Patent ID: 12247057

EXAMPLES

Example 1

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

Tissue Samples

Patients' tumor tissues were obtained from: Asterand (Detroit, MI, USA & Royston, Herts, UK); Bio-Options Inc. (Brea, CA, USA); Geneticist Inc. (Glendale, CA, USA); University Hospital Heidelberg (Heidelberg, Germany); ProteoGenex Inc. (Culver City, CA, USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Munich (Munich, Germany). Normal tissues were obtained from Asterand (Detroit, MI, USA & Royston, Herts, UK); Bio-Options Inc. (Brea, CA, USA); BioServe (Beltsville, MD, USA); Capital BioScience Inc. (Rockville, MD, USA); Centre for Clinical Transfusion Medicine Tuebingen (Tubingen, Germany); Geneticist Inc. (Glendale, CA, USA); Kyoto Prefectural University of Medicine (KPUM) (Kyoto, Japan); Osaka City University (OCU) (Osaka, Japan); ProteoGenex Inc. (Culver City, CA, USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva (Geneva, Switzerland); University Hospital Heidelberg (Heidelberg, Germany); University Hospital Tubingen (Tubingen, Germany); University Hospital Munich (Munich, Germany). 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

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.

Mass Spectrometry Analyses

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.

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 cancer samples to a baseline of normal tissue samples. Presentation profiles of peptides exemplary over-presented or exclusively presented on tumors are shown inFIGS.1A through1N.

Table 8 shows 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. 3 for hepatocellular carcinoma, peptide SEQ ID No. 11 for melanoma, ovarian cancer and uterine cancer).

