CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/104,550, filed on October 10, 2008, the entire content of which is hereby incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

[0002] The present invention generally relates to the fields of immunology and more specifically to the identification of antigens that trigger a pathogenic T-cell response in autoimmune disease and malignant disease or of potential target antigens in immunotherapy of tumors and other diseases.

BACKGROUND OF THE INVENTION

[0003] Identification of antigens that trigger pathogenic T-cell responses in autoimmune disease or of potential target antigens in immunotherapy of tumors is still a major challenge of basic and translational immunology. In autoimmune disease a special difficulty arises from the fact that, while the affected organ or tissue is well known, the respective targeT-cell types within this tissue often remain undefined. In malignant diseases, increasing evidence suggests that successful immunotherapies need to address antigens possessing an endogenous immunogenic potential and expressed in tumor tissues at sufficient concentrations to trigger complex T-cell responses composed of T-helper cells as well as of cytotoxic T-cells. Even within the same tumor type, the intraindividual repertoire of immunogenic tumor-associated antigens (TAAs) varies markedly (Lennerz et al, 2005, Proc Natl Acad Sci USA 102:16013- 16018; Sommerfeldt et al, 2006, Cancer Res 66:8258-8265). Accordingly, future cancer immunotherapy aims to individualize therapy by identifying patient-specific TAAs and epitopes (Celis et al, 1994, MoI Immunol 31 :1423-1430). [0004] To identify relevant TAAs for cancer vaccines various complex approaches have been developed. For example, elution of MHC-I-bound peptides from tumor cells and subsequent identification of corresponding TAAs by mass spectroscopy allows for identification of antigen epitopes being processed and presented by tumor cells to the immune system (Celis et al., 1994, MoI Immunol 31 :1423-1430; Cox et al, 1994, Science 264:716-719; Kramer et al, 2005, Cancer Biol Ther 4:943-948; Ramakrishna et al, 2003, Int Immunol 15:751-763; Schirle et al, 2000, Eur J Immunol 30:2216-2255; Weinschenk et al, 2002, Cancer Res 62:5818-5827). However, this method does not answer, if such epitopes are recognized by a patient's immune system and does not address MHC-II-restricted epitopes playing a major role in efficient tumor-immune-rejection and in autoimmune disease.

[0005] In contrast, the SEREX (serological analysis of recombinant cDNA expression libraries) method is based on serological screening for tumor-specific antibodies using cDNA libraries derived from human cancers (Sahin et al, 1997, Curr Opin Immunol 9:709-716; Sahin et α/., 1995, Proc Natl Acad Sd USA 92:11810-11813). Even with this analysis, it remains unclear if the identified targets of humoral immune responses also represent target antigens of CD4 ⁺ or CD8 ⁺ T-cell responses. Analogously, cDNA libraries of tumor cells were used to identify MHC-I-restricted epitopes of tumor antigens recognized by CD8 ⁺ T- cells from individual cancer patients (Lennerz et al, 2005, Proc Natl Acad Sd USA 102 : 16013 - 16018) . These methods are either indirect or restricted to selected HLA-I subtypes and represent complicated, extremely time-consuming and costly procedures.

[0006] In this regard, Applicants have investigated whether the ProteomeLab PF2D technology might represent a time-saving method enabling an individualization of patient- specific TAA analysis. This PF2D method, a two-dimensional chromatography, represents a potent tool to separate proteins of tumor proteomes and an alternative method to two- dimensional gel electrophoresis (Sheng et al, 2006, MoI Cell Proteomics 5:26-34). Fractioning by the PF2D system is based on pi and hydrophobicity of processed proteins and provides high reproducibility (Sheng et al, 2006, MoI Cell Proteomics 5:26-34; Soldi et al, 2005, Proteomics 5:2641-2647). Resulting separated matrix-free proteins are extracted as fluids and, therefore, are immediately accessible for further functional analysis, e.g. for natural processing by antigen-presenting cells such as dendritic cells (DCs).

[0007] PF2D has been applied in several studies dealing with different research topics. Most recently, the group of Lee et al. reported the establishment of a combination of PF2D with MS for rapid profiling and semiquantitative analysis of membrane protein biomarkers followed by its application for quantitative proteomic analysis of hepatocellular carcinoma. This system proved to be very efficient and versatile analytical tool for both large-scale profiling and quantification of phosphoproteins in disease biomarker discovery (Lee et al, 2008, Proteomics 8: 3371-81 ; Lee et al, 2008, Proteomics 8:2168-77). An independent study by Chahal et al reported identification of tumor- associated membrane antigens employing immunoprecipitation prior to separation with PF2D followed by MS for protein identification (Chahal et al, 2006, Biochem Biophys Res Commun 348:1055-62). ProteomeLab PF2D has been predominantly used for identifying differentially expressed proteins (Billecke et al, 2006, MoI Cell Proteomics 5:35-42; Lee et al, 2008, Proteomics, 8:3371-81; Ruelle et al, 2007, J Proteome Res, 6:2168-75; Gunther et al, 2006, J Ind Microbiol Biotechnol 33:914-20) or checking protein arrays for quality and performance (Schabacker et al, 2006, Anal Biochem 359:84-93) in various other studies dealing with proteomic profiling of bacterial proteins (Ruelle et al, 2007, J Proteome Res, 6:2168-75; Gunther et al, 2006, J Ind Microbiol Biotechnol 33:914-20), plant proteins (Pirondini et al., 2006, J Chromatogr B Analyt Technol Biomed Life Sci 833:91-100), or human serum proteins (Sheng et al, 2006; MoI Cell Proteomics 5:26-34; Levreri et al, 2005, Clin Chem Lab Med 43:1327-33). [0008] T-cell activation by antigen-pulsed DCs is based on a complex and tightly regulated process involving multiple steps such as protein uptake, cleavage into small peptide fragments, their loading into the peptide binding cleft of MHC-I and -II molecules and their presentation at the cell surface to antigen-specific T-cells. However, since PF2D technology so far was predominantly used for identifying differentially expressed proteins (Gunther et al, 2006, J Ind Microbiol Biotechnol 33:914-920; Lee et al, 2008, Proteomics 8:2168-2177; Ruelle et al, 2007, J Proteome Res 6:2168-2175), validation of protein arrays (Schabacker et al, 2006, Anal Biochem 359:84-93), hitherto it was unknown whether proteins subjected to PF2D fractionation under partially denaturing conditions, maintain features required for appropriate antigen-processing by DCs. Further, hitherto it was unclear whether concentrations of respective proteins in the eluates are sufficient for successful presentation to CD4 ⁺ and CD8 ⁺ T-cells.

[0009] To investigate preservation of immunogenicity during PF2D separation procedure, Applicants herein tested protein samples, such as samples comprising ovalbumin (OVA) processed in the PF2D system by T-cell activation assays and in a next step identified immunogenic proteins from lysates of malignant brain tumors. Applicants demonstrated herein that PF2D-separated proteins are efficiently cross-presented to OVA-specific naϊve CD8 ⁺ T-cells and that this method can also be successfully used for the identification of novel human TAAs spontaneously recognized by CD4 ⁺ and CD8 ⁺ memory T-cells. BRIEF SUMMARY OF THE INVENTION

[0010] In some aspects of the present invention, Applicants provide methods and compositions for the identification of tissue antigens which cause T-cell responses in various disease conditions. [0011] The fast and inexpensive methods described herein are suitable to identify T-cell antigens in various diseases, such as autoimmune and malignant diseases, without restriction to their expression by a certain cell type or HLA-allele.

[0012] While the present invention may be embodied in many different forms, several specific embodiments are discussed herein with the understanding that the present disclosure is to be considered only as an exemplification of the principles of the invention, and is not intended to limit the invention to the embodiments illustrated.

[0013] The present invention provides methods for identifying a T-cell immunogenic antigen expressed by a cell in a subject. In some embodiments, this method comprises the steps of (a) obtaining a sample comprising a plurality of proteins expressed in a cell from a subject, (b) contacting the plurality of proteins of the sample with a CD4 ⁺ T-cell or a CD8 ⁺ T- cell, and (c) determining whether the CD4 ⁺ T-cell or CD8 ⁺ T-cell binds to a member of the plurality of proteins in the sample. Thereby the T-cell immunogenic antigen is identified.

[0014] In some embodiments, the T-cell immunogenic antigen is an antigen which causes activation of a T-cell. In some embodiments, the T-cell immunogenic antigen is an autologous T-cell immunogenic antigen. In other embodiments, the T-cell immunogenic antigen is an allogeneic T-cell immunogenic antigen.

[0015] Various samples can be used in the method of the present invention. In some embodiments, the sample is a cell lysate. In some embodiments, the lysate is from a tumor cell, a diseased cell, a tumor tissue, or a diseased tissue. [0016] In some embodiments, the plurality of cells are fractionated prior to performing step (b). In some embodiments, fractionation comprises two-dimensional chromatography. In some embodiments, fractionation is based on pi and hydrophobicity of the plurality of proteins. In some embodiments, fractionation is performed by a PF2D system.

[0017] In some embodiments, prior to step (b) of the method for identifying a T-cell immunogenic antigen, the plurality of proteins is pulsed onto an antigen-presenting cell. In some embodiments, the antigen-presenting cell is a dendritic cell. In some embodiments, the dendritic cell is an autologous dendritic cell. In other embodiments, the dendritic cell is induced to mature.

[0018] In some embodiments, step (b) of the method for identifying a T-cell immunogenic antigen comprises coculturing the antigen-presenting cell and the CD4 ⁺ T-cell or the CD8 ⁺ T- cell.

[0019] In some embodiments, the method for identifying a T-cell immunogenic antigen expressed by a cell in a subject comprises obtaining the CD4 ⁺ T-cell or a CD8 ⁺ T-cell from a subject. In some embodiments, the subject has a disease. The disease may be a malignant disease, such as cancer. In some embodiments, the cancer is a hematologic cancer. In other embodiments, the cancer is a non-hematologic cancer. In some embodiments, a non- hematologic cancer is selected from the group consisting of lung cancer, sarcoma, gastrointestinal cancer, cancer of the genitourinary tract, liver cancer, skin cancer, gynecological cancer, bone cancer, cancer of the nervous system, and cancer of adrenal glands. In some embodiments, a non-hematologic cancer is brain cancer. In other embodiments, the disease is an autoimmune disease. In some embodiments, the autoimmune disease is characterized by expressing a disease-associated tissue antigen.

[0020] In some embodiments, the CD4 ⁺ T-cell or the CD8 ⁺ T-cell is in a blood sample obtained from the subject. In other embodiments, the CD4 ⁺ T-cell or the CD8 ⁺ T-cell expresses a binding partner on its cell surface specific for a member of the plurality of proteins in the sample.

[0021] In some embodiments, step (c) of the method for identifying a T-cell immunogenic antigen comprises a T-cell activation assay. In some embodiments, the T-cell activation assay comprises flowcytometry. In some embodiments, flowcytometry comprises using an antibody selected from the group consisting of an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD69 antibody, an anti-CD 107a antibody, an anti-CD25 antibody, an anti-CD40L. In other embodiments, the T-cell activation assay comprises measuring dilution of intracellular carboxyfluorescein succinimidyl ester (CFSE). In some embodiments, the T-cell activation assay comprises measuring calcium influx. [0022] In some embodiments, step (c) of the method for identifying a T-cell immunogenic antigen comprises an IFN-γ assay, an assay for measuring interleukin-4 secretion, an assay for measuring interleukin-10 secretion, an assay for measuring TNF-α secretion, an assay for measuring TNF-βl secretion, or an assay for measuring perforin secretion.

[0023] In some embodiments, step (c) of the method for identifying a T-cell immunogenic antigen comprises measuring of cytotoxic activity. [0024] In some embodiments, methods of the present invention comprise the step of identifying the member of the plurality of proteins bound by the CD4 ⁺ T-cell or CD8 ⁺ T-cell. In some embodiments, this is performed by mass spectroscopy.

[0025] The present invention also provides methods for identifying a patient-derived T-cell binding to a tumor-associated antigen. In some embodiments, this method comprises the steps of (a) obtaining a plurality of T-cells from a subject, (b) contacting the plurality of T- cells with a tumor-associated antigen pulsed on an antigen-presenting cell; and (c) determining if a member of the plurality of T-cells binds to the tumor-associated antigen.

[0026] In some embodiment, the tumor-associated antigen is a recombinant polypeptide or a fragment thereof. In other embodiments, the tumor-associated antigen is a synthetic polypeptide.

[0027] In some embodiments, the plurality of T-cells comprises a plurality of CD4 ⁺ T-cells or a plurality of CD8 ⁺ T-cells.

[0028] The present invention also provides methods for pulsing polypeptides onto an antigen-presenting cell. In some embodiments, this method comprises the steps of (a) obtaining a cell lysate comprising polypeptides, (b) separating the polypeptides of the cell lysate in a first dimension, (c) collecting a first fraction of polypeptides, (d) separating the first fraction of the polypeptides in a second dimension, (e) collecting a second fraction of the polypeptides, and (f) pulsing the polypeptides of the second fraction onto an antigen- presenting cell. [0029] The present invention also provides for use of a tumor-associated antigen identified by a method of the present invention for the manufacture of a cancer vaccine.

[0030] Further, the present invention provides for the use of an antigen-presenting cell prepared according to a method of the present invention in a method to reactivate a preexisting memory CD4 ⁺ or CD8 ⁺ T-cell response in a subject. [0031] The present invention also provides for methods for identifying a cancer in a subject. In some embodiments, this method comprising the step of detecting in a sample obtained from a subject an antigen selected from the group consisting of transthyretin and calprotectin S100A9. [0032] Some embodiments of the present invention are set forth in claim format directly below:

1. A method for identifying a T-cell immunogenic antigen expressed by a cell in a subject, the method comprising the steps of:

(a) obtaining a sample comprising a plurality of proteins expressed in a cell from a subject;

(b) contacting the plurality of proteins of the sample with a CD4 ⁺ T-cell or a CD8 ⁺ T-cell; and

(c) determining whether the CD4 ⁺ T-cell or CD8 ⁺ T-cell binds to a member of the plurality of proteins of the sample; whereby the T-cell immunogenic antigen is identified.

2. The method according to claim 1, wherein the T-cell immunogenic antigen is an autologous T-cell immunogenic antigen or an allogeneic T-cell immunogenic antigen.

3. The method according to any one of claims 1 or 2, wherein the sample is a cell lysate. 4. The method according to claim 3, wherein the cell lysate is from a tumor cell, a diseased cell, a tumor tissue, or a diseased tissue.

5. The method according to any one of claims 1 to 4, wherein the plurality of proteins are fractionated prior to performing step (b).

6. The method according to claim 5, wherein fractionation comprises two-dimensional chromatography.

7. The method according to claim 5, wherein fractionation is based on pi and hydrophobicity of the plurality of proteins.

8. The method according to claim 5, wherein fractionation is performed by a PF2D system. 9. The method according to any one of claims 1 to 8, wherein prior to step (b), the plurality of proteins is pulsed onto an antigen-presenting cell.

10. The method according to claim 9, wherein the antigen-presenting cell is a dendritic cell. 11. The method according to claim 10, wherein the dendritic cell is an autologous dendritic cell.

12. The method according to claim 10, wherein the dendritic cell is induced to mature.

13. The method according to any one of claims 9 to 12, wherein step (b) comprises coculturing the antigen-presenting cell and the CD4 ⁺ T-cell or the CD8 ⁺ T-cell. 14. The method according to any one of claims 1 to 13, further comprising the step of obtaining the cell from the subject.

15. The method according to claim 14, wherein the CD4 ⁺ T-cell or a CD8 ⁺ T-cell is from a subject having a disease.

16. The method according to claim 15, wherein the disease is a malignant disease. 17. The method according to claim 16, wherein the malignant disease is cancer.

18. The method according to claim 17, wherein the cancer is a hematologic cancer.

19. The method according to claim 17, wherein the cancer is a non-hematologic cancer.

20. The method according to claim 19, wherein the non-hematologic cancer is selected from the group consisting of lung cancer, sarcoma, gastrointestinal cancer, cancer of the genitourinary tract, liver cancer, skin cancer, gynecological cancer, bone cancer, cancer of the nervous system, and cancer of adrenal glands.

21. The method according to claim 19, wherein the non-hematologic cancer is brain cancer.

22. The method according to claim 15, wherein the disease is an autoimmune disease. 23. The method according to claim 22, wherein the autoimmune disease is characterized by expressing a disease-associated tissue antigen.

24. The method according to any one of claims 1 to 23, wherein the CD4 ⁺ T-cell or the CD8 ⁺ T-cell is in a blood sample obtained from the subject. 25. The method according to any one claims 1 to 24, wherein the CD4 ⁺ T-cell or the CD8 ⁺ T-cell expresses a binding partner on its cell surface specific for a member of the plurality of proteins in the sample.

26. The method according to any one of claims 1 to 25, wherein step (c) comprises a T- cell activation assay.

27. The method according to claim 26, wherein the T-cell activation assay comprises: (i) flowcytometry;

(ii) measuring dilution of intracellular carboxyfluorescein succinimidyl ester (CFSE); or (iii) measuring calcium influx.

28. The method according to claim 27, wherein flowcytometry comprises using an antibody selected from the group consisting of an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD69 antibody, an anti-CD 107a antibody, an anti-CD25 antibody, and an anti-CD40L antibody. 29. The method according to any of claims 1 to 25, wherein step (c) comprises an IFN-γ assay, an assay for measuring interleukin-4 secretion, an assay for measuring interleukin-10 secretion, an assay for measuring TNF-α secretion, an assay for measuring TNF-βl secretion, an assay for measuring perforin secretion.

30. The method according to any one of claims 1 to 25, wherein step (c) comprises measuring of cytotoxic activity.

31. The method according to any one of claims 1 to 30, further comprising the step of: (d) identifying the member of the plurality of proteins bound by the CD4 ⁺ T-cell or CD8 ⁺ T-cell.

32. The method according to claim 31 , wherein step (d) is performed by mass spectroscopy.

33. A method for identifying a patient-derived T-cell binding to a tumor-associated antigen, the method comprising the steps of:

(a) obtaining a plurality of T-cells from a subject;

(b) contacting the plurality of T-cells with a tumor- associated antigen pulsed onto an antigen-presenting cell; and (c) determining if a member of the plurality of T-cells binds to the tumor- associated antigen.

