PEPTIDES, COMBINATION OF PEPTIDES, AND CELL BASED MEDICAMENTS FOR USE IN IMMUNOTHERAPY AGAINST URINARY BLADDER CANCER AND OTHER CANCERS

Abstract

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Claims

1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-149, wherein the peptide comprises from 9 to 20 amino acids.

2. The peptide of claim 1, wherein said peptide has the ability to bind to an MHC class-I or -II molecule, and wherein said peptide, when bound to said MHC, is capable of being recognized by CD4 and/or CD8 T cells.

3. The peptide of claim 1, wherein the peptide is in the form of a pharmaceutically acceptable salt.

4. The peptide of claim 3, wherein the pharmaceutically acceptable salt is chloride salt or acetate salt.

5. The peptide of claim 1, wherein the peptide consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2-149.

6. A fusion protein comprising the peptide of claim 1 and N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii).

7. A fusion protein comprising the peptide of claim 1 and an antibody.

8. The fusion protein of claim 8, wherein the antibody binds to an dendritic cell.

9. A pharmaceutical composition comprising the peptide of claim 1.

10. The pharmaceutical composition of claim 9, further comprising an adjuvant.

11. The pharmaceutical composition of claim 10, wherein the adjuvant is selected from anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, particulate formulations with poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

12. A modified peptide comprising the peptide of claim 1, wherein said peptide comprises at least one non-peptide bond or at least one D-amino acid.

13. An in vitro method for producing activated T lymphocytes, comprising contacting in vitro T cells with antigen loaded human HLA-A*02:01 expressed on the surface of a suitable antigen-presenting cell or an artificial construct mimicking an antigen-presenting cell for a period of time sufficient to activate said T cells in an antigen specific manner, wherein said antigen is a peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2-149.

14. A method for producing a peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2-149, comprising culturing a host cell, and isolating the peptide from said host cell and/or culture medium thereof.

15. A method for killing target cells in a patient who has cancer, wherein the target cells present a peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2-149 in a complex with HLA-A*02:01, comprising administering to the patient the activated T cells produced by the method of claim 13, wherein said cancer is selected from the group of esophageal cancer, pancreatic cancer, gallbladder cancer, uterine cancer, head and neck squamous cell carcinoma, and bile duct cancer.

16. A kit comprising: a) a container comprising the pharmaceutical composition according to claim 9 in solution or in lyophilized form; b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; c) optionally, instructions for (i) use of the solution or (ii) reconstitution and/or use of the lyophilized formulation.

17. The kit according to claim 16, further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.

18. The pharmaceutical composition of claim 10, wherein the adjuvant is selected from the group consisting of IL-2, IL-12, and IL-15.

