WO2002004508A1 - Tumour-associated antigen (b345), characterised by an amino acid sequence as in seq. id. no. 4 - Google Patents

Abstract

The invention relates to the tumour-associated antigen B345 and to DNA molecules which code for the same.

Description

TUMORASSOZI IERTES ANTIGEN (B345), CHARACTERIZED BY AN AMINO ACID SEQUENCE AS IN SEQ. ID. NO .4

5 The invention relates to the chemotherapy of tumor diseases.

Normal body cells are subject to a strictly ordered system that controls the growth, cell division and death of certain cells. So share

10 body cells of an adult only if they have to replace dead cells or heal an injury. Cancer cells, on the other hand, continue to grow uncontrollably, they accumulate and form a tumor. If the tumor reaches a critical size, can

15 cancer cells can also be transported to other areas of the body via the bloodstream or the lymphatic system and form colonies there (metastases). Not all tumors are carcinogenic, because benign tumors do not metastasize and are therefore usually not life-threatening because they

20 can be surgically removed. More detailed

The following publications provide information on this topic and the aspects of tumor development discussed below: Rauscher and Vogt, 1997; Kastan, 1997; Hesketh, 1995; Pusztai, Lewis and Yap, 1995.

25 The transformation of a healthy cell into one

Cancer cells can be triggered by a number of factors, such as environmental influences, radiation, viruses or chemical reagents. However, epigenetic (methylation, Acetylations and changed chromatin structure) and genetic modifications (point mutation, deletion, amplification, translocation) play an important role.

Mutations in coding regions of genes that are involved in the regulation of cell proliferation can contribute to the transfer of a normal cell into a tumor cell, since the transformed cell has growth advantages over its healthy neighboring cell.

Cancer therefore arises from an accumulation of inherited or acquired mutations in critical proto-oncogenes or tumor suppressor genes.

Cell proliferation is under the control of various gene systems, while products of oncogenes are involved in signaling from the cell surface to the nucleus, cyclin-dependent protein kinase and its inhibitors guide the cell through the cell cycle. Disturbances in the synthesis of these proteins are often found in tumor cells. The p53 protein plays a central role in this.

RB protein-type proteins regulate the availability of crucial transcription factors.

The up-regulated genes in tumor tissues represent ^'best starting point for more detailed analysis is and because proteins verschiedenster functions are highly expressed, the approach for therapeutic interventions is very versatile. It is therefore the goal of cancer research to find further new target molecules (so-called "targets") for therapeutic interventions, which can then be used for targeted therapy with few side effects.

First and foremost, it is therefore necessary to uncover molecular changes between normal tissue and tumor at the level of gene expression ("transcription level"), which on the one hand are intended to identify new targets and on the other hand can be used to develop or find substances to inhibit malfunctions.

A whole range of different methods for

Identification and characterization of new targets, which are the starting point for the development of new therapeutic agents, are based on the creation of differential mRNA transcription profiles between tumors and normal tissues. 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 addition to immunotherapeutic approaches, targeted chemotherapy plays an essential role in the treatment of cancer. Chemotherapy is the administration of substances that interfere with metabolism, signal transduction and

Cell division processes of malignant cells are either cytostatic or cytotoxic-cytolytic. Chemotherapeutic agents can be classified based on the influence of specific targets in the tumor cell, the type of cellular interaction and the Interaction with a specific cell cycle phase, divided into different categories.

The type of cancer treatment depends on the tumor stage, the decisive factor here is whether metastases are already present and how widespread they are in the body. The application of cell poisons for cancer treatment is, in addition to operative measures and radiation therapy, an integral part of today's therapy concepts in oncology.

There are two main objectives

Chemotherapy: First and foremost, it is curing cancer; this means that the tumor disappears and no longer occurs. If a cure is no longer possible for various reasons, one tries to restrict or control the growth and spread of the tumor.

In principle, substances that are used in chemotherapy have an effect on all dividing cells. However, tumor cells show a higher sensitivity to chemotherapeutic agents than healthy cells, since mainly proliferating cells are attacked.

Each tissue has a characteristic growth behavior, which includes cell division, growth arrest, differentiation and aging and is influenced and regulated by internal and external factors.

Many of the cytotoxic chemotherapeutic agents used today only work on proliferating cells (not in the G0 phase of cell division). In doing so however, both normal and cancer cells are attacked. The destruction of normal cells can lead to strong side effects; eg destruction of the blood cell-producing tissue of the bone marrow (myelosuppression).

Chemotherapeutic agents are divided into different categories depending on how they affect specific substances within the tumor cell, the cellular processes with which the drugs interact and the cell cycle phase they influence.

This information is necessary for oncologists to decide which preparations can be combined with each other during therapy.

The genes upregulated in tumor tissues thus represent potential new target structures and since proteins of various functions are highly expressed, the approach for therapeutic interventions is very versatile.

It is therefore the goal of cancer research to find further new targets for therapeutic interventions that can then be used for targeted therapy with fewer side effects compared to the therapeutic agents currently used.

Genes upregulated in tumor tissues represent points of attack and thus potential target structures ("targets") for chemotherapy.

The object of the present invention was to provide a new protein, preferably expressed by tumor cells, which is a target molecule for the Intervention using chemotherapy methods.

This task was solved by first using RDA ("representational difference analysis") between a lung adenocarcinoma cell line (A549) and

Normal lung tissue was created using a cDNA subtraction library. In order to select the antigens overexpressed in the tumor, the cDNA clones obtained were then sequenced and compared with sequences available in databases. Among the genes that were annotated were 321 unknowns, most of which had ESTs (expressed sequence tags) in the database. After a further qualitative PCR analysis in cDNA libraries of critical normal tissues and immune-privileged tissues as well as more detailed database searches, the number of candidate clones was restricted to 59, whose ESTs do not originate from critical normal tissues.

These clones were spotted on Incyte DNA chips and with a whole range of tumor tissues and

Normal tissues hybridized for reference. The mRNA expression profile of EST fragments, which are differentially expressed in cancer and normal tissues and belong to a still unknown gene, was verified by different methods.

The length of the transcripts was determined by means of Northern blot analysis and the expression pattern in different cell systems was precisely characterized by quantitative PCR. Only unknown genes or ESTs with tumor-specific expression profiles were found followed up and subjected to a "full length cloning". Potential ORFs ("open reading frames") are converted into the corresponding amino acid sequence and analyzed for possible function prediction using in silico strategies.

The human B345 cDNA was cloned, the sequence obtained in a first cloning approach is shown in SEQ ID NO: 1. Sequence analysis of the human B345 CDNA cloned in this approach showed a continuous open reading frame from position 215 to position 2461 (excluding stop codon) which, at the nucleotide and protein levels, has no homology or identity in the known sequences of the databases. It can be concluded from the data obtained from Northern blot experiments that the B345 transcript has a length of approximately 6.5 kb. In a first approach, a B345 CDNA with 5897 bp (exclusive polyA region) was obtained as the cloned region, the presence of a polyadenylation signal and the polyA tail at the 3 'end of the sequence indicating the completeness of the cDNA in this region , Due to the fact that there was no continuous reading frame in the 5 'region of the cloned cDNA from position 1 to 214, it was initially assumed that the ATG at position 215, which is also 75% a Kozak translation initiation site (ACCATGT) (Kozak , 1987) corresponds to the start codon of B345.