34. The method according to claim 33, wherein the tumor-associated antigen is a recombinant polypeptide or a fragment thereof. 35. The method according to claim 33, wherein the tumor-associated antigen is a synthetic polypeptide.

36. The method according to claim 33, wherein the plurality of T-cells comprises a plurality of CD4 ⁺ T-cells or a plurality of CD8 ⁺ T-cells.

37. Use of a tumor- associated antigen identified by the method of any one of claims claim 1 to 36, for the manufacture of a cancer vaccine.

38. Use of an antigen-presenting cell onto which a plurality of proteins has been pulsed according to any one of claims 9 to 36, in a method to reactivate a preexisting memory CD4+ or CD8+ T-cell response in a subject.

39. A method for pulsing polypeptides onto an antigen-presenting cell, the method comprising the steps of:

(a) obtaining a cell lysate comprising polypeptides;

(b) separating said polypeptides of said cell lysate in a first dimension;

(c) collecting a first fraction of said polypeptides;

(d) separating said first fraction of said polypeptides in a second dimension; (e) collection a second fraction of said polypeptides; and

(f) pulsing said polypeptides of said second fraction onto an antigen-presenting cell.

40. Use of an antigen-presenting cell prepared according to claim 39, in a method to reactivate a preexisting memory CD4 ⁺ or CD8 ⁺ T-cell response in a subject. 41. A method for identifying a cancer in a subject, said method comprising the step of detecting in a sample obtained from said subject an antigen selected from the group consisting of transthyretin and calprotectin S100A9.

[0033] In some embodiments of the present invention, Applicants used an automated two- dimensional chromatography system, referred to as PF2D system, to fractionate the proteome of tumor tissues and tested protein fractions for recognition by pre-existing rumor-specific CD4 ⁺ T-helper cells and cytotoxic T-cells. Applying methods of the present invention to the ovalbumin (OVA)-specific, TCRtg OT-I mouse model, efficient separation, processing and cross-presentation to CD8 ⁺ T-cells by dendritic cells of OVA expressed by the Ova- transfected mouse lymphoma RMA-OVA was demonstrated. [0034] Using the methods of the present invention to identify immunogenic antigens in human tumor tissues Applicants demonstrated that MUCl -reactive T-cells from head and neck cancer patients selectively recognized PF2D-separated protein fractions that contained MUCl -protein.

BRIEF DESCRIPTION OF THE DRAWINGS [0035] Figure 1 depicts fractionation, detection and cross-presentation of recombinant ovalbumin (OVA) and OVA from OVA-overexpressing cells, (a) ID profile of 1 mg OVA. Black area corresponds to fraction 16. (b) 2D profile of fraction 16 of recombinant OVA; AU, absorbance unit. Main peak (P) was eluted in well 32. (c) Confirmation of OVA protein in well 32. (d) Differential expression map of 2D separation of fraction 16 of RMA-OVA and RMA cells. Among others, a peak at RT = 24.4 min was detected in RMA-OVA but not in RJVIA fraction and eluted to well 33. (e) Confirmation of OVA protein in well 33 of RMA- OVA cells only, (c, e) PBS served as negative and recombinant OVA as positive control (Cl = 0.5 ng, C2 = 5 ng). (f) DCs from C57BL-6 mice pulsed with fractionated recombinant OVA (W32, Fig. Ib) were used for stimulation of OVA-specific T-cells. T-cells stained with CD8- and CD69-PE to evaluate proportion of early-activated T-cells by flowcytometry showed a dose-dependent OVA-specific T-cell activation. Mean +SEM proportions of T- cells detected in triplicate wells are shown. One representative out of three independent experiments is shown, (g)-(h) DCs pulsed with proteins derived from OVA-positive 2D fraction of RMA-OVA or respective fraction from RMA cells (W33, (e)). (g) T-cell activation as determined by flowcytometry with anti-CD69 antibodies. One representative out of three independent experiments is shown, (h) Proliferative activity of purified CD8 ⁺ OVA-specific T-cells after activation with DCs loaded with fractions W33 from RMA-Ova or RMA cells or after activation with unpulsed DCs as determined by thymidine incorporation. Means +SD of two independent experiments are shown. Asterisks depict significant p-values. Further details are described in Example 2.

[0036] Figure 2 depicts identification OfMUCl ⁺ protein fractions by MUCl -reactive T- cells from patients with head and neck cancer, (a) T-cell reactivity of patient HNl against DCs pulsed with synthetic, MUCl -derived long peptides from the signaling sequence (MUC lss) or the tandem repeat region (MUC ltr) as revealed by IFN-γ ELISPOT-assay. Black bars indicate significantly increased IFN-γ spot numbers; grey bars indicate nonsignificant response; white bars indicate negative control containing hulgG-pulsed DCs. (b) T-cell reactivity of HNl against DCs pulsed with ID fraction A but not B-F of the patient's autologous tumor. Black bars indicate significantly increased IFN-γ spot numbers; grey bars indicate non-significant response; white bars indicate negative controls containing DCs only, TCs only, TCs together with unpulsed DCs or TCs cocultured with hulgG-pulsed DCs. (c) Detection of MUCl protein in PF2D fraction A by IP. (d) T-cell reactivity of patient HN2 against TU-L-pulsed DCs as revealed by IFN-γ ELISPOT-assay. Black bars indicate significantly increased IFN-γ spot numbers, white bars indicate negative controls containing DCs pulsed with autologous PBMC lysate (PB-L). (e) T-cell reactivity of HN2 against DCs pulsed with ID fractions (A-M) of the patient's autologous tumor. Black bars indicate significantly increased IFN-γ spot numbers; grey bars indicate non-significant response; white bars indicate negative controls containing DCs only, TCs only or TCs cocultured with hulgG-pulsed DCs. Corresponding results of MUCl protein detection by MUCl -specific IP are indicated for each PF2D fraction below ("+" = MUCl -positive, "-" = no detection of MUCl). MUCl was only detectable in fraction A. (f) T-cell reactivity of patient HN2 against DCs pulsed with synthetic, MUCl -derived long peptide (tandem repeat region, (MUC ltr) as revealed by IFN-γ ELISPOT-assay. Black bars indicate significantly increased IFN-γ spot numbers; white bars indicate negative control containing hulgG-pulsed DC. Further details are described in Example 3.

[0037] Figure 3 depicts recognition of autologous tumor protein fractions by T-cells from brain tumor patient NCH550 and identification of candidate target antigens, (a) DCs from brain tumor patient NCH550 pulsed with tumor cell lysate (TU-L) or autologous PBMC lysate (PB-L) as negative control were used for stimulation of blood-derived T-cells from the same patient in IFN-γ ELISPOT-assay. Additionally, purified TU-L-pulsed DCs or unstimulated PB-derived TCs served as controls. PBTCs (referred to as "Tu-L" in Figure 2a) of NCH550 reacted significantly against autologous tumor-derived antigens, (b) T-cell reactivity of NCH550 against ID separated protein fractions (F10-F21) as revealed by IFN-γ ELISPOT-assay. Black bars indicate significantly increased IFN-γ spot numbers (asterisks indicate significant p-values as follows: FlO = 0.04; F13 = 0.03; F14 = 0.05; F15 = 0.01; F18 = 0.01; F19 = 0.02; F21 = 0.002); grey bars indicate non-significant T-cell responses; open bars indicate TC and PB-L controls (two-sided T-test). (c) ID fractions recognized by patient's T-cells (FlO, 13-15, 18-19, 21) subjected to 2D separation. Obtained subtractions were tested for recognition by autologous T-cells in IFN-γ ELISPOT-assay. Black bars indicate significantly increased IFN-γ spot numbers (asterisks indicate significant p- values which were 0.01 for each); grey bars indicate non-significant T-cell responses; open bars indicate PB-L control, (d) For validation, whole tumor lysate was subjected a second time, to two-dimensional PF2D protein separation, and subtractions being recognized before (Fig. Ic; FlOe, F14i,j, F18c,j, F21c; indicated by asterisks) were tested again for recognition by autologous T-cells. Black bars indicate significantly increased IFN-γ spot numbers compared to wells containing DCs pulsed with autologous PB-L (asterisks indicate significant p-values as follows: FlOe = 0.01; FHi = 0.001; F14j = 0.05; Fl 8c = 0.02); grey bars indicate nonsignificant T-cell responses; open bars indicate PB-L control. The experiment confirmed T- cell reactivity against sub fractions FlOe, F14i, F14j and Fl 8c. (a)-(d) Asterisks depict significant p-values of two-sided unpaired Student's T-test, respectively. Mean +SEM spot numbers of 3-5 wells/test group are shown, (e) Proteins present in recognized subtractions as identified by mass spectroscopy are shown, respectively. Further details are described in Example 3.

[0038] Figure 4 depicts expression of potentially immunogenic proteins in tumor tissue of patient NCH550. (a) In a first step, expression of identified antigens was validated by RT- PCR. Total RNA was isolated from tumor tissue and respective positive controls: HepG2, cell line for TTR; HNO41, tonsil carcinoma for desmoglein-1 (DSG-I); S100A8 and S100A9, and normal skin tissue for dermcidine (DCD) and hornerin (HRNR). Expression of TTR, DSG-I, calprotectin subunits S100A8 and S100A9, as well as DCD could be confirmed in the patient's tumor tissue (NCH550), while HRNR mRNA was only detected in the positive control. MlOO is a size marker, (b) TTR (top 2 panels), calprotectin/S 100 A9 (panels 3 and 4 from top), DSG-I (panels 5 and 6 from top), and DCD (lower two panels) were further tested for their protein expression by double immunofluorescence staining in order to identify their cellular distribution pattern in the patient's tumor tissue. Tumor cells were labeled with anti-GF AP antibodies, endothelial cells with anti-CD31 antibodies and nuclei with DAPI. While TTR protein was solely found on a subpopulation of GF AP -positive tumor cells, calprotectin/S 100 A9 and DSG-I were detected on both on endothelial as well as on few tumor cells. In contrast, DCD expression was predominantly seen on CD31-postive endothelial cells rather than on tumor cells. Scale bar is 50 μm. Magnification is 4Ox. Further details are described in Example 4.

[0039] Figure 5 depicts TTR as a target antigen of autologous T-cells from tumor patient NCH550. (a) Molecular structure and amino acid sequence (AAS) of TTR and TTR precursor (protein-ID P02766, UniProtKB/Swiss-Prot). Black lines indicate synthetic polypeptides, TTR _precu i _so i ₅ TTRi, TTR ₂ , TTR ₃ , TTR ₄ used for T-cell stimulation. Amino acid residues within these synthetic peptides are shown above the black lines and are also indicated by numbers. A schematic tertiary structure of the protein is indicated by arrows (β- strand regions), wavy lines (depicting regions of variable folding) and thin lines (depicting strand sequences that link tightly folded β-strand regions). Arrows point to 5H end. (b) Recognition of synthetic polypeptides by NCH550 T-cells in IFN-γ ELISPOT-assay. Autologous PB-L, human IgG, TCs, DCs are used as negative controls. DCs pulsed by TTR ₃ resulted in significantly increased IFN-γ spot numbers (asterisks indicate significant p- values as follows: Tu-L/CD3 = 0.03; TIR ₃ /CD3 = 0.002; Tu-L/CD8 = 0.002; TIR ₃ /CD8 = O.0001 ; TIR ₃ /CD4 = 0.002). (c) Significant reactivity of total (CD3), CD8 ⁺ and CD4 ⁺ T-cells against DCs pulsed with either total autologous tumor lysate or with synthetic TTR ₃ . White bars labeled "CD3", "CD8" or "CD4" indicate unstimulated subpopulations of respective T-cells; grey bars and black bars, see Fig. 2c. (d) Amino acid sequence of the immunogenic region of TTR as revealed by IFN-γ ELISPOT-assays using synthetic polypeptides, (e) Amino acid sequence of predicted epitopes potentially presented by different HLA-I molecules of

NCH550 (HLA-AOl 01 -restricted epitopes, 1-3; HLA-A0201 -restricted epitopes, 4-13; HLA- B4101, 15 and 16; HLA-B5101 , 14). Asterisks indicate epitopes which may be presented also by other HLA-I molecules, (f) Recognition of HLA-I-restricted peptides (see (e)). DCs pulsed with autologous PB-L, human IgG, HLA-A0201 -restricted peptide from HIVgag were used as negative control antigens. Bars: see Fig. 2c. (c), (f) Asterisks depict significant p- values. Mean +SEM spot numbers of 3-5 wells/group are shown, (g) Amino acid sequence of identified immunogenic target epitopes of TTR restricted to HLA-AOlOl, HLA-A0201, and HLA-B4101 as recognized by T-cells from patient NCH550 are indicated by black lines. Numbers next to the lines correspond to peptide numbers as shown in (e)-(f). Further details are described in Example 5.

[0040] Figure 6 depicts identification of immunogenic antigens and shows that calprotectin but not desmoglein-1 or dermcidine is a target antigen of autologous T-cells. (a) Molecular structure and amino acid sequence of calprotectin subunits S100A8 (S100A8/L1L subunit; protein-ID P05109, UniProtKB/Swiss-Prot), S100A9 (S100A9/L1H subunit; protein-ID P06702, UniProtKB/Swiss-Prot) and shorter variants thereof as indicated (C-chain; protein- ID HRJ, Protein-Data-Bank). Black lines indicate the amino acid sequence and length of synthetic polypeptides used for T-cell stimulation. A schematic tertiary structure of the protein is indicated by arrows (β-strand regions), wavy lines (depicting regions of variable folding) and thin lines (depicting strand sequences that link tightly folded β-strand regions). (b) Significant recognition of S100A9 C-chain (C-ch; p = 0.002), peptide-1 (A9i; p = 0.004), and peptide-2 (A9 ₂ ; p = 0.0006) by NCH550 T-cells. Controls: see Fig. 5b. (c) Scheme and amino acid sequences of immunogenic region of S100A9 as revealed by IFN-γ ELISPOT- assays using synthetic polypeptides (a)-(b). A9 ₂ , C-chain, and A9i showed significant recognition ("pos")- Synthetic peptides 1-4 contain overlapping 20mer peptides used for further characterization of immunogenic epitopes. Peptide 1 included amino acid residues 11-30 of A9 ₂ ; peptide 2 included amino acid residues 21-40 of A9 ₂ ; peptide 3 included amino acid residues 31-50 of A9 ₂ ; and peptide 4 included amino acid residues 41-60 of A9 ₂ . (d) Significant recognition of peptide-1 by NCH550 T-cells ("1 "; p = 0,002). Controls: see Fig. 5f. The amino acid sequence of the immunogenic region is depicted beneath the figure, (e) Significant reactivity of total (CD3), CD8 ⁺ and CD4 ⁺ T-cells against DCs pulsed with total autologous Tu-L or peptide-1 ("1") is indicated by asterisks(Tu-L/CD3 = 0.03; 1/CD3 = 0.01; Tu-L/CD8 = 0.002; 1/CD8 = O.0001; 1/CD4 = <0.0001). Open bars indicate labeled "CD3" and "CD8" or "CD4" contain unstimulated subpopulations of respective T-cells. Mean +SEM spot numbers of 3-5 wells/group are shown, (f)-(h) Reactivity of NCH550 T-cells against DCs pulsed with synthetic polypeptides DSG ₆ I-Io ₀ and DSG ₂ 11-245 (DSG-I i _-2 , respectively; protein-ID Q02413, UniProtKB/Swiss-Prot) spanning two described immunogenic regions of desmoglein-1 (f)-(h) or with DCD _1-40 , DCD _{3 ]-70} and DCD ₆ I _{-1 i0} (DCDi _-3 , respectively) of dermcidine (protein-ID P81605, UniProtKB/Swiss-Prot) (g)-(h) by NCH550 T-cells. Controls: see Fig. 5b. (h) Validation of negative results; polypeptide TTR ₃ was used as a positive control (p = 0,0002). Tests were performed in three independent experiments at three different time points. In (b), (d), (e), and (h), asterisks depict significant p-values. Mean +SEM spot numbers of 3-5 wells/group are shown. Further details are described in Example 6.

[0041] Figure 7 depicts that TTR and calprotectin are frequently recognized in patients with brain tumors and can be recognized by T-cells on antigen-expressing tumor cells, (a) DCs from additional 10 different brain tumor patients (NCH) were pulsed with synthetic peptides derived from TTR _10I - _I47 (TTR), calprotectin/S100A9i _-60 (S100A9), as test antigens or with human IgG as control antigens at a concentration of 200 μg/ml and used for stimulation of blood derived T-cells from the same patients in triplicate wells in IFN-γ ELISPOT assays. Bars represent mean +SEM IFN-γ spots per well. Significant T-cell reactivity in test wells compared to control wells is demonstrated by black bars. Numbers above bars depict exact p- values compared to control wells (two sided Student's T- test). Patients arte identified as NCH654, NCH655, NCH656, NCH656b, NCH656c, NCH657, NCH658, NCH660d, NCH660h, and NCH662a. (b, c) Response of T-cells from NCH656b against calprotectin S100A9 and transthyretin expressed by COS7 tumor cells. COS7 cells were transfected with calprotectin S100A9 (a) or transthyretin (b) either alone (C- or TTR-, respectively) which served as negative controls (open bars) or together with respective HLA-I molecules expressed by NCH550 (test groups; black and grey bars). Transfected COS cells were co-cultured with T-cells from NCH656b and T-cell reactivity was evaluated by IFN-γ ELISPOT-assay. IFN-γ spot numbers secreted by T-cells were calculated by subtracting background spots derived from transfected COS7 cells alone. Black bars indicate significantly increased IFN-γ spot numbers; grey bars indicate non-significant T-cell response; and white bars indicate negative control group containing T-cells co-cultured with COS7 cells transfected with the respective tumor antigens but not with respective HLA-I molecules. P-values of significant differences compared to the negative control group are depicted above black bars. Further details are described in Example 3.

[0042] Figure 8 depicts the workflow of an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention may be understood more readily by reference to the following detailed description of embodiments of the invention, the Examples included therein, and to the Figures 1-8.

I. DEFINITIONS

[0044] AU methods described herein can be performed in any suitable order and combination unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0045] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation.

[0046] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes mixtures of compounds, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0047] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated- range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0048] The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0050] As used herein, the abbreviation "ID" refers to 1st dimensional. [0051] As used herein, the abbreviation "2D" refers to 2nd dimensional.

