IMMUNOTHERAPY WITH B*08 RESTRICTED PEPTIDES AND COMBINATION OF PEPTIDES AGAINST CANCERS AND RELATED METHODS
20220008472 · 2022-01-13
Inventors
- Gisela Schimmack (Tuebingen, DE)
- ANNIKA SONNTAG (TUEBINGEN, DE)
- Heiko Schuster (Tuebingen, DE)
- Daniel Johannes Kowalewski (Tuebingen, DE)
- Michael ROEMER (Tuebingen, DE)
- Jens Fritsche (Tuebingen, DE)
- Oliver Schoor (Tuebingen, DE)
- Toni WEINSCHENK (Tuebingen, DE)
- Harpreet Singh (Tuebingen, DE)
Cpc classification
C07K14/4748
CHEMISTRY; METALLURGY
A61K35/17
HUMAN NECESSITIES
C07K16/00
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K35/17
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
C07K14/705
CHEMISTRY; METALLURGY
Abstract
The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.
Claims
1. A peptide consisting of the amino acid sequence YVYANHFTEA (SEQ ID NO: 291) in the form of a pharmaceutically acceptable salt.
2. The peptide of claim 1, wherein said peptide has the ability to bind to an MHC class-I molecule, and wherein said peptide, when bound to said MEW, is capable of being recognized by CD8 T cells.
3. The peptide of claim 1, wherein the pharmaceutically acceptable salt is chloride salt.
4. The peptide of claim 1, wherein the pharmaceutically acceptable salt is acetate salt.
5. A composition comprising the peptide of claim 1, wherein the composition comprises an adjuvant and a pharmaceutically acceptable carrier.
6. The composition of claim 5, wherein the peptide is in the form of a chloride salt.
7. The composition of claim 5, wherein the peptide is in the form of an acetate salt.
8. The composition of claim 5 wherein the adjuvant is selected from the group consisting of 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.
9. The composition of claim 8, wherein the adjuvant is IL-2.
10. The composition of claim 8, wherein the adjuvant is IL-7.
11. The composition of claim 8, wherein the adjuvant is IL-12.
12. The composition of claim 8, wherein the adjuvant is IL-15.
13. The composition of claim 8, wherein the adjuvant is IL-21.
14. A pegylated peptide consisting of the amino acid sequence of YVYANHFTEA (SEQ ID NO: 291) or a pharmaceutically acceptable salt thereof.
15. The peptide of claim 14, wherein the pharmaceutically acceptable salt is chloride salt.
16. The peptide of claim 14, wherein the pharmaceutically acceptable salt is acetate salt.
17. A composition comprising the pegylated peptide of claim 14 or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
18. A peptide consisting of the amino acid sequence of YVYANHFTEA (SEQ ID NO: 291), wherein at least one amino acid of the peptide is a D-amino acid.
19. The peptide in the form of a pharmaceutically acceptable salt of claim 1, wherein said peptide is produced by solid phase peptide synthesis or produced by a yeast cell or bacterial cell expression system.
20. A composition comprising the peptide of claim 1, wherein the composition is a pharmaceutical composition and comprises water and a buffer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0346] The present invention will now be described in the following examples which describe preferred embodiments thereof, and with reference to the accompanying figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.
FIGURES
[0347]
[0348]
[0349]
EXAMPLES
Example 1
[0350] Identification and Quantitation of Tumor Associated Peptides Presented on the Cell Surface
[0351] Tissue Samples
[0352] Patients' tumor tissues were 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), University Hospital Bonn (Bonn, Germany), University of Geneva (Geneva, Switzerland), University Hospital Heidelberg (Heidelberg, Germany), and the University Hospital Tübingen (Tübingen, Germany).
[0353] 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 Tübingen (Tübingen, Germany), Geneticist Inc. (Glendale, Calif., USA), ProteoGenex Inc. (Culver City, Calif., USA), University Hospital Heidelberg (Heidelberg, Germany), and the University Hospital Tübingen (Tübingen, Germany).
[0354] 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.
[0355] Isolation of HLA Peptides from Tissue Samples
[0356] 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.
[0357] Mass Spectrometry Analyses
[0358] 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.
[0359] Subsequently, the peptides were separated using a two-step 180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nL per minute. The gradient was composed of Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A gold coated glass capillary (PicoTip, New Objective) was used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometers were operated in the data-dependent mode using a TOP5 strategy. In brief, a scan cycle was initiated with a full scan of high mass accuracy in the orbitrap (R=30000), which was followed by MS/MS scans also in the orbitrap (R=7500) on the 5 most abundant precursor ions with dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted by SEQUEST 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.
[0360] 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, gastro-esophageal junction 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
[0361] Table 8A and Table 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: 4 for hepatocellular carcinoma, melanoma and esophagus cancer and peptide SEQ ID NO: 11 for cholangiocellular carcinoma, head and neck squamous cell carcinoma, non-Hodgkin lymphoma, non-small cell lung cancer adenocarcinoma, non-squamous non-adenocarcinoma non-small cell lung cancer, squamous cell non-small cell lung cancer, esophagus cancer, pancreatic cancer, small cell lung cancer and uterus endometrial cancer).
