johannes, T., Schmi
ts, R., Stenner, F., Luo, G., Schobert, I., and Pfreundschuh, M. (1995),
Proc. Natl. Acad. Sci. USA 92 , 11810-11813

Sambrook, J., Fritsch, EF, and Maniatis, T. Molecular Cloning: A Labor
atory Manual. ColdSpring Harbor Laboratory Press, 1989 Schendel DJ, Gansbacher B, Oberneder R, Kriegmair M, Hofstetter A, Rieth
muller G, and Segurado OG (1993), J. Immunol. 151 , 4209-4220 Schmidt W, Buschle M, Zauner W, Kirlappos H, Mechtler K, Trska B, Birnst
iel M (1997), Proc. Natl. Acad. Sci. USA 94 , 3262-3267 Schweighoffer, T. (1997), Onc. Res. 3 , 164-176 Sette, A., Vitiello, A., Reherman, B ., Fowler, P., Nayersina, R., Kast,
WM, Melief, CJM, Oseroff, C., Yuan, L., Ruppert, J., Sidney, J., de
l Guercio, M.-F., Southwood, S., Kubo, RT, Chesmut, RW, Gray, HM,
and Chisari, FV (1994), J. Immunol. 153 , 5586-5592. Stauss, HJ, Davies, H., Sadovnikova, E., Chain, B., Horowitz, N., and
Sinclair, C. (1992), Proc. Natl. Acad. Sci. USA 89 , 7871-7875. Tepper, RI, Pattengale, PK, and Leder, P. (1989), Cell 57 , 50-512. Tighe H, Corr, M, Roman M, and Raz E (1998), Immunol. Today 19 , 89-97

Toes, RE, Hoeben, RC, Van der Voort, E., Ressing, ME, Van-der-Eb,
AJ Melief, CJM, and Offringa, R. (1997), Proc. Natl. Acad. Sci. US
.A. 94 , 14660-14665 Tuting, T., DeLeo, AB, Lotze, MT, and Storkus, WJ, (1997), Eur. J.
Immunol. 27 , 2702-2707 ,. Van den Eynde, B. and Brichard, VG (1995), Curr. Opin. Immunol. 7 , 674
-681. Van den Eynde, BJ, and van der Bruggen, P (1997), Curr. Opin. Immunol. 9 , 684-693 van der Bruggen, P., C. Traversari, P. Chomez, C. Lurquin, E . De Plaen,
B. Van den Eynde, A. Knuth, and T. Boon. (1991), Science 254 , 1643-1647 van der Burg, SH, et al., (1996), J. Immunol. 156 , 3308-3314 van Elsas , A., van der Minne, CE, Borghi, M., van der Spek, CW, Braa
kman, E., Osanto, S., and Schrier, PI (1996), CTL Recognition of an IL
-2 Producing Melanoma Vaccine. In: Immunology of human melanoma. Tumor-h
ost interaction and immunotherapy, edited by M. Maio, Amsterdam: IOS, 19
96, p. 165-173

Van Elsas, A., Aarnoudse, C., van der Minne, CE, van der Spek, CW, B
rouwenstijn, N., Osanto, S., and Schrier, PI (1997), J. Immunother. 20 , 343-353. Vaughan, TJ, et al., (1998), Nature Biotechnol. 16 , 535-539 Velculescu, VE, Zhang, L, Vogelstein, B, and Kinzler, KW (1995), Science
270 , 484-487 Wang, L., et al., (1997), Mol. Immunol. 34 , 609-618 Wang, RF (1997), Mol. Med. 3 716-731 Winter, G., et al. ., (1994), Annu. Rev. Immunol. 12 , 433-455 Woelfel, T, Schneider, J, Zum Buschenfelde, Meyer, KH, Rammensee, HG, Ro
tzschke, O, and Falk, K (1994), Int. J. Cancer 57 , 413-418 Wu, TC, Guarnieri, FG, Staveley-O'Carroll, KF, Viscidi, RP, Levi
tsky, HI, Hedrick, L., Cho, KR, August, JT, and Pardoll, DM (199
5), Proc. Natl. Acad. Sci. USA 92 , 11671-11675. Zitvogel, L., Mayordomo, JI, Tjandrawan, T., DeLeo, AB, Clarke, MR
, Lotze, MT, and Storkus, WJ (1996), J. Exp. Med. 183 , 87-97. Zhu, D., Cheng., C.-F., and Pauli, BU (1992), J. Clin .Invest. 89 , 171
8-1724.

