A*03 restricted peptides for use in immunotherapy against cancers and related methods

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 method of eliciting an immune response in a patient who has uterine cancer overexpressing ALPP polypeptide comprising the amino acid sequence of LLYGNGPGYVLK (SEQ ID NO: 3), comprising administering to said patient a population of activated T cells that selectively kill the cancer cells, wherein the activated T cells are cytotoxic T cells produced by contacting CD8+ T cells with an antigen presenting cell that presents a peptide consisting of the amino acid sequence of LLYGNGPGYVLK (SEQ ID NO: 3) in a complex with MHC class I molecule on the surface of the antigen presenting cell in vitro, for a period of time sufficient to activate said T cell.

2. The method of claim 1, wherein the population of activated T cells are administered in the form of a composition.

3. The method of claim 2, wherein the composition comprises an adjuvant.

4. The method of claim 1, wherein the uterine cancer is endometrial cancer.

5. The method of claim 3, 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.

6. The method of claim 5, wherein the adjuvant is IL-2.

7. The method of claim 5, wherein the adjuvant is IL-7.

8. The method of claim 5, wherein the adjuvant is IL-12.

9. The method of claim 5, wherein the adjuvant is IL-15.

10. The method of claim 5, wherein the adjuvant is IL-21.

11. A method of treating a patient who has uterine cancer overexpressing ALPP polypeptide comprising the amino acid sequence of LLYGNGPGYVLK (SEQ ID NO: 3), comprising administering to said patient a population of activated T cells that selectively kill the cancer cells, wherein the activated T cells are cytotoxic T cells produced by contacting CD8+ T cells with an antigen presenting cell that presents a peptide consisting of the amino acid sequence of LLYGNGPGYVLK (SEQ ID NO: 3) in a complex with MHC class I molecule on the surface of the antigen presenting cell in vitro, for a period of time sufficient to activate said T cell.

12. The method of claim 11, wherein the population of activated T cells are administered in the form of a composition.

13. The method of claim 12, wherein the composition comprises an adjuvant.

14. The method of claim 11, wherein the uterine cancer is endometrial cancer.

15. The method of claim 13, 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.

16. The method of claim 15, wherein the adjuvant is IL-2.

17. The method of claim 15, wherein the adjuvant is IL-7.

18. The method of claim 15, wherein the adjuvant is IL-12.

19. The method of claim 15, wherein the adjuvant is IL-15.

20. The method of claim 15, wherein the adjuvant is IL-21.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A through 1I show the over-presentation of various peptides in different cancer tissues (black dots). Upper part: Median MS signal intensities from technical replicate measurements are plotted as dots for single HLA-A*03 positive normal (grey dots, left part of figure) and tumor samples (black dots, right part of figure) on which the peptide was detected. Boxes display median, 25th and 75th percentile of normalized signal intensities, while whiskers extend to the lowest data point still within 1.5 interquartile range (IQR) of the lower quartile, and the highest data point still within 1.5 IQR of the upper quartile. Normal organs are ordered according to risk categories (blood cells, blood vessels, brain, liver, lung: high risk, grey dots; reproductive organs, breast, prostate: low risk, grey dots; all other organs: medium risk; grey dots). Lower part: The relative peptide detection frequency in every organ is shown as spine plot. Numbers below the panel indicate number of samples on which the peptide was detected out of the total number of samples analyzed for each organ (N=93 for normal samples, N=151 for tumor samples). If the peptide has been detected on a sample but could not be quantified for technical reasons, the sample is included in this representation of detection frequency, but no dot is shown in the upper part of the figure. Tissues (from left to right): Normal samples: blood cells; bloodvess (blood vessels); brain; heart; liver; lung; adrenal gl (adrenal gland); bile duct; bladder; bone marrow; esoph (esophagus); gall bl (gallbladder); intest. la (large intestine); intest. sm (small intestine); kidney; lymph node; nerve perith (peripheral nerve); pancreas; skin; spinal cord; spleen; stomach; thyroid; trachea. Tumor samples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); HCC (hepatocellular carcinoma); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-hodgkin lymphoma); NSCLCadeno (non-small cell lung cancer adenocarcinoma); NSCLCother (NSCLC samples that could not unambiguously be assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (urinary bladder carcinoma); UEC (uterine and endometrial cancer).

(2) FIG. 1A) Peptide: KLLEDMVEK (SEQ ID NO.: 33), FIG. 1B) Peptide: SLYDSEPRKK (SEQ ID NO.: 61), FIG. 1C) Peptide: SSFRPLLSK (SEQ ID NO.: 63), FIG. 1D) Peptide: GLSTILLYH (SEQ ID NO.: 69), FIG. 1E) Peptide: IINESLLFY (SEQ ID NO.: 76), FIG. 1F) Peptide: AVAAVLLSR (SEQ ID NO.: 79), FIG. 1G) Peptide: RIYVYVKRK (SEQ ID NO.: 178), FIG. 1H) Peptide: KVFEEPEDFLK (SEQ ID NO.: 189), and FIG. 1I) Peptide: GISNPITTSK (SEQ ID NO.: 223).

(3) FIGS. 2A through 2H show exemplary expression profile of source genes of the present invention that are over-expressed in different cancer samples. Tumor (black dots) and normal (grey dots) samples are grouped according to organ of origin. Box-and-whisker plots represent median FPKM value, 25th and 75th percentile (box) plus whiskers that extend to the lowest data point still within 1.5 interquartile range (IQR) of the lower quartile and the highest data point still within 1.5 IQR of the upper quartile. Normal organs are ordered according to risk categories. FPKM: fragments per kilobase per million mapped reads. Normal samples: blood cells; bloodvess (blood vessels); brain; heart; liver; lung; adipose (adipose tissue); adrenal gl (adrenal gland); bile duct; bladder; bone marrow; esoph (esophagus); eye; gall bl (gallbladder); head&neck; intest. la (large intestine); intest. sm (small intestine); kidney; lymph node; nerve perith (peripheral nerve); pancreas; parathyr (parathyroid gland); perit (peritoneum); pituit (pituitary); pleura; skel. mus (skeletal muscle); skin; spleen; stomach; thyroid; trachea; ureter; breast; ovary; placenta; prostate; testis; thymus; uterus. Tumor samples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); HCC (hepatocellular carcinoma); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-hodgkin lymphoma); NSCLCadeno (non-small cell lung cancer adenocarcinoma); NSCLCother (NSCLC samples that could not unambiguously be assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (urinary bladder carcinoma); UEC (uterine and endometrial cancer).

