Title:
Identification of Tumor-Associated Antigens for Diagnosis and Therapy
Kind Code:
A1


Abstract:
The invention relates to genetic products the expression of which is associated with cancer diseases. The invention also relates to the therapy and diagnosis of diseases in which the genetic products are expressed or aberrantly expressed, in particular cancer diseases.



Inventors:
Sahin, Ugur (Mainz, DE)
Türeci, Özlem (Mainz, DE)
Koslowski, Michael (Mainz, DE)
Usener, Dirk (Wiesbaden, DE)
Application Number:
12/066399
Publication Date:
08/13/2009
Filing Date:
09/06/2006
Primary Class:
Other Classes:
424/93.71, 424/138.1, 424/185.1, 435/6.11, 435/7.23, 435/29, 435/320.1, 435/325, 530/350, 530/387.3, 530/387.7, 530/391.3, 530/391.7, 536/23.5, 536/24.5, 424/93.21
International Classes:
A61K39/395; A61K31/7088; A61K35/12; A61K39/00; C07K14/00; C07K16/18; C12N5/06; C12N15/00; C12N15/11; C12Q1/02; C12Q1/68; G01N33/574
View Patent Images:



Primary Examiner:
YAO, LEI
Attorney, Agent or Firm:
MCANDREWS HELD & MALLOY, LTD (500 WEST MADISON STREET SUITE 3400, CHICAGO, IL, 60661, US)
Claims:
1. A pharmaceutical composition, comprising an agent which (I) inhibits expression or activity of a tumor-associated antigen and/or (II) has tumor-inhibiting activity, and is selective for cells expressing or abnormally expressing a tumor-associated antigen and/or (III) when administered, selectively increases the amount of complexes between an MHC molecule and a 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: (a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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).

2. The pharmaceutical composition as claimed in claim 1, wherein the agent under (II) causes induction of cell death, reduction in cell growth, damage to the cell membrane or secretion of cytokines.

3. The pharmaceutical composition as claimed in claim 1, wherein the agent under (I) or (II) is an antisense nucleic acid which hybridizes selectively with the nucleic acid coding for the tumor-associated antigen.

4. The pharmaceutical composition as claimed in claim 1, wherein the agent under (I) or (II) is an antibody which binds selectively to the tumor-associated antigen.

5. The pharmaceutical composition as claimed in claim 1, wherein 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 the tumor-associated antigen or a part thereof, (iii) an antibody which binds to the tumor-associated antigen or a part thereof, (iv) an antisense nucleic acid which hybridizes specifically with a nucleic acid coding for the tumor-associated antigen, (v) an siRNA directed against a nucleic acid coding for the tumor-associated antigen, (vi) a host cell which expresses the tumor-associated antigen or a part thereof, and (vii) isolated complexes between the tumor-associated antigen or a part thereof and an MHC molecule.

6. The pharmaceutical composition as claimed in claim 1, wherein the agent comprises two or more agents which in each case selectively inhibit expression or activity of different tumor-associated antigens, which are in each case selective for cells expressing or abnormally expressing different tumor-associated antigens or which increase the amount of complexes between MHC molecules and different tumor-associated antigens or parts thereof, with at least one of said tumor-associated antigens 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 NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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).

7. A pharmaceutical composition, 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 tumor-associated antigen, (v) an siRNA directed against a nucleic acid coding for a tumor-associated antigen, (vi) a host cell which expresses a tumor-associated antigen or a part thereof, and (vii) isolated complexes between a 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 NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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).

8. The pharmaceutical composition as claimed in claim 7, wherein the nucleic acid of (ii) is present in an expression vector.

9. The pharmaceutical composition as claimed in claim 7, wherein the host cell secretes the tumor-associated antigen or the part thereof.

10. The pharmaceutical composition as claimed in claim 7, wherein the host cell additionally expresses an MHC molecule which binds to the tumor-associated antigen or the part thereof.

11. The pharmaceutical composition as claimed in claim 10, wherein the host cell expresses the MHC molecule and/or the tumor-associated antigen or the part thereof in a recombinant manner.

12. The pharmaceutical composition as claimed in claim 10, wherein the host cell expresses the MHC molecule endogenously.

13. The pharmaceutical composition as claimed in claim 7, wherein the host cell is an antigen-presenting cell.

14. The pharmaceutical composition as claimed in claim 7, wherein the antibody is a monoclonal, chimeric or humanized antibody, or is a fragment of an antibody.

15. The pharmaceutical composition as claimed in claim 7 or 14 wherein the antibody is coupled to a therapeutic or diagnostic agent.

16. The pharmaceutical composition as claimed in claim 1 or 7, which may be used for the treatment or prevention of cancer.

17. The pharmaceutical composition as claimed in claim 16, wherein the cancer is a lung tumor, a breast tumor, a prostate tumor, a melanoma, a colon tumor, a gastric tumor, a pancreatic tumor, an ENT tumor, a renal cell carcinoma or a cervical carcinoma, a colon carcinoma or a mammary carcinoma.

18. The pharmaceutical composition as claimed in claim 1 or 7, wherein the tumor-associated antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69, a part thereof, or derivative thereof.

19. The pharmaceutical composition as claimed in claim 1 or 7, which is in the form of a vaccine.

20. The pharmaceutical composition as claimed in claim 19 for therapeutic use, prophylactic use, or combinations thereof.

21. A method of diagnosing or monitoring a disease characterized by expression or abnormal expression of a tumor-associated antigen, wherein the method comprises the step of detecting or determining the amount of: (i) of a nucleic acid which encodes for the tumor-associated antigen or of a part thereof, or (ii) of the tumor-associated antigen or of a part thereof, or (iii) of an antibody to the tumor-associated antigen or a part thereof or (iv) of T 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 NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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); or (v) combinations of (i), (ii), (iii), or (iv).

22. The method as claimed in claim 21, wherein the detection or determination of the amount 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 T lymphocytes, and (ii) detecting the formation of or determining the amount 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 T lymphocytes.

23. The method as claimed in claim 22, wherein the agent which binds specifically to the nucleic acid coding for the tumor-associated antigen or to the part thereof is an oligonucleotide or polynucleotide, which hybridizes specifically to said nucleic acid or to said part thereof.

24. The method as claimed in claim 22, wherein the agent which binds specifically to the tumor-associated antigen or the part thereof is an antibody binding specifically to said tumor-associated antigen or to said part thereof.

25. The method as claimed in claim 22, wherein the agent which binds specifically to the antibody is a protein or peptide binding specifically to said antibody.

26. The method as claimed in claim 22, wherein the agent which binds specifically to the T lymphocytes is a cell presenting the complex between the tumor-associated antigen or the part thereof and an MHC molecule.

27. The method as claimed in claim 21 wherein said monitoring of said disease comprises determining regression, course or onset of said disease in a sample from a patient who has said disease or is suspected of falling ill with said disease.

28. The method as claimed in claim 27, which comprises a detection or determination of the amount in a first sample at a first point in time and in a further sample at a second point in time and a comparison of the two samples.

29. The method as claimed in claim 22, wherein the agent is labeled in a detectable manner.

30. The method as claimed in claim 21, in which the sample comprises body fluid and/or body tissue.

31. A method of treating or preventing a disease characterized by expression or abnormal expression of a tumor-associated antigen, which method comprises the step of administration of a pharmaceutical composition as claimed in claims 1 or 7, 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 NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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).

32. A method of treating, preventing, diagnosing or monitoring a disease characterized by expression or abnormal expression of a tumor-associated antigen, wherein method comprises the step of administering an antibody binding to said tumor-associated antigen or to a part thereof and coupled to a therapeutic or 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 NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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).

33. The method as claimed in claim 24 or 32, wherein the antibody is a monoclonal, chimeric or humanized antibody, or is a fragment of an antibody.

34. A method of treating a patient having a disease characterized by expression or abnormal expression of a tumor-associated antigen, wherein the method comprises the steps of: (i) providing a sample containing immunoreactive cells, (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 said 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: (a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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).

35. The method as claimed in claim 34, wherein the host cell recombinantly expresses an MHC molecule binding to the tumor-associated antigen or to a part thereof.

36. The method as claimed in claim 34, wherein the host cell endogenously expresses an MHC molecule binding to the tumor-associated antigen or to a part thereof.

37. A method of inhibiting the development of cancer in a patient, wherein the method comprises the step of administering an effective amount of a pharmaceutical composition as claimed in claim 1 or 7.

38. The method as claimed in claim 21, or 32, in which the tumor-associated antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69, a part or derivative thereof.

39. An agent, 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 NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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).

40. The agent as claimed in claim 39, in which the protein or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69, and a part or derivative thereof.

41. The agent as claimed in claim 39, which is an antibody.

42. The agent as claimed in claim 41, in which the antibody is a monoclonal, chimeric or humanized antibody, or is a fragment of an antibody.

43. An antibody, 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 NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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).

44. The antibody as claimed in claim 43, in which the protein or polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69, and a part or derivative thereof.

45. The antibody as claimed in claim 43, which is a monoclonal, chimeric or humanized antibody, or is a fragment of an antibody.

46. A conjugate between an agent as claimed in any of claims 39-42 and a therapeutic or diagnostic agent.

47. A kit for detecting expression or abnormal expression of a tumor-associated antigen, which kit comprises agents for detecting or determining the amount (i) of a nucleic acid which codes for the tumor-associated antigen or of a part thereof, and/or (ii) of the tumor-associated antigen or of a part thereof, and/or (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, 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 NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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).

48. The method as claimed in claim 36, in which the tumor-associated antigen comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69, and a part or derivative thereof.

49. A conjugate between an antibody as claimed in any of claims 43-45 and a therapeutic or diagnostic agent.

Description:

Despite interdisciplinary approaches and exhaustive use of classical therapeutic procedures, cancers are still among the leading causes of death. More recent therapeutic concepts aim at incorporating the patient's immune system into the overall therapeutic concept by using recombinant tumor vaccines and other specific measures such as antibody therapy. A prerequisite for the success of such a strategy is the recognition of tumor-specific or tumor-associated antigens or epitopes by the patient's immune system whose effector functions are to be interventionally enhanced. Tumor cells biologically differ substantially from their nonmalignant cells of origin. These differences are due to genetic alterations acquired during tumor development and result, inter alia, also in the formation of qualitatively or quantitatively altered molecular structures in the cancer cells. Tumor-associated structures of this kind which are recognized by the specific immune system of the tumor-harboring host are referred to as tumor-associated antigens. The specific recognition of tumor-associated antigens involves cellular and humoral mechanisms which are two functionally interconnected units: CD4+ and CD8+ T lymphocytes recognize the processed antigens presented on the molecules of the MHC (major histocompatibility complex) classes II and I, respectively, while B lymphocytes produce circulating antibody molecules which bind directly to unprocessed antigens. The potential clinical-therapeutical importance of tumor-associated antigens results from the fact that the recognition of antigens on neoplastic cells by the immune system leads to the initiation of cytotoxic effector mechanisms and, in the presence of T helper cells, can cause elimination of the cancer cells (Pardoll, Nat. Med. 4:525-31, 1998). Accordingly, a central aim of tumor immunology is to molecularly define these structures. The molecular nature of these antigens has been enigmatic for a long time. Only after development of appropriate cloning techniques has it been possible to screen cDNA expression libraries of tumors systematically for tumor-associated antigens by analyzing the target structures of cytotoxic T lymphocytes (CTL) (van der Bruggen et al., Science 254:1643-7, 1991) or by using circulating autoantibodies (Sahin et al., Curr. Opin. Immunol. 9:709-16, 1997) as probes. To this end, cDNA expression libraries were prepared from fresh tumor tissue and recombinantly expressed as proteins in suitable systems. Immunoeffectors isolated from patients, namely CTL clones with tumor-specific lysis patterns, or circulating autoantibodies were utilized for cloning the respective antigens.

In recent years a multiplicity of antigens have been defined in various neoplasias by these approaches.

However, the probes utilized for antigen identification in the classical methods are immunoeffectors (circulating autoantibodies or CTL clones) from patients usually having already advanced cancer. A number of data indicate that tumors can lead, for example, to tolerization and anergization of T cells and that, during the course of the disease, especially those specificities which could cause effective immune recognition are lost from the immunoeffector repertoire. Current patient studies have not yet produced any solid evidence of a real action of the previously found and utilized tumor-associated antigens. Accordingly, it cannot be ruled out that proteins evoking spontaneous immune responses are the wrong target structures.

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

This object is achieved by the subject matter of the claims.

According to the invention, genes are identified which are selectively or aberrantly expressed in tumor cells and thus, provide tumor-associated antigens. These genes and/or their genetic products and/or the derivatives and/or fragments thereof are useful as target structures for therapeutic and diagnostic approaches.

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, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67 of the sequence listing, a part or derivative thereof, (b) a nucleic acid which hybridizes with the nucleic acid of (a) under stringent conditions, (c) a nucleic acid which is degenerate with respect to the nucleic acid of (a) or (b), and (d) a nucleic acid which is complementary to the nucleic acid of (a), (b) or (c). In a preferred embodiment, a tumor-associated antigen identified according to the invention has an amino acid sequence encoded by a nucleic acid which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67 of the sequence listing. In a further preferred embodiment, a tumor-associated antigen identified according to the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69 of the sequence listing, a part or derivative thereof.

The present invention generally relates to the use of tumor-associated antigens identified according to the invention or of parts or derivatives thereof, of nucleic acids coding for the tumor-associated antigens identified according to the invention or of parts or derivatives thereof or of nucleic acids directed against said coding nucleic acids, of antibodies or T cells directed against the tumor-associated antigens identified according to the invention or parts or derivatives thereof and/or of host cells expressing the tumor-associated antigens identified according to the invention or parts or derivatives thereof for therapy, prophylaxis, diagnosis and/or monitoring of neoplastic diseases. This may also involve the use of a combination of two or more of these antigens, nucleic acids, antibodies, T cells and/or host cells, in one embodiment also in combination with tumor-associated antigens other than those identified according to the invention, nucleic acids coding therefor or nucleic acids directed against said coding nucleic acids, antibodies or T cells directed against said tumor-associated antigens and/or host cells expressing said tumor associated antigens.

In those embodiments of the invention relating to the use of antibodies directed against the tumor-associated antigens identified according to the invention or parts or derivatives thereof also T cell receptors directed against the tumor-associated antigens identified according to the invention or parts or derivatives thereof, optionally in a complex with MHC molecules, may be used.

