# Patent

## Title
Identification of surface-associated antigens for tumor diagnosis and therapy

## Abstract

An isolated truncated desmoglein 4 (DSG4) polypeptide splice variant of the invention is characterized by an amino acid sequence that lacks a region encoded before exon 9 or beyond exon 10 of the DSG4 gene having the polynucleotide sequence of SEQ ID NO: 75. Also disclosed is a method of diagnosing a cancer, or monitoring the course thereof, in a patient. The method comprises detecting in a tissue sample of a patient the expression of a tumor-associated antigen comprising the extracellular domain of a DSG4 polypeptide encoded by a DSG4 gene having the polynucleotide sequence of SEQ ID NO: 75, or a truncated DSG4 polypeptide splice variant characterized by an amino acid sequence that lacks a region encoded before exon 9 or beyond exon 10 of the DSG4 gene.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 11/596,106, filed on Jun. 26, 2007, which is the National Stage of PCT/EP05/005104, which was filed on May 11, 2005; each of which is incorporated herein by reference in its entirety.


BACKGROUND OF THE INVENTION
Despite interdisciplinary approaches and exhaustive use of classical therapeutic procedures, cancers are still among the leading causes of death. More recent therapeutic concepts aim at incorporating the patient's immune system into the overall therapeutic concept by using recombinant tumor vaccines and other specific measures such as antibody therapy. A prerequisite for the success of such a strategy is the recognition of tumor-specific or tumor-associated antigens or epitopes by the patient's immune system whose effector functions are to be interventionally enhanced. Tumor cells biologically differ substantially from their nonmalignant cells of origin. These differences are due to genetic alterations acquired during tumor development and result, inter alia, also in the formation of qualitatively or quantitatively altered molecular structures in the cancer cells. Tumor-associated structures of this kind which are recognized by the specific immune system of the tumor-harboring host are referred to as tumor-associated antigens.
The specific recognition of tumor-associated antigens involves cellular and humoral mechanisms which are two functionally interconnected units: CD4+ and CD8+ T lymphocytes recognize the processed antigens presented on the molecules of the MHC (major histocompatibility complex) classes II and I, respectively, while B lymphocytes produce circulating antibody molecules which bind directly to unprocessed antigens.
The potential clinical-therapeutical importance of tumor-associated antigens results from the fact that the recognition of antigens on neoplastic cells by the immune system leads to the initiation of cytotoxic effector mechanisms and, in the presence of T helper cells, can cause elimination of the cancer cells (Pardoll, Nat. Med. 4:525-31, 1998). Accordingly, a central aim of tumor immunology is to molecularly define these structures. The molecular nature of these antigens has been enigmatic for a long time. Only after development of appropriate cloning techniques has it been possible to screen cDNA expression libraries of tumors systematically for tumor-associated antigens by analyzing the target structures of cytotoxic T lymphocytes (CTL) (van der Bruggen et al., Science 254:1643-7, 1991) or by using circulating autoantibodies (Sahin et al., Curr. Opin. Immunol. 9:709-16, 1997) as probes. To this end, cDNA expression libraries were prepared from fresh tumor tissue and recombinantly expressed as proteins in suitable systems. Immunoeffectors isolated from patients, namely CTL clones with tumor-specific lysis patterns, or circulating autoantibodies were utilized for cloning the respective antigens.
In recent years a multiplicity of antigens have been defined in various neoplasias by these approaches. The class of cancer/testis antigens (CTA) is of great interest here. CTA and genes encoding them (cancer/testis genes or CTG) are defined by their characteristic expression pattern [Tureci et al, Mol Med Today. 3:342-9, 1997]. They are not found in normal tissues, except testis and germ cells, but are expressed in a number of human malignomas, not tumor type-specifically but with different frequency in tumor entities of very different origins (Chen & Old, Cancer J. Sci. Am. 5:16-7, 1999). Antibodies against CTA are not found in healthy individuals but in tumor patients. This class of antigens, in particular owing to its tissue distribution, is particularly valuable for immunotherapeutic projects and is tested in current clinical patient studies (Marchand et al., Int. J. Cancer 80:219-30, 1999; Knuth et al., Cancer Chemother. Pharmacol. 46:p 46-51, 2000).
However, the probes utilized for antigen identification in the classical methods illustrated above are immunoeffectors (circulating autoantibodies or CTL clones) from patients usually having already advanced cancer. A number of data indicate that tumors can lead, for example, to tolerization and anergization of T cells and that, during the course of the disease, especially those specificities which could cause effective immune recognition are lost from the immunoeffector repertoire. Current patient studies have not yet produced any solid evidence of a real action of the previously found and utilized tumor-associated antigens. Accordingly, it cannot be ruled out that proteins evoking spontaneous immune responses are the wrong target structures.
SUMMARY OF THE INVENTION
It was the object of the present invention to provide target structures for a diagnosis and therapy of cancers.
According to the invention, this object is achieved by the subject matter of the claims.
According to the invention, a strategy for identifying and providing antigens expressed in association with a tumor and the nucleic acids coding therefor was pursued. This strategy is based on the evaluation of human protein and nucleic acid data bases with respect to potential cancer-specific antigens which are accessible on the cell surface. The definition of the filter criteria which are necessary for this together with a high throughput methodology for analysing all proteins, if possible, form the central part of the invention. Data mining first produces a list which is as complete as possible of all known genes which according to the basic principle “gene to mRNA to protein” are examined for the presence of one or more transmembrane domains. This is followed by a homology search, a classification of the hits in tissue specific groups (among others tumor tissue) and an inspection of the real existence of the mRNA. Finally, the proteins which are identified in this manner are evaluated for their aberrant activation in tumors, e.g. by expression analyses and protein chemical procedures.
Data mining is a known method of identifying tumor-associated genes. In the conventional strategies, however, transcriptoms of normal tissue libraries are usually subtracted electronically from tumor tissue libraries, with the assumption that the remaining genes are tumor-specific (Schmitt et al., Nucleic Acids Res. 27:4251-60, 1999; Vasmatzis et al., Proc. Natl. Acad. Sci. USA. 95:300-4, 1998; Scheurle et al., Cancer Res. 60:4037-43, 2000).
The concept of the invention, however, is based on utilizing data mining for electronically extracting all genes coding for cancer specific antigens which are accessible on the cell surfaces and then evaluating said genes for ectopic expression in tumors.
The invention thus relates in one aspect to a strategy for identifying genes differentially expressed in tumors. Said strategy combines data mining of public sequence libraries (“in silico”) with subsequent laboratory-experimental (“wet bench”) studies.
According to the invention, a combined strategy based on different bioinformatic scripts enabled new genes coding for cancer specific antigens which are accessible on the cell surfaces to be identified. According to the invention, these tumor-associated genes and the genetic products encoded thereby were identified and provided independently of an immunogenic action.
The tumor-associated antigens identified according to the invention have an amino acid sequence encoded by a nucleic acid which is selected from the group consisting of (a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 69, 71, 73, 75, 79, 80, 85, 87, 102, 104, 106, 108, 110, 112, a part or derivative thereof, (b) a nucleic acid which hybridizes with the nucleic acid of (a) under stringent conditions, (c) a nucleic acid which is degenerate with respect to the nucleic acid of (a) or (b), and (d) a nucleic acid which is complementary to the nucleic acid of (a), (b) or (c). In a preferred embodiment, a tumor-associated antigen identified according to the invention has an amino acid sequence encoded by a nucleic acid which is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 69, 71, 73, 75, 79, 80, 85, 87, 102, 104, 106, 108, 110, 112. In a further preferred embodiment, a tumor-associated antigen identified according to the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 61 to 68, 70, 72, 74, 76, 81, 82, 86, 88, 96 to 101, 103, 105, 107, 109, 111, 113, a part or derivative thereof.
The present invention generally relates to the use of tumor-associated antigens identified according to the invention or of parts thereof, of nucleic acids coding therefor or of nucleic acids directed against said coding nucleic acids or of antibodies directed against the tumor-associated antigens identified according to the invention or parts thereof for therapy and diagnosis. This utilization may relate to individual but also to combinations of two or more of these antigens, functional fragments, nucleic acids, antibodies, etc., in one embodiment also in combination with other tumor-associated genes and antigens for diagnosis, therapy and progress control.
The property of the tumor-associated antigens identified according to the invention that they are localized on or at the cell surface qualifies them as suitable targets or means for therapy and diagnosis. Especially suitable for this is a part of the tumor-associated antigens identified according to the invention which corresponds to the non-transmembrane portion, in particular the extracellular portion of the antigens, or is comprised thereof. Therefore, according to the invention, a part of the tumor-associated antigens identified according to the invention which corresponds to the non-transmembrane portion of the antigens or is comprised thereof, or a corresponding part of the nucleic acids coding for the antigens identified according to the invention is preferred for therapy or diagnosis. Similarly, the use of antibodies is preferred which are directed against a part of the tumor-associated antigens identified according to the invention which corresponds to the non-transmembrane portion of the antigens or is comprised thereof.
Preferred diseases for a therapy and/or diagnosis are those in which one or more of the tumor-associated antigens identified according to the invention are selectively expressed or abnormally expressed.
The invention also relates to nucleic acids and genetic products which are expressed in association with a tumor cell and which are produced by altered splicing (splice variants) of nucleic acids of the tumor-associated antigens identified according to the invention or by altered translation with utilization of alternative open reading frames. The splice variants of the invention can be used according to the invention as targets for diagnosis and therapy of tumor diseases.
Very different mechanisms may cause splice variants to be produced, for example

    
    
        utilization of variable transcription initiation sites
        utilization of additional exons
        complete or incomplete splicing out of single or two or more exons,
        splice regulator sequences altered via mutation (deletion or generation of new donor/acceptor sequences),
        incomplete elimination of intron sequences.
    
    