(4) FIG. 2A) Gene symbol: DCAF4L2, Peptide: RVYPHKTLY (SEQ ID No: 1), FIG. 2B) Gene symbol: PRAME, Peptide: RLVELAGQSLLK (SEQ ID No: 7), FIG. 2C) Gene symbol: CSAG1, Peptide: FSNNHPSTPK (SEQ ID No: 8), FIG. 2D) Gene symbol: CTAG2, Peptide: PLPRPGAVLK (SEQ ID No: 11), FIG. 2E) Gene symbol: IGF2BP1, Peptide: KVFAEHKISY (SEQ ID No: 16), FIG. 2F) Gene symbol: SOX11, Peptide: RLYYSFKNITK (SEQ ID No: 18), FIG. 2G) Gene symbol: PTHLH, Peptide: RTALLWGLKK (SEQ ID No: 90), and FIG. 2H) Gene symbol: LAMC2, Peptide: RVRALGSQY (SEQ ID No: 97).

(5) FIGS. 3A and 3B show exemplary results of peptide-specific in vitro CD8+ T cell responses of a healthy HLA-A*03+ donor. CD8+ T cells were primed using artificial APCs coated with anti-CD28 mAb and HLA-A*03 in complex with SeqID No 248 peptide (GLASRILDAK) (FIG. 3A, left panel) and SeqID No 249 peptide (ATSGVPVYK) (FIG. 3B, left panel), respectively. After three cycles of stimulation, the detection of peptide-reactive cells was performed by 2D multimer staining with A*03/SeqID No 248 (FIG. 3A) or A*03/SeqID No 249 (FIG. 3B). Right panels (FIGS. 3A and 3B) show control staining of cells stimulated with irrelevant A*03/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

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

(7) Tissue Samples

(8) Patients' tumor tissues were obtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK), BioServe (Beltsville, Md., USA), Geneticist Inc. (Glendale, Calif., USA), Leiden University Medical Center (LUMC) (Leiden, Netherlands), ProteoGenex Inc. (Culver City, Calif., USA), Tissue Solutions Ltd (Glasgow, UK), University Hospital Bonn (Bonn, Germany), University Hospital Geneva (Geneva, Switzerland), University Hospital Heidelberg (Heidelberg, Germany), University Hospital Tübingen (Tübingen, Germany), Val d'Hebron University Hospital (Barcelona, Spain).

(9) Normal tissues were obtained from Asterand (Detroit, Mich., USA & Royston, Herts, UK), BioServe (Beltsville, Md., USA), Capital BioScience Inc. (Rockville, Md., USA), Centre for Clinical Transfusion Medicine Tuebingen (Tübingen, Germany), Geneticist Inc. (Glendale, Calif., USA), ProteoGenex Inc. (Culver City, Calif., USA), Tissue Solutions Ltd (Glasgow, UK), University Hospital Heidelberg (Heidelberg, Germany), University Hospital Tübingen (Tübingen, Germany)

(10) 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.

(11) Isolation of HLA Peptides from Tissue Samples

(12) 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, the HLA-DR specific antibody L243 and the HLA DP specific antibody B7/21, CNBr-activated sepharose, acid treatment, and ultrafiltration.

(13) Mass Spectrometry Analyses

(14) 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 μm i.d.×250 mm) packed with 1.7 μm 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=30 000), 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 at a fixed false discovery rate (q≤0.05) and additional manual control. In cases where the identified peptide sequence was uncertain it was additionally validated by comparison of the generated natural peptide fragmentation pattern with the fragmentation pattern of a synthetic sequence-identical reference peptide.

(15) 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 acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-hodgkin lymphoma, lung cancer (including non-small cell lung cancer adenocarcinoma, squamous cell non-small cell lung cancer, and small cell lung cancer), ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, urinary bladder carcinoma, uterine and endometrial cancer samples to a baseline of normal tissue samples. Presentation profiles of exemplary over-presented peptides are shown in FIGS. 1A-1I.

(16) Table 8a and 8b show the presentation on various cancer entities for selected peptides, and thus the particular relevance of the peptides as mentioned for the diagnosis and/or treatment of the cancers as indicated (e.g. peptide SEQ ID No. 1 for hepatocellular carcinoma, and urinary bladder carcinoma, peptide SEQ ID No. 25 for breast cancer, non-small cell lung cancer, and renal cell carcinoma).