In a further cloning approach, additional methods were used using a standard molecular biological method, that is to say using the so-called “promoter finder DNA walking” Information about the upstream sequence of B345 was obtained.

Thus, the B345 sequence (SEQ ID NO: 1) obtained in the first cloning experiment was expanded in the 5 "region

Application of the primer extension analysis can be located precisely and is at position 201 (SEQ ID NO: 3). Through repeated sequencing also in the 3 ^{^} region, in comparison to the sequence shown in SEQ ID NO: 1, an additional base at position 2430 was found, which leads to a reading frame shift and thus shifts the stop codon from position 2729 to 2791. The cDNA obtained (SEQ ID NO: 3) has an open reading frame which codes for a potential protein with a length of 836 amino acids (SEQ ID NO: 4). The

The translation initiation site at position 283 corresponds approximately to 70% of a Kozak consensus sequence.

The promoter region 200bp upstream of the putative transcription start site contains neither a TATA nor a CCAAT box, but a unique GC box, which represents a binding site of the SP1 protein. The fact that the GC content in the 5 "region is over 60% indicates a CpG Island (Bird, 1986).

The resulting primary amino acid sequence of B345 is shown in SEQ ID NO: 4. Analysis of the hydrophilicity plot of the amino acid sequence shows that the B345 protein has two characteristic hydrophobic domains which represent a signal peptide and a helical transmembrane domain (FIG. 6). This polarized structure indicates indicates that B345 is an integral membrane protein.

The extracellular domain suggests the existence of definitely one, possibly three, CUB domains. CUB domains are found in various proteins, most of which are regulated during embryonic development. A recent publication (Gerstein et al., 2000) demonstrated that proteins containing CUB domains are the most pronounced differentially regulated proteins in C. elegans. Since genes that play a key role in embryonic development have corresponding functions, e.g. in cell division, cell proliferation or signal transmission in cancer, it can be assumed that overexpression of B345 in cells causes a change in the properties of substrate adhesion or the extracellular matrix. The B345 protein has 12 potential N-glycosylation sites found in the putative extracellular domain.

Due to its amino acid sequence, it can be assumed that the B345 protein forms a ß-sheet secondary structure, since CUB domains are known to fold as a ß-sandwich.

The intracellular domain (range 691-836) has no significant homologies. However, the entire C-terminus shows an identity (82%) over 124 amino acids with an EST (Acc No. AW063026) from human ovarian cancer cells.

Based on the functions of other proteins containing CUB domains, it can be concluded that the B345 transmembrane protein plays a role in communication, interaction and / or signal transduction with extracellular components or ligands. Furthermore, the data from the expression analysis are a strong indication that B345 is metastatic

Process of cancer, especially colon cancer, is involved.

The following methods of investigation are suitable for elucidating the physiological function and the role of B345 in metastasis:

First, cell lines, preferably human cell lines, are identified, e.g. using TaqMan PCR that do not endogenously express B345. The cells are transfected with a plasmid containing the B345 sequence and B345 expressed. Changes in the

Morphology and / or migration behavior, e.g. Using a soft agar assay (Hamburger and Salmon, 1977) or a migration assay (Liaw et al; 1995) of the cells expressing B345 versus the non-transfected cells indicate a role of B345 in the biological process responsible for this. This is a clear indication of the involvement of B345 in the interaction of tumor cells with one another and / or with the extracellular matrix and thus for a function in metastasis.

As an alternative or in addition to this functional analysis, the expression of B345 in cells which endogenously express this protein is suppressed in a complementary approach in order to also determine any changes in morphology and / or migration behavior. In addition, it may be investigated whether protein components exist that interact with B345 inter- or extracellularly (eg using the Yeast Two Hybrid System (Fields and Song, 1989).

The invention thus relates in a first aspect to a tumor-specific polypeptide with the designation B345, with the amino acid sequence given in SEQ ID NO: 4, or to a polypeptide which is encoded by a polynucleotide which, under stringent conditions, is linked to a polynucleotide of the type shown in SEQ ID NO: 3 shown sequence or a partial sequence thereof hybridized, as well as derived protein fragments or peptides.

In a further aspect, the present invention relates to an isolated DNA molecule coding for the tumor-specific polypeptide of the designation B345.

The DNA molecule according to the invention is preferably a polynucleotide of the sequence shown in SEQ ID NO: 3 or a fragment thereof or a DNA molecule which is linked to a DNA molecule of the sequence shown in SEQ ID NO: 3 or a partial sequence thereof under stringent conditions hybridized, or a fragment thereof.

“Stringent conditions” mean, for example: overnight incubation at 65 ° C.-68 ° C. with βxSSC (lxSSC = 150 mM NaCl, 15 mM trisodium citrate), 5 × Denhardt's solution, 0.2% SDS, 50 μg / ml salmon sperm -DNA, followed by 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 molecules according to the invention code for (poly) peptides of the designation B345 with the amino acid sequence shown in SEQ ID NO: or for protein fragments or peptides derived therefrom; this also includes DNA molecules or fragments which, due to the degeneration of the genetic code, have deviations from the sequence shown in SEQ ID NO: 3

In one embodiment of the invention, the invention relates to an isolated DNA molecule of the sequence shown in SEQ ID NO: 3 or a fragment thereof, or a DNA molecule which is associated with a DNA molecule of the sequence shown in SEQ ID NO: 3 or with a Partial sequence hybridized, coding for the natural B345 polypeptide or for a fragment thereof.

The B345 DNA molecules can be used in a so-called DNA vaccine for the immunotherapy of tumors.

The B345 DNA molecules of the 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 the immunotherapy of cancer based on DNA ("DNA vaccine") on B345-DNA can be used, both in vivo and ex vivo.

Examples of in vivo administration are the direct injection of "naked" DNA, either intramuscularly or by means of a gene gun, which has been shown to lead to the formation of CTLs against tumor antigens. Examples of recombinant organisms are vaccinia virus, adenovirus or Listeria monocytogenes (an overview was given by Coulie, 1997). Furthermore, synthetic carriers for nucleic acids, such as cationic lipids, microspheres, microspheres or liposomes, can be used for the in vivo administration of nucleic acid molecules, coding for B345 peptide. Various adjuvants that enhance the immune response can be co-administered, for example cytokines, either in the form of proteins or plasmids encoding them. The application can optionally be combined with physical methods, eg electroporation.

An example of ex vivo administration is the transfection of dendritic cells as described by Tuting, 1997, or other APCs _. which are used as cellular cancer vaccines.

In a further aspect, the present invention thus relates to the use of cells which express B345, either on their own or, in optionally modified form, after transfection with the corresponding coding sequence, for the production of a cancer vaccine.

As an alternative to the natural B345 CDNA or fragments thereof, modified derivatives can be used. These include sequences with modifications for a protein (fragment) or peptides with stronger ones

Coding 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, according to Art a string of beads ("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 in the endoplasmic reticulum ("ER targeting sequence") the processing and thus the presentation and ultimately the immunogenicity of the antigen can be increased.

