METHODS AND COMPOSITIONS FOR DIAGNOSIS AND TREATMENT OF CANCER

Abstract

The present invention relates to the identification of nucleic acid and amino acid sequences that are characteristic of tumor tissues, in particular tumors of the central nervous system (CNS) such as glioma, in particular glioblastoma and which represent targets for therapy or diagnosis of tumor diseases in a subject.

Claims

1. A pharmaceutical composition comprising an inhibitor of expression and/or activity of a tumor antigen, wherein said tumor antigen comprises an amino acid sequence encoded by a nucleic acid which comprises the nucleic acid sequence according to SEQ ID NO: 1 or 2 of the sequence listing or a variant of said nucleic acid sequence.

2. The pharmaceutical composition as claimed in claim 1, wherein (i) the inhibitor of expression of a tumor antigen is an inhibitory nucleic acid selectively hybridizing to the nucleic acid which comprises the nucleic acid sequence according to SEQ ID NO: 1 or 2 of the sequence listing or a variant of said nucleic acid sequence, the inhibitory nucleic acid preferably being selected from the group consisting of an anti-sense oligonucleotide, a ribozyme, iRNA, siRNA or a DNA encoding the same or (ii) the inhibitor of activity of a tumor antigen is an antibody specifically binding to said tumor antigen.

3. A pharmaceutical composition comprising a ligand of a tumor antigen or a ligand of a nucleic acid encoding a tumor antigen, wherein said tumor antigen comprises an amino acid sequence encoded by a nucleic acid which comprises the nucleic acid sequence according to SEQ ID NO: 1 or 2 of the sequence listing or a variant of said nucleic acid sequence, and wherein said ligand of a tumor antigen or ligand of a nucleic acid encoding a tumor antigen is attached to one or more therapeutic effector moieties, said therapeutic effector molecule preferably being a radiolabel, cytotoxin or cytotoxic enzyme.

4. The pharmaceutical composition as claimed in claim 3, wherein the ligand of a tumor antigen comprises an antibody specifically binding to said tumor antigen or the ligand of a nucleic acid encoding a tumor antigen is a nucleic acid selectively hybridizing to said nucleic acid.

5. A pharmaceutical composition comprising one or more agents selected from the group consisting of: (i) a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, or a derivative of said peptide, (ii) a nucleic acid which codes for a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, or a derivative of said nucleic acid, (iii) a host cell which codes for a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, (iv) a virus which codes for a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, (v) a cell presenting a peptide comprising the amino acid sequence of a tumor antigen peptide derived from a tumor antigen, or a derivative of said peptide, (vi) an antibody or T cell receptor which binds to a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, (vii) an immunoreactive cell sensitized in vitro to recognize a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, and (viii) an effector cell or stem cell transduced with a nucleic acid encoding a T cell receptor that recognizes a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, wherein said tumor antigen comprises an amino acid sequence encoded by a nucleic acid which comprises the nucleic acid sequence according to SEQ ID NO: 1 or 2 of the sequence listing or a variant of said nucleic acid sequence and said tumor antigen peptide comprises an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of said tumor antigen.

6. The pharmaceutical composition as claimed in claim 5, wherein the peptide is a WIC class I or class II presented peptide or can be processed to produce an MHC class I or class II presented peptide.

7. The pharmaceutical composition as claimed in claim 5, wherein the host cell expresses an WIC molecule which binds to the encoded peptide or a procession product thereof.

8. The pharmaceutical composition as claimed in claim 5, wherein the antibody is a monoclonal, chimeric, human or humanized antibody, or is a fragment of an antibody or a synthetic antibody.

9. The pharmaceutical composition as claimed in any of claims 5 to 8 which is in the form of a therapeutic or prophylactic tumor vaccine.

10. The pharmaceutical composition as claimed in any of claims 1 to 9 for use in treating or preventing a tumor disease.

11. A method of treating a patient having a tumor disease or being at risk of developing a tumor disease comprising administering a pharmaceutical composition as claimed in any of claims 1 to 10 to the patient, said administering preferably inducing or promoting CTL activity against a tumor characterized by presentation of a tumor antigen comprising an amino acid sequence encoded by a nucleic acid which comprises the nucleic acid sequence according to SEQ ID NO: 1 or 2 of the sequence listing or a variant of said nucleic acid sequence with class I MHC.