[0052] As used herein, the abbreviation "AAS" refers to amino acid sequence.

[0053] As used herein, the abbreviation "AU" refers to absorbance unit.

[0054] As used herein, the abbreviation "DC" refers to dendritic cell. [0055] As used herein, the abbreviation "DCD" refers to dermcidine.

[0056] As used herein, the abbreviation "DSG-I " refers to desmoglein-1.

[0057] As used herein, the abbreviation "F" refers to fraction.

[0058] As used herein, the abbreviation "HRNR" refers to hornerin.

[0059] As used herein, the abbreviation "OVA" refers to ovalbumin. [0060] As used herein, the abbreviation "P" refers to protein peak.

[0061] As used herein, the abbreviation "p" refers to p value.

[0062] As used herein, the abbreviation "PB-L" refers to PBMC lysate.

[0063] As used herein, the abbreviation "RT" refers to retention time.

[0064] As used herein, the abbreviation "SEREX" refers to serological analysis of recombinant cDNA expression libraries.

[0065] As used herein, the abbreviation "TAA" refers to tumor-associated antigen.

[0066] As used herein, the abbreviation "TC" refers to T-cell.

[0067] As used herein, the abbreviation "TTR" refers to transthyretin.

[0068] As used herein, the abbreviation "Tu-L" refers to tumor cell lysate. [0069] As used herein, the abbreviation "W" refers to well.

[0070] As used herein, the terms "activation" or "activating," when used in reference to a T-lymphocyte, has the ordinary meaning in the art of immunology and refers to characteristic changes (e.g., calcium ion influx, tyrosine kinase activation) that follow ligand-receptor interactions between a T-lymphocyte and antigen-presenting cell. T-cell activation ordinarily results in clonal expansion of antigen-reactive T-lymphocytes. [0071] As used herein, the term "administering" means the actual physical introduction of a composition into or onto (as appropriate) a host. Any and all methods of introducing the composition into or onto a host are contemplated according to the invention. Methods of the present invention are not dependent on any particular means of introduction and are not to be so construed. Means of introduction are well-known to those skilled in the art, and also are exemplified herein.

[0072] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. "Amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, e.g., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

[0073] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0074] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention .typically conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0075] Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al, Molecular Biology of the Cell (3 ^rd ed., 1994) and Cantor & Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that often form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. "Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three dimensional structure formed by independent tertiary units, usually by noncovalent association.

[0076] As used herein, the term "amount effective" or "amount sufficient" means an amount which produces the desired effect. An "amount sufficient" or "amount effective" is that amount of a given composition that exhibits the activity of interest or, which provides either a subjective relief of a symptom(s) or an objectively identifiable improvement as noted by a clinician or other qualified observer. The dosing range varies with the composition used, the route of administration and the potency of the particular composition.

[0077] The term "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody or its functional equivalent will be most critical in specificity and affinity of binding. -See, Paul, Fundamental Immunology.

[0078] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. [0079] Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases. Thus, e.g., pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' ₂ , a dimer of Fab which itself is a light chain joined to V _H -C _H I by a disulfide bond. The F(ab)' ₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' ₂ dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see, Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al, Nature 348:552-554 (1990)).

[0080] For preparation of antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, e.g., Kohler & Milstein,

Nature 256:495-497 (1975); Kozbor et al, Immunology Today 4:72 (1983); Cole et al, pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). Techniques for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)). [0081] As used herein, the term "antigen-presenting cell" refers to a vertebrate cell on which an antigen can be pulsed. In some embodiments, an antigen-presenting cell is a dendritic cell.

[0082] As used herein, the term "autoimmune disease " refers to any disease or condition caused or aggravated by humoral or T-cell based immune responses directed against a self tissue antigen. Autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body. In other words, the body attacks its own cells.

[0083] As used herein, the term "autologous" means self. "Autologous" also refers to cells, tissues or proteins that are reimplanted in the same individual where they come from. In contrast, cells or tissues transplanted from a different individual are referred to as "allogeneic," "homologous," or as an "allograft." Moreover, "autologous" refers to interactions between cells from the same individual, e.g., an interaction between a T-cell and an antigen-presenting cell from the same individual. In this regard, an autologous antigen- presenting cell can present antigens derived from an allogeneic source (e.g., obtained from cell or tissue lysate from a different individual).

[0084] "Biological sample" as used herein is a sample of biological tissue or fluid that contains nucleic acids and/or polypeptides. Such samples are typically from humans, but include tissues isolated from non-human primates, or rodents, e.g., mice, and rats. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, etc. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A "biological sample" also refers to a cell or population of cells or a quantity of tissue or fluid from an animal. Most often, the biological sample has been removed from an animal, but the term "biological sample" can also refer to cells or tissue analyzed in vivo, i.e., without removal from the animal. Typically, a "biological sample" will contain cells from the animal, but the term can also refer to noncellular biological material, such as noncellular fractions of blood, saliva, or urine, that can be used to measure the cancer-associated polynucleotide or polypeptide levels. Numerous types of biological samples can be used in the present invention, including, but not limited to, a tissue biopsy, a blood sample, a lymph sample, a buccal scrape, a saliva sample, or a nipple discharge. As used herein, a "tissue biopsy" refers to an amount of tissue removed from an animal, preferably a human, for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor. "Tissue biopsy" can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc.

[0085] "Providing a biological sample" or "obtaining a biological sample" means to obtain a biological sample for use in methods or compositions of the present invention. Most often, this will be done by removing a sample of cells from a patient, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome history, will be particularly useful. [0086] "Cancer cell," "transformed" cell or "transformation" in tissue culture, refers to spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic DNA, or uptake of exogenous DNA, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation is associated with phenotypic changes, such as immortalization of cells, aberrant growth control, nonmorphological changes, and/or malignancy (see, Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)).

[0087] As used herein, the term "CD4 ⁺ T-cell" refers to a T-helper cell. [0088] As used herein, the term "CD8 ⁺ T-cell" refers to a cytotoxic T-cell. [0089] As used herein, the term "coculturing" or grammatical equivalents thereof refers to a culture of two or more cell types together in one vial, plate, bioreactor, or similar device for cell growth (generally leading to cell-cell interactions and cell stimulation). The culture conditions may be those that foster growth and/or propagation of the two or more cell types or those that facilitate growth and/or proliferation of one, or a subset, of the two or more cells while maintaining cellular growth for the remainder.

[0090] As used herein, the term "contacting" refers to an instance of exposure of at least one substance to another substance and includes reference to placement of at least one substance to another substance in direct physical association. For example, contacting can include contacting a substance, such as a cell, to a polypeptide described herein. A cell can be contacted with the polypeptide, for example, by adding the polypeptide to the culture medium (by continuous infusion, by bolus delivery, or by changing the medium to a medium that contains the agent) or by adding the polypeptide to the extracellular fluid in vivo (by local delivery, systemic delivery, intravenous injection, bolus delivery, or continuous infusion). The duration of contact with a cell or group of cells is determined by the time the polypeptide is present at physiologically effective levels or at presumed physiologically effective levels in the medium or extracellular fluid bathing the cell. The term "contacting" is used herein interchangeably with the following: combined with, added to, mixed with, passed over, incubated with, flowed over, etc.

[0091] "Correlating an amount" means comparing an amount of a substance, molecule or marker (such as a T-cell immunogenic antigen) that has been determined in one sample to a an amount of the same substance, molecule or marker determined in another sample. The amount of the same substance, molecule or marker determined in another sample may be specific for a given cancer.

[0092] As used herein, the term "dendritic cell" refers to a type of specialized antigen- presenting cell. [0093] Synonyms of the term "determining an amount" are contemplated within the scope of the present invention and include, but are not limited to, detecting, measuring, testing or determining, the presence, absence, amount or concentration of a molecule, such as an antigen.

[0094] As used herein, the term "induce to mature" in the context of an antigen-presenting cell, such as a dendritic cell, refers to a vertebrate cell which acquires the functional capacity of an antigen-presenting cell after receiving a signal for its functional maturation. The signal induces, e.g., dendritic cell maturation.

[0095] As used herein, the term "IFN-γ assay" refers to an assay measuring the cytokine interferon (IFN)-γ, e.g., an ELISPOT analysis detecting the IFN- γ. The Enzyme-Linked ImmunoSpot (ELISPOT) assay is a very sensitive immunoassay, allowing the detection of a secreted cytokine at the single cell level. With detection levels that can be as low as one cell in 100 000, the ELISPOT is one of the most sensitive cellular assays available. Depending on the substance analyzed, the ELISPOT assay is between 20 and 200 times more sensitive than a conventional ELISA. Typically, as a first step in the ELISPOT assay, a cytokine- specific monoclonal antibody is immobilized on a solid phase, e.g., a 96-well microtiter plate with a polyvinyl-difluoride (PVDF) membrane. In the second step, T-cells to be investigated are added to the wells in the presence of antigen-presenting cells that are loaded with test or negative control antigens and incubated for a relevant time period (e.g., 4 to 40 hours) to allow cytokine production. The secreted cytokine, e.g., interferon- γ, will bind to the capture antibodies in the vicinity of the producing cells and, after removal of the cells by washing, detection anti-cytokine antibodies are added. Typically, these antibodies are either directly conjugated with enzyme or biotinylated, in which case a third step with enzyme conjugated Streptavidin is employed. Finally, a substrate is added which will form a colored, insoluble precipitate when catalyzed by the enzyme. This way a visible spot corresponding to the location of the producing T-cell is formed. The spots can be counted under a dissection microscope or with an automated ELISPOT reader and the frequency of positive cells registered.

[0096] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³² P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g. , by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide. The labels may be incorporated into a protein or antibody at any position. Any method known in the art for conjugating the antibody to the label may be employed, including those methods described by Hunter et al, Nature 144:945 (1962); David et al, Biochemistry 13:1014 (1974); Pain et al, J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).

[0097] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A cell, a protein or a nucleic acid that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid is separated from some open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene. The term "purified" in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Preferably, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. "Purify" or "purification" in other embodiments means removing at least one contaminant from the composition to be purified. Ln this sense, purification does not require that the purified compound be homogenous, e.g., 100% pure. A purified cell is the predominanT-cell species present in a preparation of cells.

[0098] As used herein, the term "lysate" refers to a solution containing the contents of lysed cells. [0099] As used herein, the term "lysis" or "lysing" refers to the breakage or disruption of the plasma membrane and optionally the cell wall of a biological organism sufficient to release at least some intracellular content, often by mechanical, viral or osmotic mechanisms that compromise its integrity.

[0100] As used herein, the term "malignant disease" refers to any type of tumor or cancer. [0101] As used herein, the term "mass spectrometer" refers to a gas phase ion spectrometer that measures a parameter which can be translated into mass-to-charge ratios of gas phase ions. Mass spectrometers generally include an inlet system, an ionization source, an ion optic assembly, a mass analyzer, and a detector. Examples of mass spectrometers are time-of- flight, magnetic sector, quadrapole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these.

[0102] As used herein, the term "metastases" or "metastatic tumor cell" refers to a metastasis from a primary tumor wherein the primary tumor is a solid, non-lymphoid tumor, as will be more apparent from the embodiments described herein.

[0103] As used herein, the term "PF2D system" or simply "PF2D" refers to the PROTEOMELab PF2D, a protein fractionation system developed and distributed by Beckman Coulter GmbH Krefeld, Germany.

[0104] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.

[0105] The term "preferentially expressed by a cell" is used herein, for purposes of the specification and claims, to mean a molecule (such as a T-cell immunogenic antigen) expressed on the surface of a cell, wherein the level of expression (measured directly or indirectly, and including by presence or by activity) of such molecule is at least 3 to 4 times that expressed by other subpopulations of cells contained in the sinusoidal area of an organ in which metastases develop. Thus, preferential expression may include detection of expression of the molecule on or by type 1 endothelial cells, and absence of detection of the same molecule on or by type 2 endothelial cells; or a log greater expression of the same molecule on or by type 1 endothelial cells as compared to expression on or by type 2 endothelial cells.

[0106] As used herein, the terms "pulse," "pulsing" or grammatical equivalents refer to loading of an antigen (such as a polypeptide) on HLA complexes on a vertebrate cell, preferably a dendritic cell.

[0107] As used herein, the term "subject" refers to an individual. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. The term "subject" can include domesticated animals, such as cats, dogs, etc. , livestock (e. g. , cattle, horses, pigs, sheep, goats, etc. ), and laboratory animals (e. g. , mouse, rabbit, rat, guinea pig, etc.). In some embodiments, a subject is a patient having a disease, disorder or a condition and is in need of treatment of such disease, disorder, or condition. [0108] As used herein, the term "T-cell" refers to a T-lymphocyte.

[0109] As used herein, the term "T-cell activation assay" refers to a functional test that detects activation of a T-cell and includes, for example, an ELISPOT assay, a thymidine uptake assay, flowcytometric analysis of CD69 expression.

[0110] As used herein, the term "T-cell immunogenic antigen" refers to any antigen that causes activation of a T-cell.

[0111] As used herein, the terms "treat," "treating," and "treatment" refer to administering a composition to a subject having an undesired disease, disorder, or condition or is at risk for developing such undesired disease, disorder, or condition. The condition can be any pathogenic disease, autoimmune disease, cancer or an inflammatory condition. The effect of the administration of the composition to the subject can have the effect of, but is not limited to, (i) reducing a symptom(s) of the condition, (ii) a reduction in the severity of the condition, or (iii) the complete ablation of the condition. The terms include: (i) preventing a disease, i.e. causing the clinical symptoms of the disease not to develop in a mammal that may be predisposed to the disease but does not yet experience any symptoms of the disease; (ii) inhibiting the disease, i.e. arresting or reducing the development of the disease or its clinical symptoms; or (iii) relieving the disease, i.e. causing regression of the disease or its clinical symptoms. The terms refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disease or undesired condition as well as those in which the disease or undesired condition is to be prevented. Hence, a mammal may have been diagnosed as having the disease or undesired condition or may be predisposed or susceptible to the disease.

[0112] "Tumor cell" refers to precancerous, cancerous, and normal cells in a tumor.

[0113] As used herein, the term "vaccine" refers to a material that is administered to a vertebrate host to immunize the host against the same or similar material. Typically, a vaccine comprises material associated with a disease state, such as viral infection, bacterial infection, and various malignancies.

II. METHODS AND COMPOSITIONS

A. Identifying A T-CeII Immunogenic Antigen

[0114] The present invention provides methods useful for the identification of a T-cell immunogenic antigen expressed by a cell in a subject. In some embodiments of these methods, the method comprises the steps of (a) obtaining a sample comprising a plurality of proteins expressed in a cell from a subject, (b) contacting the plurality of proteins of the sample with a CD4 ⁺ T-cell or a CD8 ⁺ T-cell; and (c) determining whether the CD4 ⁺ T-cell or CD8 ⁺ T-cell binds to a member of the plurality of proteins of the sample. Thereby the T-cell immunogenic antigen is identified. [0115] In some embodiments, the member of the plurality of proteins bound by the CD4 ⁺ T-cell or CD8 ⁺ T-cell is identified. In some embodiments identification of the member of the plurality of proteins is by mass spectrometry (see below). A member of the plurality of proteins to which a CD4 ⁺ T-cell or CD8 ⁺ T-cell binds is also referred to herein as a "peptide antigen." A preferred peptide antigen is a "tumor-associated antigen." A peptide antigen identified so finds use in many applications, e.g., pulsing onto antigen-presenting cells, for making a medicament, for making a vaccine and for treating a subject having a disease, disorder or condition.

[0116] In some embodiments the T-cell immunogenic antigen is an autologous T-cell immunogenic antigen. In other embodiments, the T-cell immunogenic antigen is an allogeneic T-cell immunogenic antigen. [0117] In some embodiments the CD4 ⁺ T-cell or CD8 ⁺ T-cell is an autologous CD4 ⁺ T-cell or CD8 ⁺ T-cell, i.e., it is obtained from the same individual from which the T-cell immunogenic antigen is obtained. In other embodiments, the CD4 ⁺ T-cell or CD8 ⁺ T-cell is an allogeneic CD4 ⁺ T-cell or CD8 ⁺ T-cell, i.e., it is not obtained from the same individual from which the T-cell immunogenic antigen is obtained, but rather from another individual.

[0118] In some embodiments, the CD4 ⁺ T-cells and/or the CD8 ⁺ T-cells are in a blood sample obtained from the subject. In some embodiments, the CD4 ⁺ T-cells and/or a CD8 ⁺ T- cells express a binding partner on their cell surface specific for a member of the plurality of proteins in the sample. The binding partner may be a cell-surface receptor or a cell-surface antigen.

[0119] In some embodiments, a CD4 ⁺ T-cell or a CD8 ⁺ T-cell recognizing a T-cell immunogenic antigen is obtained from a subject, preferably from a subject having a disease, disorder or condition. The disease can be any disease.

1. Malignant Disease [0120] In some embodiments, the disease is a malignant disease. In some embodiments, the malignant disease is cancer. A malignant disease includes, but is not limited to, at least one of: leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignamT-lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome, hypercalcemia of malignancy, solid tumors, CD-46 related tumors, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic disease, cancer related bone resorption, cancer related bone pain, and the like.

[0121] Cells from any cancer can be used in a method of the present invention. Cancer cells useful in a method of the present invention include, but are not limited to cells from lung cancer, breast cancer, colorectal cancer, melanoma, colon cancer, mesothelioma, ovarian cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, uterus cancer, thyroid cancer, pancreatic cancer, cervical cancer, esophageal cancer, head and neck cancer, hepatocellular carcinoma, brain tumor, vulval or testical cancer, sarcoma, leukemia, lymphoma, glioma and glioblastoma. In some embodiments, the cancer is a brain tumor. [0122] In some embodiments, cells recognizing a T-cell immunogenic antigen are cells from metastases or a metastatic tumor cell.

[0123] In some embodiments, the cancer is a hematologic cancer. A hematologic cancer includes any cancer associated with cells in the bloodstream. Examples thereof include B- and T-cell lymphomas, leukemias including but not limited to low grade/follicular non- Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade/ follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL and Waldenstrom's Macroglobulinemia, chronic leukocytic leukemia, myeloid leukemia (acute and chronic), myelogenous leukemia (acute and chronic), lymphoblastic leukemia (acute and chronic), lymphocytic leukemia (acute and chronic), monocytic leukemia, promyelocytic leukemia, multiple myeloma, myelodysplastic syndrome, Hodgkin's disease, and non- Hodgkin's lymphoma (malignanT-lymphoma). It should be clear to those of skill in the art that these lymphomas will often have different names due to changing systems of classification.