TABLE-US-00013 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 ALKLKVAEL NSCLCadeno, NSCLCsquam 2 QIMPKAGL HCC, OSCAR 3 SIQSRYISM MEL 4 QVMPKTGL HCC, MEL, OSCAR 5 STMPKILAL PRCA 6 NRQKRFVL NSCLCadeno, PACA, UBC, UEC 7 AARLRPAL CLL 8 FVRPKLVTI GBM 9 LLKGKPRAL UEC 10 MGKFKQCF UEC 11 MAPLKMLAL CCC, HNSCC, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA, SCLC, UEC 12 DFYLRSSAF NSCLCsquam 13 EPFTRPVL CLL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA, RCC, SCLC, UBC 14 ELILKRCL NSCLCadeno, NSCLCsquam 15 LLKKIEHA CCC, GBM 16 NRKPRTPF UEC 17 LLILKTVL BRCA 18 LSLVRKAL HNSCC 19 FTLLRRLSL MEL 20 ILKRFLAC GBM 21 RILKRFLAC GBM, HNSCC, MEL 22 EVRLKPIL CCC, MEL 23 DIKKTNESL BRCA, CLL, HNSCC, NHL, NSCLCsquam 24 DLVLKRCL MEL 25 HLHPKGREL HNSCC, NSCLCadeno 26 DARCKLAEL NSCLCadeno 27 WVLTNIVAL NSCLCadeno, NSCLCsquam 28 DPKSRLKSL MEL 29 ELFLHPVL CLL, MEL, NHL, NSCLCother 30 DLQKKAQAL BRCA, MEL, NSCLCsquam, OSCAR, PACA, PRCA 31 SPRVKVVTF GBM 32 NPYLKLVL CLL, NHL, NSCLCother, NSCLCsquam, SCLC 33 WIGLRNLDL CLL 34 IYRKKYIL CLL, NHL, UBC 35 ELFQRPVL CLL, NHL, NSCLCother, NSCLCsquam 36 IVKIKVQEL CLL, NHL, NSCLCother, NSCLCsquam 37 EAFSRASL PRCA 38 EVYQKIIL CCC, HNSCC, NHL, NSCLCadeno, NSCLCother 39 DAKSKIEQI HNSCC, NSCLCsquam, PACA 40 ESMLKTTL CCC, HCC 41 RGALRTLSL NSCLCadeno, UEC 42 VLRRKTLL CLL, GC, NHL, NSCLCsquam, UEC 43 DLKKLVDSL CRC, NSCLCother, UBC 44 HLTNRVLSL HNSCC, NSCLCsquam 45 RLKVALSTL CCC, CLL, MEL, NSCLCadeno, OSCAR, SCLC, UEC 46 DLRNKIIAA CCC, GBC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA, UBC 47 HSRVKLAQL GBM 48 ELALRQTV HNSCC 49 FLRVFTDSL CLL, NHL, NSCLCother, SCLC 50 TLRLLVAAL CCC, HNSCC, OSCAR, UBC 51 ERRVKVSSL UEC 52 ELILKHSL HNSCC, NHL, NSCLCadeno, NSCLCother 53 DLRKLKRQL CLL, MEL, NSCLCadeno 54 DLSRRDVSL CCC, PACA, RCC 55 VLLSRRTAL CLL, MEL, NSCLCadeno, UBC, UEC 56 ILCGSRKMPL NHL 57 MPLKHYLL HNSCC 58 LPKKMKLL PACA 59 FDFRGKTKVF NSCLCother 60 MLHIKKAEV MEL 61 SIKKELVVL GC, HNSCC, MEL, NSCLCadeno, NSCLCother, NSCLCsquam, PACA, UEC 62 FMLAKEASL NHL 63 YVKRKTNVL HNSCC 64 FILGREAGAL BRCA, CCC, HNSCC 65 NLLMRNVL NSCLCsquam 66 DLKKTRVL HNSCC, NSCLCadeno, NSCLCsquam, PACA 67 DMNTKRAIHTL CLL 68 DLKIPRYPV HNSCC, NSCLCadeno 69 ELARQRLL HNSCC, NSCLCadeno, NSCLCsquam 70 RPKGTPPL BRCA, CCC, CRC, GBM, GC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, RCC, UBC, UEC 71 HIRIKHTF HNSCC, NHL, NSCLCadeno, NSCLCsquam 72 LPLAHHIQL HNSCC 73 MFPARGVPL NSCLCsquam 74 RLKLRYEGL CLL 75 YARLKNVLL NSCLCadeno 76 HPRLKVNLL CCC, NSCLCother, OSCAR, SCLC, UBC, UEC 77 LPKLPVPPL NSCLCadeno, NSCLCsquam 78 DGHMKVFSL HNSCC, NSCLCadeno, NSCLCsquam, PRCA 79 GLARIYSF CCC 80 EVYLRMYQL BRCA, CCC, CLL, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, SCLC, UEC 81 MYRKEQYL NSCLCother, UEC 82 SIRKRPML NSCLCother 83 FVLLRSVDL BRCA, CCC, CLL, NHL, NSCLCother 84 YITRQFVQF NSCLCother 85 QKPRKKKL CLL 86 RPIHHPLVL BRCA, HCC 87 QILQHHVL GBC, HNSCC 88 MLLCLSLEL GBC, HNSCC, MEL, NSCLCadeno, NSCLCsquam, SCLC 89 VPYTKVQL BRCA, CCC, CRC, GBC, GC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, PACA, PRCA, UBC, UEC 90 TIGLGLHSL UBC 91 MPMQDIKMIL MEL, SCLC 92 TLKAMVQAW MEL 93 LMKEKIQEM NSCLCother 94 DQLLRHVM MEL, NSCLCsquam 95 SRNPRGFFL PACA, UEC 96 RPAGVFEL NSCLCadeno 97 EPVTKAEM HNSCC 98 EPVNTNVVL NSCLCother 99 NVKIRFLE HNSCC 100 VLLMGPLHL NSCLCsquam 101 FAFEKLIQF MEL, PRCA 102 IMKKIRESY MEL, NSCLCadeno, NSCLCsquam 103 IANLRVKNI UEC 104 FAFGEPREL MEL, OSCAR 105 APLKMLALV BRCA, CCC, CRC, GBM, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, RCC 106 HLHLLETSI HNSCC 107 LPKGLKDWQA SCLC 108 RSYYHPTNL HNSCC, NSCLCsquam 109 IPASHPVLTL NSCLCother 110 DAMTKHTL NHL 111 GAGLRITAPL NSCLCsquam, OC 112 NALDPLSAL GBM, NSCLCadeno, OSCAR 113 MEKGLASL HNSCC 114 PVKPKFYL MEL 115 MRILKRFLAC MEL 116 FTQNPRVQL MEL 117 VGPNGFKSL PACA, UEC 118 MAFVKHLL MEL 119 FQRVSSVSF OC 120 AQRSEMVTL NSCLCsquam 121 YSQLSISL MEL 122 ESSAQPTAL GBM, HNSCC 123 SLLPFTLSF MEL 124 HSWTRTSV PRCA 125 FPLVTPLL GBM 126 TAAEARLSL AML, SCLC 127 EPVIRTVSI HNSCC 128 HFHNRHVF HNSCC, OSCAR, UEC 129 HRILRLPAL MEL 130 LPAPYQHQL NSCLCother 131 MSTKTTSI GBM, PACA, RCC 132 IPIQAHQI GBM 133 QATPRVRIL MEL, NSCLCsquam, UEC 134 DQRSRATL HCC 135 EYLETKRLAF HNSCC 136 KMFYRKDVM GBC 137 