Altered splicing of a gene results in an altered transcript sequence (splice variant). Translation of a splice variant in the region of its altered sequence results in an altered protein which may be distinctly different in the structure and function from the original protein. Tumor-associated splice variants may produce tumor-associated transcripts and tumor-associated proteins/antigens. These may be utilized as molecular markers both for detecting tumor cells and for therapeutic targeting of tumors. Detection of tumor cells, for example in blood, serum, bone marrow, sputum, bronchial lavage, bodily secretions and tissue biopsies, may be carried out according to the invention, for example, after extraction of nucleic acids by PCR amplification with splice variant-specific oligonucleotides. According to the invention, all sequence-dependent detection systems are suitable for detection. These are, apart from PCR, for example gene chip/microarray systems, Northern blot, RNAse protection assays (RDA) and others. All detection systems have in common that detection is based on a specific hybridization with at least one splice variant-specific nucleic acid sequence. However, tumor cells may also be detected according to the invention by antibodies which recognize a specific epitope encoded by the splice variant. Said antibodies may be prepared by using for immunization peptides which are specific for said splice variant. Suitable for immunization are particularly the amino acids whose epitopes are distinctly different from the variant(s) of the genetic product, which is (are) preferably produced in healthy cells. Detection of the tumor cells with antibodies may be carried out here on a sample isolated from the patient or as imaging with intravenously administered antibodies.
In addition to diagnostic usability, splice variants having new or altered epitopes are attractive targets for immunotherapy. The epitopes of the invention may be utilized for targeting therapeutically active monoclonal antibodies or T lymphocytes. In passive immunotherapy, antibodies or T lymphocytes which recognize splice variant-specific epitopes are adoptively transferred here. As in the case of other antigens, antibodies may be generated also by using standard technologies (immunization of animals, panning strategies for isolation of recombinant antibodies) with utilization of polypeptides which include these epitopes. Alternatively, it is possible to utilize for immunization nucleic acids coding for oligo- or polypeptides which contain said epitopes. Various techniques for in vitro or in vivo generation of epitope-specific T lymphocytes are known and have been described in detail (for example Kessler J H, et al. 2001, Sahin et al., 1997) and are likewise based on utilizing oligo- or polypeptides which contain the splice variant-specific epitopes or nucleic acids coding for said oligo- or polypeptides. Oligo- or polypeptides which contain the splice variant-specific epitopes or nucleic acids coding for said polypeptides may also be used for utilization as pharmaceutically active substances in active immunotherapy (vaccination, vaccine therapy).
In a further aspect, the invention also relates to posttranslationally modified protein domains such as glycosylations or myristoylations. This kind of modifications can result in a differential recognition pattern of an antigen, e.g. by an antibody, and recognize different conditions possibly associated with a disease. In particular by using antibodies, this differentiation of an antigen can be utilized diagnostically as well as therapeutically. It has been published for tumor cells that the tumor-associated cellular degeneration can result in altered posttranslational modifications (Durand & Seta. 2000. Clin Chem 46: 795-805; Granovsky et al. 2000. Nat Med 6: 306-312). In particular, glycosylation patterns are strongly altered on tumor cells. These special epitopes according to the invention can discriminate tumor cells from non-carcinogenic cells diagnostically. If an epitope which can be modified posttranslationally is glycosylated in normal non-degenerated cells and is deglycosylated in tumor cells, this situation makes the development of a tumor specific therapeutic antibody within the scope of the invention possible.
In one aspect, the invention relates to a pharmaceutical composition comprising an agent which recognizes the tumor-associated antigen identified according to the invention and which is preferably selective for cells which have expression or abnormal expression of a tumor-associated antigen identified according to the invention. In particular embodiments, said agent may cause induction of cell death, reduction in cell growth, damage to the cell membrane or secretion of cytokines and preferably have a tumor-inhibiting activity. In one embodiment, the agent is an antisense nucleic acid which hybridizes selectively with the nucleic acid coding for the tumor-associated antigen. In a further embodiment, the agent is an antibody which binds selectively to the tumor-associated antigen, in particular a complement-activated antibody which binds selectively to the tumor-associated antigen. In a further embodiment, the agent comprises two or more agents which each selectively recognize different tumor-associated antigens, at least one of which is a tumor-associated antigen identified according to the invention. Recognition needs not be accompanied directly with inhibition of activity or expression of the antigen. In this aspect of the invention, the antigen selectively limited to tumors preferably serves as a label for recruiting effector mechanisms to this specific location. In a preferred embodiment, the agent is a cytotoxic T lymphocyte which recognizes the antigen on an HLA molecule and lyses the cell labeled in this way. In a further embodiment, the agent is an antibody which binds selectively to the tumor-associated antigen and thus recruits natural or artificial effector mechanisms to said cell. In a further embodiment, the agent is a T helper lymphocyte which enhances effector functions of other cells specifically recognizing said antigen.
In one aspect, the invention relates to a pharmaceutical composition comprising an agent which inhibits expression or activity of a tumor-associated antigen identified according to the invention. In a preferred embodiment, the agent is an antisense nucleic acid which hybridizes selectively with the nucleic acid coding for the tumor-associated antigen. In a further embodiment, the agent is an antibody which binds selectively to the tumor-associated antigen. In a further embodiment, the agent comprises two or more agents which each selectively inhibit expression or activity of different tumor-associated antigens, at least one of which is a tumor-associated antigen identified according to the invention.
The activity of a tumor-associated antigen identified according to the invention can be any activity of a protein or a peptide. Thus, the therapeutic and diagnostic methods according to the invention can also aim at inhibiting or reducing this activity or testing this activity.
The invention furthermore relates to a pharmaceutical composition which comprises an agent which, when administered, selectively increases the amount of complexes between an HLA molecule and a peptide epitope from the tumor-associated antigen identified according to the invention. In one embodiment, the agent comprises one or more components selected from the group consisting of (i) the tumor-associated antigen or a part thereof, (ii) a nucleic acid which codes for said tumor-associated antigen or a part thereof, (iii) a host cell which expresses said tumor-associated antigen or a part thereof, and (iv) isolated complexes between peptide epitopes from said tumor-associated antigen and an MHC molecule. In one embodiment, the agent comprises two or more agents which each selectively increase the amount of complexes between MHC molecules and peptide epitopes of different tumor-associated antigens, at least one of which is a tumor-associated antigen identified according to the invention.
The invention furthermore relates to a pharmaceutical composition which comprises one or more components selected from the group consisting of (i) a tumor-associated antigen identified according to the invention or a part thereof, (ii) a nucleic acid which codes for a tumor-associated antigen identified according to the invention or for a part thereof, (iii) an antibody which binds to a tumor-associated antigen identified according to the invention or to a part thereof, (iv) an antisense nucleic acid which hybridizes specifically with a nucleic acid coding for a tumor-associated antigen identified according to the invention, (v) a host cell which expresses a tumor-associated antigen identified according to the invention or a part thereof, and (vi) isolated complexes between a tumor-associated antigen identified according to the invention or a part thereof and an HLA molecule.
A nucleic acid coding for a tumor-associated antigen identified according to the invention or for a part thereof may be present in the pharmaceutical composition in an expression vector and functionally linked to a promoter.
A host cell present in a pharmaceutical composition of the invention may secrete the tumor-associated antigen or the part thereof, express it on the surface or may additionally express an HLA molecule which binds to said tumor-associated antigen or said part thereof. In one embodiment, the host cell expresses the HLA molecule endogenously. In a further embodiment, the host cell expresses the HLA molecule and/or the tumor-associated antigen or the part thereof in a recombinant manner. The host cell is preferably nonproliferative. In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte or a macrophage.
An antibody present in a pharmaceutical composition of the invention may be a monoclonal antibody. In further embodiments, the antibody is a chimeric or humanized antibody, a fragment of a natural antibody or a synthetic antibody, all of which may be produced by combinatory techniques. The antibody may be coupled to a therapeutically or diagnostically useful agent.
An antisense nucleic acid present in a pharmaceutical composition of the invention may comprise a sequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguous nucleotides of the nucleic acid coding for the tumor-associated antigen identified according to the invention.
In further embodiments, a tumor-associated antigen, provided by a pharmaceutical composition of the invention either directly or via expression of a nucleic acid, or a part thereof binds to MHC molecules on the surface of cells, said binding preferably causing a cytolytic response and/or inducing cytokine release.
A pharmaceutical composition of the invention may comprise a pharmaceutically compatible carrier and/or an adjuvant. The adjuvant may be selected from saponin, GM-CSF, CpG oligonucleotides, RNA, a cytokine or a chemokine. A pharmaceutical composition of the invention is preferably used for the treatment of a disease characterized by selective expression or abnormal expression of a tumor-associated antigen. In a preferred embodiment, the disease is cancer.
The invention furthermore relates to methods of treating or diagnosing a disease characterized by expression or abnormal expression of one of more tumor-associated antigens. In one embodiment, the treatment comprises administering a pharmaceutical composition of the invention.
In one aspect, the invention relates to a method of diagnosing a disease characterized by expression or abnormal expression of a tumor-associated antigen identified according to the invention. The method comprises (i) detection of a nucleic acid which codes for the tumor-associated antigen or of a part thereof and/or (ii) detection of the tumor-associated antigen or of a part thereof, and/or (iii) detection of an antibody to the tumor-associated antigen or to a part thereof and/or (iv) detection of cytotoxic or T helper lymphocytes which are specific for the tumor-associated antigen or for a part thereof in a biological sample isolated from a patient. In particular embodiments, detection comprises (i) contacting the biological sample with an agent which binds specifically to the nucleic acid coding for the tumor-associated antigen or to the part thereof, to said tumor-associated antigen or said part thereof, to the antibody or to cytotoxic or T helper lymphocytes specific for the tumor-associated antigen or parts thereof, and (ii) detecting the formation of a complex between the agent and the nucleic acid or the part thereof, the tumor-associated antigen or the part thereof, the antibody or the cytotoxic or T helper lymphocytes. In one embodiment, the disease is characterized by expression or abnormal expression of two or more different tumor-associated antigens and detection comprises detection of two or more nucleic acids coding for said two or more different tumor-associated antigens or of parts thereof, detection of two or more different tumor-associated antigens or of parts thereof, detection of two or more antibodies binding to said two or more different tumor-associated antigens or to parts thereof or detection of two or more cytotoxic or T helper lymphocytes specific for said two or more different tumor-associated antigens. In a further embodiment, the biological sample isolated from the patient is compared to a comparable normal biological sample.
The methods of diagnosing according to the invention can concern also the use of the tumor-associated antigens identified according to the invention as prognostic markers, in order to predict metastasis, e.g. through testing the migration behavior of cells, and therefore a worsened course of the disease, whereby among other things planning of a more aggressive therapy is made possible.
In a further aspect, the invention relates to a method for determining regression, course or onset of a disease characterized by expression or abnormal expression of a tumor-associated antigen identified according to the invention, which method comprises monitoring a sample from a patient who has said disease or is suspected of falling ill with said disease, with respect to one or more parameters selected from the group consisting of (i) the amount of nucleic acid which codes for the tumor-associated antigen or of a part thereof, (ii) the amount of the tumor-associated antigen or a part thereof, (iii) the amount of antibodies which bind to the tumor-associated antigen or to a part thereof, and (iv) the amount of cytolytic T cells or T helper cells which are specific for a complex between the tumor-associated antigen or a part thereof and an MHC molecule. The method preferably comprises determining the parameter(s) in a first sample at a first point in time and in a further sample at a second point in time and in which the course of the disease is determined by comparing the two samples. In particular embodiments, the disease is characterized by expression or abnormal expression of two or more different tumor-associated antigens and monitoring comprises monitoring (i) the amount of two or more nucleic acids which code for said two or more different tumor-associated antigens or of parts thereof, and/or (ii) the amount of said two or more different tumor-associated antigens or of parts thereof, and/or (iii) the amount of two or more antibodies which bind to said two or more different tumor-associated antigens or to parts thereof, and/or (iv) the amount of two or more cytolytic T cells or of T helper cells which are specific for complexes between said two or more different tumor-associated antigens or of parts thereof and MHC molecules.
According to the invention, detection of a nucleic acid or of a part thereof or monitoring the amount of a nucleic acid or of a part thereof may be carried out using a polynucleotide probe which hybridizes specifically to said nucleic acid or said part thereof or may be carried out by selective amplification of said nucleic acid or said part thereof. In one embodiment, the polynucleotide probe comprises a sequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguous nucleotides of said nucleic acid.
According to the invention, detection of a tumor-associated antigen or of a part thereof or monitoring the amount of a tumor-associated antigen or of a part thereof may be carried out using an antibody binding specifically to said tumor-associated antigen or said part thereof.
In certain embodiments, the tumor-associated antigen to be detected or the part thereof is present in a complex with an MHC molecule, in particular an HLA molecule.
According to the invention, detection of an antibody or monitoring the amount of antibodies may be carried out using a protein or peptide binding specifically to said antibody.
According to the invention, detection of cytolytic T cells or of T helper cells or monitoring the amount of cytolytic T cells or of T helper cells which are specific for complexes between an antigen or a part thereof and MHC molecules may be carried out using a cell presenting the complex between said antigen or said part thereof and an MHC molecule.
The polynucleotide probe, the antibody, the protein or peptide or the cell, which is used for detection or monitoring, is preferably labeled in a detectable manner. In particular embodiments, the detectable marker is a radioactive marker or an enzymic marker. T lymphocytes may additionally be detected by detecting their proliferation, their cytokine production, and their cytotoxic activity triggered by specific stimulation with the complex of MHC and tumor-associated antigen or parts thereof. T lymphocytes may also be detected via a recombinant MHC molecule or else a complex of two or more MHC molecules which are loaded with the particular immunogenic fragment of one or more of the tumor-associated antigens and by contacting the specific T cell receptor which can identify the specific T lymphocytes.
In a further aspect, the invention relates to a method of treating, diagnosing or monitoring a disease characterized by expression or abnormal expression of a tumor-associated antigen identified according to the invention, which method comprises administering an antibody which binds to said tumor-associated antigen or to a part thereof and which is coupled to a therapeutic or diagnostic agent. The antibody may be a monoclonal antibody. In further embodiments, the antibody is a chimeric or humanized antibody or a fragment of a natural antibody.
The invention also relates to a method of treating a patient having a disease characterized by expression or abnormal expression of a tumor-associated antigen identified according to the invention, which method comprises (i) removing a sample containing immunoreactive cells from said patient, (ii) contacting said sample with a host cell expressing said tumor-associated antigen or a part thereof, under conditions which favor production of cytolytic T cells against said tumor-associated antigen or a part thereof, and (iii) introducing the cytolytic T cells into the patient in an amount suitable for lysing cells expressing the tumor-associated antigen or a part thereof. The invention likewise relates to cloning the T cell receptor of cytolytic T cells against the tumor-associated antigen. Said receptor may be transferred to other T cells which thus receive the desired specificity and, as under (iii), may be introduced into the patient.
In one embodiment, the host cell endogenously expresses an HLA molecule. In a further embodiment, the host cell recombinantly expresses an HLA molecule and/or the tumor-associated antigen or the part thereof. The host cell is preferably nonproliferative. In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte or a macrophage.
In a further aspect, the invention relates to a method of treating a patient having a disease characterized by expression or abnormal expression of a tumor-associated antigen, which method comprises (i) identifying a nucleic acid which codes for a tumor-associated antigen identified according to the invention and which is expressed by cells associated with said disease, (ii) transfecting a host cell with said nucleic acid or a part thereof, (iii) culturing the transfected host cell for expression of said nucleic acid (this is not obligatory when a high rate of transfection is obtained), and (iv) introducing the host cells or an extract thereof into the patient in an amount suitable for increasing the immune response to the patient's cells associated with the disease. The method may further comprise identifying an MHC molecule presenting the tumor-associated antigen or a part thereof, with the host cell expressing the identified MHC molecule and presenting said tumor-associated antigen or a part thereof. The immune response may comprise a B cell response or a T cell response. Furthermore, a T cell response may comprise production of cytolytic T cells and/or T helper cells which are specific for the host cells presenting the tumor-associated antigen or a part thereof or specific for cells of the patient which express said tumor-associated antigen or a part thereof.
The invention also relates to a method of treating a disease characterized by expression or abnormal expression of a tumor-associated antigen identified according to the invention, which method comprises (i) identifying cells from the patient which express abnormal amounts of the tumor-associated antigen, (ii) isolating a sample of said cells, (iii) culturing said cells, and (iv) introducing said cells into the patient in an amount suitable for triggering an immune response to the cells.
Preferably, the host cells used according to the invention are nonproliferative or are rendered nonproliferative. A disease characterized by expression or abnormal expression of a tumor-associated antigen is in particular cancer.
The present invention furthermore relates to a nucleic acid selected from the group consisting of (a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 69, 71, 73, 79, 80, 85, 87, 102, 104, 106, 108, 110, 112, a part or derivative thereof, (b) a nucleic acid which hybridizes with the nucleic acid of (a) under stringent conditions, (c) a nucleic acid which is degenerate with respect to the nucleic acid of (a) or (b), and (d) a nucleic acid which is complementary to the nucleic acid of (a), (b) or (c). The invention furthermore relates to a nucleic acid, which codes for a protein or polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 61-68, 70, 72, 74, 81, 82, 86, 88, 96-101, 103, 105, 107, 109, 111, 113, a part or derivative thereof.
In a further aspect, the invention relates to a recombinant nucleic acid molecule, in particular DNA or RNA molecule, which comprises a nucleic acid of the invention.
The invention also relates to host cells which contain a nucleic acid of the invention or a recombinant nucleic acid molecule comprising a nucleic acid of the invention.
The host cell may also comprise a nucleic acid coding for a HLA molecule. In one embodiment, the host cell endogenously expresses the HLA molecule. In a further embodiment, the host cell recombinantly expresses the HLA molecule and/or the nucleic acid of the invention or a part thereof. Preferably, the host cell is nonproliferative. In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte or a macrophage.
In a further embodiment, the invention relates to oligonucleotides which hybridize with a nucleic acid identified according to the invention and which may be used as genetic probes or as “antisense” molecules. Nucleic acid molecules in the form of oligonucleotide primers or competent samples, which hybridize with a nucleic acid identified according to the invention or parts thereof, may be used for finding nucleic acids which are homologous to said nucleic acid identified according to the invention. PCR amplification, Southern and Northern hybridization may be employed for finding homologous nucleic acids. Hybridization may be carried out under low stringency, more preferably under medium stringency and most preferably under high stringency conditions. The term “stringent conditions” according to the invention refers to conditions which allow specific hybridization between polynucleotides.
In a further aspect, the invention relates to a protein or polypeptide which is encoded by a nucleic acid selected from the group consisting of (a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 69, 71, 73, 79, 80, 85, 87, 102, 104, 106, 108, 110, 112, a part or derivative thereof, (b) a nucleic acid which hybridizes with the nucleic acid of (a) under stringent conditions, (c) a nucleic acid which is degenerate with respect to the nucleic acid of (a) or (b), and (d) a nucleic acid which is complementary to the nucleic acid of (a), (b) or (c). In a preferred embodiment, the invention relates to a protein or polypeptide which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 61-68, 70, 72, 74, 81, 82, 86, 88, 96-101, 103, 105, 107, 109, 111, 113, a part or derivative thereof.
In a further aspect, the invention relates to an immunogenic fragment of a tumor-associated antigen identified according to the invention. Said fragment preferably binds to a human HLA receptor or to a human antibody. A fragment of the invention preferably comprises a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50, amino acids. In particular an immunogenic fragment according to the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 61-68, 81, 82, and 96-101, a part or derivative thereof.
In a further aspect, the invention relates to an agent which binds to a tumor-associated antigen identified according to the invention or to a part thereof. In a preferred embodiment, the agent is an antibody. In further embodiments, the antibody is a chimeric, a humanized antibody or an antibody produced by combinatory techniques or is a fragment of an antibody. Furthermore, the invention relates to an antibody which binds selectively to a complex of (i) a tumor-associated antigen identified according to the invention or a part thereof and (ii) an MHC molecule to which said tumor-associated antigen identified according to the invention or said part thereof binds, with said antibody not binding to (i) or (ii) alone. An antibody of the invention may be a monoclonal antibody. In further embodiments, the antibody is a chimeric or humanized antibody or a fragment of a natural antibody.
The invention furthermore relates to a conjugate between an agent of the invention which binds to a tumor-associated antigen identified according to the invention or to a part thereof or an antibody of the invention and a therapeutic or diagnostic agent. In one embodiment, the therapeutic or diagnostic agent is a toxin.
In a further aspect, the invention relates to a kit for detecting expression or abnormal expression of a tumor-associated antigen identified according to the invention, which kit comprises agents for detection (i) of the nucleic acid which codes for the tumor-associated antigen or of a part thereof, (ii) of the tumor-associated antigen or of a part thereof, (iii) of antibodies which bind to the tumor-associated antigen or to a part thereof, and/or (iv) of T cells which are specific for a complex between the tumor-associated antigen or a part thereof and an MHC molecule. In one embodiment, the agents for detection of the nucleic acid or the part thereof are nucleic acid molecules for selective amplification of said nucleic acid, which comprise, in particular a sequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguous nucleotides of said nucleic acid.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: PCR-analysis of the gene FLJ31461
A: Quantitative expression analysis of FLJ31461 in normal tissues (left) and in various tumors (pools consisting of 3-4 individual samples each, right) in a logarithmic representation of the relative expression (x-fold activation). In most tumors an at least 100-fold overexpression of FLJ31461 is observed in comparison to the level of expression in healthy tissues.
B: Gel image of a conventional RT-PCR-analysis of FLJ31461 in tumors of the breast, lungs and ear-, nose and throat with the appropriate normal tissues Nx; M: DNA-length marker.
C: Quantitative expression analysis in various normal tissues (left) and in breast tumors in a logarithmic representation of the relative expression (x-fold activation). In almost all breast tumors an at least 100-fold overexpression of FLJ31461 is observed in comparison to the level of expression in healthy tissues.
D: Summary of the FLJ31461-specific expression in various analysed tumors. Shown is the number of positively tested tumor samples relative to the total number of analysed tumor samples. While all investigated normal somatic tissues (3-10 tissues each, depending on tissue type) exhibit no expression of FLJ31461, the gene is expressed in many tumors with variable frequency.
FIG. 2: Protein localisation
Representation of the cellular localisation of the FLJ31461-protein. The figure shows the endogenous protein expression of the breast tumor cell-line MCF7.
FIG. 3: Immunohistochemical analysis
A: Normal tissue of testis (positive membrane localisation), colon and kidney (negative membrane localisation).
B: Detection of the FLJ31461-protein in a bronchial carcinoma, a cervical carcinoma as well as a lymphatic node metastasis of a breast tumor in an overview (left column) and in detail (right column).
C: Summary of the immunohistochemical analyses of the FLJ31461-protein. Shown is the number of positively tested tumor samples in relation to the total number of analysed tumor samples. While all investigated normal somatic tissues did not exhibit any expression of FLJ31461, the protein is detected in many of the tumors with varying frequency at the cell surface.
FIG. 4: PCR-analysis of DSG4-splice variants in normal tissues and tumors
A: The PCR on normal tissues and various tumors was carried out using DSG4-specific oligonucleotides in exons 8-12 and exons 10-12. The dominant expression of the transcript of exons 10-12 is recognisable in colon tumors, while the transcript of exons 8-12 is also clearly expressed in normal tissues. Ge: brain, Dd: duodenum, Pa: pancreas, Mi: spleen, Te: testis, He: heart, Ko: colon, LN: lymphatic node, TM: thymus, Pr: prostate, Ös: esophagus, Le: liver, PB: active PBMC, Lu: lung, Bl: bladder, Ma: stomach, Br: breast, Ut: uterus, Ov: ovary, Ni: kidney, Ha: skin, Mu: muscle.
B: Summary of the specific expression of the DSG4-exons 10-12 in various analysed tumors. Shown is the number of positively tested tumor samples relative to the total number. While almost all investigated normal somatic tissues did not exhibit any expression of DSG4, this gene-section is detectable in many of the tumors with varying frequency.
C: Quantitative expression analysis of the transcript section of the DSG4-exons 10-12 in normal tissues (left) and in tumors of the colon, stomach and the ear-nose-throat area in logarithmic representation of relative expression (x-fold activation). Most tumors exhibited an at least 50-fold over-expression of the DSG4-exons 10-12 in comparison to the expression levels in healthy tissues.
FIG. 5: Overview of the putative transcript variants of the DSG4-gene
FIG. 6: Protein localisation
A: Representation of the cellular localisation of the DSG4-protein using immunofluorescence on a DSG4-transfected cell.
B: FACS-analysis of DSG4-transfected cells with DSG4-specific antibodies (left figure) and of Mock-transfected cells with DSG4-specific antibodies (negative control, right figure). The specific, surface-specific staining is clearly visible.
FIG. 7: qPCR-analysis of DSG3-specific expression in normal tissues and in tumors.
A: Quantitative expression analysis of DSG3 in normal tissues (left side) and in various tumors (pools consisting of 3-4 individual samples each, right side) in logarithmic representation of the relative expression (x-fold activation). The distinct overexpression in esophageal tumors in comparison to most normal tissues is recognisable.
B: Quantitative expression analysis of DSG3 in various tumors of the cervix and lungs as well as in ear, nose, throat tumors in comparison to the expression in the respective normal tissues (n=3 (cervix); n=9 (lung)). Logarithmic representation.
C: Summary of the DSG3-specific expression in various analysed tumors. Shown is the number of positively tested tumor samples relative to the total number of analysed tumor samples. While all investigated normal somatic tissues (3-10 tissues each, depending on tissue type) do not show any expression of DSG3, the gene is expressed in many tumors with varying frequency.
FIG. 8: Immunohistochemical analysis
The figure shows in an overview (left) and in detail the homogenous DSG3-localisation in an ear, nose, throat tumor.
FIG. 9: qPCR-analysis of SLC6A3
A: Quantitative expression analysis of SLC6A3 in normal tissues (left) and in tumor samples (pools consisting of 3-4 individual samples each, right) in logarithmic representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of SLC6A3 in various kidney tumors in comparison to the expression in normal kidney (n=5). Logarithmic representation of the relative expression.
C: Conventional endpoint-RT-PCR-analysis of SLC6A3-specific transcripts (double determination) in kidney tumors and various normal kidney tissues. Image after gel-electrophoretic resolution of the SLC6A3-specific fragments.
D: Quantitative expression analysis of SLC6A3 in carcinomas of the breast, ovary, lung and prostate; Logarithmic representation of the relative expression (x-fold activation). “Tissue” N: normal tissue; “Tissue”: tumor tissue.
E: Conventional RT-PCR-analysis of SLC6A3 in tumors of the breast, ovary, lung and prostate after gel-electrophoretic separation in a double determination. M: DNA-length marker.
FIG. 10: qPCR-analysis of GRM8
A: Quantitative expression analysis of GRM8 in normal tissues (left) and tumor tissues (pools consisting of 3-4 individual samples each, right) in linear representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of GRM8 in various tumors of the kidney and uterus in comparison to the expression in the normal kidney and uterus, as well as relative expression in ear, nose, throat tumors, cervical tumors and melanomas. Logarithmic representation of the relative expression.
FIG. 11: qPCR-analysis of CDH17
A: Quantitative expression analysis of CDH17 in normal tissues (left) and in tumor tissues (pools consisting of 3-4 individual samples each, right) in linear representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of CDH17 in various tumors of the colon and stomach in comparison to the expression in the respective normal tissues. Logarithmic representation.
C: Quantitative expression analysis of CDH17 in various tumors of the esophagus and pancreas in comparison to the expression in the respective normal tissues. Logarithmic representation.
FIG. 12: qPCR-analysis of the ABC-transporter ABCC4
Quantitative expression analysis of ABCC4 in normal tissues (left) and tumors (pools consisting of 3-4 individual samples each, right) in linear representation of the relative expression (x-fold activation).
FIG. 13: qPCR-analysis of Villin1 (VIL1)
A: Quantitative expression analysis of VIL1 in normal tissues (left) and tumor tissues (pools consisting of 3-4 individual samples each, right) in linear representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of VIL1 in various tumors of the colon and stomach in comparison to the expression in the respective normal tissues. Logarithmic representation.
FIG. 14: qPCR-analysis of the hypothetical protein MGC34032
A: Quantitative expression analysis of MGC34032 in normal tissues (left) and various tumors (pools consisting of 3-4 individual samples each, right) in linear representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of MGC34032 in various tumors of the esophagus, pancreas and colon in comparison to the expression in the respective normal tissues. Logarithmic representation.
C: Quantitative expression analysis of MGC34032 in various tumors of the lung, ovary and kidney in comparison to the expression in the respective normal tissues. Logarithmic representation.
D: Summary of the MGC34032-specific expression in various analysed tumors. Shown is the number of positively tested tumor samples relative to the total number of the analysed tumor samples. While all investigated somatic normal tissues (3-10 tissues each, depending on tissue type) exhibit a significantly lower expression of MGC34032, the gene is overexpressed in many tumors with varying frequency.
FIG. 15: Immunohistochemical analysis
The figure shows 2 detailed views of the cellular localisation of the MGC34032-protein in human testis tissue.
FIG. 16: Expression analysis of enterokinase (PRSS7)
A: Quantitative expression analysis of PRSS7 in normal tissues (left) and various tumor tissues (pools consisting of 3-4 individual samples each, right) in linear representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of PRSS7 in various tumors of the stomach and esophagus in comparison to the expression in the respective normal tissues (stomach: n=7; esophagus: n=3).
For comparison the expression was measured in a normal duodenum (n=2). Logarithmic representation.
C: Quantitative expression analysis of PRSS7 in various pancreas and liver tumors in comparison to the expression in the respective normal tissues (n=4 for each). For comparison the expression in normal duodenum was measured (n=2). Logarithmic representation.
FIG. 17: Protein localisation
A: Representation of the cellular localisation of the PRSS7-protein on PRSS7-transfected cells.
B: Detection of the PRSS7-protein in overview (left) and in detail (right).
FIG. 18: qPCR-analysis of CLCA2
A: Quantitative expression analysis of CLCA2 in normal tissues (left) and various tumors (pools consisting of 3-4 individual samples each, right) in logarithmic representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of CLCA2 in various tumors of the lung, breast, cervix and uterus and in ear, nose and throat tumors in comparison to the expression in the respective normal tissues. Logarithmic representation.
C: Summary of the CLCA2-specific expression in various analysed tumors. Shown is the number of positively tested tumor samples relative to the number of total samples of analysed tumors. While all investigated normal somatic tissues exhibit a significantly lower expression of CLCA2, the gene is overexpressed in many tumors with varying frequency.
FIG. 19: Protein localisation
A: Representation of the localisation of the CLCA2-protein at the membrane of CLCA2-transfected cells.
B: The figure shows the immunohistochemical analysis at the CLCA2-protein.
FIG. 20: qPCR-analysis of TM4SF4
A: Quantitative expression analysis of TM4SF4 in normal tissues (left) and in various tumors (pools consisting of 3-4 individual samples each, right) in linear representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of TM4SF4 in various liver tumors in comparison to 4 different normal tissues of the liver (N0 to N3); linear representation.
C: Logarithmic representation of the relative expression of TM4SF4 in 12 different colon tumors in comparison to normal colon samples (NG: normal tissue; 6 different normal tissues were investigated).
FIG. 21: Protein analysis
A: The image shows an immunoblot with TM4SF4-specific antibodies in normal liver tissue and liver tumor tissue. Two putative glycosylation parameters are recognisable.
B: The figure shows the localisation of the TM4SF4-protein at the membrane of TM4SF4-transfected cells.
C: The immunohistochemical analysis was able to confirm the expression selectivity observed by PCR.
FIG. 22: Quantitative expression analysis of claudin19
A: Quantitative expression analysis of claudin19 in normal tissues (left) and in various tumors (pools consisting of 3-4 individual samples each, right) in logarithmic representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of claudin19 in various breast tumors and the respective normal breast tissues.
C: Conventional RT-PCR with analysis of claudin19 in various breast tumor samples as well as in a normal tissue; M: DNA-length marker.
D: Conventional RT-PCR-analysis of claudin19 in various normal tissues of the stomach and stomach tumors.
E: Conventional RT-PCR-analysis of claudin19 in various normal tissues of the liver and liver tumors; M: DNA-length marker.
FIG. 23: qRT-PCR-analysis of ALPPL2
A: Quantitative expression analysis of ALPPL2 in normal tissues (left) and in tumors (pools consisting of 3-4 individual samples each, right) in linear determination of the relative expression (x-fold activation).
B: Gel image of a conventional RT-PCR-analysis of ALPPL2 in various tumors of the colon and stomach as well as in the respective normal tissues after gel-electrophoretic separation; M: DNA-length marker.
FIG. 24: Quantitative RT-PCR-analysis of the G-protein-coupled receptor 64 (GPR64)
A: Quantitative expression analysis of GPR64 in normal tissues (left) and in tumors (pools consisting of 3-4 individual samples each, right) in linear representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of GPR64 in various tumors of the ovary and the respective normal ovary tissues.
C: Gel-image of a RT-PCR-analysis of GPR64 in various tumors of the ovary and in normal tissues; M: DNA-length marker.
FIG. 25: Quantitative RT-PCR-analysis of SLC12A1
A: Quantitative expression analysis of SLC12A1 in normal tissues (left) and in tumors (pools consisting of 3-4 individual samples, right) in linear representation of the relative expression (x-fold activation).
B: Quantitative expression analysis of SLC12A1 in 12 different kidney tumors in comparison to the expression in the normal kidney (n=3).
C: Quantitative expression analysis of SLC12A1 in tumors of the breast, ovary and prostate in comparison to the expression in the respective normal tissues (breast: n=9, ovary: n=8, prostate: n=3). Logarithmic representation.
D: Conventional RT-PCR-analysis of SLC12A1 in kidney tumors, various normal kidneys and various tumor types (breast, prostate, ovary) with the respective normal tissues.