In another aspect, the present invention relates to a recombinant DNA molecule containing B345 DNA, e.g. linked to a regulatory DNA sequence, especially a heterologous regulatory DNA sequence, e.g. a promoter or enhancer.

In a further aspect, the invention relates to antibodies against B345 or fragments thereof. Polyclonal antibodies can be obtained in a conventional manner by immunizing animals, in particular rabbits, by injection of the B 345 antigen or fragments thereof, and subsequent purification of the immunoglobulin.

Monoclonal anti-B345 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, for example by fusion with myeloma cells, and the supernatant hybridomas obtained is screened for monoclonal anti-B345 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-B345 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-B345 antibodies according to the invention can be used in immunohistochemical analyzes for diagnostic purposes, or as a therapeutic agent in cancer therapy. (An example of the successful use of a monoclonal antibody in cancer therapy is Herceptin; an antibody against the proto-oncogene HER2. Herceptin can be used in breast cancer patients who overexpress HER2.)

In a further aspect, the invention relates to the use of B345-specific antibodies in order to selectively bring any substances into or into a tumor which expresses B345. Examples of such substances are cytotoxic agents or radioactive nuclides, the effect of which is to damage the tumor on site. Due to the relatively tumor-specific expression of B345, only a few are involved Expected side effects. In another aspect, B345 antibodies can be used to visualize tumors that express B345. This is useful for the diagnosis and evaluation of the course of therapy.

Therapeutic and diagnostic uses of antibodies which are suitable for anti-B345 antibodies are described in WO 95/33771.

The protein of the designation B345 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

B345-positive tumors are used, especially in lung and colon carcinoma.

B345 or peptides, peptide equivalents and peptidomimetics can be used for the immunotherapy of cancer, e.g. in WO 00/73438, the disclosure of which is hereby incorporated by reference.

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., 1994). In order to determine the presence of tumor-specific B345 mutations, the B345 cDNA is expediently determined using probes from the isolated cDNA according to the invention cloned one or more different tumors and compared the sequences obtained with normal tissue B345 CDNA. Experiments are being carried out to show whether tumor B345 peptides from a sequence section mutated compared to normal tissue B345 have an increased immunogenicity compared to normal tissue B345 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 B345 peptides derived from regions of a tumor-expressed B345 which have tumor-specific mutations.

Due to the preferred expression of B345 in tumor cells, it can be assumed that this protein has an important function for the tumor, e.g. for emergence, infiltration and growth and thus a target for chemotherapeutic intervention.

With regard to its use as a target in targeted chemotherapy, B345 is characterized in more detail in order to develop the appropriate strategy for the intervention with this function.

As a first step in the so-called "down-stream" functional analysis of B345, a bioinformatic analysis is expediently carried out in a first step, which is trend-setting for the experimental validation of B345 as a target. The bioinformatics concepts based on similarity and modular structure represent an essential basis for this analysis. Established bioinformatics tools for determining similarities are BLAST

(http: //www.ncbi .nlm.nih.gov / BLAST, Altschul et al., 1997) or FASTA (Pearson & Lipman, 1988), the specialized databases such as Pfam (http: // www. sanger .ac. uk / Pfam, Bateman et al., 2000) and SMART (http: // smart. embl-heidelberg. de, Schultz et al., 2000), which take into account domain structures. To refine the analysis, applications such as Clustal (http: // www2. Ebi. Ac. Uk / clustalw, Higgins et al., 1996) HMMer (http: //hmmer.wustl. Edu), PSI-BLAST (Altschul et al ., 1997) and the PROSITE database

(http: //www.expasy.ch/prosite, Hofmann et al., 1999). Statistical analysis methods that are not based on homologies allow the prediction of further structure and function-relevant properties such as the secondary structure and the occurrence of

Transmembrane segments and helix-turn-helix motifs. Methods for predicting the secondary structure of proteins are available; Jpred is particularly worth mentioning (http: // barton. ebi .ac .uk / servers / jpred.html, Cuff et al., 1998). The secondary structure prediction can

Support functional hypotheses, for example if the structure of the suspected homologue is known.

According to bioinformatics analysis, B345 has a helical transmembrane domain, with both the N-terminal and the C-terminal region being hydrohpil, which suggests that this protein is a transmembrane protein. The N-terminal, The extracellular region has some CUB domains which tend to form disulfide bridges and are therefore involved in dimerization or in protein-protein interactions (Bork et al., 1993). The C-terminal, intracellular end shows homologies to a receptor kinase and to a C-kinase substrate.

Subsequently, B345 is subjected to a biochemical and biological analysis.

In a next step, the function of B345 for the tumor process is elucidated; e.g. by

Proliferation assays in vitro or in animal models that overexpress the B345 gene to be examined (constitutive or inducible) and express it as a control either in deleted (inactive) form or downregulate via antisense (see e.g. Grosveld and Kollias, 1992).

B345 can be used in screening assays to identify substances that modulate, especially inhibit, the activity of this protein. In one embodiment, such an assay may e.g. B. consist of introducing the B345 protein, or an active fragment thereof, into cells which react to the activity of B345 with proliferation or to express the corresponding B345 cDNA in the cell, and the proliferation of the cells in the presence and to determine in the absence of a test substance.

An example of test cells are cells with a low division rate, for example primary cells that have no endogenous B345. To determine the suitability of the cells for a screening assay, they are analyzed with B345-CDNA transformed, grown and tested for their ability to proliferate with standard assays, such as thymidine incorporation. Due to a significant increase in their proliferation properties after B345 expression, they can be used as test cells, for example in high throughput screening proliferation assays. Examples of proliferation assays in high throughput format, for example based on the MTS assay, are described in WO 98/00713.

Substances with an anti-proliferative effect can be used for the treatment of tumors with strong B345 expression, particularly in the case of lung and colon carcinoma.

LIST OF FIGURES:

FIG. 1A: Expression profile of B345, B452 and B540 in individual lung carcinomas and lung tumor cell lines.

1B: Expression profile of B345 in normal colon tissue and tumor cell lines.

Fig. IC: Graphical representation of the alignment of B345, B452 and B540.

Fig. 2A: Northern blot analysis of tumor cell line A549 with a 490bp long B345 PCR product

Fig. 2B: Northern blot analysis of various

Normal tissue with a 490bp B345 PCR product 2C: Northern blot analysis of various

Cancer tissue with a 318bp B345 PCR product

3 mRNA expression analysis of B345 by real-time PCR of tumor and normal tissues.

Fig. 4: mRNA expression analysis of B345 by real-time PCR of laser microscope-prepared colon tumors (LCM) as well as normal colon tissue and tumor cell lines.

Fig. 5: Graphical representation of the gene structure of B345.

