AU2017201358B2

AU2017201358B2 - Genetic products differentially expressed in tumors and the use thereof

Info

Publication number: AU2017201358B2
Application number: AU2017201358A
Authority: AU
Inventors: Michael Koslowski; Ugur Sahin; Özlem TÜRECI
Original assignee: Biontech SE
Current assignee: Biontech SE
Priority date: 2002-11-22
Filing date: 2017-02-28
Publication date: 2019-05-02
Anticipated expiration: 2023-11-21
Also published as: AU2015242979B2; AU2017201358A1; AU2015242979A1

Abstract

The invention relates to the identification of genetic products expressed in association with tumors and to coding nucleic acids for said products. Said invention also relate' to the therapy and diagnosis of disease in which the genetic products are aberrantly expressed in association with tumors, proteins, polypeptides and peptides which are expressed in association with tumors, and to the nucleic coding acids for said polypeptides, peptides and proteins.

Description

GENETIC PRODUCTS DIFFERENTIALLY EXPRESSED IN TUMORS AND

THE USE THEREOF

The present application is a divisional application from

Australian patent application number 2015242979, which is a 5 divisional application from Australian patent application 2012233044, which is a divisional from Australian patent application number 2010202440, which is a divisional from Australian patent application number 2003282101, the entire disclosures of which are incorporated herein by reference.

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 15 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 20 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 25 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 tumorassociated antigens involves cellular and humoral mechanisms 30 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.

2017201358 28 Feb 2017

The potential clinical-therapeutical importance of tumorassociated 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 5 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 10 appropriate cloning techniques has it been possible to screen cDNA expression libraries of tumors systematically for tumorassociated 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 20 autoantibodies were utilized for cloning the respective antigens .

In recent years a multiplicity of antigens have been defined in various neoplasias by these approaches. However, the probes 25 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 30 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.

It was the object of the present invention to provide target structures for a diagnosis and therapy of cancers.

2017201358 28 Feb 2017

According to the invention, subject matter of the claims.

According to the invention, providing antigens expressed this object is achieved by the a strategy for identifying and in association with a tumor and the nucleic acids coding therefor was pursued. This strategy is based on the fact that particular genes which are expressed in 15 an organ specific manner, e.g. exclusively in colon, lung or kidney tissue, are reactivated also in tumor cells of the respective organs and moreover in tumor cells of other tissues in an ectopic and forbidden manner. First, data mining produces a list as complete as possible of all known organ-specific genes which are then evaluated for their aberrant activation in different tumors by expression analyses by means of specific RT-PCR. Data mining is a known method of identifying tumorassociated 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	) ·
30
The	concept of	the	invention,	which	has proved	much	more

successful, however, is based on utilizing data mining for electronically extracting all organ-specific genes and then evaluating said genes for expression in tumors.

2017201358 28 Feb 2017

The invention thus relates in one aspect to a strategy for identifying tissue-specific genes differentially expressed in tumors. Said strategy combines data mining of public sequence libraries (in silico) with subsequent evaluating laboratoryexperimental (wet bench) studies.

According to the invention, a combined strategy based on two different bioinformatic scripts enabled new tumor genes to be 10 identified. These have previously been classified as being purely organ-specific. The finding that these genes are aberrantly activated in tumor cells allows them to be assigned a substantially new quality with functional implications.

According to the invention, these tumor-associated genes and 15 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: 1-8, 41-44, 51-59, 84, 117, and 119, a part or derivative thereof, (b) a nucleic acid which hybridizes with the nucleic acid of (a) under stringent 25 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 30 by a nucleic acid which is selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117, and 119. 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:

2017201358 29 Jan 2019

9-19, 45-48, 60-66, 85, 90-97, 100-102, 105, 106, 111-116, 118,

120, 123, 124, and 135-137, a part or derivative thereof.

The present invention generally relates to the use of tumor5 associated antigens identified according to the invention or of parts or derivatives 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 or derivatives 10 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 tumorassociated genes and antigens for diagnosis, therapy and 15 progress control.

The invention also relates to a pharmaceutical composition when used for treating or preventing a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, 20 comprising an agent which inhibits expression or activity of a tumor-associated antigen, said tumor-associated antigen having a 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 NO:

84, 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
30	the	nucleic acid of	(a) or	(b), and
	(d)	a nucleic acid	which	is complementary to	the nucleic
	acid	of	(a) ,	(b) or	(c) ,

5a

2017201358 29 Jan 2019 wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .

The invention also relates to a pharmaceutical composition when used for treating or preventing a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, comprising an agent with tumor-inhibiting activity, which is selective for cells expressing or abnormally expressing a 10 tumor-associated antigen, said tumor-associated antigen having a sequence encoded by a nucleic acid which is selected from the group consisting of:

84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .

The invention also relates to a pharmaceutical composition when used for treating or preventing a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, comprising an agent which, when administered, selectively 30 increases the amount of complexes between an HLA molecule and a tumor-associated antigen or a part thereof, said tumorassociated antigen having a sequence encoded by a nucleic acid which is selected from the group consisting of:

5b

2017201358 29 Jan 2019 (a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .

The invention also relates to a pharmaceutical composition when 15 used for treating or preventing a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, comprising one or more components selected from the group consisting of:

(i) a tumor-associated antigen or a part thereof, (ii) a nucleic acid which codes for a tumor-associated antigen or a part thereof, (iii) an antibody which binds to a tumor-associated antigen or a part thereof, (iv) an antisense nucleic acid which hybridizes specifically with a nucleic acid coding for a tumorassociated antigen, (v) a host cell which expresses a tumor-associated antigen or a part thereof, and (vi) isolated complexes between a tumor-associated antigen or a part thereof and an HLA molecule, said tumor-associated antigen having a sequence encoded by a nucleic acid which is selected from the group consisting of :

5c

The invention also relates to a method of diagnosing a cancer 15 disease characterized by a cancer cell expressing a tumorassociated antigen, which 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 of a part thereof and/or (iv) detection of cytotoxic or T helper lymphocytes which are specific to the tumor-associated antigen or to a part thereof in a biological sample isolated from a patient, with said tumor-associated antigen having a 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 NO: 84, a part or derivative thereof, (b) a nucleic acid which hybridizes with the nucleic acid of (a) under stringent conditions,

2017201358 29 Jan 2019

5d

(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) ,
wherein the cancer	disease is a cancer	or tumor	of the
pancreas or liver,	or	a metastasis	thereof,	or a
meta	stasis of a tumor of the colon or stomach.

The invention also relates to a method for determining regression, course or onset of a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, 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 of a part thereof, (iii) the amount of antibodies which bind to the tumorassociated antigen or to a part thereof, and (iv) the amount of cytolytic or cytokine-releasing T cells which are specific for a complex between the tumorassociated antigen or a part thereof and an MHC molecule, said tumor-associated antigen having a 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 NO: 84, 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

2017201358 29 Jan 2019

5e (d) a nucleic acid which is complementary to the nucleic acid of (a), (b) or (c), wherein the cancer disease is a cancer or tumor of the pancreas or liver, or a metastasis thereof, or a metastasis of a tumor of the colon or stomach.

The invention also relates to a method of treating a cancer disease characterized by expressing a tumor-associated antigen, which method comprises administering an antibody binding to said tumor-associated antigen or to a part thereof and coupled to a therapeutic agent, said tumor-associated antigen having a 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 NO: 84, 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) ,
wherein	the cancer	disease	is	a cancer	or tumor of the
colon,	pancreas,	stomach	or	liver,	or a metastasis
thereof .
The invention also relates to	a	method	of diagnosing or

monitoring a cancer disease characterized by expressing a tumor-associated antigen, which method comprises administering an antibody binding to said tumor-associated antigen or to a part thereof and coupled to a diagnostic agent, said tumorassociated antigen having a sequence encoded by a nucleic acid which is selected from the group consisting of:

5f

2017201358 29 Jan 2019 (a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 84, 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), wherein the cancer disease is a cancer or tumor of the pancreas or liver, or a metastasis thereof, or a metastasis of a tumor of the colon or stomach.

The invention also relates to a method of treating a patient 15 having a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, 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 or cytokine-releasing T cells against said tumor-associated antigen or a part thereof, and (iii) introducing the cytolytic or cytokine-releasing T cells into the patient in an amount suitable for lysing cells expressing the tumor-associated antigen or a part thereof, said tumor-associated antigen having a sequence encoded by a nucleic acid which is selected from the group consisting of:

5g

2017201358 29 Jan 2019 (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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .

The invention also relates to a method of treating a patient 10 having a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, which method comprises:

(i) identifying a nucleic acid which is expressed by cells associated with said disease, said nucleic acid being selected from the group consisting of:

(a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 84, 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), (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, 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, wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .

2017201358 29 Jan 2019

5h

The invention also relates to a method of treating a cancer disease characterized by a cancer cell expressing a tumorassociated antigen, 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, said tumor-associated antigen having a sequence encoded by a nucleic acid which is selected from the group consisting of :

acid of	(a) , (b) or (c) ,
wherein	the cancer disease	is	a cancer or tumor of the
colon,	pancreas, stomach	or	liver, or a metastasis
thereof .

The invention also relates to an agent when used for treating or preventing a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, which binds specifically to a protein or polypeptide or to a part thereof, said protein or polypeptide being 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 NO: 84, a part or derivative thereof,

5i

2017201358 29 Jan 2019 (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) ,
wherein	the cancer	disease	is	a cancer or tumor of the
colon,	pancreas	r	stomach	or	liver, or a	metastasis
thereof .
The invention also	relates to	an	antibody when	used for

treating or preventing a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, which binds selectively to a complex of:

(i) a protein or polypeptide or a part thereof and (ii) an MHC molecule to which said protein or polypeptide or said part thereof binds, with said antibody not binding to (i) or (ii) alone and said protein or polypeptide being 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 NO: 84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .

The invention also relates to a kit when used for detecting a

2017201358 09 Apr 2019 cancer cell characterized antigen, which kit comprises of the nucleic by expressing a tumor-associated agents for detection acid which codes for the tumorassociated antigen or of a part thereof, (ii) of the tumor-associated antigen or of a part thereof, (iii) of antibodies which bind to the antigen or to a part thereof, and/or tumor-associated complex between the tumor-associated antigen or a part thereof and an MHC molecule, said tumor-associated antigen having a sequence encoded by a nucleic acid which is selected from the group consisting of:

84, 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) , wherein the cancer is a cancer or tumor of the pancreas or liver, or a metastasis thereof, or a metastasis of a tumor of the colon or stomach.

5k

2017201358 09 Apr 2019 (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 of a part thereof, (iii) the amount of antibodies which bind to the tumorassociated antigen or to a part thereof, and (iv) the amount of cytolytic or cytokine-releasing T cells which are specific for a complex between the tumorassociated antigen or a part thereof and an MHC molecule, 10 said tumor-associated antigen having a sequence encoded by a nucleic acid which is selected from the group consisting of :

84, 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), wherein the cancer disease is a cancer or tumor of the pancreas or liver, or a metastasis thereof, or a metastasis of a tumor of the colon or stomach.

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.

2017201358 09 Apr 2019

Furthermore, the invention relates to genetic products, i.e. nucleic acids and proteins or peptides, which are produced by altered splicing (splice variants) of known genes or altered translation using alternative open reading frames. In this aspect the invention relates to nucleic acids which comprise a nucleic acid sequence selected from the group consisting of sequences according to SEQ ID NOs: 3-5 of the sequence listing. Moreover, in this aspect, the invention relates to proteins or peptides which comprise an amino acid sequence selected from the group consisting of the sequences according to SEQ ID NOs:

and 12-14 of the sequence listing. The splice variants of

2017201358 28 Feb 2017 the invention can be used according to the invention as targets for diagnosis and therapy of tumor diseases.

In particular, the invention relates to the amino acid sequence according to SEQ ID NO: 10 of the sequence listing which is encoded by an alternative open reading frame identified according to the invention and differs from the previously described protein sequence (SEQ ID NO: 9) in additional 85 10 amino acids at the N terminus of the protein.

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 25 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 tumorassociated proteins/antigens. These may be utilized as molecular markers both for detecting tumor cells and for 30 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

2017201358 28 Feb 2017 oligonucleotides. In particular, pairs of primers are suitable as oligonucleotides at least one of which binds to the region of the splice variant which is tumor-associated under stringent conditions. According to the invention, oligonucleotides 5 described for this purpose in the examples are suitable, in particular oligonucleotides which have or comprise a sequence selected from SEQ ID NOs: 34-36, 39, 40, and 107-110 of the sequence listing. According to the invention, all sequencedependent detection systems are suitable for detection. These 10 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 variantspecific nucleic acid sequence. However, tumor cells may also 15 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. In this aspect, the invention relates, in particular, to peptides which 20 have or comprise a sequence selected from SEQ ID NOs: 17-19,

111-115, 120, and 137 of the sequence listing and specific antibodies which are directed thereto. Suitable for immunization are particularly the amino acids whose epitopes are distinctly different from the variant(s) of the genetic 25 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 30 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

2017201358 28 Feb 2017 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 JH, 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 as pharmaceutically active substances in active immunotherapy (vaccination, vaccine therapy).

The present invention also describes proteins which differ in nature and degree of their secondary modifications in normal and tumor tissue (for example Durand & Seta, 2000; Clin. Chem. 46: 795-805; Hakomori, 1996; Cancer Res. 56: 5309-18).

The analysis of protein modifications can be done in Western blots. In particular, glycosylations which as a rule have a size of several kDa result in a higher overall mass of the target protein which can be separated in an SDS-PAGE. For the detection of specific 0- and N-glycosidic bonds protein lysates are incubated with 0- or N-glycosylases (according to the instructions of the respective manufactures, for example, PNgase, endoglycosidase F, endoglycosidase H, Roche Diagnostics) prior to denaturation using SDS. Thereafter, a Western blot is performed. If the size of target protein is reduced a specific glycosylation can be detected in this manner

2017201358 28 Feb 2017 following incubation with a glycosidase and thus, also the tumor specificity of a modification can be analyzed. Protein regions which are differentially glycosylated in tumor cells and healthy cells are of particular interest. Such differences 5 in glycosylation, however, have hitherto only been described for a few cell surface proteins (for example, Mucl).

According to the invention, it was possible to detect a differential glycosylation for Claudin-18 in tumors.

Gastrointestinal carcinomas, pancreas carcinomas, esophagus tumors, prostate tumors as well as lung tumors have a form of Claudin-18 which is glycosylated at a lower level. Glycosylation in healthy tissues masks protein epitopes of Claudin-18 which are not covered on tumor cells due to lacking glycosylation. Correspondingly it is possible according to the invention to select ligands and antibodies which bind to these

domains .	Such	ligands and antibodies according to the	invention
do not	bind	to Claudin-18 on healthy cells since	here	the
epitops	are covered due to glycosylation.
20
As has	been	described above for protein epitopes	which	are
derived	from	tumor-associated splice variants it	is	thus

possible to use the differential glycosylation to distinguish normal cells and tumor cells with diagnostic as well as 25 therapeutic intention.

In one aspect, the invention relates to a pharmaceutical composition comprising an agent which recognizes the tumorassociated antigen identified according to the invention and 30 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

2017201358 28 Feb 2017

antigen,	in particular	a complement-activated	or	toxin
conj ugated	antibody	which	binds selectively to	the	tumor-
associated	antigen.	In	a further embodiment,	the	agent

comprises two or more agents which each selectively recognize 10 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 15 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 cells labeled in this way. In a further embodiment, the agent is an antibody which binds selectively to 20 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 30 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

2017201358 28 Feb 2017 selectively inhibit expression or activity of different tumorassociated antigens, at least one of which is a tumorassociated antigen identified according to the invention.