TABLE 8Overview of presentation of selected tumor-associatedpeptides of the present invention across entities.SEQ IDNo.SequencePeptide Presentation on cancer entities3YLEKFYGLHCC6KYKDYFPVIHCC9RILRFPWQLMEL11YYSKSVGFMQWMEL, OC, UEC13HYTYILEVFGC, UEC14SYSSCYSFGC18DYIGSVEKWPRCA19ILKEDPFLFOC, RCC21SYEVRSTFCCC, OC, PRCA22TQPGDWTLFMEL23KFIISDWRFMEL24MYPDLSELLMAML, GBM, OC, UEC26KTPTNYYLFGC28YYSIISHTLHCC31QYQNVLTLWGBM, GC, HCC, MEL, NHL, PRCA, RCC, UEC32SLPDLTPTFPRCA33KSSVIASLLFGBM34MQPRMFFLFAML, GBM, HCC, MEL, UEC36KQMEDGHTLFAML, OC37QWPWQASLQFGC38KYTNWKAFLAML, HCC41VIYFMGAIFHCC, PRCA, UEC43IQMDEPMAFAML, MEL, OC44AYLSAVGTFAML, GC, MEL45KYFVPPQLFGC47KYADYFLEVGBM, OC, UEC48VFIDHPVHLKFUEC50SYPELVKMVWAML, GC, HCC51KYALLLQELAML, CLL, GBM, GC, HCC, MEL, OC, PRCA,RCC, UEC52KYMKIFHKFOC, UEC53KYITNLEDLPRCA54LLIKLLQTFAML, GBM, MEL, OC, PRCA, RCC55RWMDQRLVFGC, HCC, MEL, UEC56VYMIEPLELGBM, NHL57YPSIIQEFGBM, RCC58QFAAPLRGIYFGBM, HCC59KYSTTFFMVGBM60TYLSIFDQLAML, GC, HCC, OC61NYAENILTLAML, GBM, GC, MEL62LYQEILAQLAML, GBM, GC, HCC, MEL, PRCA, RCC63VMPSDSFFFMEL, NHL, OC, UEC64NYAIFDEGHMLGBM, GC65VYPASKMFPFIGBM, GC, HCC, MEL, NHL, OC, UEC66IYFRDSSFLAML, GC, MEL67RYPGKFYRVOC68IYQQIIQTYGC, MEL, UEC69IMPEKFEFWAML, GC, MEL, NHL, UEC70PYTNYTFDFGC, MEL71SYMVLAPVFMEL72RYEGILYTIGC, HCC, NHL, PRCA73SYIGLPLTLGC, HCC, RCC74VYDQYFITLAML, PRCA76WYGWHFPELAML, GC, HCC, RCC, UEC77AYTLLGHEFVGC, MEL, OC78TWFPKTPMLFAML, GBM, GC, HCC, MEL, UEC79RYLADLPTLGC, HCC, OC, UEC80YYSPLRDLLMEL82RFLPSPVVIAML, GC, HCC, MEL, PRCA, UEC83TYCQNIKEFAML, OC, PRCA, UEC84YVDINTFRLHCC86FVIDGFDELCRC, GC, NHL, OSCAR87TLYPYQISQLHCC, OSCAR90FLLMHPSICLL, HCC, RCC91FALPGLLHAGC, HCC, NHL, OSCAR, RCC92NLRDLLSEVHCC93TLQEKILQVCLL, GBM, NHL95ITIGVLARVCRC, PACA96HLVGGLHTVAML, BRCA, CRC, GC, MEL, OC, PRCA97VLALVNSTVCLL98LQSSGLTLLLGBM, OC, PRCA99FLKEKVPGICLL, GC, MEL, NHL100RQYPTPFQLAML, CLL, CRC, GBM, NHL, OC, RCC101FIISDWRFVLHNSCC102SLLEQAIALGC, HCC, NHL, OC, UEC103FLYYPDPVLGBC, HCC, MEL, NHL105SLLTHIPTAAML, CRC, HNSCC, MEL, OC, OSCAR, RCC,UBC, UEC106FIIDTTYPAYVCRC, OC, OSCAR107LLQGAIESVBRCA, CLL, CRC, HNSCC, MEL, NHL, UBC,UEC108MIIALSLYIAML, BRCA110LLADFQALLRCC111ALCLLLHLLAML, GBC, HCC, HNSCC, RCC113AVLTGLVEVBRCA, CLL, CRC, GC, HCC, NHL, OSCAR,RCC, UEC114ILDERQVLLAML, BRCA, CRC, GBC, GC, HCC, HNSCC,MEL, NHL, OC, OSCAR, PACA, UBC, UEC115MLLETQDALYVHNSCC116VLMEENSKLGBM117FLDPNARPLVNHL, OC118ALSSVLHSIAML, BRCA, CRC, GBC, HCC, HNSCC, MEL,NHL, OC, OSCAR, PACA, RCC, UBC119RTADITVTVAML, CRC, UBC120ALLANLPAVGBC, GC, OC, PACA121ALVDTLTGIHNSCC, NHL, UEC122ALLEMFPEITVBRCA, OC, PRCA123LMAFFLAVVCCC, HCC, NHL, OSCAR, RCC124SVASVLLYLAML, BRCA, CLL, HCC, HNSCC, NHL, OC125VLQPFLPSIAML, BRCA, CCC, CLL, CRC, HCC, HNSCC,MEL, NHL, OC, RCC126FLSTVTSVCCC, GBM, HCC, HNSCC, MEL, NHL, OSCAR,PACA, RCC, UBC, UEC127GLDGSLVFLAML, CLL, CRC, GBM, HCC, HNSCC, MEL,NHL, OC, OSCAR, UBC128FLGTTPTLAML, CLL, GBM, HNSCC, NHL, OC, UBC, UEC129VLYDKDAVYVAML, CLL, CRC, HNSCC, NHL, OC, UBC, UEC130NLWGGQGLLGVBRCA, GBC, GBM, GC, HCC, OC, PRCA, UEC131LLKEFVQRVBRCA, CRC, HCC, HNSCC, OC, OSCAR132ALWLVDPLTVCLL, CRC, GBM, HCC133MTLPVDAVISVCLL, CRC, HCC, HNSCC, NHL, PRCA, UEC134AAEIGDKSWLYGC, MEL, NHL, UEC135ASEDSVLLYGBM, GC, MEL, NHL, OSCAR, PRCA,136ATDLVVLDRYGBM, GC, HCC, MEL, NHL, OSCAR, PRCA,UEC137ATSKFMEFYGBM, MEL, OC, PRCA, UEC139ECDMAFHIYMEL, OC140ESDREELNYGC, PRCA141ESDVGVVVYGBM, GC142EVAEPSVLFDLYGC, MEL, NHL, OC, UEC144FLDSQNLSAYGC145FVDKPVAYGBM, GC, MEL146GLNTGSALSYGC, OC148GTEFTTILYGC, NHL, OSCAR, PRCA149GTEFTTVLYGC, HNSCC, MEL, OSCAR, PRCA150GTELLSLVYGC, MEL, NHL, OC, PRCA, UEC152HTDSLHLLIGC, MEL, OC154LLDISQKNLYGBM, NHL, PRCA155LLDPNPHMYPRCA156LLDSLREQYGC, NHL, OSCAR157LMDRPIFYGBM, GC, MEL, NHL159LSDTSVIQFYGBM, GC, HCC, MEL, NHL, PRCA160LTEAVLNRYOC161LVDDGTHGQYGBM, GC, MEL162LVDNSIRELQYGC, HCC, MEL, NHL, OC, UEC163NSDSSLTLREFYHCC, PRCA166NTQITDIGRYMEL167QSDPGTSVLGYGBM169RLDTPLYFSYMEL, OC170RSDDTAVYYCLL, GC, HCC, NHL, PRCA, UEC172RTDSCSSAQAQYMEL173RTEFNLNQYGBC, GC, MEL, UEC177SSDEVNFLVYGC, MEL, OC, UEC178SSDSSTLPKLGC, OC, PRCA179STAKSATWTYPRCA180STDPWIQMAYGBM, GC, MEL181TADGKTYYYGBM, HCC, MEL, PRCA182TDYHVRVYGBM,PRCA184TSAHPEDSSFYGC, NHL, PRCA186TTDIIEKYGC, MEL,187VADLHLYLYGC, MEL, OC, PRCA, RCC190VTDGINPLIDRYMEL191VTDGSLYEGVAYGC, MEL, UEC192VTEESFDSKFYGC193VTEFSLNTYGBC, GC, MEL, OC, OSCAR, UEC196WMFFVINYUEC197YADTVRPEFYOC198YLDPVQRDLYGBM, GC, HCC, MEL, NHL, OC, UEC202KLAELEGALQKMEL, OC, UEC204AVFDKFIRYGBM207RSFNGLLTMYMEL, OC210ATSGVPVYKUEC211TVNPVAIHKGBM, NHL, OC, PRCA, UEC212KAYEQVMHYUEC213LNINMTSPMGTKGBM, GC, MEL, NHL, PACA214RTMSEAALVRKGC, NHL, OC, PRCA, UEC215MMFSGPQILKLMEL216KLYAWELAFAML, GBM, MEL, OC, PRCA217RILNQILYYAML, GBM, GC, HCC, MEL, NHL, PRCA, UEC218KTLVAELLILKAML, NHL, UEC219RLRSSLVFKUEC220SPSVSQLSVLUEC235MPLKHYLLLMEL, NHL, OC, UEC237RPAATAVISLGBM, NHL, OC244FPYVRDFVMGBC, MEL, NHL, OC, UEC247RALLARLLLNHL251VPRSSGQTVBRCA, GBM, UEC255MPLLENLYLOC, UEC256SPRVPSIELNHL259RPPAAGLRGISLGBM260YPQHPGLNABRCA, GBM, GC, NHL262SAYPQRLEIGC263HPAPYGDLLUEC271MPLPWSLALPMEL273MPLLWLRGFUEC274TPYQEHVALOC275APHPPLSVVAML, BRCA, MEL276LPRAGGAFLNHL, OC, RCC, UEC277MPLFEPRVFOC, UEC278HPMIDINGIIVFUEC280VPISEEGTPVLMEL, OC, PRCA, UEC281RPRAPVTPAGBM282MPQIETRVILUEC283RPHSLSSELAML, NHL284FPVTSIFHTFMEL, OC, UEC285FPSFLTNSLAML286VPTLRSELOC288FPQKFIDLLUEC289VPENHSVALUEC290APYRPPDISLBRCA292SPQRLRGLLLNHL293RPRSALPRLLLPNHL, UEC295KPEGTRIAVNHL, UEC296MPMQDIKMUEC300VASPKHCVLOC301YMHKLLVLAML, NHL, OC, PRCA, UEC305ALKLRVAVLNHL306ILKVKVGLMEL, OC308MLKQKVEELOSCAR311EIRIRVVQMMEL, NHL, OC, PRCA313ELKKKEYEELNHL314AIISRLVALNHL, OSCAR, UBC316VIKEKALTLNHL, OC, PRCA, RCC318EAAIRSVELGBM, MEL, NHL, OC321AEMLESVIKNYNHL322KEVDPAGHSYNHL323SEFMQVIFNHL328FEYDFLLQRIUEC330KEGDLGGKQWNHL335KELEATKQYMEL, NHL337TENRYCVQLHCC342HEFSSPSHLNHL343TEFTTVLYGBM, NHL, PRCA345IEFIHPQAFGBM, GC, NHL, PRCA347ALNPYQYQYUEC348AEIQGNINHVUEC351EEVNYINTFAML, MEL, NHL, OC354TEDPTILRIGC, HCC, OC, PRCA, UEC356EEGRVYLFGBM,PRCA357RELENCFQIQUEC359DELFSIALYNHL363AELDKLTSVGBM, UEC366AENLFRAFGBM, NHL, OC367GEVHPSEMIUEC368GEFPVRVQVAML, GC, OC, UEC370YEDLSQKYGBM, NHL, OC371GELALKKKIUEC372TEGIIMKDFOC, PRCA, RCC, UEC373MEMQKSPVFNHL, OC374DEVNFLVYCCC, GBC, GBM, NHL, OC, PRCA375VYSDLHAFYYGBM, GC, HCC, MEL, PRCA376KYVKDFHKFAML, OC, PRCA, UEC377VYVGAVNRIGC, HCC, PRCA378KFLGPAEHLTFOC379NYIVPDKQIFGBM, GC, HCC, MEL, OC, PRCA380VFQEKHHVIPRCA381TYSKKHFRIMEL, OC382IYHSHHPTLAML, MEL, NHL, OC, UEC383RYKQDVERFAML, MEL, OC, PRCA, UEC384KYVKVFDKFAML, UEC385MYINEVERLGBM, MEL, OC, PRCA, UEC386VYNDHSIYVWAML, GBM, HCC, MEL, NHL, PRCA, UEC387RWLPQKNAAQFAML, GC, HCC, MEL, OC, RCC, UEC388FSIPEGALVAVAML, CCC, CLL, CRC, GBC, GC, HCC,HNSCC, MEL, NHL, OC, OSCAR, PRCA, RCC,UBC, UEC389TLMEQPLTTLAML, BRCA, CRC, HCC, HNSCC, NHL, OC,PRCA, UEC390HIMPTVHTVBRCA, CLL, GBM, HCC, HNSCC, MEL, NHL,OC, OSCAR, UBC, UEC391SLIDMRGIETVBRCA, CLL, GBM, HCC, HNSCC, OC, UBC392SLFKDQMELAML, BRCA, CLL, CRC, GBM, HCC, MEL, NHL,OC, PRCA, RCC, UBC, UEC393ILLPYLQTLAML, BRCA, CLL, CRC, GBM, HCC, HNSCC,NHL, OC394ASEAEMRLFYGBM, GC, MEL, NHL395ASEASRLAHYGBM, GC, NHL, PRCA, UEC396ASEFGNHYLYGBM, GC, MEL, UEC397ASEITSKGASLYGBM, GC, HCC, MEL, OC, PRCA398ASEQQALHTVQYGBM, GC, MEL, NHL, PRCA, UEC399ATDIPCLLYMEL400ATDISRQNEYGBM, GC, NHL, OC401DSDESYMEKSLYGC402DTDSQRLAYGBM, GC, MEL403ELDSKVEVLTYGBM, GC, MEL, OC, PRCA404ETARKFLYYGBM405ETEEGIYWRYGC406ETEQTKFWDYGBM, GC, MEL, OC, RCC, UEC407FSDNDKLYLYGBM, GC, MEL, NHL, PRCA, UEC408FTEQWTDGYGBM, GC, OC, OSCAR, PRCA,409FVDPLVTNYGBM, GC, HCC, MEL, NHL, OC, OSCAR,PRCA, RCC410GSDHQSPSSSSYGBM, GC, MEL, OC, PRCA,411GTVYEDLRYGBM, GC, HCC, MEL, NHL, OC, OSCAR412ILDEVIMGYGBM, GC, MEL, NHL, OC, OSCAR413ISDRYYTALYGBM, GC, PRCA, UEC414KTDESLTKYGC, NHL, PRCA415LLDPRSYHTYGC, NHL416LLDTAQKNLYGBM, NHL, PRCA417LLEDKHFQSYGBM, GC, HCC, MEL, NHL, OC, PRCA, UEC418LSDPSGPKSYGBM, HCC, PRCA419LSELKPMSYGBM, GC, HCC, MEL, OSCAR, PRCA, RCC,UEC420LTEDKETLQYGC, HCC, MEL, NHL, PRCA421LTELLERAAFYGC, MEL, NHL, UEC422MIDVTKSYYGBM, GC, MEL, NHL, OC, OSCAR, PRCA,UEC423NLDAVHDITVAYGC, MEL, PRCA, UEC424NLDEEKQLLYMEL, PRCA425NLDIIQQEYGBM, GC, HCC, MEL, NHL, OC, OSCAR,PRCA, UEC426NLDQATRVAYNHL427NSDEQKITEMVYGC428NSELSCQLYGBM, GC, MEL, RCC429NTEDSSMSGYLYGC, MEL,430NTEGLHHLYGBM, HCC, MEL, PRCA431NTSDMMGRMSYGC, MEL, NHL, OC,432NVDPVQHTYGBM, GC, HCC, HNSCC, MEL, NHL, OC,OSCAR, PRCA, RCC, UEC433QIDTGENLYGC, MEL434QTDCAPNNGYGBM, GC, MEL, PRCA435QTDDTWRTEYGBM, GC, MEL, NHL, PRCA436QTETGTPYMLYGBM, GC, MEL, NHL, OC, OSCAR, PRCA,UEC437STDGKHWWEYGC, MEL, NHL, PRCA438STDNFNCKYGC, HCC, MEL439TLDAGKFQIYGC, HCC, MEL, NHL, PRCA, UEC440TLDENPGVRYGBM, GC, HCC, MEL, NHL, PRCA, UEC441TLDSALNAASYYGBM, GC, MEL, NHL, PRCA, RCC442TSDFSRFTNYGBM443TTDFPSESSFEYGC, MEL, NHL, OC, PRCA, UEC444TTDTVIRSYGC, MEL, UEC445VLDQGKITEYGBM, HCC446VTAQVVGTERYGC, MEL447VVDEDHELIYGBM448YLDIPNPRYGBM, MEL, RCC449YLDRGTGNVSFYGBM, GC, HCC, MEL, PRCA, RCC450YSDDGQKWTVYMEL451YSDSLVQKGYGBM, GC, HCC, OC, PRCA, UEC452YVDAVLGKGHQYGBM, GC, MEL, NHL, OC, PRCA, UEC453AINTSIKNKPRCA454KVYTPSISKGBM, HCC, MEL, UEC455RIADIFVKKGC, MEL, NHL, OC, UEC456SMFTAILKKAML, MEL, NHL, OC, UEC457SINKPTSERGBM458GIADFVLKYAML, BRCA, GBM, MEL, NHL, UEC461RPILIIVTLNHL464YPRPGTPAAAML, GC, MEL, NHL, OC, RCC465VPRPIFSQLNHL468SPMYGQAGLRCC469YPENGVVQMAML, OC470SPNSYFRVLRCC471KPRPDVTNELNHL472NPRATDAQLAML473LPRALLSSLNHL, OC474LPRLLPALAML, NHL476AEEEIMKKINHL477QENSYQSRLOC479AEIQPQTQVUEC480GEVSGLTKDFMEL, NHL, OC481RELQHEHSLOC482TEREWADEWAML, MEL, NHL, OC, RCC483EENDQSTHKWMEL, NHL, OC, RCC, UEC484AEVGFVRFFAML, MEL, NHL, OC485SEIEDSTKQVFMEL, NHL, OC486SEDDPILQINHL, OC, UEC487AEDQLHHSFAML, NHL488TEFPIIKMYAML, MEL, NHL, OC, PRCAAML = acute myeloid leukemia,BRCA = breast cancer,CCC = bile duct cancer,GBM = brain cancer,CLL = chronic lymphocytic leukemia,CRC = colorectal carcinoma,OSCAR = esophageal cancer,GBC = gallbladder adenocarcinoma,GC = gastric cancer,HNSCC = head and neck squamous cell carcinoma,HCC = hepatocellular carcinoma,MEL = melanoma,NHL = non-Hodgkin lymphoma,OC = ovarian cancer,PACA = pancreatic cancer,PRCA = prostate cancer and benign prostate hyperplasia,RCC = renal cell carcinoma,UBC = urinary bladder cancer,UEC = uterine cancer.