VQWKPPAL NSCLCadeno 138 TQHLTVATL NSCLCsquam, OC 139 YGRIGISLF HNSCC, NSCLCadeno 140 IAVDKPITL MEL, NSCLCsquam 141 AQLKLVAL NSCLCadeno 142 YNLIYSMCL NSCLCadeno 143 DADLREQAL HNSCC, NSCLCsquam, PACA 144 IEQIRAVL HNSCC 145 MIYRKALRL NHL 146 FQTAHFYL MEL 147 HAMDGASHL MEL 148 DVNPVSLQL HNSCC, NSCLCother 149 TQKSVQVL GBM 150 MRSSYIREL UEC 151 DRHLTNRVL MEL 152 FNKLVTEL CCC, HCC 153 HAIPHYVTM MEL 154 VLKTLQEL CCC, HCC 155 LPASFPAVL NSCLCother 156 ILKEQSSSSF HNSCC, NSCLCsquam 157 QPYRFPQA HNSCC 158 DVIIKGNGL HNSCC, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, RCC 159 DLRNKIIA HNSCC, NSCLCsquam 160 LPINNTHI BRCA, CCC, CLL, GBM, HCC, MEL, NSCLCother, NSCLCsquam, UBC 161 DIVPPFSAF CCC, MEL, OSCAR, UBC 162 LFKQTKINL NSCLCsquam 163 EVMAQFKEI OSCAR, UBC 164 LPAPIPTLL NSCLCother 165 QNSLRHNL CCC 166 VLSGGRILAL HCC 167 DMKITVSL CCC 168 HVQDFTAF NSCLCsquam 169 YELNNLHAL MEL 170 SPANVRGQSL GBC 171 FPSQVPKQVL NSCLCother 172 PYEKVSRL CCC 173 YPLLKDPSL CCC, PACA, SCLC 174 HAMPSPRIL NSCLCother 175 MRFQQFLFA HNSCC 176 YVIQRQSVL NSCLCother 177 SVPVRSSPL NHL 178 IPRLAVISI GBM 179 LPLTEHEL HNSCC, NSCLCother, SCLC 180 LAVPIFVAL OC 181 SIRSSYSRF UEC 182 ILHLSAIAL BRCA 183 YVSKPGAQL GBC 184 DRLKPLKM MEL 185 MELKTVKPI CCC, GBM, NSCLCother, NSCLCsquam 186 DLISPRQPRL HNSCC 187 VPYNSVLF MEL 188 EIMEKTTL MEL, NSCLCsquam 189 APDNVLLTL BRCA, GBM, HCC, NSCLCsquam, OSCAR, SCLC 190 ELLNRIYF MEL 191 RPLKPGEVL BRCA 192 EEKHFTTL NSCLCadeno 193 LGGLRLTAL NSCLCadeno 194 RAIEHVLQV NSCLCother 195 EGNQKSVI NHL, NSCLCadeno, NSCLCsquam, PACA, UEC 196 LDLRQKVL BRCA, CCC, CLL, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 197 YKAYPSQL NSCLCadeno 198 FPLTSIIAI NSCLCother 199 IPFIHLPEI GBM, HCC 200 VAAARAVPV MEL 201 TASAMQHVL NSCLCother 202 RIPEKASFL HCC 203 DVYTQVSAF AML 204 MSPLLRSI BRCA, CCC, CRC, GBC, GC, HCC, HNSCC, MEL, NSCLCother, NSCLCsquam, PACA, UEC 205 YMQYGFLSM NSCLCsquam, OC 206 MEFPNKFNTL MEL 207 LRKRKSPE HNSCC 208 LPPPQPLSL BRCA, CLL, NHL, NSCLCother 209 SRFGKFVQL GC, HNSCC, NSCLCother, NSCLCsquam, PACA, PRCA, UEC 210 QPNTHQLL HNSCC 211 DVISKGVSL MEL 212 EEYKFPSL NSCLCsquam, UEC 213 FPSLFINQF NSCLCsquam, UEC 214 DAPRHRLL NSCLCadeno 215 NPLIEIISI SCLC 216 EARPPSPAV GBM 217 ETIKGHSVRL HNSCC 218 DNHPRLVTL MEL 219 VRNPKILIL CCC, CLL, GBM, HCC, MEL, NHL, NSCLCadeno, NSCLCsquam, PACA, PRCA, RCC, UEC 220 LAVRHLSL CCC 221 SLKEELLSL CCC 222 AQKAELIAL OC 223 DVSARKLRV SCLC 224 LPYPPQKVV GBM 225 MPKRAHWGA HNSCC 226 DIYEVAVSL CCC 227 SRFPGMSVL MEL 228 TLRAYVLAL NSCLCother 229 DTHTNTYYL HNSCC 230 DVYFHHVL HNSCC, NSCLCadeno, NSCLCsquam 231 GEKLLRPSL HNSCC 232 KLYIHRVTL MEL 233 DVKLVFVM NHL, NSCLCother 234 VFRVGISF GBC 235 SPNSLVTIL HNSCC 236 STLKKSLEI NSCLCsquam 237 LPLDSRYVTL NSCLCother 238 IPLAIARL NSCLCsquam 239 SEPVMRVTL HNSCC 240 KVIDRKVEL NSCLCadeno, NSCLCsquam 241 NAYEAPSI GBM 242 KPQSLQLVL NSCLCother 243 EGVPPGTVL NSCLCsquam 244 HALPPYITVL MEL, NHL 245 GPRGPSSGHPL MEL 246 RLLQKSKEL BRCA, CCC, HNSCC, NSCLCadeno, NSCLCsquam, SCLC, UBC 247 TPEPSVHAL NSCLCother 248 SEVNKHETAL HNSCC 249 QQIDRVVEV CCC, GBM, NHL 250 AARAPPQAL BRCA, GBC 251 DAAAFFKSV GBM 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.
TABLE-US-00014 TABLE 8B Overview of presentation of selected tumor-associated peptides of the present invention across entities. SEQ ID NO Sequence Peptide Presentation on cancer entities 289 SLAESEASL CCC, GBC, HNSCC, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, UEC 290 EEFLTPKKL PRCA 291 YVYANHFTEA BRCA, CCC, CRC, GBC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, SCLC, UBC, UEC BRCA: breast cancer; CCC: cholangiocellular carcinoma; 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; SCLC: small cell lung cancer; UBC: urinary bladder carcinoma; UEC: uterine and endometrial cancer.
Example 2
[0362] 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.
[0363] RNA Sources and Preparation
[0364] 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.
[0365] 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).
[0366] 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).
[0367] 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).
[0368] RNASeq Experiments
[0369] 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.