DETAILED DESCRIPTION OF THE INVENTION
According to the invention, genes are described which are expressed in tumor cells selectively or aberrantly and which are tumor-associated antigens.
According to the invention, these genes or their derivatives are preferred target structures for therapeutic approaches. Conceptionally, said therapeutic approaches may aim at inhibiting the activity of the selectively expressed tumor-associated genetic product. This is useful, if said aberrant respective selective expression is functionally important in tumor pathogenicity and if its ligation is accompanied by selective damage of the corresponding cells. Other therapeutic concepts contemplate tumor-associated antigens as labels which recruit effector mechanisms having cell-damaging potential selectively to tumor cells. Here, the function of the target molecule itself and its role in tumor development are totally irrelevant.
“Derivative” of a nucleic acid means according to the invention that single or multiple nucleotide substitutions, deletions and/or additions are present in said nucleic acid. Furthermore, the term “derivative” also comprises chemical derivatization of a nucleic acid on a base, on a sugar or on a phosphate of a nucleotide. The term “derivative” also comprises nucleic acids which contain nucleotides and nucleotide analogs not occurring naturally.
According to the invention, a nucleic acid is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids comprise according to the invention genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. According to the invention, a nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule.
The nucleic acids described according to the invention have preferably been isolated. The term “isolated nucleic acid” means according to the invention that the nucleic acid was (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel-electrophoretic fractionation, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid which is available for manipulation by recombinant DNA techniques.
A nucleic acid is “complementary” to another nucleic acid if the two sequences are capable of hybridizing and forming a stable duplex with one another, with hybridization preferably being carried out under conditions which allow specific hybridization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y., 1989 or Current Protocols in Molecular Biology, F. M. Ausubel et al., Editors, John Wiley & Sons, Inc., New York and refer, for example, to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodium citrate, pH 7. After hybridization, the membrane to which the DNA has been transferred is washed, for example, in 2×SSC at room temperature and then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68° C.
According to the invention, complementary nucleic acids have at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98% or at least 99%, identical nucleotides.
Nucleic acids coding for tumor-associated antigens may, according to the invention, be present alone or in combination with other nucleic acids, in particular heterologous nucleic acids. In preferred embodiments, a nucleic acid is functionally linked to expression control sequences or regulatory sequences which may be homologous or heterologous with respect to said nucleic acid. A coding sequence and a regulatory sequence are “functionally” linked to one another, if they are covalently linked to one another in such a way that expression or transcription of said coding sequence is under the control or under the influence of said regulatory sequence. If the coding sequence is to be translated into a functional protein, then, with a regulatory sequence functionally linked to said coding sequence, induction of said regulatory sequence results in transcription of said coding sequence, without causing a frame shift in the coding sequence or said coding sequence not being capable of being translated into the desired protein or peptide.
The term “expression control sequence” or “regulatory sequence” comprises according to the invention promoters, enhancers and other control elements which regulate expression of a gene. In particular embodiments of the invention, the expression control sequences can be regulated. The exact structure of regulatory sequences may vary as a function of the species or cell type, but generally comprises 5′ untranscribed and 5′ untranslated sequences which are involved in initiation of transcription and translation, respectively, such as TATA box, capping sequence, CAAT sequence, and the like. More specifically, 5′ untranscribed regulatory sequences comprise a promoter region which includes a promoter sequence for transcriptional control of the functionally linked gene. Regulatory sequences may also comprise enhancer sequences or upstream activator sequences.
Thus, on the one hand, the tumor-associated antigens illustrated herein may be combined with any expression control sequences and promoters. On the other hand, however, the promoters of the tumor-associated genetic products illustrated herein may, according to the invention, be combined with any other genes. This allows the selective activity of these promoters to be utilized.
According to the invention, a nucleic acid may furthermore be present in combination with another nucleic acid which codes for a polypeptide controlling secretion of the protein or polypeptide encoded by said nucleic acid from a host cell. According to the invention, a nucleic acid may also be present in combination with another nucleic acid which codes for a polypeptide causing the encoded protein or polypeptide to be anchored on the cell membrane of the host cell or compartmentalized into particular organelles of said cell.
In a preferred embodiment, a recombinant DNA molecule is according to the invention a vector, where appropriate with a promoter, which controls expression of a nucleic acid, for example a nucleic acid coding for a tumor-associated antigen of the invention. The term “vector” is used here in its most general meaning and comprises any intermediary vehicle for a nucleic acid which enables said nucleic acid, for example, to be introduced into prokaryotic and/or eukaryotic cells and, where appropriate, to be integrated into a genome. Vectors of this kind are preferably replicated and/or expressed in the cells. An intermediary vehicle may be adapted, for example, to the use in electroporation, in bombardment with microprojectiles, in liposomal administration, in the transfer with the aid of agrobacteria or in insertion via DNA or RNA viruses. Vectors comprise plasmids, phagemids, bacteriophages or viral genomes.
The nucleic acids coding for a tumor-associated antigen identified according to the invention may be used for transfection of host cells. Nucleic acids here mean both recombinant DNA and RNA. Recombinant RNA may be prepared by in-vitro transcription of a DNA template. Furthermore, it may be modified by stabilizing sequences, capping and polyadenylation prior to application. According to the invention, the term “host cell” relates to any cell which can be transformed or transfected with an exogenous nucleic acid. The term “host cells” comprises according to the invention prokaryotic (e.g. E. coli) or eukaryotic cells (e.g. dendritic cells, B cells, CHO cells, COS cells, K562 cells, yeast cells and insect cells). Particular preference is given to mammalian cells such as cells from humans, mice, hamsters, pigs, goats, primates. The cells may be derived from a multiplicity of tissue types and comprise primary cells and cell lines. Specific examples comprise keratinocytes, peripheral blood leukocytes, stem cells of the bone marrow and embryonic stem cells. In further embodiments, the host cell is an antigen-presenting cell, in particular a dendritic cell, monocyte or a macrophage. A nucleic acid may be present in the host cell in the form of a single copy or of two or more copies and, in one embodiment, is expressed in the host cell.
According to the invention, the term “expression” is used in its most general meaning and comprises the production of RNA or of RNA and protein. It also comprises partial expression of nucleic acids. Furthermore, expression may be carried out transiently or stably. Preferred expression systems in mammalian cells comprise pcDNA3.1 and pRc/CMV (Invitrogen, Carlsbad, Calif.), which contain a selective marker such as a gene imparting resistance to G418 (and thus enabling stably transfected cell lines to be selected) and the enhancer-promoter sequences of cytomegalovirus (CMV).
In those cases of the invention in which an HLA molecule presents a tumor-associated antigen or a part thereof, an expression vector may also comprise a nucleic acid sequence coding for said HLA molecule. The nucleic acid sequence coding for the HLA molecule may be present on the same expression vector as the nucleic acid coding for the tumor-associated antigen or the part thereof, or both nucleic acids may be present on different expression vectors. In the latter case, the two expression vectors may be cotransfected into a cell. If a host cell expresses neither the tumor-associated antigen or the part thereof nor the HLA molecule, both nucleic acids coding therefor are transfected into the cell either on the same expression vector or on different expression vectors. If the cell already expresses the HLA molecule, only the nucleic acid sequence coding for the tumor-associated antigen or the part thereof can be transfected into the cell.
The invention also comprises kits for amplification of a nucleic acid coding for a tumor-associated antigen. Such kits comprise, for example, a pair of amplification primers which hybridize to the nucleic acid coding for the tumor-associated antigen. The primers preferably comprise a sequence of 6-50, in particular 10-30, 15-30 and 20-30 contiguous nucleotides of the nucleic acid and are nonoverlapping, in order to avoid the formation of primer dimers. One of the primers will hybridize to one strand of the nucleic acid coding for the tumor-associated antigen, and the other primer will hybridize to the complementary strand in an arrangement which allows amplification of the nucleic acid coding for the tumor-associated antigen.
“Antisense” molecules or “antisense” nucleic acids may be used for regulating, in particular reducing, expression of a nucleic acid. The term “antisense molecule” or “antisense nucleic acid” refers according to the invention to an oligonucleotide which is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide or modified oligodeoxyribonucleotide and which hybridizes under physiological conditions to DNA comprising a particular gene or to mRNA of said gene, thereby inhibiting transcription of said gene and/or translation of said mRNA. According to the invention, the “antisense molecule” also comprises a construct which contains a nucleic acid or a part thereof in reverse orientation with respect to its natural promoter. An antisense transcript of a nucleic acid or of a part thereof may form a duplex with the naturally occurring mRNA specifying the enzyme and thus prevent accumulation of or translation of the mRNA into the active enzyme. Another possibility is the use of ribozymes for inactivating a nucleic acid. Antisense oligonucleotides preferred according to the invention have a sequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguous nucleotides of the target nucleic acid and preferably are fully complementary to the target nucleic acid or to a part thereof.
In preferred embodiments, the antisense oligonucleotide hybridizes with an N-terminal or 5′ upstream site such as a translation initiation site, transcription initiation site or promoter site. In further embodiments, the antisense oligonucleotide hybridizes with a 3′ untranslated region or mRNA splicing site.
In one embodiment, an oligonucleotide of the invention consists of ribonucleotides, deoxyribonucleotides or a combination thereof, with the 5′ end of one nucleotide and the 3′ end of another nucleotide being linked to one another by a phosphodiester bond. These oligonucleotides may be synthesized in the conventional manner or produced recombinantly.
In preferred embodiments, an oligonucleotide of the invention is a “modified” oligonucleotide. Here, the oligonucleotide may be modified in very different ways, without impairing its ability to bind its target, in order to increase, for example, its stability or therapeutic efficacy. According to the invention, the term “modified oligonucleotide” means an oligonucleotide in which (i) at least two of its nucleotides are linked to one another by a synthetic internucleoside bond (i.e. an internucleoside bond which is not a phosphodiester bond) and/or (ii) a chemical group which is usually not found in nucleic acids is covalently linked to the oligonucleotide. Preferred synthetic internucleoside bonds are phosphorothioates, alkyl phosphonates, phosphorodithioates, phosphate esters, alkyl phosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
The term “modified oligonucleotide” also comprises oligonucleotides having a covalently modified base and/or sugar. “Modified oligonucleotides” comprise, for example, oligonucleotides with sugar residues which are covalently bound to low molecular weight organic groups other than a hydroxyl group at the 3′ position and a phosphate group at the 5′ position. Modified oligonucleotides may comprise, for example, a 2′-O-alkylated ribose residue or another sugar instead of ribose, such as arabinose.
Preferably, the proteins and polypeptides described according to the invention have been isolated. The terms “isolated protein” or “isolated polypeptide” mean that the protein or polypeptide has been separated from its natural environment. An isolated protein or polypeptide may be in an essentially purified state. The term “essentially purified” means that the protein or polypeptide is essentially free of other substances with which it is associated in nature or in vivo.
Such proteins and polypeptides may be used, for example, in producing antibodies and in an immunological or diagnostic assay or as therapeutics. Proteins and polypeptides described according to the invention may be isolated from biological samples such as tissue or cell homogenates and may also be expressed recombinantly in a multiplicity of pro- or eukaryotic expression systems.
For the purposes of the present invention, “derivatives” of a protein or polypeptide or of an amino acid sequence comprise amino acid insertion variants, amino acid deletion variants and/or amino acid substitution variants.
Amino acid insertion variants comprise amino- and/or carboxy-terminal fusions and also insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or polypeptides. Preference is given to replacing amino acids with other ones having similar properties such as hydrophobicity, hydrophilicity, electronegativity, volume of the side chain and the like (conservative substitution). Conservative substitutions, for example, relate to the exchange of one amino acid with another amino acid listed below in the same group as the amino acid to be substituted:
1. small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly)
2. negatively charged residues and their amides: Asn, Asp, Glu, Gln
3. positively charged residues: His, Arg, Lys
4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys)
5. large aromatic residues: Phe, Tyr, Trp.
Owing to their particular part in protein architecture, three residues are shown in brackets. Gly is the only residue without a side chain and thus imparts flexibility to the chain. Pro has an unusual geometry which greatly restricts the chain. Cys can form a disulfide bridge.
The amino acid variants described above may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis (Merrifield, 1964) and similar methods or by recombinant DNA manipulation. Techniques for introducing substitution mutations at predetermined sites into DNA which has a known or partially known sequence are well known and comprise M13 mutagenesis, for example. The manipulation of DNA sequences for preparing proteins having substitutions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example.
According to the invention, “derivatives” of proteins or polypeptides also comprise single or multiple substitutions, deletions and/or additions of any molecules associated with the enzyme, such as carbohydrates, lipids and/or proteins or polypeptides. The term “derivative” also extends to all functional chemical equivalents of said proteins or polypeptides.
According to the invention, a part or fragment of a tumor-associated antigen has a functional property of the polypeptide from which it has been derived. Such functional properties comprise the interaction with antibodies, the interaction with other polypeptides or proteins, the selective binding of nucleic acids and an enzymatic activity. A particular property is the ability to form a complex with HLA and, where appropriate, generate an immune response. This immune response may be based on stimulating cytotoxic or T helper cells. A part or fragment of a tumor-associated antigen of the invention preferably comprises a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50, consecutive amino acids of the tumor-associated antigen. A part or fragment of a tumor-associated antigen is preferably a part of the tumor-associated antigen which corresponds to the non-transmembrane portion, in particular the extracellular portion of the antigen or is comprised thereof.
A part or a fragment of a nucleic acid coding for a tumor-associated antigen relates according to the invention to the part of the nucleic acid, which codes at least for the tumor-associated antigen and/or for a part or a fragment of said tumor-associated antigen, as defined above. Preferably, a part or fragment of a nucleic acid coding for a tumor-associated antigen is that part which corresponds to the open reading frame, in particular as indicated in the sequence listing.
The isolation and identification of genes coding for tumor-associated antigens also make possible the diagnosis of a disease characterized by expression of one or more tumor-associated antigens. These methods comprise determining one or more nucleic acids which code for a tumor-associated antigen and/or determining the encoded tumor-associated antigens and/or peptides derived therefrom. The nucleic acids may be determined in the conventional manner, including by polymerase chain reaction or hybridization with a labeled probe. Tumor-associated antigens or peptides derived therefrom may be determined by screening patient antisera with respect to recognizing the antigen and/or the peptides. They may also be determined by screening T cells of the patient for specificities for the corresponding tumor-associated antigen.
The present invention also enables proteins binding to tumor-associated antigens described herein to be isolated, including antibodies and cellular binding partners of said tumor-associated antigens.
According to the invention, particular embodiments ought to involve providing “dominant negative” polypeptides derived from tumor-associated antigens. A dominant negative polypeptide is an inactive protein variant which, by way of interacting with the cellular machinery, displaces an active protein from its interaction with the cellular machinery or which competes with the active protein, thereby reducing the effect of said active protein. For example, a dominant negative receptor which binds to a ligand but does not generate any signal as response to binding to the ligand can reduce the biological effect of said ligand. Similarly, a dominant negative catalytically inactive kinase which usually interacts with target proteins but does not phosphorylate said target proteins may reduce phosphorylation of said target proteins as response to a cellular signal. Similarly, a dominant negative transcription factor which binds to a promoter site in the control region of a gene but does not increase transcription of said gene may reduce the effect of a normal transcription factor by occupying promoter binding sites, without increasing transcription.
The result of expression of a dominant negative polypeptide in a cell is a reduction in the function of active proteins. The skilled worker may prepare dominant negative variants of a protein, for example, by conventional mutagenesis methods and by evaluating the dominant negative effect of the variant polypeptide.
The invention also comprises substances such as polypeptides which bind to tumor-associated antigens. Such binding substances may be used, for example, in screening assays for detecting tumor-associated antigens and complexes of tumor-associated antigens with their binding partners and in a purification of said tumor-associated antigens and of complexes thereof with their binding partners. Such substances may also be used for inhibiting the activity of tumor-associated antigens, for example by binding to such antigens.
The invention therefore comprises binding substances such as, for example, antibodies or antibody fragments, which are capable of selectively binding to tumor-associated antigens. Antibodies comprise polyclonal and monoclonal antibodies which are produced in the conventional manner.
It is known that only a small part of an antibody molecule, the paratope, is involved in binding of the antibody to its epitope (cf. Clark, W. R. (1986), The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991), Essential Immunology, 7th Edition, Blackwell Scientific Publications, Oxford). The pFc′ and Fc regions are, for example, effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc′ region has been enzymatically removed or which has been produced without the pFc′ region, referred to as F(ab′)2 fragment, carries both antigen binding sites of a complete antibody. Similarly, an antibody from which the Fc region has been enzymatically removed or which has been produced without said Fc region, referred to Fab fragment, carries one antigen binding site of an intact antibody molecule. Furthermore, Fab fragments consist of a covalently bound light chain of an antibody and part of the heavy chain of said antibody, referred to as Fd. The Fd fragments are the main determinants of antibody specificity (a single Fd fragment can be associated with up to ten different light chains, without altering the specificity of the antibody) and Fd fragments, when isolated, retain the ability to bind to an epitope.
Located within the antigen-binding part of an antibody are complementary-determining regions (CDRs) which interact directly with the antigen epitope and framework regions (FRs) which maintain the tertiary structure of the paratope. Both the Fd fragment of the heavy chain and the light chain of IgG immunoglobulins contain four framework regions (FR1 to FR4) which are separated in each case by three complementary-determining regions (CDR1 to CDR3). The CDRs and, in particular, the CDR3 regions and, still more particularly, the CDR3 region of the heavy chain are responsible to a large extent for antibody specificity.
Non-CDR regions of a mammalian antibody are known to be able to be replaced by similar regions of antibodies with the same or a different specificity, with the specificity for the epitope of the original antibody being retained. This made possible the development of “humanized” antibodies in which nonhuman CDRs are covalently linked to human FR and/or Fc/pFc′ regions to produce a functional antibody.
WO 92/04381 for example, describes production and use of humanized murine RSV antibodies in which at least part of the murine FR regions have been replaced with FR regions of a human origin. Antibodies of this kind, including fragments of intact antibodies with antigen-binding capability, are often referred to as “chimeric” antibodies.
The invention also provides F(ab′)2, Fab, Fv, and Fd fragments of antibodies, chimeric antibodies, in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain-CDR3 regions have been replaced with homologous human or nonhuman sequences, chimeric F(ab′)2-fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain-CDR3 regions have been replaced with homologous human or nonhuman sequences, chimeric Fab-fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain-CDR3 regions have been replaced with homologous human or nonhuman sequences, and chimeric Fd-fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced with homologous human or nonhuman sequences. The invention also comprises “single-chain” antibodies.
Preferably, an antibody used according to the invention is directed against one of the sequences according to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 61-68, 70, 72, 74, 76, 81, 82, 86, 88, 96-101, 103, 105, 107, 109, 111, 113, or a part or derivative thereof and/or may be obtained by immunization using these peptides.
The invention also comprises polypeptides which bind specifically to tumor-associated antigens. Polypeptide binding substances of this kind may be provided, for example, by degenerate peptide libraries which may be prepared simply in solution in an immobilized form or as phage-display libraries. It is likewise possible to prepare combinatorial libraries of peptides with one or more amino acids. Libraries of peptoids and nonpeptidic synthetic residues may also be prepared.
Phage display may be particularly effective in identifying binding peptides of the invention. In this connection, for example, a phage library is prepared (using, for example, the M13, fd or lambda phages) which presents inserts of from 4 to about 80 amino acid residues in length. Phages are then selected which carry inserts which bind to the tumor-associated antigen. This process may be repeated via two or more cycles of a reselection of phages binding to the tumor-associated antigen. Repeated rounds result in a concentration of phages carrying particular sequences. An analysis of DNA sequences may be carried out in order to identify the sequences of the expressed polypeptides. The smallest linear portion of the sequence binding to the tumor-associated antigen may be determined. The “two-hybrid system” of yeast may also be used for identifying polypeptides which bind to a tumor-associated antigen. Tumor-associated antigens described according to the invention or fragments thereof may be used for screening peptide libraries, including phage-display libraries, in order to identify and select peptide binding partners of the tumor-associated antigens. Such molecules may be used, for example, for screening assays, purification protocols, for interference with the function of the tumor-associated antigen and for other purposes known to the skilled worker.
The antibodies described above and other binding molecules may be used, for example, for identifying tissue which expresses a tumor-associated antigen. Antibodies may also be coupled to specific diagnostic substances for displaying cells and tissues expressing tumor-associated antigens. They may also be coupled to therapeutically useful substances. Diagnostic substances comprise, in a nonlimiting manner, barium sulfate, iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium tyropanoate and radio diagnostic, including positron emitters such as fluorine-18 and carbon-11, gamma emitters such as iodine-123, technetium-99m, iodine-131 and indium-111, nuclides for nuclear magnetic resonance, such as fluorine and gadolinium. According to the invention, the term “therapeutically useful substance” means any therapeutic molecule which, as desired, is selectively guided to a cell which expresses one or more tumor-associated antigens, including anticancer agents, radioactive iodine-labeled compounds, toxins, cytostatic or cytolytic drugs, etc. Anticancer agents comprise, for example, aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil, interferon-α, lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HCl, thioguanine, vinblastine sulfate and vincristine sulfate. Other anticancer agents are described, for example, in Goodman and Gilman, “The Pharmacological Basis of Therapeutics”, 8th Edition, 1990, McGraw-Hill, Inc., in particular Chapter 52 (Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner). Toxins may be proteins such as pokeweed antiviral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin or Pseudomonas exotoxin. Toxin residues may also be high energy-emitting radionuclides such as cobalt-60.
The term “patient” means according to the invention a human being, a nonhuman primate or another animal, in particular a mammal such as a cow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouse and rat. In a particularly preferred embodiment, the patient is a human being.
According to the invention, the term “disease” refers to any pathological state in which tumor-associated antigens are expressed or abnormally expressed. “Abnormal expression” means according to the invention that expression is altered, preferably increased, compared to the state in a healthy individual. An increase in expression refers to an increase by at least 10%, in particular at least 20%, at least 50% or at least 100%. In one embodiment, the tumor-associated antigen is expressed only in tissue of a diseased individual, while expression in a healthy individual is repressed. One example of such a disease is cancer, in particular seminomas, melanomas, teratomas, gliomas, colon cancer, rectal cancer, kidney cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer, endometrial cancer, cancer of the esophagus, blood cancer, liver cancer, pancreatic cancer, skin cancer, brain cancer and lung cancer, lymphomas, and neuroblastomas. Examples for this are lung tumor, breast tumor, prostate tumor, colon tumor, renal cell carcinoma, cervical carcinoma, colon carcinoma and mamma carcinoma or metastases of the above cancer types or tumors.
According to the invention, a biological sample may be a tissue sample and/or a cellular sample and may be obtained in the conventional manner such as by tissue biopsy, including punch biopsy, and by taking blood, bronchial aspirate, urine, feces or other body fluids, for use in the various methods described herein.
According to the invention, the term “immunoreactive cell” means a cell which can mature into an immune cell (such as B cell, T helper cell, or cytolytic T cell) with suitable stimulation. Immunoreactive cells comprise CD34+ hematopoietic stem cells, immature and mature T cells and immature and mature B cells. If production of cytolytic or T helper cells recognizing a tumor-associated antigen is desired, the immunoreactive cell is contacted with a cell expressing a tumor-associated antigen under conditions which favor production, differentiation and/or selection of cytolytic T cells and of T helper cells. The differentiation of T cell precursors into a cytolytic T cell, when exposed to an antigen, is similar to clonal selection of the immune system.
Some therapeutic methods are based on a reaction of the immune system of a patient, which results in a lysis of antigen-presenting cells such as cancer cells which present one or more tumor-associated antigens. In this connection, for example autologous cytotoxic T lymphocytes specific for a complex of a tumor-associated antigen and an MHC molecule are administered to a patient having a cellular abnormality. The production of such cytotoxic T lymphocytes in vitro is known. An example of a method of differentiating T cells can be found in WO-A-96/33265. Generally, a sample containing cells such as blood cells is taken from the patient and the cells are contacted with a cell which presents the complex and which can cause propagation of cytotoxic T lymphocytes (e.g. dendritic cells). The target cell may be a transfected cell such as a COS cell. These transfected cells present the desired complex on their surface and, when contacted with cytotoxic T lymphocytes, stimulate propagation of the latter. The clonally expanded autologous cytotoxic T lymphocytes are then administered to the patient.
