NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST NHL AND OTHER CANCERS

Abstract

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

Claims

1. A peptide consisting of the amino acid sequence FVIDSFEEL (SEQ ID NO: 113) 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-1 molecule, and wherein said peptide, when bound to said MHC, is capable of being recognized by CD8 T cells.

3. The peptide of claim 1, wherein the pharmaceutically acceptable is chloride salt.

4. The peptide of claim 1, wherein the pharmaceutically acceptable 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 FVIDSFEEL (SEQ ID NO: 113) or a pharmaceutically acceptable salt thereof.

15. The peptide of claim 14, wherein the pharmaceutically acceptable is chloride salt.

16. The peptide of claim 14, wherein the pharmaceutically acceptable 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 FVIDSFEEL (SEQ ID NO: 113), 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

[0387] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0388] FIGS. 1A to 1P show the over-presentation of various peptides in normal tissues (white bars) and NHL (black bars). FIG. 1A) Gene symbol: TOX2, Peptide: LLSGQLPTI (SEQ ID NO.: 1); Tissues from left to right: 3 adipose tissues, 3 adrenal glands, 15 blood cell samples, 12 blood vessels, 10 bone marrows, 7 brains, 8 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6 hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 8 lymph nodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal muscles, 11 skins, 3 small intestines, 11 spleens, 5 stomachs, 6 testes, 2 thymi, 2 thyroid glands, 9 tracheas, 7 ureters, 8 urinary bladders, 5 uteri, 6 esophagi, 18 NHL samples. The peptide has additionally been detected on 1/84 lung cancers, 1/17 chronic lymphocytic leukemias, 1/20 pancreatic cancer cell lines, 1/20 ovarian cancers and 1/16 uterus cancers. FIG. 1B) Gene symbol: TAP1, Peptide: VLQGLTFTL (SEQ ID NO.: 5); Tissues from left to right: 3 adipose tissues, 3 adrenal glands, 15 blood cell samples, 12 blood vessels, 10 bone marrows, 7 brains, 8 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6 hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 8 lymph nodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal muscles, 11 skins, 3 small intestines, 11 spleens, 5 stomachs, 6 testes, 2 thymi, 2 thyroid glands, 9 tracheas, 7 ureters, 8 urinary bladders, 5 uteri, 6 esophagi, 18 NHL samples. The peptide has additionally been detected on 4/101 lung cancers, 1/18 breast cancers, 1/17 chronic lymphocytic leukemias, 2/17 bile duct and gallbladder cancers, 2/16 melanomas, 2/20 ovarian cancers and 1/15 urinary bladder cancers. FIG. 1C) Gene symbol: SLC20A1, Peptide: ILASIFETV (SEQ ID NO.: 41); Tissues from left to right: 3 adipose tissues, 3 adrenal glands, 15 blood cell samples, 12 blood vessels, 10 bone marrows, 7 brains, 8 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6 hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 8 lymph nodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal muscles, 11 skins, 3 small intestines, 11 spleens, 5 stomachs, 6 testes, 2 thymi, 2 thyroid glands, 9 tracheas, 7 ureters, 8 urinary bladders, 5 uteri, 6 esophagi, 18 NHL samples. The peptide has additionally been detected on 10/101 lung cancers, 4/18 acute myelogenous leukemias, 1/18 breast cancers, 1/17 chronic lymphocytic leukemias, 3/20 pancreatic cancer cell lines, 2/17 bile duct and gallbladder cancers, 4/16 melanomas, 1/20 ovarian cancers, 2/19 pancreas cancers, 1/38 prostate cancers, 2/22 kidney cancers and 1/15 urinary bladder cancers. FIG. 1D) Gene symbol: COPS7B, Peptide: NLLEQFILL (SEQ ID NO.: 248); Samples from left to right: 4 cancer cell lines, 6 normal tissues (1 lymph node, 3 spleens, 1 stomach, 1 uterus), 55 cancer tissues (2 brain cancers, 1 breast cancer, 1 cecum cancer, 6 colon cancers, 6 leukocytic leukemia cancers, 2 liver cancers, 10 lung cancers, 8 lymph node cancers, 1 myeloid cell cancer, 3 ovarian cancers, 1 prostate cancer, 1 rectum cancer, 4 skin cancers, 2 stomach cancers, 3 urinary bladder cancers, 4 uterus cancers). Discrepancies regarding the list of tumor types between FIG. 1D and Table 4A might be due to the more stringent selection criteria applied in Table 4A (for details please refer to Table 4A). The normal tissue panel and the cancer cell lines and xenografts tested were the same as in FIGS. 1A to 1C. FIG. 1E) Gene symbol: KDMSB, Peptide: LLSEETPSA (SEQ ID NO.: 2); Samples from left to right: 1 primary culture, 40 cancer tissues (1 bone marrow cancer, 1 brain cancer, 2 breast cancers, 8 head and neck cancers, 4 leukocytic leukemia cancers, 1 liver cancer, 7 lung cancers, 6 lymph node cancers, 2 myeloid cell cancers, 1 ovarian cancer, 3 skin cancers, 3 urinary bladder cancers, 1 uterus cancer). FIG. 1F) Gene symbol: CDCl42, Peptide: FLLVGTQIDL (SEQ ID NO.: 10); Samples from left to right: 2 cell lines, 10 cancer tissues (2 breast cancers, 1 head and neck cancer, 1 leukocytic leukemia cancer, 1 lung cancer, 4 lymph node cancers, 1 uterus cancer). FIG. 1G) Gene symbol: HAPLN3, Peptide: GLLLLVPLL (SEQ ID NO.: 12); Samples from left to right: 16 cancer tissues (1 breast cancer, 1 colon cancer, 1 colorectal cancer, 1 esophageal cancer, 1 gallbladder cancer, 1 head and neck cancer, 2 lung cancers, 5 lymph node cancers, 2 ovarian cancers, 1 skin cancer). FIG. 1H) Gene symbol: JAK3, Peptide: HLVPASWKL (SEQ ID NO.: 13); Samples from left to right: 10 cancer tissues (1 leukocytic leukemia cancer, 1 lung cancer, 5 lymph node cancers, 1 ovarian cancer, 1 skin cancer, 1 testis cancer). FIG. 1I) Gene symbol: TMEM67, Peptide: FLGSFIDHV (SEQ ID NO.: 26); Samples from left to right: 1 cell line, 9 cancer tissues (1 brain cancer, 1 lung cancer, 1 lymph node cancer, 1 myeloid cell cancer, 2 ovarian cancers, 2 skin cancers, 1 uterus cancer). FIG. 1J) Gene symbols: PTTG1, PTTG2, Peptide: ILSTLDVEL (SEQ ID NO.: 30); Samples from left to right: 29 cancer tissues (1 bone marrow cancer, 2 colon cancers, 1 gallbladder cancer, 3 head and neck cancers, 1 kidney cancer, 5 lung cancers, 7 lymph node cancers, 1 ovarian cancer, 5 skin cancers, 2 urinary bladder cancers, 1 uterus cancer). FIG. 1K) Gene symbol: DCAKD, Peptide: VILDIPLLFET (SEQ ID NO.: 36); Samples from left to right: 2 cell lines, 20 cancer tissues (1 brain cancer, 1 breast cancer, 1 colorectal cancer, 1 head and neck cancer, 1 leukocytic leukemia cancer, 1 liver cancer, 3 lung cancers, 4 lymph node cancers, 1 myeloid cell cancer, 1 ovarian cancer, 4 skin cancers, 1 uterus cancer). FIG. 1L) Gene symbol: KDM2B, Peptide: ALLEGVKNV (SEQ ID NO.: 43); Samples from left to right: 13 cancer tissues (1 breast cancer, 1 leukocytic leukemia cancer, 1 lung cancer, 6 lymph node cancers, 3 ovarian cancers, 1 rectum cancer). FIG. 1M) Gene symbol: ACHE, Peptide: SLDLRPLEV (SEQ ID NO.: 74); Samples from left to right: 1 cell line, 2 normal tissues (1 lymph node, 1 spleen), 24 cancer tissues (3 brain cancers, 1 colon cancer, 1 gallbladder cancer, 1 kidney cancer, 1 lung cancer, 12 lymph node cancers, 1 ovarian cancer, 1 skin cancer, 2 stomach cancers, 1 testis cancer). FIG. 1N) Gene symbol: CYTB, Peptide: FLYSETWNI (SEQ ID NO.: 254); Samples from left to right: 7 cell lines, 15 cancer tissues (1 colon cancer, 2 head and neck cancers, 3 leukocytic leukemia cancers, 1 liver cancer, 6 lymph node cancers, 1 myeloid cell cancer, 1 skin cancer). FIG. 1O) Gene symbol: ACN9, Peptide: FLQEWEVYA (SEQ ID NO.: 257); Samples from left to right: 2 cell lines, 11 cancer tissues (2 leukocytic leukemia cancers, 1 liver cancer, 4 lymph node cancers, 1 myeloid cells cancer, 2 skin cancers, 1 urinary bladder cancer). FIG. 1P) Gene symbol: SMC2, Peptide: TVLDGLEFKV (SEQ ID NO.: 259); Samples from left to right: 1 primary culture, 14 cancer tissues (1 head and neck cancer, 3 leukocytic leukemia cancers, 3 lung cancers, 3 lymph node cancers, 1 myeloid cell cancer, 1 ovarian cancer, 2 skin cancers).