[0370] 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
TABLE-US-00015 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 and neck, kidney, large intestine, liver, lung, lymph node, nerve, parathyroid, pancreas, peritoneum, pituitary, pleura, skeletal muscle, skin, small intestine, spinal cord, spleen, stomach, thyroid gland, trachea, ureter, 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 very highly SEQ ID highly over- over-expressed NO Sequence over-expressed (+) expressed (++) (+++) 1 ALKLKVAEL CRC, HCC, PACA BRCA, GBC, GC, HNSCC, MEL, NHL, NSCLCsquam, NSCLCadeno, OSCAR, SCLC, OC, UEC UBC 2 QIMPKAGL CRC, NHL, PACA GBC, GC, HCC, NSCLCadeno, HNSCC, MEL, UEC NSCLCsquam, OSCAR, SCLC, UBC 3 SIQSRYISM AML, BRCA, GC, HCC, GBC, HNSCC, MEL, OC, NHL, OSCAR, UBC NSCLCadeno, SCLC, UEC NSCLCsquam, RCC 4 QVMPKTGL CRC, PACA, UEC GBC, HCC, GC, MEL, HNSCC, SCLC, UBC NSCLCadeno, NSCLCsquam, OSCAR 5 STMPKILAL PRCA 6 NRQKRFVL HCC, MEL, CRC, GBC, GC, BRCA, CCC, NSCLCother, RCC, HNSCC, PACA SCLC NSCLCadeno, NSCLCsquam, OC, OSCAR, UBC, UEC 7 AARLRPAL NHL CLL 8 FVRPKLVTI GBM, SCLC 9 LLKGKPRAL BRCA UEC 10 MGKFKQCF OC, UEC 11 MAPLKMLAL NHL 12 DFYLRSSAF BRCA, SCLC, UBC PRCA 13 EPFTRPVL NHL CLL 14 ELILKRCL GBM, MEL, OC, SCLC, AML, UEC UBC 15 LLKKIEHA GBM, MEL, OC, SCLC, AML, UEC UBC 16 NRKPRTPF UEC 17 LLILKTVL RCC PRCA 18 LSLVRKAL CCC, GC, HNSCC, NSCLCadeno, NSCLCsquam, NSCLCother, PACA, OSCAR, UBC UEC 19 FTLLRRLSL MEL 20 ILKRFLAC GBM 21 RILKRFLAC GBM 22 EVRLKPIL CCC MEL 23 DIKKTNESL AML, BRCA, CCC, CLL, SCLC CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, UBC, UEC 24 DLVLKRCL MEL 25 HLHPKGREL BRCA, GC, HNSCC, CCC, CRC, GBC MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, RCC, UBC, UEC 26 DARCKLAEL BRCA, GBC, MEL, NSCLCadeno, NSCLCsquam, OC, UBC SCLC 27 WVLTNIVAL GBM OC 28 DPKSRLKSL MEL 29 ELFLHPVL CLL, NHL 30 DLQKKAQAL PRCA 31 SPRVKWTF GBM 32 NPYLKLVL NHL CLL 33 WIGLRNLDL CLL, NHL 34 IYRKKYIL CLL, GC, OC, SCLC BRCA 35 ELFQRPVL NHL CLL 36 IVKIKVQEL NHL CLL 37 EAFSRASL PRCA 38 EVYQKIIL OC, SCLC GBC, MEL 39 DAKSKIEQI CCC, GBC, GC, OSCAR HNSCC, NSCLCadeno, NSCLCsquam, PACA, UBC 40 ESMLKTTL HCC 41 RGALRTLSL BRCA, CCC, NHL, GBC NSCLCsquam, OC, OSCAR, SCLC, UBC, UEC 42 VLRRKTLL NHL CLL 43 DLKKLVDSL GBC, GC CRC 44 HLTNRVLSL HNSCC, UBC NSCLCsquam, OSCAR 45 RLKVALSTL MEL, PRCA, UEC BRCA 46 DLRNKIIAA OSCAR HNSCC 47 HSRVKLAQL GBM 48 ELALRQTV HNSCC 49 FLRVFTDSL AML, NHL, PRCA CLL 50 TLRLLVAAL CCC, GBC, HNSCC, OSCAR NSCLCsquam, PRCA, UBC 51 ERRVKVSSL UBC UEC 52 ELILKHSL GBM 53 DLRKLKRQL MEL 54 DLSRRDVSL RCC 55 VLLSRRTAL CLL 56 ILCGSRKMPL MEL 57 MPLKHYLL BRCA, HNSCC, MEL, NHL, NSCLCadeno, OSCAR 58 LPKKMKLL CCC 59 FDFRGKTKVF CLL, CRC 60 MLHIKKAEV MEL 61 SIKKELVVL AML, GBM, OSCAR, SCLC 62 FMLAKEASL NHL 63 YVKRKTNVL HNSCC, OC, OSCAR, PRCA 64 FILGREAGAL BRCA, CCC, MEL, OC 65 NLLMRNVL HCC, HNSCC, OSCAR, SCLC 66 DLKKTRVL CCC, GBC, RCC 67 DMNTKRAIHTL CLL, OC 68 DLKIPRYPV BRCA, PRCA, UBC, UEC 69 ELARQRLL CCC, CRC, GBC, HCC, MEL 70 RPKGTPPL BRCA, CCC, CRC, GBC, GBM, GC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, SCLC, UBC, UEC 71 HIRIKHTF HNSCC, NSCLCsquam, OSCAR 72 LPLAHHIQL AML 73 MFPARGVPL SCLC 74 RLKLRYEGL CLL 75 YARLKNVLL UBC 76 HPRLKVNLL BRCA, CCC, GC, MEL, NHL, NSCLCother, NSCLCsquam, OC, RCC 77 LPKLPVPPL GBM, OC, RCC 78 DGHMKVFSL CCC 79 GLARIYSF AML 80 EVYLRMYQL MEL 81 MYRKEQYL MEL 82 SIRKRPML CCC, GBM, PACA 83 FVLLRSVDL PRCA 84 YITRQFVQF MEL, NSCLCadeno 85 QKPRKKKL BRCA, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, OC, OSCAR, UEC 86 RPIHHPLVL GBM, SCLC 87 QILQHHVL HNSCC, OC, OSCAR, UBC 88 MLLCLSLEL GBM 89 VPYTKVQL CRC, PRCA 90 TIGLGLHSL PRCA 91 MPMQDIKMIL AML, BRCA, GC, NHL, GBC, HNSCC, MEL, OC, OSCAR, UBC NSCLCadeno, SCLC, UEC NSCLCsquam, RCC 92 TLKAMVQAW AML, BRCA, GC, HCC, GBC, HNSCC, MEL, OC, NHL, OSCAR, UBC NSCLCadeno, SCLC, UEC NSCLCsquam, RCC 93 LMKEKIQEM HCC, RCC CCC, GBC, GC, CRC, HNSCC, MEL, NHL, NSCLCother, NSCLCadeno, NSCLCsquam, OC, OSCAR, SCLC PACA, UBC, UEC 94 DQLLRHVM AML, BRCA, NHL, GBC, HNSCC, MEL, UEC OSCAR, UBC NSCLCadeno, NSCLCsquam, OC, RCC, SCLC 95 SRNPRGFFL OC UEC 96 RPAGVFEL MEL, SCLC GBM 97 EPVTKAEM GBC, NHL, UEC GC, HCC, HNSCC, NSCLCadeno, MEL, SCLC NSCLCsquam, OSCAR, UBC 98 EPVNTNVVL HNSCC GBM, SCLC NSCLCother 99 NVKIRFLE NHL CLL 100 VLLMGPLHL MEL 101 FAFEKLIQF UEC BRCA, PRCA 102 IMKKIRESY AML, NSCLCother, CCC, CLL, CRC, UEC GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, RCC, SCLC, UBC 103 IANLRVKNI BRCA, CCC, HNSCC, GBC, HCC, OC, MEL, NHL, SCLC