Especially suitable for therapy, prophylaxis, diagnosis and/or monitoring is a part of the tumor-associated antigens identified according to the invention which corresponds to the non-transmembrane portion, in particular the extracellular portion of the tumor-associated antigens or is comprised thereof. Therefore, according to the invention, a part of the tumor-associated antigens identified according to the invention which corresponds to the non-transmembrane portion, in particular the extracellular portion of the tumor-associated antigens or is comprised thereof, or a corresponding part of the nucleic acids coding for the tumor-associated antigens identified according to the invention is preferred for therapy, prophylaxis, diagnosis and/or monitoring. Similarly the use of antibodies is preferred which are directed against a part of the tumor-associated antigens identified according to the invention which corresponds to the non-transmembrane portion, in particular the extracellular portion of the tumor-associated antigens or is comprised thereof.

Preferred diseases for a therapy, prophylaxis 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.

Furthermore, the invention relates to nucleic acids and proteins or peptides, which result from 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 SEQ ID NOs: 28 and 49 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 SEQ ID NOs: 29 and 50 of the sequence listing.

Altered splicing of a gene results in an altered transcript sequence (splice variant). Translation of a splice variant in the region of its altered sequence results in an altered protein which may be distinctly different in the structure and function from the original protein. Tumor-associated splice variants may produce tumor-associated transcripts and tumor-associated proteins/antigens. These may be utilized as molecular markers both for detecting tumor cells and for therapeutic targeting of tumors. Detection of tumor cells in a sample from a patient may be carried out according to the invention, for example, after extraction of nucleic acids by PCR amplification with splice variant-specific oligonucleotides.

According to the invention, all sequence-dependent detection systems are suitable for detection. These are, apart from PCR, for example gene chip/microarray systems, Northern blot, RNAse protection assays (RDA) and others. All detection systems have in common that detection is based on a specific hybridization with at least one splice variant-specific nucleic acid sequence. However, tumor cells may also be detected according to the invention by antibodies which recognize a specific epitope encoded by the splice variant. Said antibodies may be prepared by using for immunization peptides which are specific for said splice variant. Suitable for immunization are particularly the amino acid sequences which are distinctly different from the variant(s) of the genetic product, which is (are) preferably produced in healthy cells. Detection of the tumor cells with antibodies may be carried out here on a sample isolated from the patient or as imaging with intravenously administered antibodies.

In addition to diagnostic usability, splice variants having new or altered epitopes are attractive targets for immunotherapy as these epitopes may be utilized for targeting antibodies or T lymphocytes as described herein. In passive immunotherapy, antibodies or T lymphocytes which recognize splice variant-specific epitopes are adoptively transferred here. As in the case of other antigens, antibodies may be generated also by using standard technologies with utilization of polypeptides which include these epitopes. Alternatively, it is possible to utilize for immunization nucleic acids coding for peptides which contain said epitopes. Various techniques for in vitro or in vivo generation of epitope-specific T lymphocytes are known and have been described in detail (for example Kessler J H, et al. 2001, Sahin et al., 1997) and are likewise based on utilizing peptides which contain the splice variant-specific epitopes or nucleic acids coding for said peptides. Peptides which contain the splice variant-specific epitopes or nucleic acids coding for said peptides may also be used as pharmaceutically active substances in active immunotherapy (e.g. vaccination, vaccine therapy).

In one aspect, the invention relates to a pharmaceutical composition comprising an agent which recognizes a tumor-associated antigen identified according to the invention or a nucleic acid coding for the tumor-associated antigen and which is preferably selective for cells which have expression or abnormal expression of a tumor-associated antigen identified according to the invention. In a further aspect, the invention relates to a pharmaceutical composition comprising an agent which (I) inhibits expression or activity of a tumor-associated antigen identified according to the invention, and/or (II) has tumor-inhibiting or tumor-destroying activity and is selective for cells expressing or abnormally expressing a tumor-associated antigen identified according to the invention, and/or (III) when administered, selectively increases the amount of complexes between an MHC molecule and a tumor-associated antigen identified according to the invention or a part thereof, such as a peptide epitope. 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 a siRNA preferably comprising a sense RNA strand and an antisense RNA strand, wherein the sense and antisense RNA strands form an RNA duplex, and wherein the sense RNA strand comprises a nucleotide sequence substantially identical to a target sequence of about 19 to about 25 contiguous nucleotides in a nucleic acid coding for the tumor-associated antigen, preferably mRNA coding for the tumor-associated antigen. In a further embodiment, the agent is an antibody which binds selectively to the tumor-associated antigen, in particular a complement-activated or toxin conjugated antibody which binds selectively to the tumor-associated antigen. In a preferred embodiment, the antibody which binds selectively to the tumor-associated antigen is coupled to a therapeutically useful substance and/or recruits natural or artificial effector mechanisms to said cell expressing or abnormally expressing said tumor-associated antigen. In a further embodiment, the agent is a cytotoxic T lymphocyte which recognizes the tumor-associated antigen or a part thereof bound by an MHC molecule on a cell and lyses the cells labeled in this way. In a further embodiment, the agent is a T helper lymphocyte which enhances effector functions of other cells specifically recognizing said tumor-associated antigen or a part thereof.

In a further embodiment, the agent comprises two or more agents which each recognize different tumor-associated antigens and/or inhibit expression or activity of different tumor-associated antigens, and/or have tumor-inhibiting or tumor-destroying activity and are selective for cells expressing or abnormally expressing different tumor-associated antigens, and/or when administered, selectively increase the amount of complexes between MHC molecules and different tumor-associated antigens or parts thereof, wherein at least one of said different tumor-associated antigens is a tumor-associated antigen identified according to the invention. Preferably, a tumor-associated antigen selectively limited to tumors serves as a label for recruiting effector mechanisms to this specific location. The invention includes embodiments wherein the agent itself does not have an ability to inhibit activity of a tumor-associated antigen or a tumor-inhibiting or tumor-destroying activity but mediates such effect, in particular by recruiting effector mechanisms, in particular those having cell damaging potential, to a specific location, in particular a tumor or tumor cells.

The activity of a tumor-associated antigen identified according to the invention can be any activity of a protein or a peptide. In one embodiment this activity is an enzymatic activity.

According to the invention the phrase “inhibit expression or activity” includes a complete or essentially complete inhibition of expression or activity and a reduction in expression or activity.

The agent which, when administered, selectively increases the amount of complexes between an MHC molecule and a tumor-associated antigen identified according to the invention or a part thereof comprises one or more components selected from the group consisting of (i) the tumor-associated antigen or a part thereof, (ii) a nucleic acid which codes for said tumor-associated antigen or a part thereof, (iii) a host cell which expresses said tumor-associated antigen or a part thereof, and (iv) isolated complexes between peptide epitopes from said tumor-associated antigen and an MHC molecule.

The invention furthermore relates to a pharmaceutical composition which comprises one or more components selected from the group consisting of (i) a tumor-associated antigen identified according to the invention or a part thereof, (ii) a nucleic acid which codes for a tumor-associated antigen identified according to the invention or 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) an siRNA directed against a nucleic acid coding for a tumor-associated antigen identified according to the invention, (vi) a host cell which expresses a tumor-associated antigen identified according to the invention or a part thereof, and (vii) isolated complexes between a tumor-associated antigen identified according to the invention or a part thereof and an MHC molecule.

In one embodiment, a nucleic acid coding for a tumor-associated antigen identified according to the invention or a part thereof is present in the pharmaceutical composition in an expression vector and functionally linked to a promoter. In a further embodiment, a nucleic acid coding for a tumor-associated antigen identified according to the invention or a part thereof is present in the pharmaceutical composition in a virus as further described below.

In a further embodiment, a host cell present in a pharmaceutical composition of the invention secretes the tumor-associated antigen or the part thereof, expresses it on the surface and preferably additionally express an MHC molecule which binds to said tumor-associated antigen or said part thereof. In one embodiment, the host cell expresses the MHC molecule endogenously. In a further embodiment, the host cell expresses the MHC molecule and/or the tumor-associated antigen or the part thereof in a recombinant manner. The host cell is preferably nonproliferative. In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte or a macrophage.

In a further embodiment, an antibody present in a pharmaceutical composition of the invention is a monoclonal antibody. In further embodiments, the antibody is a chimeric or humanized antibody, a fragment of a natural antibody or a synthetic antibody. The antibody may be coupled to a therapeutically or diagnostically useful agent also termed therapeutic or diagnostic agent herein.

An antisense nucleic acid present in a pharmaceutical composition of the invention may comprise a sequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguous nucleotides of the nucleic acid coding for the tumor-associated antigen identified according to the invention.

In further embodiments, a tumor-associated antigen or a part thereof, provided by a pharmaceutical composition of the invention either directly or via expression of a nucleic acid, binds to MHC molecules on the surface of cells, said binding preferably causing a cytolytic response and/or inducing cytokine release.

In particular embodiments of the siRNA targeting the nucleic acid according to SEQ ID NO: 1 the sense RNA strand has the sequence of SEQ ID NO: 70 and the antisense RNA strand has the sequence of SEQ ID NO: 71, or the sense RNA strand has the sequence of SEQ ID NO: 72 and the antisense RNA strand has the sequence of SEQ ID NO: 73.

A pharmaceutical composition of the invention may comprise a pharmaceutically compatible carrier and/or an adjuvant.

A pharmaceutical composition of the invention is preferably used for the treatment or prevention of a disease characterized by selective expression or abnormal expression of a tumor-associated antigen. In a preferred embodiment, the disease is a neoplastic disease, preferably cancer.

In a preferred embodiment, the pharmaceutical composition of the invention is in the form of a vaccine which may be used therapeutically or prophylactically. Such vaccine preferably comprises a tumor-associated antigen identified according to the invention or a part thereof, and/or a nucleic acid which codes for a tumor-associated antigen identified according to the invention or a part thereof. In particular embodiments, the nucleic acid is present in a virus or host cell.

The invention furthermore relates to methods of treating, preventing, diagnosing or monitoring, i.e. determining the regression, progression, course and/or onset of, a disease characterized by expression or abnormal expression of one of more tumor-associated antigens identified according to the invention, preferably a neoplastic disease, in particular cancer. In one embodiment, the treatment or prevention comprises administering a pharmaceutical composition of the invention.

Said methods of diagnosing and/or methods of monitoring according to the invention generally concern the detection of and/or determination of the quantity of one or more parameters selected from the group consisting of (i) a nucleic acid, which codes for a tumor-associated antigen identified according to the invention, or a part thereof, (ii) a tumor-associated antigen identified according to the invention, or a part thereof (iii) an antibody against a tumor-associated antigen identified according to the invention or a part thereof, and (iv) T lymphocytes, preferably cytotoxic or T helper lymphocytes, which are specific for a tumor-associated antigen identified according to the invention or a part thereof and/or a complex between the tumor-associated antigen or a part thereof and an MHC molecule, in a biological sample isolated from a patient, preferably from a patient having said disease, being suspected of having or falling ill with said disease or having a potential for said disease. Means for accomplishing said detection and/or determination of the quantity are described herein and will be apparent to the skilled person.

Preferably, the presence of said nucleic acid, said tumor-associated antigen or said part thereof, said antibody and/or said T lymphocytes and/or a quantity of said nucleic acid, said tumor-associated antigen or said part thereof, said antibody and/or said T lymphocytes which is increased compared to a patient without said disease is indicative for the presence of said disease or a potential for a development of said disease.

The methods of diagnosing and/or monitoring of the invention also include embodiments wherein by detection or determination of the quantity of said nucleic acid, said tumor-associated antigen or said part thereof, said antibody and/or said T lymphocytes it is possible to assess and/or prognose the metastatic behavior of said disease, wherein, preferably, the presence of said nucleic acid, said tumor-associated antigen or said part thereof, said antibody and/or said T lymphocytes and/or a quantity of said nucleic acid, said tumor-associated antigen or said part thereof, said antibody and/or said T lymphocytes which is increased compared to a patient without said disease or without a metastasis of said disease is indicative for a metastatic behavior of said disease or a potential for a metastatic behavior of said disease.

In particular embodiments, said detection or determination of the quantity comprises (i) contacting a biological sample with an agent which binds specifically to said nucleic acid coding for the tumor-associated antigen or said part thereof, to said tumor-associated antigen or said part thereof, to said antibody or said part thereof or to said T lymphocytes, and (ii) detecting the formation of or determining the amount 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 part thereof, or the T lymphocytes. In one embodiment, the disease is characterized by expression or abnormal expression of two or more different tumor-associated antigens and a detection or determination of the amount comprises a detection or determination of the amount of two or more nucleic acids coding for said two or more different tumor-associated antigens or of parts thereof, of two or more different tumor-associated antigens or of parts thereof, of two or more antibodies binding to said two or more different tumor-associated antigens or to parts thereof and/or of two or more T lymphocytes specific for said two or more different tumor-associated antigens or parts thereof, or complexes thereof with MHC molecules. In a further embodiment, the biological sample isolated from the patient is compared to a comparable normal biological sample.

The methods of monitoring according to the invention preferably comprise a detection of and/or determination of the quantity of one or more of the parameters mentioned above in a first sample at a first point in time and in a further sample at a second point in time, wherein the course of the disease is determined by comparing the two samples.

According to the invention, detection of a nucleic acid or of a part thereof or determining the quantity of a nucleic acid or of a part thereof may be carried out using a oligo- or polynucleotide probe which hybridizes specifically to said nucleic acid or said part thereof or may be carried out by selective amplification of said nucleic acid or said part thereof, e.g. by means of PCR amplification. In one embodiment, the oligo- or 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 or the part thereof which is to be detected or the amount of which is to be determined in the methods of the present invention is present intracellularly, on the cell surface or in a complex with an MHC molecule. According to the invention, detection of a tumor-associated antigen or of a part thereof or determining the quantity 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.

According to the invention, detection of an antibody or determining the quantity of an antibody may be carried out using a protein or peptide binding specifically to said antibody.

According to the invention, detection of or determining the quantity of T lymphocytes which are specific for a tumor-associated antigen or a part thereof and/or a complex thereof with an MHC molecule may be carried out using a cell presenting the complex between said tumor-associated antigen or said part thereof and an MHC molecule. T lymphocytes may additionally be detected by detecting their proliferation, their cytokine production, and their cytotoxic activity triggered by specific stimulation with a complex of an MHC molecule and a tumor-associated antigen or a part thereof. T lymphocytes may also be detected with aid of a recombinant MHC molecule or a complex of two or more MHC molecules loaded with immunogenic fragments of one or more tumor-associated antigens.