[0389] FIGS. 2A to 2C show exemplary expression profiles of source genes of the present invention that are highly over-expressed or exclusively expressed in NHL in a panel of normal tissues (white bars) and 10 NHL samples (black bars). Tissues from left to right: 6 arteries, 2 blood cell samples, 2 brains, 1 heart, 2 livers, 3 lungs, 2 veins, 1 adipose tissue, 1 adrenal gland, 5 bone marrows, 1 cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gallbladders, 1 kidney, 6 lymph nodes, 4 pancreases, 2 peripheral nerves, 2 pituitary glands, 1 rectum, 2 salivary glands, 2 skeletal muscles, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 5 placentas, 1 prostate, 1 testis, 1 thymus, 1 uterus, 10 NHL samples. FIG. 2A) Gene symbol: MIXL1. FIG. 2B) Gene symbol: CCR4. FIG. 2C) Gene symbol: HIST1H1B.

[0390] FIG. 3 shows exemplary immunogenicity data: flow cytometry results after peptide-specific multimer staining.

[0391] FIGS. 4A to 4C show exemplary results of peptide-specific in vitro CD8+ T cell responses of a healthy HLA-A*02+ donor. CD8+ T cells were primed using artificial APCs coated with anti-CD28 mAb and HLA-A*02 in complex with Seq ID No 253 peptide (FIG. 4A, left panel), Seq ID No 258 peptide (FIG. 4B, left panel) and Seq ID No 260 peptide (FIG. 4C, left panel), respectively. After three cycles of stimulation, the detection of peptide-reactive cells was performed by 2D multimer staining with A*02/Seq ID No 253 (FIG. 4A), A*02/Seq ID No 258 (FIG. 4B) or A*02/Seq ID No 260 (FIG. 4C). Right panels (FIGS. 4A, 4B and 4C) show control staining of cells stimulated with irrelevant A*02/peptide complexes. Viable singlet cells were gated for CD8+ lymphocytes. Boolean gates helped excluding false-positive events detected with multimers specific for different peptides. Frequencies of specific multimer+ cells among CD8+ lymphocytes are indicated.

DETAILED DESCRIPTION OF THE INVENTION

Examples

Example 1

[0392] Identification and quantitation of tumor associated peptides presented on the cell surface Tissue samples Patients' tumor tissues were obtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK); ProteoGenex Inc. (Culver City, Calif., USA).

[0393] Normal tissues were obtained from Asterand (Detroit, Mich., USA & 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).

[0394] 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

[0395] HLA peptide pools from shock-frozen tissue samples were obtained by immune precipitation from solid tissues according to a slightly modified protocol (Falk et al., 1991; Seeger et al., 1999) using the HLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibody W6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

[0396] 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 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.

[0397] 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 NHL samples to a baseline of normal tissue samples. Presentation profiles of exemplary over-presented peptides are shown in FIGS. 1A-1P. Presentation scores for exemplary peptides are shown in Table 8.