12. A method for diagnosis, detection or monitoring of a tumor disease comprising 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 peptide comprising the amino acid sequence of a tumor antigen, (ii) a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, (iii) an antibody which binds to a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, (iv) a T cell that recognizes a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen and/or (v) a cell which presents a peptide comprising the amino acid sequence of a tumor antigen peptide derived from a tumor antigen, in a biological sample isolated from a patient, wherein said tumor antigen comprises an amino acid sequence encoded by a nucleic acid which comprises the nucleic acid sequence according to SEQ ID NO: 1 or 2 of the sequence listing or a variant of said nucleic acid sequence and said tumor antigen peptide comprises an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of said tumor antigen.

13. The method as claimed in claim 12, wherein the biological sample is from a tissue or organ wherein the cells when the tissue or organ is free of tumors do not substantially express said tumor antigen and/or a nucleic acid encoding said tumor antigen.

14. The method as claimed in claim 12 or 13, wherein the detection and/or determination of the quantity comprises: (i) contacting the biological sample with an agent which binds specifically to the nucleic acid, the peptide, the antibody, the T cell or the cell which is to be detected and/or the quantity of which is to be determined, and (ii) detecting the formation of and/or determining the quantity of a complex between the agent and the nucleic acid, the peptide, the antibody, the T cell or the cell which is to be detected or the quantity of which is to be determined.

15. The method as claimed in claim 14, wherein (i) the agent which binds specifically to the nucleic acid comprises an oligonucleotide, which hybridizes specifically to said nucleic acid, (ii) the agent which binds specifically to the peptide comprises an antibody binding specifically to said peptide, (iii) the agent which binds specifically to the antibody comprises a peptide binding specifically to said antibody, or (iv) the agent which binds specifically to the T cell comprises a cell presenting a complex between an MHC molecule and the peptide comprising the amino acid sequence of a tumor antigen peptide derived from said tumor antigen.

16. The method as claimed in claim 14 or 15, wherein the agent further comprises a detectable label.

17. A diagnostic test kit which comprises an agent which binds specifically to (i) a nucleic acid which codes for a peptide comprising the amino acid sequence of a tumor antigen, (ii) a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, (iii) an antibody which binds to a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, (iv) a T cell that recognizes a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen and/or (v) a cell which presents a peptide comprising the amino acid sequence of a tumor antigen peptide derived from a tumor antigen, wherein said tumor antigen comprises an amino acid sequence encoded by a nucleic acid which comprises the nucleic acid sequence according to SEQ ID NO: 1 or 2 of the sequence listing or a variant of said nucleic acid sequence and said tumor antigen peptide comprises an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of said tumor antigen, wherein the agent preferably further comprises a detectable label.

18. An agent which binds to a peptide comprising the amino acid sequence of a tumor antigen or of a tumor antigen peptide derived from said tumor antigen, wherein said tumor antigen comprises an amino acid sequence encoded by a nucleic acid which comprises the nucleic acid sequence according to SEQ ID NO: 1 or 2 of the sequence listing or a variant of said nucleic acid sequence and said tumor antigen peptide comprises an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of said tumor antigen, wherein the agent preferably is an antibody, more preferably a monoclonal, chimeric, human or humanized antibody, a fragment of an antibody or a synthetic antibody.

19. A conjugate between an agent as claimed in claim 18 and a therapeutic effector moiety or a detectable label.

20. The pharmaceutical composition as claimed in any of claims 1 to 10, the method as claimed in any of claims 11 to 16, the diagnostic test kit as claimed in claim 17, the agent as claimed in claim 18, or the conjugate as claimed in claim 19, wherein the tumor antigen comprise the amino acid sequence according to SEQ ID NO: 3 or 4 of the sequence listing or a variant of said amino acid sequence.

21. The pharmaceutical composition as claimed in claim 10 or 20, or the method as claimed in any of claims 11 to 16 and 20, wherein the tumor disease is a tumor disease of the central nervous system.

22. The pharmaceutical composition as claimed in claim 10, 20 or 21, or the method as claimed in any of claims 11 to 16, 20 and 21, wherein the tumor disease is a glioma, in particular a glioblastoma.

Description

FIGURES

[0246] FIG. 1: Expression of COL20A1 in different tumor tissues

COL20A1 expression was analysed using quantitative real time PCR with primers 6303 (5′-TTCACGCTCTTCAAGGACGC) and 6304 (5′-TGGAAGTCCTCGGCTGTCAT) in the indicated tumors tissues and the relative expression was calculated using HPRT (Hypoxanthine-guanine phosphoribosyltransferase) as housekeeping gene. Brain tumor refers to Glioblastoma, TNBC refers to Triple negative breast cancer.