[0124] In some embodiments, the cancer is a solid or non-hematologic cancer. Examples thereof include, but are not limited to colorectal cancer, liver cancer, breast cancer, lung cancer, head and neck cancer, stomach cancer, testicular cancer, prostate cancer, ovarian cancer, uterine cancer and others. In some embodiments, a non-hematologic cancer is selected from the group consisting of lung cancer, sarcoma, gastrointestinal cancer, cancer of the genitourinary tract, liver cancer, skin cancer, gynecological cancer, bone cancer, cancer of the nervous system, and cancer of adrenal glands. These cancers may be in the early stages (precancer), intermediate (Stages I and II) or advanced, including solid tumors that have metastasized. In some embodiments, the solid or non-hematologic cancer will be a cancer wherein B-cells elicit a protumor response, i.e. the presence of B-cells is involved in tumor development, maintenance or metastasis.

[0125] In some embodiments of the present invention, the non-hematologic cancer is a lung cancer. A lung cancer includes, but is not limited to, bronchogenic carcinoma [squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma], alveolar [bronchiolar] carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma, SCLC, and NSCLC. [0126] In other embodiments the non-hematologic cancer is a sarcoma. A sarcoma includes, but is not limited to, cancers such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma.

[0127] In yet other embodiments of the present invention, a non-hematologic cancer is a gastrointestinal cancer. A gastrointestinal cancer includes, but is not limited to cancers of esophagus [squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma], stomach [carcinoma, lymphoma, leiomyosarcoma], pancreas [ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma], small bowel [adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma], and large bowel [adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma].

[0128] In some embodiments of the present invention, a non-hematologic cancer is a cancer of the genitourinary tract. Cancers of the genitourinary tract include, but are not limited to cancers of kidney [adenocarcinoma, Wilms tumor (nephroblastoma), lymphoma, leukemia, renal cell carcinoma], bladder and urethra [squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma], prostate [adenocarcinoma, sarcoma], and testis [seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumor, fibroma, fibroadenoma, adenomatoid tumors, lipoma].

[0129] In other embodiments of the present invention, a non-hematologic cancer is a liver cancer. A liver cancer includes, but is not limited to, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0130] In some embodiments of the present invention, a non-hematologic cancer is a skin cancer. Skin cancer includes, but is not limited to, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, nevi, dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, and psoriasis.

[0131] In some embodiments of the present invention, a non-hematologic cancer is a gynecological cancer. Gynecological cancers include, but are not limited to, cancer of uterus [endometrial carcinoma], cervix [cervical carcinoma, pre-invasive cervical dysplasia], ovaries [ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid carcinoma, clear cell adenocarcinoma, unclassified carcinoma), granulosa-theca cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma and other germ cell tumors], vulva [squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma], vagina [clear cell carcinoma, squamous cell carcinoma, sarcoma botryoides (embryonal rhabdomyosarcoma), and fallopian tubes [carcinoma].

[0132] In other embodiments of the present invention, a non-hematologic cancer is a bone cancer. Bone cancer includes, but is not limited to, osteogenic sarcoma [osteosarcoma], fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant- lymphoma [reticulum cell sarcoma], multiple myeloma, malignant giant-cell tumor, chordoma, osteochondroma [osteocartilaginous exostoses], benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, and giant-cell tumors. [0133] In some embodiments of the present invention, a non-hematologic cancer is a cancer of the nervous system. Cancers of the nervous system include, but are not limited to cancers of skull [osteoma, hemangioma, granuloma, xanthoma, Paget's disease of bone], meninges [meningioma, meningiosarcoma, gliomatosis], brain [astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors], and spinal cord [neurofibroma, meningioma, glioma, sarcoma].

[0134] In some embodiments of the present invention, a non-hematologic cancer is a cancer of adrenal glands. A cancer of adrenal glands includes, but is not limited to, neuroblastoma.

2. Autoimmune Disease [0135] In some embodiments, the disease is an autoimmune disease. In some embodiments, cells recognizing a T-cell immunogenic antigen are cells obtained from a subject having an autoimmune disease. Autoimmune diseases are a major cause of immune- mediated diseases. In some embodiments, the autoimmune disease is characterized by expression of a disease-associated tissue antigen. Exemplary autoimmune diseases are Acute disseminated encephalomyelitis (ADEM), Addison's disease, Ankylosing spondylitis,

Antiphospholipid antibody syndrome (APS), Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Bullous pemphigoid, Coeliac disease, Chagas disease, Chronic obstructive pulmonary disease, Dermatomyositis, Diabetes mellitus type 1 , Endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, Hidradenitis suppurativa, Idiopathic thrombocytopenic purpura,

Interstitial cystitis, Lupus erythematosus, Morphea, Multiple sclerosis, Myasthenia gravis, Narcolepsy, Neuromyotonia, Pemphigus Vulgaris, Pernicious anaemia, Polymyositis, Primary biliary cirrhosis, Rheumatoid arthritis, Schizophrenia, Scleroderma, Sjogren's syndrome, Temporal arteritis (also known as "gianT-cell arteritis"), Vasculitis, Vitiligo, and Wegener's granulomatosis.

[0136] In some embodiments, the autoimmune disease is Diabetes mellitus type 1. In some embodiments, the autoimmune disease is Chagas disease. In some embodiments, the autoimmune disease is Graves' disease. In some embodiments, the autoimmune disease is Lupus erythematosus. In some embodiments, the autoimmune disease is Multiple sclerosis. In some embodiments, the autoimmune disease is Rheumatoid arthritis. In some embodiments, the autoimmune disease is Schizophrenia. 3. Sample

[0137] Typically, a sample used in a method of the present invention is a biological sample.

[0138] In some embodiments the sample is a cell lysate. Methods for preparing a cell lysate are well known in the art. In some embodiments, methods of the present invention comprise the step of lysing a cell containing a plurality of proteins. The step of lysing a cell (also referred to as cell lysis) can be achieved by any convenient means, including heat- induced lysis, adding a base, adding an acid, using enzymes such as proteases, using ultrasound, mechanical lysis, using osmotic shock, infection with a lytic virus, and/or expression of one or more lytic genes. Lysis is performed to release intracellular molecules, e.g., a plurality of proteins, which have been produced by the cell. Each of these methods for lysing a cell can be used as a single method or in combination. The extent of cell lysis can be observed by microscopic analysis. Using one or more of the methods described herein, typically more than 70% cell lysis is observed. Preferably, cell lysis is more than 80%, more preferably more than 90% and most preferred about 100%.

[0139] In some embodiments, the cell lysate is obtained from a tumor cell (e.g., from a cancer described herein), a diseased cell, a tumor tissue, or a diseased tissue.

[0140] In some embodiments of a method of the present invention, the plurality of proteins of the sample is contacted with a CD4 ⁺ T-cell and a CD8 ⁺ T-cell from the subject.

4. Fractionation

[0141] In some embodiments of a method the present invention, the plurality of proteins are fractionated prior to contacting the plurality of proteins of the sample with a CD4 ⁺ T-cell or a CD8 ⁺ T-cell. [0142] In some embodiments, fractionation comprises two-dimensional chromatography.

[0143] In some embodiments, fractionation of the plurality of proteins is based on pi and hydrophobicity of the plurality of proteins. In some embodiments, fractionation comprises using a system, referred to as PF2D system (ProteomeLab PF2D) and described in detail herein (see Examples and Figure 8).

[0144] The multidimensional separation system, ProteomeLab PF2D, has been introduced by Beckman Coulter for the separation/fractionation as well as quantitative comparisons of various biological and clinical samples. This system shares the advantages of 2-dimensional liquid-based protein analysis and enables high-throughput large-scale analysis for intact soluble proteins (Ruelle et al, 2007, J Proteome Res 6:2168-2175, expressly incorporated herein by reference) and membrane proteins (Shin et al, 2006, Proteomics 6:1143-50; Lee et al, 2008, Proteomics 8:2168-2177; expressly incorporated herein by reference) due to its excellent resolution, good reproducibility, high loading capacity, and full automation capabilities (Lee et al, 2008, Proteomics 8:3371-81; expressly incorporated herein by reference).

[0145] Fractionated proteins are ready to be analyzed by diverse methods according to research interest: from identification and characterization of differentially expressed proteins (Chahal et al, 2006, Biochem Biophys Res Commun 348:1055-62), through proteome profiling (Lee et al, 2008, Proteomics 8:3371-81). Applicants describe herein a novel application for the PF2D system and have shown its applicability to biological and immunological assays for functional analysis.

5. Pulsing Polypeptides onto an Antigen-Presenting Cell

[0146] In some embodiments of a method of present invention, a plurality of proteins is pulsed onto an antigen-presenting cell (APC). In some embodiments, the plurality of proteins is pulsed onto an antigen-presenting cell prior to contacting the plurality of proteins of the sample with a CD4 ⁺ T-cell or a CD8 ⁺ T-cell.. Pulsing an antigen onto an antigen-presenting cell can be performed by any method known in the art.

[0147] APCs are highly specialized cells, including macrophages, monocytes, and dendritic cells (DCs), that can process antigens and display their peptide fragments on the cell surface together with molecules required for lymphocyte activation. Generally, however, dendritic cells are superior to other antigen presenting cells for inducing a T lymphocyte mediated response (e.g., a primary immune response). DCs may be classified into subgroups, including, e.g., follicular dendritic cells, Langerhans dendritic cells, and epidermal dendritic cells. In some embodiments, an antigen-presenting cell is a dendritic cell. More preferred is an autologous dendritic cell.

[0148] DCs have been shown to be potent simulators of both T helper (Th) and cytotoxic T lymphocyte (CTL) responses (Schuler et al. , 1997, Int Arch Allergy Immunol 112:317-22). In vivo, DCs display antigenic peptides in complexes with MHC class I and MHC class II proteins. The loading of MHC class I molecules usually occurs when cytoplasmic proteins (including proteins that are ultimately transported to the nucleus) are processed and transported into the secretory compartments containing the MHC class I molecules. MHC Class II proteins are normally loaded in vivo following sampling (e.g., by endocytosis) by APCs of the extracellular milieu. DCs migrate to lymphoid organs where they induce proliferation and differentiation of antigen-specific T lymphocytes, i.e., The cells that recognize the peptide/MHC Class II complex and CTLs that recognize the peptide/MHC Class I complex. [0149] In some embodiments, DCs (or DC precursor cells) are exposed to antigenic peptide fragments ex vivo (referred to as "antigen pulsing"). DCs can also be genetically modified ex vivo to express a desired antigen, and subsequently administered to a patient to induce an anti-antigen immune response. Alternatively, the pulsed or genetically modified DCs can be cultured ex vivo with T lymphocytes (e.g., HLA -matched T lymphocytes) to activate those T cells that are specific for the selected antigen. Antigen-laden DC may be used to boost host defense against tumors. It will be appreciated that is not necessary that the target antigen (e.g., a target "tumor" antigen) be expressed naturally on the cell surface, because cytoplasmic proteins and nuclear proteins are normally processed, attached to MHC-encoded products intracellularly, and translocated to the cell surface as a peptide/MHC complex. [0150] In some embodiments, the dendritic cell is induced to mature. Methods for inducing dendritic cells to mature are known in the art and are described, e.g., by Bai et al. (2002, Int J Oncol 20(2):247-53).

[0151] In some embodiments, the step of contacting the plurality of proteins of the sample with a CD4 ⁺ T-cell or a CD8 ⁺ T-cell from the subject comprises co-culturing the antigen- presenting cell and the CD4 ⁺ T-cell or a CD8 ⁺ T-cell.

[0152] In some embodiments, polypeptides and/or polynucleotides encoding target antigens, and/or whole proteins or fragments thereof and antigen presenting cells (especially dendritic cells), are used to elicit an immune response against cells expressing or displaying the target antigen, such as cancer cells, in a subject. Typically, this involves the one or more of the following steps of (a) isolating hematopoietic stem cells, (b) genetically modifying the cells to express the target antigen and to inhibit expression of one or more immunosuppressive cytokines, (c) differentiating the precursor cells into DCs and (d) administering the DCs to the subject (e.g., human patient). In other embodiments, the this method comprises the steps of (a) isolating DCs (or isolation and differentiation of DC precursor cells), (b) genetically modifying the cells to inhibit expression of one or more immunosuppressive cytokines, (c) pulsing the cells with target antigen, and (d) administering the DCs to the subject. In another embodiment, the antigen pulsed or antigen expressing DCs are used to activate T lymphocytes ex vivo.

[0153] In some embodiments, DCs are exposed ex vivo to target antigens and allowed to process the antigen so that antigen epitopes are presented on the surface of the cell in the context of a MHC class I (or MHC class II) complex. This procedure is referred to as "antigen pulsing." The "pulsed DCs" may then be used to activate T lymphocytes.

[0154] The peptide antigen(s) used for pulsing DCs, such as a plurality of polypeptides or a single polypeptide purified according to a method described herein, includes at least one linear epitope derived from that antigen protein. Antigenic proteins or antigenic fragments thereof may be used, as they will be taken up and processed by the DCs. Alternatively, short "peptides" may be administered to the DCs. Peptide antigens useful for pulsing onto DCs include, but are not limited to, transthyretin or calprotectin subunits, described herein.

[0155] When antigenic peptides are used for pulsing, their size is typically not restricted. In some embodiments, antigenic peptides used for pulsing have at least about 6 to 8 amino acids and fewer than about 30 amino acids or fewer than about 50 amino acid residues in length. In some embodiments, the immunogenic peptide has between about 8 and 12 amino acids. In some embodiments, the immunogenic peptide has between about 8 and 15 amino acids. In some embodiments, the immunogenic peptide has between about 8 and 20 amino acids. In some embodiments, a mixture of antigenic protein fragments is used. In other embodiments, a particular peptide of defined sequence may be used, such as transthyretin or a calprotectin subunit described herein. The peptide antigens may be produced by de novo peptide synthesis, enzymatic digestion of purified or recombinant proteins, by purification of antigens from a natural source (e.g., a patient or tumor cells from a patient), or expression of a recombinant polynucleotide encoding a antigen polypeptides.

[0156] The amount of antigen used for pulsing DC will depend on the nature, size and purity of the peptide or polypeptide. In some embodiments, from about 0.05 μg/ml to about 1 mg/ml, most often from about 1 to about 100 μg/ml of peptide antigen is used. After adding the peptide antigen(s) to the cultured DC, the cells are then allowed sufficient time to take up and process the antigen and express antigen peptides on the cell surface in association with either class I or class II MHC. In some embodiments, this occurs in about 18-30 hours, most often in about 24 hours. In one exemplary embodiment, DCs are resuspended (10 ⁶ cells/ml) in RPMI media (Gibco) and cultured with transthyretin or a calprotectin subunit antigens (50 μg/ml) overnight under standard conditions (e.g., 37 ⁰ C humidified incubator, 5% CO ₂ ).

[0157] Antigens or antigenic peptide fragments can be tumor- specific antigens, antigens from an autoimmune disease or antigenic fragments thereof. Novel useful antigens described herein, include transthyretin and a calprotectin subunit. [0158] In some embodiments, a method for pulsing polypeptides onto an antigen- presenting cell comprises the steps of (a) obtaining a cell lysate comprising a plurality of polypeptides; (b) separating said plurality of polypeptides of said cell lysate in a first dimension; (c) collecting a first fraction of said plurality of polypeptides; (d) separating said first fraction of said plurality of polypeptides in a second dimension; (e) collecting a second fraction of said plurality of polypeptides; and (f) pulsing said plurality of polypeptides of said second fraction onto an antigen-presenting cell. As will be understood by one of skill in the art, upon the first separation (step (b) above), the plurality of polypeptides in the first fraction is less than the plurality of the polypeptides in the cell lysate. As will be understood by one of skill in the art, upon the second separation (step (d) above), the plurality of polypeptides in the second fraction is less than the plurality of the polypeptides in the first fraction. "Being less" has the meaning of having a lower complexity, i.e., some polypeptides are purified away and the plurality of polypeptides in the first fraction is more pure than the plurality of the polypeptides in the cell lysate. The plurality of polypeptides in the second fraction is more pure than the plurality of the polypeptides in the first fraction. Preferably, the number of polypeptide species in the second fraction is less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 10, less than 5, and most preferable, 4, 3, 2, or 1. [0159] In some embodiments, a peptide antigen, a tumor-associated antigen or an antigen associated with an autoimmune disease identified by a method of the present invention is pulsed onto an antigen-presenting cell.

6. T-CeIl Activation Assays [0160] In some embodiments of a method the present invention, determining whether the CD4 ⁺ T-cell or CD8 ⁺ T-cell binds to a member of the plurality of proteins of the sample is performed by using a T-cell activation assay.

[0161] In some embodiments, a T-cell activation assay comprises flowcytometry. Flowcytometry may be done using an antibody. In some embodiments, an antibody for flowcytometry is selected from the group consisting of an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD69 antibody, an anti-CD 107a antibody, an anti- CD25 antibody, and an anti-40L antibody.

[0162] In some embodiments, a T-cell activation assay can be performed by measuring dilution of intracellular carboxyfluorescinsuccinimidyl ester (CFSE). [0163] A T-cell activation assay may also be performed by measuring calcium influx.

7. Other Assays

[0164] In some embodiments, determining whether the CD4 ⁺ T-cell or CD8 ⁺ T-cell binds to a member of the plurality of proteins of the sample is performed by using an IFN -γ assay, an assay for measuring interleukin-4 secretion, an assay for measuring interleukin-10 secretion, an assay for measuring tumor necrosis factor (TNF)-α secretion, an assay for measuring TGF-βl secretion, or an assay for measuring perforin secretion. Theses assays are well known in the art.

[0165] In some embodiments, determining whether the CD4 ⁺ T-cell or CD8 ⁺ T-cell binds to a member of the plurality of proteins of the sample is performed by measuring cytotoxic activity.

