(12) Patent Application Publication (10) Pub. No.: US 2016/0250307 A1 Weinschenk Et Al

US 2016O250307A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0250307 A1 Weinschenk et al. (43) Pub. Date: Sep. 1, 2016

(54) NOVEL PEPTIDES AND COMBINATION OF A 6LX35/7 (2006.01) PEPTDES FOR USE IN IMMUNOTHERAPY CI2N5/0783 (2006.01) AGAINST HEPATOCELLULAR CARCINOMA (HCC) AND OTHER CANCERS (52) U.S. Cl. CPC ...... A61K 39/00II (2013.01); A61 K35/17 (71) Applicant: Immatics Biotechnologies GmbH, (2013.01); C12N5/0636 (2013.01); G0IN Tuebingen (DE) 33/6803 (2013.01); C07K 16/18 (2013.01); (72) Inventors: Toni Weinschenk, Aichwald (DE); CI2N 15/115 (2013.01); C12N 2501/998 Andrea Mahr, Tuebingen (DE); Jens (2013.01); C07K 2317/70 (2013.01); C12N Fritsche, Dusslingen (DE); Phillip 2310/16 (2013.01); A6 IK 2035/124 (2013.01) Mueller, Tuebingen (DE); Anita Wiebe, Pliezhausen (DE); Sara Kutscher, (57) ABSTRACT Tuebingen (DE) The present invention relates to peptides, proteins, nucleic (21) Appl. No.: 14/975,952 acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy (22) Filed: Dec. 21, 2015 of cancer. The present invention furthermore relates to tumor Related U.S. Application Data associated T-cell peptide epitopes, alone or in combination (60) Provisional application No. 62/096,165, filed on Dec. with other tumor-associated peptides that can for example 23, 2014. serve as active pharmaceutical ingredients of vaccine com positions that stimulate anti-tumor immune responses, or to (30) Foreign Application Priority Data stimulate T cells ex vivo and transfer into patients. Peptides Dec. 23, 2014 (GB) ...... 1423016.3 bound to molecules of the major histocompatibility complex Jan. 21, 2015 (GB) ...... 15O1017.6 (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. In par Publication Classification ticular, the present invention relates to several novel peptide sequences and their variants derived from HLA class I and (51) Int. Cl. class II molecules of human tumor cells that can be used in A6 IK39/00 (2006.01) CI2N 15/I 15 (2006.01) vaccine compositions for eliciting anti-tumor immune GOIN33/68 (2006.01) responses or as targets for the development of pharmaceuti C07K 6/8 (2006.01) cally/immunologically active compounds and cells. Patent Application Publication Sep. 1, 2016 Sheet 1 of 21 US 2016/0250307 A1

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NOVEL PEPTDES AND COMBINATION OF diabetes. As a result, healthy liver tissue gets replaced by scar PEPTIDES FOR USE IN IMMUNOTHERAPY tissue, which increases the risk of cancer development. AGAINST HEPATOCELLULAR CARCINOMA 0009 Disease management depends on the tumor stage at (HCC) AND OTHER CANCERS the time of diagnosis and the overall condition of the liver. If possible, parts of the liver (partial hepatectomy) or the whole CROSS REFERENCE TO RELATED organ (liver resection) is removed by Surgery. Especially APPLICATIONS patients with Small or completely resectable tumors are quali fied to receive a liver transplant. 0001. This application claims priority to U.S. 62/096,165, 0010. If surgery is not a treatment option, different other filed Dec. 23, 2014, GB 1423016.3, filed Dec. 23, 2014 and therapies are available at hand. For tumor ablation, a probe is GB 1501 017.6, filed Jan. 21, 2015, the content of which are injected into the liver and the tumor is destroyed by radio or incorporated herein by reference in their entireties. microwaves or cryotherapy. In embolization procedures, the blood supply of the tumor is blocked by mechanical or chemi BACKGROUND cal means. High energy radio waves can be used to destroy the 0002 1. Field of the Invention tumor in radiation therapy. 0003. The present invention relates to peptides, proteins, 0011 Chemotherapy against HCC includes combinations nucleic acids and cells for use in immunotherapeutic meth of doxorubicin, 5-fluorouracil and cisplatin for systemic ods. In particular, the present invention relates to the immu therapy and doxorubicin, floxuridine and mitomycin C for notherapy of cancer. The present invention furthermore hepatic artery infusions. However, most HCC show a high relates to tumor-associated T-cell peptide epitopes, alone or in resistance to chemotherapeutics (Enguita-German and combination with other tumor-associated peptides that can Fortes, 2014). for example serve as active pharmaceutical ingredients of 0012. Therapeutic options in advanced non-resectable vaccine compositions that stimulate anti-tumor immune HCC are limited to Sorafenib, a multi-tyrosine kinase inhibi responses, or to stimulate T cells ex vivo and transfer into tor (Changet al., 2007: Wilhelmet al., 2004). Sorafenib is the patients. Peptides bound to molecules of the major histocom only systemic drug confirmed to increase Survival by about 3 patibility complex (MHC), or peptides as such, can also be months and currently represents the only experimental treat targets of antibodies, soluble T-cell receptors, and other bind ment option for such patients (Chapiro et al., 2014; Llovet et ing molecules. In particular, the present invention relates to al., 2008). several novel peptide sequences and their variants derived 0013 Lately, a limited number of immunotherapy trials from HLA class I and class II molecules of human tumor cells for HCC have been conducted. Cytokines have been used to that can be used in vaccine compositions for eliciting anti activate Subsets of immune cells and/or increase the tumor tumor immune responses or as targets for the development of immunogenicity (Reinisch et al., 2002; Sangro et al., 2004). pharmaceutically/immunologically active compounds and Other trials have focused on the infusion of Tumor-infiltrating cells. lymphocytes or activated peripheral blood lymphocytes (Shi 0004 2. Description of Related Art et al., 2004a: Takayama et al., 1991; Takayama et al., 2000). 0005 Hepatocellular carcinoma (HCC) is one of the most 0014 So far, a small number of therapeutic vaccination common tumors in the world and accounts for about 6% of all trials have been executed. Butterfield et al. conducted two new cancer cases diagnosed worldwide. In 2012 about 782, trials using peptides derived from alpha-fetoprotein (AFP) as 000 new cases of HCC occurred in the world, making it the a vaccine or DCs loaded with AFP peptides ex vivo (Butter fifth most common cancer in men (554,000 cases) and the field et al., 2003: Butterfield et al., 2006). In two different ninth in women (228,000 cases) (http://globocaniarc.fr). studies, autologous dendritic cells (DCs) were pulsed ex vivo HCC is the most common primary liver malignancy account with autologous tumor lysate (Lee et al., 2005) or lysate of the ing for over 80% of all adult primary liver cancers. hepatoblastoma cell line HepG2 (Palmer et al., 2009). So far, 0006. The distribution of HCC varies geographically, and vaccination trials have only shown limited improvements in rates of incidence depend on gender. The age-standardized clinical outcomes. incidence rate (ASR) of HCC in men is highest in Eastern Asia (31.9) and South-Eastern Asia (22.2), intermediate in SUMMARY Southern Europe (9.5) and Northern America (9.3) and lowest 0015. In a first aspect of the present invention, the present in Northern Europe (4.6) and South-Central Asia (3.7). Inci invention relates to a peptide comprising an amino acid dent rates of HCC in women are lower than male ASRs. The sequence selected from the group consisting of SEQID NO: highest ASR in women occurs in Eastern Asia (10.2) and 1 to SEQID NO:300 or a variant sequence thereof which is Western Africa (8.1), the lowest in Northern Europe (1.9) and at least 80%, preferably at least 90%, homologous (preferably Micronesia (1.6). at least 80% or at least 90% identical) to SEQ ID NO: 1 to 0007. The overall prognosis for patients with HCC is poor. SEQID NO:300, wherein said variant binds to MHC and/or The 5-year relative survival rate (5Y-RSR) from HCC is about induces T cells cross-reacting with said peptide, or a pharma 15%, depending on the stage at the time of diagnosis. For ceutical acceptable salt thereof, wherein said peptide is not localized HCC, where the cancer is still confined to the liver, the underlying full-length polypeptide. the 5Y-RSR is about 28%. For regional and distant HCC, 0016. The present invention further relates to a peptide of were the cancer has grown into nearby or distant organs, the present invention comprising a sequence that is selected 5Y-RSRs are 7% and 2%, respectively. from the group consisting of SEQID NO: 1 to SEQID NO: 0008. The incidence of HCC is related to several risk fac 300 or a variant thereof, which is at least 80%, preferably at tors, cirrhosis being the most important one. Cirrhosis often least 88%, homologous (preferably at least 80% or at least occurs alongside alcohol abuse or HBV or HCV infections, 88% identical) to SEQID NO: 1 to SEQID NO:300, wherein but can also be caused by metabolic diseases like type II said peptide or variant thereof has an overall length of US 2016/0250307 A1 Sep. 1, 2016

between 8 and 100, preferably between 8 and 30, and most TABLE 1. preferred of between 8 and 14 amino acids. HLA-A*02 peptides according to the present invention-S = phospho serine BRIEF DESCRIPTION OF THE DRAWINGS SEQ ID Official Gene 0017. The patent or application file contains at least one No. Sequence GeneID (S) Symbol (s) drawing executed in color. Copies of this patent or patent 1. WMAPFTMTI 338 APOB application publication with color drawing(s) will be pro vided by the Office upon request and payment of the neces 2 KLOAGTVFW 1084 O ALDH1LI1 sary fee. 3 ILDDNMOKL 796.11 ACSS3 0018 FIGS. 1A through 1M show the over-presentation of various peptides in normal tissues (dark gray) and HCC (light 4. KLODFSDQL 338 APOB gray). FIG. 1A: APOB, Peptide: ALVDTLKFV (A*02) (SEQ 5 ALVEOGFTV 338 APOB ID NO:7). FIG. 1B: ALDH1 L1, Peptide: KLQAGTVFV (A*02) (SEQ. ID NO:2). FIG. 1C: C8B, Peptide: 6 KLSPTWWGL 8313 AXIN2 AYLLQPSQF (A*24) (SEQID NO:200). FIG.1D: FIG.1D) 7 ALWDTLKFW 338 APOB RAD23B Peptide: KIDEKNFVV (SEQID NO:63). FIG.1E: RAD23B Peptide: KIDEKNFVV (SEQID NO:63). FIG.1F: 8 KLLEEATISW 54808 DYM RFNG Peptide: RLPPDTLLQQV (SEQ ID NO:92). FIG. 1G: RFNG Peptide: RLPPDTLLQQV (SEQ ID NO:92). 9 ALANOKLYSW 231.95 MDN1 FIG. 1H) FLVCR1 Peptide: SVWFGPKEV (SEQ ID O SLLEEFDFHW 8615 USO1 NO:104). FIG. 1I: FLVCR1 Peptide: SVWFGPKEV (SEQ ID NO:104). FIG. 1J: IKBKAP Peptide: LLFPHPVNQV 1. SLSOELVGW 241.49 ZNF318 (SEQ ID NO:156). FIG. 1K: IKBKAP Peptide: LLFPH 2 FLAELAYDL 2719 GPC3 PVNQV (SEQID NO: 156). FIG.1L: NKD1 Peptide: FLDT PIAKV (SEQ ID NO:47). FIG. 1M: NKD1 Peptide: FLDT 3 GLIDTETAMKAW 3290 HSD11B1 PIAKV (SEQID NO:47). 4. ALADLTGTWW 233.85 NCSTN 0019 FIGS. 2A through 2F show exemplary expression profiles (relative expression compared to normal kidney) of 5 LYGHTWTV 3.47734 SLC35B2 Source genes of the present invention that are highly over expressed or exclusively expressed in HCC in a panel of 6 SLLGGNIRL 2181 ACSL3 normal tissues (dark gray) and 12 HCC samples (gray). FIG. 7 RWAS kPTSGV 866 O IRS2 2A: APOB: FIG. 2B: AMACR: FIG. 2C: ALDH1L1 FIG. 2D: FGG: FIG. 2E: C8B; FIG. 2F: HSD17B6. 8 ALYGKTEWW 57513 CASKIN2 0020 FIG.3 shows exemplary flow cytometry results after 9 FLEETKATW 338 APOB peptide-specific multimer staining. 2O KLSNVLQQV 338 APOB 0021 FIG. 4 shows exemplary flow cytometry results after peptide-specific multimer staining. CD8+ T cells were 21 QLIEVSSPITL 338 APOB primed using artificial APCs coated with anti-CD28 mAb and 22 RIAGIRGIQGW 231.67 EFR3A HLAA*24 in complex with IMA-KLHL24-001 (Seq ID No 190) peptide (4A, right panel) or IMAAPOB-006 (4B, right 23 RLYDPASGTISL 23456 ABCB10 panel, Seq ID No 218). Left panels (4A and 4B) show control 24 SLAEEKLOASW 2.194 FASN staining. 25 SLDGKAALTEL 338 APOB

DETAILED DESCRIPTION OF A PREFERRED 26 SLL.HTIYEW 854 Of NKD1 EMBODIMENT 27 TLPDFRLPEI 338 APOB

0022. The following tables show the peptides according to 28 TLODHLNSL 338 APOB the present invention, their respective SEQID NOS, and the prospective source (underlying) genes for these peptides. All 29 YIODEINTI 338 APOB peptides in Table 1 bind to HLA-A*02, peptides in Table 2 3 O YLGEGPRMW 5704 PSMC4 bind to HLA-A*24 alleles. The peptides in Table 3 have been disclosed before inlarge listings as results of high-throughput 31 YQMDIOOEL 338 APOB screenings with high error rates or calculated using algo rithms, but have not been associated with cancer at all before. 32 ALNAWRLLW 93.68 SLC9A3R1 They bind to HLA-A*02. The peptides in Table 4 are addi 33 LLHGHIWEL sf678 GPAM tional peptides that may be useful in combination with the other peptides of the invention. Peptides bind A*02 or, where 34 SLAEGTATW 54 O ATP7 indicated. A*24. The peptides in Table 5 are furthermore 35 SLQESILAQW 23644 EDC4 useful in the diagnosis and/or treatment of various malignan cies that involve an over-expression or over-presentation of 36 ILNWDGLIGW 47 ACLY the respective underlying polypeptide.

US 2016/0250307 A1 Sep. 1, 2016

TABLE 2 - continued HLA-A*24 peptides according to the present invention with SEQ ID numbers - S = phosphoserine

SEO ID No. Sequence GeneID (S) Official Gene Symbol (s)

209 WFHPROELI 1314 COPA

210 AYPAIRYLL 7818 DAP3

211 IYIPSYFDF 27 O42 DIEXF

212 WYGDWISNI 8893 EIF2B5.

213 YYNKVSTVF 8661 EIF3A.

214 IYVTSIEOI 55879 GABRO

215 IYTGNISSF 8836 GGH

216 IYADWGEEF 1OO3O2182, 11052 MIR1279, CPSF6

217 DYIPYWFKL 338 APOB

218 WYOGAIROI 338 APOB

TABLE 3 Additional peptides according to the present invention with no prior known cancer association-S = phosphoserine SEO ID Official No. Sequence GeneID (S) Gene Symbol (s)

219 GWMAGDIYSW 123 PLIN2

22O SLLEKELESW 1819 DRG2

221 ALCEENMRGW 1938 EEF2

222 LTDITKGV 1938 EEF2

223 FLFNTENKLLL 3422 ID11

224 ALASWIKEL 28.981 IFT81

225 KMDPWAYRV 5859 QARS

226 AVLGPLGLQEW 791.78 THTPA

227 ALLKWNOEL 25.813 SAMMSO

228 YLITSWELL 2182 ACSL4

229 KMFESFIESW 5576 PRKAR2A

23 O WLTEFTREW sist Os IPO9

231 RLFNDPWAMW 101.95 ALG3

232 KLAEIWKOW 85.50 MAPKAPK5

233 ALIGKLDAI 5876 RABGGTB

234 YLEPYLKEW 727947, 7381 UQCRB

235 KLFEEIREI 255.394 TCP11L2

236 ALADKELLPSW 84883 AIFM2

237 ALRGEIETW 101.28 LRPPRC

238 AMPPPPPOGV 5.885 RAD21 US 2016/0250307 A1 Sep. 1, 2016

TABLE 3 - continued Additional peptides according to the present invention with no prior known cancer association-S = phospho serine SEO ID Official No. Sequence GeneID (S) Gene Symbol (s)

239 FLIGFIPAKA 5976 UPF1

24 O FLWERPTLLW 79922 MRM1

241 FWLPLLGLHEA 55161 TMEM33

242 GLFAPWHKW 6249 CLIP1.

243 GILDNPELRW 26.263 FBXO22

244 KIAELLENW 91 OO USP10

245 KLGAVFNOV 23 450 SF3B3

246 KLISSYYNW 84928 TMEM2O 9

247 KLLDTMWDTFL 1OO527963, 11243 PMF1-BGLAP PMF1

248 KLNDLIORL 1314 COPA

249 (LLGERWAL 234.75 OPRT

250 NLAEWWERW 26.263 FBXO22

251 RLFADILNDW 64755 C16orf58

252 RTIEYLEEW 3O3O HADHA

253 RVPPPPOSV 64 64 SHC1.

254 RVOEAIAEW st 678 GPAM

255 SLFGODWKAV 26 O36 ZNF451

256 SLFOGWEFHYW 393 O BR

27 SLLEKAGPEL 54.625 PARP14

258 SLMGPWWHEW 5116 PCNT

259 TLITDGMRSW 298.94 CPSF1

26 O TLMDMRLSOV 241.48 PRPF6

261 WLFOEALWHV 21.94 FASN

262 WLPNFLPYNW 10299 MARCH6

263 WLYPSLKEI 50717, 5824 DCAF8 PEX19

264 VMODPEFLOSV 266971, 571O PIPSL, PSMD4

265 WLIEDGKWWTW 10726 NUDC

266 SLLESNKDLLL 652O SLC3A2

267 ALNENINOV 80025 PANK2

268 KLYOEVEIASW 5976 UPF1

269 YLMEGSYNKW 571.4 PSMD8

27 O SVLDOKILL 987 URB1

271 LILDKLILL 85440 DOCK7

272 QOLDSKFLEQV 6772 STAT1

273 AILETAPKEW 6238 RRBP1 US 2016/0250307 A1 Sep. 1, 2016

TABLE 3 - continued Additional peptides according to the present invention with no prior known cancer association-S = phospho serine SEO ID Official No. Sequence GeneID (S) Gene Symbol (s)

274 ALAEALKEW 55164 SHO1

27s ALIEGAGILL 104.4 O TIMM17A

276 ALLEADWNIKL 6729 SRP54

277 ALLEENSTPOL 83.933 HDAC10

278 ALTSWWWTL 1021 CDK6

279 ALWWTGMHTI 5.1479 ANKFY1

28O ATLNIIHSW 51542 WPS54

281 GLLAGDRLWEW 93.68 SLC9A3R1

282 GOFPSYLETV 54919 HEATR2

283 ILSGIGVSOV 3703 STT3A

284 KLDAFWEGW 528 ATP6V1C1

285 KLLDLSDSTSW 6093 ROCK1

286 KWLDKWFRA 375,056 MIA3

287 LIGEFLEKW 8731 RNMT

288 LLDDSLWSI 25824 PRDX5

289 LLLEEGGLWOW 733 UFD1L

290 NLIDLDDLYW st 187 THOC2

291 OLIDYEROL 11072 DUSP14

292 RIPAYEWTV 74. Of WARS

293 FLASESLIKQI 4736 RPL1 OA

294 RLIDLHTNW 23256 SCFD1

295 SLFSSPPEI 252983 STXBP4.

296 SLLSGRISTL 51133,92799 KCTD3, SHKBP1

297 TLFYSLREW 8O233 C17orf7 O

298 TMAKESSIIGW 1429 CRYZ

299 ALLRVTPFI 4 O1505 TOMMs

3 OO TLAQQPTAV 48O2 NYC

TABLE 4 Peptides useful for e.g. personalized cancer therapies-S* = 32 phospho serine

SEO ID No. Sequence GeneID (S) Official Gene Symbol (s) 3 O1 WLADFGARW 114899, 236OO C1GTNF3, AMACR

3 O2 KIOEILTOW 10643 IGF2BP3

3O3 GWYDGEEHSW 41.13 MAGEB2

US 2016/0250307 A1 Sep. 1, 2016

TABLE 4 - continued Peptides useful for e.g. personalized cancer therapies-S* = 32 phospho serine

SEO ID No. Sequence GeneID (s Official Gene Symbol (s)

34 O KVFDEVIEW 89.08 GYG2

341 YLAIGIHEL 3O34 HAL

342 AMSSKFFLW 7474 WNTSA

343 LLLPDYYLW 27O44 SND1

344 WYISSLALL 1O213 PSMD14 (A24)

345 SYNPLWLRI 259266 ASPM (A24)

346 LYOILOGIWF 983 CDK1 (A24)

347 ALNPADITW 5.1497 TH1L

348 AYKPGALTF 84883 AIFM2

0023 The present invention furthermore generally relates 0025. As shown in the following tables 5A and B, many of to the peptides according to the present invention for use in the peptides according to the present invention can also be the treatment of proliferative diseases, such as, for example, used in the immunotherapy of other indications. The tables show, for selected peptides on which additional tumor types pancreatic cancer, colon or rectal cancer, kidney cancer, brain they were found showing over-presentation (including spe cancer, and/or leukemias. cific presentation) on more than 5% of the measured tumor 0024 Particularly preferred are the peptides—alone or in samples, or presentation on more than 5% of the measured combination—according to the present invention selected tumor samples with a ratio of geometric means tumor VS from the group consisting of SEQID NO: 1 to SEQID NO: normal tissues being larger than 3. Over-presentation is defined as higher presentation on the tumor sample as com 300. More preferred are the peptides—alone or in combina pared to the normal sample with highest presentation. Normal tion—selected from the group consisting of SEQID NO: 1 to tissues against which over-presentation was tested were: adi SEQID NO: 124 (see Table 1), preferably for A*02 binding, pose tissue, adrenal gland, blood cells, blood vessel, bone and from the group consisting of SEQID NO: 187 to SEQID marrow, brain, cartilage, esophagus, eye, gallbladder, heart, NO: 218 (see Table 2) preferably for A*24 binding, and their kidney, large intestine, liver, lung, lymph node, nerve, pan uses in the immunotherapy of HCC, brain cancer, kidney creas, parathyroid gland, peritoneum, pituitary, pleura, Sali cancer, pancreatic cancer, colon or rectal cancer or leukemia, vary gland, skeletal muscle, skin, Small intestine, spleen, and preferably HCC. stomach, thyroid gland, trachea, ureter, urinary bladder. TABL E 5A Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases-S* = phosphoserine SEO ID Other relevant No. Sequence organs/diseases

1. WMAPETMTI Pancreas

6 KLSPTWWGL Colon Rectum

1O SLLEEFDFHW Kidney

14 ALADLTGTWW Kidney, Brain, Pancreas

15 LLYGHTWTW Kidney, Brain, Colon, Rectum, Pancreas

16 SLIGGNIRL Brain, Colon, Rectum

17 RWAS kPTSGV Brain

22 RIAGIRGIQGV Kidney, Colon, Rectum US 2016/0250307 A1 Sep. 1, 2016 11

TABLE 5A- continued Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases-S* = phosphoserine SEO ID Other relevant No. Sequence organs/diseases

26 SLLHTIYEW Colon, Rectum

3O YLGEGPRMW Colon, Rectum, CLL,

34 SLAEGTATW Colon, Rectum 36 ILNWDGLIGW Kidney, Brain, Colon, Rectum 39 ALDPKANFST Kidney, Brain

41 ALLELDEPLWL Pancreas

43 ALLGWWTSW Pancreas

47 FLDTPIAKW Brain, Colon Rectum

51 GLAEELWRA Brain

54 GLLDPNVKSIFV Kidney, Brain

55 GLYGRTIEL Kidney

58 ILADLNLSW Pancreas

59 ILADTFIGW Colon, Rectum, Pancreas

60 ILSPLSWAL Kidney, Pancreas

65 KLFSGDELLEV Brain, Colon Rectum

69 KLLDEWTYLEA Colon, Rectum

70 KLLDLETERILL Colon, Rectum

72 KLSEAWTSW Kidney

77 LLHEENFSW Kidney, Colon, Rectum

8O LLYEGKLTL Colon, Rectum 81 NLASFIEQVAW Kidney, Colon, Rectum, Pancreas 88 RLIDRIKTW Brain, Colon Rectum

90 RLLDWLAPLW Kidney

96 SLLEEPNWIRV Kidney

O1 SLWEGGWRGW Brain

12 WLGEWKWGW Kidney

16 WWIPAISAW Kidney

19 YLDKNLTWSW Kidney

21 YLITGNLEKL Kidney, Colon, Rectum, Pancreas 23 YLWDLDHGFAGV Brain, Colon Rectum

25 ALYGRLEWW Brain, Colon Rectum

27 WLIGSNHSL Colon, Rectum 33 ALLEMDARL Kidney, Brain, Colon, Rectum

34 ALLETNPYLL Brain

35 ALLGKIEKW Brain, Pancreas US 2016/0250307 A1 Sep. 1, 2016 12

37 ALPTWLWGW Kidney, Brain, Colon, Rectum

38 ALSQWTLLL Kidney

39 ALSSKPAEW Colon, Rectum, Pancreas

41 AMGEKSFSW Brain

44 FIOLITGV Pancreas

47 GLAPGGLAWW Brain

48 GLFAPLWFL Kidney

61 RLLDEQFAW Brain

66 SILDIWTKW Brain

69 SLFEWFHPL Kidney, Brain, Colon, Rectum

70 SLHINGVIQL Kidney 72 SLLNFLOHL Kidney, Colon, Rectum, CLL,

73 SLTSEIHFL CIL

76 TLGOIWDV Brain, Colon, Rectum, Pancreas

77 WLDEPYEKW Kidney

79 YIHNILYEW Brain

81 YLLEKFWAW Colon, Rectum

84 WLDGGOIVTV Brain 86 WLLAQIIQW Kidney, Brain, Colon, Rectum 87 SYPTEFPRF Kidney, Brain 89 AFSPDSHYLLF Kidney, Brain

91 KYPDIISRI Brain

92 SYITKPEKW Kidney, Brain

93 IYPGAFWDL Brain

94 QYASRFWOL Brain

95 RYAPPPSFSEF Brain

96 AYLKWISOI Brain 97 RWPKKSAEF Kidney, Brain

98 LYWSHPRKF Kidney

99 KFVTVOATF Brain 2O3 YYGILOEKI Kidney, Brain 2O6 KWPETPLLL Kidney, Brain

2O8 SYNPAENAWLL Brain

214 IYVTSIEOI Brain

219 GWMAGDIYSW Kidney US 2016/0250307 A1 Sep. 1, 2016 13

22O SLLEKELESW Brain

221 ALCEENMRGW Kidney, Brain, Colon, Rectum

223 FLFNTENKLLL Colon, Rectum

224 ALASWIKEL Brain

229 KMFESFIESW Kidney, Brain, Colon, Rectum 23 O WLTEFTREW Kidney, Brain, Colon, Rectum

231 RLFNDPWAMW Brain, Colon Rectum

232 KLAEIWKOW Colon, Rectum 233 ALIGKLDAI Kidney, Colon, Rectum 234 YLEPYLKEW Kidney, Brain, Colon, Rectum 236 ALADKELLPSV Kidney, Colon, Rectum, Pancreas

237 ALRGEIETW Colon, Rectum

238 AMPPPPPOGV Brain, Colon Rectum

239 FLIGFIPAKA Brain

24 O FLWERPTLLW CIL

244 KIAELLENW Brain, Colon Rectum

245 KLGAVFNOV Brain

247 KLLDTMWDTFL Colon, Rectum

248 KLNDLIORL Pancreas

249 (LLGERWAL Colon, Rectum

250 NLAEWWERW Brain, Colon, Rectum, CLL

251 RLFADILNDW Brain, Colon Rectum 255 SLFGODWKAV Kidney, Brain, Colon, Rectum

258 SLMGPWWHEW Brain

259 TLITDGMRSW Brain

26 O TLMDMRLSOV Kidney, Brain, Colon, Rectum

261 WLFOEALWHV Colon, Rectum

266 SLLESNKDLLL Colon, Rectum

268 KLYOEVEIASW Brain

269 YLMEGSYNKW Brain, Colon Rectum

27 O SVLDOKILL Kidney, Brain

271 LILDKLILL Brain, Colon Rectum 272 QQLDSKFLEQV Kidney, Brain

274 ALAEALKEW Colon, Rectum

27s ALIEGAGILL Kidney, Colon, Rectum, Pancreas US 2016/0250307 A1 Sep. 1, 2016 14

TABLE 5A- Continued Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases-S* = phosphoserine SEO ID Other relevant No. Sequence organs/diseases

276 ALLEADWNIKL Pancreas

277 ALLEENSTPOL Kidney

278 ALTSWWWTL Kidney, Brain

279 ALWTGMHTI Kidney, Brain

281 GLLAGDRLWEW Kidney

282 GOFPSYLETV Kidney, Brain, Colon, Rectum

283 ILSGIGVSOV Pancreas

285 KLLDLSDSTSW Kidney, Colon, Rectum

286 KWLDKWFRA Pancreas

287 LIGEFLEKW CIL

288 LLDDSLWSI Pancreas

289 LLLEEGGLWOW Kidney, Colon, Rectum, Pancreas

290 NLIDLDDLYW Brain, Colon, Rectum, Pancreas

291 OLIDYEROL Kidney, Colon, Rectum, Pancreas

292 RIPAYEWTV Kidney

293 FLASESLIKQI Brain, Colon Rectum

295 SLFSSPPEI Kidney, Brain

296 SLLSGRISTL Kidney

297 TLFYSLREW Kidney, Brain, Colon, Rectum

299 ALLRVTPFI CIL

3 OO TLAQQPTAV Pancreas

WLADFGARW Kidney, Colon, Rectum KIOEILTOW Kidney, Brain, Colon, Rectum, Pancreas, CLL

SLIDOFFGV Brain, Colon, Rectum, Pancreas

3. OS GWLENIFGW Kidney, Brain

KLWEFDFLGA Brain, Colon Rectum

ALLRTWWSW Kidney, Pancreas

GLIEIISNA. Brain

310 Brain, Colon Rectum

311 FLIPIYHOV Kidney, Brain

312 RLGIKPESW Brain

313 LTAPPEALLMW Kidney, Brain, Colon, Rectum, Pancreas

315 KWLDGSPIEW Kidney

316 LLREKWEFL Kidney, Brain, Colon, Rectum, Pancreas US 2016/0250307 A1 Sep. 1, 2016 15

TABLE 5A- continued

Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases-S* = phospho serine

SEO ID Other relevant No. Sequence organs/diseases

317 KLPEKWESW Brain, Colon, Rectum, Pancreas

319 KLFNEFIQL Kidney, Brain, Colon, Rectum

321 GWIAEILRGW Kidney, Brain

324 RLFETKITOW Kidney

3.25 RLSEAIWTW Brain, Pancreas

326 ALSDGWHKI Pancreas

327 GLNEEIARW Brain, Colon Rectum

328 RLEEDDGDWAM Kidney, Brain, Colon, Rectum

329 SLIEDLILL Kidney, Brain, Colon, Rectum, Pancreas

330 SMSADWPLW Brain, Colon Rectum

331 SLLAQNTSWLL Brain, Colon, Rectum, Pancreas

332 AMLAWLHTW Brain, Colon Rectum

333 GLAEDIDKGEW Kidney, Brain

334 SILTIEDGIFEW Kidney, Brain, Colon, Rectum, Pancreas, CLL,

335 SLLPWDIROYL Kidney, CLL,

336 YLPTFELTV Kidney, Brain

337 TLLAAEFLKOW Brain

338 KLFDSDPITVTV Brain

339 RLISKFDTW Brain

34 O KVFDEVIEW Brain

342 AMSSKFFLW Brain, Colon, Rectum, Pancreas

343 LLLPDYYLW Brain, Pancreas

344 WYISSLALL Brain (A24)

345 SYNPLWLRI Brain (A24)

346 LYOILOGIWF Kidney (A24)

347 ALNPADITW Brain US 2016/0250307 A1 Sep. 1, 2016 16

TABL E Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases S* = phosphoserine

SEO ID NO. Sequence Additional Entities 189 AFSPDSHYLLF NSCLC, Pro

273 AILETAPKEW Esophageal Cancer

236 ALADKELLPSW NSCLC, SCLC, GC, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer, NHL

14 ALADLTGTW NSCLC, SCLC, BRCA, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, PC

38 ALADWWHEA BRCA, OC

274 ALAEALKEW BRCA, MCC, Melanoma, OC, Uterine Cancer, AML ALANOKLYSW NSCLC, CRC, MCC, OC, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer, PC

224 ALASWIKEL SCLC, PC, Melanoma

221 ALCEENMRGW NSCLC, SCLC, MCC, Melanoma

131 ALDSGAFOSV CRC Melanoma, Gallbladder Cancer, Bille Duct Cancer

185 ALFPALRPGGF Gallbladder Cancer, Bile Duct Cancer QA

ALIEGAGILL SCLC, MCC, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, NHL 276 ALLEADWNIKL Gallbladder Cancer, Bile Duct Cancer, OC

277 ALLEENSTPOL SCLC, CLL, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, NHL

133 ALLEMDARL NSCLC, SCLC, BRCA, MCC, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML NHL

134 ALLETNPYLL Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer

135 ALLGKIEKW NSCLC, SCLC, BRCA, OC, Gallbladder Cancer, Bile Duct Cancer

233 ALIGKLDAI CLL, OC, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL

43 ALLGWWTSW SCLC, Brain Cancer, CLL, BRCA, PC

227 ALLKWNOEL Melanoma, Uterine Cancer

136 ALLNOHYOV BRCA, OC

299 ALLRVTPFI NHL, OC

32 ALNAWRLLW SCLC

267 ALNENINOV SCLC, Brain Cancer, MCC, Melanoma, Esophageal Cancer, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer

137 ALPTWLWGW NSCLC, GC, BRCA, Melanoma, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, PC

45 SCLC, Brain Cancer US 2016/0250307 A1 Sep. 1, 2016 17

TABLE 5B- continued Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases S* = phosphoserine

SEO ID NO. Sequence Additional Entities 237 ALRGEIETW SCLC, BRCA, OC, Esophageal Cancer, Urinary bladder cancer, NHL 139 ALSSKPAEW Pro, OC, Uterine Cancer 278 ALTSWWWTL NSCLC, SCLC, GC, Esophageal Cancer, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL 279 ALWTGMHTI Esophageal Cancer, Urinary bladder cancer, PC 125 ALYGRLEWW BRCA, MCC, Melanoma, OC, Urinary bladder cancer, Uterine Cancer

141 AMGEKSFSW Melanoma

238 AMPPPPPOGV NSCLC, SCLC, BRCA, MCC, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 342 AMSSKFFLW NSCLC, GC PrO BRCA, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, PC 28O ATLNIIHSW Urinary bladder cancer

226 AVLGPLGLQEW Pro, Melanoma, OC

2O2 AYGTYRSNF NSCLC

196 AYLKWISOI NSCLC

210 AYPAIRYLL NSCLC, GC 144 FIOLITGV NSCLC, Brain Cancer, Urinary bladder cancer 293 FLASESLIKQI NSCLC, Pro, MCC, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL, PC 47 FLDTPIAKW NSCLC, GC, Esophageal Cancer 223 FLFNTENKLLL Melanoma, Urinary bladder cancer, NHL

145 FLIAEYFEHW SCLC

239 FLIGFIPAKA Urinary bladder cancer, AML, NHL 128 GLAFSLNGW Melanoma, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer

147 GLAPGGLAWW NSCLC, SCLC, PrC, BRCA, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer 148 GLFAPLWFL Esophageal Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 242 GLFAPWHKW Urinary bladder cancer

52 GLFNAELLEA SCLC 281 GLLAGDRLWEW NSCLC, SCLC, OC, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 243 GILDNPELRW Urinary bladder cancer 54 GLLDPNVKSIFVN SCLC, SCLC, OC, Urinary bladder cancer

149 GLISGLDIMEW SCLC US 2016/0250307 A1 Sep. 1, 2016 18

TABLE 5B- continued Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases S* = phosphoserine

SEO ID NO. Sequence Additional Entities

150 GLSNLGIKSI Urinary bladder cancer, NHL

55 GLYGRTIEL SCLC

282 GOFPSYLETV NSCLC, CLL, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, NHL 219 GWMAGDIYSW SCLC, Gallbladder Cancer, Bile Duct Cancer, PC 151 HLAKWTAEW SCLC, OC, Urinary bladder cancer

st HLTEAIOYV NHL

58 LADLNLSW BRCA 59 LADTFIGW NSCLC, SCLC, GC, OC, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 36 LNWDGLIGW NSCLC, SCLC, PrO, Melanoma, OC, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL, PC 283 LSGIGWSOW NSCLC, Pro, BRCA, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL, PC

216 YADWGEEF NSCLC, GC Pro

211 YIPSYFDF NSCLC, Brain Cancer, GC

2O7 YLERFPIF NSCLC, GC

193 YPGAFWDL NSCLC

214 YVTSIEOI NSCLC, RCC

244 KIAELLENW NSCLC, SCLC, PrC, CLL, BRCA, Melanoma, OC, Uterine Cancer, AML NHL

62 KIAGTNAEW BRCA

63 KIDEKNFVW SCLC, Brain Cancer, Urinary bladder cancer, Uterine Cancer 64 KILEETLYW Esophageal Cancer, Urinary bladder cancer, Uterine Cancer

