NZ786786A - Neoantigens and methods of their use - Google Patents

Abstract

The field of the present invention relates to immunotherapeutic peptides, peptide binding agendas, and their use, for example, in the immunotherapy of cancer.

Description

NEOANTIGENS AND METHODS OF THEIR USE CROSS-REFERENCE The present application is a divisional application of New d Patent Application No. 746442, which is the national phase of International Application No. 2017/025462, which in turn claims priority to U.S. Provisional Application No. 62/316,530, filed March 31, 2016, U.S. Provisional Application No. 62/316,533, filed March 31, 2016, U.S. ional Application No. ,547, filed March 31, 2016, U.S. Provisional Application No. 62/316,552, filed March 31, 2016, U.S. Provisional Application No. 62/316,567, filed April 1, 2016, and U.S. Provisional Application No. 62/316,571, filed April 1, 2016. Each of the aforementioned applications are incorporated herein by cross reference in their ties.

FIELD OF THE INVENTION The field of the present invention relates to immunotherapeutic es, nucleic acids encoding the peptides, peptide binding agents, and their use, for example, in the immunotherapy of cancer. In one aspect, the invention provides neoantigenic peptides, useful alone or in combination with other tumor-associated peptides, anti-cancer, or immunomodulatory agents to treat .

BACKGROUND OF THE INVENTION Tumor vaccines are typically composed of tumor ns and immunostimulatory molecules (e.g., adjuvants, cytokines or TLR ligands) that work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. Such vaccines n either shared tissue restricted tumor antigens or a mixture of shared and patient-specific antigens in the form of whole tumor cell preparations. The shared tissue restricted tumor antigens are ideally immunogenic proteins with selective expression in tumors across many individuals and are commonly delivered to patients as synthetic es or recombinant proteins. In contrast, whole tumor cell preparations are delivered to patients as autologous irradiated cells, cell lysates, cell fusions, heat-shock protein preparations or total mRNA. Since whole tumor cells are isolated from the autologous t, the cells may include patient-specific tumor antigens as well as shared tumor antigens. Finally, there is a third class of tumor antigens, neoantigens, that has rarely been used in vaccines, which consists of proteins with tumor-specific mutations (which can be patient-specific or shared) that result in altered amino acid sequences. Such mutated ns are: (a) unique to the tumor cell as the mutation and it's corresponding protein are present only in the tumor; (b) avoid l nce and are therefore more likely to be immunogenic; (c) provide an excellent target for immune recognition including by both l and cellular immunity. However, the use of personalized neoantigens requires sequencing of each patient's genome and then production of a patient-specific neoantigen composition. Accordingly, there is still a need for developing additional cancer therapeutics.