NSCLCadeno, NSCLCsquam, OSCAR, PACA, RCC, UBC, UEC 104 FAFGEPREL SCLC HCC, MEL 105 APLKMLALV NHL 106 HLHLLETSI CRC, GBC, GC, CCC, HNSCC, NSCLCadeno, NSCLCsquam, NSCLCother, OC, UEC OSCAR, PACA, UBC 107 LPKGLKDWQA PRCA 108 RSYYHPTNL NSCLCadeno, UBC HNSCC, NSCLCsquam, OSCAR 109 IPASHPVLTL NHL CLL 110 DAMTKHTL CLL NHL 111 GAGLRITAPL GBC, GC, CCC, HNSCC, NSCLCadeno, NSCLCsquam, NSCLCother, PACA, OSCAR, UBC UEC 112 NALDPLSAL PRCA, SCLC GBM 113 MEKGLASL CCC, GC, HNSCC, NSCLCadeno, NSCLCsquam, NSCLCother, PACA, OSCAR, UBC UEC 114 PVKPKFYL MEL 115 MRILKRFLAC GBM 116 FTQNPRVQL MEL 117 VGPNGFKSL CCC, GBC, GC, HNSCC, NSCLCadeno, NSCLCsquam, NSCLCother, PACA OSCAR, UBC 118 MAFVKHLL OC, UBC GBC, HCC, MEL, UEC 119 FQRVSSVSF MEL 120 AQRSEMVTL NHL CLL 121 YSQLSISL MEL 122 ESSAQPTAL BRCA, HCC, MEL, OC, PRCA SCLC, UEC 123 SLLPFTLSF MEL 124 HSWTRTSV PRCA 125 FPLVTPLL GBM 126 TAAEARLSL SCLC 127 EPVIRTVSI MEL 128 HFHNRHVF CLL 129 HRILRLPAL MEL 130 LPAPYQHQL CCC, GBC, GC, HCC, CRC, MEL HNSCC, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, UBC 131 MSTKTTSI BRCA UBC 132 IPIQAHQI GBM 133 QATPRVRIL MEL 134 DQRSRATL HCC 135 EYLETKRLAF HNSCC, NSCLCsquam OSCAR 136 KMFYRKDVM NHL 137 VQWKPPAL NSCLCsquam NSCLCadeno, NSCLCother 138 TQHLTVATL GBC, GBM, HNSCC, CRC, GC, UBC MEL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, SCLC, UEC 139 YGRIGISLF GBC HCC 140 IAVDKPITL MEL 141 AQLKLVAL BRCA, NSCLCsquam, HNSCC OSCAR, UBC 142 YNLIYSMCL AML CCC 143 DADLREQAL CCC, GBC, GC, OSCAR HNSCC, NSCLCadeno, NSCLCsquam, PACA, UBC 144 IEQIRAVL CCC, GBC, GC, OSCAR HNSCC, NSCLCadeno, NSCLCsquam, PACA, UBC 145 MIYRKALRL PRCA 146 FQTAHFYL GBM 147 HAMDGASHL BRCA, MEL, OC PRCA 148 DVNPVSLQL CCC, GBC, GC, OSCAR HNSCC, NSCLCadeno, NSCLCother, NSCLCsquam, PACA, UBC 149 TQKSVQVL GBM 150 MRSSYIREL BRCA UEC 151 DRHLTNRVL HNSCC, UBC NSCLCsquam, OSCAR 152 FNKLVTEL HCC 153 HAIPHYVTM MEL 154 VLKTLQEL HCC 155 LPASFPAVL AML, NHL CLL 156 ILKEQSSSSF HNSCC 157 QPYRFPQA CLL, NHL 158 DVIIKGNGL GBM, MEL, UEC NSCLCsquam, OC, RCC, SCLC 159 DLRNKIIA OSCAR HNSCC 160 LPINNTHI AML, GBM, NHL CLL 161 DIVPPFSAF PRCA 162 LFKQTKINL GBM 163 EVMAQFKEI BRCA, CCC, GBC, NSCLCsquam, OC, OSCAR, UBC, UEC 164 LPAPIPTLL NHL, NSCLCadeno, OC, SCLC, UEC 165 QNSLRHNL NHL, NSCLCadeno, OC, PRCA, SCLC, UEC 166 VLSGGRILAL MEL 167 DMKITVSL RCC 168 HVQDFTAF PRCA 169 YELNNLHAL CLL 170 SPANVRGQSL BRCA, HNSCC, MEL, NHL, NSCLCadeno, OSCAR 171 FPSQVPKQVL NSCLCsquam, OC, SCLC 172 PYEKVSRL CCC 173 YPLLKDPSL CCC 174 HAMPSPRIL CCC, RCC 175 MRFQQFLFA HNSCC, MEL, PACA, PRCA 176 YVIQRQSVL NSCLCadeno, NSCLCother, NSCLCsquam 177 SVPVRSSPL HNSCC, MEL, NSCLCsquam, OSCAR, RCC 178 IPRLAVISI GBM 179 LPLTEHEL NHL 180 LAVPIFVAL GBM, HNSCC, NSCLCadeno, NSCLCsquam, OSCAR 181 SIRSSYSRF HNSCC, MEL, OSCAR 182 ILHLSAIAL AML 183 YVSKPGAQL CCC, GBC, NSCLCadeno 184 DRLKPLKM MEL, NSCLCsquam, OC, SCLC, UEC 185 MELKTVKPI AML, SCLC 186 DLISPRQPRL NSCLCsquam, SCLC 187 VPYNSVLF AML, GBM 188 EIMEKTTL MEL, NSCLCsquam, OC, SCLC, UEC 189 APDNVLLTL AML, GBC, MEL, NSCLCsquam, OC 190 ELLNRIYF MEL 191 RPLKPGEVL GBM 192 EEKHFTTL OC 193 LGGLRLTAL CRC, GC, SCLC 194 RAIEHVLQV BRCA, PRCA 195 EGNQKSVI SCLC 196 LDLRQKVL OC 197 YKAYPSQL CRC, NHL, NSCLCother, NSCLCsquam, OSCAR, SCLC 198 FPLTSIIAI NSCLCother 199 IPFIHLPEI NSCLCother 200 VAAARAVPV SCLC 201 TASAMQHVL AML, GC, NSCLCother 202 RIPEKASFL HCC 203 DVYTQVSAF AML 204 MSPLLRSI HNSCC, MEL, NSCLCsquam, OSCAR, RCC 205 YMQYGFLSM BRCA, OC, UBC 206 MEFPNKFNTL MEL 207 LRKRKSPE HCC, NHL, RCC, SCLC 208 LPPPQPLSL HCC 209 SRFGKFVQL MEL 210 QPNTHQLL BRCA, PRCA 211 DVISKGVSL GBM, SCLC 212 EEYKFPSL SCLC 213 FPSLFINQF SCLC 214 DAPRHRLL GBC, GBM, OC, RCC, SCLC, UBC, UEC 215 NPLIEIISI SCLC 216 EARPPSPAV GBM 217 ETIKGHSVRL SCLC 218 DNHPRLVTL AML 219 VRNPKILIL MEL 220 LAVRHLSL CCC 221 SLKEELLSL MEL 222 AQKAELIAL OC, RCC 223 DVSARKLRV GBM 224 LPYPPQKVV CCC, GBM, PACA 225 MPKRAHWGA BRCA, CRC, GBC, GC, HNSCC, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA 226 DIYEVAVSL MEL 227 SRFPGMSVL AML 228 TLRAYVLAL NHL 229 DTHTNTYYL CRC 230 DVYFHHVL BRCA, HNSCC, NSCLCsquam, OC, OSCAR, UBC 231 GEKLLRPSL CRC 232 KLYIHRVTL HNSCC, NSCLCsquam 233 DVKLVFVM CLL 234 VFRVGISF PRCA, SCLC 235 SPNSLVTIL BRCA, MEL 236 STLKKSLEI CLL 237 LPLDSRYVTL GBM 238 IPLAIARL GBM, PACA 239 SEPVMRVTL HNSCC, OC, OSCAR, UBC 240 KVIDRKVEL SCLC 241 NAYEAPSI CLL, OC 242 KPQSLQLVL SCLC 243 EGVPPGTVL GBM 244 HALPPYITVL GC, PACA, UBC, UEC 245 GPRGPSSGHPL GBM, OSCAR 246 RLLQKSKEL BRCA, CCC, CRC, GBC, GC, HNSCC, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA, UBC 247 TPEPSVHAL GBM, MEL, SCLC 248 SEVNKHETAL AML, BRCA, CCC, CLL, NHL, SCLC CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, UBC, UEC 249 QQIDRVVEV SCLC 250 AARAPPQAL CCC, GBC, MEL, OC 251 DAAAFFKSV CLL 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