TABLE-US-00012 TABLE 8 Presentation scores. The table lists peptides that are very highly over-presented on tumors compared to a panel of normal tissues (+++), highly over- presented on tumors compared to a panel of normal tissues (++) or over-presented on tumors compared to a panel of normal tissues (+).The panel of normal tissues considered relevant for comparison with tumors consisted of: adipose tissue, adrenal gland, artery, vein, bone marrow, brain, central and peripheral nerve, colon, rectum, small intestine incl. duodenum, esophagus, eye, gallbladder, heart, kidney, liver, lung, lymph node, mononuclear white blood cells, pancreas, parathyroid gland, peritoneum, pituitary, pleura, salivary gland, skeletal muscle, skin, spleen, stomach, thymus, thyroid gland, trachea, ureter, urinary bladder. SEQ ID Peptide No. Sequence Presentation 1 LLSGQLPTI +++ 2 LLSEETPSA +++ 3 LTIDTQYYL +++ 5 VLQGLTFTL +++ 6 TLITLPLLFL +++ 7 NLLGMIFSM +++ 8 ALYAVIEKA +++ 9 FLLDLDPLL +++ 10 FLLVGTQIDL +++ 11 GLDTVVALL +++ 12 GLLLLVPLL +++ 13 HLVPASWKL +++ 15 IIIEDLLEA +++ 16 TLIAAILYL +++ 17 VIIPLLSSV +++ 18 KLTDQPPLV +++ 19 VLEAILPLV +++ 20 YLIAGGDRWL +++ 21 ALFKEAYSL +++ 22 ALKKHLTSV +++ 23 ALVEDIINL +++ 24 AVLGFSFRL +++ 25 FLDTSNQHLL +++ 26 FLGSFIDHV +++ 27 FLNQESFDL +++ 28 FLSNANPSL +++ 29 ILSDVTQGL +++ 30 ILSTLDVEL +++ 31 KLYDEESLL +++ 32 VLNEDELPSV +++ 33 LLANIVPIAMLV +++ 34 LLWEDGVTEA +++ 35 SLSSERYYL +++ 36 VILDIPLLFET +++ 37 VLGNALEGV +++ 38 YLTAEILELAGN +++ 40 FLNSVIVDL + 41 ILASIFETV +++ 43 ALLEGVKNV + 44 FIIEEQSFL +++ 45 FILDDSALYL + 46 FLVEEIFQT ++ 47 GLLPKLTAL + 49 TILGDPQILL +++ 50 LLLDGLIYL + 53 FLREYFERL +++ 54 DIFDAMFSV +++ 55 ILVEVDLVQA ++ 56 GLQDLLFSL ++ 57 LQIGDFVSV + 60 SLLIDVITV +++ 61 SLLNKDLSL + 62 ALAPYLDLL +++ 64 FLVEVSNDV ++ 65 NLTDVSPDL +++ 67 LLATVNVAL +++ 69 TLLAFPLLL + 71 VLLDYVGNVQL +++ 72 TLQEETAVYL +++ 74 SLDLRPLEV + 75 AALKYIPSV +++ 76 ALADLVPVDVVV +++ 77 ALLDVSNNYGI +++ 78 AMEEAVAQV +++ 79 AMKEEKEQL +++ 80 YLFDEIDQA +++ 81 FIFSYITAV +++ 82 FLIDGSSSV +++ 83 FLMDDNMSNTL +++ 84 FLQELQLEHA +++ 85 GLAPAEVVVATVA +++ 86 GLATIRAYL +++ 87 GLFARIIMI +++ 88 GLFDNRSGLPEA +++ 89 GLTALHVAV +++ 90 HLDEVFLEL +++ 91 HLSSTTAQV +++ 92 KLLFEIASA +++ 93 KLLGSLQLL +++ 94 LLAGQATTAYF +++ 95 LLFDLIPVVSV +++ 96 LLLNENESLFL +++ 97 LLNFSPGNL +++ 98 MLQDGIARL +++ 99 QLYDGATALFL +++ 100 RLIRTIAAI +++ 101 SLDQSTWNV +++ 102 SLFAAISGMIL +++ 103 SLQDHLEKV +++ 104 VLLGLPLLV +++ 105 VLTPVILQV +++ 106 VLYELLQYI +++ 107 VQAVSIPEV +++ 108 YLAPENGYLM +++ 109 YLFQFSAAL +++ 110 YQYPFVLGL +++ 111 YLLDTLLSL +++ 112 FLAILPEEV +++ 113 FVIDSFEEL +++ 114 GLSDISPST +++ 115 LLIDIIHFL +++ 116 SLLDNLLTI + 117 VLATILAQL +++ 118 VLDGMIYAI +++ 119 ELCDIILRV +++ 120 VLLGTTWAL +++ 121 YLTGYNFTL +++ 122 AISEAQESV + 123 ALLSAFVQL ++ 124 FLGVVVPTV +++ 125 FVAPPTAAV +++ 126 GLSIFIYRL +++ 128 KLFDASPTFFA ++ 131 VLIEETDQL +++ 132 VLQDQVDEL +++ 133 ALEELTGFREL +++ 134 ALGRLGILSV +++ 135 ALTGLQFQL +++ 136 FIFGIVHLL +++ 137 FIQQERFFL +++ 138 NLINNIFEL + 139 FLASPLVAI +++ 140 FLFEDFVEV +++ 141 FLGELTLQL +++ 142 FLYEDSKSVRL +++ 143 TLHAVDVTL +++ 144 GLITQVDKL +++ 145 GLLHEVVSL +++ 146 GLLQQPPAL +++ 147 GLSEYQRNFL +++ 148 ICAGHVPGV +++ 149 ILNPVTTKL +++ 150 ILSEKEYKL +++ 151 ILVKQSPML +++ 152 KIMYTLVSV +++ 153 KLLKGIYAI +++ 154 KLMNIQQQL +++ 155 KLMTSLVKV +++ 156 KMLEDDLKL +++ 157 KVLEFLAKV +++ 158 KVQDVLHQV +++ 159 LLLSDSGFYL +++ 160 LLPPPSPAA +++ 161 NLMLELETV +++ 162 RLADLKVSI +++ 163 SIFDAVLKGV +++ 164 SLFDGAVISTV +++ 165 KLLEEIEFL ++ 166 SLFSEVASL +++ 167 SLFSITKSV +++ 168 SLLSPLLSV +++ 169 SSLEENLLHQV +++ 170 STIELSENSL +++ 171 TLLDVISAL +++ 172 TLQDSLEFI +++ 173 VILDSVASV +++ 174 VLVEITDVDFAA +++ 175 VMESILLRL +++ 176 YLHIYESQL +++ 177 YLYEAEEATTL +++ 178 YVLQGEFFL +++ 179 FVDTNLYFL +++ 180 GILQLVESV ++ 182 LLPPPPPVA + 183 VLFETVLTI + 185 FIAQLNNVEL + 186 FLDVSRDFV + 188 GLEDEMYEV ++ 189 SLSHLVPAL + 190 GLIELVDQL ++ 191 GLSDISAQV +++ 194 SLAPFDREPFTL +++ 195 ALIPDLNQI +++ 196 TLALAMIYL ++ 200 YLLDFEDRL + 201 YLNISQVNV ++ 203 ILDTIFHKV +++ 204 RLCDIVVNV +++ 207 GLVGLLEQA ++ 211 FIDDLFAFV +++ 212 FLIGQGAHV + 213 YINEDEYEV + 214 FLFDGSMSL ++ 215 QLFEEEIEL + 216 KVVSNLPAI +++ 217 AQFGAVLEV + 218 ALDQFLEGI + 219 ALLELENSV +++ 220 FLAEAPTAL ++ 221 FLAPDNSLLLA +++ 222 FLIETGTLL + 224 FLSPLLPLL + 225 GTYQDVGSLNIGDV +++ 226 GVIDPVPEV + 227 IIAEGIPEA + 231 IVMGAIPSV + 232 KVMEGTVAA ++ 233 MLEVHIPSV ++ 236 SLFDGFFLTA + 237 YLDRLIPQA ++ 239 VLIDDTVLL ++ 242 GILDFZVFL + 243 GLPDLDIYL +++ 244 ILEPFLPAV + 246 KLPVPLESV + 249 VLLESLVEI +++ 252 YLGDLIMAL + 253 YSDDDVPSV +++ 254 FLYSETWNI +++ 255 GMWNPNAPVFL +++ 256 ALQETPPQV +++ 257 FLQEWEVYA +++ 258 RIYPFLLMV +++ 259 TVLDGLEFKV +++ 260 RLDEAFDFV ++ 263 GLMDNEIKV +++ 264 ILTGTPPGV +++ 265 ILWHFVASL +++ 266 QLTEMLPSI +++ 267 SLLETGSDLLL +++ 268 VLFPLPTPL +++ 269 VLQNVAFSV +++ 270 VVVDSDSLAFV +++ 271 YLLDQPVLEQRL +++ 272 KLDHTLSQI +++ 273 AILLPQPPK +++ 274 KLLNLISKL +++ 275 KLMDLEDCAL +++ 276 NMISYVVHL +++ 277 FLIDLNSTHGTFL + 279 NLAGENILNPL ++ 280 SLLNHLPYL +++ 285 SITAVTPLL + 287 ILMGHSLYM ++ 289 SLLAANNLL +++ 290 IASPVIAAV +++ 291 KIIDTAGLSEA +++ 292 KLINSQISL ++ 294 KLYGPEGLELV + 296 FILEPLYKI ++ 298 ALTDVILCV + 299 RLLEEEGVSL + 302 SLAELDEKISA + 303 FVWEASHYL ++ 305 AMLAQQMQL + 307 FLLPVAVKL ++ 308 SLLDQIPEM +

Example 2

[0398] 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

[0399] 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.