BRIEF SUMMARY OF THE INVENTION ed herein is an isolated neoantigenic peptide sing a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native polypeptide, n the tope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: AxByCz, wherein each A and C represents an amino acid corresponding to the native polypeptide, y is at least 1 and B represents an amino acid substitution or insert of the native polypeptide, x + y + z is at least 8, the at least 8 contiguous amino acids comprises By, and the native ptide is encoded by a gene selected from the group consisting of: (a) ABLl. wherein AxByCz is (i) VADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGKVYEGVWKKYSLTV AVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVC, (ii) VADGLHTLHYPAPKRNKPTVYGVSPNYDKWEMERTDlTMKHKLGGGQYGVVYEGVWKKYSLTV AVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVC, (iii) LLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSATEYLEKKNFIHRDLAARN CLVGENHLVKVADFGLSRLMTGDTYTAHAGAKF. (iv) SLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIIIEFMTYGNLLDYLRECNR QEVNAVVLLYMATQISSAMEYLEKKNFIHRDLA, or (v) STVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQHGEVYEGVWKKYSLT KEDTMEVEEFLKEAAVMKEIKHPNLVQLLG: (b) ALK, wherein AxByCz is (i) SSLAMLDLLHVARDIACGCQYLEENHFIHRDIAARNCLLTCPGPGRVAKIADFGMARDIYRASYYRK GGCAMLPVKWMPPEAFMEGIFTSKTDTWSFGVLL. or (ii) QVAVKTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIGVSLQSLPRFILMELMAGGDLKSFLRETRPR PSQPSSLAMLDLLHVARDIACGCQYLEENHFI; (c) BRAF, wherein AxByCz is MIKLIDIARQTAQGMDYLHAKSIIHRDLKSNNIFLHEDLTVKIGDFGLATEKSRWSGSHQFEQLSGSIL IRMQDKNPYSFQSDVYAFGIVLYELM; (d) BTK, wherein AxByCz is MIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGSLLNYLREMRI—IRFQTQQ LLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND; (e) EEF 182, wherein AxByCz is MGFGDLKSPAGLQVLNDYLADKSYIEGYVPSQADVAVFEAVSGPPPADLCHALRWYNHIKSYEKEK ASLPGVKKALGKYGPADVEDTTGSGAT; (f) EGFR, wherein AxByCz is (i) SLNITSLGLRSLKEISDGDVIISGNKNLCYANTTNWKKLFGTSGQKTKIIRNRGENSCKATGQVCHALC SPEGCWGPEPRDCVSCRNVSRGRECVDKCNLL, or (ii) IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; (g) ERBB3, wherein AxByCz is ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRMVRGTQVYDGKFAIFV MLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDK; (h) ESRl, wherein AxByCz is (i) HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLLEMLDAHRLHAP SVEETDQSHLATAGSTSSHSLQKYYITGEA, (ii) NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLPSTLKSLEEKDHIHRVLD KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH, (iii) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLCDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, (iv) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLNDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, or (v) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE: (i) FGFR3, wherein AxByCz is HRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERCPHRPILQAGLPANQTA VLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGl (i) FRG 18, wherein AxByCz is AVKLSDSRIALKSGYGKYLGINSDELVGHSDAIGPREQWEPVFQNGKMALSASNSCFIRCNEAGDIEA EEEMIKIRSCAEKETKKKDDIPEEDKG; (k) HERZ, wherein AxByCz is GSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGLGSPYVSRLLGICLTSTV QLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCM; (1) [DH], whcrcin AxByCz is (i) RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGHI-IAYGDQYRATDFVVPGP GKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, (ii) LKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGCHAYGDQYRATDFVVPGPG KVElTYTPSDGTQKVTYLVHNFEEGGGVAMGM, (iii) RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGGHAYGDQYRATDFVVPGP GKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, or (iv) RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGSHAYGDQYRATDFVVPGPG KVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM; (m) KIT, wherein AxByCz is (i) VEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIANLLGACTIGGPTLVIT EYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYK, or (ii) VEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIANLLGACTIGGPTLVIT EYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYK; (n) MEK, wherein AxByCz is (i) ISELGAGNGGVVFKVSHKPSGLVMARKLIHLEIKPAIRNQIIRELQVLHESNSPYIVGFYGAFYSDGEIS ICMEHMDGGSLDQVLKKAGRIPEQILGKVSI. or (ii) GVVFKVSHKPSGLVMARKLIHLEIKPAIRNQIIRELQVLHECNSLYIVGFYGAFYSDGEISIC MEHMDGGSLDQVLKKAGRIPEQILGKVSIAVI; (o) MYC, wherein AxByCz is (i) MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSDLQPPAPSEDlWKKFELLPTPPLSPSRRSG LCSPSYVAVTPFSLRGDNDGG, (ii) FTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLSTPPLSPSRRSGLCSPSY VAVTPFSLRGDNDGGGGSFSTADQLEMVTELLG. or (iii) TNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPIPPLSPSRRSGLCSPSYVA VTPFSLRGDNDGGGGSFSTADQLEMVTELLGG; (p) PDGFRa, wherein AxByCz is VAVKMLKPTARSSEKQALMSELKIMTHLGPHLNlVNLLGACTKSGPIYlllEYCFYGDLVNYLHKNRD SFLSHHPEKPKKELDIFGLNPADESTRSYVILS; (q) PIK3CA, wherein AxByCz is (i) IEEHANWSVSREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSKITEQEKDFLWSHRHYC LPKLLLSVKWNSRDEVAQMYCLVKDWPP. (ii) HANWSVSREAGFSYSHAGLSNRLARDNELRENDKEQLKA]STRDPLSEITKQEKDFLWSHRHYCVTI PEILPKLLLSVKWNSRDEVAQMYCLVKD\NPPIKP, or (iii) LFINLFSMMLGSGMPELQSFDDIAYIRKTLALDKTEQEALEYFNIKQIVINDARHGGWTTKMDWIFHTI KQHALN; (r) POLE, wherein AxByCz is QRGGVITDEEETSKKIADQLDNIVDMREYDVPYHIRLSIDIETTKLPLKFRDAETDQIMMISYMIDGQG YLITNREIVSEDIEDFEFTPKPEYEGPFCVFN; (s) PTEN. wherein AxByCz is KFNCRVAQYPFEDHNPPQLELIKPFCEDLDQWLSEDDNHVAAIHCKAGKGQTGVMICAYLLHRGKF LKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSY; (t) RAC 1, wherein AxByCz is MQAIKCVVVGDGAVGKTCLLISY'ITNAFSGEYIPTVFDNYSANVMVDGKPVNLGLWDTAGQEDYD RLRPLSYPQTVGET; and (u) TP53, wherein AxByCz is (i) [RVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGSMNRRPILTIITLEDSSG NLLGRNSFEVRVCACPGRDRRTEEENLRKKGEP. (ii) NKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRHCPHHERCSDSDGLAP PQHLlRVEGNLRVEYLDDRNTFRHSVVVPYEPPEV, (iii) EGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNQRPILTIITLEDSSGNLL GRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE, (iv) EGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNWRPILTIITLEDSSGNLL GRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE. or (v) PEVGSDC'ITIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVCVCACPGRDRRTEEENLR KKGEPHHELPPGSTKRALPNNTSSSPQPKKKPL.

In embodiments, the native polypeptide is encoded by the EGFK ERBB3 or FGFR3 gene and at least one By is expressed extracellularly.