[0247] FIG. 2: Expression of COL20A1 in glioblastoma tissues

COL20A1 expression was analysed using quantitative real time PCR with primers 6303 (5′-TTCACGCTCTTCAAGGACGC) and 6304 (5′-TGGAAGTCCTCGGCTGTCAT) in 71 glioblastoma tissues and the relative expression was calculated using HPRT (Hypoxanthine-guanine phosphoribosyltransferase) as housekeeping gene.

[0248] FIG. 3: Expression of COL20A1 in normal tissues

Expression height is color coded for a number of discrete expression levels (see color code above the figure).

[0249] FIG. 4: Homology of COL20A1 to others proteins

A. Homology of COL20A1 to other collagens proteins was assessed by Blastp using as reference the NCBI Protein Reference Sequences. B. Homology of COL20A1 to COL14A1. Homology of COL20A1 to COL14A1 was assessed by Blastp showing no identical consecutive protein sequence.

[0250] FIG. 5: Predicted HLA-A*02:01 T-Cell epitopes of COL20A1

HLA-A*02:01 T-Cell epitopes were predicted using SYFPEITHI, only TCR epitopes with a score more than 20 and unique for COL20A1 are shown.

[0251] FIG. 6: COL20A1 mRNA can be translated from human cells

HEK-293 T cells were transfected with a vector containing the sequence coding for COL20A1 (lane1) or with vector alone (lane2).

EXAMPLES

[0252] The techniques and methods used herein are described herein or carried out in a manner known per se and as 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 unless specifically indicated.

Example 1: COL20A1 is a Highly Specific Biomarker for Glioblastoma

[0253] To verify the specificity of COL20A1 for glioblastoma, RNA was extracted from normal tissues or glioblastoma samples using an extraction kit for lipid-rich tissues. cDNA synthesis was performed using MMLV Reverse Transcriptase and random timers/oligo dT primers. COL20A1 expression was analysed using quantitative real time PCR with primers 6303 (5′-TTCACGCTCTTCAAGGACGC) and 6304 (5′-TGGAAGTCCTCGGCTGTCAT) and the relative expression was calculated using HPRT (Hypoxanthine-guanine phosphoribosyltransferase) as housekeeping gene.

[0254] This method is used for analysis the expression of COL20A1 in 13 different tumour entities (FIG. 1). Only glioblastoma (labeled as brain tumors in FIG. 1) show a strong expression of COL20A1, while all other tumour entities show no or very low expression of COL20A1, with the exception of few samples in testis cancer. As shown in FIG. 2, the transcript COL20A1 (NM_020882.2, NP_065933.2) is highly expressed in 50% of analysed glioblastomas if a cut off of 40000 is used, suggesting that a large number of GBM patients would benefit from a treatment targeting COL20A1. The tumor specificity of the transcript was analysed by said qRT-PCR protocol in a large set of normal tissues (n=65). COL20A1 is weekly expressed in some adult brain regions and spinal cord (FIG. 3). In contrast, ERBB2, a generally accepted target of vaccination and other immunotherapeutic approaches, shows a high expression (relative expression more than 40000) in several normal tissues, including some brain areals (FIG. 3).

Example 2: Low Homology Limits Expected Cross Reactivity of Other Collagen Family Members

[0255] The human COL20A1 open reading frame encodes a 1284 amino acid protein with a predicted molecular weight of 136 kDa. A 22 amino acid signal sequence is predicted. Homology of COL20A1 to other collagen proteins was assessed by Blast showing that the overall homology to other collagens is low, with the highest alignment score found with the collagen alpha-1(XIV) (COL14A1) and collagen alpha-1(XII) (COL12A1) (FIG. 4A). Importantly, no long stretches of amino acids are identical between COL20A1 and other collagens as showed in FIG. 4B for COL14A1 suggesting that COL20A1 can therefore be used for specific targeting and detection of the molecule (FIG. 4B).

Example 3: COL20A1 Specific Sequences Contain Predicted T Cell Epitopes

[0256] To address the targetability of COL20A1 by CD8+T-lymphocytes, HLA-A*02:01 epitopes present on the sequence were predicted using the SYFPEITHI database. 58 HLA-A*02:01 epitopes unique for COL20A1 were detected (FIG. 5) suggesting that COL20A1 could be used for T-cell based therapy.

Example 4: COL20A1 mRNA can be Translated from Human Cells

[0257] A recombinant sequence coding for COL20A1 was transfected into HEK-293 T cells and cell lysates were analysed by Western Blot with a commercial available COL20A1 specific antibody (Sigma). A band of the expected COL20A1 size can be detected in the transfected cells but not in the mock transfected cells indicating that the COL20A1 mRNA can be translated to a stable protein in human cells (FIG. 6).