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

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

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.

Total RNA from healthy human tissues for RNASeq experiments was obtained from: Asterand (Detroit, MI, USA & Royston, Herts, UK); Bio-Options Inc. (Brea, CA, USA); BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, MD, USA); Capital BioScience Inc. (Rockville, MD, USA); Geneticist Inc. (Glendale, CA, USA); Heidelberg University Hospital (Thoraxklinik, Heidelberg, Germany); Istituto Nazionale Tumori “Pascale” (Naples, Italy); ProteoGenex Inc. (Culver City, CA, USA).

Total RNA from tumor tissues for RNASeq experiments was obtained from: Asterand (Detroit, MI, USA & Royston, Herts, UK); Geneticist Inc. (Glendale, CA, USA); ProteoGenex Inc. (Culver City, CA, USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Bonn (Bonn, Germany); University Hospital Tubingen (Tubingen, Germany).

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

Gene expression analysis of—tumor and normal tissue RNA samples was performed by next generation sequencing (RNAseq) by CeGaT (Tubingen, Germany). Briefly, sequencing libraries are prepared using the Illumina HiSeq v4 reagent kit according to the provider's protocol (Illumina Inc., San Diego, CA, 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 lung cancer (including NSCLC and SCLC) are shown inFIGS.2A through2N. Expression scores for further exemplary genes are shown in Table 9.

TABLE 9Expression scores. The table lists peptides from genes that are veryhighly over- expressed in lung cancer tissues (NSCLCadeno = non-smallcell lung carcinoma adenocarcinoma; NSCLCsquam = non-small cell lungcarcinoma squamous cell; NSCLCother = non-small cell lung carcinoma,other subtypes; SCLC = small cell lung carcinoma) compared to a panelof normal tissues (+++), highly over-expressed in tumors compared toa panel of normal tissues (++) or over-expressed in tumors comparedto a panel of normal tissues (+). The baseline for this score wascalculated from measurements of the following relevant normal tissues:adipose tissue, adrenal gland, bile duct, blood cells, blood vessels,bone marrow, brain, cartilage, esophagus, eye, gallbladder, heart,head&neck, kidney, large intestine, liver, lung, lymph node, nerve,parathyroid, pancreas, pituitary, pleura, skeletal muscle, skin,small intestine, spleen, stomach, thyroid gland, trachea, urinarybladder, ureter. In case expression data for several samples of thesame tissue type were available, the arithmetic mean of all respectivesamples was used for the calculation.SEQ IDGene ExpressionNoSequenceNSCLCadenoNSCLCsquamNSCLCotherSCLC1QYDPTPLTW++++++++2VWSNVTPLKF++++++++++3YLEKFYGL+++++++++4SYEKVINYL++++++5RYMKKDYLI+++6KYKDYFPVI++++++7VQQWSVAVF+++8PFLPPAACFF+++9RILRFPWQL+++++10VWSDVTPLNF+++++11YYSKSVGFMQW++12STIRGELFFF++++13HYTYILEVF++++14SYSSCYSF++15KYALLLQDL+++16TYNPDFSSL++17YYADKKTFIVL++++18DYIGSVEKW++19ILKEDPFLF+20EFTTVLYNF+21SYEVRSTF+++22TQPGDWTLF+23KFIISDWRF+24MYPDLSELLM++25SYNGYVFYL++26KTPTNYYLF+27NYTLYPITF+28YYSIISHTL++29VYPLLSRLYW++30QYLPGWTVLF+31QYQNVLTLW+32SLPDLTPTF++33KSSVIASLLF+34MQPRMFFLF++35KYLEESVWL+36KQMEDGHTLF+37QWPWQASLQF+38KYTNWKAFL+39LIFMLANVF+40QYEPPSAPSTTF+41VIYFMGAIF+42TLPNTIYRF++43IQMDEPMAF+44AYLSAVGTF+45KYFVPPQLF+46AFPVTSIFHTF++47KYADYFLEV+48VFIDHPVHLKF+49LYISEVRNI+50SYPELVKMVW+51KYALLLQEL+52KYMKIFHKF+53KYITNLEDL+54LLIKLLQTF+55RWMDQRLVF+56VYMIEPLEL+57YPSHQEF+84YVDINTFRL++++++++++85YIDEFQSLV++86FVIDGFDEL++++++87TLYPYQISQL+++++++88VQMVITEAQKV++++89ILSTTMVTV++++++90FLLMHPSI++91FALPGLLHA++++92NLRDLLSEV++++++93TLQEKILQV++94VLPDIETLIGV++++95ITIGVLARV++96HLVGGLHTV+97VLALVNSTV+98LQSSGLTLLL+++99FLKEKVPGI+100RQYPTPFQL++101FIISDWRFVL+102SLLEQAIAL+103FLYYPDPVL+104GMLDIFWGV+105SLLTHIPTA+106FIIDTTYPAYV+107LLQGAIESV+108MIIALSLYI++109LLLGSIGLLGV+110LLADFQALL+111ALCLLLHLL+112SVSDGIHSV+113AVLTGLVEV+114ILDERQVLL++115MLLETQDALYV++116VLMEENSKL+117FLDPNARPLV+118ALSSVLHSI+119RTADITVTV++120ALLANLPAV++121ALVDTLTGI+122ALLEMFPEITV+123LMAFFLAVV+124SVASVLLYL+138DSDSCHFNY++139ECDMAFHIY+140ESDREELNY+143FIDYPKKEDY+++146GLNTGSALSY+++147GSSDSSTLPKL+148GTEFTTILY+149GTEFTTVLY+150GTELLSLVY+153KLDRSVFTAY++155LLDPNPHMY+++158LSDLLKQGY+++160LTEAVLNRY+++++++163NSDSSLTLREFY++164NTDNNLAVY+++++++165NTDPTAPPY++166NTQITDIGRY++167QSDPGTSVLGY+168QTDHPQPILDRY+++171RSDPVTLNVLY++172RTDSCSSAQAQY+174SADDIRGIQSLY++++++++++175SDVTPLTF+++++176SRTINVSNLY++177SSDEVNFLVY++178SSDSSTLPKL+179STAKSATWTY++183TLEDIATSHLY+185TSDSNLNKY++188VSDAKLDKY+189VSDSECLSRY++++++190VTDGINPLIDRY++192VTEESFDSKFY+193VTEFSLNTY+++194VVADTKMIEY+++195VVDSVGGYLY+++199YLPQHTIETY+200YSDEDVTKY+++201YVGKEHMFY++++++202KLAELEGALQK++++203KVKDTPGLGK++++++204AVFDKFIRY++205SLDGAARPK++++206KLIDLSQVMY+207RSFNGLLTMY++208GLASRILDAK++209RTQIPMSEK+210ATSGVPVYK++211TVNPVAIHK+212KAYEQVMHY+213LNINMTSPMGTK+214RTMSEAALVRK+215MMFSGPQILKL+216KLYAWELAF+217RILNQILYY+218KTLVAELLILK++219RLRSSLVFK+220SPSVSQLSVL++++++++221VPDVAQFVL++++++222NPFYPEVEL++++++223YPKDIYSSF+++++224GPQPWHAAL++++++225LPFDGPGGIL+++++226SPRMSGLLSQT+++227YPRGNHWAVGH+++228YPRGNHWAVGHL+++229VPLPAGGGTV+++230VPLPAGGGTVL+++231RPRALRDLQL++++232RPRALRDLQLL++++233KPYQGNPTF++234RAKNAGVTI++++235MPLKHYLLL++236RVRGGEDGDRAL++237RPAATAVISL++++238KPGPPWAAF++239YVPSASLFML+++240SPREVTTVL++241SARLATDAL++242SPRWLPVSL++243RPIENRILIL+++244FPYVRDFVM+++245RIREHVPQL+++246TPLPAVIVL+++247RALLARLLL+++248IPNWARQDL+++249VPSSRILQL+++250SPRDFLSGL++251VPRSSGQTV++++252SPDIRNTTV++253RVIDAVRFTL++254NPFPHLITL+++255MPLLENLYL++256SPRVPSIEL+257LPRIPFADV++++258LPRGPLASL++259RPPAAGLRGISL+260YPQHPGLNA++261APSARVGVC+++262SAYPQRLEI+263HPAPYGDLL++264RPILIHTL+265SPRQPPRLV+266HAYPPGPGL+267HPELVNHIVF+268YPLFRGINL++269APRAPRLML+270APGPRFLVT++271MPLPWSLALP+272MPLPWSLALPL+273MPLLWLRGF+++274TPYQEHVAL+275APHPPLSVV+276LPRAGGAFL++277MPLFEPRVF+++278HPMIDINGIIVF++279SPARASPAL+280VPISEEGTPVL+281RPRAPVTPA+282MPQIETRVIL++283RPHSLSSEL++284FPVTSIFHTF++285FPSFLTNSL+286VPTLRSEL+287APREEQQRSL+288FPQKFIDLL+289VPENHSVAL+290APYRPPDISL+296MPMQDIKM+++++++++297RAQLKLVAL+++298FNKRKPLSL++++299MAQFKEISL++++300VASPKHCVL++++++301YMHKLLVL++302HLLQKQTSI++303LPFPKFTV++304ELKKLYCQI+305ALKLRVAVL+306ILKVKVGL+307ILLPRTVSL++308MLKQKVEEL+309DAIQRKYSC+310LPPKKFVL+311EIRIRVVQM+312EAMLRNKEL+313ELKKKEYEEL+314AIISRLVAL+319AEMLERVIKNY++++++++320MEVDPIGHVYIF+++++++++321AEMLESVIKNY+++++++322KEVDPAGHSY++++++323SEFMQVIF++++++324TDSIHAWTF+++325QEQDVDLVQKY++++++326QEMQHFLGL+++++++++327YEIEARNQVF+++++++++328FEYDFLLQRI++++++++329NEHPSNNW+++++330KEGDLGGKQW++++++331EDAQGHIW++++332MEVPVIKI++++++333AETLSTIQI+++++334AEDEPAAAHL++++++335KELEATKQY+++++336ASSSGPMRWW++++337TENRYCVQL++338SEGSEPALLHSW++339SEPALLHSW++340TEFSLNTY+++341EEIEGKGSFTYF++342HEFSSPSHL++343TEFTTVLY+344EEATGQFHVY+345IEFIHPQAF++346VEAPGPVHVYW+++347ALNPYQYQY+348AEIQGNINHV+349AEQDMRELTY+350GECDVFKEIL+351EEVNYINTF+352NEVLTYIKF+353GEIIMQNNW+354TEDPTILRI+355SDMVRFHLF+++356EEGRVYLF++357RELENCFQIQ+358KEADIHFLI+++359DELFSIALY+360AEVPTGVII+361SENLFFASF+362SEKGVIQVY+363AELDKLTSV+364AETPIQNVI+365SEMNVNMKY+366AENLFRAF++367GEVHPSEMI++368GEFPVRVQV+369EEIERFFKL+370YEDLSQKY+371GELALKKKI+372TEGIIMKDF+373MEMQKSPVF+374DEVNFLVY++375VYSDLHAFYY++376KYVKDFHKF+377VYVGAVNRI+378KFLGPAEHLTF+388FSIPEGALVAV+389TLMEQPLTTL+401DSDESYMEKSLY+402DTDSQRLAY+405ETEEGIYWRY++409FVDPLVTNY+419LSELKPMSY+427NSDEQKITEMVY+429NTEDSSMSGYLY+432NVDPVQHTY+438STDNFNCKY++442TSDFSRFTNY+450YSDDGQKWTVY++454KVYTPSISK+455RIADIFVKK++456SMFTAILKK+457SINKPTSER+458GIADFVLKY+459RPMQQARAQL+++460MPMAGDMNGL++461RPILIIVTL+462RPFHTRATV++++++463TPKAGPTL++++++464YPRPGTPAA+465VPRPIFSQL+466APYKSVTSL++467KPFSSFTSM+468SPMYGQAGL+469YPENGVVQM+470SPNSYFRVL+471KPRPDVTNEL+472NPRATDAQL+476AEEEIMKKI++++++++++477QENSYQSRL+++++478SEIEQEIGSL++++479AEIQPQTQV+++480GEVSGLTKDF+481RELQHEHSL++482TEREWADEW+483EENDQSTHKW+484AEVGFVRFF+485SEIEDSTKQVF+486SEDDPILQI+487AEDQLHHSF+488TEFPIIKMY+489SEIGKAVGF+