Provided herein is an isolated neoantigenic peptide comprising a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native polypeptide, n the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: AxByCz. wherein each A is an amino acid corresponding to the native ptide; By is absent; each C is an amino acid d by a frameshifi of a sequence encoding the native polypeptide; x + _v + z is at least 8; the at least 8 contiguous amino acids comprises at least one Cz; and the native ptide is encoded by a gene selected from the group consisting of: (a) APC, wherein C2 is (i) AKFQQCHSTLEPNPADCRVLVYLQNQPGTKLLNFLQERNLPPKVVLRHPKVHLNTMFRRPHSCLAD VLLSVHLIVLRVVRLPAPFRVNHAVEW. (ii) APVIFQIALDKPCHQAEVKHLHHLLKQLKPSEKYLKIKHLLLKRERVDLSKLQ, or (iii) MLQFRGSRFFQMLILYYILPRKVLQMDFLVHPA; (b) , wherein C2 is (i) ALGPHSRISCLPTQTRGCILLAATPRSSSSSSSNDMlPMAlSSPPKAPLLAAPSPASRLQCINSNSRITSGQ WMAHMALLPSGTKGRCTACHTALGRGSLSSSSCPQPSPSLPASNKLPSLPLSKMYTTSMAMPILPLPQ LLLSADQQAAPRTNFHSSLAETVSLHPLAPMPSKTCHHK, (ii) AHQGFPAAKESRVIQLSLLSLLIPPLTCLASEALPRPLLALPPVLLSLAQDHSRLLQCQATRCHLGl-IPV ASRTASCILP. (iii) PlLAATGTSVRTAARTWVPRAAIRVPDPAAVPDDHAGPGAECHGRPLLYTADSSLVVTTRPQRVWST GPDSILQPAKSSPSAAAATLLPA'ITVPDPSCPTFVSAAATVSTI'I‘APVLSASILPAAIPASTSAVPGSIPL PAVDDTAAPPEPAPLLTATGSVSLPAAATSAASTLDALPAGCVSSAPVSAVPANCLFPAALPSTAGAIS RFIVVVSGILSPLNDLQ, (iv) PCRAGRRVPWAASLIHSRFLLMDNKAPAGMVNRARLHITTSKVLTLSSSSHPTPSNHRPRPLMPNLR] SSSHSLNHHSSSPLSLHTPSSHPSLHISSPRLHTPPSSRRHSSTPRASPPTHSHRLSLLTSSSNLSSQHPRR SPSRLRILSPSLSSPSKLPIPSSASLHRRSYLKIHLGLRHPQPPQ, (v) RTNPTVRMRPHCVPFWTGRILLPSAASVCPIPFEACHLCQAMTLRCPNTQGCCSSWAS, or (vi) TNQALPKIEVICRGTPRCPSTVPPSPAQPYLRVSLPEDRYTQAWAP’I‘SRTPWGAMVPRGVSMAHKVA TPGSQTIMPCPMP'ITPVQAWLEA; (c) BZM, wherein C2 is (i) RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKMSMPAV. or (ii) LCSRYSLFLAWRLSSVLQRFRFTHVIQQRMESQIS; (d) CDH I, wherein C2 is (i) RSACVTVKGPLASVGRHSLSKQDCKFLPFWGFLEEFLLC, (ii) IQWGTITAPRPIRPPFLESKQNCSHFPTPLLASEDRRETGLFLPSAAQKMKKAHFLKTWFRSNPTKTK KARFSTASLAKELTHPLLVSLLLKEKQDG, (iii) PTDPFLGLRLGLHLQKVFHQSHAEYSGAPPPPPAPSGLRFWNPSRIAHISQLLSWPQKTEERLGYSSHQ LPRK, (iv) FCCSCCFFGGERWSKSPYCPQRMTPGTTFITMMKKEAEKRTRTLT. or (v) WRRNCKAPVSLRKSVQTPARSSPARPDRTRRLPSLGVPGQPWALGAAASRRCCCCCRSPLGSARSRS PATLALTPRATRSRCPGATWREAASWAE; (e) GATA3, wherein C2 is (i) PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLW'ITPPLQHGHRHGLEPCSMLTGPPARVPA VPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH; or (ii) PRPRRCTRHPACPLDHTTPPAWSPPWVRALLDAHRAPSESPCSPFRLAFLQEQYHEA; (f) MLLZ, wherein C2 is TRRC LCLPHLNHHLFLHWRSRPCLHRKSHPHLLHLRRLYPHHLKHRPCPI-[HLKNLLCPRHLRNCPLPRI—ILK HLACLHHLRSHPCPLHLKSHPCLHHRRHLVCSI-II-ILKSLLCPLHLRSLPFPI-II-ILRI—[I—IACPHI-ILRTRLCP LCPPHLRYRAYPPCLWCHACLHRLRNLPCPHRLRSLPRPLHLRLHASPHHLRTPPHPHHLR THLLPHHRRTRSCPCRWRSHPCCHYLRSRNSAPGPRGRTCHPGLRSRTCPPGLRSHTYLRRLRSHTCP AYALCLRSHTCPPRLRDHICPLSLRNCTCPPRLRSRTCLLCLRSHACPPNLRNHTCPPSLRSHA CPPGLRNRICPLSLRSHPCPLGLKSPLRSQANALHLRSCPCSLPLGNHPYLPCLESQPCLSLGNHLCPLC PHLGSHPCRLS; (g) PTEN, wherein C2 is (i) SWKGTNWCNDMCIFITSGQIFKGTRGPRFLWGSKDQRQKGSNYSQSEALCVLL, (ii) KRTKCFTFG, (iii) PlFlQTLLLWDFLQKDLKAYTGTILMM, (iv) QKMILTKQIKTKPTDTFLQILR, (v) GFWIQSIKTITRYTIFVLKDIMTPPNLIAELHNILLKTITHHS, (vi) NYSNVQWRNLQSSVCGLPAKGEDIFLQFRTHTI‘GRQVHVL, or (vii) PQTEQDAKKGQNVSLLGKYILHTRTRGNLRKSRKWKSM; (h) TP53. wherein C2 is (i) SSQNARGCSPRGPCTSSSYTGGPCTSPLLAPVIFCPFPENLPGQLRFPSGLLAFWDSQVCDLHVLPCPQ QDVLPTGQDLPCAAVG, (ii) GAAPTMSAAQIAMVWPLLSILSEWKEICVWSIWMTETLFDIVWWCPMSRLRLALTVPPSTI'ITCVTV PAWAA, (iii) TGGPSSPSSHWKTPVVIYWDGTALRCVFVPVLGETGAQRKRISARKGSLTTSCPQGALSEHCPTTPAP LPSQRRNHWMENISPFRSVGVSASRCSES. (iv) FHTPARHPRPRHGHLQAVTAHDGGCEALPPP. (v) CCPRTILNNGSLKTQVQMKLPECQRLLPPWPLHQQLLHRRPLHQPPPGPCHLLSLPRKPTRAATVSV WASCILGQPSL, (vi) VRKHFQTYGNYFLKTTFCPPCRPKQWMI, or (vii) LARTPLPSTRCFANWPRPALCSCGLIPHPRPAPASAPWPSTSSHST; or (i) VHL wherein C2 is (i) ELQETGHRQVALRRSGRPPKCAERPGAADTGAHCTSTDGRLKISVETYTVSSQLLMVLMSLDLDTGL VPSLVSKCLILRVK, (ii) KSDASRLSGA, (iii) RTAYFCQYHTASVYSERAMPPGCPEPSQA, (iv) TRASPPRSSSAIAVRASCCPYGSTSTASRSPTQRCRLARAAASTATEVTFGSSEMQGHTMGFWLTKLN YLCHLSMLTDSLFLPISHCQCIL, (v) SSLRITGDWTSSGRSTKIWKTFQMCRKTWSG, or (vi) VGRRGVRPGRVRPGGTGRRGGDGGRAAAARAALGELARALPGHLLQSQSARRAARNIAQ LRRRAAALPNAAAWHGPPHPQLPRSPLALQRCRDTRWASG; (j) ACVRZA, wherein AxByCz is (i) GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKRQP, or (ii) GDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKTALKYIFVAVRAICVM RRWKSHSPLQIQLHLSHPITTSCSIPWCHLC; (k) C 150RF40. wherein AxByCz is TAEAVNVAIAAPPSEGEANAELCRYLSKVLELRKSDVVLDKVGLALFFFFFETKSCSVAQAGVQWRS LGSLQPPPPGFKLFSCLSFLSSWDYRRMPPCLANFCIFNRDGVSPCWSGWS; (l) CNOTI, wherein AxByCz is (i) LSVIIFFFVYIWHWALPLILNNHHICLMSSIILDCNSVRQSIMSVCFFFFSVIFSTRCLTDSRYPNICWFK, or (ii) LSVIIFFFVYIWHWALPLILNNHHICLMSSIILDCNSVRQSIMSVCFFFFCYILNTMFDR; (m) EIF2B3, wherein AxByCz or C2 is VLVLSCDLITDVALHEVVDLFRAYDASLAMLMRKGQDSIEPVPGQKGKKKQWSSVTSLEWTAQERG CSSWLMKQTWMKSWSLRDPSYRSILEYVSTRVLWMPTSTV; (n) EPHBZ, wherein AxByCz or C2 is SIQVMRAQMNQIQSVEGQPLARRPRATGRTKRCQPRDVTKKTCNSNDGKKREWEKRKQILGGGGK YKEYFLKR]LIRKAMTVLAGDKKGLGRFMRCVQSETKAVSLQLPLGR; (o) ESRPl, wherein AxByCz is (i) LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKKHEGQIC, or (ii) LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKT; (p) FAMl 1B, wherein AxB)‘Cz is GALCKDGRFRSDIGEFEWKLKEGHKKIYGKQSMVDEVSGKVLEMDISKKKHYNRKISIKKLNRMKV PLMKLITRV; (q) GBP3, wherein AxByCz is RERAQLLEEQEKTLTSKLQEQARVLKERCQGESTQLQNEIQKLQKTLKKKPRDICRIS; (r) JAKl, wherein AxByCz is (i) VNTLKEGKRLPCPPNCPDEVYQLMRKCWEFQPSNRTSFQNLIEGFEALLKTSN or (ii) SCKELADLMTRCMNYDPNQRPFFRAIMRDINKLEEQNPDIVSEKNQQLKWTPHILKSAS; (s) LMANl, wherein AxByCz is (i) DDHDVLSFLTFQLTEPGKEPPTPDKEISEKEKEKYQEEFEHFQQELDKKKRGIPEGPPRPPRAACGGNI, or (ii) DDHDVLSFLTFQLTEPGKEPPTPDKEISEKEKEKYQEEFEHFQQELDKKKRNSRRATPTSKGSLRRKY LRV; (t) MSH3, n AxByCz is (i) TKSTLIGEDVNPL]KLDDAVNVDEIMTDTSTSYLLCISENKENVRDKKKGQHFYWHCGSAACHRRGC V, or (ii) LYTKSTLIGEDVNPLIKLDDAVNVDEIMTDTSTSYLLCISENKENVRDKKRATFLLALWECSLPQARL CLlVSRTLLLVQS; (u) NDUFC2, wherein AxByCz is (i) LPPPKLTDPRLLYIGFLGYCSGLIDNLIRRRPIATAGLHRQLLYITAFFFCWILSCKT, or (ii) SLPPPKLTDPRLLYIGFLGYCSGLIDNLIRRRPIATAGLHRQLLYITAFFLLDIIL; (v) RBM27, whcrcin AxByCz is NQSGGAGEDCQIFSTPGHPKMIYSSSNLKTPSKLCSGSKSHDVQEVLKKKTGSNEVTTRYEEKKTGSV RKANRMPKDVNIQVRKKQKHETRRKSKYNEDFERAWREDLTIKR: (w) RPLZZ, wherein AxByCz is (i) MAPVKKLVVKGGKKKEASSEVHS, or (ii) MAPVKKLVVKGGKKRSKF; (x) SEC31A, wherein AxByCz is (i) MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKN, or (ii) MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKLMLLRLNLRKMCGPF; (y) SEC63. n AxByCz is (i) AEVFEKEQSICAAEEQPAEDGQGETNKNRTKGGWQQKSKGPKKTAKSKKKETFKKKTYTCAITTVK ATETKAGKWSRWE, or (ii) MAEVFEKEQSICAAEEQPAEDGQGETNKNRTKGGWQQKSKGPKKTAKSKKRNL; (z) SLC35F5, wherein AxByCz is NIMEIRQLPSSHALEAKLSRMSYPVKEQESILKTVGKLTATQVAKISFFFALCGFWQICHIKKHFQTHK LL; (aa) SMAPl, wherein AxByCz is (i) YEKKKYYDKNAIAITNISSSDAPLQPLVSSPSLQAAVDKNKLEKEKEKKKGREKERKGARKAGKTTY S, or (ii) KYEKKKYYDKNAIAITNISSSDAPLQPLVSSPSLQAAVDKNKLEKEKEKKRKRKREKRSQKSRQNHL QLKSCRRKISNWSLKKVPALKKLRSPLWIF; (bb) TFAM, wherein AxByCz is (i) IYQDAYRAEWQVYKEEISRFKEQLTPSQIMSLEKEIMDKHLKRKAMTKKKRVNTAWKTKKTSFSL, or (ii) IYQDAYRAEWQVYKEEISRFKEQLTPSQIMSLEKEIMDKHLKRKAMTKKKS; (cc) TGFBR2, wherein AxByCz is (i) KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKAW. or (ii) EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKSLVRLSSCVPVALMSAM TTSSSQKNITPAILTCC; (dd) THAPS, n AxByCz is VPSKYQFLCSDHFTPDSLDIRWGIRYLKQTAVPTIFSLPEDNQGKDPSKKNPRRKTWKMRKKYAQKP SQKNHLY; (cc) TI'K, whcrcin AxByCz is GTTEEMKYVLGQLVGLNSPNSILKAAKTLYEHYSGGESHNSSSSKTFEKKGEKNDLQLFVMSDTTYK IYWTVILLNPCGNLHLKTTSL: and (fl) XPOT, n AxByCz is QQLIRETLISWLQAQMLNPQPEKTFIRNKAAQVFALLFVTEYLTKWPKFFLTFSQ.