An agent which is used for detection or determining the quantity in the methods of the invention such as a oligo- or polynucleotide probe, an antibody, a protein or peptide or a cell is preferably labeled in a detectable manner, in particular by a detectable marker such as a radioactive marker or an enzymic marker.

In a particular aspect, the invention relates to a method of treating, preventing, diagnosing or monitoring a disease characterized by expression or abnormal expression of a tumor-associated antigen identified according to the invention, which method comprises administering an antibody which binds to said tumor-associated antigen or to a part thereof and which is coupled to a therapeutic or diagnostic agent. The antibody may be a monoclonal antibody. In further embodiments, the antibody is a chimeric or humanized antibody or a fragment of a natural antibody.

In certain embodiments, the methods of the invention of diagnosing or monitoring a disease characterized by expression or abnormal expression of a tumor-associated antigen identified according to the invention are performed with a biological sample containing or suspected of containing disseminating tumor cells or metastatic tumor cells. Such biological samples include, for example, blood, serum, bone marrow, sputum, bronchial aspirate, and/or bronchial lavage.

In one particular aspect, the invention relates to a method of treating a patient having a disease characterized by expression or abnormal expression of a tumor-associated antigen identified according to the invention, which method comprises (i) providing a sample containing immunoreactive cells, either obtained from said patient or from another individual of the same species, in particular a healthy individual, or an individual of a different species, (ii) contacting said sample with a host cell expressing said tumor-associated antigen or a part thereof, under conditions which favor production of cytolytic T cells against said tumor-associated antigen or a part thereof, and (iii) introducing the cytolytic T cells into the patient in an amount suitable for lysing cells expressing the tumor-associated antigen or a part thereof. In one embodiment, the method includes cloning of the T cell receptor of cytolytic T cells obtained and transferring the nucleic acid coding for the T cell receptor to T cells, either obtained from said patient or from another individual of the same species, in particular a healthy individual, or an individual of a different species, which T cells thus receive the desired specificity and, as under (iii), may be introduced into the patient.

In one embodiment, the host cell endogenously expresses an MHC molecule. In a further embodiment, the host cell recombinantly expresses an MHC molecule and/or the tumor-associated antigen or the part thereof. Preferably, the host cell presents the tumor-associated antigen or the part thereof by MHC molecules on its surface. The host cell is preferably nonproliferative. In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte or a macrophage.

The invention also relates to a method of treating a disease characterized by expression or abnormal expression of a tumor-associated antigen identified according to the invention, which method comprises (i) identifying cells from the patient which express abnormal amounts of the tumor-associated antigen, (ii) isolating a sample of said cells, (iii) culturing said cells, and (iv) introducing said cells into the patient in an amount suitable for triggering an immune response to the cells.

The present invention furthermore relates to a nucleic acid selected from the group consisting of (a) a nucleic acid which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, a part or derivative thereof, (b) a nucleic acid which hybridizes with the nucleic acid of (a) under stringent conditions, (c) a nucleic acid which is degenerate with respect to the nucleic acid of (a) or (b), and (d) a nucleic acid which is complementary to the nucleic acid of (a), (b) or (c). The invention furthermore relates to a nucleic acid, which codes for a protein or polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69, a part or derivative thereof.

In a further aspect, the invention relates to a recombinant nucleic acid molecule, in particular DNA or RNA molecule, which comprises a nucleic acid of the invention.

The invention also relates to host cells which contain a nucleic acid or recombinant nucleic acid molecule of the invention.

The host cell may also comprise a nucleic acid coding for a MHC molecule. In one embodiment, the host cell endogenously expresses the MHC molecule. In a further embodiment, the host cell recombinantly expresses the MHC molecule and/or the nucleic acid or recombinant nucleic acid molecule of the invention or a part thereof. Preferably, the host cell is nonproliferative. In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte or a macrophage.

In a further embodiment, the invention relates to oligonucleotides which hybridize with a nucleic acid identified according to the invention and which may be used as genetic probes or as “antisense” molecules. Nucleic acid molecules in the form of oligonucleotide primers or competent probes, which hybridize with a nucleic acid identified according to the invention or parts thereof, may be used for finding nucleic acids which are homologous to said nucleic acid identified according to the invention, e.g. by PCR amplification, Southern and Northern hybridization. Hybridization may be carried out under low stringency, more preferably under medium stringency and most preferably under high stringency conditions.

In a further aspect, the invention relates to a protein 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 consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, 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 peptide which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69, 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 MHC molecule or an antibody, preferably to a human HLA receptor or a human antibody. According to the invention, a fragment 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 peptide which has or comprises a sequence selected from the group consisting of SEQ ID NOs: 51-60, 68 and 69 of the sequence listing, 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 a protein or peptide, in particular an antibody, a T cell receptor or an MHC molecule. In further embodiments, the antibody is a monoclonal, chimeric, or humanized antibody, an antibody produced by combinatory techniques, or a fragment of an antibody. In one preferred embodiment, 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.

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 consisting of SEQ ID NOs: 51-60, 68, and 69 of the sequence listing, a part or derivative thereof.

According to the invention, the term “binding” preferably relates to a specific binding. “Specific binding” means that an agent such as an antibody binds stronger to a target such as an epitope for which it is specific compared to the binding to another target. An agent binds stronger to a first target compared to a second target if it binds to the first target with a dissociation constant (KD) which is lower than the dissociation constant for the second target. Preferably the dissociation constant (KD) for the target to which the agent binds specifically is more than 10-fold, preferably more than 20-fold, more preferably more than 50-fold, even more preferably more than 100-fold, 200-fold, 500-fold or 1000-fold lower than the dissociation constant (KD) for the target to which the agent does not bind specifically.

Such specific antibodies may, for example, be obtained by immunization using the aforementioned peptides.

The invention furthermore relates to a conjugate between an agent of the invention which binds to a tumor-associated antigen identified according to the invention or to a part thereof or an antibody of the invention and a therapeutic or diagnostic agent. In one embodiment, the therapeutic or diagnostic agent is a toxin.

In a further aspect, the invention relates to a kit for detecting expression or abnormal expression of a tumor-associated antigen identified according to the invention, which kit comprises agents for detection or determining the quantity (i) of the nucleic acid which codes for the tumor-associated antigen or of a part thereof, (ii) of the tumor-associated antigen or of a part thereof, (iii) of antibodies which bind to the tumor-associated antigen or to a part thereof, and/or (iv) of T cells which are specific for the tumor-associated antigen or a part thereof or a complex thereof with an MHC molecule. In one embodiment, the agents for detection of the nucleic acid or the part thereof are nucleic acid molecules for selective amplification of said nucleic acid, which comprise, in particular, a sequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguous nucleotides of said nucleic acid.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, a “reference” such as a reference sample or reference organism may be used to correlate and compare the results obtained in the methods of the invention from a test sample or test organism, i.e. a patient. Typically the reference organism is a healthy organism, in particular an organism which does not suffer from cancer.

A “reference value” can be determined from a reference empirically by measuring a sufficiently large number of references. Preferably the reference value is determined by measuring at least 2, preferably at least 3, preferably at least 5, preferably at least 8, preferably at least 12, preferably at least 20, preferably at least 30, preferably at least 50, or preferably at least 100 references.

“Derivative” of a nucleic acid means according to the invention that single or multiple such as at least 2, at least 4, or at least 6 and preferably up to 3, up to 4, up to 5, up to 6, up to 10, up to 15, or up to 20 nucleotide substitutions, deletions and/or additions are present in said nucleic acid. Furthermore, the term “derivative” also comprises chemical derivatization of a nucleic acid on a 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.

As used herein, the term “RNA” means a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′-position of a beta-D-ribo-furanose moiety. The term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

The nucleic acids described according to the invention have preferably been isolated. The term “isolated nucleic acid” means according to the invention that the nucleic acid was (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel-electrophoretic fractionation, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid which is available for manipulation by recombinant DNA techniques.

A nucleic acid is “complementary” to another nucleic acid if the two sequences are capable of hybridizing and forming a stable duplex with one another, with hybridization preferably being carried out under conditions which allow specific hybridization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y., 1989 or Current Protocols in Molecular Biology, F. M. Ausubel et al., Editors, John Wiley & Sons, Inc., New York and refer, for example, to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodium citrate, pH 7. After hybridization, the membrane to which the DNA has been transferred is washed, for example, in 2×SSC at room temperature and then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68° C.

According to the invention, complementary nucleic acids have at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98% or at least 99%, identical nucleotides.

The term “percentage identity” is intended to denote a percentage of nucleotides or of amino acid residues which are identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two nucleotide or amino acid sequences are conventionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out by segment or by “window of comparison” in order to identify and compare local regions of sequence similarity. The optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared and multiplying the result obtained by 100 so as to obtain the percentage identity between these two sequences.

Nucleic acids coding for tumor-associated antigens may, according to the invention, be present alone or in combination with other nucleic acids, in particular heterologous nucleic acids. In preferred embodiments, a nucleic acid is functionally linked to expression control sequences or regulatory sequences which may be homologous or heterologous with respect to said nucleic acid. A coding sequence and a regulatory sequence are “functionally” linked to one another, if they are covalently linked to one another in such a way that expression or transcription of said coding sequence is under the control or under the influence of said regulatory sequence. If the coding sequence is to be translated into a functional protein, then, with a regulatory sequence functionally linked to said coding sequence, induction of said regulatory sequence results in transcription of said coding sequence, without causing a frame shift in the coding sequence or said coding sequence not being capable of being translated into the desired protein or peptide.

The term “expression control sequence” or “regulatory sequence” comprises according to the invention promoters, enhancers and other control elements which regulate expression of a gene. In particular embodiments of the invention, the expression control sequences can be regulated. The exact structure of regulatory sequences may vary as a function of the species or cell type, but generally comprises 5′ untranscribed and 5′ untranslated sequences which are involved in initiation of transcription and translation, respectively, such as TATA box, capping sequence, CAAT sequence, and the like. More specifically, 5′ untranscribed regulatory sequences comprise a promoter region which includes a promoter sequence for transcriptional control of the functionally linked gene. Regulatory sequences may also comprise enhancer sequences or upstream activator sequences.

According to the invention, a nucleic acid may furthermore be present in combination with another nucleic acid which codes for a peptide controlling secretion of the protein or peptide 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 peptide causing the encoded protein or peptide 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 nucleic acid 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 identified according to the invention. The term “vector” is used here in its most general meaning and comprises any intermediary vehicle for a nucleic acid which enables said nucleic acid, for example, to be introduced into prokaryotic and/or eukaryotic cells and, where appropriate, to be integrated into a genome. Vectors of this kind are preferably replicated and/or expressed in the cells. An intermediary vehicle may be adapted, for example, to the use in electroporation, in bombardment with microprojectiles, in liposomal administration, in the transfer with the aid of agrobacteria or in insertion via DNA or RNA viruses. Vectors comprise plasmids, phagemids, bacteriophages or viral genomes.

The nucleic acids coding for a tumor-associated antigen identified according to the invention may be used for transfection of host cells. Nucleic acids here mean both recombinant DNA and RNA. Recombinant RNA may be prepared by in-vitro transcription of a DNA template. Furthermore, it may be modified by stabilizing sequences, capping and polyadenylation prior to application.

According to the invention, the term “host cell” relates to any cell which can be transformed or transfected with an exogenous nucleic acid. The term “host cells” comprises according to the invention prokaryotic (e.g. E. coli) or eukaryotic cells (e.g. dendritic cells, B cells, CHO cells, COS cells, K562 cells, yeast cells and insect cells). Particular preference is given to mammalian cells such as cells from humans, mice, hamsters, pigs, goats, primates. The cells may be derived from a multiplicity of tissue types and comprise primary cells and cell lines. Specific examples comprise keratinocytes, peripheral blood leukocytes, stem cells of the bone marrow and embryonic stem cells. In further embodiments, the host cell is an antigen-presenting cell, in particular a dendritic cell, monocyte or a macrophage. A nucleic acid may be present in the host cell in the form of a single copy or of two or more copies and, in one embodiment, is expressed in the host cell.

According to the invention, the term “expression” is used in its most general meaning and comprises the production of RNA or of RNA and protein. It also comprises partial expression of nucleic acids. Furthermore, expression may be carried out transiently or stably. Preferred expression systems in mammalian cells comprise pcDNA3.1 and pRc/CMV (Invitrogen, Carlsbad, Calif.), which contain a selectable marker such as a gene imparting resistance to G418 (and thus enabling stably transfected cell lines to be selected) and the enhancer-promoter sequences of cytomegalovirus (CMV).

In those cases of the invention in which a MHC molecule presents a tumor-associated antigen or a part thereof, an expression vector may also comprise a nucleic acid sequence coding for said MHC molecule. The nucleic acid sequence coding for the MHC 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 MHC molecule, both nucleic acids coding therefor may be transfected into the cell either on the same expression vector or on different expression vectors. If the cell already expresses the MHC molecule, only the nucleic acid sequence coding for the tumor-associated antigen or the part thereof can be transfected into the cell.

The invention also comprises kits for amplification of a nucleic acid coding for a tumor-associated antigen. Such kits comprise, for example, a pair of amplification primers which hybridize to the nucleic acid coding for the tumor-associated antigen. The primers preferably comprise a sequence of 6-50, in particular 10-30, 15-30 and 20-30 contiguous nucleotides of the nucleic acid and are nonoverlapping, in order to avoid the formation of primer dimers. One of the primers will hybridize to one strand of the nucleic acid coding for the tumor-associated antigen, and the other primer will hybridize to the complementary strand in an arrangement which allows amplification of the nucleic acid coding for the tumor-associated antigen.

“Antisense molecules” or “antisense nucleic acids” may be used for regulating, in particular reducing, expression of a nucleic acid. The term “antisense molecule” or “antisense nucleic acid” refers according to the invention to an oligonucleotide which is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide or modified oligo-deoxyribonucleotide and which hybridizes under physiological conditions to DNA comprising a particular gene or to mRNA of said gene, thereby inhibiting transcription of said gene and/or translation of said mRNA. According to the invention, 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 part thereof may form a duplex with the naturally occurring mRNA specifying the enzyme and thus prevent accumulation of or translation of the mRNA into the active enzyme. Another possibility is the use of ribozymes for inactivating a nucleic acid. Antisense oligonucleotides preferred according to the invention have a sequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguous nucleotides of the target nucleic acid and preferably are fully complementary to the target nucleic acid or to a part thereof.