19. The pharmaceutical composition of claim 10, further comprising a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 10, further comprising a pharmaceutically acceptable stabilizer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0339] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0340] FIGS. 1A through 1R show the over-presentation of various peptides in normal tissues (white bars) and urinary bladder cancer (black bars). FIG. 1A: Gene symbol: GATA3, Peptide: VLFNIDGQGNHV (SEQ ID NO.: 110); Tissues from left to right: 3 adipose tissues, 3 adrenal glands, 16 blood cells, 15 blood vessels, 9 bone marrows, 10 brains, 7 breasts, 6 esophagi, 2 eyes, 3 gallbladders, 6 hearts, 12 kidneys, 19 large intestines, 19 livers, 45 lungs, 7 lymph nodes, 8 nerves, 3 ovaries, 8 pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal muscles, 12 skins, 3 small intestines, 11 spleens, 5 stomachs, 4 testes, 2 thymi, 2 thyroid glands, 9 tracheas, 6 ureters, 5 uteri, 8 urinary bladders, 15 urinary bladder cancer. The peptide has additionally been detected on 3/15 urinary bladder cancers. FIG. 1B: Gene symbol: KRT7, Peptide: GLLKAYSIRTA (SEQ ID NO.: 2); Tissues from left to right: 3 adipose tissues, 3 adrenal glands, 16 blood cells, 15 blood vessels, 9 bone marrows, 10 brains, 7 breasts, 6 esophagi, 2 eyes, 3 gallbladders, 6 hearts, 12 kidneys, 19 large intestines, 19 livers, 45 lungs, 7 lymph nodes, 8 nerves, 3 ovaries, 8 pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal muscles, 12 skins, 3 small intestines, 11 spleens, 5 stomachs, 4 testes, 2 thymi, 2 thyroid glands, 9 tracheas, 6 ureters, 5 uteri, 8 urinary bladders, 15 urinary bladder cancer. The peptide has additionally been detected on 3/15 urinary bladder cancers. FIG. 1C: Gene symbol: CXorf57, Peptide: YLDPSLNSL (SEQ ID NO.: 112); Tissues from left to right: 3 adipose tissues, 3 adrenal glands, 16 blood cells, 15 blood vessels, 9 bone marrows, 10 brains, 7 breasts, 6 esophagi, 2 eyes, 3 gallbladders, 6 hearts, 12 kidneys, 19 large intestines, 19 livers, 45 lungs, 7 lymph nodes, 8 nerves, 3 ovaries, 8 pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal muscles, 12 skins, 3 small intestines, 11 spleens, 5 stomachs, 4 testes, 2 thymi, 2 thyroid glands, 9 tracheas, 6 ureters, 5 uteri, 8 urinary bladders, 15 urinary bladder cancer. The peptide has additionally been detected on 3/15 urinary bladder cancers. FIG. 1D: Gene symbol: DYNC1H, Peptide: FVFEPPPGV (SEQ ID NO.: 113); Tissues from left to right: 3 adipose tissues, 3 adrenal glands, 16 blood cells, 15 blood vessels, 9 bone marrows, 10 brains, 7 breasts, 6 esophagi, 2 eyes, 3 gallbladders, 6 hearts, 12 kidneys, 19 large intestines, 19 livers, 45 lungs, 7 lymph nodes, 8 nerves, 3 ovaries, 8 pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal muscles, 12 skins, 3 small intestines, 11 spleens, 5 stomachs, 4 testes, 2 thymi, 2 thyroid glands, 9 tracheas, 6 ureters, 5 uteri, 8 urinary bladders, 15 urinary bladder cancer. The peptide has additionally been detected on 2/15 urinary bladder cancers. FIG. 1E: Gene symbol: SNRNP70, Peptide: YLAPENGYLMEA (SEQ ID NO.: 15); Tissues from left to right: 1 cell line (1 kidney), 25 cancer tissues (2 colon cancers, 1 leukocytic leukemia cancer, 3 liver cancers, 6 lung cancers, 3 lymph node cancers, 3 prostate cancers, 2 skin cancers, 3 urinary bladder cancers, 2 uterus cancers). FIG. 1F: Peptide: GLWHGMFANV (SEQ ID NO.: 10); Tissues from left to right: 8 cell lines (1 head-and-neck, 1 ovary, 2 lymphocytes, 3 skins, 1 head-and-neck), 1 normal tissue (1 spleen), 25 cancer tissues (1 brain cancer, 1 leukocytic leukemia cancer, 1 bone marrow cancer, 1 breast cancer, 3 colon cancers, 2 liver cancers, 1 head-and-neck cancer, 1 skin cancer, 2 ovarian cancers, 2 brain cancers, 7 lung cancers, 3 urinary bladder cancers); FIG. 1G: Peptide: RLSQLEGVNV (SEQ ID NO.: 17); Tissues from left to right: 7 cell lines (1 lymphocyte, 2 skins, 4 pancreas), 1 normal tissue (1 adrenal gland), 15 cancer tissues (1 liver cancer, 2 head-and-neck cancers, 2 ovarian cancers, 1 lung cancers, 1 kidney cancer, 3 lung cancers, 4 urinary bladder cancers, 1 uterus cancer); FIG. 1H: Peptide: GLALLYSGV (SEQ ID NO.: 26); Tissues from left to right: 7 cell lines (1 urinary bladder, 3 leukocytes, 3 pancreas), 1 normal tissue (1 lung), 18 cancer tissues (1 bone marrow cancer, 1 breast cancer, 1 leukocytic leukemia cancer, 7 liver cancers, 2 skin cancers, 1 stomach cancer, 1 lung cancer, 3 urinary bladder cancers, 1 uterus cancer); FIG. 1I: Peptide: GLIDSLMAYV (SEQ ID NO.: 28); Tissues from left to right: 2 normal tissues (1 head-and-neck, 1 thymus), 27 cancer tissues (11 head-and-neck cancers, 1 skin cancer, 1 lymph node