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 10 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 15 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 20 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 25 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

2017201358 28 Feb 2017 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 15 the HLA molecule endogenously. In a further embodiment, the host cell expresses the HLA molecule and/or the tumorassociated 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 20 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 25 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 30 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.

2017201358 28 Feb 2017

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 nucleotides, 10 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 20 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 25 invention. The method comprises detection of (i) 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) 30 det ection 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

2017201358 28 Feb 2017 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 5 (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 10 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 tumorassociated antigens or of parts thereof, detection of two or 15 more antibodies binding to said two or more different tumorassociated 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 20 compared to a comparable normal biological sample.

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 tumor25 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 30 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

2017201358 28 Feb 2017 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 5 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 10 acids which code for said two or more different tumorassociated 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 tumor15 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 molecules .	antigens	or of	parts	thereof and	MHC
20
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 25 the reof 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.

In particular embodiments, the tumor-associated antigen to be detected or the part thereof is present intracellularly or on the cell surface. According to the invention, detection of a tumor-associated antigen or of a part thereof or monitoring the

2017201358 28 Feb 2017 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 further 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 10 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 15 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 25 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 30 which are loaded with the particular immunogenic fragment of one or more of the tumor-associated antigens and which can identify the specific T lymphocytes by contacting the specific T cell receptor.

2017201358 28 Feb 2017

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 5 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 10 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 15 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 tumorassociated antigen or a part thereof, under conditions which favor production of cytolytic T cells against said tumor20 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 25 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

2017201358 28 Feb 2017 monocyte or a macrophage.

In a further aspect, the invention relates to a method of 5 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 10 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
15 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 25 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 tumor30 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,

2017201358 28 Feb 2017 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 5 nonproliferative or are rendered nonproliferative. A disease characterized by expression or abnormal expression of a tumorassociated antigen is in particular cancer.

The present invention furthermore relates to a nucleic acid 10 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: 3-5, 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 15 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 20 consisting of SEQ ID NOs: 10 and 12-14, a part or derivative thereof .

In a further aspect, the invention relates to promoter sequences of nucleic acids of the invention. These sequences 25 may be functionally linked to another gene, preferably in an expression vector, and thus ensure selective expression of said gene in appropriate cells.

In a further aspect, the invention relates to a recombinant 30 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.

2017201358 28 Feb 2017

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, 10 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 20 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, polypeptide or peptide 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

2017201358 28 Feb 2017 consisting of SEQ ID NOs: 3-5, 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 or peptide which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 12-14, 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 15 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 this aspect the invention relates, in particular, to a 20 peptide which has or comprises a sequence selected from the group consisting of SEQ ID NOs: 17-19, 90-97, 100-102, 105,

106, 111-116, 120, 123, 124, and 135-137, 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 30 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.

2017201358 28 Feb 2017

In particular, the invention relates to such an agent, in particular an antibody, which specifically binds to a peptide which has or comprises a sequence selected from the group 10 consisting of SEQ ID NOs: 17-19, 90-97, 100-102, 105, 106, 111116, 120, 123, 124, and 135-137, a part or derivative thereof.

The invention furthermore relates to a conjugate between an agent of the invention which binds to a tumor-associated 15 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.
20 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 25 the reof, (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 30 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.

described which are

2017201358 28 Feb 2017

Detailed description of the invention

According to the invention, genes are expressed in tumor cells selectively or are tumor-associated antigens.

According to the invention, these products and/or their derivatives target structures for therapeutic genes and/or aberrantly and which and/or their genetic parts are preferred 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 pathogenecity and 15 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 20 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, 25 the t erm derivative also comprises chemical derivatization of a nucleic acid on a nucleotide base, on the sugar or on the phosphate. 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.

2017201358 28 Feb 2017

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, 10 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

Stringent conditions are described, for example, in Molecular

Cloning:

Edition,

Harbor,

Biology,

New York

A Laboratory Manual,

Cold Spring Harbor

New

F.M.

and

York, 1989

Ausubel et refer, for or al. ,

J. Sambrook et al.,

Laboratory press,

Current

Editors, example, to

Protocols

John Wiley

Editors, 2nd

Cold Spring in Molecular & Sons,

Inc . , hybridization at 65

C in hybridization polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM

NaH₂PO₄ chloride/0.15 M sodium mM EDTA) . SSC is 0.15 M sodium citrate, pH 7. After hybridization, the membrane to which the

DNA has been transferred is washed, for example, in 2 χ SSC at room temperature and then in 0.1-0.5 *

SSC/0.1 χ SDS at temperatures of up to 68 °C.

2017201358 28 Feb 2017

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 10 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 15 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 20 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 t erm 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 30 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'

2017201358 28 Feb 2017 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 10 he rein 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 15 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 20 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. Similarly, a combination with a nucleic acid is possible which represents a reporter gene or any tag.

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 30 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

2017201358 28 Feb 2017 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 5 with the aid of agrobacteria or in insertion via DNA or RNA viruses. Vectors comprise plasmids, phagemids or viral genomes.

The nucleic acids coding for a tumor-associated antigen identified according to the invention may be used for 10 transfection of host cells. Nucleic acids here mean both recombinant DNA and RNA. Recombinant RNA may be prepared by invitro 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 25 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 30 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

2017201358 28 Feb 2017 nucleic acids.

transiently or mammalian cells

Furthermore, expression may be stably. Preferred expression comprise pcDNA3.1 and pRc/CMV carried out systems in (Invitrogen,

Carlsbad, imparting

CA), which contain a selectable marker such as a gene resistance to G418 (and thus enabling stably transfected cell lines to be selected) and the enhancerpromoter 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 25 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 30 hybr idize 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

2017201358 28 Feb 2017 to one strand of the nucleic acid coding for the tumorassociated antigen, and the other primer will hybridize to the complementary strand in an arrangement which allows amplification of the nucleic acid coding for the tumor5 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 10 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 15 inhibiting transcription of said gene and/or translation of said mRNA. According to the invention, an 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 20 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 25 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.

2017201358 28 Feb 2017

In one embodiment, an oligonucleotide of the invention consists 5 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, 15 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 20 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, 25 carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.

The term modified oligonucleotide also comprises oligonucleotides having a covalently modified base and/or 30 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,

2017201358 28 Feb 2017 a 2'-O-alkylated ribose residue or another sugar instead of ribose, such as arabinose.

Preferably, the proteins and polypeptides described according 5 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 10 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 15 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 .
25
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 30 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

2017201358 28 Feb 2017 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 5 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 10 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, Gin

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 25 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 30 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

2017201358 28 Feb 2017 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, polypeptides or peptides also comprise single or multiple substitutions, deletions and/or additions of any molecules associated with the enzyme, such as carbohydrates, lipids 10 and/or proteins, polypeptides or peptides. The term derivative also extends to all functional chemical equivalents of said proteins, polypeptides or peptides.

According to the invention, a part or fragment of a tumor15 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 20 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 25 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 tumorassociated antigen.

A part or a fragment of a nucleic acid coding for a tumor30 associated antigen relates according to the invention to the part of the nucleic acid, which codes at least for the tumorassociated antigen and/or for a part or a fragment of said tumor-associated antigen, as defined above.

2017201358 28 Feb 2017

The isolation and identification of genes coding for tumorassociated antigens also make possible the diagnosis of a disease characterized by expression of one or more tumorassociated antigens. These methods comprise determining one or 5 more nucleic acids which code for a tumor-associated antigen and/or determining the encoded tumor-associated antigens and/or peptides in the reaction derived therefrom. The nucleic acids may be determined conventional manner, including by polymerase chain or hybridization with a labeled probe. Tumorassociated 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 tumorassociated antigens described herein to be isolated, including antibodies and cellular binding partners of said tumorassociated 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 25 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 30 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

2017201358 28 Feb 2017 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 10 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 15 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 tumorassociated antigens with their binding partners and in the purification of said tumor-associated antigens and of complexes 20 the reof 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, 25 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.

Such antibodies can recognize proteins in the native and/or denaturated 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).

2017201358 28 Feb 2017

Antisera which contain specific antibodies specifically binding to the target protein can be prepared by various standard processes; see, for example, 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 and Using Antibodies:

A Laboratory Manual: Portable Protocol NO by Edward Harlow, David Lane, Ed Harlow ISBN 0879695447. Thereby it is also possible 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 in particular relevant for the preparation of antibodies which are to be used therapeutically, but also for many diagnostic applications. In this respect, it is possible to immunize with the whole protein, with extracellular partial sequences as well as with cells which express the target molecule in physiologically 20 folded form.

Monoclonal antibodies are hybridoma technology. (for

Antibodies :

A Practical

Christopher

Manual by

Antibodies :

traditionally prepared technical details see:

Approach by Philip using the

Monoclonal

Shepherd,

Dean ISBN 0-19-963722-9; Antibodies: A Laboratory

Ed

Harlow, David Lane

Laboratory Manual:

Edward Harlow,

David Lane, Ed Harlow

ISBN: 0879693142; Using

Portable Protocol NO by

ISBN: 0879695447).

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

2017201358 28 Feb 2017

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 5 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 as Fab fragment, carries one 10 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 15 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 20 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) 25 which are separated in each case by three complementarydetermining 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

2017201358 28 Feb 2017 development of humanized antibodies in which nonhuman CDRs are covalently linked to human FR and/or Fc/pFc' regions to produce a functional antibody.

This is utilized in the so called SLAM technology, wherein B cells from whole blood are isolated and the cells are monocloned. Then, the supernatant of the single B cells is analyzed with respect to its antibody specificity. In contrast to the hybridoma technology the variable region of the antibody 10 gene is amplified using single cell PCR and cloned into a suitable vector. In this way, the provision of monoclonal antibodies is accelerated (de Wildt et al., J. Immunol. Methods 207: 61-67, 1997).

As another example, WO 92/04381 describes the 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 20 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 25 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 30 and/or light chain-CDR3 regions have been replaced with homologous human or nonhuman sequences, and chimeric Fdfragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced with homologous human or nonhuman

2017201358 28 Feb 2017 sequences. The invention also comprises single-chain antibodies .

The invention also comprises polypeptides which bind 5 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 10 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 15 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 20 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 25 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 30 peptide libraries, including phage-display libraries, in order to identify and select peptide binding partners of the tumorassociated 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

2017201358 28 Feb 2017 purposes known to the skilled worker.

The antibodies described above and other binding molecules may be used, for example, for identifying tissue which tumor-associated antigen. Antibodies may also be specific diagnostic substances for displaying cells expressing tumor-associated to therapeutically useful comprise, in a nonlimiting expresses a coupled to and tissues antigens. They may also substances. Diagnostic manner, barium sulfate, be coupled substances iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium radio diagnostic, including positron emitters and carbon-11, gamma emitters such tyropanoate and such as fluorineas iodine-123, technetium-99m, iodine-131 and indium-ill, nuclides for nuclear magnetic resonance, such as fluorine and gadolinium. According to the invention, the term therapeutically useful substance means any selectively associated therapeutic molecule which, guided to a cell which expresses as desired, is one or more tumorantigens, including anticancer agents, radioactive iodine-labeled compounds, toxins, cytostatic or cytolytic drugs, etc. Anticancer agents comprise, for example, aminoglutethimide, azathioprine, bleomycin sulfate, carmustine, chlorambucil, cisplatin, busulfan, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil, interferon-a, lomustine, mercaptopurine, methotrexate, mitotane, procarbazine

HC1, 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.

2017201358 28 Feb 2017

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, 15 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 20 expression in a healthy individual is repressed. One example of such a disease is cancer, wherein the term cancer according to the invention comprises leukemias, seminomas, melanomas, teratomas, gliomas, kidney cancer, adrenal cancer, thyroid cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer and the matastases thereof .

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, sputum, urine,

2017201358 28 Feb 2017 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 10 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 15 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 antigen20 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 25 such cytotoxic T lymphocytes in vitro is known. An example of a method of differentiating T cells can be found in WO-A-9633265. 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 30 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

2017201358 28 Feb 2017 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 5 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 10 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 15 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 20 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. 21:14031410, 1991; Hast et al., Cell 59:603-614, 1989), cells presenting the desired combined with cytotoxic complex (e.g. dendritic cells) are

T lymphocytes of the patient to be treated, resulting in a propagation of specific cytotoxic T lymphocytes .

The propagated cytotoxic 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

2017201358 28 Feb 2017 low affinity propagated, extinguished here may be too, cells individuals propagation affinity if for a since due to specific complex of this those with high affinity development of a transfer of the T cell presenting the desired combined with cytotoxic or of the kind can be have been tolerance. An alternative receptor itself. For this complex (e.g.

lymphocytes dendritic of healthy another species (e.g.

specific cytotoxic T mouse). This lymphocytes results in with high

T lymphocytes are derived from a donor organism which had no previous contact high affinity T cell receptor lymphocytes is cloned. If the with the specific complex. The of these propagated specific T high affinity T cell receptors have been cloned from another species they can a different extent. Such T cell receptors are via gene transfer, for example using retroviral be humanized to then transduced vectors, into T cells of 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,

The therapeutic aspects above start out from the fact that at least some of the abnormal cells of the patient present 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 cytotoxic the cells with a sample

T lymphocytes.

presenting the tumor-associated antigen from the patient, which contains

If the cytotoxic T lymphocytes lyse complex, it can be assumed that a is presented.

Adoptive transfer is not applied according to the the only form of therapy which can be invention. Cytotoxic T lymphocytes may

2017201358 28 Feb 2017 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 5 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 15 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 20 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 25 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 tumorassociated 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

2017201358 28 Feb 2017 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 5 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 10 (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 tumorassociated 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 20 cells of many cancer patients. Of particular interest, however, are those against which no spontaneous immune responses preexist. 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 invention may also

According to the compositions described according to the be used invention, vaccination mean an increase as vaccines the terms response to an antigen.

testing an immunizing associated antigen or for immunization.

immunization or in or activation of

It is possible to use animal effect on cancer by using an immune models for a tumora nucleic acid coding therefor. For

2017201358 28 Feb 2017 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 5 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 10 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 15 providing an antigen reservoir (extracellularly or in macrophages), activating macrophages and/or 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 20 (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. Kreig et al., Nature 374:546-9,

1995) and various water-in-oil emulsions prepared from b iologically 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 qg to about 100 qg.

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.

2017201358 28 Feb 2017

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 10 of proteins or nucleic acids. Examples of such costimulating and CD86, respectively) which are expressed on dendritic cells cells. This interaction provides a costimulation (signal for an antigen/MHC/TCR15 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 et al. , Proc.

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 Thl-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

2017201358 28 Feb 2017 the CD40 molecule expressed by dendritic cells (Ridge et al., Nature 393:474 (1998), Bennett et al., Nature 393:478 (1998),

Schonberger et al., Nature 393:480 (1998)). The mechanism of this costimulating signal probably relates to the increase in 5 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 antigenpresenting cells (tumor cells). In these situations, T helper 15 and B7-costimulating signals are not provided. This mechanism could be used in connection with therapies based on antigenpulsed dendritic cells.

The invention also provides for administration of nucleic 20 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 25 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 30 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.

2017201358 28 Feb 2017

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 5 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 10 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 15 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 20 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 25 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 30 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

2017201358 28 Feb 2017 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, CpG and cytokines and, where appropriate, other 10 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 15 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.

2017201358 28 Feb 2017

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 5 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 10 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 15 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 qg, are formulated and administered for a treatment or for generating or increasing an immune response. If the administration of nucleic acids (DNA 25 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 30 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

2017201358 28 Feb 2017 compounds. When pharmaceutically pharmaceutically 5 pharmaceutically invention.

used in medicine, compatible. However, compatible may be compatible salts and the salts should be salts which are not used for preparing are included in the

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, 10 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 20 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 25 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, 30 citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.