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

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 for HLA-A*02:01, HLA-A*24:02, HLA-A*01:01, HLA-A*03:01, HLA-B*07:02, HLA-B*08:01 and HLA-B*44:02 restricted TUMAPs of the invention, demonstrating that these peptides are T-cell epitopes against which CD8+ precursor T cells exist in humans (Table 10a and Table 10b).

In Vitro Priming of CD8+ T Cells

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, HLA-A*24, HLA-A*01, HLA-A*03, HLA-B*07, HLA-B*08 or HLA-B*44 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.

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, Nornberg, Germany) were also added to the TCM at this step.

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.

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).

pMHC used for positive and negative control stimulations were A*02:01/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 532) from modified Melan-A/MART-1) and A*02:01/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO. 533), respectively. 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 1×106CD8+ T cells with 2×105washed 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, 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 Lung Cancer (Including NSCLC and SCLC) Peptides

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 16 peptides of the invention are shown inFIGS.3A through11Btogether with corresponding negative controls. Results for 152 peptides from the invention are summarized in Table 10a and Table 10b.

TABLE 10ain vitro immunogenicity of HLA class I peptides ofthe inventionExemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention.Wells positiveSeq ID NoSequence[%]491KYLEKYYNL++492NYEDHFPLL++493TYKYVDINTF+494RYLEKFYGL+495SYNDALLTF+++496VFMKDGFFYF+498EYIRALQQL+500VWSDVTPLTF+504VYEKNGYIYF++++510KVLEHVVRV+513KLVELEHTL+515KIFEMLEGV+516YTFSGDVQL+519KIQEILTQV+520KIQEMQHFL+525RLDDLKMTV+528RLLDSVSRL+<20% = +;20%-49% = ++;50%-69% = +++;>=70% = ++++