(17) TABLE-US-00010 TABLE 8a Overview of presentation of selected tumor-associated peptides of the present invention across entities. SEQ ID No. Sequence Peptide Presentation on cancer entities 1 RVYPHKTLY HCC, UBC 2 KVMPKQTWK HCC 3 LLYGNGPGYVLK UEC 4 RGLSGIGAFR HNSCC 5 AMVPIYAAY RCC 6 SIFGLAPGK UEC 7 RLVELAGQSLLK UEC 8 FSNNHPSTPK MEL 9 HVLYPVPLESY MEL 10 KARDLRTPK CRC 11 PLPRPGAVLK NHL 12 KVLTRNIEY GBC 13 AVAFFVLPSK NHL 14 VAFFVLPSK AML 15 RLYAPVVNISRL BRCA 16 KVFAEHKISY HNSCC 17 GGLSSQGVYY CCC 18 RLYYSFKNITK NHL 19 ALAAKLEVK GBM 20 LLPTVLIKK AML, HCC, HNSCC, NSCLCother, OSCAR, UEC 21 SSLAELIAK HNSCC 22 EGLFLLGCVK NHL 23 RLSPGPRAY RCC 24 KLETGVVKK RCC 25 KTWAHCMSY BRCA, NSCLCadeno, RCC 26 QLGYQAAVLK GBM 27 SGSTLECILYK UEC 28 STLECILYK UEC 30 ILDSSLLK NHL 31 HAFFPKTHR UEC 32 LLDAEPPILY BRCA 33 KLLEDMVEK UEC 34 AIGTPLIGK CRC, NSCLCadeno, UEC 35 VLLLLSLLH NSCLCadeno 36 QIRAELMKK HNSCC, NHL 37 EVILTTKTPK MEL 38 SLFPYYNNLY NHL 39 KTQFPQLK HCC 40 ALNDRFAGY PRCA 41 RLGAARGQLR NSCLCadeno 43 TVYDSIWCNMK UEC 44 HAFPPGPNY HNSCC 45 KCYEVGMMK UEC 46 VVTGNVPLK HCC, MEL 47 ALFGNALVFY RCC 48 KGWNGQIFK AML 49 ITAPLMPLGK CCC, HNSCC 50 HSAGIFSMY NHL 51 VTADGALAMEK CCC, CRC, HNSCC, UEC 52 GMYEYGSIEK MEL 53 KQALSLVRK CCC, CRC, HNSCC, UEC 54 GVTITKTLK BRCA, MEL 55 RLSAESKDLLK GBM 56 TTYYPSPLNK RCC 57 RVLYRPQLEK HCC 58 VLYRPQLEK HCC 59 KINQYIIKK MEL 60 VVFPFPVNKR AML 61 SLYDSEPRKK AML, CCC, GBC, GBM, HNSCC, MEL, NHL, OSCAR, UBC, UEC 62 GIFPKIMPK HCC 63 SSFRPLLSK BRCA, CRC, GBM, HCC, MEL, PACA, PRCA, SCLC, UEC 64 SVLSRMLVR MEL, UEC 65 RTIEELQNK HNSCC 66 RVKEIVINK MEL 67 VVREILHAQTLK MEL, NHL 68 KGPMAGILAY AML, NHL 69 GLSTILLYH CCC, CRC, GBC, GBM, MEL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, RCC, UEC 70 VTAVASLLK MEL 71 VLYELGIIGNNK MEL 72 KLYPQCLQK NHL 73 RCFSGPYLNK BRCA, MEL, NHL 74 HSTLVALFY NHL 75 IIFVPEMNK UEC 76 IINESLLFY BRCA, CRC, GBC, MEL, NSCLCadeno, RCC, UEC 77 VVDDSQLPK CCC, CRC, MEL, NHL, NSCLCsquam, RCC 78 KTGTFVLYKS HNSCC, NHL, SCLC 79 AVAAVLLSR CCC, CLL, CRC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, RCC, UEC 80 ALCGTQLFY AML, CCC, HCC, NHL, NSCLCadeno, NSCLCsquam, UEC 81 HLFLPFSYK CCC, HCC 82 CLANYTVNK UEC 83 KLADSVMAGK BRCA, CRC, GBM, SCLC 84 QLYSPPSPSYR HNSCC, NSCLCadeno 85 IMPTFDLTK HCC 86 RVSGSGGGGAGK NHL 87 RLASVGLDAK HCC 88 GTHVWVGLYK PACA 90 RTALLWGLKK HNSCC 92 KRIPFRPLAK HCC 95 FLGLAFHPK SCLC 96 RAVSVNPGK CCC, GBC, HNSCC 97 RVRALGSQY CCC 98 VSVAGSILAK GBC 100 TTNARILAR MEL 101 HMDEFKRTQK CRC 102 RLLQHTPSAR CRC 103 SIYKKAVYR CRC 104 AVVQGLVEK HNSCC 105 QVLDLQSVK CRC 106 LLRSGLTLR HCC, UEC 107 ILNLNKMVK NHL 108 KMPILSYWPY CRC 109 KLQNLPTLLY RCC 110 IIFIPATILLK AML, HCC 111 ATSPPASVR HNSCC 112 ASLAAAVLAK MEL 113 VSIRNTLLY HCC 115 AILHPFRAK UEC 116 KGVKKELPQK UBC 117 TVFVELWLK AML 118 SLRGSFPILY CCC, MEL 119 RMGFRTLSK AML 120 TRMQKAGFLYY NSCLCsquam 121 LLPAARATSR NSCLCsquam 122 LIGPLLICK CCC, HCC, NSCLCadeno, RCC, UEC 123 SLQGLTISY GBM 124 VVYDTMIEK GBM 125 VVYDTMIEKF HNSCC 127 GLAAGALLLY NSCLCsquam 128 KIKKPLSYR UEC 129 ALARVSSVKL CRC 130 KANSGNTFKY HCC 132 ASSFRPLLSK MEL 134 SLLKPSGDYFK CRC 135 TFKSVLLNK RCC 137 TVYVAMCHKF OSCAR 138 VSLSKMLKK NHL 139 KAIIRVIPLK CRC 140 RLLAAGQVIR CCC, HCC, HNSCC, OSCAR 141 RLRDYISSLK NHL 142 SPRVYWLGLNDR NHL 143 KTLGKIAEK CCC, CRC, GBC, HNSCC, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PRCA 144 PLAMLAATCNK HCC 145 SLFEGIYTIK AML 147 KLFMPRPK GBC 148 RIGNKGIYK AML 149 TVFLSKYLKK UEC 151 KVASFTVIGY CCC, NHL 152 KIICGVHYLY CRC 154 TIASVLVAR NHL 155 ALSHAVNNY SCLC 156 VSLGIAVSK AML, CCC, HNSCC 157 SLPLQRILAMSK NSCLCadeno 158 RALGVPFVPR CCC 159 LLLLPFLLY CCC 160 RLLPGKVVWK UEC 161 MKTLPAMLGTGK HNSCC, NSCLCadeno, OSCAR 162 LLALGAAYVY UEC 163 MLYYPSVSR MEL 165 AATIISSAK RCC 166 KVIAPGVIY CCC 167 MLKQARRPLFR NSCLCadeno 168 ATNGKVLKK GBC, HNSCC 169 ATNGKVLKKR CLL, GBM, HNSCC, UEC 171 ATIGLSVSK AML, HCC 172 SLLEADPFLK AML 173 VSYNRLIK GBC, HCC, HNSCC, MEL, PRCA, RCC, UEC 174 KMMKRLMTVEK NSCLCother 175 VIGTTSSPK CCC, HCC 176 RLYDAYVNR GBM, RCC 177 ALLGVIIAKK RCC 178 RIYVYVKRK AML, HNSCC, OSCAR, UBC, UEC 179 KINPTASLK MEL 180 RLKMAQESVSK MEL 181 RVAEEILIK MEL 182 QICLPAIYK CRC, GBC, NSCLCadeno, RCC 183 KVYIPPIINK BRCA, HCC, HNSCC, MEL, UEC 184 KVLEPPLGAR AML, CLL 185 SIINFKPLTY HNSCC 186 CTLPFKIFY HNSCC, NSCLCadeno, PACA, UEC 187 KTCQVSGLLY BRCA, MEL 188 SSLPRAFQK CCC, HNSCC 189 KVFEEPEDFLK BRCA, CCC, CRC, GBC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 190 RSKWSNVFK CRC 191 SLYNLGGAK HNSCC, OSCAR, UEC 192 RSYSHVVLK BRCA, CRC, GBC, GBM 193 IVYPSATDKTK BRCA, HNSCC 194 PVLICLALSK AML, BRCA, CCC, CLL, CRC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, RCC, SCLC, UBC, UEC 195 KLQAKVLQY GBC, MEL 196 AISSTVLGK GBC, PACA, UEC 197 RIVDYLLEK CCC, HNSCC 198 FLYGAQTVY CLL 199 IVFPDVISK UEC 201 KVADFGLARLLK BRCA, CRC, NHL 202 RLFPGLYLGY HCC 205 RVYPRPPSK CCC, HCC, UEC 206 RLYEMILQR AML, BRCA, CLL, HCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, RCC, UEC 207 ATLNLFQIVSK AML 208 KTGWFTLLY BRCA, HCC 209 KILDRVLSRY AML 210 KIFQGQINK HCC 211 VSLGTPIMK AML, GBM, HNSCC, UEC 212 RTIDRSVFK MEL, UEC 213 KLYPTHACR BRCA 214 KLFTSVFGVGLK AML 215 KIWQNLRLK CCC, CRC 216 RVSSVKLISK CCC, CRC, NSCLCadeno, PRCA 217 RVYEGDGRNSLK NHL 218 KAFNQSSIFTK CCC, NHL, NSCLCadeno 219 ALERKFRQK CRC, UEC 220 ALPRQAFHSK AML 221 RLAVSTRGK NSCLCadeno 223 GISNPITTSK HCC, HNSCC, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, PRCA, RCC, UEC 224 SLYDGFLSY GBM, NSCLCadeno 225 RVYPRPPSKTY HCC, UEC 226 RVWLGKHYK BRCA, CRC, HCC, HNSCC, MEL, NSCLCother, OSCAR, SCLC, UBC, UEC AML: acute myeloid leukemia; BRCA: breast cancer; CCC: cholangiocellular carcinoma; CLL: chronic lymphocytic leukemia; CRC: colorectal cancer; GBC: gallbladder cancer; GBM: glioblastoma; GC: gastric cancer; HCC: hepatocellular carcinoma; HNSCC: head and neck squamous cell carcinoma; MEL: melanoma; NHL: non-hodgkin lymphoma; NSCLCadeno: non-small cell lung cancer adenocarcinoma; NSCLCother: NSCLC samples that could not unambiguously be assigned to NSCLCadeno or NSCLCsquam; NSCLCsquam: squamous cell non-small cell lung cancer; OC: ovarian cancer; OSCAR: esophageal cancer; PACA: pancreatic cancer; PRCA: prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC: urinary bladder carcinoma; UEC: uterine and endometrial cancer.