2017201358 28 Feb 2017

The pharmaceutical compositions may, where appropriate, also contain suitable preservatives such as benzalkonium chloride, chlorobutanol, paraben 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, 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 15 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 20 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.

Figures:

Fig. 1. GPR35 mRNA expression in colon carcinoma biopsies

RT-PCR investigations with DNA-free RNA show GPR35 expression in most of the colon carcinoma biopsies. By contrast, there is 30 no detectable expression in normal tissues. (1-Breast, 2-lung,

3-lymph nodes, 4-thymus, 5-colon, 6-15 colon carcinoma, 16-neg. control).

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Fig. 2. Quantitative PCR analysis of GUCY2C mRNA expression in normal and tumor tissues

Real-time PCR investigation with GUCY2C-specific primers (SEQ ID NO: 22-23) shows selective mRNA expression in normal ileum, colon, and in all colon carcinoma biopsies. Distinct quantities of GUCY2C transcripts were also detected in a colon carcinoma metastasis in the liver.

Fig. 3. Identification of tumor-specific GUCY2C splice variants

PCR products from normal colon tissues and colon carcinomas were cloned, and clones from both groups were checked by restriction analysis (EcoR I) and sequenced.

Fig. 4. Selective SCGB3A expression in normal lung and lung 15 carcinoma

RT-PCR analysis with gene-specific SCGB3A2 primers (SEQ ID NO: 37, 38) shows cDNA amplification exclusively in normal lung (lane 8, 14-15) and in lung carcinoma biopsies (lane 16-24). (1-Liver-N, 2-PBMC-N, 3-lymph node-N, 4-stomach20 N, 5-testis-N, 6-breast-N, 7-kidney-N, 8-lung-N, 9-thymus-N,

10-ovary-N, 11-adrenal-N, 12-spleen-N, 14-15-lung-N, 16-24-lung carcinoma, 25-negative control).

Fig. 5. Claudin-18A2.1 expression in stomach, esophagus, 25 stomach carcinoma and pancreatic carcinoma

RT-PCR analysis with claudin-18A2.1-specific primers (SEQ ID NO: 39, 40) showed according to the invention pronounced claudin-18A2.1 expression in 8/10 stomach carcinoma biopsies and in 3/6 pancreatic carcinoma biopsies. Distinct 30 expression was also detected in stomach and normal esophageal tissue. In contrast thereto, no expression was detected in the ovary and in ovarian carcinoma.

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Fig. 6. SLC13A1 expression in the kidney and renal cell carcinoma

RT-PCR analysis with SLC13Al-specific primers (SEQ ID NO: 49, 50) showed expression in 7/8 renal cell carcinoma samples.

Otherwise, transcripts within normal tissues were detected exclusively in the kidney. (1-2-kidney, 3-10-renal cell carcinoma, 11-breast, 12-lung, 13-liver, 14-colon, 15-lymph nodes, 16-spleen, 17-esophagus, 18-thymus, 19-thyroid, 20-PBMCs, 21-ovary, 22-testis).

Fig. 7. CLCA1 expression in colon, colon carcinoma and stomach carcinoma

RT-PCR investigations with CLCAl-specific primers (SEQ ID NO: 67, 68) confirmed selective expression in the colon and showed high expression in (3/7) investigated colon carcinoma and (1/3) investigated stomach carcinoma samples. The other normal tissues (NT) showed no or only very weak expression.

Fig. 8. FLJ21477 expression in the colon and colon carcinoma

RT-PCR investigations with FLJ21477-specific primers (SEQ ID NO: 69, 70) showed selective expression in the colon and additionally various levels of expression in (7/12) investigated colon carcinoma samples. The other normal tissues 25 (NT) showed no expression.

Fig. 9. FLJ20694 expression in the colon and colon carcinoma

RT-PCR investigations with FLJ20694-specific primers (SEQ ID NO: 71, 72) showed selective expression in the colon 30 and additionally various levels of expression in (5/9) investigated colon carcinoma samples. The other normal tissues (NT) showed no expression.

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Fig. 10. von Ebner expression in stomach, lung and lung carcinoma

RT-PCR investigations with von Ebner-specific primers (SEQ ID NO: 73, 74) showed selective expression in the stomach, in the lung and in (5/10) investigated lung carcinoma samples. The other normal tissues (NT) showed no expression.

Fig. 11. Plunc expression in thymus, lung and lung carcinoma RT-PCR investigations with Plunc-specific primers (SEQ ID NO: 75, 76) showed selective expression in the thymus, in the lung and in (6/10) investigated lung carcinoma samples. The other normal tissues showed no expression.

Fig. 12. SLC26A9 expression in lung, lung carcinoma and thyroid

RT-PCR investigations with SLC26A9-specific primers (SEQ ID NO: 77, 78) showed selective expression in the lung and in all (13/13) investigated lung carcinoma samples. The other normal tissues (NT) showed no expression with the exception of the thyroid.

Fig. 13. THC1005163 expression in stomach, ovary, lung and lung carcinoma

RT-PCR investigations with a THC1005163-specific primer (SEQ ID NO: 79) and a nonspecific oligo dT tag primer showed 25 expression in stomach, ovary, lung and in (5/9) lung carcinoma biopsies. The other normal tissues (NT) showed no expression.

Fig. 14. LOC134288 expression in kidney and renal cell carcinoma

RT-PCR investigations with LOC134288-specific primers (SEQ ID NO: 80, 81) showed selective expression in the kidney and in (5/8) investigated renal cell carcinoma biopsies.

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Fig. 15. THC943866 expression in kidney and renal cell carcinoma

RT-PCR investigations with THC943866-specific primers (SEQ ID NO: 82, 83) showed selective expression in the kidney and in (4/8) investigated renal cell carcinoma biopsies.

Fig. 16. FLJ21458 expression in colon and colon carcinoma

RT-PCR investigations with FLJ21458-specific primers (SEQ ID NO: 86, 87) showed selective expression in the colon and in (7/10) investigated colon carcinoma biopsies. (1—2— colon, 3-liver, 4-PBMCs, 5-spleen, 6-prostate, 7-kidney,

8-ovary, 9-skin, 10-ileum, 11-lung, 12-testis, 13-22 colon carcinoma, 23-neg. control).

Fig. 17. Cellular localization of GPR35

Immunofluorescence for detecting the cellular localization of GPR35 after transfection of a plasmid that expresses a GPR35GFP fusion protein. The arrows identify the membrane-associated fluorescence of the fluorescent GFP.

Fig. 18. Quantitative expression of GPR35

A. Quantitative RT-PCR with GPR35-specific primers

	(SEQ ID NO:	88, 89)	show	selective	expression	in	the
	intestine,	in colon	tumor	samples and	in metastases	from
25	intestinal	tumors .	The	following normal tissues	were
	analyzed:	liver, lung,	lymph nodes,	stomach,	spleen,

adrenal, kidney, esophagus, ovary, testis, thymus, skin,

	breast, pancreas, lymphocytes,	activated tube,	lymphocytes, endometrium,
prostate,	thyroid, fallopian
30	cerebellum,	brain.
B.	Prevalence	of GPR35 in colon	tumors and	metastases

thereof. GPR35 is expressed both in the tumor and in metastases in more than 90% of the cases.

GUCY2C-specific primers

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Fig. 19. Quantitative expression

Quantitative normal colonic

RT-PCR with show high and expression in colonic detectable in 11/12 carcinomas .

of GUCY2C and gastric tissue selective expression in (A) and GUCY2C-specific and gastric tumor samples (B). GUCY2C is colon carcinomas and in 7/10 stomach

Fig. 20. Quantitative expression of SCGB3A2

Quantitative RT-PCR with SCGB3A2-specific primers (SEQ ID NO: 103, 104) show selective expression in lung samples and lung tumor samples. 19/20 lung tumor samples are SCGB3A2positive, and SCGB3A2 is over-expressed by a factor of at least 10 in more than 50% of the samples. The following normal tissues were analyzed: liver, lung, lymph nodes, stomach, spleen, adrenal, kidney, esophagus, ovary, testis, thymus, skin, breast, pancreas, lymphocytes, activated lymphocytes, prostate, thyroid, fallopian tube, endometrium, cerebellum, brain.

Fig. 21. Immunofluorescence with SCGB3A2-specific antibodies

COS7 cells were transfected with a plasmid which codes for an SCGB3A2-GFP fusion protein. A. Detection of the transfected fusion protein with an SCGB3A2-specific rabbit antiserum 25 (immunization with SEQ ID NO: 105) . B. Detection of the transfected fusion protein by GFP fluorescence.

C. Superimposition of the two fluorescences from A and B. The yellow color is produced at the points where the two fluorescences are superimposed and thus demonstrates the 30 specificity of the SCGB3A2 antiserum.

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Fig. 22. Diagrammatic depiction of claudin-18 splice variants

The two claudin-18 splice variants Al and A2 differ in the N terminus and show different potential glycosylation sites.

Fig. 23. Quantitative expression of claudin-18, variant Al

Claudin-Al is highly	activated in a large number of tumor
tissues. Particularly	strong expression is found in gastric

tumors, lung tumors, pancreatic carcinomas and esophageal carcinomas .

Fig. 24. Quantitative expression of claudin-18, variant A2

Variant A2 is, like variant Al, activated in many tumors.

Fig. 25. Use of claudin-18A2-specific antibodies (extracellular 15 domain) (Top) Staining of claudin-18A2-positive gastric carcinoma cells (SNU-16) with an antibody which was produced by immunization with a peptide (SEQ ID NO: 17). Membrane staining appears particularly strongly in the cell/cell interaction regions.

A-preimmune, MeOH; B-immune serum MeOH, 5 pg/ml; (Below)

Demonstration of the specificity of the antibody by colocalization analysis in claudin-18A2-GFP-transfected 293T cells. A-Claudin-18A2 GFP; B-anti-claudin-A2;

C-superimposition.

Fig. 26. Use of claudin-18A2-specific antibodies (extracellular domain)

Membrane staining of claudin-18A2-positive gastric carcinoma cells (SNU-16) with an antibody which was produced by 30 immunization with a peptide (SEQ ID NO: 113, N-terminally located extracellular domain). A monoclonal antibody which is directed against E-cadherin was used for counterstaining. A-antibody; B-counterstaining; C-superimposition.

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Fig. 27. Use of antibodies against the C-terminal extracellular domain of claudin-18 (Left, top and below) Membrane staining of claudin-18A2positive gastric carcinoma cells (SNU-16) with an antibody 5 which was produced by immunization with a peptide (SEQ ID NO: 116, C-terminally located extracellular domain). A monoclonal antibody which is directed against E-cadherin was used for counterstaining (right top, below).

Fig. 28. Use of claudin-18A1-specific antibodies (Top) Weak to absent staining of gastric carcinoma cells (SNU-16; claudinl8A2 positive) with an antibody which was produced by immunization with a claudin-18Al-specific peptide (SEQ ID NO: 115) . A-anti-E-cadherin; B-anti-claudin-18A1; 15 C-superimposition.

(Below) Demonstration of the specificity of the antibody by colocalization analysis in claudin-18Al-GFP-transfected 293T cells. A-GFP-claudin-18Al; B-anti-claudin-18Al; Csuperimposition.

Fig. 29. Detection of claudin-18A2 in a Western blot.

Western blotting with lysates from various healthy tissues with a claudin-18A2-specific antibody directed against the epitope with SEQ ID NO: 17. 1-Stomach; 2-testis; 3-skin; 4-breast;

5-liver; 6-colon; 7-lung; 8-kidney; 9-lymph nodes.

Fig. 30. Claudin-18A2 Western blotting with samples from stomach and stomach tumors

Lysates from stomach and stomach tumors were blotted and tested 30 using a claudin-18A2-specific antibody against the epitope having SEQ ID NO: 17. Stomach tumors show a less glycosylated form of claudin-18A2. PNGase F treatment of stomach lysates leads to the formation of the low-glycosylated form.

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Left: 1-stomach No #A; 2-stomach Tu #A; 3-stomach No #B;

4-stomach Tu #B

Right: 1-stomach No #A; 2-stomach No #B; 3-stomach No #B +

PNGase F; 4-stomach Tu #C; 5-stomach Tu #D; 6-stomach Tu #D + 5 PNGase F

Fig. 31. Expression of claudin-18 in lung tumors

Low-glycosylated claudin-18A2 variants were detected in lung tumors in accordance with fig. 30. 1-Stomach No; 2-stomach Tu;

3-9-lung Tu.

Fig. 32. Immunohistochemical analysis of claudin-18 using claudin-18A2-specific antibodies in stomach tumor tissue

Fig. 33. Indirect immunofluorescence of stomach-specific Snul6 cells with a claudin-18-specific polyclonal antiserum

A. Staining with a preimmune serum generated before the immunization; B. Staining with the claudin-18-specific serum.

Fig. 34. Quantitative expression of SLC13A1

Quantitative RT-PCR with SLC13Al-specific primers (SEQ ID NO: 121, 122) show high and selective expression in normal kidney tissue (A) and SLC13Al-specific expression in renal cell carcinomas (B) . SLC13A1 transcription is detectable 25 in 5/8 renal cell carcinomas.

Fig. 35. Cellular localization of SLC13A1

Immunofluorescence to demonstrate the cellular localization of SLC13A1 after transfection of a plasmid which provides an 30 SLC1 3A1-GFP fusion protein. The membrane-associated fluorescence of the SLC13A1 fusion protein is to be seen clearly (as ring around the transfected cell).

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Fig. 36. Quantitative expression of CLCA1

Quantitative RT-PCR with CLCAl-specific primers (SEQ ID NO: 125, 126) show high and selective expression in normal colonic tissue and stomach tissue (A) and CLCAl-specific 5 expression in colonic and gastric tumor samples (B) . CLCA1 is detectable in 6/12 colon carcinomas and in 7/10 stomach carcinomas .

Fig. 37. Quantitative expression of FLJ21477

Quantitative RT-PCR with FLJ21477-specific primers (SEQ ID NO: 127, 128) show high and selective expression in normal colonic and gastric tissue and weak expression in thymus, esophagus and brain (A) and the FLJ21477-specific expression in colonic tumor samples (B). FLJ21477 is detectable 15 in 11/12 colon carcinomas.

Fig. 38. Quantitative expression of FLJ20694

Quantitative RT-PCR with FLJ20694-specific primers (SEQ ID NO: 129, 130) show high and selective expression in normal colonic and gastric tissue (A) and FLJ20694-specific overexpression in colonic and gastric tumor samples (B).

FLJ20694 is detectable in 11/12 colon carcinomas and in 7/10 stomach carcinomas.

Fig. 39. Quantitative expression of FLJ21458

Quantitative RT-PCR with FLJ21458-specific primers (SEQ ID NO: 133, 134) show selective expression in testis, gastric and intestinal tissue. In addition, FLJ21458-specific transcripts were detectable in 20/20 colonic tumors and in 7/11 30 colonic metastases. The following normal tissues were analyzed: liver, lung, lymph nodes, spleen, adrenal, kidney, esophagus, ovary, testis, thymus, skin, breast, pancreas, lymphocytes, activated lymphocytes, prostate, thyroid, fallopian tube, endometrium, cerebellum, brain.