cancer, 2 esophageal cancers, 9 lung cancers, 3 urinary bladder cancers); FIG. 1J: Peptide: SLIGGTNFV (SEQ ID NO.: 49); Tissues from left to right: 3 normal tissues (2 lymph nodes, 1 spleen), 38 cancer tissues (1 myeloid cells cancer, 2 leukocytic leukemia cancers, 1 bone marrow cancer, 1 prostate cancer, 3 breast cancers, 1 leukocytic leukemia cancers, 2 colon cancers, 7 liver cancers, 2 skin cancers, 8 lymph node cancers, 1 esophageal cancer, 4 lung cancers, 3 urinary bladder cancers, 2 uterus cancers); FIG. 1K: Peptide: ILLRDLPTL (SEQ ID NO.: 56); Tissues from left to right: 14 cell lines (9 lymphocytes, 4 begnin, 1 pancreas), 11 normal tissues (1 esophagus, 2 livers, 3 lungs, 1 parathyroid gland, 1 spleen, 1 stomach, 1 thyroid gland, 1 uterus), 72 cancer tissues (17 prostate cancers, 4 breast cancers, 2 colon cancers, 1 bile duct cancer, 1 gallbladder cancer, 1 liver cancer, 2 head-and-neck cancers, 1 skin cancer, 5 lymph node cancers, 3 ovarian cancers, 2 esophageal cancers, 1 esophagus and stomach cancer, 1 stomach cancer, 20 lung cancers, 1 pancreas cancer, 7 urinary bladder cancers, 3 uterus cancers); FIG. 1L: Peptide: GLDSSVNVQGSVL (SEQ ID NO.: 58); Tissues from left to right: 2 cell lines (2 leukocytes), 7 normal tissues (1 adrenal gland, 1 lymph node, 5 spleens), 40 cancer tissues (1 colon cancer, 1 bile duct cancer, 1 liver cancer, 2 head-and-neck cancers, 2 skin cancers, 4 lymph node cancers, 6 ovarian cancers, 1 esophagus and stomach cancer, 1 stomach cancer, 17 lung cancers, 3 urinary bladder cancers, 1 uterus cancer); FIG. 1M: Peptide: HLLDSKVPSV (SEQ ID NO.: 85); Tissues from left to right: 2 normal tissues (2 spleens), 25 cancer tissues (1 colon cancer, 2 liver cancers, 2 head-and-neck cancers, 2 ovarian cancers, 2 esophageal cancers, 1 brain cancer, 12 lung cancers, 3 urinary bladder cancers); FIG. 1N: Peptide: ALIGDDVGL (SEQ ID NO.: 99); Tissues from left to right: 6 normal tissues (2 leukocyte samples, 1 lymphocytes, 1 pancreas, 1 spleen, 1 thymus), 34 cancer tissues (1 myeloid cells cancer, 1 bone marrow cancer, 2 leukocytic leukemia cancers, 2 colon cancers, 1 colorectal cancer, 1 gallbladder cancer, 4 head-and-neck cancers, 2 skin cancers, 2 lymph node cancers, 2 ovarian cancers, 1 esophageal cancer, 1 stomach cancer, 9 lung cancers, 4 urinary bladder cancers, 1 uterus cancer); FIG. 1O: Peptide: FVFEPPPGV (SEQ ID NO.: 113); Tissues from left to right: 5 cell lines (1 skin, 1 urinary bladder, 1 ovary, 2 pancreas), 25 cancer tissues (1 prostate cancer, 1 breast cancer, 1 liver cancer, 2 head-and-neck cancers, 6 skin cancers, 1 lymph node cancer, 2 pancreas cancers, 1 brain cancer, 2 stomach cancers, 4 lung cancers, 4 urinary bladder cancers); FIG. 1P: Peptide: QLQGYLRSV (SEQ ID NO.: 121); Tissues from left to right: 10 cell lines (1 ovary, 2 primary cultures, 1 lymphocytes, 1 leukocytes, 1 kidney, 2 rectum, 2 pancreas), 7 normal tissues (1 adrenal gland, 1 leukocyte sample, 3 bone marrows, 1 colon, 1 trachea), 36 cancer tissues (1 cecum cancer, 1 myeloid cells cancer, 1 leukocytic leukemia cancer, 4 colon cancers, 1 rectum cancer, 1 bile duct cancer, 5 liver cancers, 6 head-and-neck cancers, 1 lymph node cancer, 1 esophageal cancer, 1 pancreas cancer, 1 brain cancer, 9 lung cancers, 1 urinary bladder cancer, 2 uterus cancers); FIG. 1Q: Peptide: ILEPSLYTV (SEQ ID NO.: 122); Tissues from left to right: 13 cell lines (1 kidney, 2 leukocytes, 1 skin, 9 pancreas), 10 normal tissues (1 adrenal gland, 5 colons, 1 liver, 2 lungs, 1 lymph node), 63 cancer tissues (2 myeloid cells cancers, 2 leukocytic leukemia cancers, 1 bone marrow cancer, 2 prostate cancers, 4 breast cancers, 2 leukocytic leukemia cancers, 1 colon cancer, 1 rectum cancer, 1 bile duct cancer, 7 liver cancers, 3 head-and-neck cancers, 5 skin cancers, 2 lymph node cancers, 2 ovarian cancers, 1 esophageal cancer, 1 pancreas cancer, 5 brain cancers, 4 stomach cancers, 5 lung cancers, 3 kidney cancers, 1 lung cancer, 4 urinary bladder cancers, 4 uterus cancers); FIG. 1R: Peptide: YLEPKLTQV (SEQ ID NO.: 129); Tissues from left to right: 25 cell lines (1 head-and-neck, 1 ovary, 1 PBMCs, 2 lymphocytes, 3 skins, 1 kidney, 16 pancreas), 8 normal tissues (3 adrenal glands, 1 bone marrow, 1 head-and-neck and salivary gland, 1 kidney, 1 liver, 1 spleen), 62 cancer tissues (1 myeloid cells cancer, 1 bone marrow cancer, 1 leukocytic leukemia cancer, 3 prostate cancers, 1 breast cancer, 1 colon cancer, 2 bile duct cancers, 6 liver cancers, 3 head-and-neck cancers, 5 skin cancers, 2 lymph node cancers, 3 ovarian cancers, 2 esophageal cancers, 2 brain cancers, 1 stomach cancer, 11 lung cancers, 1 kidney cancer, 4 lung cancers, 6 urinary bladder cancers, 6 uterus cancers).