(18) TABLE-US-00011 TABLE 8b Overview of presentation of selected tumor-associated peptides of the present invention across entities. SEQ Peptide Presentation ID No. Sequence on cancer entities 252 AVYNYPEGAAY UEC 253 KLSLSHKK CRC, MEL, NSCLCadeno 254 SVLDKLLFH NHL, RCC 255 VRTLPFLIK GBC, HCC 256 AVIGALLAVGATK MEL 257 LAELEGALQK BRCA, CCC 258 QVRDTLKIVFK CCC, HCC, MEL, NSCLCadeno 259 RSYVPLAH MEL 260 VIAAQLPK CCC, NSCLCadeno, OC 261 AVAQKLLSH HCC, NHL, OC, UEC 262 TMWQIPQEFVK UEC 263 RTYQLYSR CCC, MEL, OC, PRCA, UEC 264 VIFDAVTEQ GBM, HCC, MEL, NHL, NSCLCother, OSCAR, RCC, UBC 265 KIYEFRLMM HNSCC, OC, PACA 266 RNLNTPVVK CRC, OSCAR 267 VLKSITVKV HCC, MEL, NSCLCsquam 268 KVDMKDVTKI BRCA, CRC, GBM, HCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, RCC, UBC, UEC 269 KITDFNNIR AML, BRCA, GBC, GBM, HCC, HNSCC, MEL, NHL, NSCLCadeno, OC, OSCAR, PACA, RCC, UEC 270 KFKPNQYTK CRC, MEL 271 RLPLRVVKH NHL AML: acute myeloid leukemia; BRCA: breast cancer; CCC: cholangiocellular carcinoma; CLL: chronic lymphocytic leukemia CRC: colorectal cancer; GBC: gallbladder cancer; GBM: glioblastoma; GC: gastric cancer; HCC: hepatocellular carcinoma; HNSCC: head and neck squamous cell carcinoma; MEL: melanoma; NHL: non-hodgkin lymphoma NSCLCadeno: non-small cell lung cancer adenocarcinoma; NSCLCother: NSCLC samples that could not unambiguously be assigned to NSCLCadeno or NSCLCsquam; NSCLCsquam: squamous cell non-small cell lung cancer; OC: ovarian cancer; OSCAR: esophageal cancer; PACA: pancreatic cancer; PRCA: prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC: urinary bladder carcinoma; UEC: uterine and endometrial cancer.