Fig. 6: Hydrophilicity and transmembrane blot of the B345 protein

Figure 7: Potential protein structure of B345

Table overview

Tab. 1: Summary of Northern blot data from B345 in different normal tissues (1A), cancer cell lines (1B); and various

Normal tissues in comparison with the corresponding tumor tissue (IC)

Tab. 2A: Summary of the data of the quantitative PCR of B345 in various normal and cancer tissues Tab. 2B: Summary of the data of the quantitative PCR of B345 in different normal tissues and microdissected colon adenocarcinoma tissues

Explanations

+++ extremely positive

++ strongly positive

+ positive

(+) weakly positive

negative

example 1

Representative Difference Analysis (RDA) from the human adenocarcinoma cell line of the lungs (A549) and normal lung tissue

The human lung adenocarcinoma cell line A549 (CCL 185) obtained from ATCC was grown up in T150 cell culture flasks. MEM served with 10% heat-inactivated, fetal calf serum and 2 mM L-glutamine as the nutrient medium. Every 3 to 4 days the cells were cleaved by trypsinization 1: 5 to 1:10 for propagation. After approximately 80% confluence had been reached, 4 ml of a trypsin solution (data per liter: 8 g NaCl, 0.2 g KC1, 1.13 g Na ₂ HP0 ₄ - water-free, 0.2 g KH ₂ PO ₄ ,

100ml 2.5% trypsin solution, 1g EDTA Na salt; pH 7.2 - 7.4) used to harvest the cells. The 4 ml were transferred to a 15 ml falcon tube, mixed with 8 ml PBS, centrifuged at 1200 rpm in a Haereus table centrifuge (Megafuge 2.0R) for 5 min at 4 ° C, the cell pellet with 1 ml lysis buffer (10 mm Tris-HCl pH8, 140 mM NaCl, 1.5 mM MgCl ₂ , 0.5% NP40) were added, shaken vigorously and centrifuged in a 2 ml Eppendorf tube at 12,000 rpm and 4 ° C. for 5 min in a Sigma table centrifuge (Sigma 202 MK). The supernatant was transferred to a new Eppendorf tube and, after addition of 55 μl 20% SDS solution, extracted twice with twice the volume of a CHCl ₃ / phenol (1: 1 v / v) mixture and once with the single volume of CHC1 ₃ . The aqueous RNA-containing phase was mixed with 1/10 volume of 3M NaAc (pH5) and twice the volume of 96% EtOH and the RNA was precipitated overnight at -20 ° C. Starting with 1 mg of total RNA, the PolyATtract Kit (Promega) was used to isolate poly-A (+) RNA in accordance with the manufacturer's protocol. The A549 poly-A (+) RNA with a concentration of 1 mg / ml in DEPC-treated H ₂ 0 was stored in aliquots at -80 ° C.

In order to carry out the representative difference analysis (RDA; Hubank and Schatz, 1994; Diatchenko et al., 1996), the poly-A (+) RNA of the lung adenocarcinoma cell line A549 was described as "solid", that of normal lung tissue (1 mg / ml; Clontech, Palo Alto; # 6524-1) used as a "driver". The RDA was carried out using the PCR-select ™ kit (Clontech, Palo Alto) according to the manufacturer's protocol, except that a modified primer / adapter-2 oligonucleotide system was used: adapter-2-alt-l (SEQ ID NO: 31) and nested PCR primer-2-alt (SEQ ID NO: 32) and adapter-2-alt-2 (SEQ ID NO: 33). The newly generated primer / adapter sequences enable the presence of three new restriction enzyme interfaces (Kpn I, Sac I and Xho I) in the sequence of the nested PCR primer-2-alt after cloning the subtracted cDNA fragments in the pPCRII- Vector a subsequent cutting out of the respective cDNA fragments. Design a primer / adapter sequence with several available

Restriction enzyme interfaces were necessary because point mutations were often observed, particularly in the primer sequences due to the PCR amplification steps.

After the synthesis of double-stranded cDNA using oligo-dT, the cDNA obtained was digested by "tester" and "driver" with Rsal (Rsal is a 4-base-recognizing restriction enzyme and provides a statistical average of 256 bp long cDNA fragments). Identical parts of "tester cDNA" were ligated either with adapters 1 or 2 and then hybridized separately with an excess of "driver cDNA" at 65 ° C. The two approaches were then combined and subjected to a second hybridization with freshly denatured "driver cDNA". The enriched "tester" -specific cDNAs were then exponentially amplified by PCR, with primers 1 and 2 specific for the adapters. For further enrichment, an aliquot of this reaction was subjected to a second PCR with specific "nested" primers. The exponentially amplified cDNA fragments resulting from this reaction became direct ligated into the pCRII vector (Invitrogen; "TA-cloning vector") and then a third of the ligation mixture was transfected into competent E. coli (OneShot ™, Invitrogen).

712 positive transformants (blue-white selection) were obtained and cultured in 96-well blocks in LB-Amp medium (1.3 ml per well) for 48 h at 37 ° C. 750 μl of the E. coli suspensions were used per well for the preparation of the plasmid DNA (96-well mini-preparation method from QIAgen according to the manufacturer's instructions). The remaining bacterial cultures were stored as glycerol stocks at -80 ° C.

A cDNA subtraction library consisting of 712 individual clones was obtained, which was available both in the form of E. coli glycerol stock cultures and in the form of purified plasmids.

Example 2

DNA sequencing and annotation of TAA candidates

The isolated plasmid DNA of all 712 clones (see example 1) was sequenced according to the Sanger method on an ABI-377 Prism device. The sequences obtained were annotated using the BioScout software (LION, Heidelberg) and subjected to database comparisons (Genbank). From 712 clones, 678 could be sequenced and annotated. The rest (34) had either only poly (A) sequences as an insert or corresponded to a religious vector or could not be sequenced. Of the 678 annotable sequences, 357 proved to be genes with a known function. The remaining 321 represented clones encoding genes with unknown function; 59 of them did not even have entries in the human EST database. Known genes were not further treated. For those unknown

Genes for which an EST entry was available were assessed for the expression profile: all those ESTs with> 95% identity (BLAST) that belonged to the experimentally determined sequence of the subtraction libraries were checked. The annotation was divided into a) critical normal tissues, b) fetal, "dispensable" and immune-privileged tissues and c) tumors and tumor cell lines. Based on this "virtual mRNA profile" ("virtual Northern blot")

200 clones for which no ESTs were found in group a) were selected for further experimental analysis (including the 59 clones for which there was no EST entry). To further narrow the candidate clones, oligonucleotide primer pairs were designed and synthesized from the sequences determined from the 200 selected clones. First, 8 different human tissue-derived cDNA libraries (GibcoBRL "SUPERSCRIPT ™"), which are directionally cloned into pCMV-SPORT, were tested for the presence of the respective candidates by means of qualitative PCR. The cDNA libraries used here came from tissue from the heart (# 10419-018), liver (# 10422-012), leukocytes (# 10421-022), kidney (# 10420-016), lung (# 10424-018), Testis (# 10426-013), brain (# 10418-010) and fetal brain (# 10662-013). The PCR conditions were like follows: 20 μl total volume per PCR mixture contained 1 × TaqPol buffer (50 mM KC1, 10 mM Tris-HCl pH9, 0.1% Triton X-100), 1.5 mM MgCl ₂ , 0.2 mM dNTPs (Promega ), 0.025 U / μl Taq DNA polymerase (Promega), each 5 pM at specific oligonucleotide primers for B345 (B345-D,

SEQ ID NO: 34) and (B345-U, SEQ ID NO: 35) and 100 ng of the plasmid DNA to be examined in each case. As a control, specific primers for GAPDH (SEQ ID NO: 36 and 37) were used. To check the selective detection, the respective B345 were specific