8. Novel Tumor Antigens Identified Herein

[0166] As described herein, Applicants identified in patients with malignant brain tumors CD4 and CD8 T-cell responses against two novel antigens, transthyretin (TTR) and calprotectin S100A9, which were expressed on tumor and endothelial cells. Surprisingly, immunogenicity of these antigens could be confirmed in 4 out of 10 other brain tumor patients. Thus, transthyretin (TTR) and calprotectin S100A9 are useful antigens which when detected in a sample from patient, indicate that the subject has a cancer. a) Transthyretin

[0167] Transthyretin (TTR) identified herein as tumor-specific antigen is a carrier protein. It transports thyroid hormones in the plasma and cerebrospinal fluid, and also transports retinol (vitamin A) in the plasma. The protein consists of a tetramer of identical subunits.

[0168] More than 80 different mutations in the transthyretin gene have been reported. Mutations in the TTR gene have been described to cause amyloidosis involving the heart, peripheral nerves, and other organs (Jacobson et al, 1992, Hum Genet 89(3):353-6; Almeida et al, 1992, Hum Genet 1(3):211-5). Most mutations are related to amyloid deposition, affecting predominantly peripheral nerve and/or the heart, and a small portion of the gene mutations is non-amyloidogenic. The diseases caused by mutations include familial amyloidotic polyneuropathy (FAP), senile systemic amyloidosis (SSA), euthyroid hyperthyroxinaemia, amyloidotic vitreous opacities, cardiomyopathy, oculoleptomeningeal amyloidosis, meningocerebrovascular amyloidosis, carpal tunnel syndrome, etc. (e.g., Lee et al, 2009, Biochem Biophys Res Commun 388(2):256-60). FAP TTR Val30Met is a lethal autosomal dominant sensorimotor and autonomic neuropathy due to a substitution of methionine for valine at position 30 of the TTR gene (Dardiotis et al, 2009, J Neurol Sci 284(l-2):1258-62. Increased levels of transthyretin were detected in the cerebrospinal fluid of patients suffering from Guillain-Barre syndrome, an autoimmune inflammatory polyradiculoneuropathy that causes acute areflexic paralysis with a high risk of respiratory failure (Chiang et al, 2009, Clin Chim Acta 405(l-2):143-147). Each of the disease listed above can be diagnosed using methods described herein.

[0169] A human transthyretin nucleotide and amino acid sequence is available at GenBank Accession Number NM_000371. b) Calprotectin

[0170] Calprotectin S100A8 and S100A9 identified herein as tumor- specific antigens are members of the SlOO family of proteins calcium binding proteins containing 2 EF -hand calcium-binding motifs (Ravasi et al, 2004, Genomics 84:10-22). SlOO proteins typically have a mass of between 8 and 14 kDa and are located in the cytoplasm and/or nucleus of a wide range of cells and are involved in the regulation of a number of cellular processes, such as cell cycle progression and differentiation. S 100 genes include about 20 members which are located as a cluster on chromosome Iq21 (Schafer et al, 1996, Trends Biochem Sci 21:134-140; Heizmann et al, 1998, Biometals 11 :383-397; Yanamandra et al, 2009, PLOS One 4(5)e5562). The pro-inflammatory calcium-binding proteins S100A8 and S100A9 are also known as calgranulin A and calgranulin B, respectively (Yanamandra et al. , 2009, PLOS One 4(5)e5562).

[0171] A human S 100 calcium binding protein A8 (S 100 A8) nucleotide and amino acid sequence is available at GenBank Accession Number NM_002964. A human SlOO calcium binding protein A9 (S100A9) nucleotide and amino acid sequence is available at GenBank Accession Number NM_002965. [0172] Differential expression of S100A8 and S100A9 or of the heterodimer S100A8/A9 has been associated with acute and chronic inflammation (Gebhardt et al, 2006, Biochem Pharmacol 72 : 1622- 1631 ; Gebhardt et al , Oncogene 21 :4266-4276). For example, they have been implicated in rheumatoid arthritis, psoriasis and inflammatory bowel disease (Foell et al, 2004, Clin Chim Acta 344:37-51; Renaud et al, 1994, Biophys Res Commun 201:1518- 1525; Zwadio et al, 1988, Clin Exp Immunol 72:510-515; Yui et al, 2003, Biol Pharm Bull 26:753-760; Yang et al, 2001, J Leukoc Biol 69:986-994).

[0173] Increased levels of S 100A8 and S 100 A9 have been detected in some human cancers, being abundantly expressed in neoplastic cells and also in infiltrating immune cells (Gebhardt et al, 2006, Biochem Pharmacol 72:1622-1631; Emberly et al., 2004, Biochem Cell Biol 82:508-515; Heizmann et al, 2007, Subcell Biochem 45:93-138; Ott et al, 2003, Cancer Res 63 :7507-7514). In particular, enhanced secretion of S 100a8 and S 100A9 was found in human prostate cancer ells (H ermani et al, 2005, CHn Cancer Res 11 :5146-5152; Hermani et al, 2006, Exp Cell Res 312:184-197). Others have described that S100A9 was severely down-regulated in human ESCC (oesophageal squamous cell carcinoma) and that it could be positively regulated in a p53-dependent manner (see, Li et al, 2009, Biochem J, 422(2):363-72). Each of the disease listed above can be diagnosed using methods described herein.

[0174] Their expression patterns, potential cytokine-like functions, up-regulation and regulation via signaling pathways , including tumor-promoting RAGE receptor (receptor for advanced glycation end products) , suggest that S100A8/A9 may play a key role in inflammation-associated cancers (Hermani et al, 2006, Exp Cell Res 312:184-197; Hofmann et al, 199, Cell 97:889-901). Although many functions have been proposed for S100A8/A9, their roles in various biological processes still remain unknown. For the first time, herein, it is described that the S100A9 subunit is presented as tumor antigen on T-cells.

B. Identifying Patient-Derived T-CeIIs Binding To A Tumor-Associated Antigen [0175] The present invention also provides methods useful for the identification of a patient-derived T-cell binding to a tumor- associated antigen. In some embodiments of these methods, the method comprises the steps of (a) obtaining a plurality of T-cells from a subject, preferably, a patient, (b) contacting the plurality of T-cells with a tumor- associated antigen pulsed on an antigen-presenting cell, and (c) determining if a member of the plurality of T- cells binds to the tumor-associated antigen.

[0176] In some embodiments, the tumor-associated antigen is a recombinant polypeptide or a fragment thereof.

[0177] In other embodiments, the tumor-associated antigen is a synthetic polypeptide or a fragment thereof. [0178] In some embodiments, the plurality of T-cells comprises a plurality of CD4 ⁺ T-cells or a plurality of CD8 ⁺ T-cells. In other embodiments, the plurality of T-cells comprises a plurality of CD4 ⁺ T-cells and a plurality of CD8 ⁺ T-cells.

[0179] Determining if a member of the plurality of T-cells binds to the tumor-associated antigen can be performed in various ways. Useful assays are described herein. In some embodiments, the step of determining if a member of the plurality of T-cells binds to the tumor-associated antigen comprises a T-cell activation assay. A preferred T-cell activation assay is an ELISPOT assay.

C. Use Of A Tumor- Associated Antigen In The Manufacture Of A Cancer Vaccine And Methods Of Treatment [0180] The present invention also provides uses of a tumor-associated antigen identified by a method of the present invention for the manufacture of a cancer vaccine. Methods for producing a vaccine are known in the art. A vaccine composition comprising a modified antigen-presenting cell or a peptide antigen may further comprise an adjuvant. The adjuvant can be any suitable adjuvant or a combination of adjuvants. [0181] Peptides and proteins may also be used to pulse dendritic cells, e.g., autologous dendritic cells, as a means of immunization against the peptide or protein, using techniques well-known to those of skill in the art.

[0182] Thus, the invention also provides for medicaments comprising a peptide pulsed or transduced onto dendritic cells. Dendritic cells treated in this manner may display the peptide at their cell surface, thereby stimulating an immune response in a host receiving the modified dendritic cell. Alternatively, the dendritic cell may be incubated in a culture medium containing the peptide; here the peptide becomes incorporated into or on to the surface of the dendritic cell. Medicaments comprising the peptide noted above and a pharmaceutically acceptable carrier are also contemplated. These medicaments my also contain an immunostimulating agent or adjuvant, to aid in eliciting an immune response.

[0183] The invention also provides for the use of peptide-pulsed DCs for the treatment of a subject having cancer. In some embodiments, peptide-pulsed DCs are introduced into the subject (e.g., human patient) where they induce an immune response. Typically the immune response includes a CTL response against target cells bearing target antigenic peptides (e.g., in a MHC class I/peptide complex). These target cells are typically cancer cells.

[0184] When the modified DCs are to be administered to a patient they are preferably isolated or derived from precursor cells from that patient (i.e., the DCs are administered to an autologous patient). However, the cells may be infused into HLA-matched allogeneic, or HLA-mismatched allogeneic patients. In the latter case, immunosuppressive drugs may be administered to the recipient.

[0185] The cells are administered in any suitable manner, preferably with a pharmaceutically acceptable carrier (e.g., saline). Usually administration will be intravenous, but intra-articular, intramuscular, intradermal, intraperitoneal, and subcutaneous routes are also acceptable. Administration (i.e., immunization) may be repeated at time intervals.

Infusions of DC may be combined with administration of cytokines that act to maintain DC number and activity (e.g., GM-CSF, IL- 12).

[0186] The dose administered to a patient, referred to as amount effective, should be sufficient to induce an immune response. In some embodiments, the immune response is detected by an assay which measures T cell proliferation or T lymphocyte cytotoxicity. In some embodiments, a beneficial therapeutic response in the patient over time is demonstrated, e.g., inhibiting growth of cancer cells or a reduction in the number of cancer cells or the size of a tumor. Typically, 10 ⁶ to 10 ⁹ or more DCs are infused.

[0187] Modified antigen-presenting cells, displaying a tumor antigen or a autoimmune antigen on their cell surface, can be used to treat any disease, disorder or condition described herein. The antigen presenting cells can be autologous or heterologous and can be administered to a subject or patient in need of such treatment as described above. In some embodiments, the modified antigen-presenting cells are administered to a subject to reduce the size of a tumor, to reduce the number of tumors, to prevent metastasis, or to alleviate a symptom(s) associated with an autoimmune disease. [0188] In some embodiments, the compositions described herein are used as adjunctive therapy, along with, before and/or after treatment with other conventional therapies. In some embodiments, modified antigen-presenting DCs are administered to a cancer patient who is treated with a cytotoxic drug, such as cisplatin, BCNU, methotrexate, or taxol. In other embodiments, modified antigen-presenting DCs are administered to a cancer patient who is treated with an antibodies such as Herceptin ^® . In some embodiments, modified antigen- presenting DCs are administered to a cancer patient who is treated with a radiation therapy. In some embodiments, modified antigen-presenting DCs are administered to a cancer patient who is treated with a cytokine or growth factor, such as an interleukin or GM-CSF. In some embodiments, modified antigen-presenting DCs are administered to a patient having an autoimmune disease and who is treated who is treated with a drug alleviating a symptom(s) associated with autoimmune disease. In some embodiments, modified antigen-presenting DCs are administered to a cancer patient who is treated surgically to remove a cancerous tissue.

[0189] The present invention also provides methods for reactivation of a preexisting memory CD4 ⁺ or CD8 ⁺ T-cell response in a subject. In some embodiments, this method comprises administering to a subject a modified antigen-presenting cell as described herein.

D. Detecting Malignant Cells And Cells Indicative Of An Autoimmune Disease In A Subject

[0190] The present invention also provides methods for identifying a cancer or an autoimmune disease in a subject. In some embodiments, this method comprising the step of detecting in a sample obtained from a subject an antigen selected from the group consisting of transthyretin and calprotectin S100A9. [0191] Transthyretin antigen and calprotectin S100A9 antigens are detected as described herein. In some embodiments, a transthyretin antigen or a calprotectin S100A9 antigen is detected by detecting their respective mRNA. In other embodiments, a transthyretin antigen or a calprotectin S100A9 antigen is detected by detecting the respective polypeptides or a fragment thereof.

[0192] Compositions and methods of the present invention find use in a variety of ways. In some embodiments of this invention a method of detecting a diseased cell, such as a malignant-cell or a cell indicative of an autoimmune disease in a subject is provided. A "cell indicative of an autoimmune disease" can be readily identified by a skilled artisan using well defined characteristics known in the art. In some embodiments, this method comprises the steps of providing a biological sample from a subject, wherein the biological sample comprises a cell suspected of being a diseased cell and detecting a T-cell immunogenic antigen expressed by the diseased cell. In some embodiments, this method comprises comparing the level of the T-cell immunogenic antigen expression in the suspected diseased cell with the level of the T-cell immunogenic antigen expression in a cell from one or more healthy subjects or with a previously determined reference range for a level of the T-cell immunogenic antigen expression. In some embodiments of the invention, detecting the level of the T-cell immunogenic antigen expression is carried out by detecting the level of the T- cell immunogenic antigen mRNA. In other embodiments of the invention, detecting the level of the T-cell immunogenic antigen expression is carried out by detecting the level of a the T- cell immunogenic antigen polypeptide. Agents for use in this method, such as anti-T-cell immunogenic antigen antibodies are disclosed herein.

[0193] Detection of the level of a T-cell immunogenic antigen expression may be determined for a variety of reasons. Detecting the level of T-cell immunogenic antigen expression may be (i) part of screening, diagnosis or prognosis of a malignant disease or autoimmune disease in the subject; (ii) part of determining susceptibility of the subject to malignant disease or autoimmune disease; (iii) part of determining the stage or severity of a malignant disease or autoimmune disease in the subject; (iv) part of identifying a risk for the subject of developing a malignant disease or autoimmune disease; or (v) part of monitoring the effect of a drug or therapy administered to the subject diagnosed with a malignant disease or autoimmune disease. In some embodiments, a disease is a cancer. In some embodiments, a disease is an autoimmune disease. E. Identification Of Subjects At Risk Of Developing A Malignant Disease Or Autoimmune Disease

[0194] The present invention also provides a method for identifying a subject at risk of developing a disease, such as a malignant disease or autoimmune disease. Further, the present invention provides a method for identifying in a subject the stage or severity of such a disease. As shown herein, a T-cell immunogenic antigen expression, e.g., is increased in brain cancer cells (Figs. 3 and 7). Thus, amounts of T-cell immunogenic antigens are characteristic of various cancer risk states, e.g., high, medium or low. The risk of developing cancer may be determined by measuring a T-cell immunogenic antigen and then either submitting the measurements to a classification algorithm or comparing them with a reference amount and/or pattern of T-cell immunogenic antigen that is associated with a particular risk level or with a particular stage or severity of a cancer. The above considerations also apply to other malignant disease and autoimmune diseases described herein. In some embodiments, a disease is a cancer. In some embodiments, a disease is an autoimmune disease.

[0195] Using the methods of the invention, T-cell immunogenic antigen levels are determined in a biological sample from a subject for whom a risk of developing a disease (e.g., malignant disease or autoimmune disease) is to be determined. A T-cell immunogenic antigen level detected in a biological sample from the subject for whom a risk of developing a disease is to be determined that is higher than the T-cell immunogenic antigen level detected in a comparable biological sample from normal or healthy subjects or higher than a predetermined base level, indicates that the subject for whom a risk of developing the disease is to be determined has a risk of developing such disease.

F. Screening, Diagnosis Or Prognosis Of A Disease In A Subject And Identifying In A Subject The Stage Or Severity Of A Malignant Disease

Or Autoimmune Disease

[0196] In some embodiments of the present invention, a disease, such as a malignant disease or autoimmune disease, in a subject is determined as part of screening, diagnosis or prognosis of the disease in the subject. Malignant and autoimmune diseases useful in methods of the present invention are described herein. Using the methods of the invention, a T-cell immunogenic antigen level is determined in a biological sample from a subject to be screened for a disease. A T-cell immunogenic antigen level detected in a biological sample from the subject to be screened for a disease that is higher than the T-cell immunogenic antigen level detected in a comparable biological sample from normal or healthy subjects or lower than a predetermined base level, indicates that the subject screened for the disease has or is likely to have that disease. In some embodiments, a disease is a cancer. In some embodiments, a disease is an autoimmune disease. [0197] The stage or severity of a disease refers to different clinical stages of a disease, such as a tumor. Clinical stages of tumors are defined by various parameters which are well- established in the field of medicine. Some of the parameters include morphology, size of tumor, the degree in which the tumor has metastasized through a patient's body and the like.

[0198] In some embodiments of the present invention, a disease, such as a malignant disease or autoimmune disease, is determined as part of determining the course of the disease. Thus, the invention provides methods for determining the course of a disease, such as a malignant disease or autoimmune disease, in a subject. Disease course refers to changes in disease status over time, including disease progression (worsening) and disease regression (improvement). Regression, with respect to cancer, includes tumor remission, reduction or diminution in tumor size, decrease in number cancerous cells, and lessening of symptoms associated with cancer.

[0199] A composition comprising an antibody directed against a T-cell immunogenic antigen is useful for treatment of a disease, such as a malignant disease or autoimmune disease, wherein the T-cell immunogenic antigen expression is up-regulated. However, other drugs, for example, a composition comprising an inhibitor of T-cell immunogenic antigen will also be useful for treating such disease in a patient wherein a T-cell immunogenic antigen expression is up-regulated.

[0200] In some embodiments of the present invention, a disease status is determined as part of monitoring the effect of surgery (e.g., removal of tumor), the effect of a drug or a therapy administered to a subject diagnosed with a disease wherein T-cell immunogenic antigen expression is up-regulated. The effect of surgery, drug or a therapy administered to a subject having a disease may include reoccurrence of the disease, progression of the disease (worsening) and disease regression (improvement).

[0201] Using the compositions, methods and kits of the present invention, a T-cell immunogenic antigen level is determined in a biological sample from a subject at various times after surgery or at various of having been given a drug or a therapy. A T-cell immunogenic antigen level detected in a biological sample from a subject at a first time (tl ; e.g. , before giving a drug or a therapy) that is higher than the T-cell immunogenic antigen level detected in a comparable biological sample from the same subject taken at a second time (t2; e.g., after giving the drug or the therapy), indicates that the disease in the subject is regressing. Likewise, a higher T-cell immunogenic antigen level at a second time compared to a T-cell immunogenic antigen level at a first time, indicates that the disease in the subject is progressing. Similarly, a T-cell immunogenic antigen level detected in a biological sample from a subject at a first time (tl; e.g., shortly after surgery) that is lower than the T-cell immunogenic antigen level detected in a comparable biological sample from the same subject taken at a second time (t2; e.g., weeks or months after surgery), may indicate that the disease in the subject is not reoccurring. Likewise, a higher T-cell immunogenic antigen level at a second time compared to a T-cell immunogenic antigen level at a first time, may indicate that the disease in the subject is reoccurring.