In preferred embodiments, the antisense oligonucleotide hybridizes with an N-terminal or 5′ upstream site such as a translation initiation site, transcription initiation site or promoter site. In further embodiments, the antisense oligonucleotide hybridizes with a 3′ untranslated region or mRNA splicing site.

In one embodiment, an oligonucleotide of the invention consists of ribonucleotides, deoxyribonucleotides or a combination thereof, with the 5′ end of one nucleotide and the 3′ end of another nucleotide being linked to one another by a phosphodiester bond. These oligonucleotides may be synthesized in the conventional manner or produced recombinantly.

In preferred embodiments, an oligonucleotide of the invention is a “modified” oligonucleotide. Here, the oligonucleotide may be modified in very different ways, without impairing its ability to bind its target, in order to increase, for example, its stability or therapeutic efficacy. According to the invention, the term “modified oligonucleotide” means an oligonucleotide in which (i) at least two of its nucleotides are linked to one another by a synthetic internucleoside bond (i.e. an internucleoside bond which is not a phosphodiester bond) and/or (ii) a chemical group which is usually not found in nucleic acids is covalently linked to the oligonucleotide. Preferred synthetic internucleoside bonds are phosphorothioates, alkyl phosphonates, phosphorodithioates, phosphate esters, alkyl phosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.

The term “modified oligonucleotide” also comprises oligonucleotides having a covalently modified base and/or sugar. “Modified oligonucleotides” comprise, for example, oligonucleotides with sugar residues which are covalently bound to low molecular weight organic groups other than a hydroxyl group at the 3′ position and a phosphate group at the 5′ position. Modified oligonucleotides may comprise, for example, a 2′-O-alkylated ribose residue or another sugar instead of ribose, such as arabinose.

It is to be understood that all embodiments described above with respect to oligonucleotides may also apply to polynucleotides.

By “small interfering RNA” or “siRNA” as used herein is meant an isolated RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that is used to identify a target gene or mRNA to be degraded. A range of 19-25 nucleotides is the most preferred size for siRNAs.

siRNA according to the invention can comprise partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA; modifications that make the siRNA resistant to nuclease digestion (e.g., the use of 2′-substituted ribonucleotides or modifications to the sugar-phosphate backbone); or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides. Furthermore, siRNA may be modified to increase the stability thereof as described above for modified oligonucleotides, in particular by introducing one or more phosphorothioate linkages.

One or both strands of the siRNA can also comprise a 3′-overhang. As used herein, a “3′-overhang” refers to at least one unpaired nucleotide extending from the 3′-end of an RNA strand. Thus in one embodiment, the siRNA comprises at least one 3′-overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, preferably from 1 to about nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length. In the embodiment in which both strands of the siRNA molecule comprise a 3′-overhang, the length of the overhangs can be the same or different for each strand. In a most preferred embodiment, the 3′-overhang is present on both strands of the siRNA, and is 2 nucleotides in length. For example, each strand of the siRNA of the invention can comprise 3′-overhangs of dideoxythymidylic acid (“TT”) or diuridylic acid (“uu”).

In order to enhance the stability of the siRNA, the 3′-overhangs can be also stabilized against degradation. In one embodiment, the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotides in the 3′-overhangs with 2′-deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation. In particular, the absence of a 2′-hydroxyl in the 2′-deoxythymidine significantly enhances the nuclease resistance of the 3′-overhang in tissue culture medium.

The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded “hairpin” area. That is, the sense region and antisense region can be covalently connected via a linker molecule. The linker molecule can be a polynucleotide or non-nucleotide linker. Without wishing to be bound by any theory, it is believed that the hairpin area of the latter type of siRNA molecule is cleaved intracellularly by the “Dicer” protein (or its equivalent) to form a siRNA of two individual base-paired RNA molecules.

As used herein, “target mRNA” refers to an RNA molecule that is a target for downregulation.

siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol III promoters is only believed to be efficient when the first transcribed nucleotide is a purine.

siRNA according to the invention can be targeted to any stretch of approximately 19-25 contiguous nucleotides in any of the target mRNA sequences (the “target sequence”). Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T. et al., “The siRNA User Guide”, revised Oct. 11, 2002, the entire disclosure of which is herein incorporated by reference. “The siRNA User Guide” is available on the world wide web at a website maintained by Dr. Thomas Tuschl, Laboratory of RNA Molecular Biology, Rockefeller University, New York, USA, and can be found by accessing the website of the Rockefeller University and searching with the keyword “siRNA”. Thus, the sense strand of the present siRNA comprises a nucleotide sequence substantially identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA.

Generally, a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the 3′-direction) from the start codon. The target sequence can, however, be located in the 5′- or 3′-untranslated regions, or in the region nearby the start codon.

siRNA can be obtained using a number of techniques known to those of skill in the art. For example, siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. published application 2002/0086356 of Tuschl et al., the entire disclosure of which is herein incorporated by reference.

Preferably, siRNA is chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.

Alternatively, siRNA can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Such embodiments are included according to the present invention when reference is made herein to the administration of siRNA or the incorporation of siRNA into pharmaceutical compositions. Suitable promoters for expressing siRNA of the invention from a plasmid include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter.

Selection of other suitable promoters is within the skill in the art. The recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.

The siRNA expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly. The use of recombinant plasmids to deliver siRNA to cells in vivo is discussed in more detail below. siRNA can be expressed from a recombinant plasmid either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.

Selection of plasmids suitable for expressing siRNA, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art.

siRNA can also be expressed from recombinant viral vectors intracellularly in vivo. The recombinant viral vectors comprise sequences encoding the siRNA and any suitable promoter for expressing the siRNA sequences. The recombinant viral vectors can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment. siRNA can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.

The term “peptide” comprises oligo- and polypeptides and refers to substances comprising two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 more, preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acids joined covalently by peptide bonds. The term “protein” refers to large peptides, preferably to peptides with more than 100 amino acid residues, but in general the terms “peptides” and “proteins” are synonyms and are used interchangeably herein.

Preferably, the proteins and peptides described according to the invention have been isolated. The terms “isolated protein” or “isolated peptide” mean that the protein or peptide has been separated from its natural environment. An isolated protein or peptide may be in an essentially purified state. The term “essentially purified” means that the protein or peptide is essentially free of other substances with which it is associated in nature or in vivo.

Such proteins and peptides may be used, for example, in producing antibodies and in an immunological or diagnostic assay or as therapeutics. Proteins and peptides 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 peptide or of an amino acid sequence comprise amino acid insertion variants, amino acid deletion variants and/or amino acid substitution variants.

Amino acid insertion variants comprise amino- and/or carboxy-terminal fusions and also insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties such as hydrophobicity, hydrophilicity, electronegativity, volume of the side chain and the like (conservative substitution). Conservative substitutions, for example, relate to the exchange of one amino acid with another amino acid listed below in the same group as the amino acid to be substituted:

  • 1. small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly)
  • 2. negatively charged residues and their amides: Asn, Asp, Glu, Gln
  • 3. positively charged residues: His, Arg, Lys
  • 4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys)
  • 5. large aromatic residues: Phe, Tyr, Trp.

Owing to their particular part in protein architecture, three residues are shown in brackets. Gly is the only residue without a side chain and thus imparts flexibility to the chain. Pro has an unusual geometry which greatly restricts the chain. Cys can form a disulfide bridge.

The amino acid variants described above may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis (Merrifield, 1964) and similar methods or by recombinant DNA manipulation. The manipulation of DNA sequences for preparing proteins and peptides having substitutions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example.

According to the invention, “derivatives” of proteins and peptides also comprise single or multiple substitutions, deletions and/or additions of any molecules associated with the protein or peptide, such as carbohydrates, lipids and/or proteins or peptides. The term “derivative” also extends to all functional chemical equivalents of said proteins and peptides.

According to the invention, a part or fragment of a tumor-associated antigen preferably has a functional property of the protein or peptide from which it has been derived. Such functional properties comprise the interaction with antibodies, the interaction with other peptides or proteins, the selective binding of nucleic acids and an enzymatic activity. A particular property is the ability to form a complex with MHC molecules and, where appropriate, generate an immune response, preferably by stimulating cytotoxic or T helper cells. A part or fragment of a tumor-associated antigen of the invention preferably comprises a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50, consecutive amino acids of the tumor-associated antigen. A part or fragment of a tumor-associated antigen of the invention preferably comprises a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the tumor-associated antigen. A part or fragment of a tumor-associated antigen is preferably a part of the tumor-associated antigen which corresponds to the non-transmembrane portion, in particular the extracellular portion of the antigen, or is comprised thereof.

Preferred parts or fragments of a tumor-associated antigen according to the invention are in particular suitable for the stimulation of cytotoxic T-lymphocytes in vivo but also for the production of expanded and stimulated T-lymphocytes for the therapeutic adoptive transfer ex vivo.

A part or a fragment of a nucleic acid coding for a tumor-associated antigen relates according to the invention to the part of the nucleic acid, which codes at least for the tumor-associated antigen and/or for a part or a fragment of said tumor-associated antigen, as defined above. A part or fragment of a nucleic acid coding for a tumor-associated antigen is preferably that part of the nucleic acid corresponding to the open reading frame.

According to the invention, particular embodiments ought to involve providing “dominant negative” proteins or peptides derived from tumor-associated antigens. A dominant negative protein or peptide is an inactive protein or peptide variant which, by way of interacting with the cellular machinery, displaces an active protein or peptide from its interaction with the cellular machinery or which competes with the active protein or peptide, thereby reducing the effect of said active protein.

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 folded form. Monoclonal antibodies are traditionally prepared using the hybridoma technology. (for technical details see: “Monoclonal Antibodies: A Practical Approach” by Philip Shepherd, Christopher Dean ISBN 0-19-963722-9; “Antibodies: A Laboratory Manual” by Ed Harlow, David Lane ISBN: 0879693142; “Using Antibodies: A Laboratory Manual: Portable Protocol NO” by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447).

It is known that only a small part of an antibody molecule, the paratope, is involved in binding of the antibody to its epitope (cf. Clark, W. R. (1986), The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991), Essential Immunology, 7th Edition, Blackwell Scientific Publications, Oxford). The pFc′ and Fc regions are, for example, effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc′ region has been enzymatically removed or which has been produced without the pFc′ region, referred to as F(ab′)2 fragment, carries both antigen binding sites of a complete antibody. Similarly, an antibody from which the Fc region has been enzymatically removed or which has been produced without said Fc region, referred to as Fab fragment, carries one antigen binding site of an intact antibody molecule. Furthermore, Fab fragments consist of a covalently bound light chain of an antibody and part of the heavy chain of said antibody, referred to as Fd. The Fd fragments are the main determinants of antibody specificity (a single Fd fragment can be associated with up to ten different light chains, without altering the specificity of the antibody) and Fd fragments, when isolated, retain the ability to bind to an epitope.

Located within the antigen-binding part of an antibody are complementary-determining regions (CDRs) which interact directly with the antigen epitope and framework regions (FRs) which maintain the tertiary structure of the paratope. Both the Fd fragment of the heavy chain and the light chain of IgG immunoglobulins contain four framework regions (FR1 to FR4) which are separated in each case by three complementary-determining regions (CDR1 to CDR3). The CDRs and, in particular, the CDR3 regions and, still more particularly, the CDR3 region of the heavy chain are responsible to a large extent for antibody specificity.

Non-CDR regions of a mammalian antibody are known to be able to be replaced by similar regions of antibodies with the same or a different specificity, with the specificity for the epitope of the original antibody being retained. This made possible the development of “humanized” antibodies in which nonhuman CDRs are covalently linked to human FR and/or Fc/pFc′ regions to produce a functional antibody.

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 referred to as “chimeric” antibodies.

According to the invention, the term “antibody” also includes F(ab′)2, Fab, Fv, and Fd fragments of antibodies, chimeric antibodies, in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain-CDR3 regions have been replaced with homologous human or nonhuman sequences, chimeric F(ab′)2-fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain-CDR3 regions have been replaced with homologous human or nonhuman sequences, chimeric Fab-fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain-CDR3 regions have been replaced with homologous human or nonhuman sequences, and chimeric Fd-fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced with homologous human or nonhuman sequences. The term “antibody” also comprises “single-chain” antibodies.

The invention also comprises proteins and peptides which bind specifically to tumor-associated antigens. Binding substances of this kind may be provided, for example, by degenerate peptide libraries which may be prepared simply in solution in an immobilized form or as phage-display libraries. It is likewise possible to prepare combinatorial libraries of peptides with one or more amino acids. Libraries of peptoids and nonpeptidic synthetic residues may also be prepared.

Antibodies may also be coupled to specific diagnostic substances for displaying cells and tissues expressing tumor-associated antigens. They may also be coupled to therapeutically useful substances.

Diagnostic substances include any label that functions to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g. FRET (Fluorescence Resonance Energy Transfer); (iii) affect mobility, e.g. electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters, or (iv) provide a capture moiety, e.g., affinity, antibody/antigen, or ionic complexation. Suitable as label are structures, such as fluorescent labels, luminescent labels, chromophore labels, radioisotopic labels, isotopic labels, preferably stable isotopic labels, isobaric labels, enzyme labels, particle labels, in particular metal particle labels, magnetic particle labels, polymer particle labels, small organic molecules such as biotin, ligands of receptors or binding molecules such as cell adhesion proteins or lectins, label-sequences comprising nucleic acids and/or amino acid residues which can be detected by use of binding agents, etc. Diagnostic substances comprise, in a nonlimiting manner, barium sulfate, iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium tyropanoate and radio diagnostic, including positron emitters such as fluorine-18 and carbon-11, gamma emitters such as iodine-123, technetium-99m, iodine-131 and indium-111, nuclides for nuclear magnetic resonance, such as fluorine and gadolinium.

According to the invention, the term “therapeutically useful substance” means any molecule which may exert a therapeutic effect. According to the invention, a therapeutically useful substance is preferably selectively guided to a cell which expresses one or more tumor-associated antigens and includes anticancer agents, radioactive iodine-labeled compounds, toxins, cytostatic or cytolytic drugs, etc. Anticancer agents comprise, for example, aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil, interferon-α, lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HCl, thioguanine, vinblastine sulfate and vincristine sulfate. Other anticancer agents are described, for example, in Goodman and Gilman, “The Pharmacological Basis of Therapeutics”, 8th Edition, 1990, McGraw-Hill, Inc., in particular Chapter 52 (Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner). Toxins may be proteins such as pokeweed antiviral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin or Pseudomonas exotoxin. Toxin residues may also be high energy-emitting radionuclides such as cobalt-60.