In another method of selecting antigen-specific cytotoxic T lymphocytes, fluorogenic tetramers of MHC class I molecule/peptide complexes are used for detecting specific clones of cytotoxic T lymphocytes (Altman et al., Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416, 1998). Soluble MHC class I molecules are folded in vitro in the presence of β2 microglobulin and a peptide antigen binding to said class I molecule. The MHC/peptide complexes are purified and then labeled with biotin. Tetramers are formed by mixing the biotinylated peptide-MHC complexes with labeled avidin (e.g. phycoerythrin) in a molar ratio of 4:1. Tetramers are then contacted with cytotoxic T lymphocytes such as peripheral blood or lymph nodes. The tetramers bind to cytotoxic T lymphocytes which recognize the peptide antigen/MHC class I complex. Cells which are bound to the tetramers may be sorted by fluorescence-controlled cell sorting to isolate reactive cytotoxic T lymphocytes. The isolated cytotoxic T lymphocytes may then be propagated in vitro.
In a therapeutic method referred to as adoptive transfer (Greenberg, J. Immunol. 136(5):1917, 1986; Riddel et al., Science 257:238, 1992; Lynch et al., Eur. J. Immunol. 21:1403-1410, 1991; Kast et al., Cell 59:603-614, 1989), cells presenting the desired complex (e.g. dendritic cells) are combined with cytotoxic T lymphocytes of the patient to be treated, resulting in a propagation of specific cytotoxic T lymphocytes. The propagated cytotoxic T lymphocytes are then administered to a patient having a cellular anomaly characterized by particular abnormal cells presenting the specific complex. The cytotoxic T lymphocytes then lyse the abnormal cells, thereby achieving a desired therapeutic effect.
Often, of the T cell repertoire of a patient, only T cells with low affinity for a specific complex of this kind can be propagated, since those with high affinity have been extinguished due to development of tolerance. An alternative here may be a transfer of the T cell receptor itself. For this too, cells presenting the desired complex (e.g. dendritic cells) are combined with cytotoxic T lymphocytes of healthy individuals. This results in propagation of specific cytotoxic T lymphocytes with high affinity if the donor had no previous contact with the specific complex. The high affinity T cell receptor of these propagated specific T lymphocytes is cloned and can be transduced via gene transfer, for example using retroviral vectors, into T cells of other patients, as desired. Adoptive transfer is then carried out using these genetically altered T lymphocytes (Stanislawski et al., Nat Immunol. 2:962-70, 2001; Kessels et al., Nat Immunol. 2:957-61, 2001).
The therapeutic aspects above start out from the fact that at least some of the abnormal cells of the patient present a complex of a tumor-associated antigen and an HLA molecule. Such cells may be identified in a manner known per se. As soon as cells presenting the complex have been identified, they may be combined with a sample from the patient, which contains cytotoxic T lymphocytes. If the cytotoxic T lymphocytes lyse the cells presenting the complex, it can be assumed that a tumor-associated antigen is presented.
Adoptive transfer is not the only form of therapy which can be applied according to the invention. Cytotoxic T lymphocytes may also be generated in vivo in a manner known per se. One method uses nonproliferative cells expressing the complex. The cells used here will be those which usually express the complex, such as irradiated tumor cells or cells transfected with one or both genes necessary for presentation of the complex (i.e. the antigenic peptide and the presenting HLA molecule). Various cell types may be used. Furthermore, it is possible to use vectors which carry one or both of the genes of interest. Particular preference is given to viral or bacterial vectors. For example, nucleic acids coding for a tumor-associated antigen or for a part thereof may be functionally linked to promoter and enhancer sequences which control expression of said tumor-associated antigen or a fragment thereof in particular tissues or cell types. The nucleic acid may be incorporated into an expression vector. Expression vectors may be nonmodified extrachromosomal nucleic acids, plasmids or viral genomes into which exogenous nucleic acids may be inserted. Nucleic acids coding for a tumor-associated antigen may also be inserted into a retroviral genome, thereby enabling the nucleic acid to be integrated into the genome of the target tissue or target cell. In these systems, a microorganism such as vaccinia virus, pox virus, Herpes simplex virus, retrovirus or adenovirus carries the gene of interest and de facto “infects” host cells. Another preferred form is the introduction of the tumor-associated antigen in the form of recombinant RNA which may be introduced into cells by liposomal transfer or by electroporation, for example. The resulting cells present the complex of interest and are recognized by autologous cytotoxic T lymphocytes which then propagate.
A similar effect can be achieved by combining the tumor-associated antigen or a fragment thereof with an adjuvant in order to make incorporation into antigen-presenting cells in vivo possible. The tumor-associated antigen or a fragment thereof may be represented as protein, as DNA (e.g. within a vector) or as RNA. The tumor-associated antigen is processed to produce a peptide partner for the HLA molecule, while a fragment thereof may be presented without the need for further processing. The latter is the case in particular, if these can bind to HLA molecules. Preference is given to administration forms in which the complete antigen is processed in vivo by a dendritic cell, since this may also produce T helper cell responses which are needed for an effective immune response (Ossendorp et al., Immunol Lett. 74:75-9, 2000; Ossendorp et al., J. Exp. Med. 187:693-702, 1998). In general, it is possible to administer an effective amount of the tumor-associated antigen to a patient by intradermal injection, for example. However, injection may also be carried out intranodally into a lymph node (Maloy et al., Proc Natl Acad Sci USA 98:3299-303, 2001). It may also be carried out in combination with reagents which facilitate uptake into dendritic cells. Preferred tumor-associated antigens comprise those which react with allogenic cancer antisera or with T cells of many cancer patients. Of particular interest, however, are those against which no spontaneous immune responses pre-exist. Evidently, it is possible to induce against these immune responses which can lyse tumors (Keogh et al., J. Immunol. 167:787-96, 2001; Appella et al., Biomed Pept Proteins Nucleic Acids 1:177-84, 1995; Wentworth et al., Mol Immunol. 32:603-12, 1995).
The pharmaceutical compositions described according to the invention may also be used as vaccines for immunization. According to the invention, the terms “immunization” or “vaccination” mean an increase in or activation of an immune response to an antigen. It is possible to use animal models for testing an immunizing effect on cancer by using a tumor-associated antigen or a nucleic acid coding therefor. For example, human cancer cells may be introduced into a mouse to generate a tumor, and one or more nucleic acids coding for tumor-associated antigens may be administered. The effect on the cancer cells (for example reduction in tumor size) may be measured as a measure for the effectiveness of an immunization by the nucleic acid.
As part of the composition for an immunization, one or more tumor-associated antigens or stimulating fragments thereof are administered together with one or more adjuvants for inducing an immune response or for increasing an immune response. An adjuvant is a substance which is incorporated into the antigen or administered together with the latter and which enhances the immune response. Adjuvants may enhance the immune response by providing an antigen reservoir (extracellularly or in macrophages), activating macrophages and stimulating particular lymphocytes. Adjuvants are known and comprise in a nonlimiting way monophosphoryl lipid A (MPL, SmithKline Beecham), saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739), QS7, QS17, QS18 and QS-L1 (So et al., Mol. Cells 7:178-186, 1997), incomplete Freund's adjuvant, complete Freund's adjuvant, vitamin E, montanide, alum, CpG oligonucleotides (cf. Krieg et al., Nature 374:546-9, 1995) and various water-in-oil emulsions prepared from biologically degradable oils such as squalene and/or tocopherol. Preferably, the peptides are administered in a mixture with DQS21/MPL. The ratio of DQS21 to MPL is typically about 1:10 to 10:1, preferably about 1:5 to 5:1 and in particular about 1:1. For administration to humans, a vaccine formulation typically contains DQS21 and MPL in a range from about 1 μg to about 100 μg.
Other substances which stimulate an immune response of the patient may also be administered. It is possible, for example, to use cytokines in a vaccination, owing to their regulatory properties on lymphocytes. Such cytokines comprise, for example, interleukin-12 (IL-12) which was shown to increase the protective actions of vaccines (cf. Science 268:1432-1434, 1995), GM-CSF and IL-18.
There are a number of compounds which enhance an immune response and which therefore may be used in a vaccination. Said compounds comprise costimulating molecules provided in the form of proteins or nucleic acids. Examples of such costimulating molecules are B7-1 and B7-2 (CD80 and CD86, respectively) which are expressed on dendritic cells (DC) and interact with the CD28 molecule expressed on the T cells. This interaction provides a costimulation (signal 2) for an antigen/MHC/TCR-stimulated (signal 1) T cell, thereby enhancing propagation of said T cell and the effector function. B7 also interacts with CTLA4 (CD152) on T cells, and studies involving CTLA4 and B7 ligands demonstrate that B7-CTLA4 interaction can enhance antitumor immunity and CTL propagation (Zheng, P. et al., Proc. Natl. Acad. Sci. USA 95(11):6284-6289 (1998)).
B7 is typically not expressed on tumor cells so that these are no effective antigen-presenting cells (APCs) for T cells. Induction of B7 expression would enable tumor cells to stimulate more effectively propagation of cytotoxic T lymphocytes and an effector function. Costimulation by a combination of B7/IL-6/IL-12 revealed induction of IFN-gamma and Th1-cytokine profile in a T cell population, resulting in further enhanced T cell activity (Gajewski et al., J. Immunol. 154:5637-5648 (1995)).
A complete activation of cytotoxic T lymphocytes and a complete effector function require an involvement of T helper cells via interaction between the CD40 ligand on said T helper cells and the CD40 molecule expressed by dendritic cells (Ridge et al., Nature 393:474 (1998), Bennett et al., Nature 393:478 (1998), Schönberger et al., Nature 393:480 (1998)). The mechanism of this costimulating signal probably relates to the increase in B7 production and associated IL-6/IL-12 production by said dendritic cells (antigen-presenting cells). CD40-CD40L interaction thus complements the interaction of signal 1 (antigen/MHC-TCR) and signal 2 (B7-CD28).
The use of anti-CD40 antibodies for stimulating dendritic cells would be expected to directly enhance a response to tumor antigens which are usually outside the range of an inflammatory response or which are presented by nonprofessional antigen-presenting cells (tumor cells). In these situations, T helper and B7-costimulating signals are not provided. This mechanism could be used in connection with therapies based on antigen-pulsed dendritic cells or in situations in which T helper epitopes have not been defined in known TRA precursors.
The invention also provides for administration of nucleic acids, polypeptides or peptides. Polypeptides and peptides may be administered in a manner known per se. In one embodiment, nucleic acids are administered by ex vivo methods, i.e. by removing cells from a patient, genetic modification of said cells in order to incorporate a tumor-associated antigen and reintroduction of the altered cells into the patient. This generally comprises introducing a functional copy of a gene into the cells of a patient in vitro and reintroducing the genetically altered cells into the patient. The functional copy of the gene is under the functional control of regulatory elements which allow the gene to be expressed in the genetically altered cells. Transfection and transduction methods are known to the skilled worker. The invention also provides for administering nucleic acids in vivo by using vectors such as viruses and target-controlled liposomes.
In a preferred embodiment, a viral vector for administering a nucleic acid coding for a tumor-associated antigen is selected from the group consisting of adenoviruses, adeno-associated viruses, pox viruses, including vaccinia virus and attenuated pox viruses, Semliki Forest virus, retroviruses, Sindbis virus and Ty virus-like particles. Particular preference is given to adenoviruses and retroviruses. The retroviruses are typically replication-deficient (i.e. they are incapable of generating infectious particles).
Various methods may be used in order to introduce according to the invention nucleic acids into cells in vitro or in vivo. Methods of this kind comprise transfection of nucleic acid CaPO4 precipitates, transfection of nucleic acids associated with DEAE, transfection or infection with the above viruses carrying the nucleic acids of interest, liposome-mediated transfection, and the like. In particular embodiments, preference is given to directing the nucleic acid to particular cells. In such embodiments, a carrier used for administering a nucleic acid to a cell (e.g. a retrovirus or a liposome) may have a bound target control molecule. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell may be incorporated into or attached to the nucleic acid carrier. Preferred antibodies comprise antibodies which bind selectively a tumor-associated antigen. If administration of a nucleic acid via liposomes is desired, proteins binding to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation in order to make target control and/or uptake possible. Such proteins comprise capsid proteins or fragments thereof which are specific for a particular cell type, antibodies to proteins which are internalized, proteins addressing an intracellular site, and the like.
The therapeutic compositions of the invention may be administered in pharmaceutically compatible preparations. Such preparations may usually contain pharmaceutically compatible concentrations of salts, buffer substances, preservatives, carriers, supplementing immunity-enhancing substances such as adjuvants (e.g. CpG oligonucleotides) and cytokines and, where appropriate, other therapeutically active compounds.
The therapeutically active compounds of the invention may be administered via any conventional route, including by injection or infusion. The administration may be carried out, for example, orally, intravenously, intraperitonealy, intramuscularly, subcutaneously or transdermally. Preferably, antibodies are therapeutically administered by way of a lung aerosol. Antisense nucleic acids are preferably administered by slow intravenous administration.
The compositions of the invention are administered in effective amounts. An “effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of treatment of a particular disease or of a particular condition characterized by expression of one or more tumor-associated antigens, the desired reaction relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting the progress of the disease. The desired reaction in a treatment of a disease or of a condition may also be delay of the onset or a prevention of the onset of said disease or said condition.
An effective amount of a composition of the invention will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors.
The pharmaceutical compositions of the invention are preferably sterile and contain an effective amount of the therapeutically active substance to generate the desired reaction or the desired effect.
The doses administered of the compositions of the invention may depend on various parameters such as the type of administration, the condition of the patient, the desired period of administration, etc. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.
Generally, doses of the tumor-associated antigen of from 1 ng to 1 mg, preferably from 10 ng to 100 μg, are formulated and administered for a treatment or for generating or increasing an immune response. If the administration of nucleic acids (DNA and RNA) coding for tumor-associated antigens is desired, doses of from 1 ng to 0.1 mg are formulated and administered.
The pharmaceutical compositions of the invention are generally administered in pharmaceutically compatible amounts and in pharmaceutically compatible compositions. The term “pharmaceutically compatible” refers to a nontoxic material which does not interact with the action of the active component of the pharmaceutical composition. Preparations of this kind may usually contain salts, buffer substances, preservatives, carriers and, where appropriate, other therapeutically active compounds. When used in medicine, the salts should be pharmaceutically compatible. However, salts which are not pharmaceutically compatible may used for preparing pharmaceutically compatible salts and are included in the invention. Pharmacologically and pharmaceutically compatible salts of this kind comprise in a nonlimiting way those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acids, and the like. Pharmaceutically compatible salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.
A pharmaceutical composition of the invention may comprise a pharmaceutically compatible carrier. According to the invention, the term “pharmaceutically compatible carrier” refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to humans. The term “carrier” refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate application. The components of the pharmaceutical composition of the invention are usually such that no interaction occurs which substantially impairs the desired pharmaceutical efficacy.
The pharmaceutical compositions of the invention may contain suitable buffer substances such as acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.
The pharmaceutical compositions may, where appropriate, also contain suitable preservatives such as benzalkonium chloride, chlorobutanol, parabens and thimerosal.
The pharmaceutical compositions are usually provided in a uniform dosage form and may be prepared in a manner known per se. Pharmaceutical compositions of the invention may be in the form of capsules, tablets, lozenges, solutions, suspensions, syrups, elixir or in the form of an emulsion, for example.
Compositions suitable for parenteral administration usually comprise a sterile aqueous or nonaqueous preparation of the active compound, which is preferably isotonic to the blood of the recipient. Examples of compatible carriers and solvents are Ringer solution and isotonic sodium chloride solution. In addition, usually sterile, fixed oils are used as solution or suspension medium.
The present invention is described in detail by the figures and examples below, which are used only for illustration purposes and are not meant to be limiting. Owing to the description and the examples, further embodiments which are likewise included in the invention are accessible to the skilled worker.
EXAMPLES
Materials and Methods
The terms “in silico” and “electronic” refer solely to the utilization of methods based on databases, which may also be used to simulate laboratory experimental processes.
Unless expressly defined otherwise, all other terms and expressions are used so as to be understood by the skilled worker. The techniques and methods mentioned are carried out in a manner known per se and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methods including the use of kits and reagents are carried out according to the manufacturers' information.
A. Data Mining-Based Strategy for Identifying Tumor-Associated Antigens
According to the invention, public human protein and nucleic acid databases were screened with regard to cancer-specific antigens accessible on the cell surface. The definition of the screening criteria required therefor, together with high throughput methods for analyzing, if possible, all proteins, formed the central component of this strategy.
The starting point consisted of the validated protein entries (NP) and, respectively, the corresponding mRNAs (NM) which have been deposited in the RefSeq database (Pruitt et al., Trends Genet. January; 16(1):44-47, 2000) of the National Center for Biotechnology Information (NCBI). Following the fundamental principle of gene→mRNA→protein, the proteins were first studied for the presence of one or more transmembrane domains. To this end, the protein analysis program TMHMM server v. 2.0 (Krogh et al., Journal of Molecular Biology 305(3):567-580, 2001) was used and the results thereof then verified again using the program ALOM 2 (Nakai et al., Genomics 14:897-911, 1992). The prediction of further signal sequences which influenced the intracellular localisation of proteins was done using the programs PSORT II (Horton et al., Intelligent Systems for Molecular Biology 4:109-115, 1996) and iPSORT (Bannai et al., Bioinformatics, 18(2):298-305, 2002). The human NP fraction having a total of 19 110 entries provided 4634 filtered proteins.
The corresponding mRNA of each of these 4634 proteins, respectively, was then subjected to a homology search in the EST database (Boguski et al., Nat. Genet. 4(4):332-333, 1993) of the NCBI with the aid of the BLAST algorithm (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997). The screening criteria in this search were set to an e-value<10e-20 and a minimal sequence identity of 93% in such a way that the hits resulting therefrom with high probability could only be derived from the respective mRNA but not from the homologous transcripts. Almost all mRNAs provided at least one hit in the EST database wherein in some cases the number of hits exceeded 4000.
Subsequently, the tissue-specific origin of the underlying cDNA library as well as the name of the library were determined for each of these valid hits. The tissues resulting therefrom were divided into 4 different groups ranging from dispensable organs (group 3) to absolutely essential organs (group 0). Another group, group 4, consisted of any samples obtained from cancer tissue. The distribution of hits to the five groups was recorded in a table which was sorted according to the best ratio of the sum of groups 3 and 4 to the sum of groups 0-2. Those mRNAs whose EST hits originated exclusively from cancer tissue reached a top position, followed by those which can additionally be found also in tissues of dispensable organs of group 3. A further criterium for the significance of this distribution was the number of the independent cDNA libraries from which the ESTs were obtained and was recorded in a separate column of the table.
Since the transcripts determined in the first approach and the corresponding proteins are firstly hypothetic constructs, further screening criteria were used with the intention to prove the real existence of the mRNAs and consequently also of the proteins. For this purpose, each mRNA was compared to the predicted gene locus using the program “Spidey” (Wheelan et al., Genome Res. 11(11): 1952-1957, 2001). Only those transcripts which have at least one splicing process, i.e. which spread over at least 2 exons, were used for more detailed analyses.
Sequential application of all the filters mentioned led to the tumor-associated antigens of the invention which can be considered extracellularly accessible, owing to a predicted transmembrane domain and the topology related thereto. The expression profile derived from the EST data indicates, in all cases, cancer-specific expression which may at most extend only to dispensable organs.
B. Strategy of Validating the Tumor-Associated Antigens Identified by in Silico Analysis
In order to utilize the targets for immunotherapeutic purposes (antibody therapy by means of monoclonal antibodies, vaccination, T-cell receptor-mediated therapeutic approaches; cf. EP-B-0 879 282), in cancer therapy as well as for diagnostic problems, the validation of the targets identified according to the invention is of central importance. In this connection, validation is carried out by expression analysis at both RNA and protein levels.
1. Examination of RNA Expression
The identified tumor antigens are first validated with the aid of RNA which is obtained from various tissues or from tissue-specific cell lines. Since the differential expression pattern of healthy tissue in comparison with tumor tissue is of decisive importance for the subsequent therapeutic application, the target genes are preferably characterized with the aid of these tissue samples.
Total RNA is isolated from native tissue samples or from tumor cell lines by standard methods of molecular biology. Said isolation may be carried out, for example, with the aid of the RNeasy Maxi kit (Qiagen, Cat. No. 75162) according to the manufacturer's instructions. This isolation method is based on the use of chaotropic reagent guanidinium isothiocyanate. Alternatively, acidic phenol can be used for isolation (Chomczynski & Sacchi, Anal. Biochem. 162: 156-159, 1987). After the tissue has been worked up by means of guanidinium isothiocyanate, RNA is extracted with acidic phenol, subsequently precipitated with isopropanol and taken up in DEPC-treated water.
2-4 μg of the RNA isolated in this way are subsequently transcribed into cDNA, for example by means of Superscript II (Invitrogen) according to the manufacturer's protocol. cDNA synthesis is primed with the aid of random hexamers (e.g. Roche Diagnostics) according to standard protocols of the relevant manufacturer. For quality control, the cDNAs are amplified over 30 cycles, using primers specific for the p53 gene which is expressed only lowly. Only p53-positive cDNA samples will be used for the subsequent reaction steps.
The antigens are analyzed in detail by carrying out an expression analysis by means of PCR or quantitative PCR (qPCR) on the basis of a cDNA archive which has been isolated from various normal and tumor tissues and from tumor cell lines. For this purpose, 0.5 μl of cDNA of the above reaction mixture is amplified by a DNA polymerase (e.g. 1 U of HotStarTaq DNA polymerase, Qiagen) according to the protocols of the particular manufacturer (total volume of the reaction mixture: 25-50 μl). Aside from said polymerase, the amplification mixture comprises 0.3 mM dNTPs, reaction buffer (final concentration 1×, depending on the manufacturer of the DNA polymerase) and in each case 0.3 mM gene-specific forward and reverse primers.
The specific primers of the target gene are, as far as possible, selected in such a way that they are located in two different exons so that genomic contaminations do not lead to false-positive results. In a non-quantitative end point PCR, the cDNA is typically incubated at 95° C. for 15 minutes in order to denature the DNA and to activate the Hot-Start enzyme. Subsequently the DNA is amplified over 35 cycles (1 min at 95° C., 1 min at the primer-specific hybridization temperature (approx. 55-65° C.), 1 min at 72° C. to elongate the amplicons). Subsequently, 10 μl of the PCR mixture are applied to agarose gels and fractionated in the electric field. The DNA is made visible in the gels by staining with ethidium bromide and the PCR result is documented by way of a photograph.
As an alternative to conventional PCR, expression of a target gene may also be analyzed by quantitative real time PCR. Meanwhile various analytical systems are available for this analysis, of which the best known ones are the ABI 7900 HT sequence detection system (Applied Biosystems), the iCycler (Biorad) and the Light cycler (Roche Diagnostics). As described above, a specific PCR mixture is subjected to a run in the real time instruments. By adding a DNA-intercalating dye (e.g. ethidium bromide, CybrGreen), the newly synthesized DNA is made visible by specific light excitation (according to the dye manufacturers' information). A multiplicity of points measured during amplification enables the entire process to be monitored and the nucleic acid concentration of the target gene to be determined quantitatively. The PCR mixture is normalized by measuring a housekeeping gene (e.g. 18S RNA, β-actin, GAPDH). Alternative strategies via fluorescently labeled DNA probes likewise allow quantitative determination of the target gene of a specific tissue sample (see TaqMan applications from Applied Biosystems).
2. Cloning
The complete target gene which is required for further characterization of the tumor antigen is cloned according to common molecular-biological methods (e.g. in “Current Protocols in Molecular Biology”, John Wiley & Sons Ltd., Wiley InterScience). In order to clone the target gene or to analyze its sequence, said gene is first amplified by a DNA polymerase having a proof reading function (e.g. pfu, Roche Diagnostics). The amplicon is then ligated by standard methods into a cloning vector. Positive clones are identified by sequence analysis and subsequently characterized with the aid of prediction programs and known algorithms.
3. Production of Antibodies
The tumor-associated antigens identified according to the invention are characterized, for example, by using antibodies. The invention further comprises the diagnostic or therapeutic use of antibodies. Antibodies may recognize proteins in the native and/or denatured state (Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J. Immunol. Methods 234: 107-116, 2000; Kayyem et al., Eur. J. Biochem. 208: 1-8, 1992; Spiller et al., J. Immunol. Methods 224: 51-60, 1999).
Antisera comprising specific antibodies which specifically bind to the target protein may be prepared by various standard methods; cf., for example, “Monoclonal Antibodies: A Practical Approach” by Phillip Shepherd, Christopher Dean ISBN 0-19-963722-9, “Antibodies: A Laboratory Manual” by Ed Harlow, David Lane ISBN: 0879693142 and “Using Antibodies: A Laboratory Manual: Portable Protocol NO” by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447. It is also possible here to generate affine and specific antibodies which recognize complex membrane proteins in their native form (Azorsa et al., J. Immunol. Methods 229: 35-48, 1999; Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J. Immunol. Methods. 234: 107-116, 2000). This is especially important in the preparation of antibodies which are intended to be used therapeutically but also for many diagnostic applications. For this purpose, both the complete protein and extracellular partial sequences may be used for immunization.
Immunization and Production of Polyclonal Antibodies
Several immunization protocols have been published. A species (e.g. rabbits, mice) is immunized by a first injection of the desired target protein. The immune response of the animal to the immunogen can be enhanced by a second or third immunization within a defined period of time (approx. 2-4 weeks after the previous immunization). Blood is taken from said animals and immune sera obtained, again after various defined time intervals (1st bleeding after 4 weeks, then every 2-3 weeks, up to 5 takings). The immune sera taken in this way comprise polyclonal antibodies which may be used to detect and characterize the target protein in Western blotting, by flow cytometry, immunofluorescence or immunohistochemistry.
The animals are usually immunized by any of four well-established methods, with other methods also in existence. The immunization may be carried out using peptides specific for the target protein, using the complete protein, using extracellular partial sequences of a protein which can be identified experimentally or via prediction programs. Since the prediction programs do not always work perfectly, it is also possible to employ two domains separated from one another by a transmembrane domain. In this case, one of the two domains has to be extracellular, which may then be proved experimentally (see below). The immunization is provided by various commercial service providers.