TABLE-US-00016 TABLE 9B 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, peritoneum, pituitary, pleura, skeletal muscle, skin, small intestine, spinal cord, spleen, stomach, thyroid gland, trachea, ureter, 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 highly over- very highly over- NO Sequence over-expressed (+) expressed (++) expressed (+++) 289 SLAESEASL CRC, GBC, GC, CCC, HNSCC, NSCLCadeno, NSCLCsquam, NSCLCother, OC, UEC OSCAR, PACA, UBC 290 EEFLTPKKL PRCA 291 YVYANHFTEA BRCA, CCC, CRC, GBC, GC, HNSCC, MEL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, UBC BRCA: breast cancer; CCC: cholangiocellular carcinoma; CRC: colorectal cancer; GBC: gallbladder cancer; GC: gastric cancer; HNSCC: head and neck squamous cell carcinoma; MEL: melanoma; 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; UBC: urinary bladder carcinoma; UEC: uterine and endometrial cancer
Example 3
[0371] In Vitro Immunogenicity for MHC Class I Presented Peptides
[0372] 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-B*08 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).
[0373] In vitro priming of CD8+ T cells
[0374] 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-B*08 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.
[0375] 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.
[0376] 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.
[0377] 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).
[0378] pMHC used for positive and negative control stimulations were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO: 287) from modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO: 288), respectively.
[0379] 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, 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).
[0380] In Vitro Immunogenicity for 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 Peptides
[0381] 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 7 peptides of the invention are shown in
TABLE-US-00017 TABLE 10A in vitro immunogenicity of HLA class I peptides of the invention Seq ID NO Sequence Wells positive [%] 278 RAQLKLVAL + 283 MAQFKEISL ++++ 284 ELKKKEYEEL + 285 EAMLRNKEL + 286 ILLPRTVSL + Exemplary results of in vitro immunogenicity experiments conducted by the applicant for peptides of the invention. <20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++
TABLE-US-00018 TABLE 10B in vitro immunogenicity of HLA class I peptides of the invention SEQ ID NO Sequence Wells positive [%] 1 ALKLKVAEL ++ 3 SIQSRYISM ++ 5 STMPKILAL + 8 FVRPKLVTI + 9 LLKGKPRAL + 10 MGKFKQCF ++++ 11 MAPLKMLAL +++ 13 EPFTRPVL + 18 LSLVRKAL ++ 19 FTLLRRLSL ++++ 20 ILKRFLAC + 21 RILKRFLAC + 22 EVRLKPIL + 25 HLHPKGREL ++ 29 ELFLHPVL ++ 31 SPRVKWTF ++++ 33 WIGLRNLDL + 34 IYRKKYIL + 35 ELFQRPVL + 36 IVKIKVQEL ++ 37 EAFSRASL ++ 38 EVYQKIIL ++++ 40 ESMLKTTL + 41 RGALRTLSL + 42 VLRRKTLL + 45 RLKVALSTL + 47 HSRVKLAQL ++ 48 ELALRQTV + 49 FLRVFTDSL + 57 MPLKHYLL +++ 58 LPKKMKLL ++ 60 MLHIKKAEV + 62 FMLAKEASL + 63 YVKRKTNVL + 66 DLKKTRVL + 68 DLKIPRYPV +++ 71 HIRIKHTF ++ 74 RLKLRYEGL ++ 75 YARLKNVLL +++ 76 HPRLKVNLL + 78 DGHMKVFSL ++ 80 EVYLRMYQL +++ 82 SIRKRPML + 83 FVLLRSVDL +++ 86 RPIHHPLVL + 89 VPYTKVQL + Exemplary results of in vitro immunogenicity experiments conducted by the applicant for the peptides of the invention. <20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++
Example 4
[0382] Synthesis of Peptides
[0383] 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
[0384] MHC Binding Assays
[0385] 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 a1. (Rodenko et al., 2006).
[0386] 96 well MAXISorp plates (NUNC) were coated over night with 2 μg/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 NH.sub.2SO.sub.4. Absorption was measured at 450 nm. Candidate peptides that show a high exchange yield (preferably higher than 50%, most preferred higher than 75%) are generally preferred for a generation and production of antibodies or fragments thereof, and/or T cell receptors or fragments thereof, as they show sufficient avidity to the MHC molecules and prevent dissociation of the MHC complexes.
[0387] MHC:peptide binding results for 252 peptides from the invention are summarized in Table 11A, Table11B and Table 11C.