Provided herein is an ed neoantigenic peptide comprising a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native polypeptide, wherein the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence ented by: AxByCz, n each A is an amino acid corresponding to a first native polypeptide; each C is an amino acid corresponding to a second native polypeptide, or a cryptic exon or exon of a splice variant of the first native polypeptide, each B is an amino acid that is not an amino acid corresponding to the first native polypeptide, the second native polypeptide, or the cryptic exon of the first native polypeptide, and x + y + z is at least 8, wherein y is absent and the at least 8 contiguous amino acids comprises at least one Ax and at least one C2, or y is at least 1 and the at least 8 contiguous amino acids comprises at least one By, wherein: (a) the first native polypeptide is encoded by a BCR gene. the second native polypeptide is encoded by an ABL gene, and (i) y is 0. and AxByCz is ERAEWRENIREQQKKCFRSFSLTSVELQMLTNSCVKLQTVHSIPLTINKEEALQRPVASDFEPQGLSEA ARWNSKENLLAGPSENDPNLFVALYDFVASG, or (ii) y is l, and AxByCz is NSCVKLQTVHSIPLTINKEDDESPGLYGFLNVIVHSATGFKQSSKALQRPVASDFEPQGLSE KENLLAGPSENDPNLFVALYDFVASGD; (b) the first native polypeptide is encoded by a C l lorf95 gene, the second native polypeptide is encoded by an RELA gene, y is l, and AxByCz is ISNSWDAHLGLGACGEAEGLGVQGAEEEEEEEEEEEEEGAGVPACPPKGPELFPLIFPAEPAQASGPY VEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTD; (e) the first native polypeptide is encoded by a CBFB gene and the second native polypeptide is encoded by an MYHI I gene, y is 0, and AxByCz is LQRLDGMGCLEFDEERAQQEDALAQQAFEEARRRTREFEDRDRSHREEMEVHELEKSKRALETQME EELEDELQATEDAKLRLEVNMQALKGQF; (d) the first native polypeptide is encoded by a CD74 gene and the second native polypeptide is encoded by an ROSl gene, y is 0, and AxByCz is KGSFPENLRHLKNTMET]DWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKAGVPNKPGIPKLLEGS KNSIQWEKAEDNGCRJTYYILEIRKSTSNNLQNQ; (e) the first native polypeptide is encoded by an EGFR gene and the second native polypeptide is encoded by (i) an SEPT14 gene, y is 0, and AxByCz is LPQPPICTIDVYMIMVKCWMIDADSRPKFRELIlEFSKMARDPQRYLVIQLQDKFEHLKMIQQEEIRKL EEEKKQLEGEIlDFYKMKAASEALQTQLSTD; or (ii) an EGFR gene, y is l, and AxByCz is MRPSGTAGAALLALLAALCPASRALEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGP CRKVCNGIGIGEFKD; (f) the first native polypeptide is encoded by a EML4 gene, the second native ptide is d by an ALK gene. y is l. and AxByCz is SWENSDDSRNKLSKIPSTPKLIPKVTKTADKHKDVIINQAKMSTREKNSQVYRRKHQELQAMQMEL LSKLRTSTIMTDYNPNYCFAGKTSSISDL (g) the first native polypeptide is encoded by a FGFR3 gene, the second native polypeptide is encoded by an TACC3 gene, y is 0, and AxByCz is EGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDRVLTVTSTDVKATQEENRELRSRCE ELHGKNLELGKIMDRFEEVVYQAMEEVQKQKELS, (h) the first native polypeptide is encoded by a NAB gene. the second native polypeptide is encoded by an STAT6 gene, y is at least 1. and AxByCz is RDNTLLLRRVELFSLSRQVARESTYLSSLKGSRLHPEELGGPPLKKLKQEATSKSQIMSLWGLVSKMP PEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCC; (i) the second native polypeptide is encoded by an ERG, y is 0, and (i) the first native polypeptide is encoded by a NDRGl gene, and AxByCz is MSREMQDVDLAEVKPLVEKGETITGLLQEFDVQEALSVVSEDQSLFECAYGTPHLAKTEMTASSSSD YGQTSKMSPRVPQQDW or (ii) the first native ptide is encoded by a Z gene, and AxByCz is ALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDW; (j) the first native polypeptide is encoded by a PML gene, the second native polypeptide is encoded by an RARA gene, y is l, and AxByCz is (i) VLDMHGFLRQALCRLRQEEPQSLQAAVRTDGFDEFKVRLQDLSSCITQGKAIETQSSSSEEIVPSPPSP PPLPRIYKPCFVCQDKSSGYI—IYGVSACEGCKG. or (ii) RSSPEQPRPSTSKAVSPPHLDGPPSPRSPVIGSEVFLPNSNHVASGAGEAAIETQSSSSEEIVPSPPSPPPL PRIYKPCFVCQDKSSGYHYGVSACEGCKG; (k) the first native ptide is encoded by a RUNXl gene. the second native polypeptide is encoded by an CBFAZTI TI) gene, y is l, and AxByCz is VARFNDLRFVGRSGRGKSFTLTITVFTNPPQVATYHRAIKITVDGPREPRNRTEKHSTMPDSPVDVKT QSRLTPPTMPPPPTFQGAPRTSSFTPTFLTNGT; (I) the first native polypeptide is encoded by a AR-v7 gene, the cryptic exon or the exon of a splice variant is encoded by the AR-v7 gene, y is 0, and AxByCz is SCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGEKFRVGNCKHLKMTRP Provided herein is an isolated neoantigenic peptide comprising a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native polypeptide, wherein the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: AxByCz wherein each A is an amino acid corresponding to a first native polypeptide; each B is an amino acid that is not an amino acid corresponding to the first native polypeptide or the second native ptide, each C is an amino acid encoded by a frameshift of a sequence encoding a second native polypeptide; x + y + z is at least 8, wherein y is absent and the at least 8 contiguous amino acids comprises at least one C2, or y is at least I and the at least 8 contiguous amino acids ses at least one By and/or at least one Cz; and (a) the first native polypeptide is encoded by an ACO] 1997.] gene, the second native polypeptide is encoded by a LRRC69 gene, y is l, and AxByCz is MAGAPPPASLPPCSLISDCCASNQRDSVGVGPSEPGNNlKlCNESASRK (b) the first native polypeptide is encoded by an EEFlDP3 gene, the second native polypeptide is encoded by a FRY gene, y is 1, and AxByCz is HGWRPFLPVRARSRWNRRLDVTVANGRSWKYGWSLLRVPQVNGIQVLNVSLKSSSNVISY. (c) the first native polypeptide is d by a MADlLl gene, the second native polypeptide is encoded by a MAFK gene, y is 0, and AxByCz is RLKEVFQTKIQEFRKACYTLTGYQIDITTENQYRLTSLYAEHPGDCLIFKLRVPGSSVLVTVPGL, or (d) the first native ptide is encoded by a PPPlRlB gene, the second native polypeptide is encoded by a STARD3 gene, y is l, and AxByCz is AEVLKVIRQSAGQKTI‘CGQGLEGPWERPPPLDESERDGGSEDQVEDPALSALLLRPRPPRPEVGAHQ DEQAAQGADPRLGAQPACRGLPGLLTVPQPEPLLAPPSAA.