3 O2 KIOEILTOW NSCLC, SCLC, GC, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL, PC 232 KLAEIWKOW NSCLC, SCLC, BRCA, Melanoma, OC, Urinary bladder cancer

284 KLDAFWEGW BRCA, OC

152 KLDNNLDSW BRCA, Melanoma 235 KLFEEIREI NSCLC, CRC, Melanoma, Urinary bladder cancer

245 KLGAVFNOV NSCLC, SCLC, RCC, PrC, BRCA, Melanoma, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer

246 KLISSYYNW OC

69 KLLDEWTYLEA Urinary bladder cancer

70 KLLDLETERILL OC, Uterine Cancer US 2016/0250307 A1 Sep. 1, 2016 19

TABLE 5B- continued Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases S* = phosphoserine

SEO ID NO. Sequence Additional Entities

285 KLLDLSDSTSW SCLC, Uterine Cancer 247 KLLDTMWDTFL NSCLC, SCLC, RCC, Brain Cancer, CLL, BRCA, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, AML, NHL 8 KLLEEATISW SCLC, MCC, Melanoma, Urinary bladder cancer, Uterine Cancer

248 KLNDLIORL GC, Uterine Cancer

2O KLSNVLQQV SCLC

6 KLSPTWGL CLL, OC

154 KLTDHLKYW SCLC 268 KLYOEVEIASW CRC, MCC, Melanoma, Urinary bladder cancer 225 KMDPWAYRV CRC, Pro, BRCA, Urinary bladder cancer, Uterine Cancer 229 KMFESFIESW NSCLC, SCLC, PrO, OC, Urinary bladder cancer 286 KVLDKWFRA CRC, Gallbladder Cancer, Bile Duct Cancer

2O6 KWPETPLLL GC

191 KYPDIISRI NSCLC, GC 287 IGEFLEKW SCLC, RCC, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, NHL 288 LDDSLWSI Melanoma, Urinary bladder cancer, AML 156 LLFPHPVNOV NSCLC, SCLC, OC, Esophageal Cancer, Urinary bladder cancer 77 LLHEENFSW NSCLC, SCLC, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 78 LLIDDEYKW Esophageal Cancer, Urinary bladder cancer, PC 271 LLDKLILL CLL, Melanoma, OC, Urinary bladder cancer, Uterine Cancer 289 LLLEEGGLWOW NSCLC, SCLC, PrO, Melanoma, OC, Urinary bladder cancer, NHL

249 (LLGERWAL OC

37 LLPLLPPLSP SCLC, PC, MCC Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer

8O LLYEGKLTL OC

15 LYGHTWTV NSCLC, SCLC, BRCA, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, NHL, PC

222 TDITKGV BRCA, NHL

346 LYOILOGIVF NSCLC, Brain Cancer, GC

198 YWSHPRKF NSCLC

250 NLAEWWERW NSCLC, SCLC, PrC, BRCA, MCC, OC, Gallbladder Cancer, Bile Duct Cancer US 2016/0250307 A1 Sep. 1, 2016 20

TABLE 5B- continued Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases S* = phosphoserine

SEO ID NO. Sequence Additional Entities 81 NLASFIEQVAW SCLC, PrC, OC, Uterine Cancer, NHL

290 NLIDLDDLYW SCLC, PrO, MCC, OC, Urinary bladder cancer, Uterine Cancer

291 OLIDYEROL NSCLC, SCLC, Brain Cancer, BRCA, Melanoma, Esophageal Cancer, Urinary bladder cancer, NHL, PC

157 OLLPNLRAW RCC

272 QOLDSKFLEQV MCC, OC, NHL

194 QYASRFWOL NSCLC, GC

22 RIAGIRGIQGW NSCLC, PrC, BRCA, OC, NHL

292 RIPAYEWTV GC, BRCA, Melanoma, NHL

86 RLAAFYSOW AML

251 RLFADILNDW NSCLC, SCLC, PrO, Melanoma, Urinary bladder cancer, Uterine Cancer 231 RLFNDPWAMW NSCLC, SCLC, MCC, Melanoma, OC, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer

294 RLIDLHTNW Esophageal Cancer

88 RLIDRIKTW NSCLC, SCLC, OC, AML NHL

89 RLIEEIKNW SCLC

161 RLLDEQFAW SCLC, BRCA

90 RLLDWLAPLW BRCA

91 RLPDIPLROV NSCLC, CLL, Urinary bladder cancer, NHL 92 RLPPDTLLQQV Urinary bladder cancer

23 RLYDPASGTISL CLL, Melanoma, NHL

164 RMLIKLLEW SCLC, CRC

94 BRCA, OC

252 RTIEYLEEW Melanoma

17 RWAJPTSGW AML

197 NSCLC

195 RYAPPPSFSEF NSCLC

190 RYNEKCFKL NSCLC

95 SICNGWPMW Urinary bladder cancer

166 SILDIWTKW SCLC, CLL, MCC, Melanoma, Urinary bladder cancer, AML

24 SLAEEKLOASW Pro, BRCA, Urinary bladder cancer

169 SLFEWFHPL NSCLC, Gallbladder Cancer, Bille Duct Cancer

255 SLFGODWKAV NSCLC, SCLC, CLL, BRCA, MCC, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL, PC US 2016/0250307 A1 Sep. 1, 2016 21

TABLE 5B- continued Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases S* = phosphoserine

SEO ID NO. Sequence Additional Entities

256 SLFOGWEFHYW CRC, CLL, MCC, NHL

295 SLFSSPPEI NSCLC, SCLC, CRC PrC, BRCA, Melanoma, OC, Urinary bladder cancer, Uterine Cancer 17O SLHINGVIQL NSCLC, Urinary bladder cancer, Uterine Cancer, NHL 331 SLLAQNTSWLL NSCLC, RCC, GC, BRCA, Melanoma, Esophageal Cancer, Urinary bladder cancer 1O SLLEEFDFHW NSCLC, BRCA, OC, Esophageal Cancer, Uterine Cancer

96 SLLEEPNWIRV Melanoma, Gallbladder Cancer, Bile Duct Cancer

27 SLLEKAGPEL CLL, Melanoma, OC 22O SLLEKELESW NSCLC, SCLC, PrO, CLL, BRCA, OC, Esophageal Cancer, Urinary bladder cancer, NHL 266 SLLESNKDLLL SCLC, MCC Uterine Cancer 16 SLLGGNIRL GC, PrO, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, PC 26 SLLHTIYEW Pro, BRCA, Esophageal Cancer, NHL

172 SLLNFLOHL AML

97 SLLPOLIEW SCLC 296 SLLSGRISTL BRCA, Urinary bladder cancer 258 SLMGPWWHEW SCLC, MCC, Melanoma, OC, Urinary bladder cancer, NHL 35 SLQESILAQW NSCLC, SCLC, PrO, MCC, Melanoma, Urinary bladder cancer, AML 99 SLSAFLPSL OC, Esophageal Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 11 SLSOELVGV Brain Cancer, Melanoma, Uterine Cancer, NHL

173 SLTSEIHFL Uterine Cancer, NHL

1 O1 SLWEGGWRGW Melanoma

103 SMGDHLWWA BRCA, Urinary bladder cancer, NHL 330 SMSADWPLW NSCLC, SCLC, BRCA, MCC, Melanoma, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL 129 SOADVIPAV Urinary bladder cancer 27 O SVLDOKILL Melanoma, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 104 SWWFGPKEW SCLC, Melanoma, Urinary bladder cancer, PC

192 SYITKPEKW NSCLC

2O8 SYNPAENAWILL NSCLC

345 SYNPLWLRI NSCLC, RCC, GC

187 SYPTEFPRF NSCLC, Pro US 2016/0250307 A1 Sep. 1, 2016 22

TABLE 5B- continued Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases S* = phosphoserine

SEO ID NO. Sequence Additional Entities 3 OO TLAQQPTAV Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 297 TLFYSLREW BRCA, Uterine Cancer, AML 176 TLGOIWDV NSCLC, GC, Melanoma, Urinary bladder cancer, PC 259 TLITDGMRSW RCC, CRC, OC, Esophageal Cancer, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer, PC 26 O TLMDMRLSOV SCLC, PrC, CLL, OC, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 298 TMAKESSIIGW SCLC, Gallbladder Cancer, Bile Duct Cancer 11O TWGGSEILFEW SCLC, Gallbladder Cancer, Bile Duct Cancer 111 TVMDIDTSGTF SCLC, CRC, CLL, Melanoma, OC, Urinary bladder NW cancer, Gallbladder Cancer, Bile Duct Cancer, NHL

209 WFHPROELI NSCLC 261 WLFOEALWHV Urinary bladder cancer 127 WLIGSNHSL PC, BRCA, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, AML 186 WLLAQIIQW Melanoma, OC, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer

262 WLPNFLPYNW NSCLC, SCLC, Brain Cancer, GC, BRCA, MCC, Melanoma, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL, PC

114 VLSOVYSKV SCLC

23 O WLTEFTREW NSCLC, SCLC, CLL, BRCA, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 263 WLYPSLKEI RCC, BRCA, Uterine Cancer

1. WMAPFTMTI SCLC, Melanoma, NHL 264 VMODPEFLOSV SCLC, CRC, PC, CLL, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer, NHL

115 WLDDKDYFL CLI

344 WYISSLALL NSCLC, GC, CRC

265 WLIEDGKWWTW Melanoma

117 YAFPKSITV PC

178 YIFTTPKSW AML

179 YIHNILYEW CLL, NHL

341 YLAIGIHEL SCLC

234 YLEPYLKEW NSCLC, SCLC, BRCA, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL, PC 3O YLGEGPRMW NSCLC, Melanoma, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer US 2016/0250307 A1 Sep. 1, 2016

TABLE 5B- continued Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases S* = phosphoserine

SEO ID NO. Sequence Additional Entities

18O YLGPHIASWTL Melanoma

121 YLITGNLEKL NSCLC, CLL, BRCA, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL, PC

228 YLITSWELL OC, NHL 181 YLLEKFWAW NSCLC, SCLC, CLL, OC, Urinary bladder cancer, AML, NHL

269 YLMEGSYNKW NSCLC, SCLC, PrC, BRCA, MCC, Melanoma, OC, Esophageal Cancer, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer

123 YLWDLDHGFA NSCLC, SCLC, PrC, BRCA, Melanoma, OC GW Esophageal Cancer, AML, NHL

31 YOMDIOOEL SCLC

2O3 YYGILOEKI NSCLC

213 YYNKVSTVF NSCLC

0026 NSCLC—non-small cell lung cancer, SCLC-small 328, 329, 333,334, 335, 336, and 346 for the in one pre cell lung cancer, RCC-kidney cancer, CRC-colon or rectum ferred embodiment combined treatment of kidney cancer. cancer, GC=stomach cancer, HCC-liver cancer, 0030 Thus, another aspect of the present invention relates PC pancreatic cancer, PrC-prostate cancer, leukemia, to the use of at least one peptide according to the present BRCA-breast cancer, MCC=Merkel cell carcinoma, invention according to SEQID No. 14, 15, 16, 17, 36,39, 47. OC-ovarian cancer, NHL non-Hodgkin lymphoma, 51,54, 65, 88, 101, 123, 125, 133, 134, 135, 137, 141, 147, AML-acute myeloid leukemia, CLL=chronic lymphocytic 161, 166, 169, 176, 179, 184, 186, 187, 189, 191, 192, 193, leukemia. 194, 195, 196, 197, 199, 203, 206, 208, 214, 220, 221, 224, 0027 Thus, another aspect of the present invention relates 229, 230, 231, 234, 238, 239, 244, 245, 250, 251, 255, 258, to the use of at least one peptide according to the present 259, 260,268, 269, 270, 271, 272, 278, 279, 282, 295, 297, invention according to any one of SEQID No. 1, 14, 15, 41, 302, 304, 305, 306, 309, 310, 311, 312, 313,316, 317, 319, 43,58, 59, 60, 81, 121, 135,139,144, 176,236,248,275,276, 321,325, 327, 328, 329, 330, 331, 332,333,334, 336,337, 283, 286, 288, 289, 290, 291, 300, 302, 304,308, 313, 316, 338,339, 340, 342, 343, 344, 345, and 347 for the in one 317, 325, 326, 329, 331, 334, 342, and 343 for the in one preferred embodiment combined treatment of brain cancer. preferred embodiment combined—treatment of pancreatic 0031. Thus, another aspect of the present invention relates CaCC. to the use of at least one peptide according to the present 0028. Thus, another aspect of the present invention relates invention according to SEQID No. 172, 173,240,250, 287, to the use of at least one peptide according to the present 299,302,334, and 335 for the in one preferred embodiment invention according to SEQID No. 6, 15, 16, 22, 26, 30, 34, combined treatment of CLL. 36,47, 59, 65, 69,70, 77,80, 81, 88, 121, 123,125, 127, 133, 0032 Similarly, the peptides as listed in Table 5B as above 137, 139, 169, 172, 176, 181, 186, 221, 223, 229, 230, 231, can form the basis for the in one preferred embodiment 232, 233,234, 236, 237,238,244, 247, 249, 250, 251, 255, combined—treatment of the diseases as indicated. 260, 261, 266, 269,271, 274, 275,282, 285, 289, 290, 291, 0033. Thus, another aspect of the present invention relates 293,297, 301,302,304,306,310,313,316, 317, 319, 327, to the use of the peptides according to the present invention 328, 329, 330, 331, 332, 334, and 342 for the in one pre for the preferably combined treatment of a proliferative ferred embodiment combined treatment of colon cancer or disease selected from the group of HCC, brain cancer, kidney renal cancer. cancer, pancreatic cancer, colon or rectal cancer, and leuke 0029. Thus, another aspect of the present invention relates 18. to the use of at least one peptide according to the present 0034. The present invention furthermore relates to pep invention according to SEQID No. 10, 14, 15, 22, 36,39, 54. tides according to the present invention that have the ability to 55, 60, 72, 77, 81, 90,96, 112, 116, 119, 121, 133, 137, 138, bind to a molecule of the human major histocompatibility 148, 169, 170, 172, 177, 186,187, 189, 192, 197, 198, 203, complex (MHC) class-I or in an elongated form, Such as a 206, 219, 221, 229, 230, 233,234, 236, 255, 260, 270, 272, length-variant MHC class-II. 275, 277,278, 279, 281, 282,285, 289, 291, 292, 295, 296, 0035. The present invention further relates to the peptides 297, 301,302,305, 308, 311, 313, 315, 316, 319, 321, 324, according to the present invention wherein said peptides US 2016/0250307 A1 Sep. 1, 2016 24

(each) consist or consist essentially of an amino acid 0048. The present invention further relates to activated T sequence according to SEQID NO: 1 to SEQID NO: 300. cells, produced by the method according to the present inven 0036. The present invention further relates to the peptides tion, wherein said T cell selectively recognizes a cell which according to the present invention, wherein said peptide is expresses a polypeptide comprising an amino acid sequence modified and/or includes non-peptide bonds. according to the present invention. 0037. The present invention further relates to the peptides 0049. The present invention further relates to a method of according to the present invention, wherein said peptide is killing target cells in a patient which target cells aberrantly part of a fusion protein, in particular fused to the N-terminal express a polypeptide comprising any amino acid sequence amino acids of the HLA-DR antigen-associated invariant according to the present invention, the method comprising chain (Ii), or fused to (or into the sequence of) an antibody, administering to the patient an effective number of T cells as Such as, for example, an antibody that is specific for dendritic produced according to the present invention. cells. 0050. The present invention further relates to the use of 0038. The present invention further relates to a nucleic any peptide as described, the nucleic acid according to the acid, encoding the peptides according to the present inven present invention, the expression vector according to the tion. The present invention further relates to the nucleic acid present invention, the cell according to the present invention, according to the present invention that is DNA, cDNA, PNA, the activated T lymphocyte, the T cell receptor or the antibody RNA or combinations thereof. or other peptide- and/or peptide-MHC-binding molecules 0039. The present invention further relates to an expres according to the present invention as a medicament or in the sion vector capable of expressing and/or expressing a nucleic manufacture of a medicament. Preferably, the medicament is acid according to the present invention. active against cancer. 0040. The present invention further relates to a peptide 0051 Preferably, said medicament is for a cellular according to the present invention, a nucleic acid according to therapy, a vaccine or a protein based on a soluble TCR or the present invention or an expression vector according to the antibody. present invention for use in the treatment of diseases and in 0.052 The present invention further relates to a use accord medicine, in particular in the treatment of diseases including ing to the present invention, wherein said cancer cells are cancer and autoimmune/inflammatory/immune pathological HCC, brain cancer, kidney cancer, pancreatic cancer, colon or diseases. rectal cancer or leukemia, and preferably HCC cells. 0041. The present invention further relates to antibodies 0053. The present invention further relates to particular against the peptides according to the present invention or marker proteins and biomarkers based on the peptides accord complexes of said peptides according to the present invention ing to the present invention, herein called “targets that can be with MHC, and methods of making these. used in the diagnosis and/or prognosis of HCC. The present 0042. The present invention further relates to T-cell recep invention also relates to the use of these novel targets in the tors (TCRs), in particular soluble TCR (sTCRs) and cloned context of cancer treatment. TCRS engineered into autologous or allogeneic T cells, and 0054. There are two classes of MHC-molecules, MHC methods of making these, as well as NK cells or other cells class I and MHC class II. MHC molecules are composed of an bearing said TCR or cross-reacting with said TCRs. alpha heavy chain and beta-2-microglobulin (MHC class I receptors) or an alpha and a beta chain (MHC class II recep 0043. The antibodies and TCRs are additional embodi tors), respectively. Their three-dimensional conformation ments of the immunotherapeutic use of the peptides accord results in a binding groove, which is used for non-covalent ing to the invention at hand. interaction with peptides. MHC class I molecules can be 0044) The present invention further relates to a host cell found on most nucleated cells. They present peptides that comprising a nucleic acid according to the present invention result from proteolytic cleavage of predominantly endog or an expression vector as described before. The present enous proteins, defective ribosomal products (DRIPs) and invention further relates to the host cell according to the larger peptides. MHC class II molecules can be found pre present invention that is an antigen presenting cell, and pref dominantly on professional antigen presenting cells (APCs), erably is a dendritic cell. and primarily present peptides of exogenous or transmem 0045. The present invention further relates to a method for brane proteins that are taken up by APCs during endocytosis, producing a peptide according to the present invention, said and are Subsequently processed. Complexes of peptide and method comprising culturing the host cell according to the MHC class I are recognized by CD8-positive T cells bearing present invention, and isolating the peptide from said host cell the appropriate TCR (T-cell receptor), whereas complexes of or its culture medium. peptide and MHC class II molecules are recognized by CD4 0046. The present invention further relates to said method positive-helper-T cells bearing the appropriate TCR. It is well according to the present invention, wherein the antigen is known that the TCR, the peptide and the MHC are thereby loaded onto class I or II MHC molecules expressed on the present in a stoichiometric amount of 1:1:1. Surface of a Suitable antigen-presenting cell or artificial anti 0055 CD4-positive helper T cells play an important role gen-presenting cell by contacting a sufficient amount of the in inducing and Sustaining effective responses by CD8-posi antigen with an antigen-presenting cell. tive cytotoxic T cells. The identification of CD4-positive 0047. The present invention further relates to the method T-cell epitopes derived from tumor associated antigens according to the present invention, wherein the antigen-pre (TAA) is of great importance for the development of pharma senting cell comprises an expression vector capable of ceutical products for triggering anti-tumor immune responses expressing or expressing said peptide containing SEQID No. (Gnjatic S, et al. Survey of naturally occurring CD4+ T cell 1 to SEQID No.: 300, preferably containing SEQID No. 1 to responses against NY-ESO-1 in cancer patients: correlation SEQID No. 124, and SEQID No. 187 to SEQID No. 218 or with antibody responses. Proc Natl AcadSci USA. 2003 Jul. a variant amino acid sequence. 22: 100(15):8862-7). At the tumor site. Thelper cells, support US 2016/0250307 A1 Sep. 1, 2016 a cytotoxic T cell- (CTL-)friendly cytokine milieu Mortara L. usually 8-12 amino acid residues in length and usually con et al. CIITA-induced MHC class II expression in mammary tain two conserved residues (“anchors’) in their sequence that adenocarcinoma leads to a Th1 polarization of the tumor interact with the corresponding binding groove of the MHC microenvironment, tumor rejection, and specific antitumor molecule. In this way each MHC allele has a “binding motif memory. Clin Cancer Res. 2006 Jun. 1; 12(11 Pt 1):3435-43) determining which peptides can bind specifically to the bind and attract effector cells, e.g. CTLS, NK cells, macrophages, ing groove. granulocytes (Hwang ML, et al. Cognate memory CD4+ T 0064. In the MHC class I dependent immune reaction, cells generated with dendritic cell priming influence the peptides not only have to be able to bind to certain MHC class expansion, trafficking, and differentiation of secondary I molecules expressed by tumor cells, they Subsequently also CD8+ T cells and enhance tumor control. J Immunol. 2007 have to be recognized by T cells bearing specific T cell recep Nov. 1; 179(9):5829-38). tors (TCR). 0056. In the absence of inflammation, expression of MHC 0065. The current classification of tumor associated anti class II molecules is mainly restricted to cells of the immune gens comprises the following major groups: system, especially professional antigen-presenting cells a) Cancer-testis antigens: The first TAAs ever identified that (APC), e.g., monocytes, monocyte-derived cells, macroph can be recognized by T cells belong to this class, which was ages, dendritic cells. In cancer patients, cells of the tumor originally called cancer-testis (CT) antigens because of the have been found to express MHC class II molecules (Dengel expression of its members in histologically different human J, et al. Unexpected abundance of HLA class II presented tumors and, among normal tissues, only in spermatocytes/ peptides in primary renal cell carcinomas. Clin Cancer Res. spermatogonia of testis and, occasionally, in placenta. Since 2006 Jul. 15; 12(14 Pt 1):4163-70). the cells of testis do not express class I and II HLA molecules, 0057 Elongated (longer) peptides of the invention can act these antigens cannot be recognized by T cells in normal as MHC class II active epitopes. Thelper cells, activated by tissues and can therefore be considered as immunologically MHC class II epitopes, play an important role in orchestrating tumor-specific. Well-known examples for CT antigens are the the effector function of CTLs in anti-tumor immunity. MAGE family members or NY-ESO-1. T-helper cell epitopes that trigger a T-helper cell response of b) Differentiation antigens: These TAAS are shared between the TH1 type support effector functions of CD8-positive tumors and the normal tissue from which the tumor arose; killer T cells, which include cytotoxic functions directed most are found in melanomas and normal melanocytes. Many against tumor cells displaying tumor-associated peptide? of these melanocyte lineage-related proteins are involved in MHC complexes on their cell surfaces. In this way tumor the biosynthesis of melanin and are therefore not tumor spe associated T-helper cell peptide epitopes, alone or in combi cific but nevertheless are widely used for cancer immuno nation with other tumor-associated peptides, can serve as therapy. Examples include, but are not limited to, tyrosinase active pharmaceutical ingredients of vaccine compositions and Melan-A/MART-1 for melanoma or PSA for prostate that stimulate anti-tumor immune responses. CaCC. 0058. It was shown in mammalian animal models, e.g., c) Overexpressed TAAs: Genes encoding widely expressed mice, that even in the absence of CD8-positive T lympho TAAs have been detected in histologically different types of cytes, CD4-positive T cells are sufficient for inhibiting mani tumors as well as in many normal tissues, generally with festation of tumors via inhibition of angiogenesis by Secretion lower expression levels. It is possible that many of the of interferon-gamma (IFNY). epitopes processed and potentially presented by normal tis 0059. There is evidence for CD4 T cells as direct anti sues are below the threshold level for T-cell recognition, tumor effectors (Braumuller et al., 2013; Tran et al., 2014). while their overexpression in tumor cells can trigger an anti 0060 Since the constitutive expression of HLA class II cancer response by breaking previously established toler molecules is usually limited to immune cells, the possibility ance. Prominent examples for this class of TAAs are Her-2/ of isolating class II peptides directly from primary tumors neu, Survivin, Telomerase or WT1. was not considered possible. However, Dengel et al. were d) Tumor specific antigens: These unique TAAS arise from successful in identifying a number of MHC Class II epitopes mutations of normal genes (such as B-catenin, CDK4, etc.). directly from tumors (WO 2007/028574, EP1760 088 B1). Some of these molecular changes are associated with neo 0061 The antigens that are recognized by the tumor spe plastic transformation and/or progression. Tumor specific cific cytotoxic T lymphocytes, that is, the epitopes thereof, antigens are generally able to induce strong immune can be molecules derived from all protein classes, such as responses without bearing the risk for autoimmune reactions enzymes, receptors, transcription factors, etc. which are against normal tissues. On the other hand, these TAAS are in expressed and, as compared to unaltered cells of the same most cases only relevant to the exact tumor on which they origin, usually up-regulated in cells of the respective tumor. were identified and are usually not shared between many 0062 Since both types of response, CD8 and CD4 depen individual tumors. Tumor-specificity (or -association) of a dent, contribute jointly and synergistically to the anti-tumor peptide may also arise if the peptide originates from a tumor effect, the identification and characterization of tumor-asso (-associated) exon in case of proteins with tumor-specific ciated antigens recognized by either CD8+ T cells (ligand: (-associated) isoforms. MHC class I molecule+peptide epitope) or by CD4-positive e) TAAS arising from abnormal post-translational modifica T-helper cells (ligand: MHC class II molecule+peptide tions: Such TAAS may arise from proteins which are neither epitope) is important in the development of tumor vaccines. specific nor overexpressed in tumors but nevertheless become 0063 For an MHC class I peptide to trigger (elicit) a tumor associated by posttranslational processes primarily cellular immune response, it also must bind to an MHC active in tumors. Examples for this class arise from altered molecule. This process is dependent on the allele of the MHC glycosylation patterns leading to novel epitopes in tumors as molecule and specific polymorphisms of the amino acid for MUC1 or events like protein splicing during degradation sequence of the peptide. MHC-class-I-binding peptides are which may or may not be tumor specific. US 2016/0250307 A1 Sep. 1, 2016 26 f) Oncoviral proteins: These TAAS are viral proteins that may 0071. Both therapeutic and diagnostic uses against addi play a critical role in the oncogenic process and, because they tional cancerous diseases are disclosed in the following more are foreign (not of human origin), they can evoke a T-cell detailed description of the underlying proteins (polypeptides) response. Examples of such proteins are the human papilloma of the peptides according to the invention. type 16 virus proteins, E6 and E7, which are expressed in (0072. Differential expression of COL18A1 was reported cervical carcinoma. for bladder cancer, rhabdoid tumors and ovarian carcinoma 0066 For proteins to be recognized by cytotoxic T-lym and specific polymorphisms within the gene were shown to phocytes as tumor-specific or -associated antigens, and to be increase the risk for sporadic breast cancer (Fang et al., 2013; used in a therapy, particular prerequisites must be fulfilled. Gadd et al., 2010; Peters et al., 2005; Lourenco et al., 2006). The antigen should be expressed mainly by tumor cells and 0073 Changes in COPA gene expression and RNA editing not, or in comparably Small amounts, by normal healthy were shown to be associated with hepatocellular carcinoma tissues. In a preferred embodiment, the peptide should be and an experimental study revealed anti-apoptotic effects of over-presented by tumor cells as compared to normal healthy COPA in mesothelioma cells (Sudo et al., 2010; Qi et al., tissues. It is furthermore desirable that the respective antigen 2014; Wong et al., 2003). is not only present in a type of tumor, but also in high con 0074 The activity of CPB2 was shown to be significantly centrations (i.e. copy numbers of the respective peptide per reduced in acute promyelocytic leukemia (Meijers et al., cell). Tumor-specific and tumor-associated antigens are often 2000). derived from proteins directly involved in transformation of a 0075 CRP, an acute phase protein synthesized in the liver, normal cell to a tumor cell due to their function, e.g. in cell was shown to be a prognostic marker in a variety of cancer cycle control or Suppression of apoptosis. Additionally, types, amongst others renal cell carcinoma and multiple downstream targets of the proteins directly causative for a myeloma (Ljungberg, 2007: Fassas and Tricot, 2004). transformation may be upregulated and thus may be indi 0076 CRYZ is a target gene of the tumor-suppressor p53 rectly tumor-associated. Such indirect tumor-associated anti (Bansal et al., 2011). Its encoded protein zeta-crystallin was gens may also be targets of a vaccination approach (Singh shown to directly interact with the mRNA of the anti-apop Jasuja et al., 2004). It is essential that epitopes are present in totic molecule bcl-2 and to stabilize the over-expression of the amino acid sequence of the antigen, in order to ensure that bcl-2 in T-cell acute lymphocytic leukemia (Lapucci et al., Such a peptide (“immunogenic peptide'), being derived from 2010). a tumor associated antigen, leads to an in vitro or in vivo (0077. Over-expression of CSRP2 is associated with de T-cell-response. differentiation of hepatocellular carcinoma (Midorikawa et 0067 Basically, any peptide able to bind an MHC mol al., 2002). ecule may function as a T-cell epitope. A prerequisite for the 0078 CYB5A encodes an enzyme which detoxifies carci induction of an in vitro or in vivo T-cell-response is the nogenic molecules and is a prognostic factor for pancreatic presence of a T cell having a corresponding TCR and the cancer (Blanke et al., 2014; Giovannetti et al., 2014). absence of immunological tolerance for this particular (0079. Increased expression levels of CYP27A1 are asso epitope. ciated with endometrial carcinoma, breast cancer and col orectal cancer (Bergada et al., 2014; Nelson et al., 2013; 0068. Therefore, TAAS are a starting point for the devel Matusiak and Benya, 2007). opment of a T cell based therapy including but not limited to 0080. Over-expression of CYP2E1 was reported in col tumor vaccines. The methods for identifying and character orectal cancer, specific polymorphisms are associated with izing the TAAS are based on the use of T-cells that can be bladder and lung cancer and in breast cancer cells (Ye et al., isolated from patients or healthy Subjects, or they are based on 2014; Patel et al., 2014: Deng et al., 2014; Leung et al., 2013). the generation of differential transcription profiles or differ I0081 CYP2J2 is an enzyme, which was shown to be over ential peptide expression patterns between tumors and nor expressed in a variety of human cancers, including esoph mal tissues. ageal, lung, breast, stomach, liver and colon cancer (Jiang et 0069. However, the identification of genes over-expressed al., 2005; Narjoz et al., 2014). in tumor tissues or human tumor cell lines, or selectively I0082 CYP4F8 was shown to be highly expressed in pros expressed in Such tissues or cell lines, does not provide pre tate cancer (Vainio et al., 2011). CYP4F2 and CYP4F3 were cise information as to the use of the antigens being transcribed both shown to be over-expressed in pancreatic ductal adeno from these genes in an immune therapy. This is because only carcinoma and CYP4F2 alone in ovarian cancer (Gandhi et an individual Subpopulation of epitopes of these antigens are al., 2013; Alexanian et al., 2012). Suitable for Such an application since a T cell with a corre I0083 Expression of CYP4F11 was shown to be regulated sponding TCR has to be present and the immunological tol by NF-kB and p53 (Kalsotra et al., 2004; Bell and Strobel, erance for this particular epitope needs to be absent or mini 2012: Goldstein et al., 2013). mal. In a very preferred embodiment of the invention it is I0084 Genetic variants of CYPAF 12 are significantly asso therefore important to select only those over- or selectively ciated with gemcitabine response in pancreatic cancer presented peptides against which a functional and/or a pro patients (Goldstein et al., 2013; Harris et al., 2014). liferating T cell can be found. Such a functional T cell is I0085 High levels of DAP3 correlate on the one hand with defined as a T cell, which upon stimulation with a specific better responses to chemotherapy in gastric cancer and better antigen can be clonally expanded and is able to execute effec clinical outcome in breast cancer, but on the other hand over tor functions (“effector T cell’). expression of DAP3 was reported in thyroid oncocytic tumors 0070. In case of TCRs and antibodies according to the and invasive glioblastoma (Jia et al., 2014; Wazir et al., 2012: invention the immunogenicity of the underlying peptides is Jacques et al., 2009; Mariani et al., 2001). secondary. For TCRs and antibodies according to the inven I0086 PEX19 is essential for peroxisomal biogenesis, but tion the presentation is the determining factor. was also shown to directly interact with p19ARF, ultimately US 2016/0250307 A1 Sep. 1, 2016 27 leading to a retention of this factor in the cytoplasm and to an effectively and specifically inhibits cancer cell growth and inactivation of p53 tumor-suppressive function (Sugihara et cell invasive capacities (Drazkowska et al., 2013: Stefanska et al., 2001). al., 2014). I0087 DDX11, belonging to the DEAH family of DNA 0104. The hydrolytic enzyme FUCA2 was found to be helicases, is highly expressed in advanced melanoma (Bhat essential for H. pylori adhesion to human gastric cancer cells tacharya et al., 2012). (Liu et al., 2009a). 0088 NME4 is a nucleoside diphosphate kinase, over 0105 GABRO encodes the GABAA receptor theta sub expressed in colon and gastric cancer, as well as in myelod unit. GABA was shown to stimulate human hepatocellular ysplastic syndrome, in the latterdisease being associated with carcinoma growth through the over-expressed GABAA poor prognosis (Kracmarova et al., 2008: Seifert et al., 2005). receptor theta subunit (Li et al., 2012). I0089. DENND5B acts as GDP-GTP exchange factor to 0106. In squamous cell carcinoma over-expression of activate Rab-GTPases (Yoshimura et al., 2010). GALNT2 was reported to enhance the invasive potential of 0090 DIEXF was shown to mediate the non-proteasomal tumor cells by modifying O-glycosylation and EGFR activity degradation of the tumor-suppressor p53 (Tao et al., 2013). (Lin et al., 2014; Hua et al., 2012a: Wu et al., 2011). 0091 DOCK7 is a guanine nucleotide exchange factor, 0107 High levels of GGH have been associated with cel which was shown to be over-expressed in glioblastoma and to lular resistance to anti-folates, in particular methotrexate and increase glioblastoma cell invasion in response to HGF by with poor prognosis in invasive breast cancer and pulmonary activating Rac-1 (Murray et al., 2014). endocrine tumors (Schneider and Ryan, 2006; Shubbar et al., 0092. In hepatocellular carcinoma cell lines, DRG2 was 2013: He et al., 2004). shown to be down-regulated during chemotherapeutic drug 0.108 GLUL is over-expressed in human breast carcinoma induced apoptosis, and over-expression of DRG2 inhibits cells and astrocytomas (Zhuang et al., 2011; Collins et al., doxorubicin induced apoptosis in these cells (Chen et al., 1997: Christa et al., 1994: Cadoret et al., 2002). 2012a). 0109 GNPAT was reported to be implicated in growth 0093. DROSHA, one of the two critical enzymes in inhibition and apoptosis induction in metastatic melanoma microRNA biosynthesis, is over-expressed in a number of (Ofman et al., 2001; Qin et al., 2013). cancers including gastrointestinal tumors, breast cancer and 0110 Deletions in the chromosomal region of GOLGA4 cervical cancer and appears to enhance proliferation, colony have been reported in cervical carcinoma and in-frame formation and migration of tumor cells (Avery-Kiejda et al., mRNA fusion of GOLGA4 with PDGFRB in myeloprolif 2014: Havens et al., 2014: Zhou et al., 2013b). erative neoplasms (Senchenko et al., 2003; Hidalgo-Curtis et 0094 SNPs in the DUSP14 gene are associated with al., 2010). altered melanoma risk (Yang et al., 2014a: Liu et al., 2013b). 0111 GPAM is expressed in human breast cancer, which 0095. A whole exome sequencing study uncovered is associated with changes in the cellular metabolism and somatic mutations within the DYNC1H1 gene in patients better overall survival (Brockmoller et al., 2012). with intra-ductal papillary mucinous neoplasm of the pan 0112 High serum levels of GPT were reported to increase creas (Furukawa et al., 2011). the risk of gastrointestinal cancer and are associated with 0096 EEF2 protein was shown to be over-expressed in carcinogenesis and recurrence in hepatitis C virus-induced lung, esophageal, pancreatic, breast and prostate cancer, in hepatocellular carcinoma (Kunutsor et al., 2014, Tarao et al., glioblastoma multiforme and in non-Hodgkin’s lymphoma 1997: Tarao et al., 1999). and to play an oncogenic role in cancer cell growth (Oji et al., 0113 GRB14 has been shown to be up-regulated in breast 2014: Zhu et al., 2014a). cancer, where high expression was significantly associated 0097. Mutations within the gene of EFR3A were identi with better disease-free and overall survival (Huang et al., fied in colorectal adenoma samples (Boijireddy et al., 2014: 2013: Balogh et al., 2012). Zhou et al., 2013a). 0114. Single nucleotide polymorphisms in the GTF2H4 0098 EIF2B5 encodes one subunit of the translation ini gene were reported to increase the risk to develop Smoking tiation factor B. Single nucleotide polymorphisms in this relatedlung cancer and papillomavirus-induced cervical can gene were described to be associated with survival time in cer (Mydlikova et al., 2010; Buch et al., 2012; Wang et al., ovarian cancer (Goode et al., 2010). 2010). 0099 EIF3A, the eukaryotic translation initiation factor 3, 0115 Different studies suggest an important role of Subunit A is over-expressed in cancers of breast, lung, cervix, HSPA2 in disease progression of cervical cancer, renal cell esophagus, stomach and colon and was shown to be involved carcinoma and bladder cancer. Polymorphisms within the in cell cycle regulation (Dong and Zhang, 2006). gene are associated with the development of gastric cancer 0100 EIF4E is a potent oncogene elevated in up to 30% of (Singh and Suri, 2014: Ferrer-Ferrer et al., 2013; Garg et al., human malignancies, including carcinomas of the breast, 2010a; Garget al., 2010b). prostate, lung, head, and neck as well as in many leukemias 0116. HSPA8 was shown to be over-expressed in esoph and lymphomas (Carroll and Borden, 2013). ageal squamous cell carcinoma. Furthermore, HSPA8 is over 0101 ELOVL2 was shown to be over-expressed in hepa expressed in multiple myeloma and colonic carcinoma and tocellular carcinoma (Jakobsson et al., 2006: Zekri et al., BCR-ABL1-induced expression of HSPA8 promotes cell sur 2012). vival in chronic myeloid leukemia (Dadkhah et al., 2013; 0102 EPRS encodes a multifunctional aminoacyl-tRNA Wang et al., 2013a; Chatterjee et al., 2013; Kubota et al., synthetase, which was reported to be a tumor-associated anti 2010; Jose-Eneriz et al., 2008). gen in colon cancer (Line et al., 2002). 0117. MDN1 was described to be a candidate tumor sup 0103 EXOSC4 promoter activity is increased in hepato pressor gene, mutated in breast cancers of the luminal B type cellular carcinoma, due to DNA hypomethylation. EXOSC4 (Cornen et al., 2014). US 2016/0250307 A1 Sep. 1, 2016 28