[0341] FIGS. 2A through 2D show exemplary expression profiles of source genes of the present invention that are highly over-expressed or exclusively expressed in urinary bladder cancer in a panel of normal tissues (white bars) and 10 urinary bladder cancer samples (black bars). Tissue from left to right: 6 arteries, 2 blood cells, 2 brains, 1 heart, 2 livers, 3 lungs, 2 veins, 1 adipose tissue, 1 adrenal gland, 5 bone marrows, 1 cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gallbladders, 1 kidney, 6 lymph nodes, 4 pancreases, 2 peripheral nerves, 2 pituitary glands, 1 rectum, 2 salivary glands, 2 skeletal muscles, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 5 placentas, 1 prostate, 1 testis, 1 thymus, 1 uterus, 10 urinary bladder cancer samples. FIG. 2A: Gene symbol: TMPRSS4; FIG. 2B: Gene symbol: KRT7; FIG. 2C: Gene symbol: CYP4F22; FIG. 2D: Gene symbol: DHRS2.

[0342] FIG. 3 shows exemplary immunogenicity data: flow cytometry results after peptide-specific multimer staining.

[0343] FIG. 4 shows exemplary results of peptide-specific in vitro CD8+ T cell responses of a healthy HLA-A*02+ donor. CD8+ T cells were primed using artificial APCs coated with anti-CD28 mAb and HLA-A*02 in complex with SeqID No 30 peptide (A, left panel), SeqID No 50 peptide (B, left panel) and SeqID No 111 peptide (C, left panel), respectively. After three cycles of stimulation, the detection of peptide-reactive cells was performed by 2D multimer staining with A*02/SeqID No 30 (A), A*02/SeqID No 50 (B) or A*02/SeqID No 111 (C). Right panels (A, B and C) show control staining of cells stimulated with irrelevant A*02/peptide complexes. Viable singlet cells were gated for CD8+ lymphocytes. Boolean gates helped excluding false-positive events detected with multimers specific for different peptides. Frequencies of specific multimer+ cells among CD8+ lymphocytes are indicated.

EXAMPLES

Example 1

Identification and Quantitation of Tumor Associated Peptides Presented on the Cell Surface

Tissue Samples

[0344] Patients' tumor tissues were obtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK); Geneticist Inc. (Glendale, Calif., USA); ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd (Glasgow, UK); and University Hospital Tbingen (Tbingen, Germany)

[0345] Normal tissues were obtained from

Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-Options Inc. (Brea, Calif., USA); BioServe (Beltsville, Md., USA); Blutspendezentrale, Zentrum fr Klinische Transfusionsmedizin, Tbingen; Capital BioScience Inc. (Rockville, Md., USA); Geneticist Inc. (Glendale, Calif., USA); Kyoto Prefectural University of Medicine (KPUM) (Kyoto, Japan); ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva (Geneva, Switzerland); University Hospital Heidelberg (Heidelberg, Germany); University Hospital Munich (Munich, Germany); and University Hospital Tbingen (Tbingen, Germany)

[0346] Written informed consents of all patients had been given before surgery or autopsy. Tissues were shock-frozen immediately after excision and stored until isolation of TUMAPs at 70 C. or below.

Isolation of HLA Peptides from Tissue Samples

[0347] HLA peptide pools from shock-frozen tissue samples were obtained by immune precipitation from solid tissues according to a slightly modified protocol (Falk et al., 1991; Seeger et al., 1999) using the HLA-A*02-specific antibody BB7.2, the HLA-A, B, C-specific antibody W6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

[0348] The HLA peptide pools as obtained were separated according to their hydrophobicity by reversed-phase chromatography (nanoAcquity UPLC system, Waters) and the eluting peptides were analyzed in LTQ-velos and fusion hybrid mass spectrometers (ThermoElectron) equipped with an ESI source. Peptide pools were loaded directly onto the analytical fused-silica micro-capillary column (75 m i.d.250 mm) packed with 1.7 m C18 reversed-phase material (Waters) applying a flow rate of 400 nL per minute. Subsequently, the peptides were separated using a two-step 180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nL per minute. The gradient was composed of Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A gold coated glass capillary (PicoTip, New Objective) was used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometers were operated in the data-dependent mode using a TOP5 strategy. In brief, a scan cycle was initiated with a full scan of high mass accuracy in the orbitrap (R=30000), which was followed by MS/MS scans also in the orbitrap (R=7500) on the 5 most abundant precursor ions with dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted by SEQUEST and additional manual control. The identified peptide sequence was assured by comparison of the generated natural peptide fragmentation pattern with the fragmentation pattern of a synthetic sequence-identical reference peptide.

[0349] Label-free relative LC-MS quantitation was performed by ion counting i.e. by extraction and analysis of LC-MS features (Mueller et al., 2007). The method assumes that the peptide's LC-MS signal area correlates with its abundance in the sample. Extracted features were further processed by charge state deconvolution and retention time alignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MS features were cross-referenced with the sequence identification results to combine quantitative data of different samples and tissues to peptide presentation profiles. The quantitative data were normalized in a two-tier fashion according to central tendency to account for variation within technical and biological replicates. Thus each identified peptide can be associated with quantitative data allowing relative quantification between samples and tissues. In addition, all quantitative data acquired for peptide candidates was inspected manually to assure data consistency and to verify the accuracy of the automated analysis. For each peptide a presentation profile was calculated showing the mean sample presentation as well as replicate variations. The profiles juxtapose urinary bladder cancer samples to a baseline of normal tissue samples. Presentation profiles of exemplary over-presented peptides are shown in FIGS. 1A-1R. Presentation scores for exemplary peptides are shown in Table 8.