In embodiments, the isolated neoantigenic peptide comprises a sequence according to Table I. In embodiments, x + y + z is at most 500, at most 250, at most 150, at most 125, or at most 100 In embodiments, x + y + z is at least 8, at least 50, at least 100, at least 200, or at least 300. In embodiments, z is at most 500, at most 250, at most 150, at most 125, or at most 100. In embodiments, z is at least 8, at least 50, at least 100, at least 200. or at least 300. In embodiments. the isolated neoantigenic peptide is from about 8 to about 500 amino acids in . In embodiments, the isolated neoantigenic peptide is from about 8 to about 100 amino acids in length. In embodiments, the isolated neoantigenic peptide is from about 8 to about 50 amino acids in length. In embodiments, the isolated neoantigenic peptide is from about 15 to about 35 amino acids in length.

In embodiments, the isolated neoantigenic peptide is from about 8 and about 15 amino acids in length. In embodiments, the isolated neoantigenic peptide is from about 8 and about 1 1 amino acids in length. In embodiments. the isolated igenic peptide is 9 or 10 amino acids in length. In embodiments. the isolated neoantigenic peptide binds major histocompatibility x (MHC) class I. In embodiments. the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 500 nM or less. In embodiments, the isolated neoantigenic peptide binds MHC class 1 with a binding y of about 250 nM or less. In embodiments, the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 50 nM or less. In embodiments, the ed neoantigenic peptide is from about 8 and about 30 amino acids in length. In embodiments. the isolated neoantigenic peptide is from about 8 to about 25 amino acids in length. In embodiments, the isolated neoantigenic peptide is from about 15 to about 24 amino acids in length. In embodiments, the isolated neoantigenic e is from about 9 to about 15 amino acids in length. In embodiments, the isolated neoantigenic peptide binds MHC class II. In embodiments, the isolated neoantigenic e binds MHC class II with a binding y of 1000 nM or less. In embodiments, the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 500 nM or less. In embodiments, the isolated neoantigenic peptide further comprises flanking amino acids. In embodiments, the flanking amino acids are not native flanking amino acids In ments. the ed neoantigenic peptide has atotal length of at least 8, at least 9, at least 10, at least 11. at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least , at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids. In embodiments, the ed neoantigenic peptide has a total length of at most 8. at most 9. at most 10. at most 1 1. at most 12. at most 13. at most 14. at most 15. at most 16. at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids. In embodiments, the isolated neoantigenic peptide is a first neoantigenic peptide linked to at least a second neoantigenic e. In embodiments, the isolated neoantigenic peptide is linked to the at least second igenic peptide by a poly-glycine or poly—serine linker. In ments, the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 1000 nM. In embodiments, the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 500 nM. In embodiments, isolated neoantigenic peptide and the second neoantigenic peptide bind to human leukocyte antigen (HLA) -A, -B, -C, -DP, -DQ, or -DR. In embodiments, the isolated neoantigenic peptide binds a class I HLA and the second igenic peptide binds a class II HLA. In embodiments, the isolated neoantigenic peptide binds a class II I-ILA and the second igenic peptide binds a class I HLA.