0118 MIA3, also known as transport and Golgi organiza membrane, which functions in the assembly of beta-barrel tion protein 1 (TANGO), was reported to be down-regulated proteins into the outer mitochondrial membrane. A growth in colon and hepatocellular carcinomas and to play a tumor promoting chimeric mRNA (SAMM50-PARVB) was suppressive role in these entities (Arndt and Bosserhoff, detected in breast and ovarian cancer cells and in a number of 2007). In contrast, a study in oral squamous cell carcinoma samples from breast, stomach, colon, kidney and uterus can indicates an association of MIA3 expression with tumor pro cer (Plebani et al., 2012). gression, metastasis formation and clinical stage, pointing (0132) SERPINF2 encodes the major inhibitor of plasmin, towards an oncogenic action of MIA3 (Sasahira et al., 2014). which degrades fibrin and various other proteins. The plasma 0119 CPSF6 was identified as one gene within a “poised level of the plasmin-alpha 2-plasmin inhibitor complex was gene cassette' associated with significant differences in shown to be a predictor of Survival in non-Small cell lung metastatic and invasive potential of several tumor types, like carcinoma and low activity of alpha2-antiplasmin has been breast, colon, liver, lung, esophageal and thyroid cancer (Yu observed in the blood of the patients with prostatic carcinoma et al., 2008). (Zietek et al., 1996; Taguchi et al., 1996). 0120 Low levels of MPDZ expression were reported to be 0.133 Over-expression of SF3B3 is significantly corre associated with poor prognosis in breast cancer patients (Mar lated with overall survival and endocrine resistance in estro tin et al., 2004). gen receptor-positive breast cancer (Gokmen-Polar et al., 0121 NAA35, also known as MAK10, encodes the N(al 2014). pha)-acetyltransferase 35, NatC auxiliary subunit. In patients I0134) Protein levels of SHC1 are elevated in prostate, with esophageal squamous cell carcinoma a highly cancer metastatic breast, ovarian and thyroid cancer and different enriched chimeric GOLM1-MAK10 RNA was detected, isoforms and are thought to function as a primary adaptor which encodes a secreted fusion protein, potentially useful as protein for mediating the mitogenic signals of steroids at the molecular marker (Zhang et al., 2013b). non-genomic level (Alam et al., 2009; Rajendranet al., 2010). 0122 NAV2 was shown to be specifically expressed in a 0.135 AMACR is used as a biomarker in prostate cancer, group of colon cancers and treatment of colon-cancer cells since it is highly over-expressed in this entity (Wu et al., with antisense oligonucleotides for NAV2 induced apoptosis 2014). Furthermore, it is used as an immunohistochemical (Ishiguro et al., 2002). marker for the diagnosis of renal cell carcinoma (Ross et al., 0123 NCSTN over-expression is indicative of worse 2012). overall Survival in estrogen-receptor-negative breast cancer 0.136 Experimental data suggest that C1OTNF3 expres patients and high levels of Nicastrin and Notch4 were Sion may play a role in osteosarcoma tumor growth, associ detected in endocrine therapy resistant breast cancer cells, ated with activation of the ERK1/2 signaling pathway and where their activation ultimately drives invasive behavior that it is a novel anti-apoptotic adipokine that protects mes (Sarajlic et al., 2014; Lombardo et al., 2014). enchymal stem cells from hypoxia/serum deprivation-in (0.124 NKD1 protein is reduced, but NKD1 mRNA is duced apoptosis through the PI3K/Akt signaling pathway elevated in non-Small cell lung cancer, the former correlating (Hou et al., 2014; Akiyama et al., 2009). with increased invasive potential and poor prognosis (Zhang 0.137 GPC3 is expressed by most hepatocellular carcino et al., 2011). NKD1 mRNA was also found to be elevated in mas. Two therapeutic approaches for HCC that target GPC3 cells from human colon tumors (Yan et al., 2001; Zhanget al., are currently being tested in phase II clinical trials: a human 2011). ized GPC3 monoclonal antibody and a vaccine that consists 0.125. In esophageal cancer, NUDC was reported to be of two GPC3-derived peptides. The peptides used in the latter associated with nodal metastasis, whereas over-expression of study are distinct from the peptide presented in this docu NUDC in prostate cancer cells leads to a block in cell division ment. GPC3 expression has also been identified in all yolk sac (Hatakeyama et al., 2006; Lin et al., 2004). tumors, some squamous cell carcinomas of the lung and clear 0126. A study investigating the role of the Notch signaling cell carcinomas of the ovary (Filmus and Capurro, 2013; pathway in ovarian cancer reported a higher frequency of Kandil and Cooper, 2009). RFNG expression inadenoma compared to carcinoma (Gu et 0.138 MAGEB2 is classified as cancer testis antigen, since al., 2012; Hopfer et al., 2005). it is expressed in testis and placenta, and in a significant 0127 RINT1 is described as an oncogene in glioblastoma fraction of tumors of various histological types, amongst multiforme and as a moderately penetrant cancer Susceptibil others multiple myeloma and head and neck squamous cell ity gene seen in breast cancer as well as in Lynch syndrome carcinoma (Pattani et al., 2012; Van et al., 2011). related cancers (Ngeow and Eng, 2014: Quayle et al., 2012). I0139 MAPKAPK5 encodes a tumor suppressor and 0128 High expression of RORC was found to be associ member of the serine/threonine kinase family. MAPKAPK5 ated with longer metastasis-free Survival in breast cancer. was shown to be under-expressed in colorectal cancer, lead Attenuated RORC expression in somatotroph adenomas is ing to an increased activity of the myc oncoprotein and to associated with increased tumor size and a blunted clinical decrease cancer formation by Suppressing oncogenic ras response to Somatostatin treatment (Cadenas et al., 2014: activity in a murine model of hematopoietic cancer (Yoshi Lekva et al., 2013). Zuka et al., 2012: Kress et al., 2011). 0129 RPL17 was reported to promote multidrug resis 0140. Over-expression of USP14 is associated with tance by Suppressing drug-induced apoptosis (Shi et al., increased tumor cell proliferation and poor prognosis in epi 2004b). thelial ovarian, non-Small cell lung and colorectal cancer 0130 Increased expression of RPS29 was reported in gas (Wang et al., 2015; Wu et al., 2013a; Shinji et al., 2006). tric and colorectal cancer (Takemasa et al., 2012; Sun et al., 0141 C4A has been described as a biomarker for polycys 2005). tic ovary syndrome and endometrial cancer and experimental 0131 SAMM50 encodes a component of the Sorting and data Suggest that C4 can mediate cancer growth (Galazis et Assembly Machinery (SAM) of the mitochondrial outer al., 2013: Rutkowski et al., 2010). US 2016/0250307 A1 Sep. 1, 2016 29

0142 CAPZB was reported to be over-expressed in 0153. THY 1 is a candidate tumor suppressor gene in human papillomaviruses 18-positive oral squamous cell car nasopharyngeal carcinoma bearing anti-invasive activity cinomas and was identified as prostate cancer Susceptibility (Lung et al., 2010). locus (Lo et al., 2007; Nwosu et al., 2001). 0154) TIMM17A is over-expressed in 21T breast cancer 0143. Single nucleotide polymorphisms within the gene cells and mRNA expression in breast cancer tissues was cor for CFHR5 are associated with event-free survival in follicu related with tumor progression (Xu et al., 2010). lar lymphoma (Charbonneau et al., 2012). (O155 TMEM209 is widely expressed in lung cancer (Fuji 0144 CLIP1 encodes the CAP-GLY domain containing tomo et al., 2012). linker protein 1, which links endocytic vesicles to microtu 0156 TNK2 also known as ACK1 tyrosine kinase is acti bules. This gene is highly expressed in Reed-Sternberg cells vated, amplified or mutated in a wide variety of human can of Hodgkin disease and breast cancer and appears to be impli cers. The de-regulated kinase is oncogenic and its activation cated in the migration and invasion of breast cancer and correlates with progression to metastatic stage. ACK1 inhibi pancreatic cancer cells (Sun et al., 2013; Suzuki and Taka tors have shown promise in pre-clinical studies (Mahajanand hashi, 2008: Li et al., 2014a: Sun et al., 2012). Mahajan, 2013). 0145 CLU may inhibit tumor progression, whereas in (O157 TRIM55 encodes a RING zinc finger protein which advanced neoplasia, it may offer a significant Survival advan associates transiently with microtubules, myosin and titin tage in the tumor by Suppressing many therapeutic stressors during muscle sarcomere assembly and is also involved in and enhancing metastasis. CLU has been shown to play a signaling from the sarcomere to the nucleus (Pizon et al., critical role in prostate cancer pathogenesis, to regulate the 2002). aggressive behavior of human clear renal cell carcinoma cells 0158 RNA interference of Ufa 1 protein can sensitize a through modulating ERK1/2 signaling and MMP-9 expres hydroxycamptothecin-resistant colon cancer cell line sion and to confer resistance to treatment in advanced stages SW1116/HCPT to hydroxyl-camptothecin (Chen et al., of lung cancer (Trougakos, 2013; Panico et al., 2009: Takeu 2011a; Chen et al., 2011c). chi et al., 2014; Wang et al., 2014). 0159. In colorectal cancer the UGT1A1 gene is silenced 0146 The fusion gene SEC16A-NOTCH1 was reported through methylation and thus is regarded as the target point of as first recurrent fusion gene in breast cancer (Edwards and research for irinotecan (CPT-11) drug resistance and control Howarth, 2012). mechanisms for the reversal of drug resistance (Xie et al., 0147 Recurrent deletion of the SHO1 gene has been 2014). observed in prostate and cervical cancer, implicating a tumor 0160, UGT1A10 is expressed in gastric and biliary tissue suppressive role of SHQ1 (Krohn et al., 2013: Lando et al., (Strassburg et al., 1997) and its over-expression significantly 2013). increased the cytotoxicity of the antitumor agent 5-dimethy 0148. In clear cell renal cell carcinomas and bladder can laminopropylamino-8-hydroxytriazoloacridinone C-1305 cer high SLC16A1 expression is associated with poor prog (Pawlowska et al., 2013). Furthermore UGT1A10 catalyzes nostic factors and predicts tumor progression. In colorectal the glucuronidation of Xenobiotics, mutagens, and reactive cancer single nucleotide polymorphisms in the SLC16A1 metabolites and thus acts as indirect antioxidant. Xenobiotic gene may affect clinical outcomes and can be used to predict (XRE) and antioxidant (ARE) response elements were the response to adjuvant chemotherapy (Kim et al., 2015; Fei detected in the promoters of UGT1A8, UGT1A9, and et al., 2014a: Fei et al., 2014a). 0149 Glioblastoma have been shown to release glutamate UGT1A10 (Kalthoffet al., 2010). at high levels, which may stimulate tumor cell proliferation 0.161 UGT1 A8 is primarily expressed in the gastrointes and facilitates tumor invasion, and to down-regulate tinal tract (Gregory et al., 2003) and mRNA expression is SLC1A2, which correlated with higher tumor grade, impli up-regulated upon treatment with chemo-preventive agent cating its potential role in glial tumor progression. Further sulforaphane (SFN) (Wang et al., 2012). more, in gastric cancer a fusion gene of SLC1A2 with CD44 0162 UGT1A7 haplotype is associated with an increased has been detected and may represent a class of gene fusions risk of hepatocellular carcinoma in hepatitis B carriers (Kong that establish a pro-oncogenic metabolic milieu favoring et al., 2008). tumor growth and survival (Tao et al., 2011; de Groot et al., 0163 UGT1A6 is over-expressed in breast cancer cells 2005). resistant to methotrexate (de Almagro et al., 2011) and 0150 High expression of SLC3A2 is associated with induced by B-Naphthoflavone a putative chemo-preventive tumor growth, biological aggressiveness, and Survival of agent (Hanioka et al., 2012). patients with biliary tract cancer and significantly contributes 0164 UGT1A9 is mainly expressed in liver and kidneys to poor prognosis of non-small cell lung cancer patients (Gregory et al., 2003). UGT1A9 germline polymorphisms are through promoting cell proliferation via the PI3K/Akt path potential predictors for prostate cancer recurrence after pros way. Furthermore, over-expression of SLC3A2 together with tatectomy (Laverdiere et al., 2014). integrin B1, integrin B3 and Fak is associated with the pro 0.165 UGT1 A4 promoter and coding region polymor gression and liver metastases of colorectal cancer (Kaira et phisms lead to a variability in the glucuronidation of anastro al., 2014: Fei et al., 2014b; Sun et al., 2014). Zole, an aromatase inhibitor for breast cancer patients (Eda 0151. Evidences of SLC9A3R1 involvement in cancer vana et al., 2013). development are present in hepatocellular carcinoma, Schw 0166 UPF 1 is part of the nonsense-mediated mRNA annoma, glioblastoma, colorectal cancer and particularly in decay (NMD) machinery and may have a functional role in breast cancer (Saponaro et al., 2014). prostate cancer progression and metastasis (Yang et al., 0152 NFYC has been reported to promote the expression 2013). Further the UPF1 RNA surveillance gene is com of oncogenes in gastric cancer and prostate cancer cells monly mutated in pancreatic adenosquamous carcinoma (Liu (Zhang et al., 2014a: Gong et al., 2013). et al., 2014). US 2016/0250307 A1 Sep. 1, 2016 30

0167 UOCRB is a subunit of mitochondrial complex III. al., 2012). HDAC10 levels are increased in chronic lympho Inhibition of UQCRB in tumor cells suppresses hypoxia cytic leukemia (Wang et al., 2011). HDAC10-589C>T pro induced tumorangiogenesis (Jung et al., 2013). Two SNPs in moter polymorphism was significantly associated with HCC the 3' untranslated region of UQCRB are candidates as prog occurrence among chronic HBV patients as well as HCC nostic markers for colorectal cancer (Lascorz et al., 2012). acceleration among chronic HBV patients (Parket al., 2007). 0168 Copy number alterations of USO1 correlated with Reduced expression of class II histone deacetylase genes is differential gene expression in Superficial spreading mela associated with poor prognosis in lung cancer patients (Osada noma compared to nodular melanoma (Rose et al., 2011). et al., 2004). (0169. Significant reductions in both USP10 and SIRT6 0182 Low HIP1R expression is strongly associated with protein expression was detected in human colon cancers (Lin poor outcome in diffuse large B-cell lymphoma patients et al., 2013). (Wong et al., 2014). 0170 UTP18 also alters translation to promote stress 0183 HM13 is a signal peptide peptidase and affected cell resistance and growth, and is frequently gained and over viability in colorectal adenoma (Sillars-Hardebol et al., expressed in cancer (Yang et al., 2014b). 2012). 0171 VARS rs207451 1 polymorphism was associated 0.184 Serum HPR levels in patients with malignant lym with Survival in patients with triple negative type breast can phoma were significantly higher than in non-diseased control cer and thus may be considered as a prognostic factor for groups and HPR expression increased with disease progress survival in patients with early breast cancer (Chae et al., (Epelbaum et al., 1998). HPR expression parallels increased 2011). malignant potential inbreast cancers and HPR-positive breast 0172 VMP1, a stress-induced autophagy-associated pro cancers are more likely to recur after primary resection and tein, is also induced by the oncogene KRAS (Lo Re et al., are associated with shorter disease-free intervals (Shurbaji et 2012). VMP1 is over-expressed in poorly differentiated al., 1991). human pancreatic cancer as a response to chemotherapeutic 0185. A variant (rs932335) in the HSD11B1 gene is asso drugs (Gilabert et al., 2013). A significant down-regulation of ciated with colorectal cancer and breast cancer (Feigelson et VMP1 was found in human HCC tissues and closely corre al., 2008: Wang et al., 2013b). lated with multiple tumor nodes, absence of capsular forma 0186. HSD17B6 expression in tissues from prostate can tion, vein invasion and poor prognosis of HCC (Guo et al., cer patients undergoing androgen deprivation therapy (ADT) 2012). was significantly higher than that in tissues of untreated indi (0173 WDR26 protects myocardial cells against oxidative viduals (Ishizaki et al., 2013). stress (Feng et al., 2012). 0187 HSPE1 is a mitochondrial chaperonin with func (0174 ZC3H7A is part of the CCCH zinc finger protein tions in protein folding and cell signaling (NF-kappaB and family known as regulators of macrophage activation (Liang WNT signaling). Increased Hsp10 levels have been found in et al., 2008). ZC3H7A was found to have higher allele fre tumor cells in large bowel cancer, exocervical cancer, prostate quencies of functional mutations in the metastatic tumor of cancer, mantle cell lymphoma, and serous ovarian cancer. In pancreatic ductal adenocarcinoma (Zhou et al., 2012). bronchial carcinogenesis, decreased levels of Hsp10 have 0175 FASN is a fatty acid synthase and involved in the been reported (David et al., 2013). enhanced lipid synthesis in different types of cancer, includ 0188 Ovarian carcinoma xenografts transplanted into the ing breast, pancreatic, prostate, liver, ovarian, colon and flanks of nude mice and treated with paclitaxel showed a endometrial cancer (Wu et al., 2014: Zhao et al., 2013). diminished IDI1 expression compared to untreated Xenograft 0176 FGG is up-regulated in hepatocellular carcinoma as (Bani et al., 2004). well as in prostate, lung and breast cancers (Vejda et al., 2002; 0189 IGFBPL1 is a regulator of insulin-growth factors Zhu et al., 2009). and is down-regulated in breast cancer cell lines by aberrant 0177 FMO5 is a monooxygenase that is the dominant hypermethylation. Methylation in IGFBPL1 was clearly liver-specific FMO, and it is up-regulated in estrogen receptor associated with worse overall survival and disease-free sur alpha-positive breast tumors (Bieche et al., 2004; Zhang and vival (Smith et al., 2007). Cashman, 2006). 0190. The androgen-sensitive microsome-associated pro (0178 HADHA mRNA is reduced with the progression of tein IKBKAP modulated the expression of prostate epithelial de-differentiation in HCC (Tanaka et al., 2013) and in estro and neuronal markers, attenuated proliferation through an gen receptor alpha-negative breast tumors (Mamtani and androgen receptor-dependent mechanism, and co-regulated Kulkarni, 2012). androgen receptor-mediated transcription in LNCaP prostate 0179 Genetic variation in the HAL gene might play a role adenocarcinoma cells (Martinez et al., 2011). in the development of skin cancer (Welsh et al., 2008). 0191 INTS8 is part of a marker panel that discriminates 0180 HLTF is a member of the SWI/SNF family of tran gastric carcinomas from adjacent noncancerous tissues Scriptional regulators with helicase and E3 ubiquitin ligase (Cheng et al., 2013). activity and was found to be inactivated by hypermethylation (0192 AIRS2-derived peptide pIRS-21097-1105 was pre in colon, gastric, uterine, bladder and lung tumors (Debauve sented on HLA-A2(+) melanomas and breast, ovarian, and et al., 2008; Castro et al., 2010; Garcia-Baquero et al., 2014). colorectal carcinomas (Zarling et al., 2014). IRS-2 1057 DD 0181 HDAC10 is a histone deacetylase and transcrip genotype and D allele were significantly associated with tional regulator. Expression of HDAC10 was significantly HCC risk (Rashad et al., 2014). decreased in gastric cancertissues as compared with adjacent 0193 ITGA7 is the alpha chain of the laminin-1 receptor tissues (Jin et al., 2014). HDAC10 is inversely related to dimer integrin alpha-7/beta-1. ITGA7 is a tumor-suppressor lymph node metastasis in human patients with cervical squa gene that is critical for Suppressing the growth of malignant mous cell carcinoma (Song et al., 2013). HDAC10 is hyper tumors. Mutational analysis revealed ITGA7 mutations in methylated in malignant adrenocortical tumors (Fonseca et prostate cancer, hepatocellular carcinoma, Soft tissue lei US 2016/0250307 A1 Sep. 1, 2016 omyosarcoma, and glioblastoma multiforme. ITGA7 was trols (Zhang et al., 2012). Fucosylated glycoform ORM2 down-regulated in non-metastatic prostate cancer and lei levels were significantly higher inadenocarcinoma lung can omyosarcoma (Tan et al., 2013). cer cases compared to controls (Ahn et al., 2014). ORM2 is a 0194 ITIH4 was down-regulated in several tumor tissues putative biomarkers for early diagnosis of cholangiocarci including colon, stomach, ovary, lung, kidney, rectum and noma (Rucksaken et al., 2012). prostate (Hamm et al., 2008). Low serum ITIH4 levels are 0206 Increased tetrahydrobiopterin levels result in an associated with shorter survival in HBV-associated HCC enhancement of PAH activity and PAH protein in human patients (Noh et al., 2014). hepatoma cells (McGuire, 1991). 0.195 Significantly increased ITIH4 serum concentrations 0207 PARP14 is highly expressed in myeloma plasma were observed in breast cancer and ITIH4 serum levels were cells and associated with disease progression and poor Sur significantly decreased after Surgery (van, I et al., 2010). vival. PARP14 is critically involved in JNK2-dependent sur 0196. A missense mutation was identified in SHKBP1, vival. PARP14 was found to promote the survival of myeloma which acts downstream of FLT3, a receptor tyrosine kinase cells by binding and inhibiting JNK1 (Barbarulo et al., 2013). mutated in about 30% of AML cases (Greif et al., 2011). SHKBP1 is one of several potential protein biomarker can 0208 PC levels are elevated in liver tumors and lung can didates for classifying well-differentiated small intestine neu cer (Chang and Morris, 1973; Fanet al., 2009). roendocrine tumors (WD-SI-NETs) at different stage of dis 0209 Increased PCNT levels and centrosomal abnormali ease (Darmanis et al., 2013). ties have been described in a variety of hematologic malig 0.197 KLB expression is elevated in HCC tissues com nancies and Solid tumors, including AML, CML, mantle cell pared to matched non-tumor tissue (Poh et al., 2012). lymphoma, breast cancer and prostate cancer (Delaval and (0198 The LBP polymorphism rs2232596 is associated Doxsey, 2010). with a significantly increased risk of colorectal carcinoma in 0210 PIGN is a cancer chromosomal instability (CIN)- Han Chinese (Chen et al., 2011b). LBP is a candidate serum Suppressor gene that is subject to frequent copy number loss biomarker in ovarian carcinoma (Boylan et al., 2010). LBP in CIN(+) colorectal cancer (Burrell et al., 2013). was reduced significantly after treatment with chemotherapy 0211 PIPOX expression varied according to subtype of in Small-cell lung carcinoma patients (Staal-Van den Brekel A breast cancer, with HER-2 type tumors showing elevated Jet al., 1997). expression and triple negative breast cancer Subtype showing 0199 LBR mRNA expression was directly associated decreased expression. Tumoral PIPOX negativity was asso with tumor grade and Nottingham Prognostic Index in breast ciated with shorter disease-free survival (Yoon et al., 2014). cancer (Wazir et al., 2013). LBR is heavily expressed in PIPOX was reduced in prostate tumors and reduced the onco papillary thyroid carcinoma cells, but an abnormal folding of genic potential of prostate cells by metabolizing sarcosine the protein might explain its lack of immunohistochemical (Khan et al., 2013). reactivity and be associated with an anomalous folding of the 0212 Increased PSMD4 levels were detected in colon nuclear membrane (Recupero et al., 2010). cancer, myeloma and hepatocellular carcinoma (Arlt et al., 0200 LEPR dysregulation has been reported in a variety 2009; Midorikawa et al., 2002; Shaughnessy, Jr. et al., 2011). of malignant cells including colon cancer, hepatocellular car 0213 PLIN2 is significantly increased in patients with cinoma, endometrial cancer, thyroid cancer, breast cancer and clear cell and papillary renal cell carcinoma compared with lung cancer (Ntikoudi et al., 2014: Surmacz, 2013: Uddin et controls. The preoperative urinary concentrations of PLIN2 al., 2011). reflects the tumor size and stage (Morrissey et al., 2014). 0201 LIG1 single-nucleotide polymorphisms are associ PLIN2 expression is significantly higher in lung adenocarci ated with the risk of lung cancer, endometrial cancer and noma specimens than in normal tissues and lung Squamous glioma (Doherty et al., 2011; Lee et al., 2008; Liu et al., cell carcinomas (Zhang et al., 2014b). 2009b). 0214 PLK4 frequently undergoes rearrangement or loss 0202 LRPPRC expression in gastric cancer tissues is sig in human cancers, at a particularly high rate in hepatocellular nificantly higher than that in paired control tissue (Li et al., carcinomas, but also in colorectal cancer, head and neck 2014b). LRPPRC levels serve as a prognosis marker of cancer (Swallow et al., 2005). PLK4 is over-expressed in patients with prostate adenocarcinomas (PCA), and patients with high LRPPRC levels survive a shorter period after sur breast cancer (Marina and Saavedra, 2014). gery than those with low levels of LRPPRC (Jiang et al., 0215. QARS is a member of the aminoacyl-tRNA syn 2014). LRPPRC is abundantly expressed in various types of thetases (ARS) and charges tRNAs with glutamine. ARS tumors, such as lung adenocarcinoma, esophageal Squamous expression and polymorphisms are associated with breast cell carcinoma, stomach, colon, mammary and endometrial cancer and glioblastoma (He et al., 2014b; Kim et al., 2012). adenocarcinoma, and lymphoma (Tian et al., 2012). 0216. The methylated PMF1 gene is a diagnostic and 0203 MANEA expression is regulated by androgens in prognostic biomarker for patients with bladder cancer (Kan prostate cancer cells (Romanuik et al., 2009). dimalla et al., 2013). 0204 OPLAH is expressed in lung, breast, kidney, colon 0217 Several human tumors and hematologic malignan and ovary normal and tumor tissues and OPLAH levels are cies up-regulated PON2, including thyroid gland, prostate, significantly higher in normal specimens than tumors for pancreas, testis, endometrium/uterus, liver and kidney can individual patients (Srivenugopal and Ali-Osman, 1997). cer, lymphoid tissues, urinary bladder tumors, ALL and 0205 ORM2 glycoforms provide valuable information CML, and such over-expression provided resistance to dif for differentiation between primary and secondary liver can ferent chemotherapeutics (imatinib, doxorubicine, stauro cer (Mackiewicz and Mackiewicz, 1995). ORM2 levels in sporine, or actinomycin) (Witte et al., 2011). plasma were confirmed to be significantly elevated in patients 0218. PRKAR2A is a regulatory subunit of protein kinase Suffering from colorectal carcinoma compared with the con A. PRKAR2A markedly increased survival of prostate cancer US 2016/0250307 A1 Sep. 1, 2016 32 cells lines treated with Taxol and Taxotere (Zynda et al., involved in the cisplatin resistance of KCP-4 human epider 2014). PRKAR2A is over-expressed in lung adenocarcinoma moid carcinoma cells (Oiso et al., 2014). (Bidkhori et al., 2013). 0234. The expression of ABCB11 was shown to be up 0219 PRPF6 is a member of the tri-snRNP (small ribo regulated in the pancreatic ductal adenocarcinoma, one of the nucleoprotein) spliceosome complex that drives colon cancer most drug-resistant cancers. Thus it may contribute to the proliferation by preferential splicing of genes associated with generally poor treatment response of this cancer (Mohelni growth regulation (Adler et al., 2014). PRPF6 is over-ex kova-Duchonova et al., 2013). pressed in lung adenocarcinoma (Bidkhori et al., 2013). 0235. The up-regulated expression of ABCC2 in primary 0220 PSMC4 is significantly and coherently up-regulated fallopian tube carcinomas is associated with poor prognosis in prostate carcinoma cells compared with the corresponding (Halon et al., 2013). adjacent normal prostate tissue (Hellwinkel et al., 2011). 0236 ABCC6 was down-regulated in colorectal cancer of 0221 QPRT expression increases with malignancy in non-responders to palliative chemotherapy (Hlavata et al., glioma and, in recurrent glioblastomas after radiochemo 2012). In contrast, it was up-regulated in the gemcitabine therapy, QPRT expression is associated with a poor prognosis resistant human NSCLC A549 cells (Ikeda et al., 2011). (Sahm et al., 2013). QPRT is a potential marker for the immu 0237. The expression of ACACA was shown to be up nohistochemical screening of follicular thyroid nodules (Hin regulated in numerous human cancers, such as breast, pros schet al., 2009). tate and liver carcinoma and correlated with enhanced lipo 0222 RABGGTB is over-expressed in chemotherapy-re genesis of cancer cells. The various ACACA inhibitors fractory diffuse large B-cell lymphoma (Linderoth et al., showed a therapeutical effect in treatment of cancer cell lines 2008). by Suppression of cell proliferation and inducing of cell death 0223 RAD21 is over-expressed in gastrointestinal through apoptosis (Zu et al., 2013). tumors, colorectal carcinoma, advanced endometrial cancer, 0238 ACLY is aberrantly expressed in various tumors, prostate cancer and breast cancer (Atienza et al., 2005; Deb et Such as breast, liver, colon, lung and prostate cancers, and is al., 2014; Porkka et al., 2004; Supernat et al., 2012; Xu et al., correlated reversely with tumor stage and differentiation (Zu 2014). et al., 2012). 0224 RAD23B has a potential role in breast cancer pro 0239 ACSL3 is over-expressed in lung cancer and based gression (Linge et al., 2014). The single nucleotide polymor on preclinical investigation is a promising new therapeutic phism RAD23B rs1805329 was significantly associated with target in lung cancer (Pei et al., 2013). The up-regulated development and recurrence of HCC in Japanese patients expression of ACSL3 can serve as a potential biomarker of with HCV (Tomoda et al., 2012). estrogen receptor-specific breast cancer risk (Wang et al., 0225 RASAL2 is a RAS-GTPase-activating protein with 2013c). tumor Suppressor functions in estrogen receptor-positive 0240 ACSL4 is over-expressed in estrogen receptor breast cancer, ovarian cancer and lung cancer (Li and Li, negative breast tumors and androgen receptor-negative breast 2014; Huang et al., 2014). In contrast, RASAL2 is oncogenic and prostate tumors. The loss of steroid hormone sensitivity in triple-negative breast cancer and drives mesenchymal inva was associated with induction of ACSL4 expression (Monaco sion and metastasis (Feng et al., 2014a). et al., 2010). The onset up-regulation of ACSL4 was shown to 0226 Depletion of RNMT effectively and specifically occur during the transformation from adenoma to adenocar inhibits cancer cell growth and cell invasive capacities in cinoma (Cao et al., 2001). 0241 The methylation of ACSS3 was found to be associ different types of cancer, inclusing liver cancer (Stefanska et ated with at least one of the classical risk factors, namely age, al., 2014). stage or MYCN status in neuroblastoma (Decock et al., 0227. Over-expression of ROCK1 or mutations in the 2012). ROCK1 gene that lead to an elevated kinase activity have 0242. The deletion of ADSSL 1 was frequently observed in been reported for several cancers, including lung cancer, gas carcinogen-induced mouse primary lung adenocarcinomas, tric carcinoma, CML and AML (Rath and Olson, 2012). mouse and human lung adenocarcinomas cell lines and asso 0228 RPL10A is a c-Myc targeted gene and may contrib ciated with a more extensive chromosome instability pheno ute to hepatocyte transformation (Hunecke et al., 2012). type in the primary mouse lung tumors (Miller et al., 2009). 0229 Inv(3) and t(3; 3) breakpoints, which are associated 0243 AGFG2 was identified to be one of 14 prognostic with a particularly poor prognosis in myeloid leukemia or gene candidates in identifying cases of hormone receptor myelodysplasia, clusterina region that is located centromeric negative or triple-negative breast cancers likely to remain free and downstream of the RPN1 gene (Wieser, 2002). of metastatic relapse (Yau et al., 2010). 0230 RRBP1 is over-expressed in lung cancer and breast 0244 AGT is a very potent anti-angiogenic factor, which cancer (Telikicherla et al., 2012: Tsai et al., 2013). was shown to exert anti-tumoral effects in vitro and in vivo 0231 SCFD1 expression is increased in erosive gastritis, (Bouquet et al., 2006). In transgenic mice, the over-expres which is linked to gastric carcinoma (Galamb et al., 2008). sion of human AGT was shown to decrease angiogenesis and 0232 ABCB1 encodes P-glycoprotein (P-gp) which is thus delaying tumor progression of hepatocarcinoma (Vin expressed in normal cells of various organs such as intestine, cent et al., 2009). liver, kidney, brain, and placenta. P-gp overexpression and 0245 AKR1C4 encodes for a human aldo-keto reductase genetic polymorphisms have been detected in colorectal car family 1 member C4 and catalyzes the reduction of retinal cinoma, tumors derived from the adrenal gland, lung cancer dehyde to retinol (Ruiz et al., 2011). Thus, the depletion of and ALL (Zhang et al., 2013a; Fojo et al., 1987; Gervasini et retinaldehyde down-regulates the biosynthesis of retinoic al., 2006; Jamroziak et al., 2004). acid and is followed by blockage of retinoid signaling, which 0233 ABCB10 encodes for an ABC transporter of the favors tumor progression (Tang and Gudas, 2011; Ruiz et al., sub-family B (MDR/TAP). ABCB10 was shown to be 2012) US 2016/0250307 A1 Sep. 1, 2016