TABLE-US-00008 TABLE8 Presentationscores. SEQIDNo. Sequence PeptidePresentation 1 ILLQASVQV +++ 2 GLLKAYSIRTA +++ 3 YLDEIPPKFSM +++ 4 SLDVVNLLV +++ 6 SIVDFLITA +++ 7 QMFEGQILDV +++ 8 ALSFSSSAGPGLLKA +++ 9 SLVDARFQL +++ 10 GLWHGMFANV +++ 11 AMAELRVVV ++ 12 GVALTVTGV +++ 13 FLEEKEQAAL +++ 14 GLAGPVRGV +++ 15 YLAPENGYLMEA +++ 16 ILGPQGNTI +++ 17 RLSQLEGVNV + 18 SIAAYNPVV +++ 19 SLATTLTKI +++ 20 YLPDSLTQL +++ 21 TLIEDDALNGA +++ 23 YTLSKTEFL +++ 24 SLLGGITVV ++ 28 GLIDSLMAYV +++ 30 ILDISRSEV +++ 31 SLFDGIATGL +++ 33 VLFGEITRL ++ 34 ALLDEQQVNV ++ 35 KLPEPPPLA +++ 36 ALWDEFNQL +++ 37 ILSAILVHL +++ 38 TLTSIIVAV +++ 40 VIADRVVTV +++ 41 FLDDGNQMLL +++ 42 FLIDASQRV +++ 43 FLIESKLLSL +++ 44 GLAQDPKSLQL +++ 45 IIDSSPTAL +++ 46 SLFIGAEIVAV +++ 47 VLMDDTDPL +++ 48 VLMDDTDPLV +++ 49 SLIGGTNFV ++ 50 VLANRVAVV + 51 ALLDKAQINL ++ 53 ILVQVIPVV +++ 54 ALNDEINFL +++ 55 KLLETKWTL + 56 ILLRDLPTL ++ 57 GLAHFVNEI ++ 58 GLDSSVNVQGSVL +++ 59 WLSTSIPEA ++ 60 SLSDVRVIV ++ 62 GQLDFSEFL + 63 LLAGLLVGV +++ 64 GLLSQGSPL +++ 65 IITDLLRSV +++ 66 SLWEENQAL ++ 67 FLTPPLLSV +++ 68 TMIVSLAAV ++ 69 QIWDKILSV + 70 KLAEISLGV +++ 73 VLKVFLENV ++ 74 LLQEGEVYSA + 77 KVFGGFQVV ++ 80 ILLDTPLFLL +++ 81 SLDKGTLYI ++ 82 NLHNSYYSV + 83 VILDKYYFL + 84 ALDPASISV + 87 RLLELLQEA ++ 89 YLFPETEFI + 91 NLDAATYQV + 92 ALLDEQQVNVLL + 94 ALADGVPVAL ++ 96 KLTNGIWVL + 97 TVGPGLLGV ++ 98 YLIGLDPENLAL ++ 99 ALIGDDVGL ++ 100 SLQSFIHGV + 102 GLYEGLDWL +++ 103 GLYSGEVLV + 104 NAVVELVTV ++ 106 ILLDTPLFL + 107 LULAKLEKV +++ 110 VLFNIDGQGNHV +++ 111 LLDVTPKAV +++ 112 YLDPSLNSL +++ 113 FVFEPPPGV +++ 114 IITKDLFQV +++ 115 SLLDFERSL +++ 116 QLAWFDTDL + 117 YMLDIFHEVL +++ 118 RLLDFPTLLV +++ 119 SLDEKQNLV +++ 120 IIIPEIQKV ++ 121 QLQGYLRSV ++ 122 ILEPSLYTV +++ 123 NLAGVYSEV ++ 124 QIDGTLSTI ++ 125 VLDEGSASV ++ 126 SLLRVGWSV ++ 129 YLEPKLTQV ++ 130 TLTSKLYSL + 132 YILEGEPGKV ++ 133 GLDPLGYEIQL + 134 IVAPGTFEV + 135 FLLPLIIVL +++ 136 GLSEPIFQL +++ 137 ALFPHLLQPVL ++ 138 YLTNEGIQYL ++ 139 LLYPTEITV +++ 140 ALLDGRVQL ++ 141 SMFGAGLTV + 142 FLGENISNFL +++ 143 TLVTGLASV + 146 YLARIQGFQV + 147 QMLELITRL + 149 VLLRVLILL + The table lists peptides that are very highly over-presented on tumors compared to a panel of normal tissues (+++), highly over-presented on tumors compared to a panel of normal tissues (++) or over-presented on tumors compared to a panel of normal tissues (+). The panel of normal tissues considered relevant for comparison with tumors consisted of: adipose tissue, adrenal gland, blood cells, blood vessel, bone marrow, brain, esophagus, eye, gallbladder, heart, kidney, large intestine, liver, lung, lymph node, nerve, pancreas, parathyroid gland, peritoneum, pituitary, pleura, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea, ureter, urinary bladder.

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

[0350] Over-presentation or specific presentation of a peptide on tumor cells compared to normal cells is sufficient for its usefulness in immunotherapy, and some peptides are tumor-specific despite their source protein occurring also in normal tissues. Still, mRNA expression profiling adds an additional level of safety in selection of peptide targets for immunotherapies. Especially for therapeutic options with high safety risks, such as affinity-matured TCRs, the ideal target peptide will be derived from a protein that is unique to the tumor and not found on normal tissues.

RNA Sources and Preparation

[0351] Surgically removed tissue specimens were provided as indicated above (see Example 1) after written informed consent had been obtained from each patient. Tumor tissue specimens were snap-frozen immediately after surgery and later homogenized with mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRI Reagent (Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN, Hilden, Germany); both methods were performed according to the manufacturer's protocol.

[0352] Total RNA from healthy human tissues for RNASeq experiments was obtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA); Geneticist Inc. (Glendale, Calif., USA); Istituto Nazionale Tumori Pascale (Naples, Italy); ProteoGenex Inc. (Culver City, Calif., USA); and University Hospital Heidelberg (Heidelberg, Germany).

[0353] Total RNA from tumor tissues for RNASeq experiments was obtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK); Geneticist Inc. (Glendale, Calif., USA); ProteoGenex Inc. (Culver City, Calif., USA); and Tissue Solutions Ltd (Glasgow, UK).

[0354] Quality and quantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip Kit (Agilent).