G. Detection Of T-CeII Immunogenic Antigen mRNA

[0202] In some embodiments, a method of detecting a disease, such as a malignant disease or autoimmune disease, comprises determining the level of a transcript encoding a T-cell immunogenic antigen in a biological sample from a subject, such as a patient. Detecting a decrease or an increase in the level of a T-cell immunogenic antigen mRNA relative to normal indicates the presence of a disease in the subject. In some embodiments, the step of determining the level of the T-cell immunogenic antigen mRNA comprises an amplification reaction. In other embodiments, the presence of the disease is evaluated by determining in a cell the level of expression of mRNA encoding a T-cell immunogenic antigen. A T-cell immunogenic antigen mRNA level lower or higher than in a corresponding non-disease tissue indicates the presence of a disease. Methods of evaluating RNA expression of a particular gene are well known to those of skill in the art, and include, inter alia, hybridization and amplification based assays.

1. Direct Hybridization-based Assays

[0203] Methods of detecting and/or quantifying the level of a T-cell immunogenic antigen gene transcripts (mRNA or cDNA made therefrom) using nucleic acid hybridization techniques are known to those of skill in the art. For example, one method for evaluating the presence, absence, or quantity of a T-cell immunogenic antigen polynucleotides involves a Northern blot. Gene expression levels can also be analyzed by techniques known in the art, e.g., dot blotting, in situ hybridization, RNase protection, probing DNA microchip arrays, and the like. Useful hybridization probes for detecting a transthyretin nucleic acid in a sample can be made as known in the art using the transthyretin nucleotide sequences (e.g., GenBank Accession No. NM_000371). Useful hybridization probes for detecting a calprotectin S100A9 nucleic acid can be made as known in the art using the calprotectin S100A9 nucleotide sequences (e.g., GenBank Accession No. NM_002965).

2. Amplification-based Assays

[0204] In some embodiments, amplification-based assays are used to measure the expression level of a T-cell immunogenic antigen. In such an assay, the T-cell immunogenic antigen nucleic acid sequences act as a template in an amplification reaction (e.g., Polymerase Chain Reaction, or PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the level of a T-cell immunogenic antigen in the sample. Methods of quantitative amplification are well known to those of skill in the art. Detailed protocols for quantitative PCR are provided, e.g., in Innis et al (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Nucleic acid sequences for some T-cell immunogenic antigens are available from e.g., GenBank, and are sufficient to enable one of skill to routinely select primers to amplify any portion of the gene.

[0205] In some embodiments, a TaqMan based assay is used to quantify the cancer- associated polynucleotides. TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end. When the PCR product is amplified in subsequent cycles, the 5' nuclease activity of the polymerase, e.g., AmpliTaq, results in the cleavage of the TaqMan probe. This cleavage separates the 5' fluorescent dye and the 3 ' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, for example, literature provided by Perkin-Elmer).

[0206] Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see, Wu and Wallace, Genomics 4:560(1989); Landegren et al, Science 241 :1077 (1988); and Barringer et al, Gene 89:117 (1990)), transcription amplification (Kwoh et al, Proc. Natl Acad. ScL USA 86:1173(1989)), self-sustained sequence replication (Guatelli et al, Proc. Nat. Acad. ScL USA 87: 1874(1990)), dot PCR, and linker adapter PCR, etc. [0207] Useful oligonucleotides for detecting a transthyretin nucleic acid in a sample are described herein {see Table 1) and can be made as known in the art using the transthyretin nucleotide sequences (e.g., GenBank Accession No. NM_000371). Useful oligonucleotides for detecting a calprotectin S100A9 nucleic acid are described herein {see Table 1) and can be made as known in the art using the calprotectin S100A9 nucleotide sequences (e.g., GenBank Accession No. NM_002965).

H. Detection Of T-CeIl Immunogenic Antigen Polypeptide

[0208] In some embodiments, a method of detecting a disease, such as a malignant disease or autoimmune disease, comprises determining the level of a T-cell immunogenic antigen polypeptide in a biological sample from a subject, such as a patient. Detecting a decrease or an increase in the level of the T-cell immunogenic antigen polypeptide relative to normal indicates the presence of a disease in the subject.

[0209] Upregulation of a T-cell immunogenic antigen expression is indicative of and can be correlated with various diseases. Thus, a T-cell immunogenic antigen polypeptide or a T- cell immunogenic antigen polynucleotide can be used as a biomarker in the diagnosis of a disease. Exemplary biomarkers described herein include transthyretin and calprotectin. In some embodiments of the present invention, the amount of T-cell immunogenic antigen in a biological sample is determined. Typically, the amount of T-cell immunogenic antigen in a biological sample provided from a normal, healthy or non-cancer subject is correlated with the amount of T-cell immunogenic antigen in a biological sample provided from a subject having a disease or from a subject suspected of having a disease. The amount of T-cell immunogenic antigen detected in the biological sample from the subject having the disease or from the subject suspected of having the disease may be specific for a given disease.

[0210] Detection of a T-cell immunogenic antigen polypeptide can be accomplished by any specific detection method including, but not limited to, affinity capture, mass spectrometry, traditional immunoassay directed to T-cell immunogenic antigen, PAGE, or HPLC as further described herein or as known by one skilled in the art.

[0211] In some embodiments, a method for detecting a T-cell immunogenic antigen is PF2D as described herein. PF2D technology comprises separating and detecting a T-cell immunogenic antigen based on pi (first dimension) and by hydrophobicity (second dimension). The fractioning by the PF2D system works on the principle of 2D PAGE, but uses a gel-free liquid phase approach to separate proteins in two dimensions. PF2D represents a potent tool for separation of intact proteins from tumor tissue and from cells indicative of an autoimmune disease. The resulting separated matrix-free proteins are extracted as fluids and therefore are immediately accessible for further functional analysis as described herein. 1. Detection by Mass Spectrometry

[0212] In some embodiments of the present invention, a T-cell immunogenic antigen is detected and identified by mass spectrometry, a method that employs a mass spectrometer to detect gas phase ions. In general, mass spectrometry involves the ionization or vaporization of a molecule, accelerating the ions in an electric field, and passing the ions through a magnetic field. The ions are separated according to their mass as they pass through a magnetic field and are then directed into a detector for identification and analysis. Mass spectrometry data can be collected in less than one minute per sample.

[0213] In some embodiments, the mass spectrometric technique for use in the present invention is surface-enhanced laser desorption/ionization (SELDI). "SELDI" is a method of gas phase ion spectrometry in which the surface of substrate which presents an analyte, such as a T-cell immunogenic antigen polypeptide, to the energy source plays an active role in the desorption and ionization process. The SELDI technology is described in, e.g., U.S. Pat. No. 5,719,060, which is incorporated herewith by reference in its entirety. Other mass spectrometry methods suitable for detecting and identifying a T-cell immunogenic antigen polypeptide and a T-cell immunogenic antigen polynucleotide are described in U.S. Pat. Nos. 5,894,063, 6,020,208, 6,027,942, 6,528,320, U.S. Pat. Appls. Nos. 2003/0091976 and 2002/0060290, all of which are incorporated herewith by reference in their entirety.

[0214] In other embodiments, the mass spectrometric technique for the detection and identification of a T-cell immunogenic antigen polypeptide is MALDI-TOF MS. Other mass spectrometric techniques for use in the present invention are LC-ESI MS (Liquid

Chromatography-Electrospray Ionization Tandem Mass Spectrometry) as, for example, described by Song et al. {Anal. Chem. 77(2):504-510 (2005)).

[0215] Typically, an analyte, such as a T-cell immunogenic antigen polypeptide or a T-cell immunogenic antigen polynucleotide (e.g., a PCR product), to be analyzed by mass spectrometry, is attached to a probe on which it is presented to an ionization source. A probe (e.g., a biochip) is optionally formed in any suitable shape (e.g., a square, a rectangle, a circle, or the like) as long as it is adapted for use with a gas phase ion spectrometer (e.g., removably insertable into a gas phase ion spectrometer). For example, the probe can be in the form of a strip, a plate, or a dish with a series of wells at predetermined addressable locations or have other surfaces features. The probe is also optionally shaped for use with inlet systems and detectors of a gas phase ion spectrometer. For example, the probe can be adapted for mounting in a horizontally, vertically and/or rotationally translatable carriage that horizontally, vertically and/or rotationally moves the probe to a successive position without requiring repositioning of the probe by hand.

[0216] In certain embodiments, the probe substrate surface can be conditioned to bind analytes. For example, the surface of the probe substrate can be conditioned (e.g., chemically or mechanically such as roughening) to place adsorbents on the surface. The adsorbent comprises functional groups for binding with an analyte, such as a T-cell immunogenic antigen polypeptide. In some embodiments, the substrate material itself can also contribute to adsorbent properties and may be considered part of an adsorbent (See, e.g., U.S. Pat. Appl. No. 2003/0091976).

[0217] Adsorbents can be placed on the probe substrate in continuous or discontinuous patterns. If continuous, one or more adsorbents can be placed on the substrate surface. If multiple types of adsorbents are used, the substrate surface can be coated such that one or more binding characteristics vary in a one- or two-dimensional gradient. If discontinuous, plural adsorbents can be placed in predetermined addressable locations or surface features (e.g. , addressable by a laser beam of a mass spectrometer) on the substrate surface. The surface features of probes or biochips include various embodiments. For example, a biochip optionally includes a plurality of surface features arranged in, e.g., a line, an orthogonal array, a circle, or an n-sided polygon, wherein n is three or greater. The plurality of surface features typically includes a logical or spatial array. Optionally, each of the plurality of surface features comprises identical or different adsorbents, or one or more combinations thereof. For example, at least two of the plurality of surface features optionally includes identical or different adsorbents, or one or more combinations thereof. Suitable adsorbents are described in, for example, U.S. Pat. Appl. No. 2003/0091976.

[0218] The probe substrate can be made of any suitable material. Probe substrates are preferably made of materials that are capable of supporting adsorbents. For example, the probe substrate material can include, but is not limited to, insulating materials (e.g., plastic, ceramic, glass, or the like), a magnetic material, semi-conducting materials (e.g., silicon wafers), or electrically conducting materials (e.g., metals, such as nickel, brass, steel, aluminum, gold, metalloids, alloys or electrically conductive polymers), polymers, organic polymers, conductive polymers, biopolymers, native biopolymers, metal coated with organic polymers, synthetic polymers, composite materials or any combinations thereof. The probe substrate material is also optionally solid or porous.

[0219] Probes are optionally produced using any suitable method depending on the selection of substrate materials and/or adsorbents. For example, the surface of a metal substrate can be coated with a material that allows derivatization of the metal surface. More specifically, a metal surface can be coated with silicon oxide, titanium oxide, or gold. Then, the surface can be derivatized with a bifunctional linker, one end of which can covalently bind with a functional group on the surface and the other end of which can be further derivatized with groups that function as an adsorbent. In another example, a porous silicon surface generated from crystalline silicon can be chemically modified to include adsorbents for binding analytes. In yet another example, adsorbents with a hydrogel backbone can be formed directly on the substrate surface by in situ polymerizing a monomer solution that includes, e.g., substituted acrylamide monomers, substituted acrylate monomers, or derivatives thereof comprising a selected functional group as an adsorbent. Probes suitable for use in the present invention are described in, e.g., U.S. Pat. Nos. 5,617,060, 5,894,063, 6,020,208, 6,027,942, 6,528,320, WO 98/59360 and U.S. Pat. Appls. Nos. 2003/0091976 and 2002/0060290, all of which are incorporated herewith by reference in their entirety.

2. Production of Antibodies and Immunological Detection of a T-cell Immunogenic Antigen

[0220] Antibodies can also be used to detect a T-cell immunogenic antigen identified by a method of the present invention. Antibodies to known T-cell immunogenic antigen may be commercially available (e.g., Santa Cruz Biotechnology) or can be produced using well known techniques (see, e.g., Harlow & Lane, Antibodies: A Laboratory Manual (1988) and Harlow & Lane, Using Antibodies (1999); Coligan, Current Protocols in Immunology (1991); Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975)). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al, Science 246:1275-1281 (1989); Ward et al, Nature 341:544-546 (1989)). Such antibodies are typically used for diagnostic or prognostic applications, e.g., in the detection of a malignant or autoimmune disease. [0221] T-cell immunogenic antigen or a fragment thereof may be used to produce antibodies specifically reactive with the T-cell immunogenic antigen. For example, a recombinant T-cell immunogenic antigen or an antigenic fragment thereof, is isolated as described herein. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used as an immunogen. Naturally occurring protein may also be used either in pure or impure form. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.

[0222] Typically, polyclonal antisera with a titer of 10 ⁴ or greater are selected and tested for their cross reactivity against non-T-cell immunogenic antigen proteins or even other related proteins from other organisms, using a competitive binding immunoassay. Specific polyclonal antisera and monoclonal antibodies will usually bind with a K _d of at least about 0.1 mM, more usually at least about 1 μM, optionally at least about 0.1 μM or better, and optionally 0.01 μM or better. For cross-reactivity determination, typically immunoabsorbed antisera are used in a competitive binding immunoassay to compare a second protein to the T-cell immunogenic antigen protein. In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required to inhibit 50% of binding is less than 10 times the amount of the antigenic T-cell immunogenic antigen protein that is required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to the T-cell immunogenic antigen. [0223] Exemplary antibodies may be made according to methods known in the art against the transthyretin and calprotectin antigens described herein or fragments thereof. Monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies and fragments thereof raised against these antigens are useful for immunological detection of a cancer.

[0224] Once T-cell immunogenic antigen-specific antibodies are available, binding interactions with T-cell immunogenic antigen can be detected by a variety of immunoassay methods. For a review of immunological and immunoassay procedures, see, Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991). Moreover, the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra).

[0225] Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen. The labeling agent may itself be one of the moieties comprising the antibody/antigen complex. Thus, the labeling agent may be a labeled T-cell immunogenic antigen polypeptide or a labeled anti-T-cell immunogenic antigen antibody. Alternatively, the labeling agent may be a third moiety, such as a secondary antibody, that specifically binds to the antibody/ antigen complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived). Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the labeling agent. These proteins exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species {see, e.g., Kronval et al, J. Immunol. 111 :1401-1406 (1973); Akerstrom et al, J. Immunol. 135:2589-2542 (1985)). The labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art.

[0226] Commonly used assays include noncompetitive assays, e.g., sandwich assays, and competitive assays. In competitive assays, the amount of T-cell immunogenic antigen- present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) T-cell immunogenic antigen displaced (competed away) from an anti-T-cell immunogenic antigen antibody by the unknown T-cell immunogenic antigen-present in a sample. Commonly used assay formats include immunoblots, which are used to detect and quantify the presence of protein in a sample. Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules {e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques {see, Monroe et al., Amer. Clin. Prod. Rev. 5:34-41 (1986)).

[0227] The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels, enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).

[0228] The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

[0229] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to another molecule (e.g., streptavidin), which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. The ligands and their targets can be used in any suitable combination with antibodies that recognize T-cell immunogenic antigen, or secondary antibodies that recognize the anti-T-cell immunogenic antigen.

[0230] The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems that may be used, see, U.S. Patent No. 4,391 ,904.

[0231] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[0232] Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.

[0233] Although the forgoing invention has been described in some detail by way of illustration and example for clarity and understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain variations, changes, modifications and substitutions of equivalents may be made thereto without necessarily departing from the spirit and scope of this invention. As a result, the embodiments described herein are subject to various modifications, changes and the like, with the scope of this invention being determined solely by reference to the claims appended hereto. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed, altered or modified to yield essentially similar results. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. As used throughout this application, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Similarly, the words "include", "including", and "includes" mean including, but not limited to.

[0234] While each of the elements of the present invention is described herein as containing multiple embodiments, it should be understood that, unless indicated otherwise, each of the embodiments of a given element of the present invention is capable of being used with each of the embodiments of the other elements of the present invention and each such use is intended to form a distinct embodiment of the present invention. Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0235] The referenced patents, patent applications, and scientific literature, including accession numbers to GenBank database sequences, referred to herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.