0246 The expression of ALDH1L1 was shown to be metastasis, and osteolytic lesions in vivo (Feng et al., 2013). down-regulated in HCC and gliomas. The down-regulation of ATP6V1C1 was shown to be over-expressed in oral squa ALDH1L1 in those cancers was associated with poorer prog mous cell carcinoma and was associated with tumor cell nosis and more aggressive phenotype (Rodriguez et al., 2008; mobility (Otero-Rey et al., 2008). Chen et al., 2012b) 0260 ATP7B is associated with cancer resistance to cis 0247 The expression of ALG3 was shown to be enhanced platin, the widely used anti-cancer drug (Dmitriev, 2011). in esophageal squamous cell carcinoma and cervical cancer 0261 AXIN2 encodes for Axin- (axis inhibition) related (Shi et al., 2014: Choi et al., 2007). In esophageal squamous protein 2, which presumably plays an important role in the cell carcinoma the increased expression of ALG3 correlated regulation of the stability of beta-catenin in the Wnt signaling with lymph node metastasis (Shi et al., 2014). pathway (Salahshor and Woodgett, 2005). Furthermore, 0248 ANKS1A was identified as a novel target of Src AXIN2 was shown to repress the expression of the oncogene family kinases which are known to be implicated in the devel c-MYC (Rennoll et al., 2014). opment of some colorectal cancers (Emaduddin et al., 2008). 0262. In HCC, the low expression of BAAT was associ 0249 APOA1 encodes for apolipoprotein A-I, the major ated with poorer survival compared to the patients with higher protein component of high density lipoprotein (HDL) in expression of BAAT (Furutani et al., 1996). plasma. In multiple animal tumor models, APOA1 showed a 0263. A strong decrease of transcripts of BHMT and potent immune-modulatory role in the tumorigenesis and was BHMT2 was shown in HepG2 cells and in HCC samples shown to Suppress tumor growth and metastasis by Support compared to normal liver tissue (Pellanda et al., 2012). ing innate and adaptive immune processes (Zamanian-Dary 0264 C12orf44 was shown to be essential for autophagy oush et al., 2013). and interact with ULK1 in an Atgl3-dependent manner (Mer 0250 APOA2 was shown to be significantly decreased in ceret al., 2009). Autophagy has dual roles in cancer, acting as pancreatic cancer patients (Honda et al., 2012). In contrast, both a tumor Suppressor by preventing the accumulation of the increased expression of APOA2 was associated with HCC damaged proteins and organelles and as a mechanism of cell (Liu et al., 2007). survival that can promote the growth of established tumors 0251. In alpha-fetoprotein-negative HBV-related HCC, (Yang et al., 2011 b). APOB was found to be one of the 14 differentially expressed 0265 C17orf70 is a component of the Fanconi anemia proteins which could be associated with HCC progression core complex and is essential for the complex stability. The (He et al., 2014a). In advanced breast cancer, APOB was Fanconi anemia core complex plays a central role in the DNA found to be the one of six differentially expressed proteins damage response network. The Fanconi anemia core com which could predict the responsiveness to neoadjuvant che plex-mediated DNA damage response involves breast cancer motherapy and relapse-free Survival of patients (Hyung et al., susceptibility gene products, BRCA1 and BRCA2 (Ling et 2011). al., 2007). 0252. By stage III colorectal cancer patients and in human 0266 C19orf80 encodes for hepatocellular carcinoma-as melanoma cells, AQP9 was associated with increased sociated gene TD26 and was shown to be one of 5 loci with chemoresistance (Dou et al., 2013; Gao et al., 2012). highest methylation levels in HCC and lowest in control 0253) ARG1 was shown to be a sensitive and specific tissue (Ammerpohl et al., 2012). marker in distinguishing of HCC from other metastatic 0267 CCT7 was found to be a part of a protein sub tumors in liver (Sang et al., 2013). ARG1 may contribute to network, which is significantly discriminative of late stage local immune suppression in NSCLC (Rotondo et al., 2009). human colorectal cancer (Nibbe et al., 2009). 0254 The phosphorylated and thus more active form of 0268 CDK6 has been shown to regulate the activity of ARSB protein was found to be increased in peripheral leuko tumor suppressor protein Rb. CDK6 can exert its tumor cytes from patients with chronic myelogenous leukemiacom promoting function by enhancing proliferation and stimulat pared to healthy donors (Uehara et al., 1983). ing angiogenesis (Kollmann et al., 2013). The pharmacologi 0255. In ovarian cancer cells, the down-regulation of cal inhibition of CDK6 was shown to inhibit the growth ASNA1 was shown to increase the sensitivity to the chemo differentiation of abnormal leukemic cells (Placke et al., therapy drugs cisplatin, carboplatin, oxaliplatin and arsenite 2014). (Hemmingsson et al., 2009). 0269 CFH may play a role in cutaneous squamous cell 0256 ASPH was shown to be over-expressed in various carcinoma progression (Riihila et al., 2014). CFH may play a cancers and cancer cell lines (Yang et al., 2010). Immuniza key role in the resistance of complement-mediated lysis in tion with ASPH-loaded dendritic cells generated cytotoxicity various cancer cells and was shown to be over-expressed in against cholangiocarcinoma cells in vitro and significantly NSCLC, which was associated with poorer prognosis (Cuiet Suppressed intrahepatic tumor growth and metastasis (Noda al., 2011). et al., 2012). 0270. An inactivating mutation of CLPTM1 was found in 0257 ATP1A2 was found among 31 proteins which were prostate cancer cells (Rossi et al., 2005). significantly up-regulated in glioblastoma (Comet al., 2012). 0271 CMAS encodes for cytidine monophosphate In contrast, ATP1A2 was shown to be down-regulated in bone N-acetylneuraminic acid synthetase, which catalyzes the marrow-infiltrating metastatic neuroblastomas (Morandi et activation of Sialic acid and its transformation to a cytidine al., 2012). monophosphate diester. The activated Sialic acid is used for 0258 ATP1A3 was found among 31 proteins, which were N-glycosylation, a common post-translational modification significantly up-regulated in glioblastoma (Comet al., 2012). during cellular differentiation. The increased expression of 0259 ATP6V1C1 may promote breast cancer growth and sialic acid Sugars on the Surface of cancer cells is one of bone metastasis through regulation of lysosomal V-ATPase well-known tumor characteristics (Bull et al., 2014). activity. ATP6V1C1 knockdown significantly inhibited 0272 TF (Transferrin) is one of the most widely used mouse 4T1 mammary tumor cell Xenograft tumor growth, tumor-targeted ligands, because TF receptors (TFRs) are US 2016/0250307 A1 Sep. 1, 2016 34 over-expressed on malignant cells and play a key role in 2004) and in the sulfation of the 6-sulfolactosamine epitope in cellular iron uptake through the interaction with TF (Biswas a human colorectal carcinoma cell line (Kamiyama et al., et al., 2013). The expression level of TFRs has been suggested 2006). Colorectal carcinoma cell lines as well as human col to correlate with tumor stage or cancer progression (Tor orectal tissues express SLC35B2 (Kamiyama et al., 2011). torella and Karagiannis, 2014). 0283 PLOD1 expression is associated with human breast 0273 TH1L might play an important role in regulation of cancer progression (Gilkes et al., 2013). proliferation and invasion in human breast cancer, and could 0284 PRDX5 is up-regulated in many malignant tumors be a potential target for human breast cancer treatment (Zouet (Urig and Becker, 2006) and inhibition of PRDX5 could al., 2010). prevent the tumor initiation and progression, Suggesting (0274 THTPA hydrolysis might be responsible for the PRDX5 to be a promising target for cancer therapy. Its highly anti-proliferative effects of Ndrg-1. Ndrg-1 has been shown to nucleophilic and accessible selenocysteine active site might reduce the invasion and metastasis of breast, colon, prostate be the prime target for drug design (Liu et al., 2012). and pancreatic cancer (Kovacevic et al., 2008). (0285 Increased expression of PSMD8 in the peripheral 0275 SMYD3 promotes cancer invasion by epigenetic lung may be potentially informative as to what critical cell up-regulation of the metalloproteinase MMP-9 (Medjkane et populations are involved in the development of invasive can al., 2012). Expression of SMYD3 is undetectable or very cers (Zhou et al., 1996). weak in many types of normal human tissue, whereas over 0286 SNRPD1 is a core spliceosomal protein, which is expression of SMYD3 has been linked with the development up-regulated in malignant tumors. and progression of gastric, colorectal, hepatocellular, prostate (0287 Reduced expression of the SPTBN1 is associated and breast cancers (Hamamoto et al., 2006; Liu et al., 2014: with worsened prognosis in pancreatic cancer (Jiang et al., Liu et al., 2013a). 2010). 0276 A link between STAT2 and tumorigenesis was 0288 SQSTM1 functions as a signaling hub for various observed in transgenic mice lacking STAT2 (Yue et al., 2015) signal transduction pathways, such as NF-KB signaling, apo or expressing constitutively IFN-C. in the brain (Wang et al., ptosis, and Nrf2 activation, whose dysregulation is associated 2003). with Paget disease of bone and tumorigenesis (Komatsu et al., 0277 TACC3 is over-expressed in many human cancers, 2012). including ovarian cancer, breast cancer, squamous cell carci 0289 PCNA expression predicts survival in anorectal noma and lymphoma (Ma et al., 2003; Jacquemier et al., malignant melanoma (Ben-Izhak et al., 2002). A cancer-as 2005; Lauffart et al., 2005). sociated isoform of PCNA (calPCNA) was identified that 0278 SPBP is also shown to repress the transcriptional contained an unusual pattern of methyl ester groups on activity of estrogen receptor C. (ERC). Over-expression of numerous glutamic and aspartic acid residues within PCNA SPBP inhibited the proliferation of an ERC-dependent breast (Hoelz et al., 2006). cancer cell line (Gburcik et al., 2005). In the cell nucleus, 0290. Depleting SRP54 in several tumor cell lines did not SPBP displays relatively low mobility and is enriched in produce overt cellular phenotypes, such as growth arrest or chromatin dense regions, clearly indicating that it is a chro death, even in cells selected for stable reduction of SRP matin binding protein (Darvekar et al., 2012). TCF20 is components (Ren et al., 2004). important for enhanced induction of proteins involved in the 0291. At the molecular level, STAT1 inhibits the prolifera cellular defensive program against oxidative stress (Darvekar tion of both mouse and human tumor cells treated with IFN-y et al., 2014). via its ability to increase the expression of cyclin-dependent 0279) C3 is a prominent element of the inflammatory kinase inhibitor p21(Cip1, or to decrease c-myc expression tumor microenvironment (Rutkowski et al., 2010) and acti (Ramana et al., 2000). The anti-tumor activity of STAT1 is Vation can give a tumor growth advantage (Markiewski et al., further Supported by its ability to inhibit angiogenesis and 2008). Enzymatic cleavage of C3 leads to the production of tumor metastasis in mouse models (Huang et al., 2002). the anaphylatoxin C3a, an inflammatory mediator and Increased STAT1 mRNA levels were shown to be part of a chemoattractant, and C3b (Sahu et al., 1998). molecular signature associated with better prediction of the 0280 CLN3 is an anti-apoptotic gene in NT2 neuronal metastatic outcome for patients with hormone receptor nega precursor cells and a few types of cancers (Zhu et al., 2014b). tive and triple-negative breast cancers (Yau et al., 2010). It is involved in intracellular trafficking and regulation in 0292 Fine-needle aspirate samples from follicular neo neuronal and non-neuronal cells (Rakheja et al., 2008: Getty plasms demonstrated that malignant nodules over-express and Pearce, 2011) and it is implicated in several important STT3A as compared with benign disease (Patel et al., 2011). signaling pathways (Persaud-Sawin et al., 2002). CLN3 0293. A meta-analysis showed that the STXBP4/COX11 mRNA and protein are over-expressed in a number of cancer rs6504950 polymorphism is significantly correlated with cell lines including breast, colon, malignant melanoma, pros breast cancer risk (Tang et al., 2012). tate, ovarian, neuroblastoma, and glioblastoma multiforme, 0294. A peptide consisting or consisting essentially of the but not lung or pancreatic cancer cell lines (Rylova et al., amino acid sequence as indicated herein can have one or two 2002). non-anchor amino acids (see below regarding the anchor (0281) SLC13A5 is one of 7 CIMP-marker genes. CIMP motif) exchanged without that the ability to bind to a mol (CpG island methylator phenotype) of clear cell renal cell ecule of the human major histocompatibility complex (MHC) carcinomas (ccRCCs) is characterized by accumulation of class-I or-II is substantially changed or is negatively affected, DNA methylation at CpG islands and poorer patient outcome when compared to the non-modified peptide. In another (Tian et al., 2014; Arai et al., 2012). embodiment, in a peptide consisting essentially of the amino 0282 SLC35B2 is involved in coordinated transcriptional acid sequence as indicated herein, one or two amino acids can regulation during induction of Sialyl Sulfo-Lex glycan bio be exchanged with their conservative exchange partners (see synthesis during acute inflammation (Huopaniemi et al., herein below) without that the ability to bind to a molecule of US 2016/0250307 A1 Sep. 1, 2016 the human major histocompatibility complex (MHC) class-I aptamers, have been Successfully employed for targeted or-II is Substantially changed, or is negatively affected, when therapies and shown to be functional in Xenograft in vivo compared to the non-modified peptide. models. Furthermore, aptamers recognizing specific tumour 0295 The present invention further relates to a peptide cell lines have been identified. according to the present invention, wherein said peptide is 0306 DNAaptamers can be selected to reveal broad-spec modified and/or includes non-peptide bonds as described trum recognition properties for various cancer cells, and par herein below. ticularly those derived from solid tumours, while non-tu 0296. The present invention further relates to a peptide mourgenic and primary healthy cells are not recognized. If according to the present invention, wherein said peptide is the identified aptamers recognise not only a specific tumour part of a fusion protein, in particular fused to the N-terminal sub-type but rather interact with a series of tumours, this amino acids of the HLA-DR antigen-associated invariant renders the aptamers applicable as so-called broad-spectrum chain (Ii), or fused to (or into the sequence of) an antibody, diagnostics and therapeutics. Such as, for example, an antibody that is specific for dendritic 0307 Further, investigation of cell-binding behaviour cells, i.e. binds to dendritic cells. with flow cytometry showed that aptamers revealed very 0297. The present invention further relates to a nucleic good apparent affinities in the nanomolar range. acid, encoding for a peptide according to the present inven 0308 Aptamers are useful for diagnostic and therapeutic tion. The present invention further relates to the nucleic acid according to the present invention that is DNA, cDNA, PNA, purposes. Further, it could be shown that some of the aptam RNA or combinations thereof. ers are taken up by tumour cells and thus can function as 0298. The present invention further relates to an expres molecular vehicles for the targeted delivery of anti-cancer sion vector capable of expressing, expressing, and/or present agents such as siRNA into tumour cells. ing a nucleic acid according to the present invention. 0309 Aptamers can be selected against complex targets 0299 The present invention further relates to a peptide Such as cells and tissues and complexes of the peptides com according to the present invention, a nucleic acid according to prising, preferably consisting of a sequence according to any the present invention or an expression vector according to the of SEQID NO 1 to SEQID NO 300, according to the present present invention for use in medicine. invention with the MHC molecule, using the cell-SELEX 0300. The present invention further relates to antibodies as (Systematic Evolution of Ligands by Exponential enrich described further below, and methods of making them. Pre ment) technique. ferred are antibodies that are specific for the peptides of the 0310. As used herein, the term “scaffold’ refers to a mol present invention, and/or for the peptides of the present inven ecule that specifically binds to an (eg antigenic) determinant. tion when bound to their MHC. Preferred antibodies can be In one embodiment, a scaffold is able to direct the entity to monoclonal. which it is attached (e.g. a (second) antigen binding moiety) 0301 The present invention further relates to T-cell recep to a target site, for example to a specific type of tumor cell or tors (TCR), in particular soluble TCR (STCRs) targeting the tumor stroma bearing the antigenic determinant (e.g. the peptides according to the invention and/or the peptide-MHC complex of a peptide according to the application at hand). In complexes thereof, and methods of making them. another embodiment a scaffold is able to activate signaling 0302) The present invention further relates to antibodies or through its target antigen, for example a T cell receptor com other binding molecules targeting the peptides according to plex antigen. Scaffolds include but are not limited to antibod the invention and/or the peptide-MHC complexes thereof, ies and fragments thereof, antigen binding domains of an and methods of making them. antibody, comprising an antibody heavy chain variable region 0303. The present invention further relates to a host cell and an antibody light chain variable region, binding proteins comprising a nucleic acid according to the present invention comprising at least one ankyrin repeat motif and Single or an expression vector as described before. The present domain antigen binding (SDAB) molecules, apatmers, invention further relates to the host cell according to the (soluble) TCRs and (modified) cells such as allogenic or present invention that is an antigen presenting cell. The autologous T cells. present invention further relates to the host cell according to 0311 Each scaffold can comprise a labelling which pro the present invention, wherein the antigen presenting cell is a vides that the bound scaffold can be detected by determining dendritic cell. the presence or absence of a signal provided by the label. For 0304. The present invention further relates to aptamers. example, the scaffold can be labelled with a fluorescent dye or Aptamers (see for example WO 2014/191359 and the litera any other applicable cellular marker molecule. Such marker ture cited therein) are short single-stranded nucleic acid or molecules are well known in the art. For example a fluores peptide molecules, which can fold into defined three-dimen cence-labelling, for example provided by a fluorescence dye, sional structures and recognize specific target structures. can provide a visualisation of the boundaptamer by fluores They have appeared to be suitable alternatives for developing cence or laser scanning microscopy or flow cytometry. targeted therapies. Aptamers have been shown to selectively 0312. Each scaffold can be conjugated with a second bind to a variety of complex targets with high affinity and active molecule such as for example IL-21, anti-CD3, anti specificity. CD28. Polypeptide scaffolds are described, for example, in 0305 Aptamers recognizing cell surface located mol the background section of WO 2014/071978A1, and the ref ecules have been identified within the past decade and pro erences as cited therein. vide means for developing diagnostic and therapeutic 0313 The present invention further relates to a method of approaches. Since aptamers have been shown to possess producing a peptide according to the present invention, said almost no toxicity and immunogenicity they are promising method comprising culturing the host cell according to the candidates for biomedical applications. Indeed aptamers, for present invention, and isolating the peptide from the host cell example prostate-specific membrane-antigen recognizing and/or its culture medium. US 2016/0250307 A1 Sep. 1, 2016 36

0314. The present invention further relates to an in vitro cells. The isolation of T-cells from tumor-infiltrating cell method for producing activated T-cells, the method compris populations or from peripheral blood Suggests that such cells ing contacting in vitro T cells with antigen loaded human play an important role in natural immune defense against class I or II MHC molecules expressed on the surface of a cancer. CD8-positive T-cells in particular, which recognize Suitable antigen-presenting cell for a period of time Sufficient Class I molecules of the major histocompatibility complex to activate said T cells in an antigen specific manner, wherein (MHC)-bearing peptides of usually 8 to 10 amino acid resi said antigen is at least one peptide according to the present dues derived from proteins or defect ribosomal products invention. The present invention further relates to a method, (DRIPS) located in the cytosol, play an important role in this wherein the antigen is loaded onto class I or II MHC mol response. The MHC-molecules of the human are also desig ecules expressed on the Surface of a suitable antigen-present nated as human leukocyte-antigens (HLA). ing cell by contacting a sufficient amount of the antigen with 0327. The term "peptide' is used herein to designate a an antigen-presenting cell. series of amino acid residues, connected one to the other 0315. The present invention further relates to the method typically by peptide bonds between the alpha-amino and car according to the present invention, wherein the antigen-pre bonyl groups of the adjacent amino acids. The peptides are senting cell comprises an expression vector capable of preferably 9 amino acids in length, but can be as short as 8 expressing said peptide containing SEQID NO: 1 to SEQID amino acids in length, and as long as 10, 11, 12, or 13 and in NO: 300, or a variant amino acid sequence thereof. case of MHC class II peptides (elongated variants of the 0316 The present invention further relates to activated T peptides of the invention) they can be as long as 14, 15, 16, 17. cells, produced by the method according to the present inven 18, 19 or 20 amino acids in length. tion, which selectively recognize a cell which aberrantly 0328. Furthermore, the term "peptide' shall include salts expresses a polypeptide comprising an amino acid sequence of a series of amino acid residues, connected one to the other according to the present invention. typically by peptide bonds between the alpha-amino and car 0317. The present invention further relates to a method of bonyl groups of the adjacentamino acids. Preferably, the salts killing target cells in a patient which target cells aberrantly are pharmaceutical acceptable salts of the peptides, such as, express a polypeptide comprising any amino acid sequence for example, the chloride or acetate (trifluoroacetate) salts. It according to the present invention, the method comprising has to be noted that the salts of the peptides according to the administering to the patient an effective number of T cells as present invention differ substantially from the peptides in according to the present invention. their state(s) in Vivo, as the peptides are not salts in vivo. 0318. The present invention further relates to the use of 0329. The term "peptide' shall also include "oligopep any peptide described, a nucleic acid according to the present tide'. The term "oligopeptide' is used herein to designate a invention, an expression vector according to the present series of amino acid residues, connected one to the other invention, a cell according to the present invention, or an typically by peptide bonds between the alpha-amino and car activated T-cell according to the present invention as a medi bonyl groups of the adjacent amino acids. The length of the cament or in the manufacture of a medicament. oligopeptide is not critical to the invention, as long as the 0319. The present invention further relates to a use accord correct epitope or epitopes are maintained therein. The oli ing to the present invention, wherein said medicament is a gopeptides are typically less than about 30 amino acid resi vaccine, a cell, a cell population, Such as, for example, a cell dues in length, and greater than about 15 amino acids in line, sTCRs and monoclonal antibodies. length. 0320. The present invention further relates to a use accord 0330. The term “the peptides of the present invention” ing to the present invention, wherein the medicament is active shall also include the peptides consisting of or comprising a against cancer. peptide as defined above according to SEQID NO: 1 to SEQ 0321. The present invention further relates to a use accord ID NO:3OO. ing to the present invention, wherein said cancer cells are cells 0331. The term “polypeptide' designates a series of amino of HCC. acid residues, connected one to the other typically by peptide 0322 The present invention further relates to particular bonds between the alpha-amino and carbonyl groups of the marker proteins and biomarkers based on the peptides accord adjacent amino acids. The length of the polypeptide is not ing to the present invention that can be used in the diagnosis critical to the invention as long as the correct epitopes are and/or prognosis of HCC. maintained. In contrast to the terms peptide or oligopeptide, 0323 Furthermore, the present invention relates to the use the term polypeptide is meant to refer to molecules containing of these novel targets for cancer treatment. more than about 30 amino acid residues. 0324 Further, the present invention relates to a method for 0332 A peptide, oligopeptide, protein or polynucleotide producing a personalized anti-cancer vaccine for an indi coding for Such a molecule is “immunogenic' (and thus is an vidual patient using a database (herein designated also as “immunogen' within the present invention), if it is capable of “warehouse') of pre-screened tumor associated peptides. inducing an immune response. In the case of the present 0325 Stimulation of an immune response is dependent invention, immunogenicity is more specifically defined as the upon the presence of antigens recognized as foreign by the ability to induce a T-cell response. Thus, an “immunogen host immune system. The discovery of the existence of tumor would be a molecule that is capable of inducing an immune associated antigens has raised the possibility of using a hosts response, and in the case of the present invention, a molecule immune system to intervene in tumor growth. Various mecha capable of inducing a T-cell response. In another aspect, the nisms of harnessing both the humoral and cellular arms of the immunogen can be the peptide, the complex of the peptide immune system are currently being explored for cancer with MHC, oligopeptide, and/or protein that is used to raise immunotherapy. specific antibodies or TCRs against it. 0326 Specific elements of the cellular immune response 0333. A class IT cell "epitope” requires a short peptide are capable of specifically recognizing and destroying tumor that is bound to a class I MHC receptor, forming a ternary US 2016/0250307 A1 Sep. 1, 2016 37 complex (MHC class I alpha chain, beta-2-microglobulin, TABLE 6-continued and peptide) that can be recognized by a T cell bearing a matching T-cell receptor binding to the MHC/peptide com Expression frequencies F of HLA-A*02 and HLA-A*24 and the most frequent HLA-DR serotypes. Frequencies are deduced from haplotype plex with appropriate affinity. Peptides binding to MHC class frequencies Gf within the American population adapted from Mori et I molecules are typically 8-14 amino acids in length, and most al. (Mori M., et al. HLA gene and haplotype frequencies in the North typically 9 amino acids in length. American population: the National Marrow Donor Program Donor Registry. Transplantation. 1997 Oct. 15; 64(7): 1017-27) employing 0334. In humans there are three different genetic loci that the Hardy-Weinberg formula F = 1 - (1 - Gf°. Combinations of A*02 encode MHC class I molecules (the MHC-molecules of the or A*24 with certain HLA-DR alleles might be enriched or less frequent human are also designated human leukocyte antigens than expected from their single frequencies due to linkage disequilib (HLA)): HLA-A. HLA-B, and HLA-C. HLA-A*01, HLA rium. For details refer to Chanocket al. (S. J. Chanock, et al (2004) A*02, and HLA-B807 are examples of different MHC class I HLA-A, -B, -Cw, -DQA1 and DRB1 in an African American popula alleles that can be expressed from these loci. tion from Bethesda, USA Human Immunology, 65: 1223-1235). Calculated phenotype TABLE 6 Allele Population from allele frequency Expression frequencies F of HLA-A*02 and HLA-A*24 and the most A*24 India 47% frequent HLA-DR serotypes. Frequencies are deduced from haplotype A*24 South Korea 40% frequencies Gf within the American population adapted from Mori et A*24 Sri Lanka 37% al. (Mori M., et al. HLA gene and haplotype frequencies in the North A*24 China 32% American population: the National Marrow Donor Program Donor A*24:O2 India 29% Registry. Transplantation. 1997 Oct. 15; 64(7): 1017-27) employing A*24 Australia West 22% the Hardy-Weinberg formula F = 1 - (1 - Gf°. Combinations of A*02 A*24 USA 22% or A*24 with certain HLA-DR alleles might be enriched or less frequent A*24 Russia Samara 20% than expected from their single frequencies due to linkage disequilib A*24 South America 20% rium. For details refer to Chanocket al. (S. J. Chanock, etal (2004) A*24 Europe 18% HLA-A, -B, -Cw, -DQA1 and DRB1 in an African American popula tion from Bethesda, USA Human Immunology, 65. 1223-1235). 0335 The peptides of the invention, preferably when Calculated phenotype included into a vaccine of the invention as described herein Allele Population from allele frequency bind to A*02 or A*24. A vaccine may also include pan A*02 Caucasian (North America) 49.1% binding MHC class II peptides. Therefore, the vaccine of the A*02 African American (North America) 34.1% invention can be used to treat cancer in patients that are either A*02 Asian American (North America) 43.2% A*02 positive, A*24 positive or positive for A*02 and A*24, A*02 Latin American (North American) 48.3% DR1 Caucasian (North America) 19.4% whereas no selection for MHC class II allotypes is necessary DR2 Caucasian (North America) 28.2% due to the pan-binding nature of these peptides. DR3 Caucasian (North America) 20.6% 0336 Combining for example A*02 and A*24 peptides in DR4 Caucasian (North America) 30.7% one vaccine has the advantage that a higher percentage of any DRS Caucasian (North America) 23.3% DR6 Caucasian (North America) 26.7% patient population can be treated compared with addressing DR7 Caucasian (North America) 24.8% either MHC class I allele alone. While in most populations DR8 Caucasian (North America) 5.7% less than 50% of patients could be addressed by either allele DR9 Caucasian (North America) 2.1% alone, the vaccine of the invention can treat at least 60% of DR1 African (North) American 13.20% DR2 African (North) American 29.80% patients in any relevant population. Specifically, the follow DR3 African (North) American 24.80% ing percentages of patients will be positive for at least one of DR4 African (North) American 11.10% these alleles in various regions: USA 61%, Western Europe DRS African (North) American 31.10% 62%, China 75%, South Korea 77%, Japan 86% (calculated DR6 African (North) American 33.70% DR7 African (North) American 19.20% from www.allelefrequencies.net). DR8 African (North) American 12.10% 0337 As used herein, reference to a DNA sequence DR9 African (North) American S.80% includes both single stranded and double stranded DNA. DR1 Asian (North) American 6.80% DR2 Asian (North) American 33.80% Thus, the specific sequence, unless the context indicates oth DR3 Asian (North) American 9.20% erwise, refers to the single strand DNA of such sequence, the DR4 Asian (North) American 28.60% duplex of Such sequence with its complement (double DRS Asian (North) American 30.00% stranded DNA) and the complement of such sequence. The DR6 Asian (North) American 25.10% DR7 Asian (North) American 13.40% term "coding region” refers to that portion of a gene which DR8 Asian (North) American 12.70% either naturally or normally codes for the expression product DR9 Asian (North) American 18.60% of that gene in its natural genomic environment, i.e., the DR1 Latin (North) American 15.30% region coding in vivo for the native expression product of the DR2 Latin (North) American 21.20% gene. DR3 Latin (North) American 15.20% DR4 Latin (North) American 36.80% 0338. The coding region can be derived from a non-mu DRS Latin (North) American 20.00% tated (“normal'), mutated or altered gene, or can even be DR6 Latin (North) American 31.10% derived from a DNA sequence, or gene, wholly synthesized in DR7 Latin (North) American 20.20% DR8 Latin (North) American 18.60% the laboratory using methods well known to those of skill in DR9 Latin (North) American 2.10% the art of DNA synthesis. A*24 Philippines 65% 0339. In a preferred embodiment, the term “nucleotide A*24 Russia Nenets 61% A*24:O2 Japan 59% sequence” refers to a heteropolymer of deoxyribonucle A*24 Malaysia S8% otides. A*24:O2 Philippines 54% 0340. The nucleotide sequence coding for a particular peptide, oligopeptide, or polypeptide may be naturally occur US 2016/0250307 A1 Sep. 1, 2016

ring or they may be synthetically constructed. Generally, art. For example, individual clones isolated from a cDNA DNA segments encoding the peptides, polypeptides, and pro library have been conventionally purified to electrophoretic teins of this invention are assembled from cDNA fragments homogeneity. Purification of starting material or natural and short oligonucleotide linkers, or from a series of oligo material to at least one order of magnitude, preferably two or nucleotides, to provide a synthetic gene that is capable of three orders, and more preferably four or five orders of mag being expressed in a recombinant transcriptional unit com nitude is expressly contemplated. Furthermore, a claimed prising regulatory elements derived from a microbial or viral polypeptide which has a purity of preferably 99.999%, or at operon. least 99.99% or 99.9%; and even desirably 99% by weight or 0341. As used herein the term “a nucleotide coding (or greater is expressly contemplated. encoding) for a peptide' refers to a nucleotide sequence cod 0349 The nucleic acids and polypeptide expression prod ing for the peptide including artificial (man-made) start and ucts disclosed according to the present invention, as well as stop codons compatible for the biological system the expression vectors containing Such nucleic acids and/or Such sequence is to be expressed by, for example, a dendritic cellor polypeptides, may be in "enriched form'. As used herein, the another cell system useful for the production of TCRs. term "enriched' means that the concentration of the material 0342. The term “expression product” means the polypep is at least about 2, 5, 10, 100, or 1000 times its natural tide or protein that is the natural translation product of the concentration (for example), advantageously 0.01%, by gene and any nucleic acid sequence coding equivalents result weight, preferably at least about 0.1% by weight. Enriched ing from genetic code degeneracy and thus coding for the preparations of about 0.5%, 1%. 5%, 10%, and 20% by same amino acid(s). weight are also contemplated. The sequences, constructs, 0343. The term “fragment', when referring to a coding vectors, clones, and other materials comprising the present sequence, means a portion of DNA comprising less than the invention can advantageously be in enriched or isolated form. complete coding region, whose expression product retains 0350. The term “active fragment’ means a fragment, usu essentially the same biological function or activity as the ally of a peptide, polypeptide or nucleic acid sequence, that expression product of the complete coding region. generates an immune response (i.e., has immunogenic activ (0344) The term “DNA segment” refers to a DNA polymer, ity) when administered, alone or optionally with a suitable in the form of a separate fragment or as a component of a adjuvant or in a vector, to an animal. Such as a mammal, for larger DNA construct, which has been derived from DNA example, a rabbit or a mouse, and also including a human, isolated at least once in Substantially pure form, i.e., free of Such immune response taking the form of stimulating a T-cell contaminating endogenous materials and in a quantity or response within the recipient animal, such as a human. Alter concentration enabling identification, manipulation, and natively, the “active fragment may also be used to induce a recovery of the segment and its component nucleotide T-cell response in vitro. sequences by standard biochemical methods, for example, by 0351. As used herein, the terms “portion”, “segment” and using a cloning vector. Such segments are provided in the “fragment, when used in relation to polypeptides, refer to a form of an open reading frame uninterrupted by internal continuous sequence of residues, such as amino acid residues, non-translated sequences, or introns, which are typically which sequence forms a Subset of a larger sequence. For present in eukaryotic genes. Sequences of non-translated example, if a polypeptide were subjected to treatment with DNA may be present downstream from the open reading any of the common endopeptidases, such as trypsin or chy frame, where the same do not interfere with manipulation or motrypsin, the oligopeptides resulting from Such treatment expression of the coding regions. would represent portions, segments or fragments of the start 0345 The term “primer’ means a short nucleic acid ing polypeptide. When used in relation to polynucleotides, sequence that can be paired with one strand of DNA and these terms refer to the products produced by treatment of provides a free 3'-OH end at which a DNA polymerase starts said polynucleotides with any of the endonucleases. synthesis of a deoxyribonucleotide chain. 0352. In accordance with the present invention, the term 0346. The term “promoter” means a region of DNA "percent homology”, “percent identity” or “percent identi involved in binding of RNA polymerase to initiate transcrip cal, when referring to a sequence, means that a sequence is tion. compared to a claimed or described sequence after alignment 0347 The term "isolated” means that the material is of the sequence to be compared (the “Compared Sequence') removed from its original environment (e.g., the natural envi with the described or claimed sequence (the “Reference ronment, if it is naturally occurring). For example, a natu Sequence'). The Percent Identity is then determined accord rally-occurring polynucleotide or polypeptide present in a ing to the following formula: living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting Percent Identity=1001-(C/R) materials in the natural system, is isolated. Such polynucle wherein C is the number of differences between the Refer otides could be part of a vector and/or such polynucleotides or ence Sequence and the Compared Sequence over the length of polypeptides could be part of a composition, and still be alignment between the Reference Sequence and the Com isolated in that Such vector or composition is not part of its pared Sequence, wherein natural environment. 0348. The polynucleotides, and recombinant or immuno (i) each base or amino acid in the Reference Sequence that genic polypeptides, disclosed in accordance with the present does not have a corresponding aligned base or amino acid in invention may also be in “purified form. The term “purified’ the Compared Sequence and does not require absolute purity; rather, it is intended as a (ii) each gap in the Reference Sequence and relative definition, and can include preparations that are (iii) each aligned base or amino acid in the Reference highly purified or preparations that are only partially purified, Sequence that is different from an aligned base or amino acid as those terms are understood by those of skill in the relevant in the Compared Sequence, constitutes a difference and US 2016/0250307 A1 Sep. 1, 2016 39

(iii) the alignment has to start at position 1 of the aligned substitutions result in additive or synergistic effects on the sequences; and R is the number of bases or amino acids in the antigenicity of the peptide. At most, no more than 4 positions Reference Sequence over the length of the alignment with the within the peptide would simultaneously be substituted. Compared Sequence with any gap created in the Reference 0359 The peptides of the invention can be elongated by up Sequence also being counted as a base or amino acid. to four amino acids, that is 1, 2, 3 or 4 amino acids can be 0353. If an alignment exists between the Compared added to either end in any combination between 4:0 and 0:4. Sequence and the Reference Sequence for which the Percent Identity as calculated above is about equal to or greater than 0360 Combinations of the elongations according to the a specified minimum Percent Identity then the Compared invention can be depicted from Table 7: Sequence has the specified minimum Percent Identity to the Reference Sequence even though alignments may exist in C-terminus N-terminus which the herein above calculated Percent Identity is less than the specified Percent Identity. 4 O 3 O or 1 0354. The original (unmodified) peptides as disclosed 2 0 or 1 or 2 herein can be modified by the substitution of one or more 1 0 or 1 or 2 or 3 residues at different, possibly selective, sites within the pep O 0 or 1 or 2 or 3 or 4 tide chain, if not otherwise stated. Preferably those substitu tions are located at the end of the amino acid chain. Such N-terminus C-terminus Substitutions may be of a conservative nature, for example, 4 O where one amino acid is replaced by an amino acid of similar 3 O or 1 structure and characteristics, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. C-terminus N-terminus Even more conservative would be replacement of amino acids 2 0 or 1 or 2 of the same or similar size and chemical nature, Such as where 1 0 or 1 or 2 or 3 leucine is replaced by isoleucine. In studies of sequence O 0 or 1 or 2 or 3 or 4 variations in families of naturally occurring homologous pro teins, certain amino acid Substitutions are more often toler 0361. The amino acids for the elongation/extension can be ated than others, and these are often show correlation with the peptides of the original sequence of the protein or any similarities in size, charge, polarity, and hydrophobicity other amino acid(s). The elongation can be used to enhance between the original amino acid and its replacement, and Such the stability or solubility of the peptides. is the basis for defining "conservative substitutions.” 0362. The term “T-cell response' means the specific pro 0355 Conservative substitutions are herein defined as liferation and activation of effector functions induced by a exchanges within one of the following five groups: Group peptide in vitro or in vivo. For MHC class I restricted CTLs, 1-small aliphatic, nonpolar or slightly polar residues (Ala, effector functions may be lysis of peptide-pulsed, peptide Ser. Thr, Pro, Gly); Group 2-polar, negatively charged resi precursor pulsed or naturally peptide-presenting target cells, dues and their amides (Asp, ASn, Glu, Gln); Group 3-polar, secretion of cytokines, preferably Interferon-gamma, TNF positively charged residues (His, Arg, Lys); Group 4-large, alpha, or IL-2 induced by peptide, secretion of effector mol aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); and ecules, preferably granzymes or perforins induced by pep Group 5-large, aromatic residues (Phe, Tyr, Trp). tide, or degranulation. 0356. Less conservative substitutions might involve the replacement of one amino acid by another that has similar 0363 Preferably, when the T cells specific for a peptide characteristics but is somewhat different in size, such as according to the present invention are tested against the Sub replacement of an alanine by an isoleucine residue. Highly stituted peptides, the peptide concentration at which the Sub non-conservative replacements might involve Substituting an stituted peptides achieve half the maximal increase in lysis acidic amino acid for one that is polar, or even for one that is relative to background is no more than about 1 mM, prefer basic in character. Such “radical substitutions cannot, how ably no more than about 1 uM, more preferably no more than ever, be dismissed as potentially ineffective since chemical about 1 nM, and still more preferably no more than about 100 effects are not totally predictable and radical substitutions uM, and most preferably no more than about 10 uM. It is also might well give rise to serendipitous effects not otherwise preferred that the substituted peptide be recognized by T cells predictable from simple chemical principles. from more than one individual, at least two, and more pref 0357 Of course, such substitutions may involve structures erably three individuals. other than the common L-amino acids. Thus, D-amino acids 0364 Thus, the epitopes of the present invention may be might be substituted for the L-amino acids commonly found identical to naturally occurring tumor-associated or tumor in the antigenic peptides of the invention and yet still be specific epitopes or may include epitopes that differ by no encompassed by the disclosure herein. In addition, amino more than four residues from the reference peptide, as long as acids possessing non-standard R groups (i.e., R groups other they have Substantially identical antigenic activity. than those found in the common 20 amino acids of natural 0365 MHC class I molecules can be found on most cells proteins) may also be used for Substitution purposes to pro having a nucleus which present peptides that result from duce immunogens and immunogenic polypeptides according proteolytic cleavage of mainly endogenous, cytosolic or to the present invention. nuclear proteins, DRIPS, and larger peptides. However, pep 0358 If substitutions at more than one position are found tides derived from endosomal compartments or exogenous to result in a peptide with Substantially equivalent or greater sources are also frequently found on MHC class I molecules. antigenic activity as defined below, then combinations of This non-classical way of class I presentation is referred to as those substitutions will be tested to determine if the combined cross-presentation in literature. US 2016/0250307 A1 Sep. 1, 2016 40