RNAseq Experiments

[0355] Gene expression analysis oftumor and normal tissue RNA samples was performed by next generation sequencing (RNAseq) by CeGaT (Tbingen, Germany). Briefly, sequencing libraries are prepared using the Illumina HiSeq v4 reagent kit according to the provider's protocol (Illumina Inc., San Diego, Calif., USA), which includes RNA fragmentation, cDNA conversion and addition of sequencing adaptors. Libraries derived from multiple samples are mixed equimolar and sequenced on the Illumina HiSeq 2500 sequencer according to the manufacturer's instructions, generating 50 bp single end reads. Processed reads are mapped to the human genome (GRCh38) using the STAR software. Expression data are provided on transcript level as RPKM (Reads Per Kilobase per Million mapped reads, generated by the software Cufflinks) and on exon level (total reads, generated by the software Bedtools), based on annotations of the ensembl sequence database (Ensembl77). Exon reads are normalized for exon length and alignment size to obtain RPKM values. Exemplary expression profiles of source genes of the present invention that are highly over-expressed or exclusively expressed in urinary bladder cancer are shown in FIGS. 2A-2D. Expression scores for further exemplary genes are shown in Table 9.

TABLE-US-00009 TABLE9 Expressionscores. SEQIDNo. Sequence GeneExpression 1 ILLQASVQV +++ 2 GLLKAYSIRTA +++ 3 YLDEIPPKFSM ++ 4 SLDVVNLLV ++ 5 IQDPVIFYV + 6 SIVDFLITA + 8 ALSFSSSAGPGLLKA +++ 11 AMAELRVVV +++ 18 SIAAYNPVV +++ 21 TLIEDDALNGA ++ 22 SIAKEGVVGA ++ 34 ALLDEQQVNV +++ 35 KLPEPPPLA +++ 36 ALWDEFNQL +++ 37 ILSAILVHL +++ 38 TLTSIIVAV +++ 40 VIADRVVTV + 46 SLFIGAEIVAV + 50 VLANRVAVV +++ 53 ILVQVIPVV + 54 ALNDEINFL +++ 55 KLLETKWTL +++ 56 ILLRDLPTL +++ 69 QIWDKILSV +++ 76 AVVSSVNTV ++ 83 VILDKYYFL +++ 85 HLLDSKVPSV ++ 87 RLLELLQEA +++ 89 YLFPETEFI ++ 92 ALLDEQQVNVLL +++ 100 SLQSFIHGV +++ 109 RLIDDMVAQA + 110 VLFNIDGQGNHV +++ 117 YMLDIFHEVL +++ 124 QIDGTLSTI + 135 FLLPLIIVL + 139 LLYPTEITV ++ 140 ALLDGRVQL +++ 142 FLGENISNFL +++ 149 VLLRVLILL ++ The table lists peptides from genes that are very highly over-expressed in tumors compared to a panel of normal tissues (+++), highly over-expressed in tumors compared to a panel of normal tissues (++) or over-expressed in tumors compared to a panel of normal tissues (+). The baseline for this score was calculated from measurements of the following relevant normal tissues: adipose tissue, adrenal gland, artery, blood cells, bone marrow, brain, cartilage, colon, esophagus, eye, gallbladder, heart, kidney, liver, lung, lymph node, pancreas, peripheral nerve, pituitary, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, urinary bladder, and vein. In case expression data for several samples of the same tissue type were available, the arithmetic mean of all respective samples was used for the calculation.

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

[0356] In order to obtain information regarding the immunogenicity of the TUMAPs of the present invention, the inventors performed investigations using an in vitro T-cell priming assay based on repeated stimulations of CD8+ T cells with artificial antigen presenting cells (aAPCs) loaded with peptide/MHC complexes and anti-CD28 antibody. This way the inventors could show immunogenicity for HLA-A*0201 restricted TUMAPs of the invention, demonstrating that these peptides are T-cell epitopes against which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

[0357] In order to perform in vitro stimulations by artificial antigen presenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02 leukapheresis products via positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtained from the University clinics Mannheim, Germany, after informed consent.

[0358] PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium (TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 g/ml Streptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany), 20 g/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma, Nrnberg, Germany) were also added to the TCM at this step.

[0359] Generation of pMHC/anti-CD28 coated beads, T-cell stimulations and readout was performed in a highly defined in vitro system using four different pMHC molecules per stimulation condition and 8 different pMHC molecules per readout condition.

[0360] The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung et al., 1987) was chemically biotinylated using Sulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer (Perbio, Bonn, Germany). Beads used were 5.6 m diameter streptavidin coated polystyrene particles (Bangs Laboratories, Illinois, USA).

[0361] pMHC used for positive and negative control stimulations were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 206) from modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO. 207), respectively.

[0362] 800.000 beads/200 l were coated in 96-well plates in the presence of 412.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 were added subsequently in a volume of 200 l. Stimulations were initiated in 96-well plates by co-incubating 110.sup.6 CD8+ T cells with 210.sup.5 washed coated beads in 200 l TCM supplemented with 5 ng/ml IL-12 (PromoCell) for 3 days at 37 C. Half of the medium was then exchanged by fresh TCM supplemented with 80 U/ml IL-2 and incubating was continued for 4 days at 37 C. This stimulation cycle was performed for a total of three times. For the pMHC multimer readout using 8 different pMHC molecules per condition, a two-dimensional combinatorial coding approach was used as previously described (Andersen et al., 2012) with minor modifications encompassing coupling to 5 different fluorochromes. Finally, multimeric analyses were performed by staining the cells with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BD LSRII SORP cytometer equipped with appropriate lasers and filters was used. Peptide specific cells were calculated as percentage of total CD8+ cells. Evaluation of multimeric analysis was done using the FlowJo software (Tree Star, Oregon, USA). In vitro priming of specific multimer+CD8+ lymphocytes was detected by comparing to negative control stimulations. Immunogenicity for a given antigen was detected if at least one evaluable in vitro stimulated well of one healthy donor was found to contain a specific CD8+T-cell line after in vitro stimulation (i.e. this well contained at least 1% of specific multimer+ among CD8+ T-cells and the percentage of specific multimer+ cells was at least 10 the median of the negative control stimulations).