TABLE-US-00019 TABLE 11A MHC class I binding scores. Peptide SEQ ID NO Sequence exchange 1 ALKLKVAEL ++ 2 QIMPKAGL +++ 3 SIQSRYISM +++ 4 QVMPKTGL ++ 5 STMPKILAL +++ 6 NRQKRFVL ++ 7 AARLRPAL ++ 8 FVRPKLVTI +++ 9 LLKGKPRAL ++ 10 MGKFKQCF +++ 11 MAPLKMLAL +++ 12 DFYLRSSAF +++ 13 EPFTRPVL ++ 14 ELILKRCL +++ 15 LLKKIEHA ++ 16 NRKPRTPF ++ 17 LLILKTVL +++ 18 LSLVRKAL +++ 19 FTLLRRLSL +++ 20 ILKRFLAC +++ 21 RILKRFLAC +++ 22 EVRLKPIL ++ 23 DIKKTNESL ++ 24 DLVLKRCL ++++ 25 HLHPKGREL ++ 26 DARCKLAEL ++ 28 DPKSRLKSL ++ 29 ELFLHPVL ++ 30 DLQKKAQAL ++ 31 SPRVKWTF ++ 32 NPYLKLVL ++ 33 WIGLRNLDL +++ 34 IYRKKYIL ++ 35 ELFQRPVL ++ 36 IVKIKVQEL ++ 37 EAFSRASL ++ 38 EVYQKIIL +++ 39 DAKSKIEQI ++ 40 ESMLKTTL ++ 41 RGALRTLSL ++ 42 VLRRKTLL ++ 43 DLKKLVDSL +++ 44 HLTNRVLSL +++ 45 RLKVALSTL +++ 46 DLRNKIIAA +++ 47 HSRVKLAQL ++ 48 ELALRQTV ++ 49 FLRVFTDSL +++ 50 TLRLLVAAL +++ 51 ERRVKVSSL +++ 52 ELILKHSL +++ 53 DLRKLKRQL ++ 54 DLSRRDVSL ++ 55 VLLSRRTAL ++ 56 ILCGSRKMPL ++ 57 MPLKHYLL ++ 58 LPKKMKLL ++ 59 FDFRGKTKVF ++ 60 MLHIKKAEV +++ 61 SIKKELVVL ++ 62 FMLAKEASL ++ 63 YVKRKTNVL ++ 64 FILGREAGAL ++ 65 NLLMRNVL ++ 66 DLKKTRVL ++ 67 DMNTKRAIHTL ++ 68 DLKIPRYPV +++ 69 ELARQRLL ++ 70 RPKGTPPL ++ 71 HIRIKHTF +++ 72 LPLAHHIQL +++ 73 MFPARGVPL ++ 74 RLKLRYEGL +++ 75 YARLKNVLL +++ 76 HPRLKVNLL +++ 77 LPKLPVPPL +++ 78 DGHMKVFSL ++ 79 GLARIYSF ++ 80 EVYLRMYQL +++ 81 MYRKEQYL ++ 82 SIRKRPML ++ 83 FVLLRSVDL +++ 84 YITRQFVQF ++ 85 QKPRKKKL ++ 86 RPIHHPLVL ++ 87 QILQHHVL +++ 88 MLLCLSLEL ++ 89 VPYTKVQL +++ 90 TIGLGLHSL ++ 91 MPMQDIKMIL ++ 92 TLKAMVQAW +++ 93 LMKEKIQEM +++ 94 DQLLRHVM +++ 95 SRNPRGFFL ++ 96 RPAGVFEL ++ 97 EPVTKAEM +++ 98 EPVNTNVVL ++ 99 NVKIRFLE ++ 100 VLLMGPLHL ++ 101 FAFEKLIQF +++ 102 IMKKIRESY +++ 103 IANLRVKNI +++ 104 FAFGEPREL ++ 105 APLKMLALV ++ 106 HLHLLETSI ++ 107 LPKGLKDWQA ++ 108 RSYYHPTNL ++ 109 IPASHPVLTL ++ 110 DAMTKHTL +++ 111 GAGLRITAPL ++ 112 NALDPLSAL ++ 113 MEKGLASL ++ 114 PVKPKFYL ++ 115 MRILKRFLAC ++ 116 FTQNPRVQL ++ 117 VGPNGFKSL ++ 118 MAFVKHLL +++ 119 FQRVSSVSF +++ 120 AQRSEMVTL ++ 121 YSQLSISL ++ 122 ESSAQPTAL ++ 123 SLLPFTLSF ++ 124 HSWTRTSV +++ 125 FPLVTPLL +++ 126 TAAEARLSL ++ 127 EPVIRTVSI +++ 128 HFHNRHVF +++ 129 HRILRLPAL +++ 130 LPAPYQHQL ++ 131 MSTKTTSI ++ 132 IPIQAHQI +++ 133 QATPRVRIL ++ 134 DQRSRATL ++ 135 EYLETKRLAF + 136 KMFYRKDVM +++ 137 VQWKPPAL ++ 138 TQHLTVATL +++ 139 YGRIGISLF ++ 140 IAVDKPITL ++ 141 AQLKLVAL ++ 142 YNLIYSMCL +++ 143 DADLREQAL ++ 144 IEQIRAVL ++ 145 MIYRKALRL ++ 146 FQTAHFYL +++ 147 HAMDGASHL ++ 148 DVNPVSLQL ++ 149 TQKSVQVL ++ 150 MRSSYIREL ++ 151 DRHLTNRVL ++ 152 FNKLVTEL ++ 153 HAIPHYVTM ++ 154 VLKTLQEL ++ 155 LPASFPAVL ++ 156 ILKEQSSSSF ++ 157 QPYRFPQA ++ 158 DVIIKGNGL ++ 159 DLRNKIIA ++ 160 LPINNTHI ++ 161 DIVPPFSAF ++ 162 LFKQTKINL ++ 163 EVMAQFKEI ++ 164 LPAPIPTLL ++ 165 QNSLRHNL ++ 166 VLSGGRILAL ++ 167 DMKITVSL +++ 168 HVQDFTAF ++ 169 YELNNLHAL ++ 170 SPANVRGQSL ++ 171 FPSQVPKQVL ++ 172 PYEKVSRL ++ 173 YPLLKDPSL +++ 174 HAMPSPRIL ++ 175 MRFQQFLFA + 176 YVIQRQSVL ++ 177 SVPVRSSPL ++ 178 IPRLAVISI ++ 179 LPLTEHEL +++ 180 LAVPIFVAL ++ 181 SIRSSYSRF ++ 182 ILHLSAIAL ++++ 183 YVSKPGAQL ++ 184 DRLKPLKM ++ 185 MELKTVKPI ++ 186 DLISPRQPRL ++ 187 VPYNSVLF ++ 188 EIMEKTTL ++ 189 APDNVLLTL ++ 190 ELLNRIYF ++ 191 RPLKPGEVL ++ 192 EEKHFTTL ++ 193 LGGLRLTAL ++ 194 RAIEHVLQV +++ 195 EGNQKSVI ++ 196 LDLRQKVL ++ 197 YKAYPSQL ++ 198 FPLTSIIAI +++ 199 IPFIHLPEI ++ 200 VAAARAVPV ++ 201 TASAMQHVL +++ 202 RIPEKASFL ++ 203 DVYTQVSAF ++ 204 MSPLLRSI ++ 205 YMQYGFLSM ++ 206 MEFPNKFNTL ++ 207 LRKRKSPE ++ 208 LPPPQPLSL ++ 209 SRFGKFVQL ++ 210 QPNTHQLL ++ 211 DVISKGVSL ++ 212 EEYKFPSL ++ 213 FPSLFINQF ++ 214 DAPRHRLL ++ 215 NPLIEIISI ++ 216 EARPPSPAV ++ 217 ETIKGHSVRL ++ 218 DNHPRLVTL +++ 219 VRNPKILIL ++ 220 LAVRHLSL ++ 221 SLKEELLSL +++ 222 AQKAELIAL ++ 223 DVSARKLRV ++ 224 LPYPPQKVV ++ 225 MPKRAHWGA +++ 226 DIYEVAVSL +++ 227 SRFPGMSVL ++ 228 TLRAYVLAL +++ 229 DTHTNTYYL ++ 230 DVYFHHVL ++ 231 GEKLLRPSL ++ 232 KLYIHRVTL ++ 233 DVKLVFVM ++ 234 VFRVGISF ++ 235 SPNSLVTIL +++ 236 STLKKSLEI ++ 237 LPLDSRYVTL +++ 238 IPLAIARL +++ 239 SEPVMRVTL ++ 240 KVIDRKVEL ++ 241 NAYEAPSI ++ 242 KPQSLQLVL ++ 243 EGVPPGTVL +++ 244 HALPPYITVL ++ 245 GPRGPSSGHPL ++ 246 RLLQKSKEL ++ 247 TPEPSVHAL ++ 248 SEVNKHETAL ++ 249 QQIDRVVEV ++ 250 AARAPPQAL ++ 251 DAAAFFKSV ++ Binding of HLA-class I restricted peptides to HLA-B*08:01 was ranged by peptide exchange yield: ≥10% = +; ≥20% = ++; ≥50% = +++; ≥75% = ++++
TABLE-US-00020 TABLE 11B MHC class I binding scores. SEQ ID NO Sequence Peptide exchange 290 EEFLTPKKL +++ Binding of HLA-class I restricted peptides to HLA-B*44:05 was ranged by peptide exchange yield: ≥50% = +++.