0366 Since both types of response, CD8 and CD4 depen 0373 The pharmaceutical compositions comprise the dent, contribute jointly and synergistically to the anti-tumor peptides either in the free form or in the form of a pharma effect, the identification and characterization of tumor-asso ceutically acceptable salt (see also above). As used herein, “a ciated antigens recognized by either CD8-positive T cells pharmaceutically acceptable salt” refers to a derivative of the (MHC class I molecule) or by CD4-positive T cells (MHC disclosed peptides wherein the peptide is modified by making class II molecule) is important in the development of tumor acid or base salts of the agent. For example, acid salts are vaccines. It is therefore an object of the present invention, to prepared from the free base (typically wherein the neutral provide compositions of peptides that contain peptides bind form of the drug has a neutral —NH2 group) involving reac ing to MHC complexes of either class. tion with a suitable acid. Suitable acids for preparing acid 0367 Considering the severe side-effects and expense salts include both organic acids, e.g., acetic acid, propionic associated with treating cancer better prognosis and diagnos acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, tic methods are desperately needed. Therefore, there is a need malonic acid. Succinic acid, maleic acid, fumaric acid, tartaric to identify other factors representing biomarkers for cancer in acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, general and HCC in particular. Furthermore, there is a need to methane Sulfonic acid, ethane Sulfonic acid, p-toluene identify factors that can be used in the treatment of cancer in Sulfonic acid, Salicylic acid, and the like, as well as inorganic general and HCC in particular. acids, e.g., hydrochloric acid, hydrobromic acid, Sulfuric 0368. The present invention provides peptides that are acid, nitric acid phosphoric acid and the like. Conversely, useful in treating cancers/tumors, preferably HCC that over preparation of basic salts of acid moieties which may be or exclusively present the peptides of the invention. These present on a peptide are prepared using a pharmaceutically peptides were shown by mass spectrometry to be naturally acceptable base Such as Sodium hydroxide, potassium presented by HLA molecules on primary human HCC hydroxide, ammonium hydroxide, calcium hydroxide, trim samples. ethylamine or the like. 0369. The source gene/protein (also designated “full 0374. In an especially preferred embodiment, the pharma length protein’ or “underlying protein') from which the pep ceutical compositions comprise the peptides as salts of acetic tides are derived were shown to be highly overexpressed in acid (acetates), trifluoro acetates or hydrochloric acid (chlo cancer compared with normal tissues—"normal tissues in rides). relation to this invention shall mean either healthy liver cells 0375 Especially preferred is a composition and/or the use or other normal tissue cells, demonstrating a high degree of of said composition, e.g. in the form of a vaccine, comprising tumor association of the source genes (see example 2). More the peptides having a sequence according to the SEQID NOS over, the peptides themselves are strongly over-presented on 1, 2, 7, 225, 228, 301, 303, and 312 or a scaffold reactive tumor tissue "tumor tissue’ in relation to this invention against the peptides having a sequence according to the SEQ shall mean a sample from a patient suffering from HCC, but ID NOS 1, 2, 7, 225, 228,301,303, and 312 and their com not on normal tissues (see Example 1). plexes to MHC molecules. 0370 HLA-bound peptides can be recognized by the 0376. In addition to being useful for treating cancer, the immune system, specifically T lymphocytes. T cells can peptides of the present invention are also useful as diagnos destroy the cells presenting the recognized HLA/peptide tics. Since the peptides were generated from HCC cells and complex, e.g. HCC cells presenting the derived peptides. since it was determined that these peptides are not or at lower 0371. The peptides of the present invention have been levels present in normal tissues, these peptides can be used to shown to be capable of stimulating T cell responses and/or are diagnose the presence of a cancer. over-presented and thus can be used for the production of 0377 The presence of claimed peptides on tissue biopsies antibodies and/or TCRs, in particular sTCRs, according to the in blood samples can assist a pathologist in diagnosis of present invention (see Example 3). Furthermore, the peptides cancer. Detection of certain peptides by means of antibodies, when complexed with the respective MHC can be used for the mass spectrometry or other methods known in the art can tell production of antibodies and/or TCRs, in particular sTCRs, the pathologist that the tissue sample is malignant or inflamed according to the present invention, as well. Respective meth or generally diseased, or can be used as a biomarker for HCC. ods are well known to the person of skill, and can be found in Presence of groups of peptides can enable classification or the respective literature as well. Thus, the peptides of the Sub-classification of diseased tissues. present invention are useful for generating an immune 0378. The detection of peptides on diseased tissue speci response in a patient by which tumor cells can be destroyed. men can enable the decision about the benefit of therapies An immune response in a patient can be induced by direct involving the immune system, especially if T-lymphocytes administration of the described peptides or suitable precursor are known or expected to be involved in the mechanism of Substances (e.g. elongated peptides, proteins, or nucleic acids action. Loss of MHC expression is a well described mecha encoding these peptides) to the patient, ideally in combina nism by which infected of malignant cells escape immuno tion with an agent enhancing the immunogenicity (i.e. an Surveillance. Thus, presence of peptides shows that this adjuvant). The immune response originating from Such a mechanism is not exploited by the analyzed cells. therapeutic vaccination can be expected to be highly specific 0379 The peptides of the present invention might be used against tumor cells because the target peptides of the present to analyze lymphocyte responses against those peptides Such invention are not presented on normal tissues in comparable as T cell responses or antibody responses against the peptide copy numbers, preventing the risk of undesired autoimmune or the peptide complexed to MHC molecules. These lympho reactions against normal cells in the patient. cyte responses can be used as prognostic markers for decision 0372 A“pharmaceutical composition' preferably is pref on further therapy steps. These responses can also be used as erably a composition Suitable for administration to a human Surrogate markers in immunotherapy approaches aiming to being in a medical setting. Preferably, a pharmaceutical com induce lymphocyte responses by different means, e.g. Vacci position is sterile and produced according to GMP guidelines. nation of protein, nucleic acids, autologous materials, adop US 2016/0250307 A1 Sep. 1, 2016

tive transfer of lymphocytes. In gene therapy settings, lym toward a melanoma differentiation antigen. JImmunol. 2003 phocyte responses against peptides can be considered in the Sep. 1; 171(5):2197-207; and Cohen CJ, et al. Direct pheno assessment of side effects. Monitoring of lymphocyte typic analysis of human MHC class I antigen presentation: responses might also be a valuable tool for follow-up exami visualization, quantitation, and in situ detection of human nations of transplantation therapies, e.g. for the detection of viral epitopes using peptide-specific, MHC-restricted human graft versus host and host versus graft diseases. recombinant antibodies. J Immunol. 2003 Apr. 15: 17008): 0380. The peptides of the present invention can be used to 4349-61, which for the purposes of the present invention are generate and develop specific antibodies against MHC/pep all explicitly incorporated by reference in their entireties. tide complexes. These can be used for therapy, targeting 0384 Preferably, the antibody is binding with a binding toxins or radioactive Substances to the diseased tissue. affinity of below 20 nanomolar, preferably of below 10 nano Another use of these antibodies can be targeting radionu molar, to the complex, which is regarded as 'specific’ in the clides to the diseased tissue for imaging purposes Such as context of the present invention. PET. This use can help to detect small metastases or to deter 0385. It is a further aspect of the invention to provide a mine the size and precise localization of diseased tissues. method for producing a soluble T-cell receptor (sTCR) rec 0381. Therefore, it is a further aspect of the invention to ognizing a specific peptide-MHC complex. Such soluble provide a method for producing a recombinant antibody spe T-cell receptors can be generated from specific T-cell clones, cifically binding to a human major histocompatibility com and their affinity can be increased by mutagenesis targeting plex (MHC) class I or II being complexed with a HLA the complementarity-determining regions. For the purpose of restricted antigen, the method comprising: immunizing a T-cell receptor selection, phage display can be used (US genetically engineered non-human mammal comprising cells 2010/01 13300, Liddy N, et al. Monoclonal TCR-redirected expressing said human major histocompatibility complex tumor cell killing. Nat Med2012 June; 18(6): 980-987). For (MHC) class I or II with a soluble form of a MHC class I or II the purpose of stabilization of T-cell receptors during phage molecule being complexed with said HLA-restricted antigen; display and in case of practical use as drug, alpha and beta isolating mRNA molecules from antibody producing cells of chain can be linked e.g. by non-native disulfide bonds, other said non-human mammal; producing a phage display library covalent bonds (single-chain T-cell receptor), or by dimeriza displaying protein molecules encoded by said mRNA mol tion domains (see Boulter J. M. et al. Stable, soluble T-cell ecules; and isolating at least one phage from said phage receptor molecules for crystallization and therapeutics. Pro display library, said at least one phage displaying said anti tein Eng 2003 September; 16(9):707-711; Card KF, et al. A body specifically binding to said human major histocompat soluble single-chain T-cell receptor IL-2 fusion protein ibility complex (MHC) class I or II being complexed with retains MHC-restricted peptide specificity and IL-2 bioactiv said HLA-restricted antigen. ity. Cancer Immunol Immunother 2004 April; 53(4):345-357; 0382. It is a further aspect of the invention to provide an and Willcox BE, et al. Production of soluble alphabeta T-cell antibody that specifically binds to a human major histocom receptor heterodimers suitable for biophysical analysis of patibility complex (MHC) class I or II being complexed with ligand binding. Protein Sci 1999 November; 8 (11):2418 a HLA-restricted antigen, wherein the antibody preferably is 2423). The T-cell receptor can be linked to toxins, drugs, a polyclonal antibody, monoclonal antibody, bi-specific anti cytokines (see, for example, US 2013/0115,191), domains body and/or a chimeric antibody. recruiting effector cells Such as an anti-CD3 domain, etc., in 0383 Yet another aspect of the present invention then order to execute particular functions on target cells. More relates to a method of producing said antibody specifically over, it could be expressed in T cells used for adoptive trans binding to a human major histocompatibility complex fer. Further information can be found in WO 2004/033685A1 (MHC) class I or II being complexed with a HLA-restricted and WO 2004/074322A1. A combination of STCRS is antigen, the method comprising: immunizing a genetically described in WO 2012/056407A1. Further methods for the engineered non-human mammal comprising cells expressing production are disclosed in WO 2013/057586A1. said human major histocompatibility complex (MHC) class I 0386. In addition, the peptides and/or the TCRs or anti or II with a soluble form of a MHC class I or II molecule being bodies or other binding molecules of the present invention complexed with said HLA-restricted antigen; isolating can be used to Verify a pathologists diagnosis of a cancer mRNA molecules from antibody producing cells of said non based on a biopsied sample. human mammal; producing a phage display library display 0387. In order to select over-presented peptides, a presen ing protein molecules encoded by said mRNA molecules; and tation profile is calculated showing the median sample pre isolating at least one phage from said phage display library, sentation as well as replicate variation. The profile juxtaposes said at least one phage displaying said antibody specifically samples of the tumor entity of interest to a baseline of normal bindable to said human major histocompatibility complex tissue samples. Each of these profiles can then be consoli (MHC) class I or II being complexed with said HLA-re dated into an over-presentation score by calculating the stricted antigen. Respective methods for producing Such anti p-value of a Linear Mixed-Effects Model (J. Pinheiro, et al. bodies and single chain class I major histocompatibility com The nlme Package: Linear and Nonlinear Mixed Effects plexes, as well as other tools for the production of these Models. 2007) adjusting for multiple testing by False Discov antibodies are disclosed in WO 03/068201, WO 2004/ ery Rate (Y. Benjamini and Y. Hochberg. Controlling the 084798, WO 01/72768, WO 03/070752, and Cohen CJ, et al. False Discovery Rate: A Practical and Powerful Approach to Recombinant antibodies with MHC-restricted, peptide-spe Multiple Testing. Journal of the Royal Statistical Society. cific, T-cell receptor-like specificity: new tools to study anti Series B (Methodological), Vol. 57 (No. 1):289-300, 1995). gen presentation and TCR-peptide-MHC interactions. J Mol 0388 For the identification and relative quantitation of Recognit. 2003 September-October; 16(5):324-32: Denk HLA ligands by mass spectrometry, HLA molecules from berg G. etal. Selective targeting of melanoma and APCs using shock-frozen tissue samples were purified and HLA-associ a recombinant antibody with TCR-like specificity directed ated peptides were isolated. The isolated peptides were sepa US 2016/0250307 A1 Sep. 1, 2016 42 rated and sequences were identified by online nano-electro 0395. The present invention further relates to the peptides spray-ionization (nanoESI) liquid chromatography-mass according to the invention that have the ability to bind to a spectrometry (LC-MS) experiments. The resulting peptide molecule of the human major histocompatibility complex sequences were verified by comparison of the fragmentation (MHC) class-I or -II. pattern of natural TUMAPs recorded from HCC samples 0396 The present invention further relates to the peptides (N=16 A*02-positive samples including thirteen A*02:01 according to the invention wherein the peptide consists or positive samples, N=15 A*24-positive samples) with the consists essentially of an amino acid sequence according to fragmentation patterns of corresponding synthetic reference SEQID NO: 1 to SEQID NO:300. peptides of identical sequences. Since the peptides were 0397. The present invention further relates to the peptides directly identified as ligands of HLA molecules of primary according to the invention, wherein the peptide is (chemi tumors, these results provide direct evidence for the natural cally) modified and/or includes non-peptide bonds. processing and presentation of the identified peptides on pri 0398. The present invention further relates to the peptides mary cancer tissue obtained from 31 HCC patients. according to the invention, wherein the peptide is part of a 0389. The discovery pipeline XPRESIDENTR) v2.1 (see, fusion protein, in particular comprising N-terminal amino for example, US 2013–0096016, which is hereby incorpo acids of the HLA-DR antigen-associated invariant chain (Ii), rated by reference in its entirety) allows the identification and or wherein the peptide is fused to (or into) an antibody, such selection of relevant over-presented peptide vaccine candi as, for example, an antibody that is specific for dendritic cells. dates based on direct relative quantitation of HLA-restricted 0399. The present invention further relates to a nucleic peptide levels on cancer tissues in comparison to several acid, encoding the peptides according to the invention, pro different non-cancerous tissues and organs. This was vided that the peptide is not the complete (full) human pro achieved by the development of label-free differential quan tein. titation using the acquired LC-MS data processed by a pro 0400. The present invention further relates to the nucleic prietary data analysis pipeline, combining algorithms for acid according to the invention that is DNA, cDNA, PNA, sequence identification, spectral clustering, ion counting, RNA or combinations thereof. retention time alignment, charge state deconvolution and nor 04.01 The present invention further relates to an expres malization. sion vector capable of expressing a nucleic acid according to 0390 Presentation levels including error estimates for the present invention. each peptide and sample were established. Peptides exclu 0402. The present invention further relates to a peptide sively presented on tumor tissue and peptides over-presented according to the present invention, a nucleic acid according to in tumor versus non-cancerous tissues and organs have been the present invention or an expression vector according to the identified. present invention for use in medicine, in particular in the 0391 HLA-peptide complexes from HCC tissue samples treatment of HCC. were purified and HLA-associated peptides were isolated and 0403. The present invention further relates to a host cell analyzed by LC-MS (see examples). All TUMAPs contained comprising a nucleic acid according to the invention or an in the present application were identified with this approach expression vector according to the invention. on primary HCC samples confirming their presentation on 04.04 The present invention further relates to the host cell primary HCC. according to the present invention that is an antigen present ing cell, and preferably a dendritic cell. 0392 TUMAPs identified on multiple HCC tumor and 04.05 The present invention further relates to the method normal tissues were quantified using ion-counting of label according to the present invention, where—in the antigen is free LC-MS data. The method assumes that LC-MS signal loaded onto class I or II MHC molecules expressed on the areas of a peptide correlate with its abundance in the sample. Surface of a Suitable antigen-presenting cell by contacting a All quantitative signals of a peptide in various LC-MS experi Sufficient amount of the antigen with an antigen-presenting ments were normalized based on central tendency, averaged cell. per sample and merged into a bar plot, called presentation 0406. The present invention further relates to the method profile. The presentation profile consolidates different analy according to the invention, wherein the antigen-presenting sis methods like protein database search, spectral clustering, cell comprises an expression vector capable of expressing charge state deconvolution (decharging) and retention time said peptide containing SEQID NO: 1 to SEQID NO:300 or alignment and normalization. said variant amino acid sequence. 0393. The present invention relates to a peptide compris 0407. The present invention further relates to the use of ing a sequence that is selected from the group consisting of any peptide described, a nucleic acid according to the present SEQID NO: 1 to SEQID NO:300, or a variant thereof which invention, an expression vector according to the present is at least 90% homologous (preferably identical) to SEQID invention, a cell according to the present invention, or an NO: 1 to SEQID NO:300 or a variant thereofthat induces T activated cytotoxic T lymphocyte according to the present cells cross-reacting with said peptide, wherein said peptide is invention as a medicament or in the manufacture of a medi not the underlying full-length polypeptide. cament. The present invention further relates to a use accord 0394 The present invention further relates to a peptide ing to the present invention, wherein the medicament is active comprising a sequence that is selected from the group con against cancer. sisting of SEQ ID NO: 1 to SEQ ID NO: 300 or a variant 0408. The present invention further relates to a use accord thereof which is at least 90% homologous (preferably iden ing to the invention, wherein the medicament is a vaccine. tical) to SEQ ID NO: 1 to SEQ ID NO: 300, wherein said The present invention further relates to a use according to the peptide or variant has an overall length of between 8 and 100, invention, wherein the medicament is active against cancer. preferably between 8 and 30, and most preferred between 8 04.09. The present invention further relates to a use accord and 14 amino acids. ing to the invention, wherein said cancer cells are HCC cells US 2016/0250307 A1 Sep. 1, 2016

or other Solid or haematological tumor cells Such as pancre amounts. The monoclonal antibodies herein specifically atic cancer, brain cancer, kidney cancer, colon or rectal can include "chimericantibodies in which a portion of the heavy cer, or leukemia. and/or light chain is identical with or homologous to corre 0410 The present invention further relates to particular sponding sequences in antibodies derived from a particular marker proteins and biomarkers based on the peptides accord species or belonging to aparticular antibody class or Subclass, ing to the present invention, herein called “targets that can be while the remainder of the chain(s) is identical with or used in the diagnosis and/or prognosis of HCC. The present homologous to corresponding sequences in antibodies invention also relates to the use of these novel targets for derived from another species or belonging to another anti Cancer treatment. body class or Subclass, as well as fragments of such antibod 0411. The term “antibody” or “antibodies” is used herein ies, so long as they exhibit the desired antagonistic activity in abroad sense and includes both polyclonal and monoclonal (U.S. Pat. No. 4,816,567, which is hereby incorporated in its antibodies. In addition to intact or “full immunoglobulin entirety). molecules, also included in the term “antibodies' are frag 0416 Monoclonal antibodies of the invention may be pre ments (e.g. CDRs, Fv, Fab and Fc fragments) or polymers of pared using hybridoma methods. In a hybridoma method, a those immunoglobulin molecules and humanized versions of mouse or other appropriate host animal is typically immu immunoglobulin molecules, as long as they exhibit any of the nized with an immunizing agent to elicit lymphocytes that desired properties (e.g., specific binding of a HCC marker produce or are capable of producing antibodies that will spe polypeptide, delivery of a toxin to a HCC cell expressing a cifically bind to the immunizing agent. Alternatively, the lym cancer marker gene at an increased level, and/or inhibiting the phocytes may be immunized in vitro. activity of a HCC marker polypeptide) according to the inven 0417. The monoclonal antibodies may also be made by tion. recombinant DNA methods, such as those described in U.S. 0412. Whenever possible, the antibodies of the invention Pat. No. 4,816,567. DNA encoding the monoclonal antibod may be purchased from commercial sources. The antibodies ies of the invention can be readily isolated and sequenced of the invention may also be generated using well-known using conventional procedures (e.g., by using oligonucleotide methods. The skilled artisan will understand that either full probes that are capable of binding specifically to genes length HCC marker polypeptides or fragments thereof may encoding the heavy and light chains of murine antibodies). be used to generate the antibodies of the invention. A 0418. In vitro methods are also suitable for preparing polypeptide to be used for generating an antibody of the monovalent antibodies. Digestion of antibodies to produce invention may be partially or fully purified from a natural fragments thereof, particularly, Fab fragments, can be accom Source, or may be produced using recombinant DNA tech plished using routine techniques known in the art. For niques. instance, digestion can be performed using papain. Examples 0413 For example, a cDNA encoding a peptide according of papain digestion are described in WO94/29348 and U.S. to the present invention, Such as a peptide according to SEQ Pat. No. 4.342,566. Papain digestion of antibodies typically ID NO: 1 to SEQ ID NO: 300 polypeptide, or a variant or produces two identical antigenbinding fragments, called Fab fragment thereof, can be expressed in prokaryotic cells (e.g., fragments, each with a single antigen binding site, and a bacteria) or eukaryotic cells (e.g., yeast, insect, or mamma residual Fc fragment. Pepsin treatment yields a F(ab')2 frag lian cells), after which the recombinant protein can be puri ment and a pFc' fragment. fied and used to generate a monoclonal or polyclonal anti 0419. The antibody fragments, whether attached to other body preparation that specifically bind the HCC marker sequences or not, can also include insertions, deletions, Sub polypeptide used to generate the antibody according to the stitutions, or other selected modifications of particular invention. regions or specific amino acids residues, provided the activity 0414. One of skill in the art will realize that the generation of the fragment is not significantly altered or impaired com of two or more different sets of monoclonal or polyclonal pared to the non-modified antibody or antibody fragment. antibodies maximizes the likelihood of obtaining an antibody These modifications can provide for Some additional prop with the specificity and affinity required for its intended use erty, Such as to remove/add amino acids capable of disulfide (e.g., ELISA, immunohistochemistry, in vivo imaging, bonding, to increase its bio-longevity, to alter its secretory immunotoxin therapy). The antibodies are tested for their characteristics, etc. In any case, the antibody fragment must desired activity by known methods, in accordance with the possess a bioactive property, such as binding activity, regula purpose for which the antibodies are to be used (e.g., ELISA, tion of binding at the binding domain, etc. Functional or immunohistochemistry, immunotherapy, etc.; for further active regions of the antibody may be identified by mutagen guidance on the generation and testing of antibodies, see, e.g., esis of a specific region of the protein, followed by expression Harlow and Lane, Antibodies: A Laboratory Manual, Cold and testing of the expressed polypeptide. Such methods are Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., readily apparent to a skilled practitioner in the art and can 1988, new 2nd edition 2013). For example, the antibodies include site-specific mutagenesis of the nucleic acid encoding may be tested in ELISA assays or, Western blots, immuno the antibody fragment. histochemical staining of formalin-fixed cancers or frozen 0420. The antibodies of the invention may further com tissue sections. After their initial in vitro characterization, prise humanized antibodies or human antibodies. Humanized antibodies intended for therapeutic or in vivo diagnostic use forms of non-human (e.g., murine) antibodies are chimeric are tested according to known clinical testing methods. immunoglobulins, immunoglobulin chains or fragments 0415. The term “monoclonal antibody” as used herein thereof (such as Fv, Fab, Fab' or other antigen-binding sub refers to an antibody obtained from a substantially homoge sequences of antibodies) which contain minimal sequence neous population of antibodies, i.e., the individual antibodies derived from non-human immunoglobulin. Humanized anti comprising the population are identical except for possible bodies include human immunoglobulins (recipient antibody) naturally occurring mutations that may be present in minor in which residues from a complementary determining region US 2016/0250307 A1 Sep. 1, 2016 44

(CDR) of the recipient are replaced by residues from a CDR effective form. The antibodies may also be administered by of a non-human species (donor antibody) Such as mouse, rat intratumoral or peritumoral routes, to exert local as well as or rabbit having the desired specificity, affinity and capacity. systemic therapeutic effects. Local or intravenous injection is In some instances, Fv framework (FR) residues of the human preferred. immunoglobulin are replaced by corresponding non-human 0425 Effective dosages and schedules for administering residues. Humanized antibodies may also comprise residues the antibodies may be determined empirically, and making which are found neither in the recipient antibody nor in the such determinations is within the skill in theart. Those skilled imported CDR or framework sequences. In general, the in the art will understand that the dosage of antibodies that humanized antibody will comprise substantially all of at least must be administered will vary depending on, for example, one, and typically two, variable domains, in which all or the subject that will receive the antibody, the route of admin substantially all of the CDR regions correspond to those of a istration, the particular type of antibody used and other drugs non-human immunoglobulin and all or substantially all of the being administered. A typical daily dosage of the antibody FR regions are those of a human immunoglobulin consensus used alone might range from about 1 (lug/kg to up to 100 sequence. The humanized antibody optimally also will com mg/kg of body weight or more per day, depending on the prise at least a portion of an immunoglobulin constant region factors mentioned above. Following administration of an (Fc), typically that of a human immunoglobulin. antibody, preferably for treating HCC, the efficacy of the 0421 Methods for humanizing non-human antibodies are therapeutic antibody can be assessed in various ways well well known in the art. Generally, a humanized antibody has known to the skilled practitioner. For instance, the size, num one or more amino acid residues introduced into it from a ber, and/or distribution of cancer in a Subject receiving treat Source which is non-human. These non-human amino acid ment may be monitored using standard tumor imaging tech residues are often referred to as “import residues, which are niques. A therapeutically-administered antibody that arrests typically taken from an “import variable domain. Human tumor growth, results in tumor shrinkage, and/or prevents the ization can be essentially performed by Substituting rodent development of new tumors, compared to the disease course CDRs or CDR sequences for the corresponding sequences of that would occurs in the absence of antibody administration, a human antibody. Accordingly, Such "humanized' antibod is an efficacious antibody for treatment of cancer. ies are chimeric antibodies (U.S. Pat. No. 4,816,567), 0426 Because the peptides as mentioned in the Tables wherein Substantially less than an intact human variable above of the invention and thus their underlying polypeptides domain has been Substituted by the corresponding sequence are highly expressed in HCC, and are expressed at rather to from a non-human species. In practice, humanized antibodies extremely low levels in normal cells, the inhibition of a pro are typically human antibodies in which some CDR residues tein selected from the group consisting of protein products of and possibly some FR residues are substituted by residues the following genes: Preferred for the inhibition and for anti from analogous sites in rodent antibodies. bodies and/or TCRs against are GLUL, GPAM, PLIN2, 0422 Transgenic animals (e.g., mice) that are capable, SLC16A1 SLC9A3R1, PCBD1, SEC16A, AKR1C4, upon immunization, of producing a full repertoire of human ABCB11, HAL, CYP2E1, C4A, C4B, ALDH1L1, CRP, antibodies in the absence of endogenous immunoglobulin ACSL4, EEF2, HLTF, FBXO22, GALK1, TMCO1, production can be employed. For example, it has been TMEM33, ZNF318, IPO9, AMACR, C1GTNF3, CYP4F8, described that the homozygous deletion of the antibody CYP4F3, CYP4F11, CYP4F12, CYP4F2, MOCOS, A1CF, heavy chain joining region gene in chimeric and germ-line COL18A1, HPR, LBP, C19orf80, CFHR5, ITIH4, mutant mice results in complete inhibition of endogenous TMEM110, LARP4, LMF2, SLC10A5, and SLC16A11; still antibody production. Transfer of the human germ-line immu preferred for the inhibition and for antibodies and/or TCRs noglobulin gene array in Such germ-line mutant mice will against are ANKFY1, C12orf44, C16orf58, CPSF1, DCAF8, result in the production of human antibodies upon antigen PEX19, DDX11, DDX12P DECR2, NME4, DENND5B, challenge. Human antibodies can also be produced in phage DYM, EDC4, ERI3, FAM20A, FNDC3A, GPR107, GYG2, display libraries. HEATR2, IFT81, KCTD3, SHKBP1, KIAA1324L, 0423 Antibodies of the invention are preferably adminis KLHL24, MARCH6, MBTPS2, MIR1279, CPSF6, NOC4L, tered to a subject in a pharmaceutically acceptable carrier. NXF1, PANK2, PCNXL3, PIPSL, PSMD4, PSMD14, Typically, an appropriate amount of a pharmaceutically-ac SLC35B1, TCP11L2, THNSL2, THOC2, TOMM5, ceptable salt is used in the formulation to render the formu TRAPPC6B, TRIM54, TRIM55, TRIM63, UGGT2, URB1, lation isotonic. Examples of the pharmaceutically-acceptable VPS54, WIZ, ZNF451, RFTN2, SCFD1, SERINC5, carrier include Saline, Ringer's solution and dextrose solu CCT7P2, CMAS, ANKS1A, C17orf70, CCT7, CDK5RAP2, tion. The pH of the solution is preferably from about 5 to CLPTM1, and most preferred for the inhibition and for anti about 8, and more preferably from about 7 to about 7.5. bodies and/or TCRs against are APOB, FASN, and/or COPA: Further carriers include Sustained release preparations such and expression or of the activity of these markers may be as semipermeable matrices of Solid hydrophobic polymers preferably integrated into a therapeutic strategy, e.g. for treat containing the antibody, which matrices are in the form of ing or preventing HCC. shaped articles, e.g., films, liposomes or microparticles. It 0427. The principle of antisense therapy is based on the will be apparent to those persons skilled in the art that certain hypothesis that sequence-specific Suppression of gene carriers may be more preferable depending upon, for expression (via transcription or translation) may be achieved instance, the route of administration and concentration of by intra-cellular hybridization between genomic DNA or antibody being administered. mRNA and a complementary antisense species. The forma 0424 The antibodies can be administered to the subject, tion of such a hybrid nucleic acid duplex interferes with patient, or cell by injection (e.g., intravenous, intraperitoneal, transcription of the target tumor antigen-encoding genomic Subcutaneous, intramuscular), or by other methods such as DNA, or processing/transport/translation and/or stability of infusion that ensure its delivery to the bloodstream in an the target tumor antigen mRNA. US 2016/0250307 A1 Sep. 1, 2016

0428 Antisense nucleic acids can be delivered by a variety vectors, adeno-associated viral (AAV) vectors, lentiviral vec of approaches. For example, antisense oligonucleotides or tors, pseudotyped retroviral vectors. Physical transduction anti-sense RNA can be directly administered (e.g., by intra techniques can also be used, such as liposome delivery and venous injection) to a Subject in a form that allows uptake into receptor-mediated and other endocytosis mechanisms. This tumor cells. Alternatively, viral or plasmid vectors that invention can be used in conjunction with any of these or encode antisense RNA (or RNA fragments) can be introduced other commonly used gene transfer methods. into cells in vivo. Antisense effects can also be induced by 0433. The antibodies may also be used for in vivo diag sense sequences; however, the extent of phenotypic changes nostic assays. Generally, the antibody is labeled with a radio is highly variable. Phenotypic changes induced by effective nucleotide (such as '''In, TC, C, "'I, H, Por S)) so antisense therapy are assessed according to changes in, e.g., that the tumor can be localized using immunoscintiography. target mRNA levels, target protein levels, and/or target pro In one embodiment, antibodies or fragments thereof bind to tein activity levels. the extracellular domains of two or more targets of a protein 0429. In a specific example, inhibition of HCC target/ selected from the group consisting of the above-mentioned marker function by antisense gene therapy may be accom proteins, and the affinity value (Kd) is less than 1x10 uM. plished by direct administration of antisense tumor marker 0434 Antibodies for diagnostic use may be labeled with RNA to a subject. The antisense tumor marker RNA may be probes Suitable for detection by various imaging methods. produced and isolated by any standard technique, but is most Methods for detection of probes include, but are not limited readily produced by in vitro transcription using an antisense to, fluorescence, light, confocal and electron microscopy; tumor marker cDNA under the control of a high efficiency magnetic resonance imaging and spectroscopy; fluoroscopy, promoter (e.g., the T7 promoter). Administration of anti computed tomography and positron emission tomography. sense tumor marker RNA to cells can be carried out by any of Suitable probes include, but are not limited to, fluorescein, the methods for direct nucleic acid administration described rhodamine, eosinandother fluorophores, radioisotopes, gold, below. gadolinium and other lanthanides, paramagnetic iron, fluo 0430. An alternative strategy for inhibiting the function of rine-18 and other positron-emitting radionuclides. Addition a protein selected from the group consisting of the above ally, probes may be bi- or multi-functional and be detectable mentioned proteins, and most preferred of APOB, FASN, by more than one of the methods listed. These antibodies may and/or COPA, involves use of a nucleic acid (e.g. siRNA, or a be directly or indirectly labeled with said probes. Attachment nucleic acid coding for an anti-protein antibody or a portion of probes to the antibodies includes covalent attachment of thereof, which can be transferred into cancer cells or other the probe, incorporation of the probe into the antibody, and cells, leading to intracellular antibody expression and secre the covalent attachment of a chelating compound for binding tion), a protein or Small molecule, or any other compound of probe, amongst others well recognized in the art. For targeting the expression, translation, and/or biological func immunohistochemistry, the disease tissue sample may be tion of this protein. fresh or frozen or may be embedded in paraffin and fixed with 0431. In the methods described above, which include the a preservative such as formalin. The fixed or embedded sec administration and uptake of exogenous DNA into the cells of tion contains the sample are contacted with a labeled primary a subject (i.e., gene transduction or transfection), the nucleic antibody and secondary antibody, wherein the antibody is acids of the present invention can be in the form of naked used to detect the expression of the proteins in situ. DNA or the nucleic acids can be in a vector for delivering the 0435. As mentioned above, the present invention thus pro nucleic acids to the cells for inhibition of HCC marker protein vides a peptide comprising a sequence that is selected from expression. The vector can be a commercially available the group of consisting of SEQID NO: 1 to SEQID NO:300 preparation, such as an adenovirus vector (Quantum Biotech or a variant thereof which is 90% homologous to SEQID NO: nologies, Inc. (Laval, Quebec, Canada). Delivery of the 1 to SEQID NO:300, or a variant thereof that will induce T nucleic acid or vector to cells can be via a variety of mecha cells cross-reacting with said peptide. The peptides of the nisms. As one example, delivery can be via a liposome, using invention have the ability to bind to a molecule of the human commercially available liposome preparations such as Lipo major histocompatibility complex (MHC) class-I or elon fectin, Lipofectamine (GIBCO-25 BRL, Inc., Gaithersburg, gated versions of said peptides to class II. Md.). Superfect (Qiagen, Inc. Hilden, Germany) and Trans 0436. In the present invention, the term “homologous' fectam (Promega Biotec, Inc., Madison, Wis., US), as well as refers to the degree of identity (see Percent Identity above) other liposomes developed according to procedures standard between sequences of two amino acid sequences, i.e. peptide in the art. In addition, the nucleic acid or vector of this inven or polypeptide sequences. The aforementioned "homology’ tion can be delivered in vivo by electroporation, the technol is determined by comparing two sequences aligned under ogy for which is available from Genetronics, Inc. (San Diego, optimal conditions over the sequences to be compared. Such US) as well as by means of a Sonoporation machine (ImaRX a sequence homology can be calculated by creating an align Pharmaceutical Corp., Tucson, Ariz., US). ment using, for example, the ClustalW algorithm. Commonly 0432. As one example, vector delivery can be via a viral available sequence analysis Software, more specifically, Vec system, such as a retroviral vector system that can package a tor NTI, GENETYX or other analysis tools are provided by recombinant retroviral genome. The recombinant retrovirus public databases. can then be used to infect and thereby deliver to the infected 0437. A person skilled in the art will be able to assess, cells antisense nucleic acid that inhibits expression of a pro whether T cells induced by a variant of a specific peptide will tein selected from the group consisting of the above-men be able to cross-react with the peptide itself (Fong L, et al. tioned proteins. The exact method of introducing the altered Altered peptide ligand vaccination with Flt3 ligand expanded nucleic acid into mammalian cells is, of course, not limited to dendritic cells for tumor immunotherapy. Proc Natl Acad Sci the use of retroviral vectors. Other techniques are widely USA. 2001 Jul.17: 98(15):8809-14: Zaremba S, et al. Iden available for this procedure including the use of adenoviral tification of an enhancer agonist cytotoxic T lymphocyte pep US 2016/0250307 A1 Sep. 1, 2016 46 tide from human carcinoembryonic antigen. Cancer Res. polypeptide), which includes the amino acid sequences or a 1997 Oct. 15: 57(20):4570-7: Colombetti S, et al. Impact of portion or variant thereofas given. orthologous melan-A peptide immunizations on the anti-self melan-A/HLA-A2 T cell cross-reactivity. J Immunol. 2006 TABLE 8 Jun. 1; 176(11):6560-7: Appay V, et al. Decreased specific CD8+ T cell cross-reactivity of antigen recognition following Variants and motif of the peptides vaccination with Melan-A peptide. Eur J. Immunol. 2006 according to SEQ ID NO: 1, 117, and 246 July; 36(7): 1805-14). Position 0438. By a “variant of the given amino acid sequence the inventors mean that the side chains of, for example, one or two of the amino acid residues are altered (for example by SEQ ID NO. 1 W M A P T M T replacing them with the side chain of another naturally occur Variants ringamino acid residue or some other side chain) such that the peptide is still able to bind to an HLA molecule in substan tially the same way as a peptide consisting of the given amino acid sequence in consisting of SEQID NO: 1 to SEQID NO: 300. For example, a peptide may be modified so that it at least maintains, if not improves, the ability to interact with and bind to the binding groove of a suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it at least maintains, if not improves, the ability to bind to the TCR of activated T cells. 0439. These T cells can subsequently cross-react with cells and kill cells that express a polypeptide that contains the i.i.i. natural amino acid sequence of the cognate peptide as defined in the aspects of the invention. As can be derived from the i. scientific literature (Godkin A, et al. Use of eluted peptide sequence data to identify the binding characteristics of pep tides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2). Int Immunol. 1997 June; 9(6):905-11) and databases (Rammensee H. et al. SYFPEITHI: database Position for MHC ligands and peptide motifs. Immunogenetics. 1999 1. 2 3 4. 5 6 7 8 9 November; 50(3–4):213-9), certain positions of HLA binding peptides are typically anchor residues forming a core SEQ ID NO 246 K sequence fitting to the binding motif of the HLA receptor, Variants which is defined by polar, electrophysical, hydrophobic and spatial properties of the polypeptide chains constituting the binding groove. Thus, one skilled in the art would be able to modify the amino acid sequences set forth in SEQID NO: 1 to SEQ ID NO 300, by maintaining the known anchor resi dues, and would be able to determine whether such variants maintain the ability to bind MHC class I or II molecules. The variants of the present invention retain the ability to bind to the TCR of activated T cells, which can subsequently cross react with and kill cells that express a polypeptide containing the natural amino acid sequence of the cognate peptide as defined in the aspects of the invention. 0440 The amino acid residues that do not substantially contribute to interactions with the T-cell receptor can be modified by replacement with another amino acid whose incorporation does not substantially affect T-cell reactivity and does not eliminate binding to the relevant MHC. Thus, apart from the proviso given, the peptide of the invention may be any peptide (by which term the inventors include oligopep Position tide or polypeptide), which includes the amino acid sequences or a portion or variant thereofas given. 0441 Those amino acid residues that do not substantially SEQ ID NO. 117 Y A F P K. S I T W Variants L contribute to interactions with the TCR can be modified by I replacement with another amino acid whose incorporation A. does not substantially affect T-cell reactivity and does not M L eliminate binding to the relevant MHC. Thus, apart from the M I proviso given, the peptide of the invention may be any peptide M (by which term the inventors include oligopeptide or US 2016/0250307 A1 Sep. 1, 2016 47