The term “major histocompatibility complex” or “MHC” relates to a complex of genes present in all vertebrates. MHC proteins or molecules are involved in signaling between lymphocytes and antigen presenting cells in normal immune reactions by binding peptides and presenting them for recognition by T cell receptors (TCR). MHC molecules bind peptides within an intracellular processing compartment and present these peptides on the surface of antigen presenting cells for recognition by T cells. The human MHC region also termed HLA is located on chromosome 6 and includes the class I and class II region. In one preferred embodiment of all aspects of the invention an MHC molecule is an HLA molecule.

“Reduce” or “inhibit” as used herein means the ability to cause an overall decrease, preferably of 20% or greater, more preferably of 50% or greater, and most preferably of 75% or greater, in the level, e.g. in the level of protein or mRNA as compared to a reference sample (e.g., a sample not treated with siRNA). This reduction or inhibition of RNA or protein expression can occur through targeted mRNA cleavage or degradation. Assays for protein expression or nucleic acid expression are known in the art and include, for example, ELISA, western blot analysis for protein expression, and northern blotting or RNase protection assays for RNA.

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.

“Abnormal expression” means according to the invention that expression is altered, preferably increased, compared to the state in a healthy individual.

According to the invention the term “increased” or “increased amount” preferably refers to an increase by at least 10%, in particular at least 20%, at least 50% or at least 100%. The amount of a substance is also increased in a test sample such as a biological sample compared to a reference sample if it is detectable in the test sample but absent or not detectable in the reference sample.

According to the invention, the term “disease” refers to any pathological state in which tumor-associated antigens are expressed or abnormally expressed. “Abnormal expression” means according to the invention that expression is altered, preferably increased, compared to the state in a healthy individual. An increase in expression refers to an increase by at least 10%, in particular at least 20%, at least 50% or at least 100%. In one embodiment, the tumor-associated antigen is expressed only in tissue of a diseased individual, while expression in a healthy individual is repressed. One example of such a disease is cancer, wherein the term “cancer” according to the invention comprises leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer and the matastases thereof. Examples thereof are lung carcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas, or metastases of the cancer types or tumors described above. The term cancer according to the invention also comprises cancer metastases.

By “tumor” is meant an abnormal group of cells or tissue that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign or malignant.

By “metastasis” is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term “metastasis” according to the invention relates to “distant metastasis” which relates to a metastasis which is remote from the primary tumor and the regional lymph node system.

According to the invention, a biological sample may be a tissue sample, including bodily fluids, 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, feces or other body fluids. According to the invention, the term “biological sample” also includes fractions of biological samples.

According to the invention, the term “immunoreactive cell” means a cell which can mature into an immune cell (such as B cell, T helper cell, or cytolytic T cell) with suitable stimulation. Immunoreactive cells comprise CD34+ hematopoietic stem cells, immature and mature T cells and immature and mature B cells. If production of cytolytic or T helper cells recognizing a tumor-associated antigen is desired, the immunoreactive cell is contacted with a cell expressing a tumor-associated antigen under conditions which favor production, differentiation and/or selection of cytolytic T cells and of T helper cells. The differentiation of T cell precursors into a cytolytic T cell, when exposed to an antigen, is similar to clonal selection of the immune system.

The terms “T cell” and “T lymphocyte” are used interchangeably herein and include T helper cells and cytotoxic T cells which comprise cytolytic T cells.

Some therapeutic methods are based on a reaction of the immune system of a patient, which results in a lysis of antigen-presenting cells such as cancer cells which present one or more tumor-associated antigens. In this connection, for example autologous cytotoxic T lymphocytes specific for a complex of a tumor-associated antigen and an MHC molecule are administered to a patient having a cellular abnormality. The production of such cytotoxic T lymphocytes in vitro is known. An example of a method of differentiating T cells can be found in WO-A-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 cytotoxic T lymphocytes (e.g. dendritic cells). The target cell may be a transfected cell such as a COS cell. These transfected cells present the desired complex on their surface and, when contacted with cytotoxic T lymphocytes, stimulate propagation of the latter. The clonally expanded autologous cytotoxic T lymphocytes are then administered to the patient.

In another method of selecting antigen-specific cytotoxic T lymphocytes, fluorogenic tetramers of MHC class I molecule/peptide complexes are used for obtaining specific clones of cytotoxic T lymphocytes (Altman et al., Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416, 1998).

The present invention also includes therapeutic methods referred to as adoptive transfer (Greenberg, J. Immunol. 136(5):1917, 1986; Riddel et al., Science 257:238, 1992; Lynch et al., Eur. J. Immunol. 21:1403-1410, 1991; Kast et al., Cell 59:603-614, 1989), wherein cells presenting the desired complex (e.g. dendritic cells) are combined with cytotoxic T lymphocytes of the patient to be treated, resulting in a propagation of specific cytotoxic T lymphocytes. The propagated cytotoxic T lymphocytes are then administered to a patient having a cellular anomaly characterized by particular abnormal cells presenting the specific complex. The cytotoxic T lymphocytes then lyse the abnormal cells, thereby achieving a desired therapeutic effect.

Furthermore, cells presenting the desired complex (e.g. dendritic cells) may be combined with cytotoxic T lymphocytes of healthy individuals or another species (e.g. mouse) which may result in propagation of specific cytotoxic T lymphocytes with high affinity. The high affinity T cell receptor of these propagated specific T lymphocytes may be cloned and optionally humanized to a different extent, and the T cell receptors thus obtained then transduced via gene transfer, for example using retroviral vectors, into T cells of patients. Adoptive transfer may then be carried out using these genetically altered T lymphocytes (Stanislawski et al., Nat. Immunol. 2:962-70, 2001; Kessels et al., Nat. Immunol. 2:957-61, 2001).

Adoptive transfer is not the only form of therapy which can be applied according to the invention. Cytotoxic T lymphocytes may also be generated in vivo in a manner known per se. One method uses nonproliferative cells expressing the complex. The cells used here will be those which usually express the complex, such as irradiated tumor cells or cells transfected with one or both genes necessary for presentation of the complex (i.e. the antigenic peptide and the presenting MHC molecule). Another preferred form is the introduction of the tumor-associated antigen in the form of recombinant RNA which may be introduced into cells by liposomal transfer or by electroporation, for example. The resulting cells present the complex of interest and are recognized by autologous cytotoxic T lymphocytes which then propagate.

A similar effect can be achieved by combining the tumor-associated antigen or a fragment thereof with an adjuvant in order to make incorporation into antigen-presenting cells in vivo possible. The tumor-associated antigen or a fragment thereof may be represented as protein, as DNA (e.g. within a vector) or as RNA. The tumor-associated antigen is processed to produce a peptide partner for the MHC molecule, while a fragment thereof may be presented without the need for further processing. The latter is the case in particular, if these can bind to MHC molecules. Preference is given to administration forms in which the complete antigen is processed in vivo by a dendritic cell, since this may also produce T helper cell responses which are needed for an effective immune response (Ossendorp et al., Immunol Lett. 74:75-9, 2000; Ossendorp et al., J. Exp. Med. 187:693-702, 1998). In general, it is possible to administer an effective amount of the tumor-associated antigen to a patient by intradermal injection, for example. However, injection may also be carried out intranodally into a lymph node (Maloy et al., Proc Natl Acad Sci USA 98:3299-303, 2001).

The pharmaceutical compositions and methods of treatment described according to the invention may also be used for immunization or vaccination to therapeutically treat or prevent a disease described herein. According to the invention, the terms “immunization” or “vaccination” preferably relate to an increase in or activation of an immune response to an antigen. It is possible to use animal models for testing an immunizing effect on cancer by using a tumor-associated antigen or a nucleic acid coding therefor. For example, human cancer cells may be introduced into a mouse to generate a tumor, and one or more nucleic acids coding for tumor-associated antigens may be administered. The effect on the cancer cells (for example reduction in tumor size) may be measured as a measure for the effectiveness of an immunization by the nucleic acid.

As part of the composition for an immunization or a vaccination, preferably one or more tumor-associated antigens or stimulating fragments thereof are administered together with one or more adjuvants for inducing an immune response or for increasing an immune response. An adjuvant is a substance which is incorporated into the antigen or administered together with the latter and which enhances the immune response. Adjuvants may enhance the immune response by providing an antigen reservoir (extracellularly or in macrophages), activating macrophages and/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 (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 biologically degradable oils such as squalene and/or tocopherol. Preferably, the peptides are administered in a mixture with DQS21/MPL. The ratio of DQS21 to MPL is typically about 1:10 to 10:1, preferably about 1:5 to 5:1 and in particular about 1:1. For administration to humans, a vaccine formulation typically contains DQS21 and MPL in a range from about 1 μg to about 100 μg.

Other substances which stimulate an immune response of the patient may also be administered. It is possible, for example, to use cytokines in a vaccination, owing to their regulatory properties on lymphocytes. Such cytokines comprise, for example, interleukin-12 (IL-12) which was shown to increase the protective actions of vaccines (cf. Science 268:1432-1434, 1995), GM-CSF and IL-18.

There are a number of compounds which enhance an immune response and which therefore may be used in a vaccination. Said compounds comprise costimulating molecules provided in the form of proteins or nucleic acids such as B7-1 and B7-2 (CD80 and CD86, respectively).

The invention also provides for administration of nucleic acids, proteins or peptides. Proteins and peptides may be administered in a manner known per se. In one embodiment, nucleic acids are administered by ex vivo methods, i.e. by removing cells from a patient, genetic modification of said cells in order to incorporate a tumor-associated antigen and reintroduction of the altered cells into the patient. This generally comprises introducing a functional copy of a gene into the cells of a patient in vitro and reintroducing the genetically altered cells into the patient. The functional copy of the gene is under the functional control of regulatory elements which allow the gene to be expressed in the genetically altered cells. Transfection and transduction methods are known to the skilled worker. The invention also provides for administering nucleic acids in vivo by using vectors such as viruses and target-controlled liposomes. If according to the invention reference is made to the administration or incorporation into pharmaceutical compositions of nucleic acids this includes embodiments wherein the nucleic acid is present in such vectors.

In a preferred embodiment, a virus or viral vector for administering a nucleic acid coding for a tumor-associated antigen is selected from the group consisting of adenoviruses, adeno-associated viruses, pox viruses, including vaccinia virus and attenuated pox viruses, Semliki Forest virus, retroviruses, Sindbis virus and Ty virus-like particles. Particular preference is given to adenoviruses and retroviruses. The retroviruses are typically replication-deficient (i.e. they are incapable of generating infectious particles).

Methods of introducing nucleic acids into cells in vitro or in vivo comprise transfection of nucleic acid calcium phosphate precipitates, transfection of nucleic acids associated with DEAE, transfection or infection with the above viruses carrying the nucleic acids of interest, liposome-mediated transfection, and the like. In particular embodiments, preference is given to directing the nucleic acid to particular cells. In such embodiments, a carrier used for administering a nucleic acid to a cell (e.g. a retrovirus or a liposome) may have a bound target control molecule. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell may be incorporated into or attached to the nucleic acid carrier. Preferred antibodies comprise antibodies which bind selectively a tumor-associated antigen. If administration of a nucleic acid via liposomes is desired, proteins binding to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation in order to make target control and/or uptake possible. Such proteins comprise capsid proteins or fragments thereof which are specific for a particular cell type, antibodies to proteins which are internalized, proteins addressing an intracellular site, and the like.

The therapeutic compositions of the invention may be administered in pharmaceutically compatible preparations. Such preparations may usually contain pharmaceutically compatible concentrations of salts, buffer substances, preservatives, carriers, supplementing immunity-enhancing substances such as adjuvants, e.g. CpG oligonucleotides, cytokines, chemokines, saponin, GM-CSF and/or RNA and, where appropriate, other therapeutically active compounds.

The therapeutically active compounds of the invention may be administered via any conventional route, including by injection or infusion. The administration may be carried out, for example, orally, intravenously, intraperitonealy, intramuscularly, subcutaneously or transdermally. Preferably, antibodies are therapeutically administered by way of a lung aerosol. Antisense nucleic acids are preferably administered by slow intravenous administration.

The compositions of the invention are administered in effective amounts. An “effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of treatment of a particular disease or of a particular condition characterized by expression of one or more tumor-associated antigens, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing 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. According to the invention, a diagnosis or treatment of cancer may also include the diagnosis or treatment of cancer metastases which have already formed or will form. According to the invention, the term “treatment” comprises therapeutic and prophylactic treatment, i.e. prevention.

An effective amount of a composition of the invention will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors.

The pharmaceutical compositions of the invention are preferably sterile and contain an effective amount of the therapeutically active substance to generate the desired reaction or the desired effect.

The doses administered of the compositions of the invention may depend on various parameters such as the type of administration, the condition of the patient, the desired period of administration, etc. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

Generally, doses of the tumor-associated antigen of from 1 ng to 1 mg, preferably from 10 ng to 100 μg, are formulated and administered for a treatment or for generating or increasing an immune response. If the administration of nucleic acids (DNA and RNA) coding for tumor-associated antigens is desired, doses of from 1 ng to 0.1 mg are formulated and administered.

The pharmaceutical compositions of the invention are generally administered in pharmaceutically compatible amounts and in pharmaceutically compatible compositions. The term “pharmaceutically compatible” refers to a nontoxic material which does not interact with the action of the active component of the pharmaceutical composition. Preparations of this kind may usually contain salts, buffer substances, preservatives, carriers and, where appropriate, other therapeutically active compounds. When used in medicine, the salts should be pharmaceutically compatible. However, salts which are not pharmaceutically compatible may used for preparing pharmaceutically compatible salts and are included in the invention. Pharmacologically and pharmaceutically compatible salts of this kind comprise in a nonlimiting way those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acids, and the like. Pharmaceutically compatible salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

A pharmaceutical composition of the invention may comprise a pharmaceutically compatible carrier. According to the invention, the term “pharmaceutically compatible carrier” refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to humans. The term “carrier” refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate application. The components of the pharmaceutical composition of the invention are usually such that no interaction occurs which substantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions of the invention may contain suitable buffer substances such as acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may, where appropriate, also contain suitable preservatives such as benzalkonium chloride, chlorobutanol, 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, solutions, suspensions, syrups, elixirs or in the form of an emulsion, for example.