[0236] As can be appreciated from the disclosure above, the present invention has a wide variety of applications. The invention is further illustrated by the following examples, which are only illustrative and are not intended to limit the definition and scope of the invention in any way. III. EXAMPLES

Example 1: General Material And Methods a) Cell Culture and Patient Samples

[0237] Tumor cell lines (RMA, RMA-OVA, HepG2, HNO41) were grown as described (Herold-Mende et ah, 1999, Lab Invest 79:1573-1582). For tissues and blood samples used informed consent was obtained from patients according to the research proposals approved by the Institutional Review Board at the Medical Faculty Heidelberg. For PF2D analysis cells or tissues were lysed in 2ml lysis buffer (7.5 M urea, 2.5 M thiourea, 12% glycerol, 50 mM tris, 2.5% n-octylglycoside, 6.25 mM TCEP tris-(carboxyethyl) phosphine hydrochloride, 1.25 mM protease inhibitor, SIGMA- Aldrich, St.Louis; Billecke et al, 2006, MoI Cell Proteomics 5:35-42 Gunther et al, 2006, J Ind Microbiol Biotechnol 33:914-920). After centrifugation lysates were desalted and buffer was exchanged to start buffer (supplied by Beckman Coulter, Krefeld, Germany as component of ProteomeLab PF 2D kit # 390977) using PDlO sephadex G-25 columns (Amersham, Freiburg, Germany). After elution of samples with 3.5 ml start buffer (supplied by Beckman Coulter, Krefeld, Germany as component of ProteomeLab PF 2D kit # 390977), protein content was measured by Micro BCA Protein Assay Kit (ThermoScientific, Rockford, IL, USA). b) PF2D-2 Dimensional Liquid Chromatography

[0238] 1 mg ovalbumin or 2.5 mg lysate dissolved in start buffer (supplied by Beckman Coulter, Krefeld, Germany as component of ProteomeLab PF 2D kit # 390977) were injected into the ID column of the PF2D system according to the manufacturer's instructions (Beckman Coulter). During chromatofocusing the samples were fractionated first in 6 x 1.5 ml fractions starting at pH 8.5 and in a pH gradient between pH 8.5 and 4 by an interval of 0.3 pH units at a flow rate of 0.2 ml/min. Fractions were collected and the absorbance data (detected at 280 nm) were analyzed for relevant protein peaks. 200 μl of the fractions showing protein peaks were sequentially injected to the 2D RP-HPLC column of the PF2D system according to the manufacturer's instructions (Beckman Coulter) and heated to 5O ⁰ C. An additional lOOμl of these fractions were purified by using Ami con ^® Ultra-4 Centrifugal Filter Units (Millipore, Billerica, MA) according to the manufacturer's instructions. The 2nd dimension consisted of a 30 min linear gradient starting from 5 to 100% acetonitril containing 0.08% trifluoroacetic acid into distilled water containing 0.1% trifluoroacetic acid at a flow rate of 0.75 ml/min. Eluted proteins were detected by UV at 214 nm and immediately collected in 96 deep-well plates (600 μl/well). The UV absorbance data were further analyzed by ProteoVue™ and DeltaVue™ software (Beckman Coulter). Positive fractions were dried by centrifugation in a speed vac centrifuge, resolved in 10 μl 100 mM ammonium bicarbonate and stored at -2O ⁰ C before subjecting them to further T-cell activation assays and protein identification by LC-ESI-MS/MS mass spectrometry (NextGen Sciences, Ann Arbor, USA, Protagen Dortmund, Germany). c) Dot Blot

[0239] Fractions collected after 2D separation were dotted on activated PVDF membrane (Roche, Mannheim, Germany) followed by incubation with blocking buffer (5% dry milk in TBST (10x TBS-T: 876.6 g NaCl (FW 58.44), 121.1 g Tris, 40 ml HCl, 0.1% Tween), rabbit anti-ovalbumin antibody (1:1000, SIGMA- Aldrich, Muenchen, Germany) and HRP-labeled donkey α-rabbit IgG antibody and visualized by ECL-Plus luminescence staining (all Amersham). d) Primary OT-I T-CeIl Activation [0240] Dendritic cells (DCs) were isolated from spleens of C57BL-6 mice by magnetic beads labeled with anti-CD 1 Ic antibodies (CDl Ic MACS-beads, Miltenyi, Bergisch- Gladbach, Germany). Institutional guidelines for animal welfare and experimental conduct were followed. Aliquots of DCs (2 x 10 ⁴ /well) were pulsed overnight with different concentrations of PF2D-fractionated OVA protein. After 3 hrs, 20 ng/ml LPS (Sigma- Aldrich) was added to induce DC maturation. CD8 ⁺ T-cells were purified by anti-CD8 magnetic beads (Miltenyi) from spleen cells of naive OT-I mice and cocultured with OVA- pulsed DCs in a ratio of 1:5 for 6 hrs (CD69 expression) or 24 hrs (proliferative activity). e) Flowcytometry

[0241] T-cells from stimulation cultures were stained with the following anti-mouse mAbs: anti-CD69-FITC, anti-CD8-PE (all BD, Heidelberg, Germany) for 30 min on ice. Dead cells were labeled with 1 μg/ml propidium-iodide (Abeam, Cambridge, UK) and excluded from analysis. Recordings were made from minimal 5 x 10 ⁴ cells on a FACS-Calibur (BD) using Flow Jo 4.3 software (TreeStar, San Carlos, CA, USA). f) T-CeIl Proliferation

[0242] After primary T-cell activation [ ³ H]Thymidine at 1 μCi/well was added for 16 hrs of culture and [ ³ H] incorporation was measured using a liquid scintillation counter (1450 MicroBetaTM, Perkin Elmer, Wellesley; USA). g) PCR

[0243] Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) was performed as described (Dictus et al, 2007, JNeurosci Methods 161 :250-258) using 2 μg of total RNA for reverse transcription. Amplification of cDNA was performed in 40 cycles at 58 ⁰ C annealing temperature. PCR was performed with primers purchased from TIB MolBio Syntheselabor GmbH (Berlin, DE). Nucleotide sequences of PCR primers used are listed in Table 1.

[0244] Table 1. PCR Primers

h) Immunofluorescence

[0245] Double immunofluorescent staining for TTR, calprotectin S 100/A9, DSG- 1 , DCD, GFAP, and CD31 was performed as described (Dictus et al., 2007, JNeurosci Methods 161 :250-258). Antibodies and their concentrations are listed in Table 2.

[0246] Table 2. Antibodies For Immunofluorescence

i) Antigens Used for Human T-CeIl Assays

[0247] Test antigens were: lysate from cryopreserved autologous NCH550 tumor after mechanical homogenization with an ultrathurrax (IKA, Staufen, Germany) and filtration (0.22 μm filter, Millipore, Schwalbach, Germany), synthetic polypeptides from MUCl, TTR, calprotectin subunits S100A8 and S100A9, DSG-I or dermcidine and synthetic HLA-I restricted peptides from TTR (all at 200 μg/ml; all produced through solid phase chemistry and HPLC purity-tested by the core facility for peptide synthesis of the German Cancer Center in Heidelberg, Germany) or total tumor protein fractions from 1 ^st or 2 ^nd dimensional PF2D separation. Control antigens were lysates from PBMCs of NCH550, human immunoglobulin (Endobulin, Baxter, Vienna, Austria) and/or HLA- A2 -binding peptide HIVgag _77-85 . j) Immunoprecipitation (IP) [0248] Immunoprecipitation (IP) was performed with the Immunoprecipitation Starter Pack (GE Healthcare) according to the manufacturer's instructions. To neutralize the probes

5OmM Tris (pH 7,4) was added to lOOμl of the ID fractions and 200μl of the 2D fractions to a final volume of ImI. A mix of affinity-purified mouse monoclonal antibodies raised against MUCl (5μg HPMV BD Pharmingen, 5μg BM3358 Acris) was incubated with the diluted fractions for 1 hour at 4° C and under continuous agitation. After antibody incubation, an equal mix of Protein A- and Protein G labeled-beads were added and incubated for 1 hour at 4°C and under continuous agitation. After two washing steps with 5OmM Tris (pH 7.4), an additional washing step with IP wash buffer was performed. The beads were then resuspended in 40μl Red Loading Buffer (New England Biolabs) and heated to 95°C for 3 minutes. The immunoprecipitated proteins were then subjected to the Western blot analysis. k) Western Blot Analysis

[0249] 20μl of the immunoprecipitated protein solution were loaded on 6% SDS-PAGE mini-gels, analyzed, and transferred to a PVDF Western Blotting Membrane (Roche Applied Science). Blocking of the membrane was done by incubating the PVDF membrane with 5% (w/v) nonfat dry milk in TBST (0.1% (v/v) Tween, pH 7.5) for 1 hour at room temperature and under continuous agitation. The membrane was then incubated with an antibody, e.g., an affinity-purified mouse monoclonal antibody raised against MUCl (HPMV diluted 1 :200, for 1 hour at room temperature under continuous agitation. After three washes with TBST, antibody binding was visualized by incubation with peroxidase-coupled anti-rabbit IgG antibody (diluted 1 : 10,000) in 5% (w/v) BSA in TBST for 1 hour at room temperature followed by chemiluminescence detection with the enhanced chemiluminescence system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

1) Generation of Human TCs and DCs

[0250] DCs were generated as described (Choi et al, 2005, Blood 105:2132-2134). In brief, adherenT-cells from peripheral blood (PB) were cultured for 7 days in serum- free X- VIVO20 medium containing 50 ng/ml rhuGM-CSF and 1,000 U/ml IL-4. DCs were enriched using anti-CD3 and anti-CD 19-coupled magnetic beads and pulsed for 2 hrs with test or control antigens.

[0251] Human T-cells were cultured for 7days in RPMI medium containing 10% human serum from the blood group A+B+, 100 U/ml IL-2 and 60 U/ml IL-4 followed by overnight incubation without interleukins and separation from contaminating cells by anti-CD 19, anti- CDl 5, and anti-CD56 magnetic beads (Feuerer et al, 2001, Nat Med 7:452-458). m) IFN-γ ELISPOT Assay [0252] The IFN-γ ELISPOT assay was used to identify the presence of T-cells reactive against tissue lysate, fractionated proteins of tissue lysates obtained by the PF2D method, of defined candidate antigens (proteins, polypeptides thereof or small HLA-restricted peptides thereof) in patients. A positive result demonstrates the presence of tissue or antigen-reactive T-cells in tested individual and indicates an immune response against such tissue/antigen that may be caused by a disease and which might influence the course of the disease or which might indicate a disease situation. A negative result indicates the absence of a T-cell response against a diseased tissue or a defined antigen, etc. In this context, IFN-γ-producing T-lymphocytes were determined as described (Feuerer et al., 2001, Nat Med 7:452-458). Briefly, DCs were pulsed with test- (see above), or negative control antigens (e.g. lysate of autologous PBMC, human IgG, HIV, or sources of other disease unrelated antigens) and incubated in 3-5 wells/test group with autologous T-cells in a ratio of 1 :5 for 40 hrs. IFN-γ spots were measured using KS ELISPOT software (Zeiss, Jena, Germany). Spots induced by control antigens were considered as background. Test antigens were considered to be recognized by the patient's T-cells if spot numbers in the presence of test peptides were significantly higher (p<0.05) than in negative control wells. n) Expression Of S100A9 And TTR cDNAs

[0253] cDNA clones encoding human S 100A9 (IRATp970G0775D, accession no. BC047681) and human TTR (IRALρ962D2416Q, accession no. BC005310) were obtained from imaGenes, Berlin, Germany. The S100A9 and TTR full-length cDNAs were subcloned from pCMV-Sportό and pDNR-LIB vectors, respectively, into the expression vector pcDNA3.1+ (Invitrogen, Karlsruhe, Germany) and confirmed by DNA sequencing. The pcDNA3.1+ plasmids were transiently transfected into COS-7 cells cultured in 6-well plates by using Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. At day 3 after transfection, expression of S 100 A9 and TTR proteins was confirmed by immunocytochemistry using mouse anti-MRP- 14(Sl 00 A9) or sheep anti-TTR antibodies and peroxidase-labeled secondary reagents. Strong expression could be detected in 30-40% of COS-7 cells. For use in the IFN-γ ELISPOT-assay, COS-7 cells were transfected with either pcDNA3.1+/S100A9 or pcDNA3.1+/TTR together with pcDNA plasmids containing either HLA-A*0101, HLA-A*0301, HLA-B*0801, HLA-B*3501, or HLA-Cw*0702 cDNAs. o) HLA-Typing

[0254] HLA-typing of patient NCH550 was performed at the Department of Transplantation Immunology, Heidelberg, Germany as described (Heinold et al, 2007, Tissue Antigens 70:319-323). p) Statistical Analysis

[0255] Statistical evaluation was performed using an unpaired two-sided Student's T- test.

Example 2: T-CeIl Response To A Defined Antigen After PF2D Fractionation

[0256] To evaluate the influence of the PF2D separation method on the immunogenicity of proteins, OVA was chosen as a model protein recognized by OT-I CD8 ⁺ T-cells expressing an OVA-specific T-cell receptor. First, it was explored whether processing of purified OVA by using PF2D technology results in eluted proteins that can be recognized by OVA-specific T-cells, i.e., whether separation of OVA can be realized by the PF2D technology. A pure sample of recombinant OVA protein was injected to the l ^st -dimensional (ID) column; eluted fractions were monitored at 280 nm and automatically collected into a 96 deep-well plate. The 1 ^st and chromato focusing dimension of the PF2D system runs a pH gradient between pH 8.5 and 4. The ID elution profile shows protein peaks eluted between 70 and 90 min retention time (RT), corresponding to fractions 15-18 and to a pH range of 5.58-4.38 (Fig. Ia).

[0257] ID fractions were subjected to the 2nd-dimensional (2D) separation based on hydrophobicity. In the 2D profile, a major protein peak (P) was eluted at a RT of 23.5 min from ID fraction 16 and collected in well 32 (W32, Fig. Ib). Subsequent dot blot analysis confirmed eluted protein solution of W32 as OVA (Fig. Ic).

[0258] To test if OVA protein can also be separated from a complex proteome, OVA- transfected RMA cells were lysed and 2.5 mg of protein lysate were loaded to the PF2D system and run both the chromatofocusing and the reversed phase dimension. Non- transfected RMA cells were processed likewise as negative control. Comparative analysis of eluted 2D protein peaks from both cell types was performed by DeltaVue™ software. Differentially expressed peaks were identified in fraction 16 (Fig. Id) that turned out to contain OVA in the case of the RMA-OVA lysate. Additionally, 15 μl of each 2D fraction (corresponding to about 3% of the harvested fluid per fraction) were spotted o PVDF membranes and membranes were probed with anti-OVA antibodies (Fig. 1 e). Again, OVA was identified in fraction 16 at RT between 24.4 and 24.8 min by dot blot method (W33) and one of the differentially expressed peaks was confirmed as OVA. OVA was neither detected in adjacent fractions (W31-32, W34-35) nor in corresponding fractions of the PF2D separation of non-transfected RMA cells (W31-35). Altogether, after PF2D processing OVA was found in both the single protein as well as in the complex cell lysate approach in fraction 16 of ID separation and at similar RTs. Slight differences of RTs might be explained by the complexity of simultaneously present proteins in cell lysates possibly influencing each others' RT minimally.

[0259] Immunogenicity of eluted fractions was tested by T-cell activation assays. To avoid cytotoxic effects in functional assays, collected proteins were neutralized. DCs from C57BL- 6 mice were pulsed with proteins from fraction 16 separated by PF2D either from pure OVA samples or from RMA-OVA cells. After co-culture with T-cells from OT-I mice expressing the OVA-specific T-cell receptor, OVA-specific activation of T-cells was monitored analyzing the intensity of CD69 expression by CD8 ⁺ T-cells through flowcytometry and analyzing T-cell proliferation. Even DCs pulsed by 1 μg OVA induced a significant activation of more than 60% of T-cells while 5 μg OVA induced activation of nearly all T- cells (Fig. If). Accordingly, DCs from C57BL-6 mice pulsed with W33 — the 2D OVA- containing fraction of fractionated RMA-OVA lysate — induced activation of the majority of cocultured naϊve OT-I CD8 ⁺ T-cells as shown by increased CD69 expression (Fig. Ig). ³ H- thymidine uptake of CD8 ⁺ T-cells after co-culture with pulsed DCs revealed that T-cells originating from the experiment with OVA-containing RMA-OVA lysates showed a considerably (approximately two-fold) higher proliferation than the cells from OVA- free experiments (Fig. Ih). Noteworthy, proliferation rate of T-cells from the OVA-free experiment was similar to the one of T-cells co-cultured with unpulsed DCs. To Applicants' knowledge, these experiments demonstrated for the first time that proteins processed by PF2D can be cross-presented by antigen-presenting cells, such as DCs, via MHC-I molecules, allowing for their recognition by CD8 ⁺ T-cells.

Example 3: Identification Of Potential Immunogenic Proteins

[0260] To investigate if the PF2D system is suitable for identification of T-cell target antigens in human diseased tissues, it was investigated if a known tumor antigen could be recognized by autologous T-cells within separated tumor protein fractions. [0261] MUCl (mucin-1) was selected as a model tumor antigen because it is abundantly expressed in many epithelial cancers, immunogenic (Girling et al., 1989; Int J Cancer 43:1072-1076) and can be detected reliably in tumor lysates by immune precipitation (IP) using a MUCl -specific mAb. An ex vivo short-term IFN-γ ELISPOT-assay was applied exclusively allowing for the detection of pre-existing memory T-cells (Mύller-Berghaus et al., 2006, Cancer Res 66:5997-6001) using autologous DCs from the patients as antigen- presenting cells. 2x10 ⁴ DCs/triplicate well were pulsed with synthetic long peptides derived from MUCl , with autologous tumor lysate as a source of undefined TAAs or with respective negative control antigens (hulgG or autologous PBMC lysates). By this method, in head and neck cancer patients the presence of T-cells reactive against synthetic long peptides derived from the MUCl signaling sequence (pi -100, MUC lss) or from the tandem repeat region

(pl37-157, MUCltr) was tested. One patient (HNl) showing a robust T-cell response against MUC ltr but not MUC lss was identified (Fig. 2a). [0262] Next, it was tested if T-cells from a patient showing strong anti-MUCl T-cell response would be able to selectively recognize fractions of the separated tumor proteome that contained MUCl . Cryopreserved tumor lysate from HNl was separated by PF2D, the obtained protein preparations was pulsed onto autologous DCs and the reactivity of the patient's T-cells against the proteins present in these fractions was evaluated by IFN-γ

ELISPOT. A dominant T-cell reactivity against proteins in fraction A was found and only in this fraction MUCl protein was detected by IP (Figs. 2b, 2c). Thus, the T-cells of this patient selectively recognized the protein fraction that contained MUCl protein as demonstrated by immune precipitation. [0263] In order to validate this observation, and in order to test if the PF2D technique could also be used to identify de-novo MUCl -specific T-cell responses in a patient, another head and neck cancer patient with a strong T-cell response against autologous tumor lysate was identified and selected. This head and neck cancer patient (HN2) showed in an ex vivo ELISPOT-assay a strong T-cell reactivity against autologous tumor tissue-derived antigens (Fig. 2d). T-cell reactivity against separated protein fractions of the autologous tumor tissue was tested in this patient as described above. The tumor proteome was fractionated as before and a few fractions that were strongly recognized by that patient's T-cells were identified. As sown in Figure 2e, this patient's T-cells reacted against a variety of fractions, including fraction A. By IP, the presence of MUCl protein again was detected exclusively in fraction A.

[0264] In order to assess, whether MUCl in this fraction was a target antigen of the T-cells from this patient, T-cell reactivity against synthetic MUC ltr was evaluated. In other words, it was tested if the pre-existing cell repertoire of patient HN2 contained MUCl-specufic T- cells. Indeed, a strong T-cell response against MUCl in this patient was observed (Fig. 2f). These observations suggest that pre-existing, tumor antigen specific T-cells in cancer patients can identify PF2D-separated protein fractions containing the respective tumor antigen. Thus, this method is well suitable for identification of so far unknown, immunogenic tumor antigens.