Example 2

(19) Expression Profiling of Genes Encoding the Peptides of the Invention

(20) 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.

(21) RNA Sources and Preparation

(22) 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.

(23) Total RNA from healthy human tissues for RNASeq experiments was obtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-Options Inc. (Brea, Calif., USA); Geneticist Inc. (Glendale, Calif., USA); ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd (Glasgow, UK). Total RNA from tumor tissues for RNASeq experiments was obtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, Md., USA); Geneticist Inc. (Glendale, Calif., USA); Istituto Nazionale Tumori “Pascale” (Naples, Italy); ProteoGenex Inc. (Culver City, Calif., USA); University Hospital Heidelberg (Heidelberg, Germany).

(24) 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).

(25) RNAseq Experiments

(26) Gene expression analysis of—tumor and normal tissue RNA samples was performed by next generation sequencing (RNAseq) by CeGaT (Tübingen, 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.

(27) Exemplary expression profiles of source genes of the present invention that are highly over-expressed or exclusively expressed in acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-hodgkin lymphoma, lung cancer (including non-small cell lung cancer adenocarcinoma, squamous cell non-small cell lung cancer, and small cell lung cancer), ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, urinary bladder carcinoma, uterine and endometrial cancer are shown in FIGS. 2A-2H. Expression scores for further exemplary genes are shown in Table 9a and 9b.