Compositions suitable for parenteral administration usually comprise a sterile aqueous or nonaqueous preparation of the active compound, which is preferably isotonic to the blood of the recipient. Examples of compatible carriers and solvents are Ringer solution and isotonic sodium chloride solution. In addition, usually sterile, fixed oils are used as solution or suspension medium.

The present invention is described in detail by the figures and examples below, which are used only for illustration purposes and are not meant to be limiting. Owing to the description and the examples, further embodiments which are likewise included in the invention are accessible to the skilled worker.

Figures:

FIG. 1. ISC-468 mRNA expression

    • A. RT-PCR investigations with ISC-468-specific primers showed no significant expression within all tested normal tissues except placenta.
    • B. ISC-468 mRNA expression in head and neck, liver, kidney and colon carcinomas.
    • C. ISC-468 mRNA expression in breast, ovarian and stomach carcinomas.

FIG. 2. Quantitative PCR analysis of ISC-468 mRNA expression in normal control tissues and breast cancers

Real-time PCR investigation with ISC-468-specific primers showed selective mRNA expression in normal testis, placenta, stomach and PBMC, and in all breast carcinoma biopsies.

FIG. 3. Specific ISC-507 expression in normal testis and prostate carcinoma

RT-PCR analysis with gene-specific ISC-507 primers shows cDNA amplification exclusively in normal testis (A) and in prostate carcinoma biopsies (B).

FIG. 4. Quantitative expression of ISC-507

Quantitative RT-PCR with ISC-507-specific primers showed selective expression in testis, lymph node and prostate samples and prostate cancer samples.

FIG. 5. ISC-466 expression in normal testis and various tumor samples

RT-PCR analysis with ISC-466-specific primers showed no expression within normal tissues except placenta (A), but expression in head and neck carcinoma biopsies and in kidney carcinoma biopsies (B). Distinct expression was also detected in breast and lung carcinoma cell lines, as well as in ovarian carcinoma cell lines (C and D).

FIG. 6. ISC-518 mRNA expression

RT-PCR analysis with ISC-518-specific primers showed no expression within normal tissues except testis.

FIG. 7. Quantitative expression of ISC-518

Quantitative RT-PCR showed high and selective expression in normal testis and in one liver carcinoma-pool.

FIG. 8. ISC-477 expression in normal and tumor tissues

RT-PCR investigations with ISC-477-specific primers showed selective expression in placenta and ovary normal tissue (A) and high expression in investigated stomach carcinomas (B), breast, colon and lung carcinomas (C), as well as in ovarian and pancreas carcinoma samples (D).

FIG. 9. ISC-489 mRNA expression

RT-PCR investigations with ISC-489-specific primers showed selective expression in placenta control tissue and additionally various levels of expression in lung carcinoma samples (A, C), stomach carcinomas (B, C), head and neck tumors (C) and liver carcinoma samples (C).

FIG. 10. ISC-461 expression in normal testis and various tumor samples

RT-PCR investigations with ISC-461-specific primers showed selective expression in placenta control tissue and additionally various levels of expression in breast carcinomas and melanomas (B), as well as in breast carcinoma, lung carcinoma and melanoma cell lines (C) and ovarian carcinoma cell lines (D).

FIG. 11. ISC-465 mRNA expression in placenta and cancer derived samples

RT-PCR investigations with ISC-465-specific primers showed selective expression in placenta (A) and in some cell lines derived from breast cancer, melanoma, lung cancer or stomach cancer (B).

FIG. 12. Quantitative expression of Mem-030

  • A. Quantitative RT-PCR with Mem-030-specific primers showed a significant overexpression in all investigated head and neck carcinoma samples. The following normal tissues were analyzed: bladder, brain, bone marrow, cervix, colon, duodenum, heart, lung, lymph node, breast, muscle, ovary, PBMC, PBMC-activated, placenta, prostate, retina, spleen, stomach, testis, thymus and tonsil.
  • B. Prevalence of Mem-030 in esophageal, liver, uterus carcimomas and melanoma derived tissues.

FIG. 13. Quantitative expression of Mem-055

Quantitative RT-PCR with Mem-055-specific primers show high and selective expression in normal control tissues and a significant overexpression in stomach and lung cancer derived tissues (A). Mem-055 is also overexpressed in liver carcinomas, ovarian carcinomas and breast cancer samples (B).

FIG. 14. Mem-062 mRNA expression

RT-PCR analysis with Mem-064-specific primers showed selective expression in testis and weak expression in lung cancer derived tissues (A). Strong, significant expression levels of Mem-064 transcripts were detectable in various ovarian tumors (B).

FIG. 15. Specific Mem-068 expression in normal testis and renal cell carcinomas

RT-PCR analysis with gene-specific Mem-068 primers shows cDNA amplification in normal testis, weak in placenta (A), in renal cell carcinomas and in stomach cancers (B).

FIG. 16. Mem-071 expression in normal testis and various tumor samples

RT-PCR analysis with Mem-071-specific primers showed no expression within normal tissues except testis (A). Distinct expression was also detected in renal cell carcinoma samples and in stomach cancers (B).

FIG. 17. Mem-072 mRNA expression

RT-PCR analysis with Mem-072-specific primers showed no expression within normal tissues (A) and significant expression in various lung cancer samples (A+B).

FIG. 18. Mem-106 expression in normal and tumor tissues

RT-PCR investigations with Mem-106-specific primers showed no expression within normal tissues except in testis (A) and high expression were investigated in ovarian- and prostate carcinomas, as well as in melanomas and colon cancer cell lines (B).

FIG. 19. Mem-131 mRNA expression

RT-PCR investigations with Mem-131-specific primers showed no significant expression within all tested normal tissues except activated PBMC.Mem-131 mRNA expression in breast- and lung-carcinomas.Mem-131 mRNA expression in lung- and ovarian carcinomas.

FIG. 20. ISC-468 mRNA expression

(A) RT-PCR and B) Real-time PCR (investigation with ISC-468-specific primers showed selective mRNA expression in normal testis, placenta, and in 80% of breast carcinoma biopsies.

FIG. 21. Immunofluorescence analysis of ISC-468 expression

(A) Specificity of anti-ISC-468 antibodies were confirmed by staining of ISC-468-eGFP transfected cells. (B) Staining of MeOH-fixed cells either transfected with ISC-468-specific RNAI duplexes, or non-silencing control duplexes. (C) Staining of non-fixed cells either transfected with ISC-468-specific RNAi duplexes, or non-silencing control duplexes.

FIG. 22. Immunochistochmical analysis of ISC-468 expression

No expression was detectable in normal breast tissue (A) 100×, (B) 200×. In contrast, strong and homogeneous membran-staining was observed in breast carcinoma specimens (C) 100×, (D) 200×.

FIG. 23. RNAi-induced knock-down of ISC-468 mRNA expression

Transfection of cells with ISC-468-specific siRNA duplexes resulted in distinct knock-down of ISC-468 mRNA expression compared to control cells.

FIG. 24. Cell proliferation analysis

Transfection of cells with ISC-468-specific siRNA duplexes resulted in distinct impairement of cell proliferation compared to control cells.

FIG. 25. Cell cycle analysis

Transfection of cells with ISC-468-specific siRNA duplexes resulted in G1/S arrest in (A) MCF-7 and (B) BT-549 breast carcinoma cells compared to control cells.

FIG. 26. AKT phosphorylierung

Transfection of cells with ISC-468-specific siRNA duplexes resulted in distinct impairement of AKT phosphorylation compared to control cells.

FIG. 27. Antibody-mediated proliferation inhibition

Incubation of MCF-7 breast carcinoma cells with ISC-468 specific antibodies resulted in reduced proliferation compared to cells incubated with an irrelevant control antibody.

FIG. 28. Cell proliferation analysis

Transfection of cells with ISC-468-specific siRNA duplexes resulted in distinct impairement of (A) chemotaxis, (B) chemokinesis, and (C) invasion compared to control cells.

FIG. 29. Estrogen receptor correlation

Expression levels of ISC-468 mRNA in breast carcinoma samples correlates with the estrogen receptor state. Shown are the median, 10th, and 90th percentiles with error bars.

FIG. 30. 17β-estradiol treatment

ISC-468 mRNA expression was induced by treatment of estrogen receptor positive breast carcinoma cell line MCF-7 with 100 nM 17β-estradiol. No induction was seen in estrogen receptor negative cell line MDA-MB-231.

FIG. 31. Sequences

The sequences to which reference is made herein are shown.

EXAMPLES

Material and Methods

The techniques and methods mentioned herein are carried out in a manner known per se and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methods including the use of kits and reagents are carried out according to the manufacturers' information.

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 4 μg of total RNA was carried out in a 20 μl 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 ((SEQ ID NO:33,34)), hybridization temperature 67° C.).

An archive of first strand cDNA was prepared from a number of normal tissues and tumor entities. For expression studies, 0.5 μl of these cDNAs was amplified in a 30 μl reaction mixture, using GOI-specific primers (see below) and 1 U of HotStarTaq DNA polymerase (Qiagen). Each reaction mixture contained 150 μM dNTPs, 0.3 μM of each primer and 3 μl of 10× 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. After 15 minutes at 95° C. to activate the HotStarTaq DNA polymerase, 35 cycles of PCR were carried out (0.5 min at 94° C., 0.5 min at the particular hybridization temperature, 0.5 min at 72° C. and final elongation at 72° C. for 6 min). 20 μl of this reaction were fractionated and analyzed on an ethidium bromide-stained agarose gel.

Preparation of Random Hexamer-Primed cDNA 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 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 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 QuantiTect SYBR Green PCR kit (Qiagen, Hilden) was used in accordance with the manufacturer's instructions. The cDNA was synthesized with random primers (Invitrogen) using the protocol described above. Each 5 μl portions of the diluted cDNA were employed in a total volume of 30 μl 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 30 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 (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.

Cell Proliferation Analysis

24 h after transfection with siRNA duplexes 1×104 cells were cultured in medium supplemented with varying concentrations of FCS for 48 h. Proliferation was analyzed by measuring the incorporation of BrdU into newly synthesized DNA strands using the DELFIA cell proliferation Kit (Perkin Elmer) according to the manufacturer's instructions on a Wallac Victor2 multi-label counter (Perkin Elmer).

Cell Cycle Analysis and Apoptosis

Cells were cultured in medium supplemented with FCS in varying concentrations, harvested after 48 h and stained with propidiumiodide prior to flowcytometric DNA content analysis. Apoptotic cells and cells in S/G2/M phases of the cell cycle were quantified using CellQuest-Software (Becton Dickinson).

Cell Migration

Cell migration assays were conducted in transwell chambers with 8.0 μm pore membranes (BD Biosciences) with cells cultured in serum-free medium for 12 h prior to the experiments. For siRNA experiments cells were transferred to serum-free conditions 24 h after transfection with siRNA duplexes as described above. 4×104 cells in 400 μl serum-free culture medium were added to the upper chamber. The bottom chambers contained 800 μl culture medium supplemented with either FCS, PDGF-BB (Sigma-Aldrich) or SDF-1α/CXCL12 (R&D Systems) as chemoattractants. After 24 hours cells that had migrated to the bottom side of the membrane were fixed in ice-cold methanol; membranes were excised, placed on microscope slides and mounted with Hoechst (Dako) for fluorescence microscopy. Cells in five random visual fields (100× magnification) were counted for each membrane. All experiments were done in triplicates. Effects on chemokinesis of cells was analyzed using the same experimental setup with (i) no chemoattractant added to the upper and lower chamber and (ii) with chemoattractant added to both the upper and lower chamber.

In Vitro Invasion Assay

In vivo invasion assays were conducted in transwell chambers with 8.0 μm pore membranes (BD Biosciences) with cells cultured in serum-free medium for 12 h prior to the experiments. Upper chambers were prepared with 100 μl of Matrigel (BD Biosciences) diluted to 1 mg/ml in serum free medium. Chambers were incubated at 37° C. for 5 h for gelling. For siRNA experiments cells were transferred to serum-free conditions 24 h after transfection with siRNA duplexes as described above. 1×105 cells in 400 μl serum-free culture medium were added to the upper chamber. The bottom chambers contained 800 μl culture medium supplemented with FCS as chemoattractant. After 24 hours invaded cells at the bottom side of the membrane were fixed in ice-cold methanol; membranes were excised, placed on microscope slides and mounted with Hoechst (Dako) for fluorescence microscopy. Cells in five random visual fields (100× magnification) were counted for each membrane. All experiments were done in triplicates.

Example 1

Identification of ISC-468 as Therapeutic and Diagnostic Cancer Target

ISC-468 (SEQ ID NO:1) encodes a protein of 212 amino acids (SEQ ID NO:2) and with a molecular weight of 23.6 kDa.

It has been previously described as placenta-specific protein expressed during pregnancy (Fant et al., Mol Reprod Dev. 63:430-6, 2002)

The protein is predicted to have a cleavable signal peptide from aa 1-23, followed by a short putative transmembrane domain (aa 25-47) as analysed by bioinformatics tools (TMpred, SOUSI). The remaining protein is predicted to be extracellular and can therefore be used according to the invention as target structure for monoclonal antibodies.

According to the invention, a gene-specific primer pair (SEQ ID NO:3, 4) for ISC-468 was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. As expected, placenta was confirmed as the only healthy tissue expressing this gene (FIG. 1). No significant expression, whatsoever, was detected in any other normal organ tissue. Most surprisingly, when cancer specimen were investigated, we found high and significant levels of expression in a number of different tumor types, including colon, pancreatic, esophageal, stomach, lung, breast, ovarian, head&neck, kidney, prostate and liver carcinomas (FIGS. 1 and 2 as well as tab. 1). Quantitative real-time RT-PCR analysis of ISC-468 expression in 60 breast carcinoma samples revealed that 80% of all samples expressed significant levels of ISC-468 (FIG. 20A,B).

TABLE 1
ISC-468 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma+
MyocardiumPancreatic+
Skeletal musclecarcinoma
MyocardiumEsophageal+
Stomachcarcinoma
ColonStomach carcinoma+
PancreasLung cancer+
KidneyBreast cancer+++
LiverOvarian carcinoma+
TestisHead & Neck Cancer+
ThymusKidney cancer+
BreastProstate carcinoma+
OvaryLiver carcinoma++
Uterus
Skin
Lung
Placenta+++
Lymph nodes
Spleen
PBMC
Prostate

The selective and high expression of ISC-468 transcripts in tumors was not previously known and can be utilized according to the invention for molecular diagnostic methods such as RT-PCR for detecting disseminating tumor cells in the serum and bone marrow and for detecting metastases in other tissues. This molecule can be further used as specific target for therapeutic approaches.