Fig. 40. Immunofluorescence with FLJ21458-specific antibodies

2017201358 28 Feb 2017 an FLJ21458-GFP were transfected with a plasmid which codes for fusion protein. A: detection of the transfected with fusion protein an FLJ21458-specific rabbit antiserum

(immunization	wit:	h SEQ ID NO:	136) .	B: detection of	the
transfected	fusion protein	by	GFP fluorescence.	C:
superimposition of	the two fluorescences from A and B.	The
yellow color	is	produced at	the	points where the	two
fluorescences	are	superimposed	and	thus demonstrates	the
specificity of	the	FLJ21458 antis	erum.

endogenously synthesize (Below) Analysis of Snul6 cells which

FLJ21458. A: protein detection using an

FLJ21458-specific rabbit antiserum (immunization with

B: detection of the membrane protein

E-cadherin.

C:

superimposition of the two fluorescences from A and B.

The yellow color is produced at the points where the two fluorescences are superimposed, and demonstrates the membrane localization of FLJ21458.

Fig. 41. Sequences

The sequences to which reference is made herein are shown.

2017201358 28 Feb 2017

Examples :

Material and methods

The terms in silico, electronic and virtual cloning 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 10 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 15 are carried out according to the manufacturers' information.

Datamining-based strategy for determining new tumor-associated genes

Two in silico strategies, namely GenBank keyword search and the 20 cDNAxProfHer, were combined. Utilizing the NCBI ENTREZ Search and Retrieval System (http://www.ncbi.nlm.nih.gov/Entrez), a GenBank search was carried out for candidate genes annotated as being specifically expressed in specific tissues (Wheeler et al., Nucleic Acids Research 28:10-14, 2000).

Carrying out queries with keywords such as colon-specific gene, stomach-specific gene or kidney-specific gene, candidate genes (GOI, genes of interest) were extracted from the databases. The search was restricted to part of the total information of these databases by using the limits homo 30 sapiens, for the organism, and mRNA, for the type of molecule .

The list of the GOI found was curated by determining different names for the same sequence and eliminating such redundancies.

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All candidate genes obtained by the keyword search were in turn studied with respect to their tissue distribution by the electronic Northern (eNorthern) method. The eNorthern is based on aligning the sequence of a GOI with an EST (expressed sequence tag) database (Adams et al., Science 252:1651, 1991) (http://www.ncbi.nlm.nih.gov/BLAST). The tissue origin of each EST which is found to be homologous to the inserted GOI can be determined and in this way the sum of all ESTs produces a preliminary assessment of the tissue distribution of the GOI.

Further studies were carried out only with those GOI which had no homologies to EST from non organ-specific normal tissues.

This evaluation also took into account that the public domain contains wrongly annotated cDNA libraries (Scheurle et al., Cancer Res. 60:4037-4043, 2000) (www.fau.edu/cmbb/publications/ 15 cancergenes6.htm).

The second datamining method utilized was the cDNA xProfiler of the NCBI Cancer Genome Anatomy Project (http://cgap.nci.nih.gov/Tissues/xProfiler) (Hillier et al., Genome Research 6:807-828, 1996; Pennisi, Science 276:102320 1024, 1997). This allows pools of transcriptomes deposited in databases to be related to one another by logical operators. We have defined a pool A to which all expression libraries prepared for example from colon were assigned, excluding mixed libraries. All cDNA libraries prepared from normal tissues 25 other than colon were assigned to pool B. Generally, all cDNA libraries were utilized independently of underlying preparation methods, but only those with a size > 1000 were admitted. Pool B was digitally subtracted from pool A by means of the BUT NOT operator. The set of GOI found in this manner was also 30 subj ected to eNorthern studies and validated by a literature research.

This combined datamining includes all of the about 13 000 fulllength genes in the public domain and predicts out of these genes having potential organ-specific expression.

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All other genes were first evaluated in normal tissues by means of specific RT-PCR. All GOI which had proved to be expressed in non-organ specific normal tissues had to be regarded as false5 positives and were excluded from further studies. The remaining ones were studied in a large panel of a wide variety of tumor tissues. The antigens depicted below proved here to be activated in tumor cells.

RNA extraction, preparation of poly-d(T) primed cDNA and conventional RT-PCR analysis

Total RNA was extracted from native tissue material by using guanidium isothiocyanate as chaotropic agent (Chomczynski & Sacchi, Anal. Biochem. 162:156-9, 1987). After extraction with acidic phenol and precipitation with isopropanol, said RNA was dissolved in DEPC-treated water.

First strand cDNA synthesis from 2-4 qg of total RNA was carried out in a 20 μΐ reaction mixture by means of Superscript II (Invitrogen), according to the manufacturer's information.

The primer used was a dT(18) oligonucleotide. Integrity and quality of the cDNA were checked by amplification of p53 in a 30 cycle PCR (sense CGTGAGCGCTTCGAGATGTTCCG, antisense CCTAACCAGCTGCCCAACTGTAG, hybridization temperature 67°C).

An archive of first strand cDNA was prepared from a number of 25 normal tissues and tumor entities. For expression studies,

0.5 μΐ of these cDNAs was amplified in a 30 μΐ reaction mixture, using GOI-specific primers (see below) and 1 U of HotStarTaq DNA polymerase (Qiagen). Each reaction mixture contained 0.3 mM dNTPs, 0.3 μΜ of each primer and 3 μΐ of 30 10 x reaction buffer.

The primers were selected so as to be located in two different exons, and elimination of the interference by contaminating genomic DNA as the reason for false-positive results was confirmed by testing nonreverse-transcribed DNA as template.

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After 15 minutes at 95 °C to activate the HotStarTaq DNA polymerase, 35 cycles of PCR were carried out (1 min at 94 °C, 1 min at the particular hybridization temperature, 2 min at 72 °C and final elongation at 72 °C for 6 min) .

2 0 μΐ of this reaction were fractionated and analyzed on an ethidium bromide-stained agarose gel.

The following primers were used for expression analysis of the corresponding antigens at the hybridization temperature 10 indicated.

GPR35 (65°C)

Sense : 5’-AGGTACATGAGCATCAGCCTG-3’

Antisense : 5’-GCAGCAGTTGGCATCTGAGAG-3’

GUCY2C (62°C)

Sense: 5'-GCAATAGACATTGCCAAGATG-3'

Antisense : 5'-AACGCTGTTGATTCTCCACAG-3'

SCGB3A2 (66 °C)

Sense : 5'-CAGCCTTTGTAGTTACTCTGC-3'

Antisense: 5'-TGTCACACCAAGTGTGATAGC-3'

Claudinl8A2 (68°C)

Sensei : 5'-GGTTCGTGGTTTCACTGATTGGGATTGC-3'

Antisensei : 5'-CGGCTTTGTAGTTGGTTTCTTCTGGTG-3'

Sense2: 5'- TGTTTTCAACTACCAGGGGC-3'

Antisense2: 5'- TGTTGGCTTTGGCAGAGTCC-3'

Claudinl8Al (64°C)

Sense : 5'-GAGGCAGAGTTCAGGCTTCACCGA-3'

Antisense: 5'- TGTTGGCTTTGGCAGAGTCC-3'

SLC13A1 (64°C)

Sense: 5'-CAGATGGTTGTGAGGAGTCTG-3'

Antisense : 5'-CCAGCTTTAACCATGTCAATG-3'

CLCA1 (62 °C)

Sense : 5'-ACACGAATGGTAGATACAGTG-3'

Antisense : 5'-ATACTTGTGAGCTGTTCCATG-3'

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FLJ21477 (68°C)

Sense: 5'- ACTGTTACCTTGCATGGACTG-3'

Antisense: 5'- CAATGAGAACACATGGACATG-3'

FLJ20694 (64°C)

Sense: 5'- CCATGAAAGCTCCATGTCTA-3'

Antisense: 5'- AGAGATGGCACATATTCTGTC

Ebner (70°C)

Sense: 5'-ATCGGCTGAAGTCAAGCATCG-3'

Antisense : 5'-TGGTCAGTGAGGACTCAGCTG-3'

Plunc (55°C)

Sense : 5'-TTTCTCTGCTTGATGCACTTG-3'

Antisense : 5'-GTGAGCACTGGGAAGCAGCTC-3'

SLC26A9 (67°C)

Sense: 5'-GGCAAATGCTAGAGACGTGA-3'

Antisense: 5'-AGGTGTCCTTCAGCTGCCAAG-3'

THC1005163 (60°C)

Sense: 5'- GTTAAGTGCTCTCTGGATTTG-3'

LOC134288 (64°C)

Sense : 5'-ATCCTGATTGCTGTGTGCAAG-3'

Antisense: 5'-CTCTTCTAGCTGGTCAACATC-3'

THC943866 (59°C)

Sense: 5'-CCAGCAACAACTTACGTGGTC-3'

Antisense : 5'-CCTTTATTCACCCAATCACTC-3'

FLJ21458 (62°C)

Sense: 5'-ATTCATGGTTCCAGCAGGGAC-3’

Antisense : 5'-GGGAGACAAAGTCACGTACTC-3’

Preparation of random hexamer-primed eDNA and quantitative real-time PCR

The expression of several genes was quantified by real-time PCR. The PCR products were detected using SYBR Green as intercalating reporter dye. The reporter fluorescence of SYBR Green is suppressed in solution and the dye is active only after binding to double-stranded DNA fragments. The increase in

2017201358 28 Feb 2017 the SYBR Green fluorescence as a result of the specific amplification using GOI-specific primers after each PCR cycle is utilized for quantification. Expression of the target gene is quantified absolutely or relative to the expression of a 5 control gene with constant expression in the tissues to be investigated. Expression was measured after standardization of the samples against 18s RNA as so-called housekeeping gene using the ΔΔ-Ct method (PE Biosystems, USA). The reactions were carried out in duplicates and determined in triplicates. The 10 QuantiTect SYBR Green PCR kit (Qiagen, Hilden) was used in accordance with the manufacturer's instructions. The cDNA was synthesized using the high capacity cDNA Archive Kit (PE Biosystems, USA) with use of hexamer primers in accordance with the manufacturer's instructions. Each 5 μΐ portions of the 15 diluted cDNA were employed in a total volume of 25 μΐ for the

PCR: sense primer 300 nM, antisense primer 300 nM; initial denaturation 95°C for 15 min; 95°C for 30 sec; annealing for sec; 72°C for 30 sec; 40 cycles. The sequences of the primers used are indicated in the respective examples.

Cloning and sequence analysis

Cloning of full-lengths and gene fragments took place by conventional methods. To ascertain the sequence, corresponding antigenes were amplified using the proofreading polymerase pfu 25 (Stratagene). After completion of the PCR, adenosine was ligated by means of HotStarTaq DNA polymerase to the ends of the amplicon in order to clone the fragments in accordance with the manufacturer's instructions into the TOPO-TA vector. The sequencing was carried out by a commercial service. The sequences were analysed using conventional prediction programs and algorithms.

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Western blotting

Cells from cell culture (endogenous expression of the target gene or synthesis of the target protein after transfection of an expression vector which encodes the target protein) or 5 tissue samples which might contain the target protein are lysed in a 1% SDS solution. The SDS denatures the proteins present in the lysate. The lysates of an experimental mixture are fractionated according to size by electrophoresis on 8-15% denaturing polyacrylamide gels (containing 1% SDS) depending on 10 the expected protein size (SDS polyacrylamide gel electrophoresis, SDS-PAGE). The proteins are then transferred by the semi-dry electroblotting method (Biorad) to nitrocellulose membrane (Schleicher & Schull) on which the desired protein can be detected. For this purpose, the membrane 15 is initially blocked (e.g. with milk powder) and then incubated with the specific antibody in a dilution of 1:20-1:200 (depending on the specificity of the antibody) for 60 minutes.

After a washing step, the membrane is incubated with a second antibody coupled to a marker (e.g. enzymes such as peroxidase 20 or alkaline phosphatase) which recognizes the first antibody.

After a further washing step, subsequently the target protein is visualized in a color or chemiluminescence reaction on the membrane by means of an enzyme reaction (e.g. ECL, Amersham Bioscience). The result is documented by photographing with a 25 suitable camera.

Analysis of protein modifications usually takes place by Western blotting. Glycosilations, which usually have a size of several kDa, lead to a larger total mass of the target protein, 30 which can be fractionated in the SDS-PAGE. To detect specific

0- and N-glycosidic linkages, protein lysates from tissues or cells are incubated before denaturation by SDS with 0- or N-glycosidases (in accordance with their respective manufacturer's instructions, e.g. PNgase, endoglycosidase F,

2017201358 28 Feb 2017 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 glycosilation 5 and, in this way, also analyse the tumor specificity of a modification. The exact position of the glycosilated amino acid can be predicted with algorithms and prediction programs.

Immunofluorescence

Cells of established cell lines which either synthesize the target protein endogenously (detection of the RNA in RT-PCR or of the protein by Western blotting) or else have been transfected with plasmid DNA before the IF are used. 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. 1997; 75: 441-7) . The transfected plasmid may in the immunofluorescence encode the unmodified protein or else couple various amino acid markers to the target 20 protein. The most important markers are, for example, the fluorescing green fluorescent protein (GFP) in its various differentially fluorescing forms and short peptide sequences of 6-12 amino acids for which high-affinity and specific antibodies are available. Cells which synthesize the target 25 protein are fixed with paraformaldehyde, saponin or methanol.

The cells can then if required be permeabilized by incubation with detergents (e.g. 0.2% Triton X-100). After the fixation/permeabilization, the cells are incubated with a primary antibody which is directed against the target protein 30 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. fluorescin, Texas Red, Dako) which binds to the first antibody. The cells labeled in this way are then covered with a layer of glycerol and analysed with the aid

2017201358 28 Feb 2017 of a fluorescence microscope according to the manufacturer's instructions. Specific fluorescence emissions are achieved in this case by specific excitation depending on the substances employed. The analysis normally allows reliable localization of 5 the target protein, the antibody quality and the target protein being confirmed in double stainings to stain in addition to the target protein also the coupled amino acid markers or other marker proteins whose localization has been described in the literature. GFP and its derivatives represent a special case 10 that can be directly excited and itself fluoresces, so that no antibodies are necessary for the detection.

Immunohi s tochemi s try

IHC serves specifically for (1) being able to estimate the 15 amount of target protein in tumor and normal tissues, (2) analysing how many cells in the tumor and healthy tissue synthesize the target gene, and/or (3) defining the cell type in a tissue (tumor, healthy cells) in which the target protein is detectable.

Different protocols must be used depending on the individual antibody (e.g. Diagnostic Immunohistochemistry by David J., MD Dabbs ISBN: 0443065667 or in Microscopy,

Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy ISBN: 0306467704).

Immunohistochemistry (IHC) on specific tissue samples serves to detect protein in the corresponding tissue. The aim of this method is to identify the localization of a protein in a functionally intact tissue aggregate. IHC serves specifically 30 for (1) being able to estimate the amount of target protein in tumor and normal tissues, (2) analysing how many cells in tumor and healthy tissue synthesize 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

2017201358 28 Feb 2017 protein of a target gene can 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 Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy ISBN: 0306467704.

It should be noted that, because of the properties of 10 antibodies, different protocols have to be used (an example is described below) in order to obtain a valid result.

Ordinarily, histologically defined tumor tissues and, as reference, comparable healthy tissues are employed in the IHC.

It is moreover 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.

Fixed tissue (e.g. fixation with aldehyde-containing substances, formaldehyde, paraformaldehyde or in alcoholic tissue pieces with a thickness of

1-10 pm are applied to a glass support. Paraffin-embedded samples are deparaffinated for example with xylene.

are washed with incubation with for 1-18 hours,

The samples

TBS-T and blocked in serum. This is the first antibody (dilution: 1:2 with affinity-purified followed by antibodies normally being used. A washing second antibody which step is followed is coupled to an by incubation with a alkaline phosphatase (alternative: for example peroxidase), and is directed against the first antibody, for about 30-60 minutes.

by color reaction using color substrates which

This is followed are converted by the bound enzymes (cf. for example, Shi et al., J. Histochem.