TABLE 8 - continued 0448. In addition, the peptide or variant may be modified further to improve stability and/or binding to MHC molecules Variants and motif of the peptides in order to elicit a stronger immune response. Methods for according to SEQ ID NO: 1, 117, and 246 Such an optimization of a peptide sequence are well known in M A. the art and include, for example, the introduction of reverse L L peptide bonds or non-peptide bonds. L I L 0449 In a reverse peptide bond amino acid residues are L A. not joined by peptide (—CO. NH ) linkages but the pep W L tide bond is reversed. Such retro-inverso peptidomimetics W I may be made using methods known in the art, for example W W A. such as those described in Meziere et al (1997) J. Immunol. T L 159, 3230-3237, incorporated herein by reference. This T I approach involves making pseudopeptides containing T changes involving the backbone, and not the orientation of T A. Q L side chains. Meziere etal (1997) show that for MHC binding Q I and T helper cell responses, these pseudopeptides are useful. Q Retro-inverse peptides, which contain NH CO bonds Q A. instead of CO. NH peptide bonds, are much more resistant to proteolysis. 0442 Longer peptides may also be suitable. It is also 0450 A non-peptide bond is, for example, —CH NH, possible that MHC class I epitopes, although usually between —CHS , —CHCH , —CH=CH- , —COCH , 8 and 11 amino acids long, are generated by peptide process —CH(OH)CH , and CHSO-. U.S. Pat. No. 4,897,445 ing from longer peptides or proteins that include the actual provides a method for the Solid phase synthesis of non-pep epitope. It is preferred that the residues that flank the actual tide bonds (—CH2 NH) in polypeptide chains which epitope are residues that do not Substantially affect pro involves polypeptides synthesized by standard procedures teolytic cleavage necessary to expose the actual epitope dur and the non-peptide bond synthesized by reacting an amino ing processing. aldehyde and an amino acid in the presence of NaCNBH. 0443 Accordingly, the present invention provides pep 0451 Peptides comprising the sequences described above may be synthesized with additional chemical groups present tides and variants of MHC class I epitopes, wherein the pep at their amino and/or carboxy termini, to enhance the stability, tide or variant has an overall length of between 8 and 100, bioavailability, and/or affinity of the peptides. For example, preferably between 8 and 30, and most preferred between 8 hydrophobic groups such as carbobenzoxyl, dansyl, or t-bu and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in case of tyloxycarbonyl groups may be added to the peptides amino the elongated class II binding peptides the length can also be termini. Likewise, an acetyl group or a 9-fluorenylmethoxy 15, 16, 17, 18, 19, 20, 21 or 22 amino acids. carbonyl group may be placed at the peptides amino termini. 0444 Of course, the peptide or variant according to the Additionally, the hydrophobic group, t-butyloxycarbonyl, or present invention will have the ability to bind to a molecule of an amido group may be added to the peptides carboxy ter the human major histocompatibility complex (MHC) class I mini. or II. Binding of a peptide or a variant to a MHC complex may 0452 Further, the peptides of the invention may be syn be tested by methods known in the art. thesized to alter their steric configuration. For example, the 0445. In a particularly preferred embodiment of the inven D-isomer of one or more of the amino acid residues of the tion the peptide consists or consists essentially of an amino peptide may be used, rather than the usual L-isomer. Still acid sequence according to SEQ ID NO: 1 to SEQ ID NO: further, at least one of the amino acid residues of the peptides 3OO. of the invention may be substituted by one of the well-known 0446 “Consisting essentially of shall mean that a peptide non-naturally occurring amino acid residues. Alterations according to the present invention, in addition to the sequence such as these may serve to increase the stability, bioavailabil according to any of SEQID NO: 1 to SEQID NO 300 or a ity and/or binding action of the peptides of the invention. variant thereof contains additional N- and/or C-terminally 0453 Similarly, a peptide or variant of the invention may located Stretches of amino acids that are not necessarily form be modified chemically by reacting specific amino acids ing part of the peptide that functions as an epitope for MHC either before or after synthesis of the peptide. Examples for molecules epitope. Such modifications are well known in the art and are Summa 0447 Nevertheless, these stretches can be important to rized e.g. in R. Lundblad, Chemical Reagents for Protein provide an efficient introduction of the peptide according to Modification, 3rded. CRC Press, 2005, which is incorporated the present invention into the cells. In one embodiment of the herein by reference. Chemical modification of amino acids present invention, the peptide is part of a fusion protein which includes but is not limited to, modification by acylation, comprises, for example, the 80 N-terminal amino acids of the amidination, pyridoxylation of lysine, reductive alkylation, HLA-DR antigen-associated invariant chain (p33, in the fol trinitrobenzylation of amino groups with 2,4,6-trinitroben lowing “Ii') as derived from the NCBI, GenBank Accession Zene sulphonic acid (TNBS), amide modification of carboxyl number X00497. In other fusions, the peptides of the present groups and Sulphydryl modification by performic acid oxida invention can be fused to an antibody as described herein, or tion of cysteine to cysteic acid, formation of mercurial deriva a functional part thereof, in particular into a sequence of an tives, formation of mixed disulphides with other thiol com antibody, so as to be specifically targeted by said antibody, or, pounds, reaction with maleimide, carboxymethylation with for example, to or into an antibody that is specific for dendritic iodoacetic acid or iodoacetamide and carbamoylation with cells as described herein. cyanate at alkaline pH, although without limitation thereto. In US 2016/0250307 A1 Sep. 1, 2016 48 this regard, the skilled person is referred to Chapter 15 of and histidine), trityl derivative (in the case of cysteine) and Current Protocols In Protein Science, Eds. Coligan et al. 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in (John Wiley and Sons NY 1995-2000) for more extensive the case of arginine). Where glutamine or asparagine are methodology relating to chemical modification of proteins. C-terminal residues, use is made of the 4,4'-dimethoxyben 0454 Briefly, modification of e.g. arginyl residues in pro Zhydryl group for protection of the side chain amido func teins is often based on the reaction of vicinal dicarbonyl tionalities. The Solid-phase Support is based on a polydim compounds such as phenylglyoxal, 2,3-butanedione, and 1.2- ethyl-acrylamide polymer constituted from the three cyclohexanedione to form an adduct. Another example is the monomers dimethylacrylamide (backbone-monomer), reaction of methylglyoxal with arginine residues. Cysteine bisacryloylethylene diamine (cross linker) and acryloylsar can be modified without concomitant modification of other cosine methyl ester (functionalizing agent). The peptide-to nucleophilic sites Such as lysine and histidine. As a result, a resin cleavable linked agent used is the acid-labile 4-hy large number of reagents are available for the modification of droxymethyl-phenoxyacetic acid derivative. All amino acid cysteine. The websites of companies such as Sigma-Aldrich derivatives are added as their preformed symmetrical anhy (http://www.sigma-aldrich.com) provide information on spe dride derivatives with the exception of asparagine and cific reagents. glutamine, which are added using a reversed N,N-dicyclo 0455 Selective reduction of disulfide bonds in proteins is hexyl-carbodiimide/1 hydroxybenzotriazole mediated cou also common. Disulfide bonds can be formed and oxidized pling procedure. All coupling and deprotection reactions are during the heat treatment of biopharmaceuticals. Wood monitored using ninhydrin, trinitrobenzene Sulphonic acid or ward's Reagent K may be used to modify specific glutamic isotin test procedures. Upon completion of synthesis, pep acid residues. N-(3-(dimethylamino)propyl)-N'-ethylcarbo tides are cleaved from the resin Support with concomitant diimide can be used to form intra-molecular crosslinks removal of side-chain protecting groups by treatment with between a lysine residue and a glutamic acid residue. For 95% trifluoroacetic acid containing a 50% scavenger mix. example, diethylpyrocarbonate is a reagent for the modifica Scavengers commonly used include ethandithiol, phenol, tion of histidyl residues in proteins. Histidine can also be anisole and water, the exact choice depending on the constitu modified using 4-hydroxy-2-nonenal. The reaction of lysine ent amino acids of the peptide being synthesized. Also a residues and other C.-amino groups is, for example, useful in combination of solid phase and solution phase methodologies binding of peptides to Surfaces or the cross-linking of pro for the synthesis of peptides is possible (see, for example, teins/peptides. Lysine is the site of attachment of poly(ethyl Bruckdorfer et al., 2004, and the references as cited therein). ene)glycol and the major site of modification in the glycosy 0461 Trifluoroacetic acid is removed by evaporation in lation of proteins. Methionine residues in proteins can be vacuo, with subsequent trituration with diethyl ether afford modified with e.g. iodoacetamide, bromoethylamine, and ing the crude peptide. Any scavengers present are removed by chloramine T. a simple extraction procedure which onlyophilization of the 0456 Tetranitromethane and N-acetylimidazole can be aqueous phase affords the crude peptide free of Scavengers. used for the modification of tyrosyl residues. Cross-linking Reagents for peptide synthesis are generally available from via the formation of dityrosine can be accomplished with e.g. Calbiochem-Novabiochem (Nottingham, UK). hydrogen peroxide/copper ions. 0462 Purification may be performed by any one, or a 0457 Recent studies on the modification of tryptophan combination of techniques such as re-crystallization, size have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl exclusion chromatography, ion-exchange chromatography, bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)- hydrophobic interaction chromatography and (usually) 3H-indole (BPNS-skatole). reverse-phase high performance liquid chromatography 0458 Successful modification of therapeutic proteins and using e.g. acetonitril/water gradient separation. peptides with PEG is often associated with an extension of 0463 Analysis of peptides may be carried out using thin circulatory half-life while cross-linking of proteins with glu layer chromatography, electrophoresis, in particular capillary taraldehyde, polyethylene glycol diacrylate and formalde electrophoresis, solid phase extraction (CSPE), reverse hyde is used for the preparation of hydrogels. Chemical modi phase high performance liquid chromatography, amino-acid fication of allergens for immunotherapy is often achieved by analysis after acid hydrolysis and by fast atom bombardment carbamylation with potassium cyanate. (FAB) mass spectrometric analysis, as well as MALDI and 0459. A peptide or variant, wherein the peptide is modified ESI-Q-TOF mass spectrometric analysis. or includes non-peptide bonds is a preferred embodiment of 0464 A further aspect of the invention provides a nucleic the invention. Generally, peptides and variants (at least those acid (for example a polynucleotide) encoding a peptide or containing peptide linkages between amino acid residues) peptide variant of the invention. The polynucleotide may be, may be synthesized by the Fmoc-polyamide mode of solid for example, DNA, cDNA, PNA, RNA or combinations phase peptide synthesis as disclosed by Lukas et al. (Solid thereof, either single- and/or double-stranded, or native or phase peptide synthesis under continuous-flow conditions. stabilized forms of polynucleotides, such as, for example, Proc Natl AcadSci USA. May 1981: 78(5): 2791-2795), and polynucleotides with a phosphorothioate backbone and it references as cited therein. Temporary N-amino group pro may or may not contain introns so long as it codes for the tection is afforded by the 9-fluorenylmethyloxycarbonyl peptide. Of course, only peptides that contain naturally occur (Fmoc) group. ring amino acid residues joined by naturally occurring pep 0460 Repetitive cleavage of this highly base-labile pro tide bonds are encodable by a polynucleotide. A still further tecting group is done using 20% piperidine in N,N-dimeth aspect of the invention provides an expression vector capable ylformamide. Side-chain functionalities may be protected as of expressing a polypeptide according to the invention. their butyl ethers (in the case of serine threonine and 0465. A variety of methods have been developed to link tyrosine), butyl esters (in the case of glutamic acid and aspar polynucleotides, especially DNA, to vectors for example via tic acid), butyloxycarbonyl derivative (in the case of lysine complementary cohesive termini. For instance, complemen US 2016/0250307 A1 Sep. 1, 2016 49 tary homopolymer tracts can be added to the DNA segment to 0473. Many expression systems are known, including bac be inserted to the vector DNA. The vector and DNA segment teria (for example E. coli and Bacillus subtilis), yeasts (for are then joined by hydrogen bonding between the comple example Saccharomyces cerevisiae), filamentous fungi (for mentary homopolymeric tails to form recombinant DNA example Aspergillus spec.), plant cells, animal cells and molecules. insect cells. Preferably, the system can be mammalian cells 0466 Synthetic linkers containing one or more restriction such as CHO cells available from the ATCC Cell Biology sites provide an alternative method of joining the DNA seg Collection. ment to vectors. Synthetic linkers containing a variety of 0474. A typical mammalian cell vector plasmid for con restriction endonuclease sites are commercially available stitutive expression comprises the CMV or SV40 promoter from a number of Sources including International Biotech with a suitable poly A tail and a resistance marker, Such as nologies Inc. New Haven, Conn., USA. neomycin. One example is pSVL available from Pharmacia, 0467 A desirable method of modifying the DNA encod Piscataway, N.J., USA. An example of an inducible mamma ing the polypeptide of the invention employs the polymerase lian expression vector is pMSG, also available from Pharma chain reaction as disclosed by Saiki R K, et al. (Diagnosis of cia. Useful yeast plasmid vectors are pRS403-406 and sickle cell anemia and beta-thalassemia with enzymatically pRS413-416 and are generally available from Stratagene amplified DNA and nonradioactive allele-specific oligo Cloning Systems, La Jolla, Calif. 92037, USA. Plasmids nucleotide probes. N Engl J Med. 1988 Sep. 1; 319(9):537 pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating 41). This method may be used for introducing the DNA into plasmids (Ylps) and incorporate the yeast selectable markers a Suitable vector, for example by engineering in Suitable HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are restriction sites, or it may be used to modify the DNA in other Yeast Centromere plasmids (Ycps). CMV promoter-based useful ways as is known in the art. If viral vectors are used, vectors (for example from Sigma-Aldrich) provide transient poX- or adenovirus vectors are preferred. or stable expression, cytoplasmic expression or secretion, and 0468. The DNA (or in the case of retroviral vectors, RNA) N-terminal or C-terminal tagging in various combinations of may then be expressed in a suitable host to produce a polypep FLAG, 3xFLAG, c-myc or MAT. These fusion proteins allow tide comprising the peptide or variant of the invention. Thus, for detection, purification and analysis of recombinant pro the DNA encoding the peptide or variant of the invention may tein. Dual-tagged fusions provide flexibility in detection. be used in accordance with known techniques, appropriately 0475. The strong human cytomegalovirus (CMV) pro modified in view of the teachings contained herein, to con moter regulatory region drives constitutive protein expression struct an expression vector, which is then used to transforman levels as high as 1 mg/L in COS cells. For less potent cell appropriate host cell for the expression and production of the lines, protein levels are typically -0.1 mg/L. The presence of polypeptide of the invention. Such techniques include those the SV40 replication origin will result in high levels of DNA disclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530. replication in SV40 replication permissive COS cells. CMV 901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, vectors, for example, can contain the pMB1 (derivative of 4,757,006, 4,766,075, and 4,810,648. pBR322) origin for replication in bacterial cells, the b-lacta 0469. The DNA (or in the case of retroviral vectors, RNA) mase gene forampicillin resistance selection in bacteria, hCH encoding the polypeptide constituting the compound of the polyA, and the fl origin. Vectors containing the pre-pro invention may be joined to a wide variety of other DNA trypsin leader (PPT) sequence can direct the secretion of sequences for introduction into an appropriate host. The com FLAG fusion proteins into the culture medium for purifica panion DNA will depend upon the nature of the host, the tion using ANTI-FLAG antibodies, resins, and plates. Other manner of the introduction of the DNA into the host, and vectors and expression systems are well known in the art for whether episomal maintenance or integration is desired. use with a variety of host cells. 0470 Generally, the DNA is inserted into an expression 0476. In another embodiment two or more peptides or vector, Such as a plasmid, in proper orientation and correct peptide variants of the invention are encoded and thus reading frame for expression. If necessary, the DNA may be expressed in a Successive order (similar to “beads on a string linked to the appropriate transcriptional and translational constructs). In doing so, the peptides or peptide variants may regulatory control nucleotide sequences recognized by the be linked or fused together by stretches of linkeramino acids, desired host, although Such controls are generally available in Such as for example LLLLLL, or may be linked without any the expression vector. The vector is then introduced into the additional peptide(s) between them. These constructs can host through standard techniques. Generally, not all of the also be used for cancer therapy, and may induce immune hosts will be transformed by the vector. Therefore, it will be responses both involving MHC I and MHC II. necessary to select for transformed host cells. One selection 0477 The present invention also relates to a host cell technique involves incorporating into the expression vector a transformed with a polynucleotide vector construct of the DNA sequence, with any necessary control elements, that present invention. The host cell can be either prokaryotic or codes for a selectable trait in the transformed cell, such as eukaryotic. Bacterial cells may be preferred prokaryotic host antibiotic resistance. cells in Some circumstances and typically are a strain of E. 0471 Alternatively, the gene for such selectable trait can coli such as, for example, the E. coli strains DH5 available be on another vector, which is used to co-transform the from Bethesda Research Laboratories Inc., Bethesda, Md., desired host cell. USA, and RR1 available from the American Type Culture 0472 Host cells that have been transformed by the recom Collection (ATCC) of Rockville, Md., USA (No ATCC binant DNA of the invention are then cultured for a sufficient 31343). Preferred eukaryotic host cells include yeast, insect time and under appropriate conditions known to those skilled and mammalian cells, preferably vertebrate cells such as in the art in view of the teachings disclosed herein to permit those from a mouse, rat, monkey or human fibroblastic and the expression of the polypeptide, which can then be recov colon cell lines. Yeast host cells include YPH499, YPH500 ered. and YPH501, which are generally available from Stratagene US 2016/0250307 A1 Sep. 1, 2016 50

Cloning Systems, La Jolla, Calif. 92037, USA. Preferred 0482. A further aspect of the invention provides a method mammalian host cells include Chinese hamster ovary (CHO) of producing a peptide or its variant, the method comprising cells available from the ATCC as CCL61, NIH Swiss mouse culturing a host cell and isolating the peptide from the host embryo cells NIH/3T3 available from the ATCC as CRL cell or its culture medium. 1658, monkey kidney-derived COS-1 cells available from the 0483. In another embodiment the peptide, the nucleic acid ATCC as CRL 1650 and 293 cells which are human embry or the expression vector of the invention are used in medicine. onic kidney cells. Preferred insect cells are Sf9 cells which For example, the peptide or its variant may be prepared for can be transfected with baculovirus expression vectors. An intravenous (i.v.) injection, Sub-cutaneous (s.c.) injection, overview regarding the choice of suitable host cells for intradermal (i.d.) injection, intraperitoneal (i.p.) injection, expression can be found in, for example, the textbook of intramuscular (i.m.) injection. Preferred methods of peptide Paulina Balbás and Argelia Lorence “Methods in Molecular injection includes.c., i.d., i.p., i.m., and i.v. Preferred methods Biology Recombinant Gene Expression, Reviews and Proto of DNA injection include i.d., i.m., s.c., i.p. and i.v. Doses of cols. Part One, Second Edition, ISBN 978-1-58829-262-9, e.g. between 50 ug and 1.5 mg, preferably 125 ug to 500 ug, and other literature known to the person of skill. of peptide or DNA may be given and will depend on the 0478 Transformation of appropriate cell hosts with a respective peptide or DNA. Dosages of this range were Suc DNA construct of the present invention is accomplished by cessfully used in previous trials (Walteretal Nature Medicine well-known methods that typically depend on the type of 18, 1254-1261 (2012)). vector used. With regard to transformation of prokaryotic host 0484 Another aspect of the present invention includes an cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. in vitro method for producing activated T cells, the method Sci. USA 69, 2110, and Sambrook et al (1989) Molecular comprising contacting in vitro T cells with antigen loaded Cloning, A Laboratory Manual, Cold Spring Harbor Labora human MHC molecules expressed on the surface of a suitable tory, Cold Spring Harbor, N.Y. Transformation of yeast cells antigen-presenting cell for a period of time sufficient to acti is described in Sherman etal (1986) Methods InYeast Genet vate the T cell in an antigen specific manner, wherein the ics, A Laboratory Manual, Cold Spring Harbor, N.Y. The antigen is a peptide according to the invention. Preferably a method of Beggs (1978) Nature 275, 104-109 is also useful. Sufficient amount of the antigen is used with an antigen With regard to vertebrate cells, reagents useful in transfecting presenting cell. such cells, for example calcium phosphate and DEAE-dext 0485 Preferably the mammalian cell lacks or has a ran or liposome formulations, are available from Stratagene reduced level or function of the TAP peptide transporter. Cloning Systems, or Life Technologies Inc., Gaithersburg, Suitable cells that lack the TAP peptide transporter include Md. 20877, USA. Electroporation is also useful for trans T2, RMA-S and Drosophila cells. TAP is the transporter forming and/or transfecting cells and is well known in the art associated with antigen processing. for transforming yeast cell, bacterial cells, insect cells and 0486 The human peptide loading deficient cell line T2 is vertebrate cells. available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA under Cata 0479. Successfully transformed cells, i.e. cells that con logue No CRL 1992; the Drosophila cell line Schneider line tain a DNA construct of the present invention, can be identi 2 is available from the ATCC under Catalogue No CRL fied by well-known techniques such as PCR. Alternatively, 19863; the mouse RMA-S cell line is described in Karre et al. the presence of the protein in the Supernatant can be detected (Ljunggren, H.-G., and K. Karre. 1985. J. Exp. Med. 162: using antibodies. 1745). 0480. It will be appreciated that certain host cells of the 0487. Preferably, before transfection the host cell invention are useful in the preparation of the peptides of the expresses substantially no MHC class I molecules. It is also invention, for example bacterial, yeast and insect cells. How preferred that the stimulator cell expresses a molecule impor ever, other host cells may be useful in certain therapeutic tant for providing a co-stimulatory signal for T-cells Such as methods. For example, antigen-presenting cells, such as den any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acid dritic cells, may usefully be used to express the peptides of the sequences of numerous MHC class I molecules and of the invention such that they may be loaded into appropriate MHC co-stimulator molecules are publicly available from the Gen molecules. Thus, the current invention provides a host cell Bank and EMBL databases. comprising a nucleic acid or an expression vector according 0488. In case of a MHC class I epitope being used as an to the invention. antigen, the T cells are CD8-positive T cells. 0481. In a preferred embodiment the host cell is an antigen 0489. If an antigen-presenting cell is transfected to presenting cell, in particular a dendritic cell or antigen pre express Such an epitope, preferably the cell comprises an senting cell. APCs loaded with a recombinant fusion protein expression vector capable of expressing a peptide containing containing prostatic acid phosphatase (PAP) were approved SEQ ID NO: 1 to SEQID NO: 300, or a variant amino acid by the U.S. Food and Drug Administration (FDA) on Apr. 29, sequence thereof. 2010, to treat asymptomatic or minimally symptomatic meta 0490. A number of other methods may be used for gener static HRPC (Sipuleucel-T) (Small E J, et al. Placebo-con ating T cells in vitro. For example, autologous tumor-infil trolled phase III trial of immunologic therapy with trating lymphocytes can be used in the generation of CTL. sipuleucel-T (APC8015) in patients with metastatic, asymp Plebanski et al. (Induction of peptide-specific primary cyto tomatic hormone refractory prostate cancer. J. Clin Oncol. toxic T lymphocyte responses from human peripheral blood. 2006 Jul. 1; 24(19):3089-94. Rini et al. Combination immu EurJImmunol. 1995 June; 25(6):1783-7) make use of autolo notherapy with prostatic acid phosphatase pulsed antigen gous peripheral blood lymphocytes (PLBs) in the preparation presenting cells (provenge) plus bevacizumab in patients with of T cells. Furthermore, the production of autologous T cells serologic progression of prostate cancer after definitive local by pulsing dendritic cells with peptide or polypeptide, or via therapy. Cancer. 2006 Jul. 1; 107(1):67-74). infection with recombinant virus is possible. Also, B cells can US 2016/0250307 A1 Sep. 1, 2016 be used in the production of autologous T cells. In addition, invention also provides a method of killing target cells in a macrophages pulsed with peptide or polypeptide, or infected patient whose target cells aberrantly express a polypeptide with recombinant virus, may be used in the preparation of comprising an amino acid sequence of the invention, the autologous T cells. S. Walter et al. 2003 (Cutting edge: pre method comprising administering to the patient an effective determined avidity of human CD8 T cells expanded on cali number of T cells as defined above. brated MHC/anti-CD28-coated microspheres. J Immunol. 0497. By “aberrantly expressed the inventors also mean 2003 Nov. 15; 171 (10):4974-8) describe the in vitro priming that the polypeptide is over-expressed compared to normal of T cells by using artificial antigen presenting cells (aAPCs), levels of expression or that the gene is silent in the tissue from which is also a suitable way for generating T cells against the which the tumor is derived but in the tumor it is expressed. By peptide of choice. In the present invention, a APCs were gen “over-expressed the inventors mean that the polypeptide is erated by the coupling of preformed MHC:peptide complexes present at a level at least 1.2-fold of that present in normal to the surface of polystyrene particles (microbeads) by biotin: tissue; preferably at least 2-fold, and more preferably at least streptavidin biochemistry. This system permits the exact con 5-fold or 10-fold the level present in normal tissue. trol of the MHC density on aAPCs, which allows to selec 0498 T cells may be obtained by methods known in the tively elicit high- or low-avidity antigen-specific T cell art, e.g. those described above. responses with high efficiency from blood samples. Apart 0499 Protocols for this so-called adoptive transfer of T from MHC-peptide complexes, aAPCs should carry other cells are well known in the art. Reviews can be found in: proteins with co-stimulatory activity like anti-CD28 antibod Gattinoni L, et al. Adoptive immunotherapy for cancer: build ies coupled to their surface. Furthermore such a APC-based ing on success. Nat Rev Immunol. 2006 May: 6(5):383-93. systems often require the addition of appropriate soluble fac Review. and Morgan RA, et al. Cancer regression in patients tors, e.g. cytokines, like interleukin-12. after transfer of genetically engineered lymphocytes. Sci 0491 Allogeneic cells may also be used in the preparation ence. 2006 Oct. 6; 314(5796): 126-9). of T cells and a method is described in detail in WO97/26328, 0500 Any molecule of the invention, i.e. the peptide, incorporated herein by reference. For example, in addition to nucleic acid, antibody, expression vector, cell, activated T drosophila cells and T2 cells, other cells may be used to cell, T-cell receptor or the nucleic acid encoding it is useful for present antigens such as CHO cells, baculovirus-infected the treatment of disorders, characterized by cells escaping an insect cells, bacteria, yeast, vaccinia-infected target cells. In immune response. Therefore any molecule of the present addition plant viruses may be used (see, for example, Porta et invention may be used as medicament or in the manufacture al. (1994) Development of cowpea mosaic virus as a high of a medicament. The molecule may be used by itself or yielding system for the presentation of foreign peptides. combined with other molecule(s) of the invention or (a) Virology. 1994 Aug. 1: 202(2):949-55) which describes the known molecule(s). development of cowpea mosaic virus as a high-yielding sys 0501 Preferably, the medicament of the present invention tem for the presentation of foreign peptides. is a vaccine. It may be administered directly into the patient, 0492. The activated T cells that are directed against the into the affected organ or systemically i.d., i.m., s.c., i.p. and peptides of the invention are useful in therapy. Thus, a further i.v., or applied ex vivo to cells derived from the patient or a aspect of the invention provides activated T cells obtainable human cell line which are subsequently administered to the by the foregoing methods of the invention. patient, or used in vitro to select a subpopulation of immune 0493 Activated T cells, which are produced by the above cells derived from the patient, which are then re-administered method, will selectively recognize a cell that aberrantly to the patient. If the nucleic acid is administered to cells in expresses a polypeptide that comprises an amino acid vitro, it may be useful for the cells to be transfected so as to sequence of SEQID NO: 1 to SEQID NO 300. co-express immune-stimulating cytokines, such as interleu 0494 Preferably, the T cell recognizes the cell by interact kin-2. The peptide may be substantially pure, or combined ing through its TCR with the HLA/peptide-complex (for with an immune-stimulating adjuvant (see below) or used in example, binding). The T cells are useful in a method of combination with immune-stimulatory cytokines, or be killing target cells in a patient whose target cells aberrantly administered with a suitable delivery system, for example express a polypeptide comprising an amino acid sequence of liposomes. The peptide may also be conjugated to a suitable the invention wherein the patient is administered an effective carrier Such as keyhole limpet haemocyanin (KLH) or man number of the activated T cells. The T cells that are adminis nan (see for example WO95/18145). The peptide may also be tered to the patient may be derived from the patient and tagged, may be a fusion protein, or may be a hybrid molecule. activated as described above (i.e. they are autologous T cells). The peptides whose sequence is given in the present invention Alternatively, the T cells are not from the patient but are from are expected to stimulate CD4 or CD8 T cells. However, another individual. Of course, it is preferred if the individual stimulation of CD8T cells is more efficient in the presence of is a healthy individual. By “healthy individual' the inventors help provided by CD4 T-helper cells. Thus, for MHC Class I mean that the individual is generally in good health, prefer epitopes that stimulate CD8 T cells the fusion partner or ably has a competent immune system and, more preferably, is sections of a hybrid molecule suitably provide epitopes which not suffering from any disease that can be readily tested for, stimulate CD4-positive T cells. CD4- and CD8-stimulating and detected. epitopes are well known in the art and include those identified 0495. In vivo, the target cells for the CD8-positive T cells in the present invention. according to the present invention can be cells of the tumor 0502. In one aspect, the vaccine comprises at least one (which sometimes express MHC class II) and/or stromal cells peptide having the amino acid sequence set forth SEQID No. Surrounding the tumor (tumor cells) (which sometimes also 1 to SEQ ID No. 300, and at least one additional peptide, express MHC class II; (Dengel et al., 2006)). preferably two to 50, more preferably two to 25, even more 0496 The T cells of the present invention may be used as preferably two to 20 and most preferably two, three, four, five, active ingredients of a therapeutic composition. Thus, the six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, US 2016/0250307 A1 Sep. 1, 2016 52 fifteen, sixteen, seventeen or eighteen peptides. The peptide cell migration to lymphoid tissues (e.g., TNF-), accelerating (s) may be derived from one or more specific TAAS and may the maturation of dendritic cells into efficient antigen-pre bind to MHC class I molecules. senting cells for T-lymphocytes (e.g., GM-CSF, IL-1 and 0503. In another aspect, the vaccine comprises at least one IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated peptide having the amino acid sequence set forth in SEQID herein by reference in its entirety) and acting as immunoad NO: 1 to SEQIDNO:300, and at least one additional peptide, juvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) preferably two to 50, more preferably two to 25, even more (Gabrilovich, 1996 Production of vascular endothelial preferably two to 20 and most preferably two, three, four, five, growth factor by human tumors inhibits the functional matu six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, ration of dendritic cells Nat Med. 1996 October; 2(10): 1096 fifteen, sixteen, seventeen or eighteen peptides. The peptide 103). (s) may be derived from one or more specific TAAS and may 0506 CpG immunostimulatory oligonucleotides have bind to MHC class I molecules. also been reported to enhance the effects of adjuvants in a 0504 The polynucleotide may be substantially pure, or vaccine setting. Without being bound by theory, CpG oligo contained in a suitable vector or delivery system. The nucleic nucleotides act by activating the innate (non-adaptive) acid may be DNA, cDNA, PNA, RNA or a combination immune system via Toll-like receptors (TLR), mainly TLR9. thereof. Methods for designing and introducing such a CpG triggered TLR9 activation enhances antigen-specific nucleic acid are well known in the art. An overview is pro humoral and cellular responses to a wide variety of antigens, vided by e.g. (Pascolo et al., Human peripheral blood mono including peptide or protein antigens, live or killed viruses, nuclear cells transfected with messenger RNA stimulate anti dendritic cell vaccines, autologous cellular vaccines and gen-specific cytotoxic T-lymphocytes in vitro. Cell Mol Life polysaccharide conjugates in both prophylactic and therapeu Sci. 2005 August: 62(15):1755-62). Polynucleotide vaccines tic vaccines. More importantly it enhances dendritic cell are easy to prepare, but the mode of action of these vectors in maturation and differentiation, resulting in enhanced activa inducing an immune response is not fully understood. Suit tion of TH1 cells and strong cytotoxic T-lymphocyte (CTL) able vectors and delivery systems include viral DNA and/or generation, even in the absence of CD4 T cell help. The TH1 RNA, such as systems based on adenovirus, vaccinia virus, bias induced by TLR9 stimulation is maintained even in the retroviruses, herpes virus, adeno-associated virus or hybrids presence of vaccine adjuvants such as alum or incomplete containing elements of more than one virus. Non-viral deliv Freund's adjuvant (IFA) that normally promote a TH2 bias. ery systems include cationic lipids and cationic polymers and CpG oligonucleotides show even greater adjuvant activity are well known in the art of DNA delivery. Physical delivery, when formulated or co-administered with other adjuvants or Such as via a "gene-gun' may also be used. The peptide or in formulations such as microparticles, nanoparticles, lipid peptides encoded by the nucleic acid may be a fusion protein, emulsions or similar formulations, which are especially nec for example with an epitope that stimulates T cells for the essary for inducing a strong response when the antigen is respective opposite CDR as noted above. relatively weak. They also accelerate the immune response 0505. The medicament of the invention may also include and enable the antigen doses to be reduced by approximately one or more adjuvants. Adjuvants are Substances that non two orders of magnitude, with comparable antibody specifically enhance or potentiate the immune response (e.g., responses to the full-dose vaccine without CpG in some immune responses mediated by CD8-positive T cells and experiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 helper-T (TH) cells to an antigen, and would thus be consid describes the combined use of CpG oligonucleotides, non ered useful in the medicament of the present invention. Suit nucleic acid adjuvants and an antigen to induce an antigen able adjuvants include, but are not limited to, 1018 ISS, specific immune response. A CpGTLR9 antagonist is dSLIM aluminum salts, AMPLIVAX(R), AS15, BCG, CP-870, 893, (double Stem Loop Immunomodulator) by Mologen (Berlin, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived Germany) which is a preferred component of the pharmaceu from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiqui tical composition of the present invention. Other TLR binding mod (ALDARAR), residuimod, ImuPact IMP321, Interleu molecules such as RNA binding TLR 7, TLR8 and/or TLR9 kins as IL-2, IL-13, IL-21, Interferon-alpha or -beta, or pegy may also be used. lated derivatives thereof, IS Patch, ISS, ISCOMATRIX, 0507. Other examples for useful adjuvants include, but are ISCOMs, JuvImmune(R), LipoVac, MALP2, MF59, mono not limited to chemically modified CpGs (e.g. CpR, Idera), phosphoryl lipid A. Montanide IMS 1312, Montanide ISA dsRNA analogues such as Poly(I:C) and derivates thereof 206, Montanide ISA 50V, Montanide ISA-51, water-in-oil (e.g. AmpliCenR), HiltonolR), poly-(ICLC), poly(IC-R), poly and oil-in-water emulsions, OK-432, OM-174, OM-197-MP (I:C12U), non-CpG bacterial DNA or RNA as well as immu EC, ONTAK, Osp.A, PepTel(R) vector system, poly(lactid co noactive Small molecules and antibodies such as cyclophos glycolid) IPLG-based and dextran microparticles, talactof phamide, Sunitinib, Bevacizumab(R), celebrex, NCX-4016, errin SRL172, Virosomes and other Virus-like particles, sildenafil, tadalafil. Vardenafil, Sorafenib, temozolomide, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, QS21 stimulon, which is derived from saponin, mycobacte ZD2171, AZD2171, anti-CTLA4, other antibodies targeting rial extracts and synthetic bacterial cell wall mimics, and key structures of the immune system (e.g. anti-CD40, anti other proprietary adjuvants such as Ribis Detox, Quil, or TGFbeta, anti-TNFalpha receptor) and SC58175, which may Superfos. Adjuvants such as Freund's or GM-CSF are pre act therapeutically and/or as an adjuvant. The amounts and ferred. Several immunological adjuvants (e.g., MF59) spe concentrations of adjuvants and additives useful in the con cific for dendritic cells and their preparation have been text of the present invention can readily be determined by the described previously (Allison and Krummel, 1995 The Yin skilled artisan without undue experimentation. and Yang of T cell costimulation. Science. 1995 Nov. 10; 0508 Preferred adjuvants are anti-CD40, imiquimod, 270(5238):932-3). Also cytokines may be used. Several residuimod, GM-CSF, cyclophosphamide, Sunitinib, bevaci cytokines have been directly linked to influencing dendritic Zumab, interferon-alpha, CpG oligonucleotides and deri US 2016/0250307 A1 Sep. 1, 2016