The following peptides, inter alia, were selected for producing ISC-468 specific antibodies according to the invention: SEQ ID NO:58, 59, 60, 68, 69, 2. Specificity of the antibodies was confirmed by immunofluorescence analysis of ISC-468-eGFP transfected cells (FIG. 21A).

The subcellular localization of ISC-468 in endogenously expressing breast carcinoma cell lines MCF-7 and BT-549 was analyzed by immunofluorescence analyses. Staining of either MeOH-fixed (FIG. 21B) or non-fixed (FIG. 21C) cells revealed that ISC-468 is localized at the plasma membranes of the expressing cells. Specificity of the staining was confirmed by RNAi-induced knock-down of ISC-468 expression, resulting in the loss of plasma mebrane staining.

Furthermore, ISC-468 specific antibodies were used for immunohistochemical analysis of ISC-468 expression in clinical samples of normal breast and breast carcinomas. Expression of ISC-468 was not detectable in normal breast specimens (FIG. 22A,B). In contrast, breast carcinoma specimens showed strong and homogeneous expression of ISC-468 (FIG. 22C,D). Signals were accentuated at the plasmamembrane of the expressing cancer cells, confirming that ISC-468 is a membrane protein selectively expressed in cancer cells.

The extracellular domains of ISC-468 can be used according to the invention as target structure for immunodiagnosis and therapy by means of monoclonal antibodies. In addition, ISC-468 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).

RNAi-induced knock-down of ISC-468 expression was achieved by transfection of cells with siRNA duplexes specifically targeting ISC-468 mRNA (SEQ ID NOs: 70-73). Transfection of endogenously expressing breast carcinoma cell lines MCF-7 and BT-549 resulted in stable and specific reduction of ISC-468 mRNA expression (FIG. 23).

To gain insight into the physiological role of ISC-468 expression several RNAi-based in vitro cell assays were performed. Transfection of breast carcionoma cell lines MCF-7 and BT-549 with siRNA duplexes resulted in a distinct reduction of cell proliferation compared to the respective controls, as analyzed in a BrdU-based proliferation assay (FIG. 24). FACS-based cell cycle analysis showed that the abrogation of cell proliferation resulted from a G1/S arrest (FIG. 25A,B). Additionally, it could be shown that RNAi-induced knock-down of ISC-468 profoundly affects the AKT signaling pathway in endogenously expressing cancer cells by inhibition of AKT phosphorylation (FIG. 26). Furthermore, proliferation of MCF-7 cells was attenuated when cells were incubated with ISC-468 specific antibodies generated against ISC-468 specific peptides (SEQ ID NO:68,69) compared to an irrelevant control antibody (FIG. 27). These results indicate that ISC-468 is a critical factor for the proliferation of cancer cells presumably by mediating growth factor-induced activation of the AKT signaling pathway and others. ISC-468 itself might represent a receptor, co-receptor or membrane-bound chaperone for growth-factors, chemokines or other substances.

Furthermore, the impact of ISC-468 expression on the migratory ability of cancer cells was analyzed. RNAi-induced knock-down of ISC-468 expression in breastcarcinoma cell lines MCF-7 and BT-549 resulted in distinct impairment of chemotaxis, chemokinesis and invasion of the cells, as assessed in transwell migration assays (FIG. 28A,B,C). Chemotaxis, chemokinesis and invasion are critical factors for the metastasis of cancer cells to other organs. Therefore, expression of ISC-468 in cancer cells might be a positive factor for cancer cell metastasis.

In breast carcinomas, it could be shown that expression of ISC-468 is correlated with the estrogen-receptor state of the tumor. Quantitative real-time RT-PCR analysis of ISC-468 expression in 60 breast carcinoma samples revealed that estrogen-receptor positive breast carcinomas showed significantly higher levels of ISC-468 expression than receptor-negative tumors (FIG. 29). Accordingly, expression of ISC-468 could be induced in estrogen-receptor positive breast carcinoma cell line MCF-7 by treatment with 178-estradiol (FIG. 30).

Example 2

Identification of ISC-507 as Therapeutic and Diagnostic Cancer Target

ISC-507 (SEQ ID NO:5) encodes a 754 aa protein (SEQ ID NO:6) with a molecular weight of 85.6 kDa. ISC-507 is member of a family of zinc-binding proteins with disintegrin and metalloprotease activities that can function as adhesion proteins and/or endopeptidases. Members of this family have been described as involved in a number of biologic processes, including fertilization, neurogenesis, muscle development, and immune response (Seals et al., Genes Dev. 17(1):7-30, 2003)

ISC-507 has one transmembrane domain (aa 671-687), a large N-terminal extracellular and a shorter C-terminal cytoplasmatic region.

ISC-507 expression has been reported to be specifically restricted to mammalian epididymis, the small gland adjacent to the testicle, which is critically involved in maturation of sperm. According to literature, ISC-507 is transferred from the epididymis to the sperm surface and redistributed in the sperm head during acrosome reaction (Adachi et al., Mol Reprod Dev. 64:414-21, 2003).

RT-PCR investigations with ISC-507 specific primers (SEQ ID NO:7, 8) confirmed selective expression in the testis and absence of ISC-507 from any other normal tissue (tab. 2, FIG. 3), except weak expression in prostate and lymph node derived tissues (tab. 2, FIG. 4).

However and most surprisingly, we observed expression of ISC-507 in a significant number of prostate cancers (FIGS. 3,4). This protein had not been reported before to be involved in cancer.

TABLE 2
ISC-507 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma
CerebellumPancreatic
Myocardiumcarcinoma
Skeletal muscleEsophageal
Myocardiumcarcinoma
StomachStomach carcinoma
ColonLung cancer
PancreasBreast cancer
KidneyOvarian carcinoma
LiverUterus carcinoma
Testis+++Head & Neck Cancer
ThymusKidney cancer
BreastProstate carcinoma+++
OvaryLiver carcinoma
Uterus
Skin
Lung
Placenta
Lymph nodes+
Spleen
PBMC
Prostate+

The absence from toxicity relevant normal tissues and the frequent and significant expression of ISC-507 in prostate cancers 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 detection of metastases in other organs by means of RT-PCR. In addition, the extracellular domains of ISC-507 can be used according to the invention as target structure for immunodiagnosis and therapy by means of monoclonal antibodies. In addition, ISC-507 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).

Antibodies for detecting ISC-507 could be produced with following peptides and proteins: SEQ ID NO:51, 52, 53, 54, 55, 6, 56 and 57.

According to the invention an antibody which binds to ISC-507 might be useful for therapeutic or diagnostic purposes.

Example 3

Identification of ISC-466 as Therapeutic and Diagnostic Cancer Target

ISC-466 (SEQ ID NO:9) encodes a 426 aa protein (SEQ ID NO:10) with a molecular weight of 48.2 kDA. It belongs to the family of pregnancy-specific glycoproteins. The human pregnancy-specific glycoproteins (PSGs) are a group of molecules that are mainly produced by the placental syncytiotrophoblasts during pregnancy and are part of the immunoglobulin superfamily (Beauchemin et al., Exp Cell Res. 252(2):243-9, 1999)

As other PSGs, ISC-466 as well has been reported to be restricted to placenta.

According to the invention, a gene-specific primer pair (SEQ ID NO:11, 12) for ISC-466 was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. The RT-PCR analysis reveals expression of ISC-466 transcripts in normal placenta, and weak expression in thymus and ovary (tab. 3, FIG. 5A). No significant expression was detected in any other normal organ tissue. Most surprisingly, when cancer cell lines were investigated, we found high and significant levels of expression in a number of tumor types, including breast cancer (FIG. 5C), lung cancer (FIG. 5C), ovarian carcinoma (FIG. 5D) and head and neck- and kidney carcinomas (FIG. 5B).

TABLE 3
ISC-466 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma+
Thymus+Pancreatic+
Myocardiumcarcinoma
Skeletal muscleEsophageal+
Myocardiumcarcinoma
StomachStomach carcinoma
ColonLung cancer++
PancreasBreast cancer+++
KidneyOvarian carcinoma++
LiverCervix carcinoma
Testis+++Head & Neck Cancer+++
ThymusKidney cancer++
BreastProstate carcinoma+
Ovary+Liver carcinoma
UterusMelanoma+
Skin
Lung
Placenta
Lymph nodes
Spleen
PBMC
Prostate

In contrast to the observation, that ISC-466 is involved by colorectal carcinomas (Salahshor et al., BMC Cancer. 5:66, 2005), our investigations reveal ISC-466 according to the invention as diagnostic and therapeutic marker for head & neck, breast, ovarian, prostate cancer and melanoma.

Example 4

Identification of ISC-518 as Therapeutic and Diagnostic Cancer Target

ISC-518 (SEQ ID NO:13) encodes a 237 aa translation product (SEQ ID NO:14). However, no data with regard to tissue distribution and no connection to cancer is available so far.

ISC-518 is a hypothetical, bioinformaticly predicted gene/protein. Sequence analyses revealed that the protein has a transmembrane domain (aa 102-118). The extracellular C-terminus features a functional domain, which occurs in cell-surface glycoproteins.

According to the invention, a gene-specific primer pair (SEQ ID NO:15, 16) for ISC-518 was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. The only normal tissue we found to express this gene was testis, whereas no significant expression of ISC-518 was detectable in any other normal organ (FIG. 6). Most surprisingly, when cancer specimen were investigated, we found high and significant levels of expression in hepatocarcinomas (FIG. 7)

TABLE 4
ISC-518 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma
CerebellumPancreatic
Myocardiumcarcinoma
Skeletal muscleEsophageal
Myocardiumcarcinoma
StomachStomach carcinoma+
ColonLung cancer+
PancreasBreast cancer+
KidneyOvarian carcinoma+
LiverUterus carcinoma
Testis+++Head & Neck Cancer
ThymusKidney cancer
BreastProstate carcinoma+
OvaryLiver carcinoma++
Uterus
Skin
Lung
Placenta
Lymph nodes
Spleen
PBMC
Prostate

Bioinformatic investigations showed that the protein encoded by ISC-518 represents a cell surface molecule. The previous unknown selective expression of this surface molecule makes it a target for therapeutic purposes and for developing diagnostic methods for the detection of tumor cells and therapeutic methods for the elimination of tumor cells.

Example 5

Identification of ISC-477 as Therapeutic and Diagnostic Cancer Target

ISC-477 (SEQ ID NO:17) encodes a 130 aa translation product (SEQ ID NO:18). ISC-477 is a hypothetical protein. No data with regard to tissue distribution and no connection to cancer was publicly available. Structural analysis reveals a hydrophobic region, which might be a transmembrane region or signal peptide.

According to the invention, a gene-specific primer pair (SEQ ID NO: 19, 20) for ISC-477 was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. The only normal tissues we found to express this gene were placenta and ovary. In contrast, no significant expression of ISC-477 was detectable in any other normal organ (FIG. 8A). Most surprisingly, when cancer specimens were investigated, we found high and significant levels of expression in lung, ovarian, colon and stomach cancer (FIG. 8A-D). Expression levels are clearly higher than expression in normal ovary.

TABLE 5
ISC-477 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma++
CerebellumPancreatic+
Myocardiumcarcinoma
Skeletal muscleEsophageal
Myocardiumcarcinoma
StomachStomach carcinoma++
ColonLung cancer+++
PancreasBreast cancer++
KidneyOvarian carcinoma++
LiverKidney cancer
TestisProstate carcinoma
ThymusLiver carcinoma
Breast
Ovary++
Uterus
Skin
Lung
Placenta+++
Lymph nodes
Spleen
PBMC
Prostate

Example 6

Identification of ISC-489 as Therapeutic and Diagnostic Cancer Target

ISC-489 (SEQ ID NO:21) encodes a 363 aa translation product (SEQ ID NO:22). The protein is a newly described member of the family of G-protein coupled receptors. However, no data with regard to tissue distribution and no connection to cancer was publicly available.

According to the invention, a gene-specific primer pair (SEQ ID NO:23,24) for ISC-489 was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. The only normal tissues we found to express this gene were placenta and esophagus (weak expression). In contrast, no significant expression of ISC-489 was detectable in any other normal organ (FIG. 9A). Most surprisingly, when cancer specimens were investigated, we found high and significant levels of expression in head and neck, and stomach cancers (FIG. 9B, 9C).

As member of the G-protein coupled receptor family, ISC-489 is an integral membrane protein with 7 transmembrane domains and several extraxcellular loops, which can be targeted on the cell surface.

TABLE 6
ISC-489 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma+
Esophagus+Stomach carcinoma++
MyocardiumLung cancer+
Skeletal muscleBreast cancer
MyocardiumOvarian carcinoma
StomachHead & Neck Cancer+++
ColonKidney cancer+
PancreasProstate carcinoma
KidneyLiver carcinoma+
Liver
Testis
Thymus
Breast
Ovary
Uterus
Skin
Lung
Placenta
Lymph nodes+++
Spleen
PBMC
Prostate

The pronounced expression and unexpected high incidence of ISC-489 in head and neck carcinomas make this protein according to the invention a highly interesting diagnostic and therapeutic marker.

Example 7

Identification of ISC-461 as Therapeutic and Diagnostic Cancer Target

ISC-461 (SEQ ID NO:25) encodes a 419 aa protein (SEQ ID NO:26) with a molecular weight of 47.1 kDA.

It belongs to the family of pregnancy-specific glycoproteins. The human pregnancy-specific glycoproteins (PSGs) are a group of molecules that are mainly produced by the placental syncytiotrophoblasts during pregnancy and are part of the immunoglobulin superfamily (Beauchemin et al., Exp Cell Res. 252(2):243-9, 1999).

As other PSGs, ISC-461 as well has been reported to be restricted to placenta.

According to the invention, a gene-specific primer pair (SEQ ID NO:11, 27) for ISC-461 was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. As expected, placenta was confirmed as expressing this gene, besides weak expression in testis and ovary (FIGS. 10A and 10B). No significant expression, whatsoever, was detected in any other normal organ tissue. Most surprisingly, when cancer derived tissues and cancer cell lines were investigated, we found high and significant levels of expression in a number of tumor types, including breast cancer (FIG. 10C), ovarian carcinoma (FIG. 10D) and melanoma (FIG. 10B, 10C).