Primer pairs of oligonucleotide primers (SEQ ID NO: 34) and (SEQ ID NO: 35) were also tested in parallel for the isolated plasmid with the B345 "original fragment" (originally isolated cDNA fragment from B345). The detectability of fragments of the expected length with a strong signal in one of the critical normal tissues (heart, liver, lung, kidney and leukocytes), but not in the cDNA libraries from immune-privileged tissues (brain, fetal brain and testis) under these PCR Conditions (1 cycle: 3 '94 ° C; 35 cycles: 1' 94 ° C - 1 '55 ° C - 1' 72 ° C; 1 cycle: 7 '72 ° C) was defined as the elimination criterion. This qualitative PCR analysis reduced the number of candidates to 56; Clone B345 was in this pre-selected candidate group. Example 3

Expression analysis by cDNA chip hybridization

For the design of a cDNA chip, a number of clones were selected from the dBEΞT database via nucleotide sequence search, the most diverse

Functional categories, such as apoptosis to cell cycle regulation, belong. A total of 1299 IMAGE clones (of which 1024 already known genes) were ordered and sequenced for control. Microtiter plates with bacteria containing sequences of approximately 800 bp from the 3 "end of the gene in the vector were sent to Incyte Pharmaceuticals, Inc. (USA) and spotted there on 60 chips. In addition to these clones, 120 EST identified by RDA were also found The DNA chips produced in this way were then hybridized with Cy3-labeled cDNA from normal tissue, tumor tissue and cell lines together with Cy5-labeled cDNA from a mixture of nine different normal tissues and the two signals for normalizing the expression values were compared. The calculations were carried out in part in S-Plus or in Microsoft Excel The evaluation of the chip experiments revealed a very similar expression profile for B345, B540 and B452 when hybridizing with lung cancer samples of cell lines and patient material (see FIG. 1A). Such a tumor-associated expression profile could be used for B345 also when comparing colonic adenocarcinoma with normal colonic tissue be shown (see Fig. 1B). The sequence alignment of B345, B540 and B452 clearly showed an overlap of the individual EST fragments. It could therefore be assumed that the three clones are ESTs from one and the same gene. The resulting DNA segment covers a length of 843 bp (see FIG. IC) and was used in further experiments to search public databases. The search results did not provide any significant homology to known DNA or protein sequences, which indicates that B345 is a gene that has not been known for a long time.

Example 4

Expression analysis of B345 using Northern blots

B345 is a gene which, according to DNA chip analyzes, is up-regulated in tumor tissues (see FIGS. 1A and 1B, Tab. La and Tab. 1B).

In order on the one hand to confirm the transcription profile obtained and on the other hand to determine the length of the mRNA to be expected for the full size cloning, a Northern blot analysis was carried out for B345 using human cell lines and the "Human Multiple Tissue Northern Blot" (Clontech and Invitrogen) , The 490 bp and 318 bp long PCR products labeled with [α- ³² P] dCTP (NEN, Boston) (primers (SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 7 and SEQ ID NO: 8)). The hybridization took place at 68 ° for 2 h; visualization using standard autoradiography (Hyperfilm, Amersham). 2A, 2B and 2C and Tab. La and Tab. 1B, IC show the result of this analysis: Fig. 2A from cell line A549, Fig. 2B from 12 normal tissues (peripheral blood lymphocytes (PBL), lungs, placenta, small intestine, liver, kidney, spleen , Thymus, colon, skeletal muscle, heart and brain) and FIG. 2C of 8 cancer cell lines (promyelocytic leukemia HL60, HeLa-S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma (Raji), colon adenocarcinoma SW480, lung adenocarcinoma A549 and melanoma G361) The B345 transcript is 6.5 kb in length.

Example 5

Analysis of the expression profile of B345 at the RNA level using quantitative RT-PCR (real time PCR or TaqMan analysis)

In order to carry out a more precise quantification of the mRNA expression in the different normal and tumor tissues, the "real time PCR" was used, which allows the RNA concentration to be calculated in comparison to an external standard.

The RNA was isolated from frozen tissue using trizole in accordance with Gibco's manufacturer's protocol. To remove any contaminating DNA, the prepared RNA was digested with DNAase I as follows: 3 μg of total RNA were mixed with 20 μl of 5 × AMV buffer (Promega), 1 μl of RNasin (Promega) and 2 μl of DNase I (Boehringer Mannheim) incubated for a total of 80 μl at 37 ° C. for 15 minutes. 120 μl phenol: chloroform: Isoamyl alcohol (25: 24: 1) was added, mixed on a vortex mixer and centrifuged briefly. The aqueous phase was removed, mixed with 120 ul chloroform: isoamyl alcohol (24: 1) and centrifuged as before. The purified RNA was ethanol-precipitated and dissolved in water.

The total RNA was then transcribed with reverse transcriptase (Superscript, Gibco, BRL) into cDNA: 1 μl of oligo dT primer (Promega) was added to 3 μg of total RNA and water was added to it

Brought final volume of 10 ul. After incubation for 5 minutes at 70 ° C, the solution was cooled for 5 minutes at room temperature. 5 ul RT reaction buffer (5x, Gibco, BRL), 2.5 ul dNTPs (10 mM each, Boehringer Mannheim), 1 ul RNasin (lOU / ul, Promega), 1.5 ul

Superscript (10 U / μl, Gibco, BRL) and 5 μl water were added and incubated for 1 hour at 42 ° C. and the reaction was ended by incubating for 3 minutes at 95 ° C.

To produce a cDNA pool of a certain tissue or tumor type, 3 to 10 different individual preparations from different patients were mixed in equal proportions.

The quantitative determination of the "household genes" ß-actin, GAPDH and tubulin in cDNA pools was carried out as follows:

A) ß-Actin-TaqMan PCR (Perkin Elmer)

For details on the principle of the TaqMan method, see manufacturer information (Perkin Elmer). A TaqMan PCR Run included samples of β-actin control sequence with 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ copies / μl (Perkin Elmer) to determine the standard curve, a negative control without DNA and the cDNA pools to be quantified. All samples were analyzed in triplicate. For a 25 μl

The reaction mixture was 1 ul cDNA, 2.5 ul lOx buffer A (Perkin Elmer), 4 ul MgCl ₂ (25 mM, (Perkin Elmer)), 0.5 ul per nucleotide (10 mM dATP, dCTP, dGTP; 20 mM dUTP ), 0.125 μl TaqMan probe (20 μM; TaqMan probe for ß-actin (SEQ ID NO: 20 fluorescence-labeled at the 5 'end with 6-carboxyfluorescein and with

6-carboxytetramethylrhodamine at the 3 'end), 1 μl per ß-actin-specific primer (20 μM each, forward primer SEQ ID NO: 21 and reverse primer SEQ ID NO: 22), 0.25 μl AmpErase uracil N-glycosylase " UNG "(1 U / μl, Perkin Elmer), and 0.125 μl AmpliTaq Gold (5 U / μl, Perkin Elmer) mixed, transferred to MicroAmp. Optical Tubes (Perkin Elmer) and sealed with MicroAmp Optical Caps. The PCR was carried out as follows: a cycle with 2 minutes 50 ° C for the UNG reaction, a cycle 10 minutes 95 ° C to activate the AmpliTaq, 40 cycles with 15 seconds each 95 ° and 1 minute 60 ° C. The samples were then kept at 25 ° C. The data are evaluated with the program "Sequence Detection System 1.5bl" * (PE Applied Biosystems), the fluorescence signals of the cDNA samples to be quantified being compared in principle with the signals of the control plasmid dilutions of known concentration.