vates, poly-(I:C) and derivates, RNA, sildenafil, and particu described above. The label may further indicate that the for late formulations with PLG or virosomes. mulation is useful or intended for Subcutaneous administra 0509. In a preferred embodiment, the pharmaceutical tion. composition according to the invention the adjuvant is 0516. The container holding the formulation may be a selected from the group consisting of colony-stimulating fac multi-use vial, which allows for repeat administrations (e.g., tors, such as Granulocyte Macrophage Colony Stimulating from 2-6 administrations) of the reconstituted formulation. Factor (GM-CSF, SargramoStim), cyclophosphamide, imi The kit may further comprise a second container comprising quimod, residuimod, and interferon-alpha. a Suitable diluent (e.g., sodium bicarbonate Solution). 0510. In a preferred embodiment, the pharmaceutical 0517. Upon mixing of the diluent and the lyophilized for composition according to the invention the adjuvant is mulation, the final peptide concentration in the reconstituted selected from the group consisting of colony-stimulating fac formulation is preferably at least 0.15 mg/mL/peptide (-75 tors, such as Granulocyte Macrophage Colony Stimulating ug) and preferably not more than 3 mg/mL/peptide (=1500 Factor (GM-CSF, SargramoStim), cyclophosphamide, imi ug). The kit may further include other materials desirable quimod and residuimod. In a preferred embodiment of the from a commercial and user standpoint, including other buff pharmaceutical composition according to the invention, the ers, diluents, filters, needles, Syringes, and package inserts adjuvant is cyclophosphamide, imiquimod or residuimod. with instructions for use. Even more preferred adjuvants are Montanide IMS 1312, 0518 Kits of the present invention may have a single Montanide ISA 206, Montanide ISA 50V, Montanide ISA container that contains the formulation of the pharmaceutical 51, poly-ICLC (HiltonolR) and anti-CD40 mAB, or combi compositions according to the present invention with or with nations thereof. out other components (e.g., other compounds or pharmaceu 0511. This composition is used for parenteral administra tical compositions of these other compounds) or may have tion, Such as Subcutaneous, intradermal, intramuscular or oral distinct container for each component. administration. For this, the peptides and optionally other 0519 Preferably, kits of the invention include a formula molecules are dissolved or Suspended in a pharmaceutically tion of the invention packaged for use in combination with the acceptable, preferably aqueous carrier. In addition, the com co-administration of a second compound (such as adjuvants position can contain excipients. Such as buffers, binding (e.g. GM-CSF), a chemotherapeutic agent, a natural product, agents, blasting agents, diluents, flavors, lubricants, etc. The a hormone orantagonist, an anti-angiogenesis agent or inhibi peptides can also be administered together with immune tor, an apoptosis-inducing agent or a chelator) or a pharma stimulating substances, such as cytokines. An extensive list ceutical composition thereof. The components of the kit may ing of excipients that can be used in Such a composition, can be pre-complexed or each component may be in a separate be, for example, taken from A. Kibbe, Handbook of Pharma distinct container prior to administration to a patient. The ceutical Excipients, 3rd Ed., 2000, American Pharmaceutical components of the kit may be provided in one or more liquid Association and pharmaceutical press. The composition can Solutions, preferably, an aqueous solution, more preferably, a be used for a prevention, prophylaxis and/or therapy of sterile aqueous solution. The components of the kit may also adenomateous or cancerous diseases. Exemplary formula be provided as solids, which may be converted into liquids by tions can be found in, for example, EP2112253. addition of suitable solvents, which are preferably provided 0512. The present invention provides a medicament that in another distinct container. useful in treating cancer, in particular HCC and other malig 0520. The container of a therapeutic kit may be a vial, test nancies. tube, flask, bottle, Syringe, or any other means of enclosing a 0513. The present invention is further directed at a kit Solid or liquid. Usually, when there is more than one compo comprising: nent, the kit will contain a second vial or other container, which allows for separate dosing. The kit may also contain (a) a container containing a pharmaceutical composition as another container for a pharmaceutically acceptable liquid. described above, in solution or in lyophilized form: Preferably, a therapeutic kit will contain an apparatus (e.g., (b) optionally a second container containing a diluent or one or more needles, Syringes, eye droppers, pipette, etc.), reconstituting solution for the lyophilized formulation; and which enables administration of the agents of the invention (c) optionally, instructions for (i) use of the Solution or (ii) that are components of the present kit. reconstitution and/or use of the lyophilized formulation. 0521. The present formulation is one that is suitable for 0514. The kit may further comprise one or more of (iii) a administration of the peptides by any acceptable route such as buffer, (iv) a diluent, (v) a filter. (vi) a needle, or (v) a syringe. oral (enteral), nasal, ophthal, Subcutaneous, intradermal, The container is preferably a bottle, a vial, a Syringe or test intramuscular, intravenous or transdermal. Preferably, the tube; and it may be a multi-use container. The pharmaceutical administration is s.c., and most preferably i.d. administration composition is preferably lyophilized. may be by infusion pump. 0515 Kits of the present invention preferably comprise a 0522 Since the peptides of the invention were isolated lyophilized formulation of the present invention in a suitable from HCC, the medicament of the invention is preferably container and instructions for its reconstitution and/or use. used to treat HCC. Suitable containers include, for example, bottles, vials (e.g. 0523 The present invention further includes a method for dual chamber vials), Syringes (such as dual chamber Syringes) producing a personalized pharmaceutical for an individual and test tubes. The container may beformed from a variety of patient comprising manufacturing a pharmaceutical compo materials such as glass or plastic. Preferably the kit and/or sition comprising at least one peptide selected from a ware container contain/s instructions on or associated with the house of pre-screened TUMAPs, wherein the at least one container that indicates directions for reconstitution and/or peptide used in the pharmaceutical composition is selected use. For example, the label may indicate that the lyophilized for suitability in the individual patient. In one embodiment, formulation is to be reconstituted to peptide concentrations as the pharmaceutical composition is a vaccine. The method US 2016/0250307 A1 Sep. 1, 2016 54 could also be adapted to produce T cell clones for down immunogenicity assays were performed using human T cells stream applications, such as TCR isolations, or soluble anti from healthy donors as well as from HCC patients. bodies and other treatment options. 0527. It is important to realize that the immune response 0524. A “personalized pharmaceutical shall mean spe triggered by the vaccine according to the invention attacks the cifically tailored therapies for one individual patient that will cancer in different cell-stages and different stages of devel only be used for therapy in Such individual patient, including opment. Furthermore different cancer associated signaling actively personalized cancer vaccines and adoptive cellular pathways are attacked. therapies using autologous patient tissue. 0528. This is an advantage over vaccines that address only 0525. As used herein, the term “warehouse' shall refer to one or few targets, which may cause the tumor to easily adapt a group of peptides that have been pre-screened for immuno to the attack (tumor escape). Furthermore, not all individual genicity and/or over-presentation in a particular tumor type. tumors express the same pattern of antigens. Therefore, a The term “warehouse' is not intended to imply that the par combination of several tumor-associated peptides ensures ticular peptides included in the vaccine have been pre-manu that every single tumor bears at least some of the targets. The factured and stored in a physical facility, although that pos composition was specifically designed in Sucha way that each sibility is contemplated. It is expressly contemplated that the HLA-A*02 and/or HLA-A*24-positive tumor is expected to peptides may be manufactured de novo for each individual express several of the antigens and cover several independent ized vaccine produced, or may be pre-manufactured and pathways necessary for tumor growth and maintenance. For stored. The warehouse (e.g. in the form of a database) is each of the peptide subsets specific for the two HLA class I composed of tumor-associated peptides which were highly alleles (A*02 and A*24) this is independently ensured based overexpressed in the tumor tissue of HCC patients with vari on the underlying experimental analyses. Thus, the vaccine ous HLA-A HLA-B and HLA-C alleles. It may contain MHC can easily be used “off-the-shelf for a larger patient popula class I and MHC class II peptides or elongated MHC class I tion. This means that a pre-selection of patients to be treated peptides. In addition to the tumor associated peptides col with the vaccine can be restricted to HLA typing, does not lected from several HCC tissues, the warehouse may contain require any additional biomarker assessments for antigen HLA-A*02 and HLA-A*24 marker peptides. These peptides expression, but it is still ensured that several targets are simul allow comparison of the magnitude of T-cell immunity taneously attacked by the induced immune response, which is induced by TUMAPS in a quantitative manner and hence important for efficacy (Banchereau et al., 2001; Walter et al., allow important conclusion to be drawn on the capacity of the 2012). vaccine to elicit anti-tumor responses. Secondly, they func 0529. In an aspect, the peptides are pre-screened for tion as important positive control peptides derived from a immunogenicity before being included in the warehouse. By “non-self antigen in the case that any vaccine-induced T-cell way of example, and not limitation, the immunogenicity of responses to TUMAPs derived from “self antigens in a the peptides included in the warehouse is determined by a patient are not observed. And third, it may allow conclusions method comprising in vitro T-cell priming through repeated to be drawn, regarding the status of immunocompetence of stimulations of CD8+ T cells from healthy donors with arti the patient. ficial antigen presenting cells loaded with peptide/MHC com 0526 TUMAPs for the warehouse are identified by using plexes and anti-CD28 antibody. an integrated functional genomics approach combining gene 0530. This method is preferred for rare cancers and expression analysis, mass spectrometry, and T-cell immunol patients with a rare expression profile. In contrast to multi ogy (XPresident(R)). The approach assures that only peptide cocktails with a fixed composition as currently devel TUMAPs truly present on a high percentage of tumors but not oped the warehouse allows a significantly higher matching of or only minimally expressed on normal tissue, are chosen for the actual expression of antigens in the tumor with the vac further analysis. For initial peptide selection, HCC samples cine. Selected single or combinations of several “off-the from patients and blood from healthy donors were analyzed shelf peptides will be used for each patient in a multitarget in a stepwise approach: approach. In theory an approach based on selection of e.g. 5 1. HLA ligands from the malignant material were identified different antigenic peptides from a library of 50 would by mass spectrometry already lead to approximately 17 million possible drug prod 2. Genome-wide messenger ribonucleic acid (mRNA) uct (DP) compositions. expression analysis was used to identify genes over-ex 0531. In an aspect, the peptides are selected for inclusion pressed in the malignant tissue (HCC) compared with a range in the vaccine based on their suitability for the individual of normal organs and tissues patient based on the method according to the present inven 3. Identified HLA ligands were compared to gene expression tion as described herein, or as below. data. Peptides over-presented or selectively presented on 0532. The HLA phenotype, transcriptomic and pepti tumor tissue, preferably encoded by selectively expressed or domic data is gathered from the patient's tumor material, and over-expressed genes as detected in step 2 were considered blood samples to identify the most suitable peptides for each suitable TUMAP candidates for a multi-peptide vaccine. patient containing “warehouse' and patient-unique (i.e. 4. Literature research was performed in order to identify mutated) TUMAPs. Those peptides will be chosen, which are additional evidence supporting the relevance of the identified selectively or over-expressed in the patients tumor and, where peptides as TUMAPs possible, show strong in vitro immunogenicity if tested with 5. The relevance of over-expression at the mRNA level was the patients’ individual PBMCs. confirmed by redetection of selected TUMAPs from step 3 on 0533 Preferably, the peptides included in the vaccine are tumor tissue and lack of (or infrequent) detection on healthy identified by a method comprising: (a) identifying tumor tissues. associated peptides (TUMAPs) presented by a tumor sample 6. In order to assess, whether an induction of in vivo T-cell from the individual patient; (b) comparing the peptides iden responses by the selected peptides may be feasible, in vitro tified in (a) with a warehouse (database) of peptides as US 2016/0250307 A1 Sep. 1, 2016

described above; and (c) selecting at least one peptide from from the warehouse that correlates with a tumor-associated the warehouse (database) that correlates with a tumor-asso peptide identified in the patient; and (d) optionally, selecting ciated peptide identified in the patient. For example, the at least one peptide identified de novo in (a) confirming its TUMAPs presented by the tumor sample are identified by: immunogenicity. (al)) comparing expression data from the tumor sample to 0536. In one exemplary embodiment, the peptides expression data from a sample of normal tissue correspond included in the vaccine are identified by: (a) identifying ing to the tissue type of the tumor sample to identify proteins tumor-associated peptides (TUMAPs) presented by a tumor that are over-expressed or aberrantly expressed in the tumor sample from the individual patient; and (b) selecting at least sample; and (a2) correlating the expression data with one peptide identified de novo in (a) and confirming its immu sequences of MHC ligands bound to MHC class I and/or class nogenicity. II molecules in the tumor sample to identify MHC ligands 0537. Once the peptides for a personalized peptide based derived from proteins over-expressed or aberrantly expressed vaccine are selected, the vaccine is produced. The vaccine by the tumor. Preferably, the sequences of MHC ligands are preferably is a liquid formulation consisting of the individual identified by eluting bound peptides from MHC molecules peptides dissolved in 33% DMSO. isolated from the tumor sample, and sequencing the eluted 0538 Each peptide to be included into a product is dis ligands. Preferably, the tumor sample and the normal tissue solved in DMSO. The concentration of the single peptide are obtained from the same patient. Solutions has to be chosen depending on the number of pep 0534. In addition to, or as an alternative to, selecting pep tides to be included into the product. The single peptide tides using a warehousing (database) model, TUMAPs may DMSO solutions are mixed in equal parts to achieve a solu be identified in the patient de novo, and then included in the tion containing all peptides to be included in the product with vaccine. As one example, candidate TUMAPs may be iden a concentration of ~2.5 mg/ml per peptide. The mixed solu tified in the patient by (al) comparing expression data from tion is then diluted 1:3 with water for injection to achieve a the tumor sample to expression data from a sample of normal concentration of 0.826 mg/ml perpeptide in 33% DMSO. The tissue corresponding to the tissue type of the tumor sample to diluted solution is filtered through a 0.22 Lumsterile filter. The identify proteins that are over-expressed or aberrantly final bulk solution is obtained. expressed in the tumor sample; and (a2) correlating the 0539 Final bulk solution is filled into vials and stored at expression data with sequences of MHC ligands bound to -20°C. until use. One vial contains 700 uL solution, contain MHC class I and/or class II molecules in the tumor sample to ing 0.578 mg of each peptide. Of this, 500 uL (approx. 400 ug identify MHC ligands derived from proteins over-expressed per peptide) will be applied for intradermal injection. or aberrantly expressed by the tumor. As another example, 0540. The present invention will now be described in the proteins may be identified containing mutations that are following examples which describe preferred embodiments unique to the tumor sample relative to normal corresponding thereof, nevertheless, without being limited thereto. For the tissue from the individual patient, and TUMAPs can be iden purposes of the present invention, all references as cited tified that specifically target the mutation. For example, the herein are incorporated by reference in their entireties. genome of the tumor and of corresponding normal tissue can (0541. In the Figures, be sequenced by whole genome sequencing: For discovery of 0542 FIG. 1 shows the over-presentation of various pep non-synonymous mutations in the protein-coding regions of tides in normal tissues (dark gray) and HCC (light gray). FIG. genes, genomic DNA and RNA are extracted from tumor 1A) APOB, Peptide: ALVDTLKFV (A*02) (SEQID NO: 7), tissues and normal non-mutated genomic germline DNA is tissues from left to right; 1 adipose tissues, 3 adrenal glands, extracted from peripheral blood mononuclear cells (PBMCs). 2 arteries, 2 bone marrows, 7 brains, 3 breasts, 13 colons, 4 The applied NGS approach is confined to the re-sequencing esophagi, 2 gallbladders, 3 GI tracts, 3 hearts, 16 kidneys, 4 of protein coding regions (exome re-sequencing). For this leukocyte samples, 45 lungs, 1 lymph node, 1 ovary, 7 pan purpose, exonic DNA from human samples is captured using creas, 1 peripheral nerve, 1 pituitary gland, 3 pleuras, 1 pros Vendor-supplied target enrichment kits, followed by sequenc tate, 6 recti, 3 Sceletal muscles, 1 serous membrane, 3 skins, ing with e.g. a HiSeq2000 (Illumina). Additionally, tumor 4 spleens, 7 stomachs, 1 testis, 2 thymi, 3 thyroid glands, 2 mRNA is sequenced for direct quantification of gene expres uteri, 2 veins, and 20 livers: FIG. 1B) ALDH1L1, Peptide: sion and validation that mutated genes are expressed in the KLQAGTVFV (A*02) (SEQID NO: 2), tissues from left to patients tumors. The resultant millions of sequence reads are right: 1 adipose tissues, 3 adrenal glands, 2 arteries, 2 bone processed through software algorithms. The output list con marrows, 7 brains, 3 breasts, 13 colons, 4 esophagi, 2 gall tains mutations and gene expression. Tumor-specific Somatic bladders, 3 GI tracts, 3 hearts, 16 kidneys, 4 leukocyte mutations are determined by comparison with the PBMC samples, 45 lungs, 1 lymph node, 1 ovary, 7 pancreas, 1 derived germline variations and prioritized. The denovo iden peripheral nerve, 1 pituitary gland, 3 pleuras, 1 prostate, 6 tified peptides can then be tested for immunogenicity as recti, 3 Sceletal muscles, 1 serous membrane, 3 skins, 4 described above for the warehouse, and candidate TUMAPs spleens, 7 stomachs, 1 testis, 2 thymi, 3 thyroid glands, 2 possessing Suitable immunogenicity are selected for inclu uteri, 2 veins, and 20 livers: FIG. 1C) C8B, Peptide: sion in the vaccine. AYLLQPSQF (A*24) (SEQID NO:200), tissues from left to 0535 In one exemplary embodiment, the peptides right: including 2 adrenal glands, 1 artery, 4 brains, 1 breast, included in the vaccine are identified by: (a) identifying 5 colons, 1 hearts, 13 kidneys, 9 lungs, 3 pancreas, 2 recti, 3 tumor-associated peptides (TUMAPs) presented by a tumor skins, 1 spleen, 12 stomachs, 1 thymus, 2 uteri, and 9 livers; sample from the individual patient by the method as described FIG.1D) RAD23BPeptide: KIDEKNFVV (SEQID NO: 63) above; (b) comparing the peptides identified in a) with a 1 serous membrane, 1 adipose tissue, 3 adrenal glands, 2 warehouse of peptides that have been prescreened for immu arteries, 2 bone marrows, 7 brains, 3 breasts, 13 colons, 2 nogenicity and overpresentation in tumors as compared to gallbladders, 3 GI tracts, 3 hearts, 12 kidneys, 4 leukocytes, corresponding normal tissue; (c) selecting at least one peptide 19 livers, 43 lungs, 1 lymph node, 1 ovary, 6 pancreases, 1 US 2016/0250307 A1 Sep. 1, 2016 56 peripheral nerve, 1 pituitary gland, 3 pleuras, 1 prostate, 6 (5 brain cancers, 6 colon cancers, 1 esophageal cancer, 6 liver rectums, 3 skeletal muscles, 3 skins, 4 spleens, 5 stomachs, 1 cancers, 9 lung cancers, 1 ovarian cancer, 1 prostate cancer, 4 testis, 2 thymi, 3 thyroid glands, 2 uteri, 2 veins, 4 esophagi; rectum cancers, 2 stomach cancers) (from left to right). FIG.1E) RAD23BPeptide: KIDEKNFVV (SEQID NO: 63), 0543 FIG. 2 shows exemplary expression profiles (rela shown are only samples on which the peptide was presented: tive expression compared to normal kidney) of Source genes 5 cell-lines, 1 normal tissue (1 adrenal gland), 16 cancer of the present invention that are highly over-expressed or tissues (2 brain cancers, 4 liver cancers, 5 lung cancers, 1 exclusively expressed in HCC in a panel of normal tissues (dark gray) and 12 HCC samples (gray). FIG. 2A) APOB, rectum cancer, 1 urinary bladder cancer, 3 uterus cancers) tissues from left to right: 1 adrenal gland, 1 artery, 1 bone (from left to right); FIG.1F) RFNGRLPPDTLLQQV (SEQ marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 IDNO:92), shown are only samples on which the peptide was heart, 3 kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1 lymph presented: 1 serous membrane, 1 adipose tissue, 3 adrenal node, 1 ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivary glands, 2 arteries, 2 bone marrows, 7 brains, 3 breasts, 13 gland, 1 skeletal muscle, 1 skin, 1 Small intestine, 1 spleen, 1 colons, 2 gallbladders, 3 GI tracts, 3 hearts, 12 kidneys, 4 stomach, 1 testis, 1 thymus, 1 thyroid gland, 1 urinary blad leukocytes, 19 livers, 43 lungs, 1 lymph node, 1 ovary, 6 der, 1 uterine cervix, 1 uterus, 1 vein; FIG. 2B) AMACR, pancreases, 1 peripheral nerve, 1 pituitary gland, 3 pleuras, 1 tissues from left to right: 1 adrenal gland, 1 artery, 1 bone prostate, 6 rectums, 3 skeletal muscles, 3 skins, 4 spleens, 5 marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 stomachs, 1 testis, 2 thymi, 3 thyroid glands, 2 uteri, 2 veins, heart, 3 kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1 lymph 4 esophagi; FIG. 1G) RFNG Peptide: RLPPDTLLQQV node, 1 ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivary (SEQ ID NO: 92), shown are only samples on which the gland, 1 skeletal muscle, 1 skin, 1 Small intestine, 1 spleen, 1 peptide was presented: 2 cell-lines, 2 normal tissues (2 adre stomach, 1 testis, 1 thymus, 1 thyroid gland, 1 urinary blad nal glands), 17 cancertissues (1 brain cancer, 1 breast cancer, der, 1 uterine cervix, 1 uterus, 1 vein; FIG. 2C) ALDH1L1, 1 esophageal cancer, 5 liver cancers, 4 lung cancers, 1 ovarian tissues from left to right: 1 adrenal gland, 1 artery, 1 bone cancer, 1 prostate cancer, 2 urinary bladder cancers, 1 uterus marrow, 1 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 cancer) (from left to right); FIG.1H) FLVCR1 Peptide: SVW heart, 3 kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1 lymph FGPKEV (SEQ ID NO: 104) 1 serous membrane, 1 adipose node, 1 ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivary tissue, 3 adrenal glands, 2 arteries, 2 bone marrows, 7 brains, gland, 1 skeletal muscle, 1 skin, 1 Small intestine, 1 spleen, 1 3 breasts, 13 colons, 2 gallbladders, 3 GI tracts, 3 hearts, 12 stomach, 1 testis, 1 thymus, 1 thyroid gland, 1 urinary blad kidneys, 4 leukocytes, 19 livers, 43 lungs, 1 lymph node, 1 der, 1 uterine cervix, 1 uterus, 1 vein; FIG. 2D) FGG, tissues ovary, 6 pancreases, 1 peripheral nerve, 1 pituitary gland, 3 from left to right: 1 adrenal gland, 1 artery, 1 bone marrow, 1 pleuras, 1 prostate, 6 rectums, 3 skeletal muscles, 3 skins, 4 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 spleens, 5 stomachs, 1 testis, 2 thymi, 3 thyroid glands, 2 kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1 lymph node, 1 uteri, 2 veins, 4 esophagi; FIG. 1I) FLVCR1 Peptide: SVW ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 FGPKEV (SEQ ID NO: 104), shown are only samples on skeletal muscle, 1 skin, 1 Small intestine, 1 spleen, 1 stomach, which the peptide was presented: 9 cell lines, 1 normal tissue 1 testis, 1 thymus, 1 thyroid gland, 1 urinary bladder, 1 uterine (1 Small intestine), 16 cancertissues (1 brain cancer, 1 breast cervix, 1 uterus, 1 vein; FIG. 2E) C8B, tissues from left to cancer, 5 liver cancers, 5 lung cancers, 1 skin cancer, 1 stom right: 1 adrenal gland, 1 artery, 1 bone marrow, 1 brain ach cancer, 1 urinary bladder cancer, 1 uterus cancer) (from (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 kidneys, 1 left to right); FIG. 1J) IKBKAP Peptide: LLFPHPVNQV leukocyte sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1 (SEQ ID NO: 156) 1 serous membrane, 1 adipose tissue, 3 pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 skeletal adrenal glands, 2 arteries, 2 bone marrows, 7 brains, 3 breasts, muscle, 1 skin, 1 Small intestine, 1 spleen, 1 stomach, 1 testis, 13 colons, 2 gallbladders, 3 GI tracts, 3 hearts, 12 kidneys, 4 1 thymus, 1 thyroid gland, 1 urinary bladder, 1 uterine cervix, leukocytes, 19 livers, 43 lungs, 1 lymph node, 1 ovary, 6 1 uterus, 1 vein; and FIG.2F) HSD17B6, tissues from left to pancreases, 1 peripheral nerve, 1 pituitary gland, 3 pleuras, 1 right: including 1 adrenal gland, 1 artery, 1 bone marrow, 1 prostate, 6 rectums, 3 skeletal muscles, 3 skins, 4 spleens, 5 brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 stomachs, 1 testis, 2 thymi, 3 thyroid glands, 2 uteri, 2 veins, kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1 lymph node, 1 4 esophagi; FIG. 1K) IKBKAP Peptide: LLFPHPVNQV ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivary gland, 1 (SEQ ID NO: 156), shown are only samples on which the skeletal muscle, 1 skin, 1 Small intestine, 1 spleen, 1 stomach, peptide was presented: 7 cell-lines, 2 primary cultures, 1 1 testis, 1 thymus, 1 thyroid gland, 1 urinary bladder, 1 uterine normal tissue (1 colon), 34 cancer tissues (1 bone marrow cervix, 1 uterus, and 1 vein. cancer, 1 breast cancer, 1 colon cancer, 2 esophageal cancers, 0544 FIG.3 shows exemplary flow cytometry results after 2 leukocytic leukemia cancers, 4 liver cancers, 11 lung can peptide-specific multimer staining. Further explanations see cers, 3 lymph node cancers, 5 ovarian cancers, 4 urinary example 4. bladder cancers) (from left to right); FIG.1L) NKD1 Peptide: 0545 FIG. 4 shows exemplary flow cytometry results after FLDTPIAKV (SEQID NO: 47), 1 serous membrane, 1 adi peptide-specific multimer staining. Further explanations see pose tissue, 3 adrenal glands, 2 arteries, 2 bone marrows, 7 example 4. brains, 3 breasts, 13 colons, 2 gallbladders, 3 GI tracts, 3 hearts, 12 kidneys, 4 leukocytes, 19 livers, 43 lungs, 1 lymph EXAMPLES node, 1 ovary, 6 pancreases, 1 peripheral nerve, 1 pituitary Example 1 gland, 3 pleuras, 1 prostate, 6 rectums, 3 skeletal muscles, 3 skins, 4 spleens, 5 stomachs, 1 testis, 2 thymi, 3 thyroid Identification and Quantitation of Tumor Associated glands, 2 uteri, 2 veins, 4 esophagi; FIG.1M) NKD1 Peptide: Peptides Presented on the Cell Surface FLDTPIAKV (SEQID NO: 47), shown are only samples on (0546 Tissue Samples which the peptide was presented: 1 other disease (encepha (0547 Patients tumor tissues were obtained from Univer locele), 2 normal tissues (1 lung, 1 spleen), 35 cancer tissues sitätsklinik für Allgemeine, Viszeralund Transplantation US 2016/0250307 A1 Sep. 1, 2016 57

Schirurgie, Tübingen, Germany; Istituto Nazionale Tumori 0553 Presentation profiles of exemplary over-presented “Pascale'. Molecular Biology and Viral Oncology Unit, Via peptides are shown in FIG. 1. Mariano, Naples, Italy; Bio-Options Inc., Brea, Calif., USA; 0554 Presentation scores for exemplary peptides are ProteoGenex Inc., Culver City, Calif., USA; Asterand shown in Table 8. Europe, Royston Herts, United Kingdom. Written informed consents of all patients had been given before Surgery. Tissues TABLE 8 were shock-frozen immediately after Surgery and stored until isolation of TUMAPs at -70° C. or below. Presentation scores. 0548 Isolation of HLA Peptides from Tissue Samples SEQ ID 0549 HLA peptide pools from shock-frozen tissue No. Sequence PeptidePresentation samples were obtained by immune precipitation from Solid tissues according to a slightly modified protocol (Falk, K., 1. WMAPFTMTI ------1991; Seeger, F. H. T., 1999) using the HLA-A*02-specific 2 KLOAGTVFW ------antibody BB7.2, the HLA-A. -B, -C-specific antibody W6/32, CNBr-activated Sepharose, acid treatment, and ultra 4. KLODFSDQL ------filtration. 5 ALVEOGFTV ------0550 Mass Spectrometry Analyses 0551. The HLA peptide pools as obtained were separated 6 KLSPTWWGL ------according to their hydrophobicity by reversed-phase chroma- 7 ALWDTLKFW ------tography (nano Acquity UPLC system, Waters) and the elut ing peptides were analyzed in LTQ Velos and fusion hybrid 8 KLLEEATISW -- mass spectrometers (ThermoElectron) equipped with an ESI source. Peptide pools were loaded directly onto the analytical 9 ALANOKLYSW -- fused-silica micro-capillary column (75 um i.d.x250 mm) 1O SLLEEFDFHW ------packed with 1.7 um C18 reversed-phase material (Waters) applying a flow rate of 400 mL per minute. Subsequently, the 11 SLSOELVGV -- peptides were separated using a two-step 180 minute-binary 12 FLAELAYDL ------gradient from 10% to 33% B at a flow rate of 300 nL per minute. The gradient was composed of Solvent A (0.1% for- 14 ALADLTGTWW ------mic acid in water) and solvent B (0.1% formic acid in aceto- 15 LYGHTWTV -- nitrile). A gold coated glass capillary (PicoTip, New Objec tive) was used for introduction into the nanoESI source. The 16 SLLGGNIRL ---- LTQ-Orbitrap mass spectrometers were operated in the data dependent mode using a TOPS strategy. In brief, a scan cycle 17 RWAS kPTSGV -- was initiated with a full scan of high mass accuracy in the 19 FLEETKATW ------orbitrap (R-30 000), which was followed by MS/MS scans also in the orbitrap (R-7500) on the 5 most abundant precur- 2O KLSNVLQQV ------sor ions with dynamic exclusion of previously selected ions. 21 QLIEVSSPITL ------Tandem mass spectra were interpreted by SEQUEST and additional manual control. The identified peptide sequence 22 RIAGIRGIQGW ------was assured by comparison of the generated natural peptide fragmentation pattern with the fragmentation pattern of a 23 RLYDPASGTISL -- synthetic sequence-identical reference peptide. 24 SLAEEKLOASW ------0552 Label-free relative LC-MS quantitation was per formed by ion counting i.e. by extraction and analysis of 25 SLDGKAALTEL ------LC-MS features (Mueller et al. 2007a). The method assumes 26 SLLHTIYEW ------that the peptide's LC-MS signal area correlates with its abun dance in the sample. Extracted features were further pro- 27 TLPDFRLPEI ------cessed by charge State deconvolution and retention time 28 TLODHLNSL ------alignment (Mueller et al. 2007b; Sturm et al. 2008). Finally, all LC-MS features were cross-referenced with the sequence 29 YIODEINTI ------identification results to combine quantitative data of different samples and tissues to peptide presentation profiles. The 3O YLGEGPRMW ---- quantitative data were normalized in a two-tier fashion 31 YOMDIQOEL ---- according to central tendency to account for variation within technical and biological replicates. Thus each identified pep- 32 ALNAWRLLW ------tide can be associated with quantitative data allowing relative 33 LLHGHIWEL -- quantification between samples and tissues. In addition, all quantitative data acquired for peptide candidates was 34 SLAEGTATW ------inspected manually to assure data consistency and to Verify the accuracy of the automated analysis. For each peptide a 38 ALADWWHEA -- presentation profile was calculated showing the mean sample 39 ALDPKANFST ------presentation as well as replicate variations. The profiles jux tapose HCC samples to a baseline of normal tissue samples. US 2016/0250307 A1 Sep. 1, 2016 58

TABLE 8 - continued TABLE 8 - continued

Presentation scores. Presentation scores.

SEO ID SEQ ID No. Sequence PeptidePresentation No. Sequence PeptidePresentation

4 O ALLAEGITWWW -- 94 RMSDWWKGW ------

42 ALLGGNWRMML ------96 SLLEEPNWIRV ----

44 ALQDAIRQL -- 97 SLLPOLIEW ----

47 FLDTPIAKW -- 98 SLLSPEHLOYL ----

49 FLYPEKDEPT ------99 SLSAFLPSL ------

51 GLAEELWRA -- O1 SLWEGGWRGW ------

52 GLFNAELLEA -- O3 SMGDHLWWWA ------

53 GLIHLEGDTW ------O7 TLGOFYOEV ------

54 GLLDPNWKSIFW ------O8 TLLKKISEA ------

55 GLYGRTIEL -- O9 TLYALSHAW --

56 GWLPGLWGW -- 11 TVMDIDTSGTFNV --

f HLTEAIOYV ---- 13 WLMDKLWEL ----

58 ILADLNLSW -- 14 VLSOVYSKV ------

59 ILADTFIGW ---- 16 WWWIPAISAW ------

6 O ILSPLSWAL -- 17 YAFPKSITV ------

61 KIADFELPTI ------19 YLDKNLTWSW --

62 KIAGTNAEW ---- 2O YLGEEYWKA ------

66 KLHEEIDRW ---- 21 YLITGNLEKL --

67 KLKETIOKL ------22 YLSOAADGAKVL ------

70 KLLDLETERILL ---- 23 YLWDLDHGFAGW ----

71. KLLDNWDSW ------24 LLIDWWTYL ------

72 KLSEAWTSW -- 26 TLLDSPIKW ----

7s KOMEPLHAV -- 27 WLIGSNHSL --

76 LADIGGDPFAA ------28 GLAFSLNGW --

77 LLHEENFSW -- 29 SOADVIPAV --

79 LLSTGYEA ------3O ALDAGAWYTL ----

81 NLASFIEOVAW ---- 31 ALDSGAFOSV ----

82 NWFDGLWRW -- 32 ALHEEWWGW --

83 QLHDFVMSL ------33 ALLEM DARL --

84 OLTPVLVSV ---- 34 ALLETNPYLL ----

85 RILPKWLEW -- 35 ALLGKIEKW --

86 RLAAFYSOW ------37 ALPTWLWGW ----

88 RLIDRIKTW ------39 ALSSKPAEW --

89 RLIEEIKNW ------42 AWIGGLIYW ----

91 RLPDIPLROV -- 44 FIOLITGV --

93 RLYTMDGITV ------46 FLWTEOAHTV -- US 2016/0250307 A1 Sep. 1, 2016 59

TABLE 8 - Continued TABLE 8 - Continued

Presentation scores. Presentation scores.

SEO ID SEQ ID No. Sequence PeptidePresentation No. Sequence PeptidePresentation

47 GLAPGGLAWW -- 2 OO AYLLOPSQF ------

48 GLFAPLWFL ------2O1 AYWNTFHNI ------

51 HLAKWTAEW -- 2O2 AYGTYRSNF ------

54 KLTDHLKYW -- 2O3 YYGILOEKI ------

61 RLLDEOFAW -- 2O4. KYRLTYAYE ----

62 RLMSALTOW ---- 2O5 WYGLORNLL --

63 RLTESWLYL ---- 2O6 KWPETPLLL ------

64 RMLIKLLEW -- 2O8 SYNPAENAWILL ----

67 SLAESSFDW ---- 210 AYPAIRYLL ----

68 SLAWLWPIW -- 211 IYIPSYFDF ----

69 SLFEWFHPL -- 212 WYGDWISNI ------

70 SLHINGVIQL -- 213 YYNKVSTVF --

71. SLIPAWLTW -- 214 IYVTSIEOI ------

72 SLLNFLOHL -- 217 DYIPYWFKL ------

73 SLTSEIHFL -- 218 WYOGAIROI --

74 TLAELGAWOW -- 219 GWMAGDIYSW --

76 TLGOIWDV -- 22O SLLEKELESW ----

77 WLDEPYEKW -- 221 ALCEENMRGW --

79 YIHNILYEW ---- 224 ALASWIKEL --

8O YLGPHIASWTL ---- 225 KMDPWAYRV --

81 YLLEKFWAW -- 226 AVLGPLGLQEW --

84 WVLDGGOIVTV -- 227 ALLKVNOEL --

85 ALFPALRPGGFOA ---- 228 YLITSWELL ----

86 WLLAQIIQW -- 229 KMFESFIESW ----

87 SYPTEFPRF -- 23 O WLTEFTREW --

88 RYSAGWDAKF -- 231 RLFNDPWAMW ----

89 AFSPDSHYLLF ------233 ALIGKDDAI --

90 RYNEKCFKL ------234 YLEPYLKEW --

91 KYPDIISRI ---- 236 ALADKELLPSW ----

92 SYITKPEKW -- 237 ALRGEIETW ------

93 IYPGAFWDL ------238 AMPPPPPOGV ----

94 QYASRFWOL ------239 FLIGFIPAKA --

95 RYAPPPSFSEF ------24 O FLWERPTLLW ------

96 AYLKWISOI ------241 FWLPLIGLHEA ----

97 RWPKKSAEF -- 242 GLFAPWHKW --

98 LYWSHPRKF -- 243 GILDNPELRW ------US 2016/0250307 A1 Sep. 1, 2016 60

TABLE 8 - Continued TABLE 8 - continued

Presentation scores. Presentation scores.