(28) TABLE-US-00012 TABLE 9a Expression scores. 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, bile duct, blood cells, blood vessels, bone marrow, brain, esophagus, eye, gallbladder, heart, head&neck, kidney, large intestine, liver, lung, lymph node, nerve, parathyroid, pancreas, pituitary, peritoneum, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, urinary bladder. 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. Seq Gene Expression in tumor samples ID very highly No Sequence over-expressed(+) highly over-expressed(++) over-expressed(+++) 1 RVYPHKTLY BRCA, GBM, MEL, GBC HCC NSCLCadeno, OSCAR, SCLC 2 KVMPKQTWK MEL, NSCLCadeno, HCC PACA, PRCA, SCLC 3 LLYGNGPGYVLK OC UEC 4 RGLSGIGAFR NHL CLL 5 AMVPIYAAY NSCLCadeno, RCC NSCLCsquam, SCLC 6 SIFGLAPGK OC UEC 7 RLVELAGQSLLK AML, BRCA, GC, GBC, HNSCC, MEL, OC, HCC, NHL, OSCAR, NSCLCadeno, SCLC, UEC UBC NSCLCsquam, RCC 8 FSNNHPSTPK CCC, GBC, GBM, GC, HNSCC, HCC, MEL, NHL, OC, PACA NSCLCadeno, SCLC NSCLCsquam, OSCAR, UBC 9 HVLYPVPLESY AML, BRCA, NHL, GBC, HNSCC, MEL, OC, UEC OSCAR, UBC NSCLCadeno, NSCLCsquam, RCC, SCLC 10 KARDLRTPK GC, HNSCC, HCC, OC, SCLC GBC, MEL NSCLCsquam, UBC 11 PLPRPGAVLK GC, HNSCC, HCC, MEL, SCLC GBC NSCLCsquam, OC, UBC 12 KVLTRNIEY MEL, NSCLCadeno, GBM, HNSCC, SCLC NSCLCother NSCLCsquam, OC, OSCAR, UBC, UEC 13 AVAFFVLPSK NHL 14 VAFFVLPSK NHL 15 RLYAPVVNISRL OC BRCA 16 KVFAEHKISY CCC, NSCLCother, CRC, GC, HCC, GBC PACA, RCC, UBC HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, SCLC 17 GGLSSQGVYY GBC, SCLC PACA, UEC GC 18 RLYYSFKNITK CLL, HNSCC, MEL, BRCA, SCLC GBM NHL, NSCLCadeno, NSCLCsquam, OC, PACA, RCC, UBC 19 ALAAKLEVK OC UEC 20 LLPTVLIKK BRCA, CRC, GBC, HNSCC, NSCLCsquam, SCLC GBM, GC, HCC, OC, OSCAR, UBC MEL, NSCLCadeno, NSCLCother, PACA, PRCA, RCC 21 SSLAELIAK CRC, GBM, MEL, HCC, NHL NSCLCadeno, RCC, SCLC 22 EGLFLLGCVK MEL 23 RLSPGPRAY BRCA, RCC NSCLCadeno, UEC 24 KLETGVVKK GBM, SCLC 25 KTWAHCMSY BRCA, RCC NSCLCadeno, UEC 26 QLGYQAAVLK BRCA UEC 27 SGSTLECILYK BRCA UEC 28 STLECILYK BRCA UEC 29 KVLSILSRLK CRC HCC, NHL 30 ILDSSLLK GBC, HCC, HNSCC, MEL OSCAR, SCLC 31 HAFFPKTHR HCC, OC BRCA, CCC, CRC, GBC, GC, HNSCC, NSCLCadeno, NSCLCsquam, OSCAR, PACA, UBC, UEC 32 LLDAEPPILY BRCA, OC, UEC 33 KLLEDMVEK OC, UEC 34 AIGTPLIGK NSCLCadeno NHL, OC 35 VLLLLSLLH GBC, SCLC, UEC HCC 36 QIRAELMKK BRCA, CRC, GBC, CCC, HCC HNSCC, MEL, OC, SCLC 37 EVILTTKTPK GBM 38 SLFPYYNNLY GBC, NHL, SCLC NSCLCsquam, OC, OSCAR, UEC 39 KTQFPQLK MEL, NHL, BRCA, GBC, HCC, NSCLCother, NSCLCadeno, OC, NSCLCsquam, SCLC OSCAR, UBC 40 ALNDRFAGY PRCA 41 RLGAARGQLR PRCA 42 RVCMTVDSLVNK MEL 43 TVYDSIWCNMK OC, UEC 44 HAFPPGPNY NSCLCadeno, UBC HNSCC, NSCLCsquam, OSCAR 45 KCYEVGMMK BRCA, OC, UEC 46 VVTGNVPLK GBC, HCC, OC, MEL SCLC 47 ALFGNALVFY NSCLCsquam, RCC OSCAR 48 KGWNGQIFK AML 49 ITAPLMPLGK GBC, GC, CCC, HNSCC, NSCLCadeno, NSCLCsquam, OSCAR NSCLCother, OC, PACA, UBC, UEC 50 HSAGIFSMY CLL NHL 51 VTADGALAMEK CCC, GC, HNSCC, NSCLCsquam, NSCLCadeno, OSCAR NSCLCother, PACA, UBC, UEC 52 GMYEYGSIEK MEL 53 KQALSLVRK CCC, GC, HNSCC, NSCLCsquam, NSCLCadeno, OSCAR NSCLCother, PACA, UBC, UEC 54 GVTITKTLK MEL 55 RLSAESKDLLK OC, PACA, RCC, SCLC UEC 56 TTYYPSPLNK GBM RCC 57 RVLYRPQLEK HCC 58 VLYRPQLEK HCC 59 KINQYIIKK MEL 60 VVFPFPVNKR GBM, MEL, OC, AML, UEC SCLC, UBC 61 SLYDSEPRKK GBM, SCLC, UEC AML 62 GIFPKIMPK HCC 63 SSFRPLLSK BRCA, HCC, MEL, PRCA OC, SCLC, UEC 64 SVLSRMLVR BRCA, HCC, MEL, PRCA OC, SCLC, UEC 65 RTIEELQNK NSCLCsquam HNSCC, OSCAR 66 RVKEIVINK MEL 67 VVREILHAQTLK CLL 68 KGPMAGILAY MEL 69 GLSTILLYH MEL 70 VTAVASLLK MEL 71 VLYELGIIGNNK MEL 72 KLYPQCLQK NHL CLL 73 RCFSGPYLNK CLL 74 HSTLVALFY PRCA 75 IIFVPEMNK CRC UEC 76 IINESLLFY MEL 77 VVDDSQLPK NHL 78 KTGTFVLYKS NSCLCadeno, CCC NSCLCother, NSCLCsquam, OC, SCLC 79 AVAAVLLSR CLL 80 ALCGTQLFY CLL 81 HLFLPFSYK HCC 82 CLANYTVNK OC, OSCAR, UEC AML 83 KLADSVMAGK CRC, GBM 84 QLYSPPSPSYR NSCLCadeno, NSCLCother, NSCLCsquam 85 IMPTFDLTK HCC, MEL 86 RVSGSGGGGAGK CLL, HNSCC, MEL, BRCA, SCLC GBM NHL, NSCLCadeno, NSCLCsquam, OC, PACA, RCC, UBC 87 RLASVGLDAK CLL NHL 88 GTHVWVGLYK CRC, NSCLCsquam, BRCA, CCC, GBC, GC, OC, UBC, UEC HNSCC, NSCLCadeno, OSCAR, PACA 89 VIYVICRHK NSCLCadeno, UEC RCC 90 RTALLWGLKK CCC, NSCLCother, HNSCC, NSCLCsquam, UBC OSCAR, RCC 91 IMKRQVKCITK HNSCC, SCLC NSCLCsquam, OC, OSCAR, UBC 92 KRIPFRPLAK BRCA, HCC NSCLCadeno, OC 93 SVEGLSRRLK CCC, NSCLCother, HNSCC, NSCLCsquam, UBC OSCAR, RCC 94 GMTLLCEALK PACA PRCA 95 FLGLAFHPK BRCA, OC, PRCA HCC, NSCLCadeno 96 RAVSVNPGK NHL 97 RVRALGSQY GBC, GC, CCC, HNSCC, NSCLCadeno, NSCLCsquam, OSCAR NSCLCother, OC, PACA, UBC, UEC 98 VSVAGSILAK SCLC GBM 99 RTYTCQVTY NHL 100 TTNARILAR GBC, GC, HNSCC, MEL, OSCAR, NSCLCsquam UBC 101 HMDEFKRTQK CCC, GBC, GC, HNSCC, NSCLCsquam, NSCLCadeno, OSCAR NSCLCother, PACA, UBC 102 RLLQHTPSAR HCC, NSCLCadeno, CRC, GBC, GC, RCC NSCLCsquam, PACA, SCLC 103 SIYKKAVYR NSCLCother, HNSCC OSCAR 104 AVVQGLVEK CCC, GC, HNSCC, NSCLCsquam, NSCLCadeno, OSCAR NSCLCother, PACA, UBC 105 QVLDLQSVK CRC, GBC, GBM, AML, BRCA, HCC, HNSCC, MEL, NSCLCother, NSCLCadeno, OC, NSCLCsquam, SCLC OSCAR, UBC, UEC 106 LLRSGLTLR NHL, OSCAR CCC 107 ILNLNKMVK MEL, NSCLCother NHL 108 KMPILSYWPY MEL, NSCLCother NHL 109 KLQNLPTLLY OC, SCLC 110 IIFIPATILLK PRCA HCC 111 ATSPPASVR MEL 112 ASLAAAVLAK BRCA HCC 113 VSIRNTLLY AML 114 SLLTVSGAWAK NHL 115 AILHPFRAK PACA, UEC GC 116 KGVKKELPQK CCC, NSCLCadeno, GBC, HNSCC, OC, NSCLCother, OSCAR NSCLCsquam, SCLC, UBC 117 TVFVELWLK BRCA, OC, SCLC CCC, HCC 118 SLRGSFPILY CLL, NHL 119 RMGFRTLSK AML 120 TRMQKAGFLYY AML 121 LLPAARATSR GC, NSCLCadeno, CCC, HNSCC, OSCAR NSCLCother, NSCLCsquam, OC, PACA, UBC, UEC 122 LIGPLLICK CCC HCC 123 SLQGLTISY GBM 124 VVYDTMIEK GBM 125 VVYDTMIEKF GBM 126 KILETSLK MEL 127 GLAAGALLLY NHL CLL 128 KIKKPLSYR CCC, HNSCC, OC, UEC PACA, UBC 129 ALARVSSVKL GBM, OC CCC 130 KANSGNTFKY AML GBM 131 RVDSKQRYY HCC, NSCLCother, GBM NSCLCsquam, SCLC 132 ASSFRPLLSK BRCA, HCC, MEL, PRCA OC, SCLC, UEC 133 SFRPLLSK BRCA, HCC, MEL, PRCA OC, SCLC, UEC 134 SLLKPSGDYFK GC, HNSCC, CRC NSCLCsquam, OSCAR, UBC 135 TFKSVLLNK CLL, GC, OC, SCLC BRCA 136 ALSRMSQQY NSCLCother 137 TVYVAMCHKF HCC SCLC 138 VSLSKMLKK NHL CLL 139 KAIIRVIPLK HCC CRC 140 RLLAAGQVIR MEL 141 RLRDYISSLK NHL CLL 142 SPRVYWLGLNDR CLL, NHL 143 KTLGKIAEK BRCA, SCLC, UEC OC 144 PLAMLAATCNK GBM, SCLC PRCA 145 SLFEGIYTIK AML 146 TLLSYELAFK AML UBC 147 KLFMPRPK GBC, OC, UEC HCC, MEL 148 RIGNKGIYK CRC, GBC, GC, NHL HNSCC 149 TVFLSKYLKK GC UEC 150 LLLAAVTVK CCC, GBC, GC, PACA, UBC NSCLCadeno, OC 151 KVASFTVIGY HCC AML 152 KIICGVHYLY MEL 153 LASSPAGHK MEL 154 TIASVLVAR MEL 155 ALSHAVNNY HCC 156 VSLGIAVSK GBM, MEL, NHL, HCC NSCLCadeno, OC 157 SLPLQRILAMSK BRCA, CCC, GBC HNSCC, MEL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, UBC 158 RALGVPFVPR CRC, MEL, OC, CCC, GBC RCC, UEC 159 LLLLPFLLY PRCA 160 RLLPGKVVWK UEC 161 MKTLPAMLGTGK NHL CLL 162 LLALGAAYVY CRC NSCLCother 163 MLYYPSVSR MEL 164 RLAQYTIER GBC, GC, UEC PACA 165 AATIISSAK CLL 166 KVIAPGVIY BRCA, CRC, GBC, CCC GC, HNSCC, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA, UBC 167 MLKQARRPLFR BRCA, HCC, OC HNSCC, PRCA, SCLC, UEC 168 ATNGKVLKK OSCAR, UBC HNSCC 169 ATNGKVLKKR OSCAR, UBC HNSCC 170 LISGGSLRKL HCC NHL 171 ATIGLSVSK HCC 172 SLLEADPFLK OC, OSCAR, UEC AML 173 VSYNRLIK GC, HNSCC, CRC OSCAR, UBC 174 KMMKRLMTVEK GBC, HNSCC, OC, NSCLCsquam OSCAR, UEC 175 VIGTTSSPK NSCLCother, OC, SCLC, UEC 176 RLYDAYVNR GBM, RCC 177 ALLGVIIAKK RCC 178 RIYVYVKRK HNSCC, OC, OSCAR, PRCA 179 KINPTASLK MEL 180 RLKMAQESVSK MEL 181 RVAEEILIK SCLC 182 QICLPAIYK CCC, GBC, NSCLCadeno, RCC 183 KVYIPPIINK MEL 184 KVLEPPLGAR AML 185 SIINFKPLTY HCC 186 CTLPFKIFY AML, GBM 187 KTCQVSGLLY MEL, NSCLCsquam, SCLC 188 SSLPRAFQK BRCA, CCC, CRC, GBC, GBM, GC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, SCLC, UBC, UEC 189 KVFEEPEDFLK NHL 190 RSKWSNVFK CLL, CRC, NHL 191 SLYNLGGAK HNSCC, OSCAR 192 RSYSHVVLK CCC 193 IVYPSATDKTK BRCA, GBC, GBM, HCC, RCC 194 PVLICLALSK HCC, HNSCC, SCLC, UBC 195 KLQAKVLQY OC, SCLC 196 AISSTVLGK CCC, GBC, RCC 197 RIVDYLLEK MEL 198 FLYGAQTVY CLL 199 IVFPDVISK GBM, SCLC 200 RVLPPLTRILK NSCLCsquam 201 KVADFGLARLLK BRCA, CLL, OC, PRCA 202 RLFPGLYLGY HCC 203 IVAFIPLSNK UEC 204 VAFIPLSNK UEC 205 RVYPRPPSK BRCA, HCC, PRCA 206 RLYEMILQR AML 207 ATLNLFQIVSK AML, OC 208 KTGWFTLLY BRCA 209 KILDRVLSRY GBM, HNSCC, SCLC 210 KIFQGQINK HCC 211 VSLGTPIMK NSCLCsquam, OC 212 RTIDRSVFK NSCLCsquam, SCLC 213 KLYPTHACR SCLC 214 KLFTSVFGVGLK AML 215 KIWQNLRLK GBM, OC CCC 216 RVSSVKLISK GBM, OC CCC 217 RVYEGDGRNSLK CLL, NHL 218 KAFNQSSIFTK CLL 219 ALERKFRQK UEC 220 ALPRQAFHSK GBM, GC AML 221 RLAVSTRGK BRCA, CCC, CRC, GBC, NSCLCadeno GBM, GC, HNSCC, MEL, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, SCLC, UBC, UEC 222 RSNPYFQNK GBC HCC 223 GISNPITTSK BRCA, NSCLCadeno, OC, SCLC 224 SLYDGFLSY GBM, NSCLCother, OC, UEC 225 RVYPRPPSKTY BRCA, HCC, PRCA 226 RVWLGKHYK OC AML: acute myeloid leukemia; BRCA: breast cancer; CCC: cholangiocellular carcinoma; CLL: chronic lymphocytic leukemia; CRC: colorectal cancer; GBC: gallbladder cancer; GBM: glioblastoma GC: gastric cancer; HCC: hepatocellular carcinoma; HNSCC: head and neck squamous cell carcinoma; MEL: melanoma; NHL: non-hodgkin lymphoma; NSCLCadeno: non-small cell lung cancer adenocarcinoma; NSCLCother: NSCLC samples that could not unambiguously be assigned to NSCLCadeno or NSCLCsquam; NSCLCsquam: squamous cell non-small cell lung cancer; OC: ovarian cancer; OSCAR: esophageal cancer; PACA: pancreatic cancer; PRCA: prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC: urinary bladder carcinoma; UEC: uterine and endometrial cancer