B) GAPDH-TaqMan PCR The following primers or probes were used for the quantification of GAPDH, which was used to normalize the RNAs used, such as β-actin or tubulin. A TaqMan probe for GAPDH (SEQ ID NO: 23) was used at the 5 'end

Tetrachlorofluorescein and at the 3 'end labeled with carboxymethylrhodamine (Forward GAPDH primer: SEQ ID NO: 24 and reverse primer: SEQ ID NO: 25). The reactions were carried out as described above.

C) Tubulin-SybrGreen PCR (Perkin Elmer)

Principle of the SybrGreen PCR see manufacturer information (Perkin Elmer). A SybrGreen PCR run contained samples of tubulin control plasmid with 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ and 10 ^e copies / μl (Perkin. Elmer) to determine the standard curve, a negative control without DNA and the cDNA pools to be quantified , All samples were analyzed in triplicate. For a 25 μl reaction mixture, 1 μl cDNA, 2.5 μl lOx SybrGreen buffer (Perkin Elmer), 3.5 μl MgCl2 (25 mM, Perkin Elmer)), 0.5 μl per primer (20 μM each, Perkin Elmer, Tubulin Forward (SEQ ID NO: 26); Tubulin reverse (SEQ ID NO: 27), 0.25 μl AmpErase uracil N-glycosylase "UNG" (1 U / μl, Perkin Elmer), and 0.25 μl AmpliTaq Gold ( 5 U / μl, Perkin Elmer) mixed, transferred to MicroAmp Optical Tubes (Perkin Elmer) and sealed with MicroAmp Optical Caps The PCR was carried out as follows: a cycle at 2 minutes at 50 ° C. for the UNG reaction, a cycle at 10 minutes 95 ° C to activate the AmpliTaq, 40 cycles with 15 seconds each 95 ° and 1 minute 60 ° C. The samples were then kept at 25 ° C. The data are evaluated with the program "Sequence Detection System 1.5bl "(PE Applied Biosystems), whereby in principle the fluorescence signals of the cDNA samples to be quantified were compared with the signals of the control plasmid dilutions of known concentration.

D) B345-TaqMan PCR

The quantitative TaqMan PCR analysis of B345 was carried out as described for the "household genes". However, there were B345 specific primers (SEQ ID NO: 28 and SEQ ID NO: 29) (200 ng / μl) and a B345 specific probe (SEQ ID NO: 30, 20 μM), which were at the 5 'end with tetrachlorofluorescein and is labeled at the 3 'end with carboxymethylrhodamine. The PCR product from B345 with the primers SEQ ID NO: 28 and SEQ ID NO: 29 with a known copy number was used as standard.

Fig. 3 illustrates the TaqMan expression analysis (Fig. 3A: β-actin; Fig. 3B: tubulin). It was shown that B345 is expressed higher in colon cancer tissue than in normal tissue (see Table 2a). Now, however, both the normal tissue and the tumor tissue represent a very heterogeneous mixture of different cell types. Furthermore, the proportion of tumor cells in the tumor tissue varies greatly from about 30 to 80%. In order to minimize this biological heterogeneity, the epithelial cells of the large intestine, which are the original cells of the adenocarcinoma, and the cancer cells or cancer areas were specifically prepared by laser microdissection. Tissue sections of 10 μm thickness were made with the Kyromikrotome from Leica, Jung CM1800 and onto one with polyethylene coated slides applied (Böhm et al., 1997). The sections, dried at room temperature for about 30 minutes, were incubated with Mayer's hematoxylin (SIGMA DIAGNOSTICS) and then washed under running water for five minutes to remove non-specifically bound dye. After drying for five minutes at 37 ° C, the laser microdissection was carried out. The laser microscope from PALM (PALM GmbH, Bernried, Germany) was used for this and about 2000 to 5000 cells were prepared. The cDNA obtained by reverse transcription was also analyzed here by real time PCR. The result shows that the B345 expression in colon carcinoma cell lines and in patient material is many times higher than that of the normal colon tissue. The expression level of GAPDH was determined for normalization (see FIG. 4 and Table 2B).

Example 6

a) Cloning of the cDNA of B345

Searching databases for sequences of gene fragments (ESTs, expressed sequence tags) which can be used for the "in silico" cloning of B345 resulted in an overlapping EST contig of approximately 1500 bp. The polyA region at one end gave Reference to the orientation of the DNA section with respect to 5 ^Λ - 3 ^, orientation, which is essential in the design of new primers for the amplification of B345-specific cDNA fragments.

First, the potential 3 ^Λ end described by the database ^{analysis was} verified by experimental approaches. RNA from the lung carcinoma cell line Calu 6 (AACC No.HTB56) was reverse transcribed using the primer (SEQ ID NO: 9) and the resulting single-stranded cDNA was PCR by means of the gene-specific primer SEQ ID NO: 5 and the adapter primer SEQ ID NO: 10 amplified.

For a 25 ul PCR approach, 1 ul of the cDNA pool with 2.5 ul lOxTaq buffer (Promega), 1.5 ul MgCl ₂ (25 mM, Promega), 0.5 ul dNTPs (10 mM each, Boehringer Mannheim) , 1 μl primer mixture (20 μM each), 0.15 μl Taq polymerase (Promega) mixed in water. The PCR was carried out as follows: 1 × 94 ° C. for 3 minutes; 30x 94 ° C 30 seconds,

55 ° C 30 seconds, 72 ° C 1 minute; keep at 4 ° C. The PCR was analyzed on a 1.2% agarose gel.

The two primers were then used to sequence the purified PCR product. The determined Sequences showed high homology with the "in silico" cloned DNA section (including the PolyA tract).

Since the cloning of 5 'end sequences is usually a very complex process, various methods were used in the following to solve the problem.

Calu 6 was also used as the starting cell line. After the reverse transcription of the RNA with the primer SEQ ID NO: 9 and the second-strand synthesis, a linker consisting of the two oligos SEQ ID NO: 11 and SEQ ID NO: 12 was ligated to the double-strand cDNA (Abe et al., 1992) , The resulting LoneLinker cDNA library was then linearly amplified with the gene-specific primer SEQ ID NO: 6 over 35 cycles. An aliquot of the B345-enriched cDNA could then be further amplified with the primers SEQ ID NO: 13 and LLEcoRIA SEQ ID NO: 11. After gel electrophoresis of an aliquot and Southern analysis with the gene-specific oligo SEQ ID NO: 14, a 5 kb band was localized. Subsequently, this fragment was sequentially sequenced and aligned with the sequence of the EST account.