Cytochem. 39: 741-748, 1991; Shin et al., Lab. Invest. 64: 69375

2017201358 28 Feb 2017

702, 1991). To demonstrate the antibody specificity, the reaction can be blocked by previous addition of the immunogen.

Immunization (See also 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).

The process for preparing antibodies is described briefly below, and details can be found in the cited publications. Firstly, animals (e.g. rabbits) are immunized by a first injection of the desired target protein. The animal's immune response to the immunogen can be enhanced by a second or third immunization within a defined period (about 2-4 weeks after the preceding immunization). Again after various defined periods (first bleeding after 4 weeks, then about every 2 weeks with a total of up to 5 samplings), blood is taken from the animals, and an immune serum is obtained therefrom.

The animals are usually immunized by one of four wellestablished methods, with other methods also being available. It is moreover possible to immunize with peptides which are specific for the target protein, with the complete protein or with extracellular partial sequences of a protein which can be identified experimentally or via prediction programs.

(1) In the first case, peptides (length: 8-12 amino acids) conjugated to KLH (keyhole limpet hemocyanin) are synthesized by a standardized in vitro method, and these peptides are used for the immunization.

Usually, 3 immunizations are carried out with a concentration of 5-1000 pg/immunization. The immunization can also be carried out as service from service providers.

(2) Alternatively, the immunization can be carried

2017201358 28 Feb 2017 out with recombinant cloned DNA expression synthesized particular

Invitrogen, of the vector, proteins. For this purpose, target gene is cloned into the an and the target protein is in analogy manufacturer

Clontech, in vitro, in bacteria

S. pombe), in insect to the cells

After synthesis in one of protein is purified, usually taking methods .

use for molecular place

It is also the conditions of the

Roche Diagnostics, or in mammalian cells.

the systems, the target the purification in this case by standardized chromatographic possible in this connection to immunization proteins which have a anchor as aid for purification tag, Qiagen; FLAG tag, Roche Diagnostics;

number of protocols

Gst fusion are to be found for example

Molecular Biology,

Interscience .

a cell line protein also be in the Current Protocols in

John Wiley & Sons Ltd.,

Wiley which synthesizes the desired endogenously is available, this cell line used to produce the specific this case, the immunization takes inj ections inj ection can antiserum.

place in

In

1-3 in each case with about 1-5 χ 10⁷ cells.

immunization can also take place by of DNA (DNA immunization) . For this purpose, the target gene is initially cloned into an expression vector so that the target sequence is under the control of a strong eukaryotic promoter promoter).

Subsequently, 5-100 pg of DNA are 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

2017201358 28 Feb 2017 taken up by the animal's cells, the target gene is expressed, and the animal finally develops an immune response to the target gene (Jung et al., Mol Cells

12:41-49, 2001; Kasinrerk et al., Hybrid Hybridomics

21:287-293, 2002) .

Quality control of the polyclonal serum or antibody

Assays based on cell culture with subsequent Western blotting are most suitable for demonstrating specificity (various 10 variations are described for example in Current Protocols in

Protein Chemistry, John Wiley & Sons Ltd., Wiley InterScience). For the demonstration, cells are transfected with a cDNA, which is under the control of a strong eukaryotic promoter (e.g. cytomegalovirus promoter), for the target 15 protein. 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) . It is also possible alternatively to use cell lines which express the 20 target gene endogenously (demonstration by target gene-specific

RT-PCR) . As control, in the ideal case homologous genes are also transfected in the experiment, in order to be able to demonstrate in the following Western blot the specificity of the analysed antibody.

In the subsequent Western blot, cells from cell culture or tissue samples which might contain the target protein are lysed in a 1% SDS solution, and the proteins are denatured thereby.

The lysates are fractionated according to size by 30 electrophoresis on 8-15% 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

2017201358 28 Feb 2017 & Schull) . 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 about 1:20-1:200, depending on the specificity of the 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 in a color or chemiluminescent reaction to visualize the target protein on 10 the membrane (e.g. ECL, Amersham Bioscience) . An antibody with a high specificity for the target protein should in the ideal case recognize only the desired protein itself.

Various methods are used to confirm the membrane localization 15 of the target protein identified in the in silico approach. An important and well-established method using the antibodies described above is immunofluorescence (IF) . Cells of established cell lines which either synthesize the target protein (detection of the RNA in an RT-PCR or of the protein in 20 a Western blot) or else have been transfected with plasmid DNA are used for this. 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. 75:44125 7, 1997) . The plasmid transfected into the cells can in the immunofluorescence encode the unmodified protein or else couple various amino acid markers to the target protein. The principal markers are, for example, the fluorescent green fluorescent protein (GFP) in its various differentially fluorescent forms, 30 short peptide sequences of 6-12 amino acids for which highaffinity and specific antibodies are available, or the short amino acid sequence Cys-Cys-X-X-Cys-Cys which can bind via its cysteine specific fluorescent substances (Invitrogen). Cells which synthesize the target protein are fixed for example with

2017201358 28 Feb 2017 paraformaldehyde or methanol. The cells can 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 5 against one of the coupled markers. After a washing step, the mixture is incubated with a second antibody which is coupled to a fluorescent marker (e.g. fluorescin, Texas Red, Dako) and which binds to the first antibody. The cells labeled in this way are then covered with a layer of glycerol and analysed with 10 the aid of a fluorescence microscope according to the manufacturer's instructions. 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 15 the target protein being confirmed in double stainings to stain 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. GFP and its derivatives represents a special case, being excitable directly and 20 themselves fluorescing. The membrane permeability, which can be controlled through the use of detergents, permits demonstration in the immunofluorescence of whether an immunogenic epitope is located inside or outside the cell. The prediction of the selected proteins can thus be supported experimentally. An 25 alt ernative 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 analysed in a flow cytometer in accordance with the manufacturer's instructions. Only extracellular 30 epitopes can be recognized by the antibody to be analysed in this method. A difference from immunofluorescence is that it is possible to distinguish between dead and living cells by use of, for example, propidium iodide or Trypan blue, and thus avoid false-positive results.

2017201358 28 Feb 2017

Affinity purification

Purification of the polyclonal sera took place in the case of the peptide antibodies entirely, or in the case of the antibodies against recombinant proteins in part, as service by the contracted companies. For this purpose, in both cases, the appropriate peptide or recombinant protein was covalently bonded to a matrix, and the latter was, after the coupling, equilibrated with a native buffer (PBS: phosphate buffered 10 saline) and then incubated with the crude serum. After a further PBS washing step, the antibody was eluted with 100 mM glycine, pH 2.7, and the eluate was immediately neutralized in 2M TRIS, pH 8. The antibodies purified in this way could then be employed for specific detection of the target proteins both 15 by Western blotting and by immunofluorescence.

Preparation of EGFP transfectants

For the immunofluorescence microscopy of heterologously expressed tumor-associated antigens, the complete ORF of the 20 antigens was cloned in pEGFP-Cl and pEGFP-N3 vectors (Clontech). CHO and NIH3T3 cells cultivated on slides were transfected with the appropriate plasmid constructs using Fugene transfection reagent (Roche) in accordance with the manufacturer's instructions and, after 12-24 h, analysed by 25 immunofluorescence microscopy.

Example 1: Identification of GPR35 as diagnostic and therapeutic cancer target

GPR35 (SEQ ID NO:1) and its translation product (SEQ ID NO: 9) 30 have been described as putative G protein-coupled receptor. The sequence is published in Genbank under accession No. AF089087.

This transcript codes for a protein of 309 amino acids with a molecular weight of 34 kDa. It was predicted that GPR35 belongs to the superfamily of G protein-coupled receptors with

2017201358 28 Feb 2017 transmembrane domains (O'Dowd et al., Genomics 47:310-13, 1998) . In order to confirm the predicted localization of GPR35 in the cell, the protein was fused to eGFP as reporter molecule and, after transfection of the appropriate plasmid, expressed 5 heterologously in 293 cells. The localization was then analysed in a fluorescence microscope. It was confirmed according to the invention that GPR35 is an integral transmembrane molecule (fig. 17) . Investigation to date on human GPR35 (see, inter alia, Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M,

Hinokio Y, Lindner TH, Mashima H, Schwarz PE, del Bosque-Plata

L, Horikawa Y, Oda Y, Yoshiuchi I, Colilla S, Polonsky KS, Wei

S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL, Bell GI Nat Genet. 2000 Oct;

26(2):163-75) suggested that GPR35 is activated in many healthy 15 tissues. The reading frame of the gene comprises a single exon.

According to the invention, a gene-specific primer pair (SEQ ID NO:20, 21) for GPR35 was used in RT-PCR analyses to amplify cDNA in the colon and in colon carcinoma (13/26) . By contrast, no significant expression is detectable in other 20 normal tissues. Because of the particular fact that GPR35 consists of a single exon, genomic DNA impurities cannot be detected with intron-spanning primers. In order to preclude genomic contamination of the RNA samples, therefore, all RNAs were treated with DNAse. GPR35 transcripts were detected 25 according to the invention only in the colon, in the rectum, in the testis and in colon carcinomas using DNA-free RNA.

2017201358 28 Feb 2017

Tab. 1 GPR35 expression in normal tissues

Normal tissue	Expression
Brain	-
Cerebellum	-
Myocardium	-
Skeletal muscle	-
Rectum	++
Stomach	-
Colon	++
Pancreas	-
Kidney	-
Testis	-
Thymus	-
Mammary glands	-
Ovary	-
Uterus	n. d.
Skin	-
Lung	-
Thyroid	-
Lymph nodes	-
Spleen	-
PBMC	-
Adrenal	-
Esophagus	-
Small intestine	+
Prostate	-

(nd = not determined)

The selective and high expression of GPR35 transcripts in

2017201358 28 Feb 2017 normal colonic tissue and was not previously known in colon carcinoma biopsies (fig. 1) and can be utilized according to the invention for molecular diagnostic methods such as RT-PCR for detecting disseminating tumor cells in marrow and for detecting metastases

Quantitative RT-PCR with specific also confirms that GPR35 is a specific differentiation antigen the in primers highly which serum and bone other tissues.

selective intestineis also contained in intestinal tumors and in intestinal tumor metastases. In some intestinal tumors, it is in fact overexpressed by one log compared with normal intestine (fig. 18). Antibodies were produced by immunizing rabbits for detecting GPR35 protein. The following peptides were used to propagate these antibodies:

SEQ ID NO:90 GSSDLTWPPAIKLGC (AA 9-23)

SEQ ID NO:91: DRYVAVRHPLRARGLR (AA 112-127)

SEQ ID NO :92: VAPRAKAHKSQDSLC (C terminus)

SEQ ID NO:93 CFRSTRHNFNSMR (extracell, domain 2)

Stainings with these antibodies for example in a Western blot 20 confirm the expression in tumors. All 4 extracellular domains of GPR35 (position of the predicted extracellular domains in the sequence of SEQ ID NO: 9 AA 1-22 (SEQ ID NO: 94); AA 81-94 (SEQ ID NO:95); AA 156-176 (SEQ ID NO:96); AA 280-309 (SEQ ID NO:97)) can be used according to the invention as 25 target structures of monoclonal antibodies. These antibodies bind specifically to the cell surface of tumor cells and can be used both for diagnostic and for therapeutic methods.

Overexpression of GPR35 in tumors provides additional support for such a use. In addition, the sequences coding for proteins 30 can be used according to the invention as vaccine (RNA, DNA, peptide, protein) for inducing tumor-specific immune responses (T-cell and B-cell-mediated immune responses). In addition, it has surprisingly been found that a further start codon exists

5' in front of the generally known start codon and expresses an

2017201358 28 Feb 2017

N-terminally extended protein.

It has thus been protein which ubiquitously, is gastrointestinal found according to the invention that GPR35, a was previously described tumor-associated overexpressed, tumors, especially in tumors

GPR35 is therefore structure for the

Investigation to as expressed selectively in of the colon.

suitable in particular as molecular target diagnosis and treatment of these date of human GPR35, cf., for

Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M,

Hinokio Y, Lindner TH, Mashima H, Schwarz PE,

L, Horikawa Y, Oda Y, Yoshiuchi I, Colilla S,

S, Concannon P, Iwasaki N, Schulze J, Baier

Groop L, Boerwinkle E, Hanis CL, Bell Gl healthy tissues.

the invention tumors .

example,

Hara M, del Bosque-Plata

Polonsky KS, Wei

LJ, Bogardus C,

Nat Genet. 2000 suggested that GPR35 is activated in many

By contrast, showed that significantly detectable in contrast thereto, is colon tumors. In sequence, according the investigations according to

GPR35 is surprisingly not most normal tissues and, in highly activated in primary and metastatic addition, besides the described GPR35 to the invention a novel translation variant which makes use found (SEQ

GPR35 is a very large very well of an alternative start codon has been member of the group protein family whose structure and function has been investigated. GPCR are outstandingly suitable as target structures for the development of pharmaceutically active substances, because the methods necessary therefor (e.g.

receptor expression, purification, ligand screening, mutagenizing, functional inhibition, selection of agonistic and antagonistic ligands, radiolabeling of ligands) is very well developed and described in detail, cf., for example, G

2017201358 28 Feb 2017

Protein-Coupled Receptors by Tatsuya Haga, Gabriel Berstein and Gabriel Bernstein ISBN: 0849333849 and in Identification and Expression of G-Protein Coupled Receptors Receptor

Biochemistry and Methodology by Kevin R. Lynch ASIN:

0471183105. Realization according to the invention that GPR35 is undetectable in most healthy tissues but undergoes tumorassociated expression on the cell surface, enables it to be used as tumor-associated target structure for example for pharmaceutically active ligands, especially in conjugation for 10 example with radioactive molecules as pharmaceutical substances. It is possible in a particular embodiment to use radiolabeled ligands which bind to GPR35 for detecting tumor cells or for treating colon tumors in vivo.

Example 2: Identification of GUCY2C in hepatic and ovarian tumors and novel GUCY2C splice variants as diagnostic and therapeutic cancer targets

Guanylate cyclase 2C (SEQ ID NO:2; translation product: SEQ ID NO:11) - a type I transmembrane protein - belongs to the 20 family of natriuretic peptide receptors. The sequence is published in Genbank under the accession number NM_004963. Binding of the peptides guanylin and uroguanylin or else heatstable enterotoxins (STa) increases the intracellular cGMP concentration, thus inducing signal transduction processes 25 inside the cell.

Recent investigations indicate that expression of GUCY2C also extends to extraintestinal regions such as, for example, primary and metastatic adenocarcinomas of the stomach and of the esophagus (Park et al., Cancer Epidemiol Biomarkers Prev.

11: 739-44, 2002) . A splice variant of GUCYC which is found both in normal and transformed tissue of the intestine comprises a 142 bp deletion in exon 1, thus preventing translation of a GUCY2C-like product (Pearlman et al., Dig.

2017201358 28 Feb 2017

Dis. Sci. 45:298-05, 2000) . The only splice variant described to date leads to no translation product.

The aim according to the invention was to identify tumor5 associated splice variants for GUCY2C which can be utilized both for diagnosis and for therapy.

RT-PCR investigations with a GUCY2C-specific primer pair (SEQ ID NO:22, 23, 98, 99) show pronounced expression of GUCY2C transcripts in normal colon and stomach, and weak expression in 10 liver, testis, ovary, thymus, spleen, brain and lung (tab. 2, fig. 19). Expression in colon and stomach was at least 50 times higher than in all other normal tissues. Marked GUCY2C transcript levels were detected in colon carcinoma and stomach carcinoma (tab. 2) .