In Vitro Immunogenicity for Urinary Bladder Cancer Peptides

[0363] For tested HLA class I peptides, in vitro immunogenicity could be demonstrated by generation of peptide specific T-cell lines. Exemplary flow cytometry results after TUMAP-specific multimer staining for 2 peptides of the invention are shown in FIG. 3 together with corresponding negative controls. Exemplary flow cytometry results after TUMAP-specific multimer staining for 3 peptides of the invention are shown in FIG. 4 together with corresponding negative controls. Results for 25 peptides from the invention are summarized in Table 10A. Additional results for 26 peptides from the invention are summarized in Table 10B.

TABLE-US-00010 TABLE10A invitroimmunogenicityofHLAclass Ipeptidesoftheinvention. SEQIDNo. Sequence Wells 155 VLSSGLTAA + 156 KLVEFDFLGA + 159 SLIEDLILL + 160 SLLGGNIRL ++ 162 TLLAAEFLKQV + 164 ALADLTGTVV + 167 SLWGGDVVL ++ 168 LTAPPEALLMV + 170 GLIEIISNA + 172 LLYGHTVTV ++ 173 FVFSFPVSV ++ 176 SMSGYDQVL + 177 NLLQVLEKV + 178 ALNEEAGRLLL + 182 YLAPFLRNV ++ 184 LMTKEISSV + 186 VLYPHEPTAV + 189 ALLRTVVSV + 192 ALNPADITV ++ 193 ALVQDLAKA ++ 198 NLIEKSIYL + 200 YLNVQVKEL + 203 FLIPYAIML + 204 GVYDGEEHSV + 205 KIVDFSYSV ++ Exemplary results of in vitro immunogenicity experiments conducted by the applicant for the peptides of the invention. <20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++

TABLE-US-00011 TABLE10B InvitroimmunogenicityofHLAclassIpeptides oftheinvention. SEQIDNO: Sequence Wellspositive[%] 4 SLDVVNLLV + 5 IQDPVIFYV +++ 6 SIVDFLITA + 9 SLVDARFQL + 10 GLWHGMFANV ++ 14 GLAGPVRGV + 15 YLAPENGYLMEA + 16 ILGPQGNTI + 17 RLSQLEGVNV + 23 YTLSKTEFL + 24 SLLGGITVV ++ 27 STTNGGILTV + 29 ALSSPPPTV + 30 ILDISRSEV ++++ 41 FLDDGNQMLL + 50 VLANRVAVV ++++ 53 ILVQVIPVV ++ 57 GLAHFVNEI + 59 WLSTSIPEA + 60 SLSDVRVIV + 75 RVISSVISV ++ 111 LLDVTPKAV +++ 114 IITKDLFQV + 126 SLLRVGWSV ++ 146 YLARIQGFQV ++ 148 TLGVIPESV + Exemplary results of in vitro immunogenicity experiments conducted by the applicant for HLA-A*02 restricted peptides of the invention. Results of in vitro immunogenicity experiments are indicated. Percentage of positive wells and donors (among evaluable) are summarized as indicated <20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++

Example 4

Synthesis of Peptides

[0364] All peptides were synthesized using standard and well-established solid phase peptide synthesis using the Fmoc-strategy. Identity and purity of each individual peptide have been determined by mass spectrometry and analytical RP-HPLC. The peptides were obtained as white to off-white lyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPs are preferably administered as trifluoro-acetate salts or acetate salts, other salt-forms are also possible.

Example 5

MHC Binding Assays

[0365] Candidate peptides for T cell based therapies according to the present invention were further tested for their MHC binding capacity (affinity). The individual peptide-MHC complexes were produced by UV-ligand exchange, where a UV-sensitive peptide is cleaved upon UV-irradiation, and exchanged with the peptide of interest as analyzed. Only peptide candidates that can effectively bind and stabilize the peptide-receptive MHC molecules prevent dissociation of the MHC complexes. To determine the yield of the exchange reaction, an ELISA was performed based on the detection of the light chain (2m) of stabilized MHC complexes. The assay was performed as generally described in Rodenko et al. (Rodenko et al., 2006).

[0366] 96 well MAXISorp plates (NUNC) were coated over night with 2 ug/ml streptavidin in PBS at room temperature, washed 4 and blocked for 1 h at 37 C. in 2% BSA containing blocking buffer. Refolded HLA-A*02:01/MLA-001 monomers served as standards, covering the range of 15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction were diluted 100 fold in blocking buffer. Samples were incubated for 1 h at 37 C., washed four times, incubated with 2 ug/ml HRP conjugated anti-2m for 1 h at 37 C., washed again and detected with TMB solution that is stopped with NH.sub.2SO.sub.4. Absorption was measured at 450 nm. Candidate peptides that show a high exchange yield (preferably higher than 50%, most preferred higher than 75%) are generally preferred for a generation and production of antibodies or fragments thereof, and/or T cell receptors or fragments thereof, as they show sufficient avidity to the MHC molecules and prevent dissociation of the MHC complexes.