    
    
        (1) In the first case, peptides (length: 8-12 amino acids) are synthesized by in vitro methods (possibly carried out by a commercial service), and said peptides are used for immunization. Normally 3 immunizations are carried out (e.g. with a concentration of 5-100 μg/immunization).
        (2) Alternatively, immunization may be carried out using recombinant proteins. For this purpose, the cloned DNA of the target gene is cloned into an expression vector and the target protein is synthesized, for example, cell-free in vitro, in bacteria (e.g. E. coli), in yeast (e.g. S. pombe), in insect cells or in mammalian cells, according to the conditions of the particular manufacturer (e.g. Roche Diagnostics, Invitrogen, Clontech, Qiagen). It is also possible to synthesize the target protein with the aid of viral expression systems (e.g. baculovirus, vacciniavirus, adenovirus). After it has been synthesized in one of said systems, the target protein is purified, normally by employing chromatographic methods. In this context, it is also possible to use for immunization proteins which have a molecular anchor as an aid for purification (e.g. His tag, Qiagen; FLAG tag, Roche Diagnostics; GST fusion proteins). A multiplicity of protocols can be found, for example, in “Current Protocols in Molecular Biology”, John Wiley & Sons Ltd., Wiley InterScience. After the target protein has been purified, an immunization is carried out as described above.
        (3) If a cell line is available which synthesizes the desired protein endogenously, it is also possible to use this cell line directly for preparing the specific antiserum. In this case, immunization is carried out by 1-3 injections with in each case approx. 1−5×107 cells.
        (4) The immunization may also be carried out by injecting DNA (DNA immunization). For this purpose, the target gene is first cloned into an expression vector so that the target sequence is under the control of a strong eukaryotic promoter (e.g. CMV promoter). Subsequently, DNA (e.g. 1-10 μg per injection) is transferred as immunogen using a gene gun into capillary regions with a strong blood flow in an organism (e.g. mouse, rabbit). The transferred DNA is taken up by the animal's cells, the target gene is expressed, and the animal finally develops an immune response to the target protein (Jung et al., Mol. Cells 12: 41-49, 2001; Kasinrerk et al., Hybrid Hybridomics 21: 287-293, 2002).

Production of Monoclonal Antibodies

    
    