[0400] Total RNA from healthy human tissues for RNASeq experiments was obtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA); Geneticist Inc. (Glendale, Calif., USA); Istituto Nazionale Tumori “Pascale” (Naples, Italy); ProteoGenex Inc. (Culver City, Calif., USA); University Hospital Heidelberg (Heidelberg, Germany). Total RNA from tumor tissues for RNASeq experiments was obtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK); ProteoGenex Inc. (Culver City, Calif., USA).

[0401] 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

[0402] 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. Exemplary expression profiles of source genes of the present invention that are highly over-expressed or exclusively expressed in NHL are shown in FIGS. 2A-2C. Expression scores for further exemplary genes are shown in Table 9.

TABLE-US-00013 TABLE 9 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, artery, blood cells, bone marrow, brain, cartilage, colon, esophagus, eye, gallbladder, heart, kidney, liver, lung, lymph node, pancreas, pituitary, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, urinary bladder, and vein. In case expression data for several samples of the same tissue type were available, the arithmetic mean of all respective samples was used for the calculation. Gene SEQ ID No Sequence Expression   9 FLLDLDPLL ++  21 ALFKEAYSL +  25 FLDTSNQHLL ++  30 ILSTLDVEL ++  38 YLTAEILELAGN ++  43 ALLEGVKNV +  55 ILVEVDLVQA +  56 GLQDLLFSL +  61 SLLNKDLSL +  91 HLSSTTAQV ++ 102 SLFAAISGMIL +++ 106 VLYELLQYI +++ 112 FLAILPEEV ++ 113 FVIDSFEEL +++ 116 SLLDNLLTI + 133 ALEELTGFREL + 135 ALTGLQFQL +++ 142 FLYEDSKSVRL +++ 143 TLHAVDVTL +++ 146 GLLQQPPAL + 148 ICAGHVPGV +++ 155 KLMTSLVKV +++ 157 KVLEFLAKV +++ 158 KVQDVLHQV +++ 159 LLLSDSGFYL ++ 160 LLPPPSPAA +++ 161 NLMLELETV +++ 162 RLADLKVSI +++ 167 SLFSITKSV +++ 170 STIELSENSL ++ 174 VLVEITDVDFAA + 175 VMESILLRL +++ 178 YVLQGEFFL +++ 183 VLFETVLTI + 192 GMAAEVPKV + 199 SLNSTTWKV +++ 202 ALAAGGYDV +++ 222 FLIETGTLL ++ 225 GTYQDVGSLNIGDV ++ 229 ILSPWGAEV ++ 238 YQYGAVVTL ++ 256 ALQETPPQV + 260 RLDEAFDFV ++ 268 VLFPLPTPL + 276 NMISYVVHL +++ 294 KLYGPEGLELV +++ 297 ILQNGLETL +++ 298 ALTDVILCV +++

Example 3

[0403] 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 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 Vitro Priming of CD8+ T Cells

[0404] 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.

[0405] 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.

[0406] 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.

[0407] 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).

[0408] 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.

[0409] 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 μI. 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).

In Vitro Immunogenicity for NHL Peptides

[0410] For tested HLA class I peptides, in vitro immunogenicity could be demonstrated by generation of peptide specific T-cell lines. Exemplary flow cytometry results after TUMAP-specific multimer staining for 2 peptides of the invention are shown in FIG. 3 together with corresponding negative controls. Results for ten peptides from the invention are summarized in Tables 10A and 10B.

TABLE-US-00014 TABLE 10A in vitro immunogenicity of HLA class I peptides of the invention Exemplary results of in vitro immunogenicity experiments conducted by the applicant for the peptides of the invention. <20% = +; 20%-49% = ++; 50%-69% = +++; > = 70% = ++++ Seq ID Sequence wells 319 SLYKGLLSV ++ 320 LLWGNLPEI ++ 321 KLLAVIHEL ++ 322 TLTNIIHNL ++ 323 ILVDWLVQV ++ 324 LLYDAVHIV ++ 325 FLFVDPELV +++ 326 KLTDVGIATL ++++ 327 MLFGHPLLVSV ++ 328 ILFPDIIARA ++++