TABLE-US-00021 TABLE 11C MHC class I binding scores. Peptide SEQ ID NO Sequence exchange 291 YVYANHFTEA ++++ Binding of HLA-class I restricted peptides to HLA-A*02:01 was ranged by peptide exchange yield: ≥75% = ++++.
Example 6
[0388] Absolute Quantitation of Tumor Associated Peptides Presented on the Cell Surface
[0389] The generation of binders, such as antibodies and/or TCRs, is a laborious process, which may be conducted only for a number of selected targets. In the case of tumor-associated and—specific peptides, selection criteria include but are not restricted to exclusiveness of presentation and the density of peptide presented on the cell surface. In addition to the isolation and relative quantitation of peptides as described in Example 1, the inventors did analyze absolute peptide copies per cell as described in US20160187351, the content of which is incorporated by reference in its entirety. The quantitation of TUMAP copies per cell in solid tumor samples requires the absolute quantitation of the isolated TUMAP, the efficiency of the TUMAP isolation process, and the cell count of the tissue sample analyzed. An overview on our experimental approach is described below.
[0390] Peptide quantitation by nano LC-MS/MS For an accurate quantitation of peptides by mass spectrometry, a calibration curve was generated for each individual peptide using two different isotope labeled peptide variants (one or two isotope-labeled amino acids are included during TUMAP synthesis). These isotopes labeled variants differ from the tumor-associated peptide only in their mass but show no difference in other physicochemical properties (Anderson et al., 2012). For the peptide calibration curve, a series of nano LC-MS/MS measurements was performed to determine the ratio of MS/MS signals of titrated (singly isotope-labeled peptide) to constant (doubly isotope labeled peptide) isotope labeled peptides.
[0391] The doubly isotope labeled peptide, also called internal standard, was further spiked to each MS sample and all MS signals were normalized to the MS signal of the internal standard to level out potential technical variances between MS experiments.
[0392] The calibration curves were prepared in at least three different matrices, i.e. HLA peptide eluates from natural samples similar to the routine MS samples, and each preparation was measured in duplicate MS runs. For evaluation, MS signals were normalized to the signal of the internal standard and a calibration curve was calculated by logistic regression.
[0393] For the quantitation of tumor-associated peptides from tissue samples, the respective samples were also spiked with the internal standard; the MS signals were normalized to the internal standard and quantified using the peptide calibration curve.
[0394] Efficiency of Peptide/MHC Isolation
[0395] As for any protein purification process, the isolation of proteins from tissue samples is associated with a certain loss of the protein of interest. To determine the efficiency of TUMAP isolation, peptide/MHC complexes were generated for all TUMAPs selected for absolute quantitation. To be able to discriminate the spiked from the natural peptide/MHC complexes, single-isotope-labelled versions of the TUMAPs were used, i.e. one isotope-labelled amino acid was included in TUMAP synthesis. These complexes were spiked into the freshly prepared tissue lysates, i.e. at the earliest possible point of the TUMAP isolation procedure, and then captured like the natural peptide/MHC complexes in the following affinity purification. Measuring the recovery of the single-labelled TUMAPs therefore allows conclusions regarding the efficiency of isolation of individual natural TUMAPs.
[0396] The efficiency of isolation was analyzed in a small set of samples and was comparable among these tissue samples. In contrast, the isolation efficiency differs between individual peptides. This suggests that the isolation efficiency, although determined in only a limited number of tissue samples, may be extrapolated to any other tissue preparation. However, it is necessary to analyze each TUMAP individually as the isolation efficiency may not be extrapolated from one peptide to others.
[0397] Determination of the Cell Count in Solid, Frozen Tissue
[0398] In order to determine the cell count of the tissue samples subjected to absolute peptide quantitation, the inventors applied DNA content analysis. This method is applicable to a wide range of samples of different origin and, most importantly, frozen samples (Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During the peptide isolation protocol, a tissue sample is processed to a homogenous lysate, from which a small lysate aliquot is taken. The aliquot is divided in three parts, from which DNA is isolated (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). The total DNA content from each DNA isolation is quantified using a fluorescence-based DNA quantitation assay (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) in at least two replicates.
[0399] In order to calculate the cell number, a DNA standard curve from aliquots of isolated healthy blood cells from several donors, with a range of defined cell numbers, has been generated. The standard curve is used to calculate the total cell content from the total DNA content from each DNA isolation. The mean total cell count of the tissue sample used for peptide isolation is then extrapolated considering the known volume of the lysate aliquots and the total lysate volume.
[0400] Peptide Copies Per Cell
[0401] With data of the aforementioned experiments, the inventors calculated the number of TUMAP copies per cell by dividing the total peptide amount by the total cell count of the sample, followed by division through isolation efficiency. Copy cell number for selected peptides are shown in Table 12.
TABLE-US-00022 TABLE 12 Absolute copy numbers. The table lists the results of absolute peptide quantitation in tumor samples. The median number of copies per cell are indicated for each peptide: <25 = +. The number of samples, in which evaluable, high quality MS data are available, is indicated. Copies per Number SEQ ID Peptide cell of NO Code (median) samples 291 COL6A3-039 + 9
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