Monoclonal antibodies are traditionally produced with the aid of the hybridoma technology (technical details: see “Monoclonal Antibodies: A Practical Approach” by Philip Shepherd, Christopher Dean ISBN 0-19-963722-9; “Antibodies: A Laboratory Manual” by Ed Harlow, David Lane ISBN: 0879693142, “Using Antibodies: A Laboratory Manual: Portable Protocol NO” by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447). A new method which is also used is the “SLAM” technology. Here, B cells are isolated from whole blood and the cells are made monoclonal. Subsequently the supernatant of the isolated B cell is analyzed for its antibody specificity. In contrast to the hybridoma technology, the variable region of the antibody gene is then amplified by single-cell PCR and cloned into a suitable vector. In this manner production of monoclonal antibodies is accelerated (de Wildt et al., J. Immunol. Methods 207:61-67, 1997).
4. Validation of the Targets by Protein-Chemical Methods Using Antibodies
The antibodies which can be produced as described above can be used to make a number of important statements about the target protein. Specifically the following analyses of validating the target protein are useful:
Specificity of the Antibody
Assays based on cell culture with subsequent Western blotting are most suitable for demonstrating the fact that an antibody binds specifically only to the desired target protein (various variations are described, for example, in “Current Protocols in Proteinchemistry”, John Wiley & Sons Ltd., Wiley InterScience). For the demonstration, cells are transfected with a cDNA for the target protein, which is under the control of a strong eukaryotic promoter (e.g. cytomegalovirus promoter; CMV). A wide variety of methods (e.g. electroporation, liposome-based transfection, calcium phosphate precipitation) are well established for transfecting cell lines with DNA (e.g. Lemoine et al., Methods Mol. Biol. 75: 441-7, 1997). As an alternative, it is also possible to use cell lines which express the target gene endogenously (detection via target gene-specific RT-PCR). As a control, in the ideal case, homologous genes are cotransfected in the experiment, in order to be able to demonstrate in the following Western blot the specificity of the analyzed antibody.
In the subsequent Western blotting, cells from cell culture or tissue samples which might contain the target protein are lysed in a 1% strength SDS solution, and the proteins are denatured in the process. The lysates are fractionated according to size by electrophoresis on 8-15% strength denaturing polyacrylamide gels (contain 1% SDS) (SDS polyacrylamide gel electrophoresis, SDS-PAGE). The proteins are then transferred by one of a plurality of blotting methods (e.g. semi-dry electroblot; Biorad) to a specific membrane (e.g. nitrocellulose, Schleicher & Schüll). The desired protein can be visualized on this membrane. For this purpose, the membrane is first incubated with the antibody which recognizes the target protein (dilution approx. 1:20-1:200, depending on the specificity of said antibody), for 60 minutes. After a washing step, the membrane is incubated with a second antibody which is coupled to a marker (e.g. enzymes such as peroxidase or alkaline phosphatase) and which recognizes the first antibody. It is then possible to make the target protein visible on the membrane in a color or chemiluminescent reaction (e.g. ECL, Amersham Bioscience). An antibody with a high specificity for the target protein should in the ideal case only recognise the desired protein itself.
Localization of the Target Protein
Various methods are used to confirm the membrane localization, identified in the in silico approach, of the target protein. An important and well-established method using the antibodies described above is immunofluorescence (IF). For this purpose, cells of established cell lines which either synthesize the target protein (detection of the RNA by RT-PCR or of the protein by Western blotting) or else have been transfected with plasmid DNA are utilized. A wide variety of methods (e.g. electroporation, liposome-based transfection, calcium phosphate precipitation) are well established for transfection of cell lines with DNA (e.g. Lemoine et al., Methods Mol. Biol. 75: 441-7, 1997). The plasmid transfected, in immunofluorescence, may encode the unmodified protein or else couple different amino acid markers to the target protein. The principle markers are, for example, the fluorescent green fluorescent protein (GFP) in various differentially fluorescent forms, short peptide sequences of 6-12 amino acids for which high-affinity and specific antibodies are available, or the short amino acid sequence Cys-Cys-X-X-Cys-Cys which can bind via its cysteines specific fluorescent substances (Invitrogen). Cells which synthesize the target protein are fixed, for example, with paraformaldehyde or methanol. The cells may then, if required, be permeabilized by incubation with detergents (e.g. 0.2% Triton X-100). The cells are then incubated with a primary antibody which is directed against the target protein or against one of the coupled markers. After a washing step, the mixture is incubated with a second antibody coupled to a fluorescent marker (e.g. fluorescein, Texas Red, Dako), which binds to the first antibody. The cells labeled in this way are then overlaid with glycerol and analyzed with the aid of a fluorescence microscope according to the manufacturer's information. Specific fluorescence emissions are achieved in this case by specific excitation depending on the substances employed. The analysis usually permits reliable localization of the target protein, the antibody quality and the target protein being confirmed in double stainings with, in addition to the target protein, also the coupled amino acid markers or other marker proteins whose localization has already been described in the literature being stained. GFP and its derivatives represent a special case, being excitable directly and themselves fluorescing. The membrane permeability which may be controlled through the use of detergents, in immunofluorescence, allows demonstration of whether an immunogenic epitope is located inside or outside the cell. The prediction of the selected proteins can thus be supported experimentally. An alternative possibility is to detect extracellular domains by means of flow cytometry. For this purpose, cells are fixed under non-permeabilizing conditions (e.g. with PBS/Na azide/2% FCS/5 mM EDTA) and analyzed in a flow cytometer in accordance with the manufacturer's instructions. Only extracellular epitopes can be recognized by the antibody to be analyzed in this method. A difference from immunofluorescence is that it is possible to distinguish between dead and living cells by using, for example, propidium iodide or Trypan blue, and thus avoid false-positive results.
Another important detection is by immunohistochemistry (IHC) on specific tissue samples. The aim of this method is to identify the localization of a protein in a functionally intact tissue aggregate. IHC serves specifically for (1) being able to estimate the amount of target protein in tumor and normal tissues, (2) analyzing how many cells in tumor and healthy tissues express the target gene, and (3) defining the cell type in a tissue (tumor, healthy cells) in which the target protein is detectable. Alternatively, the amounts of protein of a target gene may be quantified by tissue immunofluorescence using a digital camera and suitable software (e.g. Tillvision, Till-photonics, Germany). The technology has frequently been published, and details of staining and microscopy can therefore be found, for example, in “Diagnostic Immunohistochemistry” by David J., MD Dabbs ISBN: 0443065667 or in “Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy” ISBN: 0306467704. It should be noted that, owing to the properties of antibodies, different protocols have to be used (an example is described below) in order to obtain a meaningful result.
Normally, histologically defined tumor tissues and, as reference, comparable healthy tissues are employed in IHC. It is also possible to use as positive and negative controls cell lines in which the presence of the target gene is known through RT-PCR analyses. A background control must always be included.
Formalin-fixed (another fixation method, for example with methanol, is also possible) and paraffin-embedded tissue pieces with a thickness of 4 μm are applied to a glass support and deparaffinated with xylene, for example. The samples are washed with TBS-T and blocked in serum. This is followed by incubation with the first antibody (dilution: 1:2 to 1:2000) for 1-18 hours, with affinity-purified antibodies normally being used. A washing step is followed by incubation with a second antibody which is coupled to an alkaline phosphatase (alternative: for example peroxidase) and directed against the first antibody, for approx. 30-60 minutes. This is followed by a color reaction using said alkaline phosphatase (cf., for example, Shi et al., J. Histochem. Cytochem. 39: 741-748, 1991; Shin et al., Lab. Invest. 64: 693-702, 1991). To demonstrate antibody specificity, the reaction can be blocked by previous addition of the immunogen.
Analysis of Protein Modifications
Secondary protein modifications such as, for example, N- and O-glycosylations or myristilations may impair or even completely prevent the accessibility of immunogenic epitopes and thus call into question the efficacy of antibody therapies. Moreover, it has frequently been demonstrated that the type and amount of secondary modifications differ in normal and tumor tissues (e.g. Durand & Seta, 2000; Clin. Chem. 46: 795-805; Hakomori, 1996; Cancer Res. 56: 5309-18). The analysis of these modifications is therefore essential to the therapeutic success of an antibody. Potential binding sites can be predicted by specific algorithms.
Analysis of protein modifications usually takes place by Western blotting (see above). Glycosylations which usually have a size of several kDa, especially lead to a larger total mass of the target protein, which can be fractionated in SDS-PAGE. To detect specific O- and N-glycosidic bonds, protein lysates are incubated prior to denaturation by SDS with O- or N-glycosylases (in accordance with their respective manufacturer's instructions, e.g. PNgase, endoglycosidase F, endoglycosidase H, Roche Diagnostics). This is followed by Western blotting as described above. Thus, if there is a reduction in the size of a target protein after incubation with a glycosidase, it is possible to detect a specific glycosylation and, in this way, also analyze the tumor specificity of a modification.
Functional Analysis of the Target Gene
The function of the target molecule may be crucial for its therapeutic usefulness, so that functional analyses are an important component in the characterization of therapeutically utilizable molecules. The functional analysis may take place either in cells, in cell culture experiments or else in vivo with the aid of animal models. This involves either switching off the gene of the target molecule by mutation (knockout) or inserting the target sequence into the cell or the organism (knockin). Thus it is possible to analyze functional modifications in a cellular context firstly by way of the loss of function of the gene to be analyzed (loss of function). In the second case, modifications caused by addition of the analyzed gene can be analyzed (gain of function).
a. Functional Analysis in Cells
Transfection. In order to analyze the gain of function, the gene of the target molecule must be transferred into the cell. For this purpose, cells are transfected with a DNA which allows synthesis of the target molecule. Normally, the gene of the target molecule here is under the control of a strong eukaryotic promoter (e.g. cytomegalovirus promoter; CMV). A wide variety of methods (e.g. electroporation, liposome-based transfection, calcium phosphate precipitation) are well established for transfecting cell lines with DNA (e.g. Lemoine et al., Methods Mol. Biol. 75: 441-7, 1997). The gene may be synthesized either transiently, without genomic integration, or else stably, with genomic integration after selection with neomycin, for example.
RNA interference (siRNA). An inhibition of expression of the target gene, which may induce a complete loss of function of the target molecule in cells, may be generated by the RNA interference (siRNA) technology in cells (Hannon, G J. 2002. RNA interference. Nature 418: 244-51; Czauderna et al. 2003. Nucl. Acid Res. 31: 670-82). For this purpose, cells are transfected with short, double-stranded RNA molecules of approx. 20-25 nucleotides in length, which are specific for the target molecule. An enzymic process then results in degradation of the specific RNA of the target gene and thus in an inhibition of the function of the target protein and consequently enables the target gene to be functionally analyzed.
Cell lines which have been modified by means of transfection or siRNA may subsequently be analyzed in different ways. The most common examples are listed below.
1. Proliferation
A multiplicity of methods for analyzing cell proliferation are established and are commercially supplied by various companies (e.g. Roche Diagnostics, Invitrogen; details of the assay methods are described in the numerous application protocols). The number of cells in cell culture experiments can be determined by simple counting or by colorimetric assays which measure the metabolic activity of the cells (e.g. wst-1, Roche Diagnostics). Metabolic assay methods measure the number of cells in an experiment indirectly via enzymic markers. Cell proliferation may be measured directly by analyzing the rate of DNA synthesis, for example by adding bromodeoxyuridine (BrdU), with the integrated BrdU being detected colorimetrically via specific antibodies.
2. Apoptosis and Cytotoxicity
A large number of assay systems for detecting cellular apoptosis and cytotoxicity are available. A decisive characteristic is the specific, enzyme-dependent fragmentation of genomic DNA, which is irreversible and results in any case in death of the cell. Methods for detecting these specific DNA fragments are commercially obtainable. An additional method available is the TUNEL assay which can detect DNA single-strand breaks also in tissue sections. Cytotoxicity is mainly detected via an altered cell permeability which serves as marker of the vitality state of cells. This involves on the one hand the analysis of markers which can typically be found intracellularly in the cell culture supernatant. On the other hand, it is also possible to analyze the absorbability of dye markers which are not absorbed by intact cells. The best-known examples of dye markers are Trypan blue and propidium iodide, a common intracellular marker is lactate dehydrogenase which can be detected enzymatically in the supernatant. Different assay systems of various commercial suppliers (e.g. Roche Diagnostics, Invitrogen) are available.
3. Migration Assay
The ability of cells to migrate is analyzed in a specific migration assay, preferably with the aid of a Boyden chamber (Corning Costar) (Cinamon G., Alon R. J. Immunol. Methods. 2003 February; 273(1-2):53-62; Stockton et al. 2001. Mol. Biol. Cell. 12: 1937-56). For this purpose, cells are cultured on a filter with a specific pore size. Cells which can migrate are capable of migrating through this filter into another culture vessel below. Subsequent microscopic analysis then permits determination of a possibly altered migration behavior induced by the gain of function or loss of function of the target molecule.
b. Functional Analysis in Animal Models
A possible alternative of cell culture experiments for the analysis of target gene function are complicated in vivo experiments in animal models. Compared to the cell-based methods, these models have the advantage of being able to detect faulty developments or diseases which are detectable only in the context of the whole organism. A multiplicity of models for human disorders are available by now (Abate-Shen & Shen. 2002. Trends in Genetics S1-5; Matsusue et al. 2003. J. Clin. Invest. 111:737-47). Various animal models such as, for example, yeast, nematodes or zebra fish have since been characterized intensively. However, models which are preferred over other species are animal models such as, for example, mice (Mus musculus) because they offer the best possibility of reproducing the biological processes in a human context. For mice, on the one hand transgenic methods which integrate new genes into the mouse genome have been established in recent years (gain of function; Jegstrup I. et al. 2003. Lab Anim. 2003 January; 37(1):1-9). On the other hand, other methodical approaches switch off genes in the mouse genome and thus induce a loss of function of a desired gene (knockout models, loss of function; Zambrowicz B P & Sands A T. 2003. Nat. Rev. Drug Discov. 2003 January; 2(1):38-51; Niwa H. 2001. Cell Struct. Funct. 2001 June; 26(3):137-48); technical details have been published in large numbers.
After the mouse models have been generated, alterations induced by the transgene or by the loss of function of a gene can be analyzed in the context of the whole organism (Balling R, 2001. Ann. Rev. Genomics Hum. Genet. 2:463-92). Thus it is possible to carry out, for example, behavior tests as well as to biochemically study established blood parameters. Histological analyses, immunohistochemistry or electron microscopy enable alterations to be characterized at the cellular level. The specific expression pattern of a gene can be detected by in-situ hybridization (Peters et al. 2003. Hum. Mol. Genet. 12:2109-20).
Example 1
Identification of the Hypothetical Protein FLJ31461 as Diagnostic and Therapeutic Cancer Target
Using gene prediction programs, FLJ31461 (SEQ ID NO: 1) filed under the gene bank accession number NM—152454 was determined as putative functionally not previously characterised gene on chromosome 15 (15q25.3). Two possible open reading frames result from the sequence deposited with the gene bank. The first reading frame encodes a protein with a length of 136 amino acids. The gene product (SEQ ID NO: 2) which was deposited in the RefSeq data bank of the NCBI under number NP—689667, accordingly has a calculated molecular weight of about 15 kDa. The second reading frame encodes a protein with a length of 100 amino acids (nucleotide sequence: SEQ ID NO: 69; amino acid sequence: SEQ ID NO: 70).
In sequence analyses of the gene FLJ31461 cloned by us, we were surprised to find the insertion of a nucleotide in the coding region in comparison to the sequences deposited in the databases. This results in a shifting of the reading frame. Two completely new open reading frames, which cannot be derived from the sequences already deposited in sequence databases, are the result. Hereby the new reading frame (SEQ ID NO: 71) encodes a new hypothetical protein with a length of 96 amino acids (SEQ ID NO: 72). SEQ ID NO: 73 encodes a hypothetical protein with the length of 133 amino acids (SEQ ID NO: 74). Because we have to assume, that the original depositions with the databases are incorrect, we have focussed further investigations on SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74.
In accordance with the invention, after the establishment of FLJ31461-specific quantitative RT-PCR (primer with the SEQ ID NO: 31, 32, 91, 92, 93, 94) the quantity of gene-specific transcripts was investigated in healthy tissue and in carcinoma samples (FIG. 1). With the exception of the testis, FLJ31461 cannot be detected in any of the normal tissues investigated by us (FIG. 1A). FLJ31461 is therefore with great probability a strongly gamete-specific gene-product. Surprisingly, we found during the analysis of tumors that FLJ31461 is switched on in many tumor types, while it is below the detection limit in the corresponding normal tissues (FIG. 1A-D). This does not only apply to virtually all breast tumors investigated by us (FIG. 1C) and also a series of lung tumors and nose-throat carcinomas, but also other neoplasias with varying frequency (FIG. 1D).
FLJ31461 is therefore a highly specific molecular marker for tumor tissues, which may be used diagnostically as well as therapeutically. As a typical representative of the class of so-called cancer/testis-antigens, which due to their selective tissue distribution serve as markers, this gene product can for example guarantee the precise targeting of tumor cells without damage to the normal tissues. Cancer/testis-genes are regarded as attractive target structures for targeted therapies and are already tested for specific immunotherapeutic approaches in cancerous diseases in phase I/II studies (i.e. Scanlan M J, Gure A O, Jungbluth A A, Old U, Chen Y T. 2002. Immunol. Rev. 2002 October; 188: 22-32).
In order to confirm these data on protein level, specific antibodies or immune sera have been generated by immunisation of animals. The protein topology was predicted by analysis of the transmembrane domains of SEQ ID NO: 72 and SEQ ID NO: 74 with bioinformatics tools (TMHMM, TMPRED). In this way for SEQ ID NO: 72 for example two transmembrane domains were predicted; the N-terminus and C-terminus of the protein are extracellular.
In accordance with the invention, peptide epitopes were chosen for immunisation, particularly extracellular peptide epitopes, which are specific for both protein variants.
Amongst others, the following peptides were selected for immunization in order to produce antibodies: SEQ ID NO: 61, 62, 96, 97.
By way of example the data for the antibody produced by immunisation using SEQ ID NO: 96, are shown. The specific antibody may be used under various fixation conditions for immunofluorescence investigations. In comparative staining of RT-PCR-positive as well as negative cell-lines, the respective protein is in well detectable quantity specific amongst others in those breast carcinoma cell-lines that were typed positive using quantitative RT-PCR (FIG. 2). The endogenous protein in this case presents membrane-localised.
Such antibodies are suitable for immunohistochemical staining of human tissue sections. To a large extent we were able to confirm the tissue distribution found on transcript level. While we observed hardly any reactivity of the antibody in normal tissue with the exception of testis tissue (FIG. 3A), antibodies against FLJ31461 stain various human tumor preparations, amongst these the tumors of breast and lung (FIG. 3B). The staining of the cells occurs accentuated at the membranes, which indicates a localisation of the protein at the cell surface. Surprisingly, we found that particularly metastases of tumors (FIG. 3B) express this protein particularly frequently and in a high proportion of cells.
These data indicate on one hand, that this gene found by us indeed does form a protein, that this protein is highly specific for human tumors and that it is present on the surface membrane of such tumor cells. Therefore this protein is accessible particularly for therapeutic antibodies. Likewise, our data prove, that specific antibodies against this protein may be produced. These antibodies bind selectively via the marker FLJ31461 to tumor cells.
In accordance with the invention such antibodies may be used for diagnostic purposes for example immunohistology. In particular, such antibodies may be used therapeutically. The produced antibodies can also be used directly for the production of chimeric or humanised recombinant antibodies. This can also be done directly with antibodies obtained from rabbits (cf. J Biol Chem. 2000 May 5; 275(18):13668-76 by Rader C, Ritter G, Nathan S, Elia M, Gout I, Jungbluth A A, Cohen L S, Welt S, Old L J, Barbas C F 3rd “The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies”). In order to achieve this, lymphocytes were taken from immunised animals. FLJ31461 is also a highly attractive target for immunotherapeutic procedures, such as vaccines or the adoptive transfer of antigen-specific T-lymphocytes.
Example 2
Identification of DSG4 (Desmoglein 4) as Diagnostic and Therapeutic Cancer Target
Gene DSG4 (desmoglein 4; SEQ ID NO: 75) with its translation product (SEQ ID NO: 76) is a member of the desmosomal cadherin-family. The gene consists of 16 exons and is located on chromosome 18 (18q12). The derived amino acid sequence encodes a precursor protein with a length of 1040 amino acids. The processed protein (N-terminally truncated by 49 amino acids) has a length of 991 amino acids and without modifications a molecular weight of about 108 kDa. It must be assumed that DSG4 is a glycosylised type 1 cell surface protein, just like other desmogleins. DSG4 was able to be detected as constituent of desmosomes (Kljuic et al. 2003. Cell 113: 249-260). Desmosomes are complex intercellular connections, which provide epithelial tissues (such as the epidermis) with mechanical stability. Auto-antibodies against other members of the desmoglein-family appear to contribute to the loss of cell-cell-contacts in the epidermis by binding to desmosomes and appear to contribute to the skin disease Pemphigus vulgaris. It has been described that DSG4 is not expressed in most healthy tissues. Significant expression has to date only been reported for salivary gland, testis, prostate and skin (Whittock, Bower 2003. J Invest Derm 120: 523-530). A connection with tumor diseases has not been discussed previously.
In accordance with the invention, the expression was investigated on healthy tissues and tumors using DSG4-specific oligonucleotides. Several DSG4-specific primer pairs were used for RT-PCR-investigations in accordance with the invention. These are: DSG4 primer pair SEQ ID NO: 77, 78 (exon 10 and exon 12), DSG4-primer pair SEQ ID NO: 83, 84 (exon 1 and exon 5), DSG4-primer pair SEQ ID NO: 89, 90 (exon 5 and exon 8) and DSG4-primer pair SEQ ID NO: 95, 78 (exon 8 and exon 12).
The investigation using all primer pairs confirmed that DSG4 is not expressed in most normal tissues. Depending on the primer pair however different expression patters were observed (FIG. 4B). With primer pairs SEQ ID NO: 95, 78 (exons 8-12) no expression was detected in normal tissue, with the exception of a very slight expression in prostate and skin. Surprisingly, DSG4 can be detected using this primer pair in a series of tumors. These are in particular tumors of the stomach, as well as carcinomas of the mouth, nose and throat area (FIG. 4A).
With primer pairs SEQ ID NO: 77, 78 (exons 10-12) even the expression in the above mentioned normal tissues of prostate and skin was less pronounced. Surprisingly, with this primer pair a more pronounced expression was observed in tumors (FIG. 4A). On one hand these tumors are those, which were conspicuous in investigations using the first primer pair, such as tumors of the stomach and carcinomas of the mouth, nose and throat area, but also other types of cancer (FIG. 4B, C). In particular in all intestinal tumors we detected a significant and high expression, which we were not able to detect using the first primer pair. The expression in the various tumors was manifold above that in the highest expressing toxicity-relevant normal tissue (FIG. 4B).
On the basis of these investigations, it appears that apart from the full-length transcript SEQ ID NO: 75 and the protein derived therefrom (SEQ ID NO: 76) also truncated variants of DSG4 exist, which lack regions before exon 9 (FIG. 5).
An extended analysis of the gene locus of DSG4 showed, that various variants of the molecule must be expected having a deletion before exon 9 (FIG. 5). These are the transcripts SEQ ID NO: 85, 87, 108, 110 and 112 and their altered protein products SEQ ID NO: 86, 88, 109, 111 and 113. The full-length transcript may also be modified in the regions beyond exon 10 and lead to variant transcripts SEQ ID NO: 102, 104, 106 and proteins SEQ ID NO: 103, 105, 107.
The variants truncated before exon 9 are even more tumor-selective than the full-length variant and can be found in additional tumor types, such as the colon carcinoma, in which the full-length variant is not expressed. Because the transmembrane domain is located in exon 12, the region amplified by primers SEQ ID NO: 77, 78 is extracellular and therefore should be accessible to antibodies. This truncated extracellular region contains the DSG4-gene sections exons 10, 11 and 12. Therefore transcripts containing exons 10, 11 and 12 (SEQ ID NO: 79) of DSG4, are particularly suitable as diagnostic and therapeutic cancer targets. These regions of DSG4 code for a domain (SEQ ID NO: 81), which is extracellular. Therefore DSG4-polynucleotides, which comprise exons 10, 11, 12 (SEQ ID NO: 75, 79, 80, 85, 87, 106, 112) and the polypeptides they encode (SEQ ID NO: 76, 81, 82, 86, 88, 107, 113) are particularly useful as target structure of monoclonal antibodies in accordance with the invention.
Accordingly, we have immunised animals with epitopes from the region of the full-length molecule (SEQ ID NO: 75) and from the extracellular area of the truncated molecule (SEQ ID NO: 81), respectively.
We were able to generate antibodies, which stain the DSG4 on the surface of cells transfected with DSG4. Specific antibodies are then able to specifically detect this protein using immunofluorescence (FIG. 6A) and flow cytometry (FIG. 6B) at the surface.
The pronounced expression and high incidence of this molecule for the presented tumor indications make this protein, and particularly its truncated variant, a highly interesting diagnostic and therapeutic marker in accordance with the invention. This also includes the detection of disseminated tumor cells in the serum, bone marrow and urine, as well as the detection of metastases in other organs using RT-PCR in accordance to the invention.
The extracellular domain of DSG4, particularly the part close to the cell membrane, may be utilised as target structure of monoclonal antibodies for therapy as well as immune diagnosis in accordance with the invention.
Furthermore, DSG4 can be used in accordance with the invention as vaccine (RNA, DNA, protein, peptides) for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions). In accordance with the invention, this comprises also the development of so-called “small compounds”, which modulate the biological activity of DSG4 and can be used for the therapy of tumors.
Example 3
Identification of DSG3 (Desmoglein3) as Diagnostic and Therapeutic Cancer Target
The gene DSG3 (desmoglein3; SEQ ID NO: 3) and its translation product (SEQ ID NO: 4) is a member of the desmosomal cadherin-family, which is published at the NCBI under accession number NM—001944 (nucleotide sequence) or NP—001935 (protein sequence). The gene consists of 15 exons and is located on chromosome 18 (18q12.1-q12.2). The derived amino acid sequence encodes a protein with 999 amino acids and a hypothetical size of about 130 kDa. DSG3 is a glycosylated type 1 cell surface protein and is able to be detected in desmosomes (Silos et al. J. Biol. Chem. 271: 17504-17511, 1996). Desmosomes are complex intracellular connections connecting the keratin filaments of adjacent cells in order to provide epithelial tissues (such as for example the epidermis) with mechanical stability. The desmosomal cadherines desmoglein and desmocollin are calcium-dependent adhesion molecules. Auto-antibodies against desmoglein3 and the resulting loss of cell-cell-contacts in the epidermis are involved in the skin disease Pemphigus vulgaris (Amagai et al., 1991. Cell 67: 869-877). This was also proven in animal models (Koch et al, 1997. J Cell Biol 5: 1091-1102).
In accordance with the invention, after establishment of a DSG3-specific quantitative RT-PCR (primer pair SEQ ID NO: 33, 34) the quantity of gene-specific transcripts was investigated in healthy tissues and carcinoma samples (FIG. 7; methods: compare Materials and Methods, Section B.1.). Our investigations demonstrated a differential distribution of the expression in normal tissues. DSG3 transcripts are hardly found in normal tissues. The only normal tissues expressing significant transcript quantities are the esophagus, skin and thymus (FIG. 7a). In all other analysed tissues, particularly brain, heart, liver, pancreas, PBMC, lung, mamma, ovary, kidney, spleen, colon, lymphatic node, uterus, bladder and prostate, transcription is low or not detectable (FIG. 7A). Surprisingly, we have been able to prove a significant, to date not described expression of DSG3 in some tumor types.
In quantitative RT-PCR-analyses of tumors DSG3-specific transcripts were proven amongst others in tumors of the nose-throat area (“head neck cancer”) in a quantity, which exceeded that of the highest expressing toxicity-relevant tissue (FIG. 7B). But also other tumors, such as carcinomas of the esophagus (FIG. 7C), express this protein.
We have stained sections of human tissues with DSG3-specific antibodies and were able to confirm the tumor-selectivity observed in the PCR (FIG. 8).
The pronounced expression and high incidence of this molecule in the described tumor-indications make this protein a highly interesting diagnostic and therapeutic marker in accordance with the invention. This includes in accordance with the invention the detection of disseminated tumor cells in the serum, bone marrow and urine, as well as the detection of metastases in other organs using RT-PCR.
The extracellular domain of the type I membrane protein desmoglein3 (SEQ ID NO: 4, amino acids 1-611) located on the N-terminus can be used in accordance with the invention as target structure of monoclonal antibodies for therapy as well as immune diagnosis. Furthermore, in accordance with the invention, DSG3 can be used as vaccine (RNA, DNA, protein, peptides) for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions). In accordance with the invention this comprises also the development of so-called “small compounds”, which modulate the biological activity of DSG3 and can be used for the therapy of tumors.
Example 4
Identification of the Transporter SLC6A3 (Solute Carrier Family 6) as Diagnostic and Therapeutic Cancer Target
The gene SLC6A3 (SEQ ID NO: 5) and its translation product (SEQ ID NO: 6) is a member of the sodium-neurotransmitter symporter family (SNF-family) and is deposited under accession number NM—001044 (nucleotide sequence) or NP—001035 (protein sequence). The gene consists of 16 exons and is located on chromosome 5 (5p15.3). The SLC6A3-gene encodes a glycoprotein with a length of 620 amino acids. SLC6A3 is an integral membrane protein with a total of 12 transmembrane domains, which as homo-oligomer represents part of an ion-transporter complex (Hastrup et al., 2003. J Biol Chem 278: 45045-48).
In accordance with the invention, after the establishment of a SLC6A3-specific quantitative RT-PCR (primer pair SEQ ID NO: 35, 36) the distribution of SLC6A3-specific transcripts was investigated in healthy tissue and carcinoma samples (FIG. 9; methods: compare Materials and Methods, Section B.1.). In most normal tissues SLC6A3 is only little or not at all expressed, a moderate expression of SLC6A3 was found only in thymus, spleen, ovary, pancreas as well as kidney. A significant, about 100-fold increased overexpression of SLC6A3 was detected in kidney carcinomas (FIG. 9A). A detailed analysis of the various kidney tissues using quantitative (FIG. 9B) and conventional RT-PCR (FIG. 9C) demonstrated, that SLC6A3 was expressed in 7/12 kidney cell carcinomas and overexpressed in 5/12 samples in comparison to non-tumorigenic samples. A significantly lower but detectable SLC6A3-specific expression was also demonstrated in some tumor tissues of other carcinomas. Particularly in some mamma carcinomas, ovarian carcinomas, bronchial carcinomas and prostate carcinomas SLC6A3-specific transcripts were detected (FIGS. 9D and 9E).
In accordance with the invention, the various extracellular domains of SLC6A3 can be used as target structures of monoclonal therapeutic antibodies. The following sequence regions with respect to SEQ ID NO: 6 are predicted as extracellular for SLC6A3 (based on an analysis using the software TMHMM2): amino acids 89-97, 164-237, 288-310, 369-397, 470-478, 545-558. The peptides listed under SEQ ID NO: 63 and 64 were used for the production of SLC6A3-specific antibodies.
Example 5
Identification of GRM8 as Diagnostic and Therapeutic Cancer Target