[Sequence list]

[Brief description of drawings]

FIG. 1: C42 transfection in tumor and normal tissues: semi-quantitative RT-PCR of RNA from various tissues.

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

FIG. 3: Binding assay of 5 selected C42 CTL peptides to HLA-A * 0201.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 14/47 C07K 16/30 16/30 G01N 33/48 M G01N 33/48 33/53 D 33/53 C12P 21/08 // C12P 21/08 C12N 15/00 ZNAA (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), AE, 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) Invention Heider Karl Heinz Austria Aar 2000 Stockerau Johann Strauss Promenade 4-11 (72) Inventor König Ulrich Austria Aer 1180 Vienna Hokegasse 17-17 (72) Inventor Sommerbelbel Wolfgang Austria Aar 3002 Purkersdorf Linzerstraßer 19 House 4 Reference) 2G045 AA24 AA25 DA78 FB03 4B024 AA01 BA36 CA04 DA02 GA11 HA17 4B064 AG27 AG31 CA10 DA05 4C085 AA03 AA04 AA14 AA19 BB01 CC02 DD62 4H045 AA10 AA11 AA30 BA10 CA41 DA50 DA75 DA76 DA86 FA74EA22 EA22

Claims (20)

[Claims] 1. A tumor-associated antigen designated C42, which comprises the amino acid sequence defined by SEQ ID NO: 2. 2. An immunogenic protein fragment or peptide, which is derived from the tumor-associated antigen according to claim 1. 3. The immunogenicity according to claim 1, which induces a humoral immune response.
(Poly) peptide. 4. The immunogenic (poly) peptide according to claim 1 or 2, which is presented by an MHC molecule and induces a cell-mediated immune response or a degradation product thereof. 5. The method according to claim 4, which is selected from the group of peptides of SEQ ID NOs: 40 to 89.
The immunogenic peptide described. 6. The method according to any one of claims 1-5, wherein the (poly) peptide induces an immune response against tumor cells of a patient expressing C42, for immunotherapy of cancer in vivo or ex vivo. An immunogenic (poly) peptide according to the item. 7. An immunogenic (poly) peptide derived from a portion of the tumor-associated antigen C42 expressed in tumor, which exhibits one or more tumor-specific mutations. 8. A parenteral, topical, oral or topical preparation containing at least one immunogenic (poly) peptide according to any one of claims 1 to 7 as an active ingredient. Composition for active administration. 9. The pharmaceutical composition according to claim 8, which contains various immunogenic peptides derived from C42. 10. The pharmaceutical composition according to claim 9, which comprises one or more C42-derived peptides in a mixture with peptides derived from other tumor-associated antigens. 11. A pharmaceutical composition according to claims 8-10, characterized in that the peptide binds to at least two different HLA types. 12. An isolated DNA molecule encoding the immunogenic protein or fragment thereof of the tumor-associated antigen of claim 1. 13. The DNA molecule according to claim 12, which encodes an immunogenic polypeptide designated as C42 having the amino acid sequence of SEQ ID NO: 2 or a protein fragment or peptide derived therefrom. 14. A polynucleotide having the sequence shown in SEQ ID NO: 1, or containing such a polynucleotide, or having the sequence shown in SEQ ID NO: 1 under stringent conditions. 14. The DNA molecule according to claim 13, which is a polynucleotide that hybridizes with the polynucleotide that contains, or contains such a polynucleotide. 15. Recombinant DNA comprising the DNA molecule according to any one of claims 12-14.
molecule. 16. The method according to claim 12, wherein the C42 (poly) peptide expressed by the DNA molecule induces an immune response against tumor cells of a C42-expressing patient, for immunotherapy of cancer. DNA molecule. 17. Use of cells expressing the antigen of claim 1 for the preparation of cancer vaccines. 18. An antibody against the (poly) peptide according to any one of claims 1 to 5. 19. The antibody according to claim 18, which is monoclonal. 20. A method for treating and diagnosing a cancer associated with C42 expression.
Or the antibody according to 19.