In order to check the resulting sequence from the LLcDNA cloning, two fragments were analyzed by means of PCR (SEQ ID NO: 15 and SEQ ID NO: 16 or SEQ ID NO: 6 and SEQ ID

NO: 17) amplified and used for the screening of lambda gtlO cDNA phage libraries. Positive plaques were isolated and amplified using the gtlO-specific primers (SEQ ID NO: 18 and SEQ ID NO: 19) PCR. Subsequent sequencing and that Alignment with the sequences led to the assumption that this is a differential splice product. The splice donor, acceptor and lariat sequence could subsequently be found. Differential splice products were searched for in various cell lines by means of PCR using a suitable primer combination, only one product being found in all screened cell lines and leading to the gene structure shown in FIG. 5. The cDNA mentioned has an open reading frame (ORF) which codes for a potential protein with a length of 749 amino acids. The translation initiation site at position 215 corresponds to approximately 75% of a Kozak consensus sequence. The results obtained in this experiment prompted that in a further experiment (Example 6b)

To determine exactly the transcription initiation site by primer extension to ensure that the 5 'end determined here is the actual 5' end of B345. The amino acid sequence of B345 derived from the cDNA (SEQ ID NO: 1) obtained in this cloning experiment is shown in SEQ ID NO: 2.

b) second cloning attempt; Determination of the 5 ^Λ and promoter region of B345

With the help of promoter Finder DNA Walking Kit (Clontech) ^"and subsequent primer extension reaction, the 5 ^Λ region and the promoter region as well as the accurate transcription initiation site were determined. The 5 'region was performed using a genomic DNA library from Clontech with B345 specific primers ( SEQ ID NO: 38 or nested SEQ ID NO: 39) and adapter primer in the kit amplified. The primer extension reaction was carried out to determine the exact start of transcription. For this, the primer SEQ ID NO: 40 at the 5 "end was labeled using 10 U of the T4 polynucleotide kinase (Promega) and 3μl [γ- ³² P] ATP, (3000 Ci / mmol) according to standard protocols (Sa brook et al The labeled oligonucleotide was purified by precipitation and 10,000 cpm oligonucleotide in a total volume of 20 μl to 25 μg total RNA of the Colo 205 cell line (ATCC: CCL-222) were used for the primer extension reaction.

The RNA of the cell line was reverse transcribed with the radioactively labeled primer and applied to a 10% polyacryamide gel. To determine the exact band length, a PCR fragment of nt 1000 - nt 1362 with ³⁵ S-labeled nucleotides was sequenced and also applied. The fragment of 209 nucleotides resulting from the elongation of the reverse primer specifies the start of transcription exactly at position 201 B345 sequence obtained in Example 6a expanded in the 5 "region and a new start codon at position 283 determined. Through repeated sequencing also in the 3 "region, in comparison to the sequence shown in SEQ ID NO: 1, an additional base at position 2430 was found, which leads to a reading frame shift and thus shifts the stop codon from position 2729 to 2791 The experimentally obtained cDNA (SEQ ID NO: 3) has an open reading frame which codes for a potential protein with a length of 836 amino acids (SEQ ID NO: 4). Example 7

Bioinformatics analysis on the function of B345

The resulting primary amino acid sequence of B345 is shown in SEQ ID NO: 4. Analysis of the hydrophilicity plot of the amino acid sequence by the method of Kyte and Doolittle (1982) shows that the B345 protein has two characteristic hydrophobic domains (amino acids pos. 1 - 29 and 666 - 691), which represent a signal peptide and a helical transmembrane domain ( Fig. 6). This polarized structure indicates that B345 is an integral membrane protein. The transmembrane helix connects an approximately 666 amino acid long extracellular and a short (145 amino acid) intracellular part (see FIG. 7).

The extracellular domain also has clear indications for the existence of a CUB domain at positions 220-350 as well as indications for two possible further CUB domains in the region of amino acids 425-660. CUB domains are found in various proteins, most of which are regulated during embryonic development. In addition, CUB domains are sometimes found in EGF (epidermal growth factor) -like domains. A recent publication (Gerstein et al., 2000) demonstrated that proteins containing CUB domains are the most pronounced differentially regulated proteins in C. elegans. Since genes that have a key role in embryonic development also perform analogous functions in cancer, it can be assumed overexpression of B345 in cells causes a change in the properties of the substrate adhesion or the extracellular matrix. The protein also has 12 potential N-glycosylation sites found in the predicted extracellular domain, which is consistent with the predicted orientation of the protein.

A BLAST hit (E-value: 5.8 x 10 ^{~ 2} ) for the amino acid range from 235 to 282 of B345 identified a complement-activating component of the _RA-reactive factor (RARF) from mus musculus. The alignment is located within CUB Domain 1 of B345.

The CUB domains 2 and 3 (range 425-535 and 545-660) have marginal homologies to the human and fugu

Procollagen C-Proteinase Enhancer Protein (PCOLCE). These regions occur in the area of PCOLCE, which contains a CUB domain tandem repeat (E-values: 0.5 (human) and 2.7 (Fugu)). CUB domains sometimes occur in repeats.

The B345 protein presumably forms a ß-sheet secondary structure, since CUB domains are known to fold as a ß-sandwich.

The intracellular domain (range 691-836) has no significant homologies. However, the entire C-terminus was aligned with an EST (AW063026) of human ovarian cancer cells (82% identity over 124 amino acids). Example 8

Determination of the exact gene structure of B345

Bac clones were first searched in public databases (BLAST search) which contained the B345 gene. Bac clones Ac068625 and Ac010170 contained one

Much of the gene. With intron-spanning primers, splice acceptors and donor sequences in Colo 205 cDNA and genomic DNA were searched for as templates. The PCR protocol was carried out as follows: Ix 95 ° C for 2 minutes, 35x 95 ° C for 15 seconds, 68 ° C for 3 minutes and then held at 4 ° C. The PCR was analyzed on a 1.2% agarose gel and the lengths of the PCR products of the 2 templates were compared with the same primer combinations. The B345 was found to consist of 8 exons separated by 7 introns (Fig. 5).

The chromosomal location of the gene was determined using fluorescence in situ hybridization (FISH). The human, digoxigenin-labeled B345 probe was used together with the biotin-labeled probe from B47a2 (Knight et al., 1997), which is located on the sub-telomeric region of the chromosome arm 3p, with metaphase chromosomes from two "normal" individuals hybridizes (Lichter et al., 1988). The hybridized digoxygenin probe was detected using anti-sheep dig (Boehringer Mannheim FRG) and rabbit anti-sheep FITC-labeled antibodies. The sample labeled with biotin, on the other hand, was visualized with mouse anti-biotin and rabbit anti-mouse (TRITC) and then stained with DAPI. The FISH results show that a majority of the metaphases have unique signals at one or have two chromatids of chromosome 3 in the region p21-p23. The co-localization of the B47a2 (TRITC) probe on the same chromosomal arm served as confirmation of the position.

Tab. 1A

Tab. 1B

Tab. IC

Tab. 2A

Tab. 2 B

literature

Abe, K., Rapid isolation of desired sequences from lone linker PCR amplified cDNA mixture: application to identification and recovery of expressed sequences in cloned genomic DNA. Mamm. Genomes 2,252-259

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402 (1997).