TABLE 7
ISC-461 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma
CerebellumPancreatic+
Myocardiumcarcinoma
Skeletal muscleEsophageal+
Myocardiumcarcinoma
StomachStomach carcinoma+
ColonLung cancer+
PancreasBreast cancer++
KidneyOvarian carcinoma++
LiverKidney cancer
Testis+Prostate carcinoma
ThymusLiver carcinoma
BreastMelanoma++
Ovary+
Uterus
Skin
Lung
Placenta+++
Lymph nodes
Spleen
PBMC
Prostate

A further aim according to the invention was to identify splice variants for ISC-461 which can be utilized both for diagnosis and for therapy.

On investigation of splice variants we could identify a splice form (SEQ ID NO:28) and the protein encoded thereby (SEQ ID NO:29).

Example 8

Identification of ISC-465 as Therapeutic and Diagnostic Cancer Target

ISC-465 (SEQ ID NO:30) encodes a 419 aa protein (SEQ ID NO:31) with a molecular weight of 47.0 kDA.

It belongs to the family of pregnancy-specific glycoproteins. The human pregnancy-specific glycoproteins (PSGs) are a group of molecules that are mainly produced by the placental syncytiotrophoblasts during pregnancy and are part of the immunoglobulin superfamily (Beauchemin et al., Exp Cell Res. 252(2):243-9, 1999).

As other PSGs, ISC-465 as well has been reported to be restricted to placenta.

According to the invention, a gene-specific primer pair (SEQ ID NO:11, 32) for ISC-465 was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. As expected, placenta was confirmed as expressing this gene, besides weak expression in normal ovary (FIG. 11A). No significant expression, whatsoever, was detected in any other normal organ tissue. Most surprisingly, when cancer derived tissues and cancer cell lines were investigated, we found high and significant levels of expression in a number of tumor types (FIG. 11A, 11B), especially breast cancer (FIG. 11B).

TABLE 8
ISC-461 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma
CerebellumPancreatic+
Myocardiumcarcinoma
Skeletal muscleEsophageal+
Myocardiumcarcinoma
StomachStomach carcinoma+
ColonLung cancer+
PancreasBreast cancer++
KidneyOvarian carcinoma++
LiverKidney cancer
Testis+Prostate carcinoma
ThymusLiver carcinoma
BreastMelanoma+
Ovary+
Uterus
Skin
Lung
Placenta+++
Lymph nodes
Spleen
PBMC
Prostate

The selective and high expression of ISC-465 transcripts in tumors was not previously known and can be utilized according to the invention for molecular diagnostic methods such as RT-PCR for detecting disseminating tumor cells in the serum and bone marrow and for detecting metastases in other tissues. This molecule can be further used as specific target for therapeutic approaches.

Example 9

Identification of Mem-030 as Therapeutic and Diagnostic Cancer Target

Mem-030 (SEQ ID NO:35) encodes a 592 aa protein (SEQ ID NO:36) with a molecular weight of 67.9 kDA. Mem-030 belongs to the GBP-proteins, which are large GTPases being able to bind GTP, GDP, and GMP and to catalyze the hydrolysis of GTP to GDP, as well as GMP (Cheng et al., J. Biol. Chem. 260:15834-9, 1985). GTPases play an important role in cell proliferation, differentiation, signal transduction, and intracellular protein transportation and are interferon inducible (Boehm et al., J. Immunol. 161(12):6715-23, 1998).

Also, Mem-030 counteracts the proliferative effect of inflammatory cytokines like IFN-g, interleukin 1-b (IL-1b), and tumor necrosis factor-a (TNF-a) 1 on endothelial cells (Guenzi et al., EMBO J. 20(20):5568-77, 2001).

According to the invention, a gene-specific primer pair (SEQ ID NO:37, 38) for Mem-030 was used in real time RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. Mem-030 show an ubiquitous expression pattern (FIG. 12A, tab. 9).

Most surprisingly, when cancer derived tissues and cancer cell lines were investigated, we found high and significant levels of overexpression in a number of tumor types (FIG. 12A, 12B), especially head and neck carcinomas.

TABLE 9
Mem-030 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
Brain+Colon carcinoma+
Myocardium+Pancreatic+
Skeletal muscle+carcinoma
Myocardium+Esophageal++
Stomach+carcinoma
Colon+Stomach carcinoma+
Pancreas+Lung cancer+
Kidney+Breast cancer+
Liver+Ovarian carcinoma+
Testis+Uterus carcinoma++
Thymus+Head & Neck Cancer+++
Breast+Kidney cancer+
Ovary+Prostate carcinoma+
Uterus+Liver carcinoma++
Skin+Melanoma++
Lung+
Placenta+
Lymph nodes+
Spleen+
PBMC+
Prostate+

Due to bioinformatics and literature analysis, a homologous gene of Mem-030 might be also an attractive therapeutic target (SEQ ID NO:39) and encodes a 586 aa protein (SEQ ID NO:40) with a molecular weight of 66.6 kDA.

Bioinformatic investigations showed that both proteins represent cell surface molecules. The previously unknown selective overexpression of this surface molecule makes it a target for therapeutic purposes and for developing diagnostic methods for the detection of tumor cells and therapeutic methods for the elimination of tumor cells.

Example 10

Identification of Mem-055 as Therapeutic and Diagnostic Cancer Target

Mem-055 (SEQ ID NO:41) encodes a 250 aa protein (SEQ ID NO:42) with a molecular weight of 27.9 kDA.

The protein encoded by this gene is a lysosomal thiol reductase that at low pH can reduce protein disulfide bonds. The enzyme is expressed constitutively in antigen-presenting cells and induced by gamma-interferon in other cell types. This enzyme has an important role in MHC class II-restricted antigen processing (Arunachalam et al. Proc Natl Acad Sci U S A. 97(2):745-50, 2000).

The localization of Mem-055 and the protein topology was predicted by analysis of the putative signal sequences and transmembrane domains with bioinformatic tools (TMPRED, SOUSI). Mem-055 might have an extracellular C-terminus.

According to the invention, a gene-specific primer pair (SEQ ID NO:43, 44) for Mem-055 was used in real time RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. Mem-055 show an ubiquitous expression pattern (FIG. 13A, tab. 10).

Most surprisingly, when Mem-055 expression within cancer derived tissues was investigated, we found high and significant levels of overexpression in a number of tumor types (FIG. 13A, 13B), especially stomach cancers.

TABLE 10
Mem-055 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
Brain+Colon carcinoma+
Myocardium+Pancreatic+
Skeletal muscle+carcinoma
Myocardium+Esophageal+
Stomach+carcinoma
Colon+Stomach carcinoma+++
Pancreas+Lung cancer++
Kidney+Breast cancer++
Liver+Ovarian carcinoma++
Testis+Uterus carcinoma+
Thymus+Head & Neck Cancer+
Breast+Kidney cancer+
Ovary+Prostate carcinoma+
Uterus+Liver carcinoma++
Skin+Melanoma+
Lung+
Placenta+
Lymph nodes+
Spleen+
PBMC+
Prostate+

Mem-055 is a target structure for therapeutic and diagnostic purposes, because of the putative extracellular domain and the unexpected overexpression in different carcinoma types.

Example 11

Identification of Mem-062 as Therapeutic and Diagnostic Cancer Target

Mem-062 (SEQ ID NO:45) encodes a 271 aa protein (SEQ ID NO:46) with a molecular weight of 30.7 kDA.

By a computer-based screening method Mem-062 could previously be identified and was described as testis, prostate and placenta specifically expressed (Bera et al., Biochem Biophys Res Commun.312(4):1209-15, 2003)

According to the invention, a gene-specific primer pair (SEQ ID NO:47, 48) for Mem-062 was used in RT-PCR analyses. Mem-062 surprisingly showed a cancer-testis specific expression pattern (FIG. 14A, tab. 11). No expression was detected in any other normal organ tissue. Most surprisingly, when cancer derived tissues were investigated, we found significant levels of Mem-62 expression (FIG. 14B), especially in ovarian carcinomas.

TABLE 11
Mem-062 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma+
MyocardiumPancreatic
Skeletal musclecarcinoma
MyocardiumEsophageal
Stomachcarcinoma
ColonStomach carcinoma
PancreasLung cancer
KidneyBreast cancer
LiverOvarian carcinoma++
TestisUterus carcinoma
ThymusHead & Neck Cancer
BreastKidney cancer
OvaryProstate carcinoma
UterusLiver carcinoma
SkinMelanoma
Lung
Placenta
Lymph nodes
Spleen
PBMC
Prostate

Alternative splicing results in an alternative transcript (SEQ ID NO:49) and its corresponding translation product (SEQ ID NO:50).

Example 12

Identification of Mem-068 as Therapeutic and Diagnostic Cancer Target

Mem-068 (SEQ ID NO:61) is a newly identified cDNA clone.

By a bioinformatic prediction approach (Genscan) Mem-068 could be described as multiple exon gene on chromosome 9 (SEQ ID NO:62). The deduced protein sequence (SEQ ID NO:63) has 751 aa and forms a protein with a molecular weight of 82.4 kDA.

According to the invention, a gene-specific primer pair for Mem-068 was used in RT-PCR analyses. Mem-068 show surprisingly a cancer-testis specific expression pattern (FIG. 15A, tab. 12). No expression was detected in any other normal organ tissue except placenta (weak expression). Most surprisingly, when cancer derived tissues were investigated, we found significant levels of Mem-068 expressed (FIG. 15B), especially in renal cell carcinomas and in stomach cancers.

TABLE 12
Mem-068 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma+
BreastRenal cell carcinoma++
ColonStomach carcinoma+
KidneyLung cancer+
LiverBreast cancer
LungOvarian carcinoma
Lymph nodesMelanoma
OvaryProstate carcinoma
Pancreas
Placenta+
PBMC
PBMC activated
Prostate
Skeletal muscle
Skin
Stomach
Spleen
Testis+
Uterus

According to the transmembrane prediction programme TMpred Mem-068 might be expressed at the cell surface, which makes it an interesting target for therapeutic or diagnostic purposes.

Example 13

Identification of Mem-071 as Therapeutic and Diagnostic Cancer Target

Mem-071 (SEQ ID NO:64) is a new cDNA clone, which is encoded in 2 exons on chromosome 1.

According to the invention, a gene-specific primer pair for Mem-071 was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. The only normal tissues we found to express this gene was testis (FIG. 16A). In contrast, when cancer specimen were investigated, we found high and significant levels of expression in renal cell carcinomas and stomach cancers (FIG. 16B).

TABLE 13
Mem-071 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma
BreastRenal cell carcinoma++
ColonStomach carcinoma+
KidneyLung cancer
LiverBreast cancer
LungOvarian carcinoma
Lymph nodesMelanoma
OvaryProstate carcinoma
Pancreas
Placenta
PBMC
PBMC activated
Prostate
Skeletal muscle
Skin
Stomach
Spleen
Testis+
Uterus

The unexpected high incidence of Mem-071 in renal cell carcinomas make this protein according to the invention a highly interesting diagnostic and therapeutic marker.

Example 14

Identification of Mem-072 as Therapeutic and Diagnostic Cancer Target

Mem-072 (SEQ ID NO:65) is a newly identified gene, which is encoded in 3 exons on chromosome 16.

According to the invention, a gene-specific primer pair for Mem-072 was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. No expression within all tested normal tissues could be found (FIG. 17A, tab. 14). When cancer derived tissues and cancer cell lines were investigated, we found high and significant levels of expression in lung cancer samples (FIG. 17B).

TABLE 14
Mem-072 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BrainColon carcinoma
BreastRenal cell carcinoma
ColonStomach carcinoma
KidneyLung cancer++
LiverBreast cancer
LungOvarian carcinoma
Lymph nodesMelanoma
OvaryProstate carcinoma
Pancreas
Placenta
PBMC
PBMC activated
Prostate
Skeletal muscle
Skin
Stomach
Spleen
Testis
Uterus

The selective and high expression of Mem-072 in lung tumors was not previously known and can be utilized according to the invention for molecular diagnostic methods such as RT-PCR for detecting disseminating tumor cells in the serum and bone marrow and for detecting metastases in other tissues. This molecule can be further used as specific target for therapeutic approaches.

Example 15

Identification of Mem-106 as Therapeutic and Diagnostic Cancer Target

Mem-106 (SEQ ID NO:66) is a newly identified cDNA, which is intronless encoded on chromosome 2.

According to the invention, a gene-specific primer pair for Mem-106 was used in RT-PCR analyses. Mem-106 surprisingly showed a cancer-testis specific expression pattern (FIG. 18A, tab. 15). No expression was detected in any other normal organ tissue. Most surprisingly, when cancer derived tissues were investigated, we found significant levels of Mem-106 expression (FIG. 18B), especially in ovarian carcinomas.

TABLE 15
Mem-106 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BreastColon carcinoma+
ColonRenal cell carcinoma
KidneyStomach carcinoma
LiverLung cancer
LungBreast cancer
Lymph nodesOvarian carcinoma++
OvaryMelanoma++
PancreasProstate carcinoma
Placenta
PBMC
PBMC activated
Prostate
Skeletal muscle
Skin
Stomach
Spleen
Testis++
Uterus

Mem-106 is a target structure for therapeutic and diagnostic purposes, because of the unexpected overexpression in different carcinoma types.

Example 16

Identification of Mem-131 as Therapeutic and Diagnostic Cancer Target

Mem-131 (SEQ ID NO:67) is a newly identified cDNA clone. Mem-131 is a 2 exone gene on chromosome 15.

According to the invention, a gene-specific primer pair for Mem-131 was used in RT-PCR analyses to amplify cDNA derived from a comprehensive panel of normal and tumor tissues. The RT-PCR analysis reveals expression of Mem-131 transcripts only in normal activated PBMCs (tab. 16, FIG. 19A). No significant expression was detected in any other normal organ tissue. Most surprisingly, when cancer samples were investigated, we found high and significant levels of expression in a number of tumor types, including breast cancer (FIG. 19B), lung cancer (FIGS. 19B+C) and ovarian carcinoma (FIG. 19C).

TABLE 16
Mem-131 expression in normal and tumor tissues
Normal tissuesExpressionTumor typeExpression
BreastLung cancer++
DuodenumBreast cancer++
BladderOvarian carcinoma++
Skin
Brain
Bone marrow
Colon
Liver
Lung
Lymph node
Stomach
Spleen
Myocard
Kidney
Esophagus
Ovary
Pancreas
PBMC
PBMC activated++
Placenta
Muscle
Testis
Thymus

Our investigations reveals Mem-131 according to the invention as diagnostic and therapeutic marker for lung, breast and ovarian cancers.