In embodiments, the isolated neoantigenic peptide further comprises a modification which increases in vivo ife, cellular targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, antigen presentation, or a combination thereof. In embodiments, the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, tion, alylation I-IESylation, recombinant PEG mimetics, Fe fusion, albumin fiision, nanoparticle attachment, nanoparticulate encapsulation, cholesterol fusion, iron fusion, acylation. amidation. glycosylation. side chain oxidation. orylation. biotinylation. the addition of a surface active material, the addition of amino acid mimetics, or the addition of unnatural amino acids. In embodiments, the ed neoantigenic peptide finther comprises a ation which increases ar targeting to antigen presenting cells. In embodiments, the antigen presenting cells are dendritic cells. In embodiments, the dendritic cells are targeted using DEC205, XCRI, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD1 lc, CD83, TSLP receptor, Clec9a. or CDla . In embodiments. the dendritic cells are targeted using the CD141, DEC205, Clec9a, or XCRl . In ments, the dendritic cells are autologous cells.

In embodiments, one or more of the dendritic cells are bound to a T cell. In embodiments, the T cell is an autologous T cell. In embodiments, the isolated neoantigenic peptide is not a isolated neoantigenic peptide listed in Table 2. In embodiments, the isolated neoantigenic peptide is linked to at least one additional neoantigenic e listed in Table l or 2.

Provided herein is an in vivo delivery system sing an isolated neoantigenic peptide described herein. In ments, the delivery system includes cell-penetrating peptides, nanoparticulate encapsulation, virus like particles, liposomes, or any ation thereof. In embodiments, the cell-penetrating peptide is TAT peptide, herpes simplex virus VP22, transportan, Antp, or any combination thereof.

Provided herein is a cell comprising an isolated neoantigenic peptide described herein. In embodiments, the cell is an n presenting cell. In embodiments, the cell is a dendritic cell. In embodiments, the cell is an autologous cell. In embodiments, the cell is bound to a T cell. In embodiments, the T cell is an autologous T cell.

Provided herein is a composition comprising an isolated neoantigenic peptide bed herein.