[0265] To further investigate if the PF2D system is suitable for identification of T-cell target antigens in human diseased tissues, exemplary tumor patients containing tumor tissue- reactive memory T-cells in their peripheral blood were searched and identified. By this method spontaneous T-cell reactivity against tumor tissue antigens in 11 different patients (4 patients with brain tumors, 2 patients with colorectal carcinomas and 5 patients with head and neck cancer) were analyzed. 10 of these patients contained tumor-reactive T-cells in their blood, while one patient was negative (data not shown). Among those patients, a patient, referred to herein as NCH550, was identified having an astrocytoma WHOIII containing tumor-reactive T-cells in his blood at a frequency of approximately 200/10 ⁵ total T-cells (Fig. 3a).

[0266] An ex vivo short-term IFN-γ ELISPOT-assay was applied exclusively allowing for the detection of pre-existing memory T-cells (Mύller-Berghaus et al, 2006, Cancer Res 66:5997-6001) using autologous DCs from the patients as antigen-presenting cells. 2 x 10 ⁴ DCs/triplicate well were pulsed with autologous tumor lysate as a source of TAAs.

[0267] 2.5 mg of a tumor lysate from patient NCH550 were subjected to the PF2D ID separation to identify major immunogenic fractions. 100 μl of 12 ID fractions displaying protein peaks in UV-spectrum were purified by ultrafiltration and used to pulse autologous DCs. Autologous PBMC lysate (PB-L) served as negative control antigen. 7 fractions (FlO, F13-15, F18-19, and F21) were significantly recognized by autologous T-cells (indicated by asterisks in Fig. 3b). These fractions were transferred to the 2D fractionation resulting in 40 subtractions each. Again, only subtractions displaying protein peaks in UV-spectrum were further tested for T-cell recognition resulting in up to two immunogenic fractions originally derived from the ID fractions FlO, F14, F18, and F21 (Fig. 3c). Subtractions of the highly immunogenic fraction 15 did not cause significant T-cell stimulation which might be due to an even distribution of multiple, weakly immunogenic antigens or to a loss of the antigenic determinant during the second separation procedure. For validation, the fractionation procedure was repeated with freshly prepared tumor lysate. Respective reactivity of newly obtained PBT-cells against identified immunogenic 2D fractions demonstrated stable T-cell reactivity in the patient against 4 out of 6 fractions (indicated by asterisks in Fig. 3d).

[0268] In order to evaluate the applicability of this method to patients with lower frequencies of tumor reactive T-cells, PF2D fractionation and immunogenicity testing was performed with fractionated tumor tissue antigens from the other 10 tested patients. Among those 10 patients, in all 9 patients showing tumor reactive T-cells against non-separated whole tumor tissue lysate in the pre-test, significant T-cell reactivity against one or more tumor protein fractions was detected. In contrast, the patient without detectable T-cell reactivity against tumor tissue lysate did not show any significant response against any separated tumor protein fraction (data not shown). Thus, this approach is broadly applicable to many cases and tumors.

[0269] The proteins that were recognized by T-cells in the exemplary patient HCH550 were identified. Mass spectrometric identification of the remaining proteins within these 6 fractions resulted in the identification of several proteins (Fig. 3e) within each fraction. Some of them, such as keratins were equally present in all wells, irrespective of their immunogenicity, suggesting exogenous contamination of the test wells which is a well- known phenomenon in proteomics research (Keller et ah, 2008, Anal Chim Acta 627:71-81). This was also the likely explanation for the presence of dermcidine, which was detectable in most fractions, including non-immunogenic fraction F18-j. Others can be considered as house-keeping antigens (actin) or unspecific, blood-derived contaminations of the lysate (hemoglobins) and therefore were not further tested for antigenicity. The remaining identified proteins were different isoforms of transthyretin (TTR), the calprotectin subunits S100A8 and S100A9, desmoglein-1, hornerin and lipocalin-1 (Fig. 3e). [0270] The above experiments were repeated in 10 additional brain tumor patients, referred to as NCH654, NCH655, NCH656, NCH656b, NCH656c, NCH657, NCH658, NCH660d, nch660h, and NCH662a. Surprisingly, immunogenicity of these antigens could be confirmed in 4 out of those 10 brain tumor patients (Figure 7A). These result confirmed the usefulness of the methods of the present invention. Example 4: Detection Of Expression of Antigens By RT-PCR And

Immunofluorescence

[0271] The expression of the antigens identified in Example 3 was evaluated by RT-PCR and immunofluorescence in the tumor tissue of patient NCH550. mRNA expression of TTR, calprotectin/S100A8, calprotectin/S100A9, dermcidine, and desmoglein-1 was confirmed. (Fig. 4a). Expression of hornerin was not detected (Fig. 4a). Thus, presence of hornerin was considered to result from sample handling.

[0272] Subsequent protein analysis by immunofluorescence revealed heterogeneous expression of TTR on GF AP -positive tumor cells but not on endothelial cells while calprotectin/S100A9 and desmoglein-1 were found on a minor subpopulation of both cell types. In contrast, dermcidine, by immunofluorescence was only detectable on endothelial cells (Fig. 4b). [0273] Taken together the data from Examples 3 and 4 demonstrated the identification of TTR, calprotectin/SlOOAδ, calprotectin/S100A9, desmoglein-1, and dermcidine as potential immunogenic tumor-derived proteins.

Example 5: Immunogenicity And Epitope Characterization Of Potential TAAs Identified Bv PF2D

[0274] To evaluate if identified proteins were the target antigens of the observed T-cell responses in patient NCH550, large, overlapping polypeptides of the respective antigens were synthesized and used as test antigens in IFN-γ ELISPOT-assays with patient-derived T-cells. Because proteins instead of cell lysates were analyzed, human IgG was used as an additional negative control antigen for DC pulsing. Amino acid sequences of the different TTR subunits and the resulting T-cell responses against the respective synthetic peptides are shown in Figures 5a-b. To exclude the possibility that T-cells responded against potential mutated epitopes of tumor cell-derived TTR, the complete TTR gene from tumor-derived RNA of patient NCH550 was sequenced. No mutations were detected (data not shown). Further, TTR] _0I-I47 (TTR3) was identified as the most immunogenic region of TTR (Fig. 5b). A more detailed analysis of TTR3 recognition by ex vivo purified CD4 ⁺ or CD8 ⁺ T-cells revealed the presence of TTR3 -specific T-cells in both T-cell populations (Fig. 5 c), with a particularly strong CD8 T-cell response. Interestingly, TTR ₃ recognition was also even stronger than recognition of whole tumor lysate. This may be attributed most likely to much higher concentrations of the synthetic peptide. Based on the observed T-cell responses to TTR peptides the immunogenic region could be confined to an epitope of 19 amino acids (Fig. 5d). On the basis of well established algorithms for prediction of HLA-allele-restricted epitopes (Keller et al, 2008, Anal Chim Acta 627:71-81), 16 different peptides were identified as potential candidates for presentation by the patient's HLA-I alleles HLA- A*0101, HLA-A*0201, HLA-B*4101 and HLA-B*5101 to CD8 ⁺ T-cells (Fig. 5e, Table 3).

[0275] Table 3. Potential HLA-I restricted epitopes of TTRioi-i ₂ o( _a igispfliehaewftand) according to the epitope prediction algorithm SYFPEITHI (Koch et al, 1996, APMIS 104:115-125).

Length = number of amino acids. Epitopes with potential binding capacity to more than one HLA-I allele are underlined.

[0276] These peptides were synthesized and used as test antigens in ELISPOT-assays. As additional appropriate negative control antigen, an HLA-A*0201 -restricted peptide derived from HIVgag was used. Five out of the 16 tested peptides, restricted to at least 2 HLA-I alleles caused a significant T-cell response, demonstrating that the repertoire of TTR-specifϊc CD8 ⁺ T-cells was polyvalent (Figs. 5f-g).

Example 6: Detection Of Immunogenic Antigens

[0277] Analogously, it was evaluated if calprotectin was the immunogenic antigen in fractions Fl 4-1, F 14-j and F18-c. Calprotectin consists of two homologous subunits, S100A8 and S100A9 and some shorter variants. Large overlapping peptides of these subunits were generated (Fig. 6a) and their recognition by the patient's T-cells was tested using the IFN-γ ELISPOT assay. T-cell recognition was found only for the calprotectin/S 100A9 (Fig. 6b), predominantly for S100A9i _-60 . To define the immunogenic epitope in more detail, overlapping 20-mer peptides of the respective region were synthesized (Fig. 6c). Using these peptides in the assay of above S 1 OO A9 _{1 1 -30} was identified as the immunogenic epitope (Fig. 6d; immunogenic region: RNIETIINTFH QYSVKLGHP). This epitope was recognized by CD8 ⁺ as well as CD4 ⁺ T-cells of patient NCH550 (Fig. 6e).

[0278] Desmoglein-1 and dermcidine were also tested for recognition by patient's T-cell repertoire. Neither of these antigens was recognized in repeated testing (Figs. 5f-h). Example 7: T-CeIl Responses Against TTR and S100A9 Are Common

In Brain Tumor Patients

[0279] Next it was assessed if the two antigens that were identified as potential major target antigens of tumor-specific T-cells in one malignant brain tumor might also represent major target antigens in other brain tumor patients. To this end ex vivo IFN-γ ELISPOT assays were performed to test the specific reactivity of blood-derived T-cells from 10 additional brain tumor patients (9 gliomas and 1 medulloblastoma) against synthetic peptides derived from the most immunogenic epitopes of TTR ₍ ioi-i4 ₇ ) and SlOOA9( _1- eo), using human IgG as a negative control antigen. As shown in Figure 7a TTR-specific T-cell reactivity was detected in 4 patients and S100A9-specific T-cells was detected in 3 patients. Interestingly, the latter patients also showed significant T-cell reactivity against TTR. Therefore, TTR and S100A9 appear to be common target antigens in brain tumors.

[0280] The proteome of DCs can differ from that of tumor cells, leading to a potentially different repertoire of peptides presented by both cell types. Moreover, spontaneous T-cell responses recognizing tumor antigens presented by DCs but not by tumor cells- or tumor stroma cells might be functionally irrelevant. Since a particularly strong T-cell response against DC-presented TTR and S100A9 was observed in patient NCH656b (Fig. 7a) it was assessed in this patient whether these S100A9- and TTR-specific T-cells were also able to recognize both antigens when they were expressed by tumor cells. Therefore, COS7 tumor cells were transfected with the respective antigens with (test group) or without (negative control group) cotransfection of the respective HLA-I molecules expressed by patient

NCH656b and co-cultured them with the patient's T-cells in an IFN-γ ELISPOT-assay. As shown in Figures 7b-c, significant T-cell responses against both antigens was obtained: S100A9-specific T-cell responses were restricted to HLA-A02 and HLA-B08 (Fig. 7b), while TTR-specifϊc T-cells were restricted to B08, B51 and CwO7 (Fig. 7c). Thus, the observed T- cell responses were able to recognize candidate TAAs also when these were endogenously processed and presented by tumor cells. Example 8: Discussion

[0281] Applicants herein describe, in particular in Examples 1 -6, a new, unbiased, inexpensive, and quick approach to identify candidate T-cell immunogenic antigens expressed by various cell types, including stroma cells in tissues on the basis of a patient's own repertoire of antigen-specific memory CD4 ⁺ T-helper and CD8 ⁺ cytotoxic T-cells. An embodiment of this method is shown in Figure 8. Protein separation, identification and a series of 4-5 subsequent ELISPOT assays to identify and verify T-cell target antigens in a patient's tissue can be performed within 2 months. Other methods known in the art typically take more than a year. This new approach became technically feasible by the recent introduction of the automated PF2D system for two-dimensional protein separation. This system provides liquid protein fractions which are directly accessible for identification and for transfer to functional assays. Although the fractionation process occurs under partially denaturing conditions, antigen-uptake, antigen-processing and cross-presentation by DCs were not impaired. This is in accordance with previous observations that denaturation of proteins increases their uptake, processing and presentation by DCs and thereby their overall immunogenicity (Koch et al, 1996, APMIS, 104:115-125; Schirmbeck et al, 1995, J Immunol 155:4676-4684; Schirmbeck et al, 1994, Eur J Immunol 24:2068-2072). Importantly, amounts of purified proteins as obtained from the separation process were sufficient to allow recognition by memory T-cells. Purified and fractionated OVA induced activation of naϊve OT-I T-cells at concentrations of 1 μg/ml. Fractionated OVA from RMA- OVA cells was still recognized at about 10 to 50-fold lower concentrations. Compared to naϊve T-cells that were used for OVA experiments, memory T-cells responding to antigens in the human situation need much lower amounts of antigen for activation (Zinkernagel et al, 1996, Annu Rev Immunol 14:333-367). As demonstrated by the identification of two new immunogenic antigens in an astrocytoma WHOIII patient (NCH550), the data presented herein demonstrate that methods of the present invention possess a sufficient sensitivity to identify any major immunogenic determinant in a tissue. Methods of the present invention are sensitive enough to detect approximately three specific T-cells in a mixture of about 100,000 T-cells. [0282] Since methods described herein are based on the processing of proteins by the immunoproteasome of DCs, the possibility that respective T-cell responses are restricted to antigen-presentation by professional antigen-presenting cells such as DCs or macrophages could not be excluded and thus, antigens presented by tumor cells or tissue stroma cells may not be directly targeted. In order to address this, testing T-cell reactivity against MHC- deficient tumor cell lines transiently transfected with respective HLA-molecules and one of the newly identified antigens TTR and S100A9, respectively, was performed. The results obtained confirmed the relevance of the postulated tumor-associated antigens in a HLA- restricted fashion. [0283] In contrast to other approaches, the methods of the present invention allow an immediate detection of CD4 and CD8 T-cell responses ex vivo against intact proteins independent of HLA subtypes, the cellular source of the antigen, and the current state of mRNA expression within the tissue. The latter point is of importance, since many approaches of antigen identification are based on differential gene expression without recognizing the more relevant differences in protein expression (Zinkernagel et al., 1996, Annu Rev Immunol 14:3330367). Additionally, the observation that even different isoforms and modifications of proteins can be separated by PF2D technology (Sheng et al, 2006, MoI Cell Proteomics 5:26-34) in combination with the findings presented herein makes this newly introduced system highly suitable in quest for T-cell target antigens. Subsequent testing of T- cell responses against tumor cells transfected with candidate antigens and HLA molecules further provides helpful information regarding the potential functional relevance of such response and the suitability of the identified antigens as candidates for therapeutic vaccination.

[0284] Using methods of the present invention, four different proteins, TTR, calprotectin, desmoglein-1 , and dermcidine, which were expressed by tumor cells in the respective tumor tissue on mRNA and on protein level (TTR, calprotectin/S 100 A9, , DSG- 1 ;) or by endothelial cells (calprotectin/S 100 A9, , DSG-I, dermcidine) were identified in immunogenic protein fractions. Of these, only TTR and calprotectin/S 100 A9, were target antigens of spontaneous CD4 and CD8 T-cell responses demonstrating that spontaneous anti-tumor immune responses are not exclusively directed against tumor cell-associated antigens but can be induced against tumor-stroma-associated antigens as well. Anti-stroma cell immune responses have been hitherto largely unrecognized but may play a fundamental role in immunopathology. [0285] TTR, a low molecular weight protein, is involved in the transport of thyroxin and retinol by interacting with retinol-binding protein (Hou et al, 2007, FEftSJ274:1637-1650; Irace and Edelhoch, 1978, Biochemistry 17:5729-5733; Peterson, 1971, J Biol Chem 246:44- 49). Under normal conditions it is mainly found in the liver and to a lesser extent in the pancreas, the brain and the blood (Shmueli et al, 2003, C R Biol 326:1067-1072). TTR- defϊcient mice have lower levels of retinol and retinol-binding protein which in turn was shown to be associated with malignant transformation of the ovarian epithelium (Roberts et al, 2002, DNA Cell Biol 21 :11-19; van Bennekum et al, 2001, J Biol Chem 276:1107-1113). Accordingly, an upregulation of TTR in lung adenocarcinoma patients with brain metastasis has been reported (Marchi et al, 2008, Cancer 112:1313-1324; Park et al, 2009, J

Neurooncol 94(1 ):31-9). This is in agreement with the findings described herein that an increased TTR expression is part of the high-grade proteomics signature of WHOIV gliomas.

[0286] Calprotectin is a Ca ²⁺ -binding protein that is overexpressed in a variety of human tumors including breast, lung, gastric, and pancreatic cancer (El-Rifai et al, Cancer Res 62:6823-6826; Seth et al, Anticancer Res 23:2043-2051; Shen et al, 2004, Cancer Res

64:9018-9026). While in the undiseased organism it is predominantly found in the blood, a strong upregulation occurs in acute inflammatory processes like appendicitis (Shmueli et al, 2003, C R Biol 326:1067-1072; Cross et al, 2005, Histopathology 46:256-269; Newton and Hogg, 1998, J Immunol 160:1427-1435). Regarding its functional activity in tumors it has been shown that it contributes to the invasive phenotype in gastric cancer cells, has chemotactic properties, and is a potent pro-inflammatory agonist (Yong and Moon, 2007, Arch Pharm Res 30:75-81; Newton and Hogg, 1998, J Immunol 160:1427-1435). Both proteins, TTR and calprotectin, might be considered as auto-antigens, since their expression is also found in healthy tissues. Still, the presence of high numbers of antigen-reactive type-1 CD4 ⁺ and CD8 ⁺ memory T-cells in the peripheral blood of the patient NCH550 and 10 additional brain tumor patients (see Figure 8) suggest a constant triggering of T-cell responses against these antigens which might be associated with their expression in the tumor tissue. Interestingly, both brain tumor tissue-associated antigens appear to represent common target antigens of tumor-specific T-cells since pre-existing T-cell responses with specificity against one or both antigens were detected in 4 of 10 additional patients.

[0287] Applicants demonstrated that proteins fractionated by PF2D can be efficiently taken up by DCs, processed, and cross-presented to antigen-specific T-cells and are suitable to specifically re-activate pre-existing memory CD4 and CD8 T-cell responses in patients. Therefore, natural repertoires of tumor-specific T-cells can be exploited by the PF2D technique to identify in tissues antigens or individual immunogenic antigens of potential clinical relevance.