The gene GRM8/GluR8 or “metabotrophic glutamate receptor 8” (SEQ ID NO: 7) and its translation product (SEQ ID NO: 8) belongs to the family of glutamate receptors. The gene consists of 10 exons and is located on chromosome 7 (7q31.3-q32.1). The protein encoded by the GRM8 gene has a length of 908 amino acids, its calculated molecular weight is 102 kDa. Prediction programs predict 7 transmembrane domains. The protein exhibits a high homology (67% to 70% similarity) with GluR4 and GluR7 (Scherrer et al., 1996. Genomics 31: 230-233).
L-glutamate is an important neurotransmitter in the central nervous system and activates ionotrophic as well as metabotrophic glutamate receptors. GRM8-specific transcripts were to date only detected in the brain or glia-cells. However, to date no investigations comparing transcript or protein on a quantitative level of a larger number of tissues have been reported (Wu et al., 1998. Brain Res. 53: 88-97).
In accordance with the invention, after establishment of a GRM8-specific quantitative RT-PCR (primer pair SEQ ID NO: 37, 38) the distribution of GRM8-specific transcripts was investigated in healthy tissue and carcinoma samples (FIG. 10; methods: compare Materials and Methods, Section B.1.). Our investigations demonstrated a differential distribution of the expression in various normal tissues. We also found GRM8-transcripts selectively not only in the brain, but also in lesser quantities in the tissues of the stomach, intestinum, bladder, ovary, lung and pancreas. In most other normal tissues GRM8 is significantly less expressed or not at all detectable. In some tumors we were able to detect a significant, not previously described expression of GRM8. Particularly carcinomas of the colon, cervix and kidney cells exhibited a more than 10-fold overexpression in comparison to all other normal tissues and are also distinctly above the expression level of brain tissue (FIGS. 10A and 10B).
In accordance with the invention, the extracellular domains of GRM8 can be used as target structures of therapeutic monoclonal antibodies. With respect to SEQ ID NO: 8, the amino acids 1-582, 644-652, 717-743 and 806-819 are extracellularly localised.
Example 6
Identification of Cadherin 17 (CDH17) as Diagnostic and Therapeutic Cancer Target
The gene CDH17 (SEQ ID NO: 9) and its translation product (SEQ ID NO: 10) is a member of the cadherin-family. The gene consists of 18 exons and is located on chromosome 8 (8q22.1). It encodes a type 1 transmembrane protein with a length of 832 amino acids, which without secondary modifications has a calculated molecular weight of 92.1 kDa and which has one transmembrane domain. Cadherin 17 was cloned as proton-dependent peptide transporter by Dantzig et al. (Science 264: 430-433, 1994). The calcium-dependent glycoprotein cadherin 17 contains 7 cadherin-domains in the extracellular region (Gessner et al., Ann N Y Acad Sci.; 915:136-43, 2000). The intracellular domain does not exhibit any homology with other cadherins. Expression investigations were available only sporadically and not in the form of quantitatively comparative transcript or protein investigations of a larger number of different tissues.
In accordance with the invention, after the establishment of a CDH17-specific quantitative RT-PCR (primer pair SEQ ID NO: 39, 40) the distribution of CDH17-specific transcripts was investigated in healthy tissue as well as carcinoma samples (FIG. 11; methods: compare Materials and Methods, Section B.1.). In most normal tissues CDH17 is not at all detectable (FIG. 11A). We found significant transcript quantities selectively in stomach and intestinal tissues, far less expression in bladder, spleen, lymph nodes, thymus, prostate and esophagus. Surprisingly, we detected a distinct, not previously described CDH17-specific expression in tumors. For CDH17 in intestinal tumors an at least 2-10-fold overexpression was measured in comparison to normal tissues. CDH17 is also strongly expressed in stomach and esophagus tumors (FIGS. 11B and 11C).
The pronounced expression and high incidence of this molecule for the described tumor indications make this protein a highly interesting diagnostic and therapeutic marker in accordance with the invention. This includes in accordance with the invention the detection of disseminated tumor cells in serum, bone marrow and urine, as well as the detection of metastases in other organs using RT-PCR.
In accordance with the invention, the extracellular domain of CDH17 can be used as target structure of monoclonal antibodies for therapy as well as immune diagnosis. With respect to SEQ ID NO: 10, the amino acids 1-785 are localised extracellularly (prediction occurred using the software TMHMM2).
Furthermore, CDH17 can be used as vaccine (RNA, DNA, protein, peptides) for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions) in accordance with the invention. This includes in accordance with the invention also the development of so-called “small compounds”, which modulate the biological activity of CDH17 and can be used for the therapy of tumors.
Example 7
Identification of ABCC4 as Diagnostic and Therapeutic Cancer Target
The gene ABCC4 (SEQ ID NO: 11) and its translation product (SEQ ID NO: 12) encode an ABC transporter (ATP-binding-cassette). The gene consists of 31 exons and is located on chromosome 13 (13q31). It encodes a protein with a length of 1325 amino acids, which without modifications has a calculated molecular weight of about 149 kDa. ABCC4 is an integral membrane protein. The topology of ABCC4 is not yet clarified, prediction programs predict 12-14 transmembrane domains. ABC-transporters transport various molecules through extra- and intracellular membranes. ABCC4 is a member of the so-called MRP-family, of multi-drug-resistance proteins. The specific function of ABCC4 is not yet clarified, however it appears that the transporter plays a role in the cellular detoxification, which is made responsible for the chemotherapeutic resistance of many tumors.
The tissue distribution of this gene product over the various organs of the human body has not yet been investigated. In accordance with the invention, after establishment of an ABCC4-specific quantitative RT-PCR (primer pair SEQ ID NO: 41, 42) specific transcripts were investigated in healthy tissue and in carcinoma samples (FIG. 12; methods: compare Materials and Methods, Section B.1.). Our comparative investigations on all normal tissues confirm the published ubiquitous expression of ABCC4. ABCC4 was detected in all tested normal tissues. Surprisingly, we found, however, that in a number of tumors an overexpression of the transcript exceeding the expression for normal tissues was observed. In this respect, ABCC4 is found in 2-15-fold increased quantity in comparison to all analysed normal tissues for example in tumors of the kidney and prostate as well as bronchial tumors (FIG. 12).
The pronounced expression and high incidence of this molecule for the described tumor indications make this protein a highly interesting diagnostic and therapeutic marker in accordance with the invention. This includes in accordance with the invention the detection of disseminated tumor cells in serum, bone marrow and urine, as well as the detection of metastases in other organs with the aid of RT-PCR.
In accordance with the invention, the extracellular domains of ABCC4 can be used as target structures of monoclonal antibodies for therapy as well as immune diagnosis. The exact localisation of the extracellular domains is still unknown. With respect to SEQ ID NO: 12, the software TMHMM2 predicts the amino acids 114-132, 230-232, 347-350, 730-768, 879-946 and 999-1325 as extracellular.
Furthermore, ABCC4 may be used as vaccine (RNA, DNA, protein, peptides) in accordance with the invention for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions). This includes in accordance with the invention also the development of so-called “small compounds”, which modulate the biological activity of ABCC4 and can be used for the therapy of tumors.
Example 8
Identification of VIL1 as Diagnostic and Therapeutic Cancer Target
The gene VIL1 or “Villin1” (SEQ ID NO: 13) and its translation product (SEQ ID NO: 14) are encoded by a gene consisting of 19 exons on chromosome 2 (2q35-q36). The gene encodes a protein with 826 amino acids, which without modifications has a calculated molecular weight of about 92 kDa. Villin is the structural main component of microvilli in cells of the gastro-intestinal and urogenital epithelia. It represents a calcium-regulated, actin-binding protein.
Pringault et al. (EMBO J. 5: 3119-3124, 1986) cloned villin1 and were able to prove the existence of two transcripts (2.7 kb and 3.5 kb). These variants arise due to the use of alternative polyadenylation signals in the last exon. VIL1-specific transcripts were previously described in a multitude of tissues such as brain, heart, lung, intestine, kidney and the liver. However, previously no comprehensive quantitatively comparative transcript or protein investigations on a larger number of tissues were carried out, which might have given information regarding the usefulness of VIL1 for therapeutic purposes.
In accordance with the invention, after establishment of a VIL1-specific quantitative RT-PCR (primer pair SEQ ID NO: 43, 44) the distribution of the specific transcripts in healthy tissue and carcinoma samples were investigated (FIG. 13; methods: compare Materials and Methods, Section B.1.). Our comparative investigations regarding all normal tissues demonstrate a differential distribution of the VIL1-specific expression. In almost all normal tissues VIL1-specific transcripts are not detectable (FIG. 13A). In particular our findings disprove the previously described expression in brain, heart, breast, ovary, lymph nodes, esophagus, skin, thymus, bladder and muscle. We only found VIL1-transcripts in stomach and intestine and a lower expression in pancreas, liver and PBMCs.
Surprisingly, however, we detected a significant, but previously not described VIL1-specific overexpression in tumors. For example in carcinomas of the colon and stomach a 5- to 10-fold overexpression was observed in comparison to all analysed normal tissues (FIGS. 13A and 13B). A significant VIL1-specific expression is also detectable in tumors of the pancreas, stomach and liver as well as bronchial tumors.
The pronounced expression and high incidence of this molecule for the described tumor indications make this protein in accordance with the invention a highly interesting diagnostic and therapeutic marker. This includes in accordance with the invention the detection of disseminated tumor cells in serum, bone marrow and urine, as well as the detection of metastases in other organs with the aid of RT-PCR.
In accordance with the invention, it can be used as vaccine (RNA, DNA, protein, peptides) for the induction of tumors-specific immune responses (T-cell and B-cell mediated immune reactions). In accordance with the invention, this also includes the development of so-called “small compounds”, which modulate the biological activity of VIL1 and can be used for the therapy of tumors.
Example 9
Identification of MGC34032 as Diagnostic and Therapeutic Cancer Target
The translation product (SEQ ID NO: 16) of gene MGC34032 (SEQ ID NO: 15) is a hypothetical protein with currently unknown function. The gene consists of 28 exons and is located on chromosome 1 (1p31.1). The gene encodes a protein with a length of 719 amino acids which has a calculated molecular weight of about 79 kDa. Prediction programs consistently predict 8 transmembrane domains. Homologies are not known, publications regarding MGC34032 do not exist.
In accordance with the invention, after establishment of a MGC34032-specific quantitative RT-PCR (primer pair SEQ ID NO: 45, 46) the distribution of specific transcripts was investigated in healthy tissue and carcinoma samples (FIG. 14; methods: compare Materials and Methods, Section B.1.). We found MGC34032-transcripts in all tested normal tissues. The comparison of transcript quantities in normal tissues with those found in tumors, however, showed surprisingly, that various tumor-types exhibited a significant, not previously described 5- to 10-fold overexpression of this gene product. These are particularly carcinomas of the esophagus, colon, ovary, lung and kidney cells as well as ear-nose-throat carcinomas (FIG. 14A-D).
In order to produce MGC34032-specific antibodies the peptides listed under SEQ ID NO: 98 and 99 were used. These antibodies were able stain MGC34032 at the cell surface (FIG. 15A).
The pronounced expression and high incidence of this molecule for the described tumor indications make this protein in accordance with the invention a highly interesting diagnostic and therapeutic marker. This also includes in accordance with the invention the detection of disseminated tumor cells in serum, bone marrow and urine, as well as the detection of metastases in other organs with the aid of RT-PCR.
The extracellular domains of MGC34032 may be used in accordance with the invention as target structures of monoclonal antibodies for therapy as well as immune diagnosis. With respect to SEQ ID NO: 16, the amino acids 62-240, 288-323, 395-461 and 633-646 are extracellularly localised (prediction occurred with the aid of the TMHMM2-software).
Furthermore, MGC34032 may be used in accordance with the invention as vaccine (RNA, DNA, protein, peptides) for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions). This includes in accordance with the invention also the development of so-called “small compounds”, which modulate the biological activity of MGC34032 and may be used for the therapy of tumors.
Example 10
Identification of the Serine Protease PRSS7 (Enterokinase) as Diagnostic and Therapeutic Cancer Target
The gene PRSS7 (SEQ ID NO: 17) and its translation product (SEQ ID NO: 18) belong to the family of serine proteases. The gene consists of 25 exons and is located on chromosome 21 (21q21). The gene encodes a protein with a length of 1019 amino acids, which is further processed after translation. The active enzyme consists of 2 peptide chains, connected by a disulfide-bridge, which are derived from a common precursor molecule through proteolytic cleavage. The heavy chain consists of 784 amino acids. The light chain consisting of 235 amino acids exhibits a distinct homology to known serine proteases. Prediction programs predict one transmembrane domain for PRSS7. PRSS7 is particularly formed in the apical cells and enterocytes of the small intestine and therefore aids in the initial activation of the proteolytic enzymes of the pancreas (such as trypsin, chymotrypsin and carboxypeptidase) (Imamura and Kitamoto, Am J Phsyiol Gastrointest Liver Physiol 285: G1235-G1241, 2003). To date this protein had not been associated with human tumors.
In accordance with the invention, after establishment of a PRSS7-specific quantitative RT-PCR (primer pair SEQ ID NO: 47, 48) the distribution of specific transcripts was investigated in healthy tissue and carcinoma samples (FIG. 16; methods: compare Materials and Methods, Section B.1.). In most analysed tissues we were not able to detect PRSS7-specific expression at all or only to a very small extent (FIG. 16A). Relevant expression was only found in the duodenum (FIG. 16B).
PRSS7 is expressed by various tumor types. In a part of the investigated stomach carcinomas a distinct overexpression was detected in comparison to normal stomach tissue (FIG. 16B). Furthermore, carcinomas of the esophagus, liver as well as pancreas expressed PRSS7, in part the gene was distinctly overexpressed in some tumor samples in comparison to the corresponding normal tissues (FIGS. 16B and 16C).
The pronounced expression and high incidence of this molecule for the described tumor indications make this protein in accordance with the invention a highly interesting diagnostic and therapeutic marker. This also includes in accordance with the invention the detection of disseminated tumor cells in serum, bone marrow and urine as well as the detection of metastases in other organs with the aid of RT-PCR.
We have stained cells transfected by PRSS7, as well as sections of human tissues with PRSS7-specific antibodies and were able to confirm the predicted protein topology on the membrane (FIGS. 17A and 17B).
The extracellular part of PRSS7 can be used in accordance with the invention as target structure of monoclonal antibodies for therapy as well as immune diagnosis. With respect to SEQ ID NO: 18, the amino acids starting from amino acid residue 50 are extracellularly localised. Furthermore, in accordance with the invention, PRSS7 can be used as vaccine (RNA, DNA, protein, peptides) for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions). This includes in accordance with the invention also the development of so-called “small compounds”, which modulate the biological activity of PRSS7 and may be used in the therapy of tumors.
Example 11
Identification of CLCA2 as Diagnostic and Therapeutic Cancer Target
The gene CLCA2 or “calcium activated chloride channel 2” (SEQ ID NO: 19) belongs to the family of chloride ion transporters. The gene consists of 14 exons and is located on chromosome 1 (1p31-p22). The gene encodes a protein with a length of 943 amino acids, which has a calculated molecular weight of about 120 kDa. Experimentally, 5 transmembrane domains as well as a large, N-terminally localised extracellular domain were detected. CLCA2 is an ion-transporter (Gruber, 1999. Am J Physiol 276, C1261-C1270).
CLCA2-transcripts were previously described in the lung, trachea and the mammary gland (Gruber, 1999. Am J Physiol 276, C1261-C1270), as well as in the tissues of testis, prostate and uterus (Agnel, 1999. FEBS Letters 435, 295-301). Comparative investigations in a comprehensive collection of tissues were not previously available.
In accordance with the invention, after establishment of a CLCA2-specific quantitative RT-PCR (primer pair SEQ ID NO: 49, 50) the distribution of specific transcripts was investigated in almost all healthy tissues of the human body and in tumor samples (FIG. 18; methods: compare Materials and Methods, Section B.1.). We found a differential expression of CLCA2 in normal tissues. In most analysed tissues transcription is not detectable. Only in the esophagus, skin, pancreas, and significantly less in thymus, bladder, colon and prostate were we able to detect expression. Surprisingly, we found in some tumor types significant, not previously described expression of CLCA2. In particular tumors of the nose-throat area, as well as breast, esophagus, ovary and pancreas carcinomas as well as bronchial carcinomas exhibited a CLCA2-specific expression increased by a factor of 10 to 1000 in comparison to the corresponding normal tissues (FIG. 18).
The pronounced expression and high incidence of this molecule for the described tumor indications make this protein in accordance with the invention a highly interesting diagnostic and therapeutic marker. This includes in accordance with the invention also the detection of disseminated tumor cells in serum, bone marrow and urine as well as the detection of metastases in other organs with the aid of RT-PCR.
The two extracellular domains (with respect to SEQ ID NO: 20; amino acids 1-235, 448-552 and 925-943) may be used in accordance with the invention as target structures of monoclonal antibodies for therapy as well as in immune diagnosis.
By immunization using CLCA2-specific peptides (SEQ ID NO: 100, SEQ ID NO: 101) antibodies could be produced staining CLCA2 on the cell surface. Cells transfected by CLCA2 express this protein on the cell membrane (FIG. 19A). The tumor selectivity could be confirmed in immunofluorescence using the specific antibody (FIG. 19B).
Furthermore, CLCA2 may be used in accordance with the invention as vaccine (RNA, DNA, protein, peptides) for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions). This includes in accordance with the invention also the development of so-called “small compounds”, which modulate the biological activity of CLCA2 and may be used for the therapy of tumors.
Example 12
Identification of TM4SF4 (“Transmembrane 4 Superfamily Member 4”) as Diagnostic and Therapeutic Cancer Target
The gene TM4SF4 (SEQ ID NO: 21) and its translation product (SEQ ID NO: 22) is a member of the tetraspanin family (Hemler, 2001. J Cell Biol 155, 1103-07). The gene consists of 5 exons and is located on chromosome 3 (3q25).
The gene encodes a protein with a length of 202 amino acids and a calculated molecular weight of about 21.5 kDa. Prediction programs consistently predict 4 transmembrane domains for TM4SF4. The protein is N-glycosylated in the region of the second extracellular domain and is located in the cell membrane. It is described that the degree of N-glycosylation has an effect on the regulation of the cell proliferation and that it is inhibited with increasing glycosylation (Wice & Gordon, 1995. J Biol Chem 270, 21907-18). Tetraspanines form complexes with various members of the group of integrins. These high-molecular multi-complexes are ascribed a multitude of important functions in the cell. For example, they fulfil functions in the cell-cell-adhesion and in intercellular contacts, in the signal transduction and in cell motility (Bereditschevski, 2001. J Cell Sci 114, 4143-51).
TM4SF4-transcripts are described in the periportal area of the liver as well as in specific sections of the intestine, but were not previously analysed in other tissues and in particular not in tumors (Wice & Gordon, 1995. J Biol Chem 270, 21907-18). In accordance with the invention, after establishment of a TM4SF4-specific quantitative RT-PCR (primer pair SEQ ID NO: 51, 52) the distribution of specific transcripts in healthy tissue and in carcinoma samples was investigated (FIG. 20; methods: compare Materials and Methods, Section B.1.). Our investigations showed a differential distribution of the expression in normal tissues. TM4SF4-specific transcripts were mainly found in samples of normal liver tissue. In several other normal tissues (amongst others pancreas) we found a distinctly lower expression (at least 10-fold). Expression was not detectable in the brain, heart muscle, skeletal muscles, skin, breast tissue, ovary, PBMC, spleen, lymph nodes and cervix. Contrary to the published prediction, that TM4SF4 is down-regulated in tumor tissue (Wice & Gordon, 1995. J Biol Chem 270, 21907-18), at least comparable TM4SF4-specific expression was shown in various tumors; in part TM4SF4 was overexpressed in tumors (FIG. 20A). In a detailed expression analysis we were also able to prove contrary to published data, that TM4SF4 is not suppressed in liver tumors (FIG. 20B). In addition, the gene was overexpressed in 4/12 colon tumor samples in comparison to normal colon tissue (FIG. 20C).
In order to produce TM4SF4-specific antibodies, the peptides listed under SEQ ID NO: 65 and 66 were used. These antibodies were able to recognise the TM4SF4-protein in various sizes, which represent putative glycosylation patters (FIG. 21A). Furthermore, the surface localisation of TM4SF4 could be confirmed with the aid of immunofluorescence (FIG. 21B) and the tumor-selectivity observed in the PCR could be confirmed with the aid of immunohistological staining of human tissues (FIG. 21C).
In summary, TM4SF4 can be characterised as a membrane protein, whose expression is limited to cell-subpopulations of a few selected normal tissues. TM4SF4 is particularly detectable in the periportal hepatocytes in the liver and in the apical membrane of the epithelia of the gastro-intestinal tract. In the case of apical protein localisation, the protein is not accessible in normal cells to antibodies, because in the intestinal epithelium it faces the lumen and therefore is not connected to the vascular system. In intestinal tumors, however, these molecules, which are not accessible in healthy tissue, are no longer compartmented due to uncontrolled proliferation and the neovascularisation of the tumor, and are therefore accessible for therapeutic antibodies.
The two extracellular domains of TM4SF4 therefore may be used in accordance with the invention as target structures of monoclonal antibodies. With respect to SEQ ID NO: 22, the amino acids 23-45 and 110-156 are located extracellularly (prediction was performed using the software TMHMM2). For the peptides with the SEQ ID NO: 65 and 66 polyclonal antibodies were already successfully generated (Wice & Gordon, 1995. J Biol Chem 270:21907-18). For therapeutic approaches for the development of tumor-specific antibodies the peptides SEQ ID NO: 67 and SEQ ID NO: 68 are suitable, which each contain a conserved motive “NXS/T” for posttranslational N-glycosylations, whereby “X” represents any amino acid except proline.
Example 13
Identification of CLDN19 as Diagnostic and Therapeutic Cancer Target
The gene CLDN19 or claudin19 (SEQ ID NO: 23) with its translation product (SEQ ID NO: 24) is a member of the claudin family.
The gene encodes a protein with a length of 224 amino acids which has a calculated molecular weight of about 21.5 kDa. Prediction programs consistently predict for claudin19 the 4 transmembrane domains characteristic for the family of claudins. Claudin19 to date has not been functionally characterised in greater detail. Functions have been described for other members of the claudin-family. Accordingly, claudins play an important role in cell-cell-adhesion and in intercellular contacts. They are part of large molecule complexes and so form membrane pores (“tight junctions”) for cell-cell-contacts.
In accordance with the invention, after establishment of a CLDN19-specific quantitative RT-PCR (primer pair SEQ ID NO: 53, 54) the distribution of specific transcripts was investigated in healthy tissue and carcinoma samples (FIG. 22; methods: compare Materials and Methods, Section B.1.). Surprisingly, we found a differential distribution of the expression in normal tissues. In the majority of normal tissues (in particular in the brain, heart muscle, skeletal muscle, liver, pancreas, PBMCs, lung, breast tissue, ovary, spleen, colon, stomach, lymph nodes, esophagus, skin and prostate) CLDN19 is not detectable. Only in normal tissue of the bladder, thymus and testis we were able to detect CLDN19-transcripts. The comparative investigation of tumor tissues showed surprisingly that CLDN19 is expressed by various tumors. These are particularly carcinomas of kidney, stomach, liver and breast, which in comparison to corresponding normal tissues exhibit an up to 10-fold overexpression. CLDN19 has not previously been described in the context of human tumors (FIG. 22A-22E).
The pronounced expression and high incidence of this molecule for the described tumor indications make this protein in accordance with the invention a highly interesting diagnostic and therapeutic marker. In accordance with the invention, this includes the detection of disseminated tumor cells in serum, bone mark and urine, as well as the detection of metastases in other organs with the aid of RT-PCR.
The two extracellular domains (amino acids 28-76 and 142-160 with respect to SEQ ID NO: 24) of CLDN19 may be used in accordance with the invention as target structures of monoclonal antibodies for the therapy and immune diagnosis.
Furthermore, CLDN19 may be used in accordance with the invention as vaccine (RNA, DNA, protein, peptides) for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions). In accordance with the invention, this includes the development of so-called “small compounds”, which modulate the biological activity of CLDN19 and may be used for the therapy of tumors.
Example 14
Identification of ALPPL2 as Diagnostic and Therapeutic Cancer Target
The gene ALPPL2 or “stem cell-specific alkaline phosphatase” or GCAP (SEQ ID NO: 25) encodes a protein (SEQ ID NO: 26) belonging to the family of alkaline phosphatases (AP). This consists of four very homologous members in total (homology: 90-98%). The gene codes for a transcript with a length of 2486 bp and consists of 11 exons. ALPPL2 is located on chromosome 2 (2q37.1) in the vicinity of its closely related family members ALPP and ALPI.
The derived protein has a length of 532 amino acids and a calculated molecular weight of about 57.3 kDa. ALPPL2 is glycosylated and located in the plasma membrane as homodimer via a GPI-anchor. The exact physiological function of the enzyme is not known. For osteosarcomas or Paget's disease the alkaline phosphatase enzyme activity is used as tumor marker (Millán, 1995. Crit Rev Clin Lab Sci 32, 1-39). However, this determination is non-specific and independent from the actual underlying molecule. It is not clear, which of the three above mentioned phosphatases or possibly even other currently not known phosphatases result in this activity.
ALPPL2 has been used previously only as diagnostic marker “in situ” for the diagnosis of gamete tumors (Roelofs et al., 1999. J Pathol 189, 236-244).
In accordance with the literature concerning a limited initial set of tissue types, ALPPL2 is expressed in testis and in the thymus as well as in some stem cell tumors (LeDu, 2002. J Biol Chem 277, 49808-49814). In accordance with the invention after establishment of an ALPPL2-specific quantitative RT-PCR (primer pair SEQ ID NO: 55, 56) the distribution of this gene product was investigated in healthy tissue and in carcinoma samples, whereby a comprehensive diversity of tissues was investigated, which amongst others also represented all body tissues (FIG. 23; methods: compare Materials and Methods B.1.). We detected no protein in most normal tissues (particularly in the brain, heart muscle, skeletal muscle, liver, pancreas, PBMCs, breast tissue, ovary, spleen, colon, stomach, lymph nodes, esophagus, skin and prostate). We demonstrated expression in normal tissues of testis and lung, and very low levels in the thymus and colon. The comparative investigation of tumors, however, surprisingly showed that ALPPL2 is expressed in significant quantities by various tumor types, particularly in carcinomas of the colon, stomach, pancreas, ovary and lung, but also in carcinomas of the nose-throat area (FIGS. 23A and 23B).
The pronounced expression and high incidence of this molecule for the described tumor indications make this protein in accordance with the invention a highly interesting diagnostic and therapeutic marker. This includes in accordance with the invention the detection of disseminated tumor cells in the serum, bone marrow and urine, as well as the detection of metastases in other organs with the aid of RT-PCR.
The entire ALPPL2-protein (SEQ ID NO: 26) is extracellularly located and therefore can be used in accordance with the invention as a target structure for developing monoclonal antibodies for therapy as well as immune diagnosis.
Furthermore, ALPPL2 in accordance with the invention can be used as vaccine (RNA, DNA, protein, peptides) for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions). This includes in accordance with the invention also the development of so-called “small compounds”, which modulate the biological activity of ALPPL2 and can be used in the therapy of tumors.
Example 15
Identification of GPR64 as Diagnostic and Therapeutic Cancer Target
The gene GPR64 or “G-protein coupled receptor 64” (SEQ ID NO: 27) and its translation product (SEQ ID NO: 28) belongs to a large group of 7-transmembrane receptors. The gene encodes a transcript with a length of 3045 bp and consists of 27 exons. GPR64 is located on the chromosome (Xp22). The gene encodes a protein with a length of 987 amino acids which has a calculated molecular weight of about 108 kDa. The N-terminal region represents an extracellular domain, which is strongly glycosylated. The exact physiological function of this protein is not known.
GPR64 has been investigated to date in only a small number of normal tissues, amongst which only the tissue of the epididymis was found to express this gene (Osterhoff, 1997. DNA Cell Biol 16, 379-389). In accordance with the invention we have established a GPR64-specific RT-PCR (primer pair SEQ ID NO: 57, 58) and have investigated the distribution of this gene product in a comprehensive collection of healthy tissues (FIG. 24; methods: compare Materials and Methods, Section B.1.). In many normal tissues GPR64 is not detectable at all, some exhibit a low expression. Surprisingly, the investigation of this protein in tumors exhibited an overexpression, which was many times higher than that of the relevant normal tissues. For example, we found significant overexpression in almost half of the ovary carcinomas (FIG. 24A to 24C).
The pronounced expression and high incidence of this molecule in the described tumor indications make this protein in accordance with the invention a highly interesting diagnostic and therapeutic marker. This includes in accordance with the invention the detection of disseminated tumor cells in serum, bone marrow and urine, as well as the detection of metastases in other organs with the aid of RT-PCR.
The four extracellular domains of GPR64 may be used in accordance with the invention as target structures of monoclonal antibodies for therapy as well as immune diagnosis. With respect to SEQ ID NO: 28, the amino acids 1-625, 684-695, 754-784 and 854-856 are located extracellularly.
Furthermore, GPR64 can be used in accordance with the invention as vaccine (RNA, DNA, protein, peptides) for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions). This also includes in accordance with the invention the development of so-called “small compounds”, which modulate the biological activity of GPR64 and may be used for the therapy of tumors.
Example 16
Identification of the Sodium/Potassium/Chloride Transporter SLC12A1 (Solute Carrier Family 12) as Diagnostic and Therapeutic Cancer Target
The gene SLC12A1 (SEQ ID NO: 29) encodes a translation product (SEQ ID NO: 30) and belongs to the family of sodium-potassium-chloride-co-transporters. The gene consists of 26 exons and is located on chromosome 15 (15q15-q21.1). It encodes a protein with a length of 1099 amino acids which has a calculated molecular weight without secondary modifications of about 120 kDa. SLC12A1 is an integral membrane protein with 10 transmembrane domains. SLC12A1 mediates the reabsorption of sodium chloride in the Henle-Schleife and is the target point of many clinically relevant diuretic agents (Quaggin et al., Mammalian Genome 6: 557-561, 1995). Correspondingly, this molecule is principally accessible as target structure for medicaments, in other words it is “druggable”.
In accordance with the invention, after establishment of a SLC12A1-specific quantitative RT-PCR (primer pair SEQ ID NO: 59, 60) the distribution of specific transcripts in healthy tissue and in carcinoma samples was investigated (FIG. 25). We confirmed that in normal tissues the expression of SLC12A1 is first and foremost limited to normal kidney tissue, as has also been described in the literature. In all other normal tissues SLC12A1-specific transcripts are detectable in only very small quantities or not all (FIG. 25A). Surprisingly, in the comparative analysis of tumors we found an expression of SLC12A1. Especially in carcinomas of the kidney, breast, ovary and prostate (FIG. 25A to 25C) we found unexpectedly an up to 1,000,000-fold over-expression in comparison to the corresponding normal tissues (FIG. 25B to 25D). Previously, SLC12A1 has not been described in the context of tumor diseases.
The pronounced expression and high incidence of this molecule for the described tumor indications make this protein in accordance with the invention a highly interesting diagnostic and therapeutic marker. This includes in accordance with the invention the detection of disseminated tumor cells in serum, bone marrow and urine, as well as the detection of metastases in other organs with the aid of RT-PCR. The extracellular domains of SLC12A1 may be used in accordance with the invention as target structures of monoclonal antibodies for therapy and also immune diagnosis. With respect to SEQ ID NO: 30, the amino acids 1-181, 234-257, 319-327, 402-415, 562-564 and 630-1099 are located extracellularly.
Furthermore, SLC12A1 can be used in accordance with the invention as vaccine (RNA, DNA, protein, peptides) for the induction of tumor-specific immune responses (T-cell and B-cell mediated immune reactions). This includes in accordance with the invention also the development of so-called “small compounds”, which modulate the biological activity of SLC12A1 and may be used for the therapy of tumors.