These results were specified by a quantitative PCR analysis and showed pronounced GUCY2C expression in normal colon, ileum, and in almost all colon carcinoma samples investigated (fig. 2, 19B) . A massive overexpression was detectable in some colon carcinoma samples. In addition, expression is found in 7/10 stomach tumors. We 20 also surprisingly found that the gene is activated in many other previously undescribed tumors, inter alia ovarian, breast, liver and prostate tumors (fig. 19B, tab. 2).

Table 2: GUC2C expression in normal and tumor tissues

Normal tissues	Expression
Brain	+
Cerebellum
Myocardium
Skeletal muscle
Myocardium

Tumor type	Expression
Colon carcinoma	+ + +
Pancreatic	-
carcinoma
Esophageal	-
carcinoma
Stomach	+ + +
carcinoma
Bronchial	-

2017201358 28 Feb 2017

Normal tissues	Expression

Stomach
Colon
Pancreas
Kidney	-
Liver	+
Testis	++
Thymus	+
Breast	-
Ovary	+
Uterus	+
Skin
Lung	+
Thyroid
Lymph nodes	-
Spleen	+
PBMC	-
Prostate	-

Tumor type	Expression
carcinoma
Mammary carcinoma	- +
Ovarian carcinoma	+
Endometrial carci
ENT tumors
Renal cell carcinoma
Prostate carcinoma	+
Liver carcinoma	+

The following primer pairs were used to detect splice variants in colonic tissue and colon carcinoma tissue: GUCY2C118s/GUCY2C-498as (SEQ ID NO:24, 29); GUCY2C-621s/GUCY2C-1140as (SEQ ID NO:25, 30); GUCY2C-1450s/GUCY2C-1790as (SEQ ID NO:26,

31); GUCY2C-1993s/GUCY2C-2366as (SEQ ID NO:27,32);

GUCY2C-2717s/GUCY2C-3200as (SEQ ID NO:28,33);

GUCY2C-118s/GUCY2C-1140as (SEQ ID NO:24,30);

GUCY2C-621S/GUCY2C-1790as (SEQ ID NO:25,31);

2017201358 28 Feb 2017

GUCY2C-1450s/GUCY2C-2366as (SEQ ID NO:26, 32);

GUCY2C-1993s/GUCY2C-3200as (SEQ ID NO:27, 33).

On investigation of splice variants in colon carcinoma tissue, three previously unknown forms were identified according to the invention.

a) A deletion of exon 3 (SEQ ID NO: 3) which leads to a variant of GUCY2C which is only 111 amino acids long and in which the asparagine at position 111 is replaced by a proline.

b) A deletion of exon 6 (SEQ ID NO:4) which results in an expression product 258 amino acids long. This would generate a C-terminal neoepitope comprising 13 amino acids .

c) A variant in which the nucleotides at positions 1606-1614, and the corresponding amino acids L(536), L(537) and Q(538), are deleted (SEQ ID NO:5).

The splice variants according to the invention with deletions respectively in exon 3 and exon 6 (SEQ ID NO:3, 4) are distinguished in particular by the translation products (SEQ ID NO:12, 13) having no transmembrane domain. The result in the case of exon 6 deletion is a C-terminal neoepitope of 13 amino acids which shows no homology whatsoever with previously known proteins. This neoepitope is thus predestined to be a target structure for immunotherapy. The splice variant of the invention with base deletions at positions 1606-1614 (SEQ ID NO: 5) and its translation product (SEQ ID NO: 14) likewise comprises a neoepitope. Antibodies for detecting GUCY2C protein were produced by immunizing rabbits. The following peptides were used to propagate these antibodies: SEQ ID NO:100: HNGSYEISVLMMGNS (AA 31-45)

SEQ ID NO:101: NLPTPPTVENQQRLA (AA 1009-1023)

Such antibodies can in principle be used for diagnostic and therapeutic purposes.

2017201358 28 Feb 2017

In particular, the extracellular domain of GUCY2C (position of the predicted extracellular domain from the sequence of SEQ ID NO:11: ΆΆ 454-1073 (SEQ ID NO:102)) can be used according to the invention as target structure of monoclonal antibodies. However, the structural prediction is somewhat ambiguous and not yet verified experimentally, so that an alternative membrane orientation is also conceivable. In this case, amino acids 1-431 would be outside the cell and be 10 suitable as starting point for monoclonal antibodies. These antibodies bind specifically to the cell surface of tumor cells and can be used both for diagnostic and for therapeutic methods. Overexpression of GUCY2C, especially in the colon tumors, provides additional support for such a use. Sequences 15 coding for proteins can moreover be used according to the invention as vaccine (RNA, DNA, peptides, protein) for inducing tumor-specific immune responses (T-cell- and B-cell-mediated immune responses).

It is moreover possible in accordance with the cellular 20 function of the GUCY2C molecule to develop according to the invention substances, especially small molecules, which modulate the function of the enzyme on tumor cells. The product of the enzymic reaction, cGMP, is a known cellular signal molecule with a wide variety of functions (Tremblay et al. Mol 25 Cell Biochem 230, 31).

Example 3: Identification of SCGB3A2 as diagnostic and therapeutic cancer target

SCGB3A2 (SEQ ID NO:6) (translation product: SEQ ID NO:15) belongs to the secretoglobin gene family. The sequence is published in GenBank under accession number NM_054023. SCGB3A2 (UGRP1) is a homodimeric secretory protein with a size of 17 kDa, which is expressed exclusively in the lung and in the spiracles (Niimi et al., Am J Hum Genet 70:718-25, 2002) . RT

2017201358 28 Feb 2017

PCR investigations with a primer pair (SEQ ID NO:37, 38) confirmed selective expression in normal lung tissue. Lung- and trachea-specific genes, e.g. for surfactant proteins, are highly downregulated in malignant tumors during 5 dedifferentiation and are normally undetectable in lung tumors.

It was surprisingly found that SCGB3A2 is active in primary and metastatic lung tumors. The investigations according to the invention showed that SCGB3A2 is strongly and frequently expressed in bronchial carcinomas (fig. 4). All the other 23 10 normal tissues tested, apart from lung and trachea, show no expression (cf. fig. 20).

This was additionally confirmed in a specific quantitative RT-PCR (SEQ ID NO:103, 104) (fig. 20) which additionally shows overexpression by at least one log in more than 50% of 15 bronchial carcinomas.

The selective and high expression of SCGB3A2 in normal lung tissue and in lung carcinoma biopsies can be used according to the invention for molecular diagnostic methods such as RT-PCR for detecting disseminating tumor cells in blood and bone 20 marrow, sputum, bronchial aspirate or lavage and for detecting metastases in other tissues, e.g. in local lymph nodes. In the healthy lung, SCGB3A2 is secreted by specialized cells exclusively into the bronchi. Accordingly, it is not to be expected that SCGB3A2 protein will be detectable in body fluids 25 outside the respiratory tract in healthy individuals. By contrast, in particular metastatic tumor cells secrete their protein products directly into the bloodstream. One aspect of the invention therefore relates to detection of SCGB3A2 products in serum or plasma of patients via a specific antibody 30 assay as diagnostic finding for lung tumors.

Antibodies for detecting SCGB3A2 protein were produced by immunizing rabbits. The following peptides were used to propagate these antibodies:

2017201358 28 Feb 2017

SEQ ID NO:105: LINKVPLPVDKLAPL

SEQ ID NO:106: SEAVKKLLEALSHLV

An SCGB3A2-specific reaction was detectable in immunofluorescence (fig. 21). As expected for a secreted 5 protein, the distribution of SCGB3A2 in the cell was assignable to the endoplasmic reticulum and secretion granules (fig. 21A).

To check the specificity, the cells were transfected in parallel with a plasmid that synthesizes an SCGB3A2-GFP fusion protein. Protein detection took place in this case via the 10 autofluorescent GFP (green fluorescent protein) (fig. 21B) .

Superimposition of the two fluorescence diagrams shows unambiguously that the immune serum specifically recognizes SCGB3A2 protein (fig. 21C).

Such antibodies can be used according to the invention for 15 example in the form of immunoassays for diagnostic and therapeutic purposes.

Example 4: Identification of claudin-18Al and claudin-18A2 splice variants as diagnostic and therapeutic cancer targets

The claudin-18 gene codes for a surface membrane molecule having 4 transmembrane domains and intracellular N terminus and C terminus. Niimi and colleagues (Mol. Cell. Biol. 21:7380-90, 2001) describe two splice variants of the murine and human claudin-18 which have been described as expressed selectively in lung tissue (claudin-18Al) and in stomach tissue (claudin-18A2), respectively. These variants differ in the N terminus (fig. 22).

It was investigated according to the invention how far the splice variants claudin-18A2 (SEQ ID NO:7) and claudin-18Al 30 (SEQ ID NO:117), and their respective translation products (SEQ ID NO: 16 and 118), can be used as markers or therapeutic target structures for tumors. A quantitative PCR able to distinguish between the two variants was established by selecting Al-specific (SEQ ID NO:109 & 110) and A2-specific

2017201358 28 Feb 2017 (SEQ ID NO: 107 & 108) primer pairs. The A2 splice variant was additionally tested with a second primer pair in a conventional PCR (SEQ ID NO: 39 & 40) . The Al variant is described to be active only in normal lung. However, it was surprisingly found according to the invention that the Al variant is also active in the gastric mucosa. Stomach and lung are the only normal tissues showing significant activation. All other normal tissues are negative for claudin-Al. On investigating tumors, it was surprisingly found that claudin-Al is highly activated in a large number of tumor tissues. Particularly strong expression is to be found in stomach tumors, lung tumors, pancreatic carcinomas, esophageal carcinomas (fig. 23), ENT tumors and prostate carcinomas. The claudin-Al expression levels in ENT, prostate, pancreatic and esophageal tumors are

100-10 000 higher than the levels in the corresponding normal tissues. The oligonucleotides used to investigate the claudin-A2 splice variant specifically enable this transcript to be amplified (SEQ ID NO:39 & 40 and 107 & 108) .

Investigation revealed that the A2 splice variant is expressed in none of the more than 20 normal tissues investigated apart from gastric mucosa and to a small extent also testis tissue. We have found that the A2 variant is also, like the Al variant, activated in many tumors (depicted by way of example in fig. 24) . These include stomach tumors (8/10), pancreatic tumors (6/6), esophageal carcinomas (5/10) and liver carcinomas. Although no activation of claudin-18A2 is detectable in healthy lung, it was surprisingly found that some lung tumors express the A2.1 splice variant.

Table 3A. Expression of claudin-18A2 in normal and tumor

2017201358 28 Feb 2017 tissues

Normal tissue	Expression
Brain	-
Cerebellum	-
Myocardium	-
Skeletal muscle
Endometrium	-
Stomach	+++
Colon	-
Pancreas	-
Kidney	-
Liver	-
Testis	+
Thymus	-
Breast	-
Ovary	-
Uterus	-
Skin	-
Lung	-
Thyroid	-
Lymph nodes	-
Spleen	-
PBMC	-
Esophagus	-

Tumor type	Expression
Colon carcinoma
Pancreatic carcinoma	+ +
Esophageal carcinoma	+ +
Gastric carcinoma
Bronchial carcinoma	+ +
Breast carcinoma
Ovarian carcinoma
Endometrial carcinoma	n. i .
ENT tumors	+ +
Renal cell carcinoma
Prostate carcinoma

Table 3B. Expression of claudin-18Al in normal and tumor

2017201358 28 Feb 2017 tissues

Normal tissue	Expression
Brain	-
Cerebellum	-
Myocardium	-
Skeletal muscle
Endometrium	-
Stomach	+++
Colon	-
Pancreas	-
Kidney	-
Liver	-
Testis	+
Thymus	-
Breast	-
Ovary	-
Uterus	-
Skin	-
Lung	+++
Thyroid	-
Lymph nodes	-
Spleen	-
PBMC	-
Esophagus	-

Tumor type	Expression
Colon carcinoma
Pancreatic carcinoma	+ +
Esophageal carcinoma	+ +
Gastric carcinoma
Bronchial carcinoma	+ +
Breast carcinoma	+
Ovarian carcinoma	n. i .
Endometrial carcinoma	n. i .
ENT tumors	+ +
Renal cell carcinoma
Prostate carcinoma	+ +

Conventional PCR as independent control investigation also

2017201358 28 Feb 2017 confirmed the oligonucleotides results specific amplification of according half of expression expression of the quantitative

NO:39, the A2 splice variant.

PCR.

The this permit

It was shown to the invention that the of is carcinomas and tested pancreatic carcinomas not detectable in other tissues

PCR. In particular, there is no expression showed strong

By contrast, by conventional in lung, liver, blood, lymph nodes, breast tissue and kidney tissue (tab. 3).

The splice variants thus represent highly specific molecular markers gastrointestinal tract as well as prostate carcinomas and metastases according to the invention for tumors of the upper lung tumors, ENT tumors, thereof. These molecular markers can be used according to the invention for detecting tumor cells. Detection of the tumors is possible according to the invention with

40, the oligonucleotides

Particularly described suitable oligonucleotides are primer pairs of which at least one binds under stringent conditions to a segment of the transcript which specific for one the other

In order to confirm these data at the protein level, claudinspecific antibodies and immune sera were generated by immunizing animals. The plasma membrane localization of claudin-18 and the protein topology was confirmed by analysis of the transmembrane domains with bioinformatic tools (TMHMM,

TMPRED) and immunofluorescence investigations of cells which expressed claudin-18 fusion proteins tagged with enhanced GFP. Claudin-18 has two extracellular domains. The N-terminal extracellular domain differs in sequence in the two splice variants (SEQ ID NO: 111 for Al and SEQ ID NO: 112 for A2) . The

2017201358 28 Feb 2017

C-terminal extracellular domain is identical for both variants (SEQ ID NO:137) . To date, no antibodies which bind to the extracellular domains of claudin-18 have yet been described. According to the invention, peptide epitopes which are located 5 extracellularly and are specific for variant Al or A2 or occur in both variants were selected for the immunization. Both variants of claudin-18 have no conventional glycosylation motifs and the glycosylation of the protein was therefore not to be expected. Nevertheless, account was taken in the 10 selection of the epitopes that epitopes which comprise asparagine, serine, threonine are potentially glycosylated in rare cases even without conventional glycosylation sites. Glycosylation of an epitope may prevent the binding of an antibody specific for this epitope. Inter alia, epitopes were 15 selected according to the invention so that the antibodies generated thereby permit the glycosylation status of the antigen to be distinguished. The following peptides, inter alia, were selected for producing antibodies for the immunization:

SEQ ID NO:17: DQWSTQDLYN (N-terminal extracellular domain, A2specific, binding independent of glycosylation)

SEQ ID NO:18: NNPVTAVFNYQ (N-terminal extracellular domain, A2specific, binding mainly to unglycosylated form, N37)

SEQ ID NO:113:	STQDLYNNPVTAVF	(N-terminal	extracellular	domain,
25	A2-specific, binding only to non-glycosylated form, N37)
	SEQ ID NO:114:	DMWSTQDLYDNP	(N-terminal	extracellular	domain,
	Al-specific)
	SEQ ID NO:115:	CRPYFTILGLPA	(N-terminal	extracellular	domain,
	mainly specific	for Al)
30	SEQ ID NO:116:	TNFWMSTANMYTG	(C-terminal	extracellular	domain,
	recognizes both	Al and A2).