TABLE 10bin vitro immunogenicity of HLA class I peptides ofthe inventionExemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention.Seq ID NoSequenceWells positive [%]HLA136ATDLVVLDRY“+”A*01143FIDYPKKEDY“+”A*01153KLDRSVFTAY“++++”A*01160LTEAVLNRY“++”A*01164NTDNNLAVY“+”A*01173RTEFNLNQY“++++”A*01174SADDIRGIQSLY“+”A*01185TSDSNLNKY“+”A*01187VADLHLYLY“+++”A*01189VSDSECLSRY“++”A*01193VTEFSLNTY“+”A*01201YVGKEHMFY“+”A*01395ASEASRLAHY“++”A*01398ASEQQALHTVQY“+”A*01405ETEEGIYWRY“+”A*01430NTEGLHHLY“+”A*01436QTETGTPYMLY“+”A*01451YSDSLVQKGY“+”A*01452YVDAVLGKGHQY“+”A*0184YVDINTFRL“+”A*0285YIDEFQSLV“++”A*0287TLYPYQISQL“++”A*0288VQMVITEAQKV“++”A*0289ILSTTMVTV“+++”A*0293TLQEKILQV“+”A*0294VLPDIETLIGV“+++”A*0295ITIGVLARV“++++”A*0296HLVGGLHTV“+++”A*0297VLALVNSTV“+++”A*02101FIISDWRFVL“+”A*02125VLQPFLPSI“++++”A*02127GLDGSLVFL“++”A*02128FLGTTPTL“+”A*02129VLYDKDAVYV“++”A*02130NLWGGQGLLGV“+”A*02131LLKEFVQRV“++++”A*02132ALWLVDPLTV“+++”A*02133MTLPVDAVISV“+”A*02392SLFKDQMEL“+”A*02393ILLPYLQTL“+”A*02205SLDGAARPK“++”A*03208GLASRILDAK“+”A*03209RTQIPMSEK“+”A*03210ATSGVPVYK“++++”A*03214RTMSEAALVRK“+”A*03218KTLVAELLILK“+”A*031QYDPTPLTW“+”A*242VWSNVTPLKF“+”A*244SYEKVINYL“+”A*246KYKDYFPVI“+”A*248PFLPPAACFF“+”A*2410VWSDVTPLNF“+”A*2411YYSKSVGFMQW“++”A*2413HYTYILEVF“+++”A*2415KYALLLQDL“+++”A*2416TYNPDFSSL“+”A*2459KYSTTFFMV“++”A*2460TYLSIFDQL“+”A*2461NYAENILTL“++”A*2462LYQEILAQL“+”A*2465VYPASKMFPFI“+”A*2466IYFRDSSFL“++”A*2472RYEGILYTI“+”A*2476WYGWHFPEL“+”A*2479RYLADLPTL“+”A*2483TYCQNIKEF“+”A*24375VYSDLHAFYY“+”A*24379NYIVPDKQIF“+”A*24380VFQEKHHVI“+”A*24383RYKQDVERF“+++”A*24384KYVKVFDKF“+++”A*24386VYNDHSIYVW“++”A*24220SPSVSQLSVL“++++”B*07221VPDVAQFVL“++”B*07222NPFYPEVEL“+”B*07223YPKDIYSSF“++”B*07224GPQPWHAAL“++”B*07225LPFDGPGGIL“++++”B*07226SPRMSGLLSQT“+++”B*07228YPRGNHWAVGHL“++”B*07231RPRALRDLQL“++”B*07232RPRALRDLQLL“+++”B*07233KPYQGNPTF“+”B*07237RPAATAVISL“+”B*07241SARLATDAL“+++”B*07242SPRWLPVSL“++++”B*07244FPYVRDFVM“+”B*07245RIREHVPQL“++”B*07248IPNWARQDL“++++”B*07249VPSSRILQL“+++”B*07250SPRDFLSGL“+”B*07252SPDIRNTTV“+”B*07274TPYQEHVAL“+”B*07285FPSFLTNSL“++++”B*07292SPQRLRGLLL“+++”B*07293RPRSALPRLLLP“++”B*07294GPTPNTGAAL“+++”B*07460MPMAGDMNGL“++”B*07462RPFHTRATV“++”B*07463TPKAGPTL“+”B*07473LPRALLSSL“++”B*07474LPRLLPAL“+++”B*07320MEVDPIGHVYIF“+”B*44322KEVDPAGHSY“++”B*44323SEFMQVIF“+”B*44325QEQDVDLVQKY“+”B*44326QEMQHFLGL“+”B*44328FEYDFLLQRI“+”B*44329NEHPSNNW“+”B*44330KEGDLGGKQW“+”B*44331EDAQGHIW“++”B*44333AETLSTIQI“+”B*44334AEDEPAAAHL“+”B*44337TENRYCVQL“++”B*44338SEGSEPALLHSW“+”B*44339SEPALLHSW“++”B*44342HEFSSPSHL“+”B*44476AEEEIMKKI“+”B*44477QENSYQSRL“+”B*44297RAQLKLVAL“+”B*08298FNKRKPLSL“+”B*08299MAQFKEISL“++++”B*08300VASPKHCVL“+”B*08303LPFPKFTV“++”B*08305ALKLRVAVL“+”B*08306ILKVKVGL“+”B*08307ILLPRTVSL“+”B*08308MLKQKVEEL“+”B*08311EIRIRVVQM“+”B*08312EAMLRNKEL“+”B*08313ELKKKEYEEL“+”B*08314AIISRLVAL“++”B*08315DIYQRALNL“+”B*08316VIKEKALTL“+”B*08318EAAIRSVEL“++”B*08<20% = +;20%-49% = ++;50%-69% = +++;>=70% = ++++

Example 4

Synthesis of Peptides

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.

Example 5

MHC Binding Assays

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 (β2m) of stabilized MHC complexes. The assay was performed as generally described in Rodenko et al. (Rodenko et al., 2006).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-β2m for 1 h at 37° C., washed again and detected with TMB solution that is stopped with NH2SO4. 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 11MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-A*01 was ranged bypeptide exchange yield:Seq ID NoSequencePeptide exchange134AAEIGDKSWLY“++++”135ASEDSVLLY“++++”136ATDLVVLDRY“++++”137ATSKFMEFY“++++”138DSDSCHFNY“++++”139ECDMAFHIY“++++”140ESDREELNY“+++”141ESDVGVVVY“+++”142EVAEPSVLFDLY“+++”143FIDYPKKEDY“+++”144FLDSQNLSAY“+++”145FVDKPVAY“+++”146GLNTGSALSY“++”147GSSDSSTLPKL“++”148GTEFTTILY“++++”149GTEFTTVLY“++++”150GTELLSLVY“++++”151HSDLKVGEY“+++”152HTDSLHLLI“+++”153KLDRSVFTAY“+++”154LLDISQKNLY“+++”155LLDPNPHMY“++++”156LLDSLREQY“+++”157LMDRPIFY“++++”158LSDLLKQGY“++++”159LSDTSVIQFY“++++”160LTEAVLNRY“+++”161LVDDGTHGQY“+++”162LVDNSIRELQY“+++”163NSDSSLTLREFY“+++”164NTDNNLAVY“++++”165NTDPTAPPY“+++”166NTQITDIGRY“+++”167QSDPGTSVLGY“+++”168QTDHPQPILDRY“++++”169RLDTPLYFSY“++++”170RSDDTAVYY“++++”171RSDPVTLNVLY“++++”172RTDSCSSAQAQY“+++”173RTEFNLNQY“++++”174SADDIRGIQSLY“++++”176SRTINVSNLY“+++”177SSDEVNFLVY“++++”178SSDSSTLPKL“++”179STAKSATWTY“++++”180STDPWIQMAY“++++”181TADGKTYYY“+++”182TDYHVRVY“+++”183TLEDIATSHLY“++++”184TSAHPEDSSFY“+++”185TSDSNLNKY“++++”186TTDIIEKY“+++”187VADLHLYLY“+++”188VSDAKLDKY“+++”189VSDSECLSRY“++++”190VTDGINPLIDRY“+++”191VTDGSLYEGVAY“++++”192VTEESFDSKFY“++++”193VTEFSLNTY“++++”194VVADTKMIEY“+”195VVDSVGGYLY“++++”196WMFFVINY“+”197YADTVRPEFY“+++”198YLDPVQRDLY“++++”199YLPQHTIETY“++”200YSDEDVTKY“++++”201YVGKEHMFY“++++”394ASEAEMRLFY“++++”395ASEASRLAHY“++++”396ASEFGNHYLY“++++”397ASEITSKGASLY“++++”398ASEQQALHTVQY“++++”399ATDIPCLLY“++++”400ATDISRQNEY“+++”401DSDESYMEKSLY“++++”402DTDSQRLAY“+++”403ELDSKVEVLTY“+++”404ETARKFLYY“++++”405ETEEGIYWRY“++++”406ETEQTKFWDY“++++”407FSDNDKLYLY“++++”408FTEQWTDGY“+++”409FVDPLVTNY“++++”410GSDHQSPSSSSY“++++”411GTVYEDLRY“++++”412ILDEVIMGY“++++”413ISDRYYTALY“++++”414KTDESLTKY“+++”415LLDPRSYHTY“+++”416LLDTAQKNLY“++++”417LLEDKHFQSY“++++”418LSDPSGPKSY“+++”419LSELKPMSY“++++”420LTEDKETLQY“++++”421LTELLERAAFY“++++”422MIDVTKSYY“++++”423NLDAVHDITVAY“++++”424NLDEEKQLLY“+++”425NLDIIQQEY“++++”426NLDQATRVAY“+++”427NSDEQKITEMVY“++++”428NSELSCQLY“++++”429NTEDSSMSGYLY“++++”430NTEGLHHLY“++++”431NTSDMMGRMSY“++++”432NVDPVQHTY“+++”433QIDTGENLY“++++”434QTDCAPNNGY“++++”435QTDDTWRTEY“++++”436QTETGTPYMLY“++++”437STDGKHWWEY“++++”438STDNFNCKY“+++”439TLDAGKFQIY“+++”440TLDENPGVRY“+++”441TLDSALNAASYY“++++”442TSDFSRFTNY“++++”443TTDFPSESSFEY“++++”444TTDTVIRSY“+++”445VLDQGKITEY“+++”446VTAQVVGTERY“++++”447VVDEDHELIY“+++”448YLDIPNPRY“+++”449YLDRGTGNVSFY“++++”450YSDDGQKWTVY“++++”451YSDSLVQKGY“++++”452YVDAVLGKGHQY“++++”>10% = +;>20% = ++;>50 = +++;>75% = ++++