## Claims

1. A method of detecting and treating a head neck cancer which is characterized by expressing a tumor-associated antigen having the amino acid sequence shown in SEQ ID NO:4, which method comprises:
(A) detecting or quantifying in a biological sample isolated from a patient said tumor-associated antigen by:
(i) isolating a biological sample from tissue in the head or neck region of a patient suspected of having head neck cancer,
(ii) contacting the biological sample with a first antibody which binds specifically to the tumor-associated antigen,
(iii) detecting or quantifying the formation of a complex between the first antibody and the tumor-associated antigen,
(iv) comparing the presence or quantity of the complex in the biological sample from the patient to the presence or quantity of the complex in a comparable normal biological sample that also has been contacted with the antibody,
(v) concluding from an increase in the amount of the complex in the biological sample from the patient relative to the normal biological sample that the patient is affected by a head neck cancer, and

(B) treating the so-affected patient with a second antibody which specifically binds to an extracellular portion of said tumor-associated antigen and which is coupled to a therapeutic agent.

2. The method of claim 1, wherein the biological sample comprises body fluid, body tissue, or a combination thereof.

3. The method of claim 1, wherein the biological sample comprises ear, nose or throat tissue.

4. The method of claim 1, wherein the first antibody is labeled with a detectable marker.

5. The method of claim 4, wherein the detectable marker is a radioactive marker or an enzymic marker.

6. The method of claim 1, wherein the first and second antibodies are monoclonal antibodies.

7. The method of claim 4, wherein the detectable marker is selected from the group consisting of barium sulfate, iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, and sodium tyropanoate.

8. The method of claim 5, wherein the detectable marker is a radioactive marker selected from the group consisting of fluorine-18, carbon-11, iodine-123, technetium-99, iodine-131, and indium-111.

9. The method of claim 1, wherein the therapeutic agent is an anticancer agent, a radioactive iodine-labeled compound, a toxin, a cytostatic drug, or a cytolytic drug.

10. The method of claim 1, wherein the therapeutic agent is an anticancer agent selected from the group consisting of aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil, interferon-α, lomustine, mercaptopurine, methoxtrexate, mitotane, procarbazine HCl, thioguanine, vinblastine sulfate, and vincristine sulfate.

11. The method of claim 9, wherein the therapeutic agent is a toxin selected from the group consisting of pokeweed antiviral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin, diptheria exotoxin and Pseudomonas exotoxin.