The data for the A2-specific antibody produced by immunization with SEQ ID NO: 17 are shown by way of example. The specific

2017201358 28 Feb 2017 antibody can be utilized under various fixation conditions for immunofluorescence investigations. With comparative stainings of RT-PCR-positive and negative cell lines, in an amount which is readily detectable, the corresponding protein can be 5 specifically detected in the gastric carcinoma cell lines typed as positive (fig. 25) . The endogenous protein is membranelocated and forms relatively large focal aggregates on the membrane. This antibody was additionally employed for protein detection in Western blotting. As expected, protein is detected only in stomach and in no other normal tissue, not even lung (fig. 29). The comparative staining of stomach tumors and adjacent normal stomach tissue from patients surprisingly revealed that claudin-18 A2 has a smaller mass weight in all stomach tumors in which this protein is detected (fig. 30, 15 left) . It was found according to the invention in a series of experiments that a band also appears at this level when lysate of normal stomach tissue is treated with the deglycosylating agent PNGase F (fig. 30, right). Whereas exclusively the glycosylated form of the A2 variant is detectable in all normal stomach tissues, A2 is detectable as such in more than 60% of the investigated gastric carcinomas, in particular exclusively in the deglycosylated form. Although claudin-18 level, it previously is is in not to the the A2 variant of detected in normal lung be found in bronchial even at the protein carcinomas, as also quantitative RT-PCR. Once again, only the deglycosylated variant is present (fig. 31). Antibodies which recognize the extracellular domain of the claudin-18-A2 splice variant have been produced according to the invention. In addition, antibodies which selectively recognize the N-terminal domain of the claudin-18-Al splice variant antibodies which bind to both variants in the region of the

C-terminal extracellular domain (fig. 27) have been produced.

It is possible according to the invention to use such antibodies for diagnostic purposes, e.g. immunohistology (fig.

2017201358 28 Feb 2017

32), but also for therapeutic purposes as explained above. A further important aspect relates to differentially glycosylated domains of claudin-18. Antibodies which exclusively bind to non-glycosylated epitopes have been produced according to the invention. Claudin-18 itself is a highly selective differentiating antigen for stomach tissue (A2) and for the lung and stomach (Al). Since it is evidently affected by changes in the glycosylation machinery in tumors, a particular, deglycosylated, variant of A2 is produced in tumors. This can 10 be utilized diagnostically and therapeutically. Immune sera such as the one described here (against peptide of SEQ ID NO:17) can be utilized diagnostically for example in Western blotting. Antibodies which are entirely unable to bind to the glycosylated epitope as obtained for example by immunization with peptide of SEQ ID NO: 113 (figure 26), can distinguish tumor tissue from normal tissue in the binding. It is possible in particular to employ such antibodies therapeutically because they are highly selective. The produced antibodies can be used directly also for producing chimeric or 20 humanized recombinant antibodies. This can also take place directly with antibodies obtained from rabbits (concerning this, see J Biol Chem. 2000 May 5;275(18):13668-76 by Rader C, Ritter G, Nathan S, Elia M, Gout I, Jungbluth AA, Cohen LS, Welt S, Old LJ, Barbas CF 3rd. The rabbit antibody repertoire 25 as a novel source for the generation of therapeutic human antibodies) . For this purpose, lymphocytes from the immunized animals were preserved. The amino acids 1-47 (SEQ ID NO:19 and 120) also represent particularly good epitopes for immunotherapeutic methods such as vaccines and the adoptive 30 transfer of antigen-specific T lymphocytes.

2017201358 28 Feb 2017

Example 5: Identification of SLC13A1 as diagnostic and therapeutic cancer target

SLC13A1 belongs to the family of sodium sulfate cotransporters.

The human gene is, in contrast to the mouse homolog of this gene, selectively expressed in the kidney (Lee et al., Genomics 70:354-63). SLC13A1 codes for a protein of 595 amino acids and comprises 13 putative transmembrane domains. Alternative splicing results in 4 different transcripts (SEQ ID NO:41-44) and its corresponding translation products (SEQ ID NO:45-48).

It was investigated whether SLC13A1 can be used as marker for kidney tumors. Oligonucleotides (SEQ ID NO:49, 50) which enable specific amplification of SLC13A1 were used for this purpose.

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Table 4. Expression of SLC13A1 in normal and tumor tissues

2017201358 28 Feb 2017

Normal tissue	Expression
Brain	-
Cerebellum	nd
Myocardium	nd
Skeletal muscle	nd
Myocardium	-
Stomach	-
Colon	-
Pancreas	nd
Kidney	+ + +
Liver	-
Testis	+
Thymus	-
Breast	-
Ovary	-
Uterus	nd
Skin	nd
Lung	-
Thyroid	-
Lymph nodes	-
Spleen	-
PBMC	-
S i gmo i d	-
Esophagus	-

Tumor type	Expression
Colon carcinoma	nd
Pancreatic carcinoma	nd
Esophageal carcinoma	nd
Gastric carcinoma	nd
Bronchial carcinoma	nd
Breast carcinoma	nd
Ovarian carcinoma	nd
Endometrial carcinoma	nd
ENT tumors	nd
Renal cell carcinoma
Prostate carcinoma	nd

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2017201358 28 Feb 2017

RT-PCR investigations with an SLC13Al-specific primer pair (SEQ ID NO:49, 50) confirmed virtually selective expression in the kidney, and showed according to the invention a high expression in virtually all (7/8) investigated renal cell carcinoma biopsies (tab. 4, fig. 6) . Quantitative RT-PCR with specific primers (SEQ ID NO:121, 122) also confirmed these data (fig. 34). Weak signals were detectable in the following normal tissues: colon, stomach, testis, breast, liver and brain.

Expression in renal carcinomas was, however, at least 100 times 10 higher than in all other normal tissues.

In order to analyse the subcellular localization of SLC13A1 in the cell, the protein was fused to eGFP as reporter molecule and, after transfection of the appropriate plasmid, expressed heterologously in 293 cells. The localization was then analysed 15 under the fluorescence microscope. Our data impressively confirmed that SLC13A1 is an integral transmembrane molecule ( fig. 35) .

Antibodies for detecting the SLC13A1 protein were produced by immunizing rabbits. The peptides of SEQ ID NO:123 and 124 were 20 used for propagating these antibodies. Such antibodies can in principle be used for diagnostic and therapeutic purposes.

The SLC13A1 protein has 13 transmembrane domains and 7 extracellular regions. These extracellular domains of SLC13A1 in particular can be used according to the invention as target 25 structures for monoclonal antibodies. SLC13A1 is involved as channel protein in the transport of ions. The extracellular domains of SLC13A1 in the healthy kidney are directed polarically in the direction of the urinary tract (luminally).

However, high molecular weight monoclonal antibodies employed 30 therapeutically are not excreted into the urinary tract, so that no binding to SLC13A1 takes place in the healthy kidney.

By contrast, the polarity of SLC13A1 is abolished in tumor cells, and the protein is available for antibody targeting directly via the bloodstream. The pronounced expression and

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2017201358 28 Feb 2017 high incidence of SLC13A1 in renal cell carcinomas make this protein according to the invention a highly interesting diagnostic and therapeutic marker. This includes according to the invention the detection of disseminated tumor cells in 5 serum, bone marrow, urine, and detection of metastases in other organs by means of RT-PCR. It is additionally possible to use the extracellular domains of SLC13A1 according to the invention as target structure for immunodiagnosis and therapy by means of monoclonal antibodies. SLC13A1 can moreover be employed 10 according to the invention as vaccine (RNA, DNA, protein, peptides) for inducing tumor-specific immune responses (T and B cell-mediated immune responses). This includes according to the invention also the development of so-called small compounds which modulate the biological activity of SLC13A1 and can be 15 employed for the therapy of renal tumors.

Example 6: Identification of CLCA1 as diagnostic and therapeutic cancer target

CLCA1 (SEQ ID NO:51; translation product: SEQ ID NO:60) belongs to the family of Ca⁺⁺-activated Cl channels. The sequence is published in Genbank under the accession No. NM_001285. CLCA1 is exclusively expressed in the intestinal crypt epithelium and in the goblet cells (Gruber et al., Genomics 54:200-14, 1998).

It was investigated whether CLCA1 can be used as marker for colonic and gastric carcinoma. Oligonucleotides (SEQ ID NO:67,

68) which enable specific amplification of CLCA1 were used for this purpose. RT-PCR investigations confirmed selective according to the investigated colonic with this primer set expression in invention high the colon, and showed expression in gastric carcinoma samples (fig. 7) . The other normal tissues showed no or only very weak expression. This was additionally confirmed with a specific quantitative RT-PCR (SEQ ID NO:125, 126), in which case no expression could be detected in the normal tissues

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2017201358 28 Feb 2017 analyzed (fig. 36) . Of the tumor samples investigated in this experiment, 6/12 colonic carcinoma samples and 5/10 gastric carcinoma samples were positive for CLCA1. Overall, expression of the gene in tumors appears to be dysregulated. Besides 5 samples with very strong expression, CLCA1 was markedly downregulated in other samples.

The protein is predicted to have 4 transmembrane domains with a total of 2 extracellular regions. These extracellular domains of CLCA1 in particular can be used according to the invention 10 as target structures for monoclonal antibodies.

The pronounced expression and high incidence of CLCA1 in gastric and colonic carcinomas make this protein according to the invention an interesting diagnostic and therapeutic marker. This includes according to the invention the detection of disseminated tumor cells in serum, bone marrow, urine, and detection of metastases in other organs by means of RT-PCR. It is additionally possible to use the extracellular domains of CLCA1 according to the invention as target structure for immunodiagnosis and therapy by means of monoclonal antibodies.

CLCA1 can moreover be employed according to the invention as vaccine (RNA, DNA, protein, peptides) for inducing tumorspecific immune responses (T and B cell-mediated immune responses). This includes according to the invention also the development of so-called small compounds which modulate the b iological activity as transport proteins of CLCA1 and can be employed for the therapy of gastrointestinal tumors.

Example 7: Identification of FLJ21477 as diagnostic and therapeutic cancer target

FLJ21477 (SEQ ID NO:52) and its predicted translation product (SEQ ID NO:61) was published as hypothetical protein in Genbank under the accession No. NM_025153. It is an integral membrane protein having ATPase activity and 4 transmembrane domains, which is accordingly suitable for therapy with specific

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2017201358 28 Feb 2017 antibodies. RT-PCR investigations with FLJ21477-specific primers (SEQ ID NO:69, 70) showed selective expression in the colon, and additionally various levels of expression in (7/12) investigated colonic carcinoma samples (fig. 8). The other normal tissues showed no expression. This was confirmed additionally by a specific quantitative RT-PCR (SEQ ID NO:127, 128) . FLJ21477-specific expression was detectable both in colon (fig. 37A) and in 11/12 of colonic carcinomas. Besides the expression in colon tissue, expression was additionally 10 detectable in stomach tissue. In addition, under the conditions of the quantitative RT-PCR, the expression detectable in brain, thymus and esophagus was distinctly weaker compared with colon and stomach (fig. 37A). It was moreover additionally possible to detect FLJ21477-specific expression in the following tumor 15 samples: stomach, pancreas, esophagus and liver.

The protein is predicted to have 4 transmembrane domains with a total of 2 extracellular regions. These extracellular domains of FLJ21477 in particular can be used according to the invention as target structures for monoclonal antibodies.

The expression and the high incidence of FLJ21477 for gastric and colonic carcinomas make this protein according to the invention a valuable diagnostic and therapeutic marker. This includes according to the invention the detection of disseminated tumor cells in serum, bone marrow, urine, and the 25 detection of metastases in other organs by means of RT-PCR. In addition, the extracellular domains of FLJ21477 can be used according to the invention as target structure for immunodiagnosis and therapy by means of monoclonal antibodies.

In addition, FLJ21477 can be employed according to the 30 invention as vaccine (RNA, DNA, protein, peptides) for inducing tumor-specific immune responses (T and B cell-mediated immune responses).

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2017201358 28 Feb 2017

Example 8: Identification of therapeutic cancer target

FLJ20694 (SEQ ID NO:53) and (SEQ ID NO:62) were published

FLJ20694 as diagnostic and its translation product as hypothetical protein in

Genbank under accession No. NM 017928.

This protein is an integral transmembrane molecule (transmembrane domain

ΆΑ 33-54), very probably with thioredoxin function. RT-PCR investigations with FLJ20694-specific primers (SEQ ID NO:71,

72) showed selective expression in the colon, and additionally 10 various levels of expression in (5/9) investigated colonic carcinoma samples (fig. 9) . The other normal tissues showed no expression. This was additionally confirmed by a specific quantitative RT-PCR (SEQ ID NO:129, 130) (fig. 38). FLJ29694 expression was undetectable in any other normal tissue apart 15 from colon and stomach (not analysed in the first experiment).

The protein is predicted to have one transmembrane domain with an extracellular region. These extracellular domains of FLJ20694 in particular can be used according to the invention as target structures for monoclonal antibodies.

In addition, FLJ20694 can be employed according to the invention as vaccine (RNA, DNA, protein, peptides) for inducing tumor-specific immune responses (T and B cell-mediated immune responses). This includes according to the invention also the development of so-called small compounds which modulate the 25 b iological activity of FLJ20694 and can be employed for the therapy of gastrointestinal tumors.

Example 9: Identification of von Ebner's protein (c20orfll4) as diagnostic and therapeutic cancer target von Ebner's protein (SEQ ID NO:54) and its translation product (SEQ ID NO:63) were published as Plunc-related protein of the upper airways and of the nasopharyngeal epithelium in Genbank under the accession No. AF364078. It was investigated according to the invention whether von Ebner's protein can be used as

106

2017201358 28 Feb 2017 marker of lung carcinoma. Oligonucleotides (SEQ ID NO:73, 74) which enable specific amplification of Ebner's protein were used for this purpose. RT-PCR investigations with this primer set showed selective expression in the lung and in (5/10) 5 investigated lung carcinoma samples (fig. 10) . In the group of normal tissues there was also expression in the stomach. The other normal tissues showed no expression.

Example 10: Identification of Plunc as diagnostic and therapeutic cancer target

Plunc (SEQ ID NO:55) and its translation product (SEQ ID NO:64) were published in Genbank under the accession No. NM_016583.

Human Plunc codes for a protein of 256 amino acids and shows

72% homology with the murine Plunc protein (Bingle and Bingle, 15 Biochem Biophys Acta 1493:363-7, 2000). Expression of Plunc is confined to the trachea, the upper airways, nasopharyngeal epithelium and salivary gland.

It was investigated according to the invention whether Plunc can be used as marker of lung carcinoma. Oligonucleotides 20 (SEQ ID NO:75, 76) which enable specific amplification of Plunc were used for this purpose.

RT-PCR investigations with this primer set showed selective expression in the thymus, in the lung and in (6/10) investigated lung carcinoma samples (fig. 11) . Other normal 25 tissues showed no expression.

Example 11: Identification of SLC26A9 as diagnostic and therapeutic cancer target

SLC26A9 (SEQ ID NO:56) and its translation product (SEQ ID NO:65) were published in Genbank under the accession

No. NM_134325. SLC26A9 belongs to the family of anion exchangers. Expression of SLC26A9 is confined to the bronchiolar and alveolar epithelium of the lung (Lohi et al., J

Biol Chem 277:14246-54, 2002).

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2017201358 28 Feb 2017

It was investigated whether SLC2 6A9 can be used as marker of lung carcinoma. Oligonucleotides (SEQ ID NO:77, 78) which enable specific amplification of SLC26A9 were used for this purpose. RT-PCR investigations with SLC26A9-specific primers 5 (SEQ ID NO:77, 78) showed selective expression in the lung and in all (13/13) investigated lung carcinoma samples (fig. 12).