(29) TABLE-US-00013 TABLE 9b Expression scores. The table lists peptides from genes that are 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, bile duct, blood cells, blood vessels, bone marrow, brain, esophagus, eye, gallbladder, heart, head&neck, kidney, large intestine, liver, lung, lymph node, nerve, parathyroid, pancreas, pituitary, peritoneum, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, urinary bladder. 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. Gene Expression in tumor samples Seq ID over- highly over- No Sequence expressed(+) expressed(++) 252 AVYNYPEGAAY BRCA, OC, UEC 253 KLSLSHKK HCC, HNSCC, BRCA, OC, NSCLCsquam, PRCA, SCLC OSCAR, PACA, UEC 254 SVLDKLLFH NSCLCadeno NHL, OC 255 VRTLPFLIK PRCA, UEC BRCA 256 AVIGALLAVGATK MEL 257 LAELEGALQK GBC, NSCLCsquam, BRCA, MEL, OC, SCLC NSCLCadeno, UBC 258 QVRDTLKIVFK CCC HCC 259 RSYVPLAH MEL 260 VIAAQLPK BRCA UEC 261 AVAQKLLSH BRCA, OC, SCLC, PRCA UEC 262 TMWQIPQEFVK BRCA UEC 263 RTYQLYSR GC, NSCLCsquam, OC, SCLC, UEC 264 VIFDAVTEQ CLL, NHL 265 KIYEFRLMM BRCA, GBC, GBM, HCC, RCC 266 RNLNTPVVK CRC 267 VLKSITVKV NHL, OC, SCLC, UBC 268 KVDMKDVTKI RCC, SCLC 269 KITDFNNIR GBC, GC, PACA 270 KFKPNQYTK CCC 271 RLPLRVVKH NSCLCadeno NHL BRCA: breast cancer; CCC: cholangiocellular carcinoma; CLL: chronic lymphocytic leukemia; CRC: colorectal cancer; GBC: gallbladder cancer; GBM: glioblastoma; GC: gastric cancer; HCC: hepatocellular carcinoma; HNSCC: head and neck squamous cell carcinoma; MEL: melanoma; NHL: non-hodgkin lymphoma; NSCLCadeno: non-small cell lung cancer adenocarcinoma; NSCLCsquam: squamous cell non-small cell lung cancer; OC: ovarian cancer; OSCAR: esophageal cancer; PACA: pancreatic cancer; PRCA: prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC: urinary bladder carcinoma; UEC: uterine and endometrial cancer