TABLE-US-00015 TABLE 10B In vitro immunogenicity of HLA class I peptides of the invention Exemplary results of in vitro immunogenicity experiments conducted by the applicant for HLA-A*02 restricted peptides of the invention. Results of in vitro immunogenicity experiments are indicated. Percentage of positive wells and donors (among evaluable) are summarized as indicated <20% = +; 20%-49% = ++; 50%-69% = +++; > = 70% = ++++ SEQ ID NO: Sequence Wells positive [%] 1 LLSGQLPTI ″+++″ 2 LLSEETPSA ″+″ 3 LTIDTQYYL ″+″ 5 VLQGLTFTL ″+++″ 7 NLLGMIFSM ″++++″ 8 ALYAVIEKA ″+″ 9 FLLDLDPLL ″++″ 12 GLLLLVPLL ″+++″ 13 HLVPASWKL ″+++″ 17 VIIPLLSSV ″++″ 19 VLEAILPLV ″+″ 21 ALFKEAYSL ″+″ 22 ALKKHLTSV ″++++″ 24 AVLGFSFRL ″++++″ 25 FLDTSNQHLL ″+″ 26 FLGSFIDHV ″+″ 27 FLNQESFDL ″+″ 28 FLSNANPSL ″++++″ 29 ILSDVTQGL ″+″ 30 ILSTLDVEL ″++″ 33 LLANIVPIAMLV ″+″ 35 SLSSERYYL ″++++″ 36 VILDIPLLFET ″++″ 37 VLGNALEGV ″+″ 40 FLNSVIVDL ″+++″ 41 ILASIFETV ″+++″ 42 YLQDLVERA ″+++″ 43 ALLEGVKNV ″++″ 44 FIIEEQSFL ″+″ 46 FLVEEIFQT ″+″ 47 GLLPKLTAL ″++″ 51 SLLGNSPVL ″+++″ 52 VLLEDVDAAFL ″+″ 53 FLREYFERL ″+++″ 57 LQIGDFVSV ″++++″ 59 RLHREVAQV ″+″ 60 SLLIDVITV ″+++″ 61 SLLNKDLSL ″+″ 62 ALAPYLDLL ″++″ 66 KLAPIPVEL ″++″ 67 LLATVNVAL ″+″ 68 QIAAFLFTV ″+++″ 73 YLGEEYPEV ″+″ 74 SLDLRPLEV ″++″ 253 YSDDDVPSV ″+++″ 254 FLYSETWNI ″+″ 256 ALQETPPQV ″+″ 258 RIYPFLLMV ″++++″ 260 RLDEAFDFV ″++++″ 261 FLPETRIMTSV ″+″ 262 LMGPVVHEV ″++″

Example 4

Synthesis of Peptides

[0411] All peptides were synthesized using standard and well-established solid phase peptide synthesis using the Fmoc-strategy. Identity and purity of each individual peptide have been determined by mass spectrometry and analytical RP-HPLC. The peptides were obtained as white to off-white lyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPs are preferably administered as trifluoro-acetate salts or acetate salts, other salt-forms are also possible.

Example 5

MHC Binding Assays

[0412] 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).

[0413] 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 NH.sub.2SO.sub.4. Absorption was measured at 450 nm. Candidate peptides that show a high exchange yield (preferably higher than 50%, most preferred higher than 75%) are generally preferred for a generation and production of antibodies or fragments thereof, and/or T cell receptors or fragments thereof, as they show sufficient avidity to the MHC molecules and prevent dissociation of the MHC complexes.