Bateman, A., Birney, E., Durbin, R., Eddy, S.R., Howe, K.L. and Sonnhammer, E.L. The Pfam Protein Families Database. Nucleic Acids Res. 28, 263-266 (2000).

Bird, A.P. (1986). CpG-rich islands and the function of DNA methylation. Nature 321: 209-213.

Bork, P. et al. (1993). The CUB domain. A widespread module in developmentally regulated proteins. J. Mol. Biol. 231: 539-545

Boulianne, G.L., et al. , (1984) Nature 312: 643-646

Böhm et al., A.J. of Pathology 151: 63-67, 1997

Brüggemann, M. and Neuberger, M.S., (1996), Immunol. Today 17: 391-397

Cuff, JA, Clamp, ME, Siddiqui, AS, Finlay, M. and Barton, GJ Jpred: a consensus secondary structure prediction server. Bioinformatics 14, 892-893 (1998).

Diatchenko, L., Lau, YF, Campbell, AP, Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, ED, and Siebert, PD (1996) Proc. Natl. Acad. Be. U.S.A. 93, 6025-6030.

Fields S, Song 0 (1989) A novel genetic system to detect protein-protein interactions. Nature 20; 340 (6230): 245-6

Gaudi C, Kremer F, Angevin E, Scott V, Triebe1 F (1999), J Immunol 162: 1730-1738

Gerstein, M. and Jansen, R. (2000). The current excitement in bioinformatics-analysis of whole-genome expression data: how does it relate to protein structure and function. Curr. Opin. Struct. Biol. 10: 574-584.

Graziano, R.F., et al. , (1995), J. Immunol. 155: 4996-5002

Griffiths, A.D., et al. , (1994), EMBO J. 13: 3245-3260

Grosveld, F. and Kollias, G. Transgenic Animals, Academic Press (1992)

Hamburger, A.W. and Salmon, S.E. (1977). Primary bioassay of human tumor stem cells. Science. 197 (4302): 461-463.

Hesketh, R., (1995), The oncogene, Academic Press Higgins, DG, Thompson, JD and Gibson, TJ Using CLUSTAL for Multiple Sequence Alignments. Methods Enzymol. 266, 383-402 (1996).

Hogan KT, Eisinger DP, Cupp SBC, Lekstrom KJ, Deacon DD, Shabanowitz J, Hunt DF, Engelhard VH, Slingluff CL, Ross MM (1998), Cancer Res 58: 5144-5150

Hofmann, K., Buchner, P., Fälquet, L. and Bairoch, A. The PROSITE database, its Status in 1999. Nucleic Acids Res. 27, 215-219 (1999).

Hubank, M. and Schatz, D.G., (1994), Nucleic. Acids. Res. 22, 5640-5648.

Jakobovits, A., (1995), Curr. Opin. Biotechnol. 6: 561-566

Kasten, M.B., (1997), Genetic Instability and Tumorigenesis, Springer Verlag

Knight, S.J., Horsley, S.W., Regan, R., Lawrie, N.M., Mäher, E.J., Cardy, D.L., Flint, J., and Kearney, L. (1997). Development and clinical application of an innovative fluorescence in situ hybridization technique which detects submicroscopic rearrangements involving telomeres. Eur. J. Hum. Genet. 5: 1-8.

Koehler, G. and Milstein, C. (1975) Nature 265, 495-497

Kozak, M (1987), An analysis of 5 "noncoding sequences from 99 vertebrates messenger RNA's. Nuc.Ac.Res. Vol. 15: 8125-8147 Kruif, J., et al. , (1995), Proc. Natl. Acad. Be. USA 92: 3938-3942

Kyte, J and Doolittle, RF (1982) J. Mol. Biol. 157: 105-132

Liaw, L., Skinner, M.P., Raines, E.W., Ross, R.,

Cheresh, D.A., Schwartz, S.M., and Giachelli, C.M .. (1995). The adhesive and migration effects of osteopontin are mediated via distinctive cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro. J. Clin. Invest. 95 (2): 713-724.

Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and Ward, D.C. (1988). Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80: 224-

234. Mandruzzato S, Brasseur F, Andry G, Boon T, van der Bruggen P (1997), J Exp Med 186: 785-793

McGuinnes, B.T., et al. , (1996), Nature Biotechnol. 14, 1149

Neuberger, M.S., et al. , (1984) Nature 312: 604-608

Pearson, W.R. and Lipman, D.J. Improved tools for biological sequence comparison. Proc. Natl. Acad. Be. USA 85: 2444-2448 (1988).

Pusztal et al. , 1996, cell proliferation in cancer, Oxford medical publications Rauscher, FJ et al. , (1997), Chromosomal translocations and oncogenic transcription factors, Springer Verlag

Riechmann, L., et al. , (1988) Nature 332: 323-327

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989).

"Molecular Cloning: A laboratory Manual" 2 ^nd ed, Cold Spring Laboratory Press, Cold Spring Harbor, NY..

Schultz, J., Copley, R.R., Doerks, T., Ponting, C.P. and Bork, P. SMART: A Web-based tool for the study of genetically mobile domains. Nucleic Acids Res. 28, 231-234 (2000).

Toes, R.E., Hoeben, R.C., Van der Voort, E., Ressing, M.E., Van-der-Eb, A.J. Melief, C.J.M., and Offringa, R. (1997), Proc. Natl. Acad. Be. U.S.A. 94 (26): 14660-14665

Velculescu, VE, Zhang, L, Vogelstein, B, and Kinzler, KW (1995), Science 270: 484-487

Winter, G., et al. , (1994), Annu. Rev. Immunol. 12, 433-455

Woelfel, T, Schneider, J, Zum Buschenfelde, Meyer, KH, Rammenee, HG, Rotzschke, 0, and Falk, K (1994), Int. J. Cancer 57: 413-418

Wu, T.C., Guarnieri, F.G., Staveley-0 'Carroll, K.F., Viscidi, R.P., Levitsky, H.I., Hedrick, L., Cho, K.R., August, J.T., and Pardoll, D.M. (1995) Proc. Natl. Acad. Be. U.S.A. 92 (25): 11671-11675.

Claims

claims

1. Tumor-associated antigen of the designation B345, characterized in that it has the amino acid sequence defined in SEQ ID NO: 4 or contains it as a partial sequence, or a fragment thereof.

2. Isolated DNA molecule, coding for the tumor-associated antigen defined in claim 1 or for fragments thereof.

3. DNA molecule according to claim 2, characterized in that it is a polynucleotide with the sequence shown in SEQ ID NO: 3 or contains this sequence or that it is or contains a polynucleotide that with a polynucleotide of SEQ ID NO : 3 sequence hybridized under stringent conditions, or a fragment thereof.

4. Recombinant DNA molecule containing a DNA molecule according to claim 2 or 3.

5. Pharmaceutical composition for the

Immunotherapy for cancer, containing as active component the tumor-associated antigen of the name B345 or one or more fragments thereof.

6. Pharmaceutical composition for the

Immunotherapy for cancer, containing as active component a DNA molecule according to one of claims 2 to 4.

7. Antibodies against the polypeptide defined in claim 1.

8. Antibody according to claim 7, characterized in that it is monoclonal.

9. Antibody according to claim 7 or 8 for the therapy and diagnosis of cancers associated with the expression of B345.