TABLE 12MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-A*02 was ranged bypeptide exchange yield:Seq ID NoSequencePeptide exchange84YVDINTFRL“++++”85YIDEFQSLV“++++”86FVIDGFDEL“++++”87TLYPYQISQL“++++”88VQMVITEAQKV“++++”89ILSTTMVTV“++++”91FALPGLLHA“+++”92NLRDLLSEV“+++”93TLQEKILQV“++++”94VLPDIETLIGV“++++”95ITIGVLARV“++++”96HLVGGLHTV“++++”97VLALVNSTV“++++”98LQSSGLTLLL“++++”99FLKEKVPGI“++++”100RQYPTPFQL“++++”101FIISDWRFVL“+++”102SLLEQAIAL“++++”103FLYYPDPVL“++++”104GMLDIFWGV“++++”105SLLTHIPTA“++++”106FIIDTTYPAYV“++++”107LLQGAIESV“++++”109LLLGSIGLLGV“++++”110LLADFQALL“++++”111ALCLLLHLL“+”112SVSDGIHSV“++++”113AVLTGLVEV“++++”114ILDERQVLL“++++”115MLLETQDALYV“++++”116VLMEENSKL“++++”117FLDPNARPLV“++++”118ALSSVLHSI“++++”119RTADITVTV“++++”120ALLANLPAV“++++”121ALVDTLTGI“++++”122ALLEMFPEITV“++++”123LMAFFLAVV“++”124SVASVLLYL“++++”125VLQPFLPSI“++++”126FLSTVTSV“++++”127GLDGSLVFL“++++”128FLGTTPTL“++++”129VLYDKDAVYV“++++”130NLWGGQGLLGV“++++”131LLKEFVQRV“++++”132ALWLVDPLTV“++++”133MTLPVDAVISV“++++”388FSIPEGALVAV“++++”389TLMEQPLTTL“++++”390HIMPTVHTV“++++”391SLIDMRGIETV“++++”392SLFKDQMEL“++++”393ILLPYLQTL“++++”>10% = +;>20% = ++;>50 = +++;>75% = ++++

TABLE 13MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-A*03 was ranged bypeptide exchange yield:Seq ID NoSequencePeptide exchange202KLAELEGALQK“++++”203KVKDTPGLGK“++++”204AVFDKFIRY“++++”205SLDGAARPK“++++”206KLIDLSQVMY“++++”207RSFNGLLTMY“++++”208GLASRILDAK“++++”209RTQIPMSEK“++++”210ATSGVPVYK“++++”211TVNPVAIHK“++++”212KAYEQVMHY“++++”214RTMSEAALVRK“++++”215MMFSGPQILKL“++++”216KLYAWELAF“+++”217RILNQILYY“++++”218KTLVAELLILK“+++”219RLRSSLVFK“++++”453AINTSIKNK“++++”454KVYTPSISK“++++”455RIADIFVKK“++++”456SMFTAILKK“++++”457SINKPTSER“++++”458GIADFVLKY“++++”>10% = +;>20% = ++;>50 = +++;>75% = ++++

TABLE 14MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-A*24 was ranged bypeptide exchange yield:Seq ID NoSequencePeptide exchange1QYDPTPLTW“++++”2VWSNVTPLKF“++++”3YLEKFYGL“++”4SYEKVINYL“++++”5RYMKKDYLI“++++”6KYKDYFPVI“++++”7VQQWSVAVF“+++”8PFLPPAACFF“++++”10VWSDVTPLNF“++++”11YYSKSVGFMQW“++++”12STIRGELFFF“+”13HYTYILEVF“++++”14SYSSCYSF“+++”15KYALLLQDL“++++”16TYNPDFSSL“+++”17YYADKKTFIVL“+++”18DYIGSVEKW“++++”19ILKEDPFLF“+”20EFTTVLYNF“++++”21SYEVRSTF“+++”22TQPGDWTLF“++++”23KFIISDWRF“++++”24MYPDLSELLM“++++”25SYNGYVFYL“++++”26KTPTNYYLF“++++”27NYTLYPITF“++++”28YYSIISHTL“++++”29VYPLLSRLYW“++++”30QYLPGWTVLF“++++”31QYQNVLTLW“++++”32SLPDLTPTF“++++”33KSSVIASLLF“+++”34MQPRMFFLF“++++”35KYLEESVWL“++++”36KQMEDGHTLF“++”37QWPWQASLQF“++++”38KYTNWKAFL“++++”39LIFMLANVF“+”40QYEPPSAPSTTF“+++”42TLPNTIYRF“++++”43IQMDEPMAF“+”44AYLSAVGTF“++++”45KYFVPPQLF“++++”46AFPVTSIFHTF“++++”47KYADYFLEV“++++”48VFIDHPVHLKF“++++”49LYISEVRNI“++++”50SYPELVKMVW“++++”51KYALLLQEL“++++”52KYMKIFHKF“++++”53KYITNLEDL“++++”54LLIKLLQTF“+++”55RWMDQRLVF“++++”56VYMIEPLEL“++++”57YPSIIQEF“++++”58QFAAPLRGIYF“++++”59KYSTTFFMV“++”60TYLSIFDQL“+++”61NYAENILTL“+++”62LYQEILAQL“++++”63VMPSDSFFF“++++”64NYAIFDEGHML“++++”65VYPASKMFPFI“++++”66IYFRDSSFL“++++”67RYPGKFYRV“++++”68IYQQIIQTY“++++”69IMPEKFEFW“++++”70PYTNYTFDF“++++”71SYMVLAPVF“++++”72RYEGILYTI“++++”73SYIGLPLTL“+++”74VYDQYFITL“++++”75NYIYSISVF“++++”76WYGWHFPEL“+++”77AYTLLGHEFV“+”78TWFPKTPMLF“++++”79RYLADLPTL“++++”80YYSPLRDLL“++++”81LYPEGLRLL“++++”82RFLPSPVVI“++++”83TYCQNIKEF“++++”375VYSDLHAFYY“++++”376KYVKDFHKF“++++”377VYVGAVNRI“++++”378KFLGPAEHLTF“++++”379NYIVPDKQIF“+++”380VFQEKHHVI“+++”381TYSKKHFRI“++++”382IYHSHHPTL“+++”383RYKQDVERF“+++”384KYVKVFDKF“++++”385MYINEVERL“++++”386VYNDHSIYVW“++++”387RWLPQKNAAQF“+++”>10% = +;>20% = ++;>50 = +++;>75% = ++++

