identification and quantitation of tumor associated peptides presented on the cell surface tissue samples patients &# 39 ; tumor tissues were obtained from : asterand ( detroit , mich ., usa & amp ; royston , herts , uk ); proteogenex inc . ( culver city , calif ., usa ). normal tissues were obtained from asterand ( detroit , mich ., usa & amp ; royston , herts , uk ); bio - options inc . ( brea , calif ., usa ); bioserve ( beltsville , md ., usa ); 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 tübingen ( tübingen , 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 . 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 . 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 tops 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 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 . 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 &# 39 ; 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 nhl samples to a baseline of normal tissue samples . presentation profiles of exemplary over - presented peptides are shown in fig1 a - 1p . presentation scores for exemplary peptides are shown in table 8 . 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 . 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 &# 39 ; s protocol . total rna from healthy human tissues for rnaseq experiments was obtained from : asterand ( detroit , mich ., usa & amp ; 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 ); university hospital heidelberg ( heidelberg , germany ). total rna from tumor tissues for rnaseq experiments was obtained from : asterand ( detroit , mich ., usa & amp ; royston , herts , uk ); proteogenex inc . ( culver city , calif ., usa ). 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 ). 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 &# 39 ; 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 &# 39 ; 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 nhl are shown in fig2 a - 2c . expression scores for further exemplary genes are shown in table 9 . 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 10a ). 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 . 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 . 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 * 0201 / mla - 001 ( peptide elagigiltv ( seq id no . 329 ) from modified melan - a / mart - 1 ) and a * 0201 / ddx5 - 001 ( yllpaivhi from ddx5 , seq id no . 330 ), 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 × 10 6 cd8 + t cells with 2 × 10 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 ). 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 fig3 together with corresponding negative controls . results for ten peptides from the invention are summarized in tables 10a and 10b . 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 & gt ; 50 %. all tumaps are preferably administered as trifluoro - acetate salts or acetate salts , other salt - forms are also possible . 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 . benjamini , y . et al ., journal of the royal statistical society . series b ( methodological ), vol . 57 ( 1995 ): 289 - 300 card , k . f . et al ., cancer immunol immunother . 53 ( 2004 ): 345 - 357 cohen , c . j . et al ., j mol recognit . 16 ( 2003a ): 324 - 332 cohen , s . n . et al ., proc . natl . acad . sci . u . s . a 69 ( 1972 ): 2110 - 2114 coligan , j . e . et al ., current protocols in protein science ( 1995 ) fong , l . et al ., proc . natl . acad . sci . u . s . a 98 ( 2001 ): 8809 - 8814 gnjatic , s . et al ., proc natl . acad . sci . u . s . a 100 ( 2003 ): 8862 - 8867 green , m . r . et al ., molecular cloning , a laboratory manual 4th ( 2012 ) jung , g . et al ., proc natl acad sci usa 84 ( 1987 ): 4611 - 4615 ljunggren , h . g . et al ., j exp . med . 162 ( 1985 ): 1745 - 1759 longenecker , b . m . et al ., ann n . y . acad . sci . 690 ( 1993 ): 276 - 291 lukas , t . j . et al ., proc . natl . acad . sci . u . s . a 78 ( 1981 ): 2791 - 2795 mueller , l . n . et al ., j proteome . res 7 ( 2008 ): 51 - 61 mumberg , d . et al ., proc . natl . acad . sci . u . s . a 96 ( 1999 ): 8633 - 8638 national cancer institute , ( 6 - 5 - 2015 ), www . cancer . gov palomba , m . l ., curr . oncol rep . 14 ( 2012 ): 433 - 440 pinheiro , j . et al ., nlme : linear and nonlinear mixed effects models ( cran . r - project . org / packe = nlme ) ( 2015 ) sherman , f . et al ., laboratory course manual for methods in yeast genetics ( 1986 ) small , e . j . et al ., j clin oncol . 24 ( 2006 ): 3089 - 3094 teufel , r . et al ., cell mol life sci . 62 ( 2005 ): 1755 - 1762
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 , or peptides as such , can also be targets of antibodies , soluble t - cell receptors , and other binding molecules .