SEO ID SEQ ID No. Sequence PeptidePresentation No. Sequence PeptidePresentation

244 KIAELLENW -- 288 LLDDSLWSI --

245 KLGAVFNOV -- 289 LLLEEGGLWOW ----

248 KLNDLIORL -- 290 NLIDLDDLYW ----

249 LLIGERWAL ------292 RIPAYEWTV --

250 NLAEWWERW ---- 293 FLASESLIKQI ----

251 RLFADILNDW ---- 295 SLFSSPPEI ----

252 RTIEYLEEW -- 297 TLFYSLREW --

253 RVPPPPOSV -- 298 TMAKESSIIGW ----

255 SLFGODWKAV ------299 ALLRVTPFI --

256 SLFOGVEFHYW -- 3 O1 WLADFGARW ------

27 SLLEKAGPEL ------3 O2 KIOEIL TOW ------

258 SLMGPWWHEW -- 3O3 GWYDGEEHSW --

26 O TLMDMRLSOV ---- 3O4. SLIDOFFGV ------

261 WLFOEALWHV ---- 3. OS GWLENIFGW --

263 WLYPSLKEI -- 3O8 ALLRTWWSW --

264 VMODPEFLOSV ---- 3 O9 GLIEIISNA. --

265 WLIEDGKWWTW ---- 310 SLWGGDWWL --

266 SLLESNKDLLL -- 311 FLIPIYHOV --

267 ALNENINOV -- 312 RLGIKPESW ------

268 KLYOEVEIASW -- 313 TAPPEALLMW --

269 YLMEGSYNKW -- 314 YLAPFLRNW --

27 O SVLDOKILL ---- 315 KVLDGSPIEW --

271 LILDKLILL -- 316 LREKWEFL --

272 QOLDSKFLEOW -- 317 KLPEKWESW ----

273 AILETAPKEW ---- 3.18 KLNEINEKI --

274 ALAEALKEW -- 319 KLFNEFIQL --

27s ALIEGAGILL ---- 32O GLADNTWIAKW --

276 ALLEADWNIKL -- 322 ILYDIPDIRL --

277 ALLEENSTPOL -- 324 RLFETKITOW ----

278 ALTSWWWTL -- 326 ALSDGWHKI ----

279 ALWTGMHTI -- 327 GLNEEIARW ----

281 GLLAGDRLWEW -- 328 RLEEDDGDWAM --

282 GOFPSYLETV ---- 329 SLIEDLILL ------

283 ILSGIGVSOV -- 330 SMSADWPLW ----

284 KLDAFWEGW -- 332 AMLAWLHTW --

286 KWLDKWFRA -- 334 SILTIEDGIFEW -- US 2016/0250307 A1 Sep. 1, 2016 61

TABLE 8 - continued 0560 Microarray Experiments

Presentation scores. 0561 Gene expression analysis of all tumor and normal tissue RNA samples was performed by Affymetrix Human SEO ID Genome (HG) U133A or HG-U133 Plus 2.0 oligonucleotide No Sequence PeptidePresentation microarrays (Affymetrix, Santa Clara, Calif., USA). All steps 335 SLLPWDIRQYL ---- were carried out according to the Affymetrix manual. Briefly, double-stranded cDNA was synthesized from 5-8 ug of total 336 YLPTFELTV -- RNA, using SuperScript RTII (Invitrogen) and the oligo-dT T7 primer (MWG Biotech, Ebersberg, Germany) as 337 TLLAAEFLKOW -- described in the manual. In vitro transcription was performed 338 KLFDSDPITVTV ------with the BioArray High Yield RNA Transcript Labelling Kit (ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the 34 O KVFDEVIEW -- U133A arrays or with the GeneChip IVT Labelling Kit (Af. 342 AMSSKFFLW -- fymetrix) for the U133 Plus 2.0 arrays, followed by crNA fragmentation, hybridization, and staining with Streptavidin 343 LLLPDYYLW -- phycoerythrin and biotinylated anti-streptavidin antibody 345 SYNPLWLRI ------(Molecular Probes, Leiden, Netherlands). Images were (A24) scanned with the Agilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-Chip Scanner 3000 (U133 346 LYOILOGIWF ------Plus 2.0), and data were analyzed with the GCOS software (A24) (Affymetrix), using default settings for all parameters. For 347 ALNPADITW -- normalization, 100 housekeeping genes provided by Affyme trix were used. Relative expression values were calculated The table lists peptides that are very highly over-presented on tumors Compared to a panel of normal tissues (+++), highly from the signal log ratios given by the Software and the over-presented on tumors Compared to a panel of normal tissues (++) or over-presented on tumors Compared to a panel of normal normal kidney sample was arbitrarily set to 1.0. Exemplary tissues (+). expression profiles of Source genes of the present invention S* = phospho serine that are highly over-expressed or exclusively expressed in HCC are shown in FIG. 2. Expression scores for further Example 2 exemplary genes are shown in Table 9.

Expression Profiling of Genes Encoding the Peptides TABLE 9 of the Invention 0555. Over-presentation or specific presentation of a pep Expression SCC eS. tide on tumor cells compared to normal cells is sufficient for Gene its usefulness in immunotherapy, and some peptides are SEQ ID No Sequence Expression tumor-specific despite their source protein occurring also in normal tissues. Still, mRNA expression profiling adds an 1. WMAPFTMTI ------additional level of safety in selection of peptide targets for immunotherapies. Especially for therapeutic options with 2 KLOAGTVFW ---- high safety risks, such as affinity-matured TCRs, the ideal 3 ILDDNMOKL -- target peptide will be derived from a protein that is unique to the tumor and not found on normal tissues. 4. KLODFSDQL ------0556 RNA Sources and Preparation 5 ALVEOGFTV ------0557. Surgically removed tissue specimens were provided as indicated above (see Example 1) after written informed 7 ALVDTLKFW ------consent had been obtained from each patient. Tumor tissue 10 SLLEEFDFHW -- specimens were Snap-frozen immediately after Surgery and later homogenized with mortar and pestle under liquid nitro 13 GLIDTETAMKAW ------gen. Total RNA was prepared from these samples using TRI Reagent (Ambion, Darmstadt, Germany) followed by a 19 FLEETKATW ------cleanup with RNeasy (QIAGEN. Hilden, Germany); both 2O KLSNVLQOW ------methods were performed according to the manufacturers protocol. 21 QLIEVSSPITL ------0558 Total RNA from healthy human tissues was 25 SLDGKAALTEL ------obtained commercially (Ambion, Huntingdon, UK; Clon tech, Heidelberg, Germany; Stratagene, Amsterdam, Nether 27 TLPDFRLPEI ------lands: BioChain, Hayward, Calif., USA). The RNA from several individuals (between 2 and 123 individuals) was 28 TLODHLNSL ------mixed such that RNA from each individual was equally 29 YIODEINTI ------weighted. 0559 Quality and quantity of all RNA samples were 31 YQMDIQOEL ------assessed on an Agilent 2100 Bioanalyzer (Agilent, Wald 38 ALADWWHEA -- bronn, Germany) using the RNA 6000 Pico LabChip Kit (Agilent). US 2016/0250307 A1 Sep. 1, 2016 62

TABLE 9 - continued TABLE 9 - continued Expression scores. Expression scores.

Gene Gene SEQ ID No Sequence Expression SEQ ID No Sequence Expression

39 ALDPKANFST -- O7 TLGOFYOEV ------

41 ALLELDEPLWL ------O9 TLYALSHAW ------

42 ALLGGNWRMML -- 1O TWGGSEILFEW ------

44 ALQDAIRQL -- 13 WLMDKLWEL ------

45 ALQDQLWLW ---- 14 VLSOVYSKV ------

46 AMAEMKWL ---- 16 WWIPAISAW ----

48 FLLEOPEIQV -- 17 YAFPKSITV --

49 FLYPEKDEPT ------19 YLDKNLTWSW ----

SO FTIPKLYOL ------2O YLGEEYWKA ------

52 GLFNAELLEA ------24 LLIDWWTYL ------

53 GLIHLEGDTW ------26 TLLDSPIKW ------

55 GLYGRTIEL ------29 SOADVIPAV ----

6 O ILSPLSWAL -- 3O ALDAGAWYTL ----

61 KIADFELPTI ------32 ALHEEWWGW ----

62 KIAGTNAEW -- 41 AMGEKSFSW --

66 KLHEEIDRW ------42 AWIGGLIYW ------

67 KLKETIOKL ------45 FLIAEYFEHW ----

68 KLIAATWILL ------46 FLWTEOAHTV ----

73 KLTLWIISW ------48 GLFAPLWFL --

74 KLYDLELIW ------49 GLISGLDIMEW ------

76 LLADIGGDPFAA -- 54 KLTDHLKYW ------

81 NLASFIEOVAW -- st OLLPNLRAW --

82 NWFDGLWRW ------58 RIISGLWKW ----

83 QLHDFVMSL ------60 RLLAKIIICL ------

84 OLTPVLVSV ---- 63 RLTESWLYL ----

85 RILPKWLEW ---- 65 RVIEHVEOV ----

87 RLFEENDWNL ------68 SLAWLWPIW ------

9 O RLLDWLAPLW -- 72 SLLNFLOHL --

93 RLYTMDGITV ------73 SLTSEIHFL --

94 RMSDWKGW -- 7s TLFEHLPHI ----

95 SICNGWPMW ---- 77 WLDEPYEKW ----

97 SLLPOLIEW ------82 YLLHFPMAL ------

1OO SLWGDIGNWNM ------83 YLYNNEEOVGL ------

103 SMGDHLWWA -- 87 SYPTEFPRF --

105 SWYDGKLLI -- 88 RYSAGWDAKF ------

106 TLAAIIHGA ---- 92 SYITKPEKW -- US 2016/0250307 A1 Sep. 1, 2016 63

TABLE 9 - Continued and exchanged with the peptide of interest as analyzed. Only peptide candidates that can effectively bind and stabilize the Expression SCC eS. peptide-receptive MHC molecules prevent dissociation of the Gene MHC complexes. To determine the yield of the exchange SEQ ID No Sequence Expression reaction, an ELISA was performed based on the detection of the light chain (B2m) of stabilized MHC complexes. The 193 IYPGAFWDL -- assay was performed as generally described in Rodenko et al. 2OO AYLLOPSQF ------(Rodenko B. Toebes M. Hadrup SR, van Esch WJ. Molenaar AM, Schumacher TN, Ovaa H. Generation of peptide-MHC 2O4. KYRLTYAYF ------class I complexes through UV-mediated ligand exchange. Nat 2O6 KWPETPLLL -- Protoc. 2006; 1(3): 1120-32). 215 IYTGNISSF ------0563 96 well MAXISorp plates (NUNC) were coated over night with 2 ug/ml streptavidin in PBS at room tempera 217 DYIPYWEKL ------ture, washed 4x and blocked for 1 h at 37° C. in 2% BSA 218 WYOGAIROI ------containing blocking buffer. Refolded HLA-A*0201/MLA 001 monomers served as standards, covering the range of 228 YLITSWELL -- 15-500 ng/ml. Peptide-MHC monomers of the UV-exchange 233 ALIGKIDAT -- reaction were diluted 100 fold in blocking buffer. Samples were incubated for 1 hat 37°C., washed four times, incubated 249 LLIGERWAL -- with 2 ug/ml HRP conjugated anti-B2m for 1 h at 37° C. 255 SLFGODWKAV -- washed again and detected with TMB solution that is stopped with NH2SO4. Absorption was measured at 450 nm. Candi 259 TLITDGMRSW -- date peptides that show a high exchange yield (preferably 263 WLYPSLKEI -- higher than 50%, most preferred higher than 75%) are gen erally preferred for a generation and production of antibodies 273 AILETAPKEW -- or fragments thereof, and/or T cell receptors or fragments 27s ALIEGAGILL -- thereof, as they show sufficient avidity to the MHC molecules and prevent dissociation of the MHC complexes. 286 KVLDKWFRA --

296 SLLSGRISTL -- TABLE 10A

298 TMAKESSIIGW -- MHC class I binding scores

3O1 WLADFGARW ---- Sed ID Peptide code Peptide exchange

3O2 KIOEILTOW -- 2 GPC3-001 ------5 APOB-001 ------315 KVLDGSPIEW ---- 7 APOB-002 ------1 APOB-003 ------3.18 KLNEINEKI ------3 HSD11B1-001 ------32O GLADNTWIAKW -- 227 SAMM-OO1 ------4 APOB-004 ------324 RLFETKITOW ---- 232 MAPKAPKS-001 ------O USO-001 ------327 GLNEEIARW -- 304 USP14-001 ------219 ADF-012 ------336 YLPTFELTV -- 223 IDI1-001 ------341 YLAIGIHEL ---- 224 IFT81-001 ------4 NCST-001 ------345 SYNPLWLRI (A*24) ---- 228 ACSL4-001 ------230 IPO9-001 ------The table lists peptides from genes that are very highly 5 SLC3SB2-001 ------overexpressed in tumors Compared to a panel of normal tissues (+++), highly overexpressed in tumors Compared to a panel of 6 ACSL3-001 ------normal tissues (++) or overexpressed in tumors Compared to a 303 MAGEB2-OO1 ------panel of normal tissues (+). 226 THTOO1 ------8 DYM-OO1 ------Example 3 6 AXIN2-001 ------225 QAR-001 ------UV-Ligand Exchange/Peptide Binding to HLA-A*02 2 ALDH1L1-OO1 ------and HLA-A24 221 EEF2-001 ------220 DRG2-001 ------0562 Candidate peptides for T cell based therapies 301 C1OTNF3-001 ------according to the present invention were further tested for their 11 ZNF318-001 ------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, US 2016/0250307 A1 Sep. 1, 2016 64

TABLE 1 OB TABLE 1 OB - continued - MHC class I binding scores - MHC class I binding scores SEO ID No Sequence Peptide exchange SEO ID No Sequence Peptide exchange

1. WMAPFTMTI ------38 ALADWWHEA ------2 KLOAGTVFW ------39 ALDPKANFST ------3 ILDDNMOKL ------4 O ALLAEGITWW ------4. KLODFSDQL ------41 ALLELDEPLWL ------5 ALVEOGFTV ------42 ALLGGNWRMML ------6 KLSPTWWGL ------43 ALLGWWTSW v. 7 ALWDTLKFW ------44 ALQDAIRQL ------8 KLLEEATISW ------45 ALQDQLWLW ------9 ALANOKLYSV ------46 AMAEMKWWL ------O SLLEEFDFHW ------47 FLDTPIAKW v. 1. SLSOELVGW ------48 FLLEOPEIQV ------2 FLAELAYDL ------49 FLYPEKDEPT v. 3 GLIDTETAMKAW ------SO FTIPKLYOL v. 4. ALADLTGTWW ------51 GLAEELWRA v. 5 LYGHTWTV ------52 GLFNAELLEA ------6 SLLGGNIRL ------53 GLIHLEGDTW ------7 RWAS kPTSGV ------54 GLLDPNWKSIFW ------8 ALYGKTEWW ------55 GLYGRTIEL ------9 FLEETKATW ------56 GWLPGLWGW ------2O KLSNVLQQV ------f HLTEAIOYV ------21 QLIEVSSPITL ------58 ILADLNLSW ------22 RIAGIRGIQGW ------59 ILADTFIGW ------23 RLYDPASGTISL ------6 O ILSPLSWAL ------24 SLAEEKLOASW ------61 KIADFELPTI ------25 SLDGKAALTEL ------62 KIAGTNAEW v. 26 SLLHTIYEW ------63 KIDEKNWW ------27 TLPDFRLPEI ------64 KILEETLYW ------28 TLODHLNSL ------65 KLFSGDELLEW ------29 YIODEINTI ------66 KLHEEIDRW ------3O YLGEGPRMW ------67 KLKETIOKL ------31 YOMDIQOEL ------68 KLIAATWILL v. 32 ALNAWRLLW ------69 KLLDEWTYLEA ------33 LLHGHIWEL ------70 KLLDLETERILL ------34 SLAEGTATW ------71. KLLDNWDSW ------35 SLQESILAQW ------72 KLSEAWTSW ------36 ILNWDGLIGW ------74 KLYDLELIW ------37 LLLPLLPPLSP ------US 2016/0250307 A1 Sep. 1, 2016 65

TABLE 1 OB - continued TABLE 1 OB - continued - MHC class I binding scores - MHC class I binding scores SEO ID No Sequence Peptide exchange SEO ID No Sequence Peptide exchange

7s KQMEPLHAV v. 3 WLMDKLWEL ------76 LLADIGGDPFAA ------4. VLSOVYSKV ------77 LLHEENFSW ------5 WWLDDKDYFL ------78 LLIDDEYKW ------6 WWWIPAISAW ------8O LLYEGKLTL ------7 YAFPKSITV ------81 NLASFIEOVAW ------8 YLDDEKNWGL ------82 NWFDGLWRW ------9 YLDKNLTWSW ------83 QLHDFVMSL ------2O YLGEEYWKA v. 84 OLTPVLVSV ------21 YLITGNLEKL ------85 RILPKWLEW ------22 YLSOAADGAKVL v. 86 RLAAFYSOW ------23 YLWDLDHGFAGW ------87 RLFEENDWNL ------24 LLIDWWTYL ------88 RLIDRIKTW v. 25 ALYGRLEWW ------89 RLIEEIKNW v. 26 TLLDSPIKW ------9 O RLLDWLAPLW ------27 WLIGSNHSL ------91 RLPDIPLROV ------28 GLAFSLNGW ------92 RLPPDTLLOOW ------29 SOADVIPAV ------93 RLYTMDGITV v. 3 O ALDAGAWYTL ------94 RMSDWWKGW ------31 ALDSGAFOSV ------95 SICNGVPMW ------32 ALHEEWWGW ------96 SLLEEPNWIRW ------33 ALLEMDARL ------97 SLLPOLIEW ------34 ALLETNPYLL ------98 SLLSPEHLOYL ------35 ALLGKIEKW ------99 SLSAFLPSL ------36 ALLNOHYOW ------OO SLWGDIGNWNM v. 37 ALPTWLWGW ------O1 SLWEGGWRGW ------38 ALSQWTLLL ------O2 SLWSWARGW ------39 ALSSKPAEW ------O3 SMGDHLWWA ------4 O ALTSISAGW ------O4 SWWFGPKEW ------41 AMGEKSFSW ------O5 SWYDGKLLI ------42 AWIGGLIYW ------O6 TLAAIIHGA ------45 FLIAEYFEHW v. O7 TLGOFYOEV ------46 FLWTEOAHTV v. O8 TLLKKISEA ------47 GLAPGGLAWW ------O9 TLYALSHAW ------48 GLFAPLWFL ------10 TWGGSEILFEW ------49 GLISGLDIMEW ------11 TVMDIDTSGTFNV ------SO GLSNLGIKSI ------12 WLGEWKWGW ------US 2016/0250307 A1 Sep. 1, 2016 66

TABLE 1 OB - continued TABLE 1 OB - continued - MHC class I binding scores - MHC class I binding scores SEO ID No Sequence Peptide exchange SEO ID No Sequence Peptide exchange

51 HLAKWTAEW ------9 O RYNEKCFKL ------52 KLDNNLDSW ------91 KYPDIISRI ------54 KLTDHLKYW ------92 SYITKPEKW ------56 LLFPHPVNOV ------93 IYPGAFWDL ------f OLLPNLRAW ------95 RYAPPPSFSEF ------58 RIISGLWKW v. 96 AYLKWISOI ------59 RLFPDGIWTW ------97 RWPKKSAEF ------6 O RLLAKIICL v. 98 LYWSHPRKF ------61 RLLDEOFAW ------99 KFVTVOATF ------62 RLMSALTOW- v. 2OO AYLLOPSQF ------63 RLTESWLYL ------2O1 AYWNTFHNI ------64 RMLIKLLEW ------2O2 AYGTYRSNF ------65 RVIEHVEOV ------2O3 YYGILOEKI ------66 SILDIWTKW ------2O5 WYGLORNLL ------67 SLAESSFDW ------2O6 KWPETPLLL ------68 SLAWLWPIW ------2O7 IYLERFPIF ------69 SLFEWFHPL ------2O8 SYNPAENAWLL ------70 SLHINGVIQL ------209 WFHPROELI ------71. SLIPAWLTW ------21 O AYPAIRYLL ------72 SLLNFLOHL ------211 IYIPSYFDF ------73 SLTSEIHFL ------212 WYGDWISNI ------74 TLAELGAWOW ------213 YYNKVSTVF ------7s TLFEHLPHI ------214 IYVTSIEOI ------76 TLGOIWDV ------215 IYTGNISSF ------77 WLDEPYEKW ------216 IYADWGEEF ------78 YIFTTPKSW ------217 DYIPYWFKL ------79 YIHNILYEW ------218 WYOGAIROI ------8O YLGPHIASWTL ------219 GWMAGDIYSW ------81 YLLEKFWAW ------22 O SLLEKELESW ------82 YLLHFPMAL ------221 ALCEENMRGW ------83 YLYNNEEOVGL v. 222 LTDITKGV v. 84 WVLDGGOIVTV ------223 FLFNTENKLLL ------85 ALFPALRPGGFOA ------224 ALASWIKEL ------86 WLLAQIIQW ------225 KMDPWAYRV ------87 SYPTEFPRF ------226 AVLGPLGLQEW ------88 RYSAGWDAKF ------227 ALLKVNOEL ------89 AFSPDSHYLLF ------US 2016/0250307 A1 Sep. 1, 2016 67

TABLE 1 OB - continued TABLE 1 OB - continued

MHC class I bindind scores MHC class I bindind scores

SEO ID No Sequence Peptide exchange SEO ID No Sequence Peptide exchange

228 YLITSWELL ------265 WLIEDGKWWTW ------229 KMFESFIESW ------

23 O WLTEFTREW ------266 SLLESNKDLLL ------

231 RLFNDPWAMW ------267 ALNENINOV ------

232 KLAEIWKOW ------268 KLYOEVEIASW ------

233 ALIGKIDAT ------269 YLMEGSYNKW ------

234 YLEPYLKEW ------27 O SVLDOKILL ------

235 KLFEEIREI ------271 LILDKLILL ------

236 ALADKELLPSW ------272 QOLDSKFLEQV ------

237 ALRGEIETW ------273 AILETAPKEW ------

238 AMPPPPPOGV v. 274 ALAEALKEW ------239 FLIGFIPAKA ------27s ALIEGAGILL ------24 O FLWERPTLLW ------276 ALLEADWNIKL ------241 FWLPLIGLHEA v. 277 ALLEENSTPOL ------242 GLFAPWHKW ------278 ALTSWWWTL ------243 GILDNPELRW ------279 ALWTGMHTI ------244 KIAELLENW ------28O ATLNIIHSW ------245 KLGAVFNOV ------281 GLLAGDRLWEW ------246 KLISSYYNW ------

247 KLLDTMWDTFL ------282 GOFPSYLETV ------

248 KLNDLIORL ------283 ILSGIGVSOV ------

249 (LLGERWAL ------284 KLDAFWEGW ------

25 O NLAEWWERW ------285 KLLDLSDSTSW ------

251 RLFADILNDW ------286 KWLDKWFRA ------

252 RTIEYLEEW ------287 LIGEFLEKW ------

253 RVPPPPOSV v. 288 LLDDSLWSI ------

254 RVOEAIAEW ------29 O NLIDLDDLYW ------

255 SLFGODWKAV ------291 OLIDYEROL ------256 SLFOGVEFHYW ------292 RIPAYEWTV v. 257 SLLEKAGPEL ------293 FLASESLIKQI v. 258 SLMGPWWHEW ------294 RLIDLHTNW ------259 TLITDGMRSW- v. 295 SLFSSPPEI ------26 O TLMDMRLSOV ------296 SLLSGRISTL ------261 WLFOEALWHV ------

262 WLPNFLPYNW ------297 TLFYSLREW ------

263 WLYPSLKEI ------298 TMAKESSIIGW v.

264 VMODPEFLOSV ------299 ALLRVTPFI ------US 2016/0250307 A1 Sep. 1, 2016

TABL E 1 OB - continued modified Melan-A/MART-1) and A*0201/DDX5-001 (YLL PAIVHI from DDX5), respectively. MHC class I binding scores (0571 800,000 beads/200ul were coated in 96-well plates SEO ID No Sequence Peptide exchange in the presence of 4x12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 were added subsequently in a volume of 200 ul. Stimulations were initiated in 96-well 3OO TLAQQPTAV v. plates by co-incubating 1x10 CD8+ T cells with 2x10 3O2 KIOEIL TOW ------washed coated beads in 200 ul TCM supplemented with 5 ng/ml IL-12 (PromoCell) for 3 days at 37° C. Half of the Binding of HLA-Class I restricted peptides to HLA-A*02 or HLA-A*24 depending frompeptide sequence was classified by medium was then exchanged by fresh TCM supplemented peptide exchange yield: with 80U/ml IL-2 and incubating was continued for 4 days at 2.10% = +; 2.20% = ++; 37° C. This stimulation cycle was performed for a total of 250 = +++; three times. For the pMHC multimer readout using 8 different 2.75% = ++++. pMHC molecules per condition, a two-dimensional combi S* = phospho serine. natorial coding approach was used as previously described (Andersenet al., 2012) with minor modifications encompass ing coupling to 5 different fluorochromes. Finally, multimeric Example 4 analyses were performed by staining the cells with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC In Vitro Immunogenicity for MHC Class I Presented antibody clone SK1 (BD, Heidelberg, Germany) and fluores Peptides cent pMHC multimers. For analysis, a BD LSRII SORP 0564. In order to obtain information regarding the immu cytometer equipped with appropriate lasers and filters was nogenicity of the TUMAPs of the present invention, the used. Peptide specific cells were calculated as percentage of inventors performed investigations using an in vitro T-cell total CD8+ cells. Evaluation of multimericanalysis was done priming assay based on repeated Stimulations of CD8+ T cells using the FlowJo software (Tree Star, Oreg., USA). In vitro with artificial antigen presenting cells (aAPCs) loaded with priming of specific multimer+ CD8+ lymphocytes was peptide/MHC complexes and anti-CD28 antibody. This way detected by comparing to negative control stimulations. the inventors could show immunogenicity for 22 HLA Immunogenicity for a given antigen was detected if at least A*0201 restricted TUMAPs of the invention so far, demon one evaluable in vitro stimulated well of one healthy donor strating that these peptides are T-cell epitopes against which was found to contain a specific CD8+ T-cell line after in vitro CD8+ precursor T cells exist in humans (Table 11). stimulation (i.e. this well contained at least 1% of specific multimer+ among CD8+ T-cells and the percentage of spe 0565. In Vitro Priming of CD8+ T Cells cific multimer+ cells was at least 10x the median of the 0566 In order to perform in vitro stimulations by artificial antigen presenting cells loaded with peptide-MHC complex negative control stimulations). (pMHC) and anti-CD28 antibody, the inventors first isolated 0572 In Vitro Immunogenicity for HCC Peptides CD8+ T cells from fresh HLA-A*02 leukapheresis products 0573 For tested HLA class I peptides, in vitro immuno via positive selection using CD8 microbeads (Miltenyi Bio genicity could be demonstrated by generation of peptide spe tec, Bergisch-Gladbach, Germany) of healthy donors cific T-cell lines. Exemplary flow cytometry results after obtained from the University clinics Mannheim, Germany, TUMAP-specific multimer staining for three peptides of the invention are shown in FIG. 3 and FIG. 4 together with after informed consent. corresponding negative controls. Results for 22 peptides from 0567 PBMCs and isolated CD8+ lymphocytes were incu the invention are summarized in Table 11 A. bated in T-cell medium (TCM) until use consisting of RPMI Glutamax (Invitrogen, Karlsruhe, Germany) Supplemented with 10% heat inactivated human AB serum (PAN-Biotech, TABLE 11A Aidenbach, Germany), 100U/ml Penicillin/100 ug/ml Strep in vitro immunogenicity of HLA class I peptides of the invention tomycin (Cambrex, Cologne, Germany), 1 mM sodium pyru vate (CC Pro, Oberdorla, Germany), 20 ug/ml Gentamycin Seq ID Peptide ID wells donors (Cambrex). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Ger 225 QAR-001 ------many) and 10 U/ml IL-2 (Novartis Pharma, Nürnberg, Ger 1 APOB-003 ------many) were also added to the TCM at this step. 2 ALDH1L1-OO1 ------301 C1OTNF3-001 ------0568 Generation of pMHC/anti-CD28 coated beads, 15 SLC3SB2-001 ------T-cell stimulations and readout was performed in a highly 16 ACSL3-001 ------defined in vitro system using four different pMHC molecules 12 GPC3-001 ------7 APOB-002 ------per stimulation condition and 8 different pMHC molecules 303 MAGEB2-OO1 ------per readout condition. 227 SAMM-OO1 ------0569. The purified co-stimulatory mouse IgG2a anti 4 APOB-004 ------226 THTOO1 ------human CD28 Ab 9.3 (Jung et al., 1987) was chemically 6 AXIN2-001 ------biotinylated using Sulfo-N-hydroxysuccinimidobiotin as 232 MAPKAPKS-001 ------recommended by the manufacturer (Perbio, Bonn, Ger 10 USO-001 ------many). Beads used were 5.6 um diameter streptavidin coated 304 USP14-001 ------219 ADF-012 ------polystyrene particles (Bangs Laboratories, Illinois, USA). 224 IFT81-001 ------0570 pMHC used for positive and negative control stimu 11 ZNF318-001 ------lations were A*0201/MLA-001 (peptide ELAGIGILTV from US 2016/0250307 A1 Sep. 1, 2016 69

TABLE 11 A-continued (0574) Exemplary Results of Peptide-Specific In Vitro CD8+ T Cell Responses of a Healthy HLA-A*02+Donor in vitro immunogenicity of HLA class I peptides of the invention (FIG. 3) (0575 CD8+ T cells were primed using artificial APCs Seq ID Peptide ID wells donors coated with anti-CD28 mAb and HLA-A*02 in complex with 14 NCSTOO1 ------IMA-APOB-002 (Seq ID No 7) peptide (A, right panel) or 228 ACSL4-001 ------IMA-APOB-003 (B, right panel, Seq ID No 1), or IMA 230 IPO9-001 ------ALDH1L1-001 (C. right panel, Seq ID No 2), respectively. After three cycles of stimulation, the detection of peptide Exemplary results of in vitro immunogenicity experiments conducted by the applicant for the peptides of the invention. reactive cells was performed by 2D multimer staining with A*02/APOB-002 (A) or A*02/APOB-003 (B), or A*02/ ALDH1L1-001. Left panels (A, B, C) show control staining of cells stimulated with irrelevant A*02/peptide complexes. TABLE 11B Viable singlet cells were gated for CD8+ lymphocytes. Bool in vitro immunogenicity of additional HLA class I ean gates helped excluding false-positive events detected peptides of the invention with multimers specific for different peptides. Frequencies of specific multimer+ cells among CD8+ lymphocytes are indi Wells Donors cated. SEQ ID No Sequence positive 3 positive 3 (0576 Exemplary Results of Peptide-Specific In Vitro 87 SYPTEFPRF wn ------' CD8+ T Cell Responses of a Healthy HLA-A*24+Donor (FIG. 4) 89 AFSPDSHYLLF wn v. (0577 CD8+ T cells were primed using artificial APCs 9 O RYNEKCFKL wn v. coated with anti-CD28 mAb and HLA-A*24 in complex with IMA-KLHL24-001 (Seq ID No 190) peptide (A, right panel) 91 KYPDIISRI wn ------' or IMA-APOB-006 (B, right panel, Seq ID No 218), respec tively. After three cycles of stimulation, the detection of pep 92 SYITKPEKW wn ------tide-reactive cells was performed by 2D multimer staining 93 IYPGAFWDL wn ------with A*24/KLHL24-001 (A) or A*24/APOB-006 (B). Left panels (A and B) show control staining of cells stimulated 94 OYASRFWOL w ------' with irrelevant A*24/peptide complexes. Viable singlet cells 96 AYLKWISOI wn ------were gated for CD8+ lymphocytes. Boolean gates helped excluding false-positive events detected with multimers spe 97 RWPKKSAEF --- v. cific for different peptides. Frequencies of specific multimer+ 98 LYWSHPRKF wn v. cells among CD8+ lymphocytes are indicated.

99 KFVTVOATF wn v. Example 5 2O1 AYWNTFHNI v. v. Syntheses of Peptides 2O2 AYGTYRSNF wn ------' 0578 All peptides were synthesized using standard and well-established solid phase peptide synthesis using the 2O3 YYGILOEKI wn ------Fmoc-strategy. Identity and purity of each individual peptide 2O5 WYGLORNLL wn ------have been determined by mass spectrometry and analytical RP-HPLC. The peptides were obtained as white to off-white 2O7 IYLERFPIF v. ------' lyophilizates (trifluoro acetate salt) in purities of >50%. All 2O8 SYNPAENAWLL wn ------TUMAPs are preferably administered as trifluoro-acetate salts or acetate salts, other salt-forms are also possible. 209 WFHPROELI v. ------'

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SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 348

SEO ID NO 1 LENGTH: 9 TYPE PRT ORGANISM: Homo sapiens

<4 OOs SEQUENCE: 1 US 2016/0250307 A1 Sep. 1, 2016 75

- Continued

Wal Met Ala Pro Phe Thir Met Thir Ile 1. 5

<210s, SEQ ID NO &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2 Llys Lieu. Glin Ala Gly Thr Val Phe Val 1. 5

<210s, SEQ ID NO &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 3 Ile Lieu. Asp Asp Asn Met Gln Lys Lieu. 1. 5

<210s, SEQ ID NO &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 4. Llys Lieu. Glin Asp Phe Ser Asp Gln Lieu. 1. 5

<210s, SEQ ID NO &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 5 Ala Lieu Wall Glu Gln Gly Phe Thr Val 1. 5

<210s, SEQ ID NO &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6 Llys Lieu. Ser Pro Thr Val Val Gly Lieu 1. 5

<210s, SEQ ID NO &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 7 Ala Lieu Val Asp Thr Lieu Lys Phe Val 1. 5

<210s, SEQ ID NO &211s LENGTH: 10 212. TYPE: PRT &213s ORGANISM: Homo sapiens US 2016/0250307 A1 Sep. 1, 2016 76

- Continued

<4 OOs, SEQUENCE: 8 Llys Lieu. Lieu. Glu Glu Ala Thir Ile Ser Val 1. 5 1O

<210s, SEQ ID NO 9 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 9 Ala Lieu Ala Asn Gln Llys Lieu. Tyr Ser Val 1. 5 1O

<210s, SEQ ID NO 10 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 10 Ser Lieu. Lieu. Glu Glu Phe Asp Phe His Val 1. 5 1O

<210s, SEQ ID NO 11 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens < 4 OO SEQUENCE: 11 Ser Lieu. Ser Glin Glu Lieu Val Gly Val 1. 5

<210s, SEQ ID NO 12 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 12 Phe Lieu Ala Glu Lieu Ala Tyr Asp Lieu. 1. 5

<210s, SEQ ID NO 13 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 13 Gly Lieu. Ile Asp Thr Glu Thir Ala Met Lys Ala Val 1. 5 1O

<210s, SEQ ID NO 14 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 14 Ala Lieu Ala Asp Lieu. Thr Gly Thr Val Val 1. 5 1O

<210s, SEQ ID NO 15 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 2016/0250307 A1 Sep. 1, 2016 77

- Continued

<4 OOs, SEQUENCE: 15 Lieu. Lieu. Tyr Gly His Thr Wall Thir Wall 1. 5

<210s, SEQ ID NO 16 &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 16 Ser Lieu. Lieu. Gly Gly Asn. Ile Arg Lieu. 1. 5

<210s, SEQ ID NO 17 &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 17 Arg Val Ala Ser Pro Thir Ser Gly Val 1. 5

<210s, SEQ ID NO 18 &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 18 Ala Lieu. Tyr Gly Lys Thr Glu Val Val 1. 5

<210s, SEQ ID NO 19 &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 19 Phe Lieu. Glu Glu Thr Lys Ala Thr Val 1. 5

<210s, SEQ ID NO &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2O Llys Lieu. Ser Asn Wall Lieu. Glin Glin Wall 1. 5

<210s, SEQ ID NO 21 &211s LENGTH: 11 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 21

Gln Lieu. Ile Glu Wall Ser Ser Pro Ile Thir Lieu. 1. 5 1O

<210s, SEQ ID NO 22 &211s LENGTH: 11 212. TYPE: PRT US 2016/0250307 A1 Sep. 1, 2016 78

- Continued <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 22 Arg Ile Ala Gly Ile Arg Gly Ile Glin Gly Val 1. 5 1O

<210s, SEQ ID NO 23 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 23 Arg Lieu. Tyr Asp Pro Ala Ser Gly. Thir Ile Ser Lieu. 1. 5 1O

<210s, SEQ ID NO 24 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 24 Ser Lieu Ala Glu Glu Lys Lieu. Glin Ala Ser Val 1. 5 1O

<210s, SEQ ID NO 25 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM; Homo sapiens <4 OOs, SEQUENCE: 25 Ser Lieu. Asp Gly Lys Ala Ala Lieu. Thr Glu Lieu. 1. 5 1O

<210s, SEQ ID NO 26 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 26 Ser Leu Lieu. His Thr Ile Tyr Glu Val 1. 5

<210s, SEQ ID NO 27 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 27 Thr Lieu Pro Asp Phe Arg Lieu Pro Glu Ile 1. 5 1O

<210s, SEQ ID NO 28 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 28 Thir Lieu. Glin Asp His Lieu. Asn. Ser Lieu. 1. 5

<210s, SEQ ID NO 29 &211s LENGTH: 9