The other normal tissues showed no expression, with the exception of the thyroid. It was possible in quantitative RT-PCR experiments with the primers of SEQ ID NO: 131 and 132 10 firstly to confirm these results, and to obtain additional information. It was possible in pooled samples of 4-5 tumor tissues to detect high expression levels for SLC26A9-specific RNA in lung, colon, pancreas and stomach tumors. SLC26A9 is member of a family of transmembrane anion transporters. In the 15 healthy lung, the protein is luminally directed in the direction of the airways and thus not directly available to IgG antibodies from the blood. By contrast, the polarity of the protein is abolished in tumors. It is therefore possible according to the invention to address SLC26A9 as therapeutic 20 target using monoclonal antibodies in the defined tumors, inter alia lung tumors, gastric carcinomas, pancreatic carcinomas.

The pronounced, high expression and high incidence of SLC26A9 for lung, stomach, pancreatic and esophageal carcinomas make this protein according to the invention an excellent diagnostic 25 and therapeutic marker. This includes according to the invention the detection of disseminated tumor cells in serum, bone marrow and urine, and detection of metastases in other organs by means of RT-PCR. In addition, the extracellular domains of SLC2 6A9 can be used according to the invention as 30 target structure for immunodiagnosis and therapy by means of monoclonal antibodies. It is additionally possible to employ SLC26A9 according to the invention as vaccine (RNA, DNA, protein, peptides) for inducing tumor-specific immune responses (T and B cell-mediated immune responses). This includes

108

2017201358 28 Feb 2017 according to the invention also the development of so-called small compounds which modulate the biological activity of SLC26A9 and can be employed for the therapy of lung tumors and gastrointestinal tumors.

Example 12: Identification of THC1005163 as diagnostic and therapeutic cancer target

THC1005163 (SEQ ID NO:57) is a gene fragment from the TIGR gene index. The gene is defined only in the 3' region, while an ORF 10 is lacking. RT-PCR investigations took place with a THC1005163specific primer (SEQ ID NO:79) and an oligo dTi₈ primer which had a specific tag of 21 specific bases at the 5' end. This tag was examined using database search programs for homology with known sequences. This specific primer was initially employed in 15 the cDNA synthesis in order to preclude genomic DNA contaminations. RT-PCR investigations with this primer set showed expression in the stomach, ovary, lung and in (5/9) lung carcinoma biopsies (fig. 13) . Other normal tissues showed no expression.

Example 13: Identification of LOC134288 as diagnostic and therapeutic cancer target

LOC134288 (SEQ ID NO:58) and its predicted translation product (SEQ ID NO:66) were published in Genbank under accession No. 25 XM_059703.

It was investigated according to the invention whether LOC134288 can be used as marker of renal cell carcinoma.

Oligonucleotides (SEQ ID NO:80, 81) which enable specific amplification of LOC134288 were used for this purpose. RT-PCR 30 investigations showed selective expression in the kidney and in (5/8) investigated renal cell carcinoma biopsies (fig. 14).

109

2017201358 28 Feb 2017

Example 14: Identification of THC943866 as diagnostic and therapeutic cancer target

THC 943866 (SEQ ID NO:59) is a gene fragment from the TIGR gene index. It was investigated whether THC943866 can be used as 5 marker of renal cell carcinoma. Oligonucleotides (SEQ ID NO:82,

83) which enable specific amplification of THC943866 were used for this purpose.

RT-PCR investigations with THC943866-specific primers (SEQ ID NO:82, 83) showed selective expression in the kidney 10 and in (4/8) investigated renal cell carcinoma biopsies (fig. 15).

Example 15: Identification of FLJ21458 as diagnostic and therapeutic cancer target

FLJ21458 (SEQ ID NO:84) and its predicted translation product (SEQ ID NO:85) were published in Genbank under the accession No. NM_034850. Sequence analyses revealed that the protein represents a new member of the butyrophillin family. Structural analyses revealed that it represents a type 1 transmembrane 20 protein with an extracellular immunoglobulin domain.

Oligonucleotides (SEQ ID NO:86, 87) which enable specific amplification of FLJ21458 were used for investigating expression. RT-PCR investigations with FLJ21458-specific primers (SEQ ID NO:86, 87) showed selective expression in colon and in (7/10) investigated colonic carcinoma biopsies (fig. 16, tab. 5) . Quantitative RT-PCR with specific primers (SEQ ID NO:133, 134) confirmed this selective expression profile (fig. 39) . It was additionally possible in the experiment to detect FLJ21458 gastrointestinal-specifically in the colon, and in stomach, in the rectum and cecum and in testis. 7/11 colon metastasis samples were also positive in the quantitative PCR. FLJ21458-specific expression was extended to other tumors, and a protein-specific expression was detectable in stomach, pancreas and liver tumors (tab. 5). Antibodies for

110

2017201358 28 Feb 2017 detecting FLJ21458 protein were produced by immunizing rabbits.

The following peptides were used to propagate these antibodies: SEQ ID NO:135: QWQVFGPDKPVQAL

SEQ ID NO:136: AKWKGPQGQDLSTDS

An FLJ21458-specific reaction was detectable in immunofluorescence (fig. 40) . To check the specificity of the antibodies, 293 cells were transfected with a plasmid that codes for an FLJ21458-GFP fusion protein. Specificity was demonstrated on the one hand by colocalization investigations 10 using the FLJ21458-specific antibody, and on the other hand via the autofluorescent GFP. Superimposition of the two fluorescent diagrams showed unambiguously that the immune serum specifically recognises FLJ21458 protein (fig. 40a) . Owing to the overexpression of the protein, the resultant cell staining 15 was diffuse and did not allow unambiguous protein localization.

For this reason, a further immunofluorescence experiment was carried out with the stomach tumor-specific cell line Snul6 which expresses FLJ21458 endogenously (fig. 41B) . The cells were stained with the FLJ21458-specific antiserum and with 20 another antibody which recognizes the membrane protein Ecadherin. The FLJ21458-specific antibody stains the cell membranes at least weakly and is thus evidence that FLF21458 is localized in the cell membrane.

Bioinformatic investigations showed that the protein encoded by

FLJ21458 represents a cell surface molecule and has an immunoglobulin supermolecule domain. Selective expression of this surface molecule makes it a good target for developing diagnostic methods for the detection of tumor cells and 30 therapeutic methods for the elimination of tumor cells.

The pronounced expression and high incidence of FLJ21458 for gastric and colonic carcinomas make this protein according to the invention a highly interesting diagnostic and therapeutic marker. This includes according to the invention the detection

111

2017201358 28 Feb 2017 of disseminated tumor cells in serum, bone marrow and urine, and the detection of metastases in other organs by means of RT-PCR. It is additionally possible to employ the extracellular domains of FLJ21458 according to the invention as target 5 structure for immunodiagnosis and therapy by means of monoclonal antibodies. It is additionally possible to employ FLJ21458 according to the invention as vaccine (RNA, DNA, protein, peptides) for inducing tumor-specific immune responses (T and B cell-mediated immune responses). This includes 10 according to the invention also the development of so-called small compounds which modulate the biological activity of FLJ21458 and can be employed for the therapy of gastrointestinal tumors.

112

Table 5 FLJ21458 expression in normal and tumor tissues

2017201358 28 Feb 2017

Normal tissue	Expression
Brain	-
Cerebellum	-
Myocardium	nd
Skeletal muscle
Myocardium	-
Stomach	++
Colon	+++
Pancreas	-
Kidney	-
Liver	-
Testis	++
Thymus	nd
Breast	nd
Ovary	-
Uterus	-
Skin	-
Lung	-
Thyroid	nd
Lymph nodes	-
Spleen	-
PBMC	-
Adrenal	nd
Esophagus	-
Small intestine
Prostate	-

Tumor type	Expression
Colonic carcinoma	7/10
Pancreatic carcinoma	5/6
Esophageal carcinoma	nd
Gastric carcinoma	8/10
Bronchial carcinoma	nd
Breast carcinoma	nd
Ovarian carcinoma	nd
Endometrial carcinoma	nd
ENT tumors	nd
Renal cell carcinoma	nd
Prostate carcinoma	nd
Colonic metastases	7/11
Liver carcinoma	5/8

112a

2017201358 29 Jan 2019

Throughout the description and claims of the specification, the word comprise and variations of the word, such as comprising and comprises, is not intended to exclude other additives, components, integers or steps.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as admission that the document or matter was known or that the information it contains was part of the common general knowledge as at the 10 priority date of any of the claims.

Claims

Claims

1. A pharmaceutical composition when used for treating or preventing a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, comprising an agent which inhibits expression or activity of a tumorassociated antigen, said tumor-associated antigen having a 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 NO: 84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
2. A pharmaceutical composition when used for treating or preventing a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, comprising an agent with tumor-inhibiting activity, which is selective for cells expressing or abnormally expressing a tumorassociated antigen, said tumor-associated antigen having a 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 NO: 84, a part or derivative thereof,

114

2017201358 09 Apr 2019 (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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
3. A pharmaceutical composition when used for treating or preventing a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, comprising an agent which, when administered, selectively increases the amount of complexes between an HLA molecule and a tumorassociated antigen or a part thereof, said tumorassociated antigen having a 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 NO: 84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
4. A pharmaceutical composition when used for treating or preventing a cancer disease characterized by a cancer cell

115

2017201358 09 Apr 2019 expressing a tumor-associated antigen, comprising one or more components selected from the group consisting of:

(i) a tumor-associated antigen or a part thereof, (ii) a nucleic acid which codes for a tumor-associated antigen or a part thereof, (iii) an antibody which binds to a tumor-associated antigen or a part thereof, (iv) an antisense nucleic acid which hybridizes specifically with a nucleic acid coding for a tumorassociated antigen, (v) a host cell which expresses a tumor-associated antigen or a part thereof, and (vi) isolated complexes between a tumor-associated antigen or a part thereof and an HLA molecule, said tumor-associated antigen having a 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 NO: 84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
5. A method of diagnosing a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, which method comprises

116

2017201358 09 Apr 2019 (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 of a part thereof and/or (iv) detection of cytotoxic or T helper lymphocytes which are specific to the tumor-associated antigen or to a part thereof in a biological sample isolated from a patient, with said tumor-associated antigen having a 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 NO: 84, 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), wherein the cancer disease is a cancer or tumor of the pancreas or liver, or a metastasis thereof, or a metastasis of a tumor of the colon or stomach.
6. The method as claimed in claim 5, in which the 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 the tumor-associated antigen or the part thereof, to the antibody or to the cytotoxic or T helper lymphocytes, and

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2017201358 09 Apr 2019 (ii) detecting the formation of a complex between the agent and the nucleic acid or the part thereof, the tumorassociated antigen or the part thereof, the antibody or the cytotoxic or T helper lymphocytes.
7. A method for determining regression, course or onset of a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, 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 of a part thereof, (iii) the amount of antibodies which bind to the tumorassociated antigen or to a part thereof, and (iv) the amount of cytolytic or cytokine-releasing T cells which are specific for a complex between the tumorassociated antigen or a part thereof and an MHC molecule, said tumor-associated antigen having a 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 NO: 84, 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),

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2017201358 09 Apr 2019 wherein the cancer disease is a cancer or tumor of the pancreas or liver, or a metastasis thereof, or a metastasis of a tumor of the colon or stomach.
8. The method as claimed in any of claims 5 to 7, wherein (a) the nucleic acid or the part thereof is detected or its amount is monitored using a polynucleotide probe which hybridizes specifically to said nucleic acid or to said part thereof, (b) the nucleic acid or the part thereof is detected or its amount is monitored by selectively amplifying said nucleic acid or said part thereof, (c) the tumor-associated antigen or the part thereof is detected or its amount is monitored using an antibody binding specifically to said tumor-associated antigen or to said part thereof, and (e) the antibody is detected or its amount is monitored

using a protein antibody. or peptide binding specifically to said The method as claimed in claim 8, in which the polynucleotide probe comprises a sequence of 6-50

contiguous nucleotides of the nucleic acid coding for the tumor-associated antigen.
10. A method of treating a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, which method comprises administration of a pharmaceutical composition as claimed in any of claims 1-4, said tumorassociated antigen having a 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 NO: 84, a part or derivative thereof,

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2017201358 09 Apr 2019 (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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
11. A method of treating a cancer disease characterized by expressing a tumor-associated antigen, which method comprises administering an antibody binding to said tumorassociated antigen or to a part thereof and coupled to a therapeutic agent, said tumor-associated antigen having a 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 NO: 84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
12. A method of diagnosing or monitoring a cancer disease characterized by expressing a tumor-associated antigen, which method comprises administering an antibody binding to said tumor-associated antigen or to a part thereof and

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2017201358 09 Apr 2019 coupled to a diagnostic agent, said tumor-associated antigen having a 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 NO: 84, 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) , wherein the ' cancer disease is a cancer or tumor of the pancreas or liver, or a metastasis thereof, or a metastasis of a tumor of the colon or stomach.
13. A method of treating a patient having a cancer disease characterized by a cancer cell expressing a tumorassociated antigen, 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 or cytokine-releasing T cells against said tumor-associated antigen or a part thereof, and (iii) introducing the cytolytic or cytokine-releasing T cells into the patient in an amount suitable for lysing cells expressing the tumor-associated antigen or a part thereof, said tumor-associated antigen having a sequence encoded by a nucleic acid which is selected from the group consisting of:

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2017201358 09 Apr 2019 (a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
14. A method of treating a patient having a cancer disease characterized by a cancer cell expressing a tumorassociated antigen, which method comprises:

(i) identifying a nucleic acid which is expressed by cells associated with said disease, said nucleic acid being selected from the group consisting of:

(a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 84, 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), (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, and (iv) introducing the host cells or an extract thereof into the patient in an amount suitable for increasing the

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2017201358 09 Apr 2019 immune response to the patient's cells associated with the disease, wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
15. A method of treating a cancer disease characterized by a cancer cell expressing a tumor-associated antigen, 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, said tumor-associated antigen having a 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 NO: 84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
16. A method of inhibiting the development of cancer in a patient, which method comprises administering an effective

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2017201358 09 Apr 2019 amount of a pharmaceutical composition as claimed in any of claims 1-4.
17. An agent when used for treating or preventing a cancer disease characterized by a cancer cell expressing a tumorassociated antigen, which binds specifically to a protein or polypeptide or to a part thereof, said protein or polypeptide being 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 NO: 84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
18. An antibody when used for treating or preventing a cancer disease characterized by a cancer cell expressing a tumorassociated antigen, which binds selectively to a complex of :

(i) a protein or polypeptide or a part thereof and (ii) an MHC molecule to which said protein or polypeptide or said part thereof binds, with said antibody not binding to (i) or (ii) alone and said protein or polypeptide being encoded by a nucleic acid selected from the group consisting of:

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2017201358 09 Apr 2019 (a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 84, 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), wherein the cancer disease is a cancer or tumor of the colon, pancreas, stomach or liver, or a metastasis thereof .
19.

A kit when used for detecting a cancer cell characterized by expressing a tumor-associated antigen, which kit comprises agents for detection of the nucleic acid which codes for the tumorassociated 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 complex between the tumor-associated antigen or a part thereof and an MHC molecule, said tumor-associated antigen having a 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 NO:

84, 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

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2017201358 09 Apr 2019 acid of (a), (b) or (c), wherein the cancer is a cancer or tumor of the pancreas or liver, or a metastasis thereof, or a metastasis of a tumor of the colon or stomach.
20. The kit as claimed in claim 19, in which the agents for detection of the nucleic associated antigen or of molecules for selective acid.

acid which codes for the tumora part thereof are nucleic acid amplification of said nucleic