TABLE-US-00012 TABLE11 MHCclassIbindingscores. SEQIDNo Sequence Peptideexchange 1 ILLQASVQV ++++ 2 GLLKAYSIRTA +++ 3 YLDEIPPKFSM ++++ 4 SLDVVNLLV ++++ 5 IQDPVIFYV ++++ 6 SIVDFLITA +++ 7 QMFEGQILDV +++ 8 ALSFSSSAGPGLLKA +++ 9 SLVDARFQL ++++ 10 GLWHGMFANV ++++ 11 AMAELRVVV ++++ 12 GVALTVTGV ++++ 13 FLEEKEQAAL +++ 14 GLAGPVRGV +++ 15 YLAPENGYLMEA +++ 16 ILGPQGNTI +++ 17 RLSQLEGVNV +++ 18 SIAAYNPVV ++++ 19 SLATTLTKI +++ 20 YLPDSLTQL ++++ 21 TLIEDDALNGA +++ 23 YTLSKTEFL ++++ 24 SLLGGITVV ++++ 25 SLDSSGFSL +++ 26 GLALLYSGV ++++ 27 STTNGGILTV +++ 28 GLIDSLMAYV ++++ 29 ALSSPPPTV +++ 30 ILDISRSEV +++ 31 SLFDGIATGL ++++ 32 YQAPDIDVQL +++ 33 VLFGEITRL +++ 34 ALLDEQQVNV +++ 35 KLPEPPPLA +++ 36 ALWDEFNQL +++ 37 ILSAILVHL +++ 38 TLTSIIVAV +++ 39 AMASHLTST +++ 40 VIADRVVTV +++ 41 FLDDGNQMLL ++++ 42 FLIDASQRV +++ 43 FLIESKLLSL ++++ 44 GLAQDPKSLQL +++ 45 IIDSSPTAL ++ 47 VLMDDTDPL +++ 48 VLMDDTDPLV +++ 49 SLIGGTNFV +++ 50 VLANRVAVV +++ 52 SLATLEGIQL +++ 53 ILVQVIPVV +++ 54 ALNDEINFL +++ 55 KLLETKVVTL ++++ 56 ILLRDLPTL +++ 57 GLAHFVNEI ++++ 58 GLDSSVNVQGSVL +++ 59 WLSTSIPEA ++++ 60 SLSDVRVIV ++++ 62 GQLDFSEFL +++ 63 LLAGLLVGV +++ 64 GLLSQGSPL +++ 65 IITDLLRSV ++++ 66 SLWEENQAL +++ 67 FLTPPLLSV ++++ 68 TMIVSLAAV +++ 69 QIWDKILSV +++ 70 KLAEISLGV ++ 71 LLSEDFVSV ++ 72 SLFTGLRSI +++ 73 VLKVFLENV ++++ 74 LLQEGEVYSA +++ 75 RVISSVISV +++ 76 AVVSSVNTV +++ 77 KVFGGFQVV ++++ 78 FIPDFAVAI ++++ 79 FLDPATPRV +++ 80 ILLDTPLFLL +++ 81 SLDKGTLYI +++ 82 NLHNSYYSV +++ 83 VILDKYYFL ++++ 84 ALDPASISV +++ 85 HLLDSKVPSV +++ 86 FLIJLIISV +++ 87 RLLELLQEA ++++ 88 ALASLENHV +++ 89 YLFPETEFI +++ 90 GMTELYFQL +++ 91 NLDAATYQV +++ 92 ALLDEQQVNVLL +++ 93 LLDLIQTKV +++ 94 ALADGVPVAL +++ 95 YLIGQHVTA ++++ 96 KLTNGIVVVL +++ 97 TVGPGLLGV +++ 98 YLIGLDPENLAL +++ 99 ALIGDDVGL +++ 100 SLQSFIHGV +++ 101 ILDEMRAQL +++ 102 GLYEGLDWL +++ 103 GLYSGEVLV ++++ 105 TLFPSKIGV +++ 106 ILLDTPLFL +++ 107 LULAKLEKV ++ 108 YLDPNQRDL +++ 109 RLIDDMVAQA +++ 110 VLFNIDGQGNHV +++ 111 LLDVTPKAV +++ 112 YLDPSLNSL +++ 113 FVFEPPPGV ++++ 114 IITKDLFQV +++ 115 SLLDFERSL +++ 116 QLAWFDTDL +++ 117 YMLDIFHEVL +++ 118 RLLDFPTLLV +++ 119 SLDEKQNLV +++ 120 IIIPEIQKV +++ 121 QLQGYLRSV +++ 122 ILEPSLYTV ++++ 123 NLAGVYSEV ++++ 124 QIDGTLSTI +++ 126 SLLRVGWSV ++++ 127 KLNATNIEL +++ 128 KLWGQSIQL ++++ 129 YLEPKLTQV +++ 130 TLTSKLYSL ++++ 131 ILTSIQSLL ++++ 132 YILEGEPGKV ++++ 133 GLDPLGYEIQL ++++ 134 IVAPGTFEV +++ 135 FLLPLIIVL +++ 136 GLSEPIFQL +++ 137 ALFPHLLQPVL ++++ 138 YLTNEGIQYL +++ 139 LLYPTEITV +++ 140 ALLDGRVQL ++++ 141 SMFGAGLTV ++++ 142 FLGENISNFL +++ 143 TLVTGLASV +++ 144 YLAGEAPTL +++ 145 ALYPGQLVQL +++ 146 YLARIQGFQV ++++ 147 QMLELITRL +++ 148 TLGVIPESV ++++ Binding of HLA-class I restricted peptides to HLA-A*02:01 was ranged by peptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++

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