TABLE-US-00016 TABLE 11 MHC class I binding scores. Binding of HLA-class I restricted peptides to HLA-A*02:01 was ranged by peptide exchange yield: ≥10% = +; ≥20% = ++; ≥50 = +++; ≥75% = ++++ SEQ ID NO: Sequence Peptide exchange 1 LLSGQLPTI ″+++″ 2 LLSEETPSA ″+++″ 3 LTIDTQYYL ″+++″ 4 TLLGFFLAKV ″++″ 5 VLQGLTFTL ″+++″ 6 TLITLPLLFL ″+++″ 7 NLLGMIFSM ″++++″ 8 ALYAVIEKA ″+++″ 9 FLLDLDPLL ″+++″ 10 FLLVGTQIDL ″+++″ 11 GLDTVVALL ″+++″ 12 GLLLLVPLL ″+++″ 13 HLVPASWKL ″+++″ 14 LLSDPTPGA ″++″ 15 IIIEDLLEA ″++++″ 16 TLIAAILYL ″++″ 17 VIIPLLSSV ″+++″ 18 KLTDQPPLV ″+++″ 19 VLEAILPLV ″++++″ 21 ALFKEAYSL ″+++″ 22 ALKKHLTSV ″+++″ 23 ALVEDIINL ″++++″ 24 AVLGFSFRL ″+++″ 25 FLDTSNQHLL ″+++″ 26 FLGSFIDHV ″+++″ 27 FLNQESFDL ″+++″ 28 FLSNANPSL ″+++″ 29 ILSDVTQGL ″+++″ 30 ILSTLDVEL ″+++″ 31 KLYDEESLL ″++++″ 32 VLNEDELPSV ″+++″ 33 LLANIVPIAMLV ″++++″ 34 LLWEDGVTEA ″+++″ 35 SLSSERYYL ″+++″ 36 VILDIPLLFET ″+++″ 37 VLGNALEGV ″+++″ 38 YLTAEILELAGN ″++″ 39 QLLPQGIVPAL ″+++″ 40 FLNSVIVDL ″++++″ 41 ILASIFETV ″++++″ 42 YLQDLVERA ″++++″ 43 ALLEGVKNV ″+++″ 44 FIIEEQSFL ″+++″ 45 FILDDSALYL ″+++″ 46 FLVEEIFQT ″+++″ 47 GLLPKLTAL ″+++″ 48 KILDEDLYI ″+++″ 49 TILGDPQILL ″++++″ 50 LLLDGLIYL ″+++″ 51 SLLGNSPVL ″++++″ 52 VLLEDVDAAFL ″++++″ 53 FLREYFERL ″++++″ 54 DIFDAMFSV ″+″ 55 ILVEVDLVQA ″++++″ 56 GLQDLLFSL ″+++″ 57 LQIGDFVSV ″++++″ 58 QLAPFLPQL ″+++″ 59 RLHREVAQV ″+++″ 60 SLLIDVITV ″+++″ 61 SLLNKDLSL ″++++″ 62 ALAPYLDLL ″++++″ 63 ALIEEAYGL ″+++″ 64 FLVEVSNDV ″++++″ 65 NLTDVSPDL ″+++″ 66 KLAPIPVEL ″++++″ 67 LLATVNVAL ″++++″ 68 QIAAFLFTV ″++++″ 69 TLLAFPLLL ″++++″ 70 VLIEILQKA ″++++″ 71 VLLDYVGNVQL ″++++″ 72 TLQEETAVYL ″++″ 73 YLGEEYPEV ″+++″ 74 SLDLRPLEV ″++++″ 75 AALKYIPSV ″+++″ 76 ALADLVPVDVVV ″++++″ 77 ALLDVSNNYGI ″++++″ 78 AMEEAVAQV ″+++″ 79 AMKEEKEQL ″++″ 80 YLFDEIDQA ″+++″ 81 FIFSYITAV ″++″ 82 FLIDGSSSV ″+++″ 83 FLMDDNMSNTL ″+++″ 84 FLQELQLEHA ″+++″ 85 GLAPAEVVVATVA ″+++″ 86 GLATIRAYL ″+++″ 87 GLFARIIMI ″++″ 88 GLFDNRSGLPEA ″+++″ 89 GLTALHVAV ″+++″ 90 HLDEVFLEL ″+++″ 91 HLSSTTAQV ″++″ 92 KLLFEIASA ″+++″ 93 KLLGSLQLL ″++++″ 94 LLAGQATTAYF ″+++″ 95 LLFDLIPVVSV ″+++″ 96 LLLNENESLFL ″+++″ 97 LLNFSPGNL ″+″ 98 MLQDGIARL ″+++″ 99 QLYDGATALFL ″++″ 100 RLIRTIAAI ″+++″ 101 SLDQSTWNV ″++++″ 102 SLFAAISGMIL ″+++″ 103 SLQDHLEKV ″+++″ 104 VLLGLPLLV ″+++″ 105 VLTPVILQV ″+++″ 106 VLYELLQYI ″++++″ 107 VQAVSIPEV ″+++″ 108 YLAPENGYLM ″+++″ 109 YLFQFSAAL ″+++″ 110 YQYPFVLGL ″++++″ 111 YLLDTLLSL ″+++″ 112 FLAILPEEV ″+++″ 113 FVIDSFEEL ″+++″ 114 GLSDISPST ″++″ 115 LLIDIIHFL ″++++″ 116 SLLDNLLTI ″+++″ 117 VLATILAQL ″++++″ 118 VLDGMIYAI ″+++″ 119 ELCDIILRV ″+++″ 120 VLLGTTWAL ″+++″ 121 YLTGYNFTL ″+++″ 122 AISEAQESV ″++″ 123 ALLSAFVQL ″+++″ 124 FLGVVVPTV ″+++″ 125 FVAPPTAAV ″+++″ 127 HLMEENMIVYV ″+++″ 128 KLFDASPTFFA ″+++″ 129 SLFEASQQL ″+++″ 130 VIFSYVLGV ″+++″ 131 VLIEETDQL ″++″ 132 VLQDQVDEL ″++″ 133 ALEELTGFREL ″++″ 134 ALGRLGILSV ″+++″ 135 ALTGLQFQL ″+++″ 136 FIFGIVHLL ″+++″ 137 FIQQERFFL ″+++″ 138 NLINNIFEL ″++++″ 139 FLASPLVAI ″++++″ 140 FLFEDFVEV ″+++″ 141 FLGELTLQL ″+++″ 142 FLYEDSKSVRL ″+++″ 143 TLHAVDVTL ″+++″ 144 GLITQVDKL ″+++″ 145 GLLHEVVSL ″+++″ 146 GLLQQPPAL ″+++″ 147 GLSEYQRNFL ″+++″ 148 ICAGHVPGV ″+++″ 149 ILNPVTTKL ″+++″ 150 ILSEKEYKL ″+++″ 151 ILVKQSPML ″+++″ 152 KIMYTLVSV ″++″ 153 KLLKGIYAI ″+++″ 154 KLMNIQQQL ″+++″ 155 KLMTSLVKV ″+++″ 156 KMLEDDLKL ″+++″ 157 KVLEFLAKV ″+++″ 158 KVQDVLHQV ″+++″ 159 LLLSDSGFYL ″+++″ 160 LLPPPSPAA ″+++″ 161 NLMLELETV ″+++″ 162 RLADLKVSI ″++++″ 163 SIFDAVLKGV ″++++″ 164 SLFDGAVISTV ″+++″ 165 KLLEEIEFL ″+++″ 167 SLFSITKSV ″+++″ 168 SLLSPLLSV ″+++″ 169 SSLEENLLHQV ″+++″ 171 TLLDVISAL ″++++″ 172 TLQDSLEFI ″++++″ 173 VILDSVASV ″++++″ 174 VLVEITDVDFAA ″++++″ 175 VMESILLRL ″+++″ 176 YLHIYESQL ″+++″ 177 YLYEAEEATTL ″+++″ 178 YVLQGEFFL ″+++″ 179 FVDTNLYFL ″+++″ 180 GILQLVESV ″+++″ 181 LLFDQNDKV ″+++″ 182 LLPPPPPVA ″++++″ 183 VLFETVLTI ″+++″ 184 AVLGTSWQL ″+++″ 185 FIAQLNNVEL ″+″ 186 FLDVSRDFV ″+++″ 187 FLNSFVFKM ″++″ 188 GLEDEMYEV ″++″ 189 SLSHLVPAL ″+++″ 190 GLIELVDQL ″+++″ 191 GLSDISAQV ″+++″ 192 GMAAEVPKV ″++″ 193 SLADSMPSL ″++″ 194 SLAPFDREPFTL ″++″ 195 ALIPDLNQI ″+++″ 197 YLLTDNVVKL ″++″ 198 GLLSAVSSV ″+++″ 199 SLNSTTWKV ″+++″ 200 YLLDFEDRL ″++++″ 201 YLNISQVNV ″+++″ 202 ALAAGGYDV ″++″ 203 ILDTIFHKV ″+++″ 204 RLCDIVVNV ″++″ 205 TLFYESPHL ″+++″ 206 SAVSGQWEV ″++″ 207 GLVGLLEQA ″++++″ 208 FLAVSLPLL ″+++″ 209 FLLDTISGL ″+++″ 210 FLAEQFEFL ″+++″ 211 FIDDLFAFV ″+++″ 212 FLIGQGAHV ″+++″ 213 YINEDEYEV ″++″ 214 FLFDGSMSL ″+++″ 215 QLFEEEIEL ″++″ 216 KVVSNLPAI ″++″ 217 AQFGAVLEV ″+++″ 218 ALDQFLEGI ″+++″ 219 ALLELENSV ″++″ 220 FLAEAPTAL ″++″ 221 FLAPDNSLLLA ″++++″ 222 FLIETGTLL ″+++″ 223 FLQDIPDGLFL ″++″ 224 FLSPLLPLL ″++″ 226 GVIDPVPEV ″++″ 227 IIAEGIPEA ″++″ 228 IIAEYLSYV ″++″ 229 ILSPWGAEV ″++++″ 230 IMDDDSYGV ″++″ 231 IVMGAIPSV ″+++″ 232 KVMEGTVAA ″++″ 233 MLEVHIPSV ″+++″ 234 NLQRTVVTV ″++″ 235 SLDVYELFL ″+++″ 236 SLFDGFFLTA ″++++″ 237 YLDRLIPQA ″+++″ 238 YQYGAVVTL ″+++″ 239 VLIDDTVLL ″+++″ 240 ALVPTPALFYL ″+++″ 241 FIPDFIPAV ″++″ 242 GILDFZVFL ″++++″ 243 GLPDLDIYL ″++++″ 244 ILEPFLPAV ″+++″ 245 KLIQLPVVYV ″+++″ 246 KLPVPLESV ″+++″ 247 KVLEMETTV ″+++″ 248 NLLEQFILL ″+++″ 249 VLLESLVEI ″++++″ 250 VLTNVGAAL ″+++″ 251 VLYELFTYI ″+++″ 252 YLGDLIMAL ″+++″ 253 YSDDDVPSV ″++++″ 254 FLYSETWNI ″++++″ 255 GMWNPNAPVFL ″++++″ 256 ALQETPPQV ″+++″ 257 FLQEWEVYA ″++++″ 258 RIYPFLLMV ″+++″ 259 TVLDGLEFKV ″++++″ 260 RLDEAFDFV ″++++″ 261 FLPETRIMTSV ″++++″ 262 LMGPVVHEV ″++++″ 263 GLMDNEIKV ″+++″ 264 ILTGTPPGV ″+++″ 265 ILWHFVASL ″++++″ 266 QLTEMLPSI ″++++″ 267 SLLETGSDLLL ″+++″ 268 VLFPLPTPL ″++++″ 269 VLQNVAFSV ″++++″ 270 VVVDSDSLAFV ″++++″ 271 YLLDQPVLEQRL ″++++″ 272 KLDHTLSQI ″++++″ 273 AILLPQPPK ″++″ 274 KLLNLISKL ″++++″ 275 KLMDLEDCAL ″++++″ 276 NMISYVVHL ″++″ 277 FLIDLNSTHGTFL ″+++″ 278 FLLFINHRL ″+++″ 279 NLAGENILNPL ″+++″ 280 SLLNHLPYL ″++++″ 281 TLQTVPLTTV ″++++″ 282 YLLEQGAQV ″++++″ 283 ALMPVTPQA ″+++″ 284 KLQEQIHRV ″+++″ 285 SITAVTPLL ″+++″ 286 HLTEDTPKV ″+++″ 287 ILMGHSLYM ″++++″ 288 RLAPEIVSA ″+++″ 289 SLLAANNLL ″++++″ 290 IASPVIAAV ″+++″ 291 KIIDTAGLSEA ″+++″ 292 KLINSQISL ″+++″ 293 GLAMVEAISYV ″++++″ 294 KLYGPEGLELV ″++++″ 295 SLAAVSQQL ″+++″ 296 FILEPLYKI ″++++″ 297 ILQNGLETL ″+++″ 298 ALTDVILCV ″++++″ 299 RLLEEEGVSL ″+++″ 300 IVLERNPEL ″+++″ 301 LQFDGIHVV ″+++″ 302 SLAELDEKISA ″+++″ 303 FVWEASHYL ″++++″ 304 ALIRLDDLFL ″+++″ 305 AMLAQQMQL ″+++″ 306 AQVALVNEV ″+++″ 307 FLLPVAVKL ″+++″ 308 SLLDQIPEM ″+++″ 309 SLSFVSPSL ″+++″ 310 VMAEAPPGV ″+++″ 311 YLHRQVAAV ″+++″ 314 LIDDKGTIKL ″++″

REFERENCE LIST

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