Example 3

(30) In Vitro Immunogenicity for MHC Class I Presented Peptides

(31) 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*03 restricted TUMAPs of the invention, demonstrating that these peptides are T-cell epitopes against which CD8+ precursor T cells exist in humans (see Table 10).

(32) In Vitro Priming of CD8+ T Cells

(33) 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.

(34) 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 μg/ml Streptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma, Nürnberg, Germany) were also added to the TCM at this step.

(35) 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.

(36) 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 μm diameter streptavidin coated polystyrene particles (Bangs Laboratories, Illinois, USA).

(37) pMHC used for positive and negative control stimulations were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 250) from modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO. 251), respectively.

(38) 800.000 beads/200 μl were coated in 96-well plates in the presence of 4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 were added subsequently in a volume of 200 μl. Stimulations were initiated in 96-well plates by co-incubating 1×10.sup.6 CD8+ T cells with 2×10.sup.5 washed coated beads in 200 μl 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, Oreg., 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).

(39) In Vitro Immunogenicity for Peptides

(40) 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 FIGS. 3A and 3B together with corresponding negative controls. Results for 6 peptides from the invention are summarized in Table 10.

(41) TABLE-US-00014 TABLE 10 in vitro immunogenicity of HLA class I peptides of the invention Exemplary results of in vitro immunogenicity experiments conducted by the applicant for the peptides of the invention. Seq ID Wells positive No Sequence [%] 232 AVAPPTPASK + 233 KTYETNLEIKK + 234 VVFPFPVNK + 237 VLYGPAGLGK + 248 GLASRILDAK + 249 ATSGVPVYK ++++ <20% = +; 20%-49% = ++; 50%-69% = +++; >= 70% = ++++

Example 4

(42) Synthesis of Peptides

(43) 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 lyophilizes (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.

REFERENCE LIST

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