TABLE 15MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-B*07 was ranged bypeptide exchange yield:Seq ID NoSequencePeptide exchange220SPSVSQLSVL“++++”221VPDVAQFVL“++++”222NPFYPEVEL“+++”223YPKDIYSSF“++++”224GPQPWHAAL“++++”225LPFDGPGGIL“++++”226SPRMSGLLSQT“++++”227YPRGNHWAVGH“++++”228YPRGNHWAVGHL“++++”229VPLPAGGGTV“+++”230VPLPAGGGTVL“+++”231RPRALRDLQL“++++”232RPRALRDLQLL“++++”233KPYQGNPTF“++++”234RAKNAGVTI“++++”235MPLKHYLLL“++++”236RVRGGEDGDRAL“++++”237RPAATAVISL“+++”238KPGPPWAAF“++++”239YVPSASLFML“++++”240SPREVTTVL“++++”241SARLATDAL“++++”242SPRWLPVSL“++++”243RPIENRILIL“++++”244FPYVRDFVM“++++”245RIREHVPQL“+++”246TPLPAVIVL“++++”247RALLARLLL“+++”248IPNWARQDL“++++”249VPSSRILQL“++++”250SPRDFLSGL“++++”251VPRSSGQTV“++++”252SPDIRNTTV“++++”253RVIDAVRFTL“+++”254NPFPHLITL“++++”255MPLLENLYL“++++”256SPRVPSIEL“++++”257LPRIPFADV“++++”258LPRGPLASL“++++”259RPPAAGLRGISL“++++”260YPQHPGLNA“+++”261APSARVGVC“+++”262SAYPQRLEI“+”263HPAPYGDLL“++++”265SPRQPPRLV“++++”267HPELVNHIVF“++”268YPLFRGINL“++++”269APRAPRLML“++++”270APGPRFLVT“++++”271MPLPWSLALP“+++”272MPLPWSLALPL“++++”273MPLLWLRGF“++”274TPYQEHVAL“+++”275APHPPLSVV“+++”276LPRAGGAFL“+++”278HPMIDINGIIVF“++”279SPARASPAL“+++”280VPISEEGTPVL“+++”281RPRAPVTPA“++++”282MPQIETRVIL“+++”283RPHSLSSEL“++++”284FPVTSIFHTF“++”285FPSFLTNSL“++++”286VPTLRSEL“++++”287APREEQQRSL“+++”288FPQKFIDLL“++”289VPENHSVAL“++++”290APYRPPDISL“++++”291SPQRLRGLL“++++”292SPQRLRGLLL“+++”293RPRSALPRLLLP“++++”294GPTPNTGAAL“++++”295KPEGTRIAV“++++”459RPMQQARAQL“+++”460MPMAGDMNGL“++++”462RPFHTRATV“++++”463TPKAGPTL“++++”464YPRPGTPAA“++++”465VPRPIFSQL“++++”466APYKSVTSL“++++”467KPFSSFTSM“++++”468SPMYGQAGL“++++”469YPENGVVQM“++”470SPNSYFRVL“++++”471KPRPDVTNEL“++++”472NPRATDAQL“++++”473LPRALLSSL“++++”474LPRLLPAL“++++”475RPHKPGLYL“++++”>10% = +;>20% = ++;>50 = +++;>75% = ++++

TABLE 16MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-B*08 was ranged bypeptide exchange yield:Seq ID NoSequencePeptide exchange296MPMQDIKM“++++”297RAQLKLVAL“++++”298FNKRKPLSL“+++”299MAQFKEISL“++++”300VASPKHCVL“+++”301YMHKLLVL“++++”302HLLQKQTSI“+++”303LPFPKFTV“++++”304ELKKLYCQI“++++”305ALKLRVAVL“++++”306ILKVKVGL“++++”307ILLPRTVSL“++++”308MLKQKVEEL“++++”309DAIQRKYSC“+++”310LPPKKFVL“++++”311EIRIRVVQM“++++”312EAMLRNKEL“++++”313ELKKKEYEEL“++++”314AIISRLVAL“++++”315DIYQRALNL“++++”316VIKEKALTL“+++”317LVKVKVLL“++++”318EAAIRSVEL“++++”>10% = +;>20% = ++;>50 = +++;>75% = ++++

TABLE 17MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-B*44 was ranged bypeptide exchange yield:Seq ID NoSequencePeptide exchange319AEMLERVIKNY“++++”320MEVDPIGHVYIF“++++”321AEMLESVIKNY“+++”322KEVDPAGHSY“++”323SEFMQVIF“+++”324TDSIHAWTF“+++”325QEQDVDLVQKY“++”326QEMQHFLGL“+++”327YEIEARNQVF“+++”328FEYDFLLQRI“++++”329NEHPSNNW“+++”330KEGDLGGKQW“+++”331EDAQGHIW“+++”332MEVPVIKI“++”333AETLSTIQI“+++”334AEDEPAAAHL“++”335KELEATKQY“++”336ASSSGPMRWW“++”337TENRYCVQL“++++”338SEGSEPALLHSW“+++”339SEPALLHSW“+++”340TEFSLNTY—341EEIEGKGSFTYF“+++”342HEFSSPSHL“+++”343TEFTTVLY“++”344EEATGQFHVY“+++”345IEFIHPQAF“++++”346VEAPGPVHVYW“++++”347ALNPYQYQY“+++”348AEIQGNINHV“+++”349AEQDMRELTY“+++”350GECDVFKEIL“+++”351EEVNYINTF“+++”352NEVLTYIKF“++++”353GEIIMQNNW“++++”354TEDPTILRI“+++”355SDMVRFHLF“++”356EEGRVYLF“+++”357RELENCFQIQ“+++”358KEADIHFLI“++++”359DELFSIALY“++++”360AEVPTGVII“+++”361SENLFFASF“++++”362SEKGVIQVY“+++”363AELDKLTSV“+++”364AETPIQNVI“+++”365SEMNVNMKY“++++”366AENLFRAF“++”367GEVHPSEMI“+++”368GEFPVRVQV“++”369EEIERFFKL“+++”370YEDLSQKY“+”371GELALKKKI“++”372TEGIIMKDF“++”373MEMQKSPVF“+++”374DEVNFLVY“+”476AEEEIMKKI“++”477QENSYQSRL“+++”478SEIEQEIGSL“+++”479AEIQPQTQV“+++”480GEVSGLTKDF“++”481RELQHEHSL“+++”482TEREWADEW“+++”483EENDQSTHKW“+++”484AEVGFVRFF“++++”485SEIEDSTKQVF“+++”486SEDDPILQI“+++”487AEDQLHHSF“+++”488TEFPIIKMY“++”489SEIGKAVGF“++++”>10% = +;>20% = ++;>50 = +++;>75% = ++++

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