In embodiments, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90. or at least 100 of the isolated neoantigenic peptides comprising a tumor-specific neoepitope according to Table l or 2. In embodiments, the composition comprises from about 2 to about 20 neoantigenic peptides, or from about 2 to about 30 neoantigenic peptides. In ments, the neoantigen is specific for an individual subject's tumor. In embodiments, the composition further comprises at least I, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 I, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24. or at least 25. at least 26. at least 27. at least 28. at least 29. at least 30. at least 40. at least 50. at least 60. at least 70, at least 80, at least 90, or at least 100 additional neoantigenic peptides. In embodiments, the ition comprises from about 4 to about 20 additional neoantigenic peptides, from about 4 to about 30 additional neoantigenic peptides. 1n embodiments, at least onof the additional neoantigenic peptides is specific for an individual subject's tumor. In embodiments, the subject specific neoantigenic e is selected by identifying sequence differences between the genome, exome, and/or transcriptome of the subject's tumor sample and the genome. exome. and/or transcriptome of a non-tumor sample. In embodiments. the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or ating tumor cells.

In embodiments, the sequence differences are determined by Next Generation Sequencing.

Provided herein is an isolated cleotide encoding an ed neoantigenic peptide described . In embodiments, the polynucleotide is DNA. In embodiments, the polynucleotide is RNA. In embodiments, the RNA is a self-amplifying RNA. In embodiments, the RNA is modified to increase ity, increase cellular ing, increase translation efficiency, adjuvanticity. cytosol ibility, and/or decrease cytotoxicity. In embodiments, the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, codon optimization, increased GC-content, incorporation of modified nucleosides, incorporation of 5'-cap or cap analog, and/or incorporation of an unmasked poly-A sequence.

Provided herein is a cell sing the polynucleotide described herein.

Provided herein is a vector comprising the polynucleotide described herein. In embodiments, the polynucleotide is operably linked to a promoter. In embodiments, the vector is a self-amplifying RNA replicon. plasmid, phage. transposon, cosmid, virus, or virion. In embodiments. the vector is derived from an adeno-associated virus, herpesvirus, lentivirus, or a pseudotype f.

Provided herein is an in vivo delivery system comprising the ed polynucleotide bed herein.

In embodiments, the delivery system es spherical nucleic acids, viruses, virus-like particles, plasmids, bacterial plasmids, or nanoparticles. ed herein is a cell comprising a vector or ry system described herein. In embodiments, the cell is an antigen presenting cell. In embodiments. the cell is a dendritic cell. In ments. the cell is an immature dendritic cell.

Provided herein is a composition comprising at least one polynucleotide described herein. In embodiments, the composition ses at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28. at least 29. at least 30, at least 40. at least 50. at least 60. at least 70, at least 80, at least 90. or at least 100 of the isolated polynucleotides. In embodiments, the composition comprises from about 2 and about 20 of the isolated polynucleotides, or from about 2 to about 30 of the isolated polynucleotides. In embodiments, the neoantigenic es are encoded by a vector comprising one or more of the the isolated polynucleotides.

In embodiments, the composition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16. at least 17. at least 18. at least 19. at least 20. at least 21. at least 22. at least 23. at least 24. at least 25. at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 additional neoantigenic poly-nucleotides encoding for additional neoantigenic peptides. 1n embodiments, one or more of the additional neoantigenic peptides are encoded by a vector comprising one or more of the additional neoantigenic polynucleotides. In embodiments, the ition comprises from about 4 to about 20 additional neoantigenic polynucleotides, or from about 4 to about 30 additional neoantigenic pol_\-'nucleotides. In embodiments. the ed cleotides and the additional neoantigenic polynucleotides are linked. In embodiments, the polynucleotides are linked using nucleic acids that encode a poly-glycine or poly-serine linker. In embodiments, at least one of the additional neoantigenic peptide is specific for an individual t '5 tumor. In embodiments, the subject specific neoantigenic peptide is selected by identifying sequence ences between the genome, exome, and/or transcriptome of the subject‘s tumor sample and the genome, cxome, and/or transcriptome of a non-tumor . In embodiments. the s are fresh or formalin-fixed paraffin embedded tumor tissues. freshly isolated cells. or circulating tumor cells. In embodiments. the sequence differences are determined by Next Generation Sequencing.

Provided herein is a T cell receptor (TCR) capable of binding at least one neoantigenic peptide described herein or an MHC-peptide complex comprising at least one neoantigenic peptide described herein.

The In embodiments, the MHC of the MHC-peptide is MHC class I or class II. In ments, TCR is a bispecific TCR fimher comprising a domain sing an antibody or antibody fragment capable of g an n. In embodiments. the antigen is a T cell-specific antigen. In embodiments. the n is CD3. In embodiments. the antibody or antibody fragment is an anti-CD3 scFv. ed herein is a chimeric antigen receptor comprising: (i) a T cell activation molecule; (ii) a transmembrane region; and (iii) an n recognition moiety capable of binding at least one neoantigenic peptide described hereinor an MHC-peptide complex comprising at least one neoantigenic peptide described herein. In embodiments, CD3-zeta is the T cell activation molecule. In ments, the chimeric antigen receptor r comprises at least one costimulatory signaling . The In embodiments. the signaling domain is CD28, 4—IBB, ICOS, 0X40, ITAM, or PC epsilon RI-gamma. In embodiments, the n recognition moiety is capable of binding the isolated neoantigenic peptide in the context of MHC class I or class 11. In embodiments, the CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CDl37, 0X40, CD27, CD40L, Tim-3, AZaK or PD-l transmembrane region. In embodiments, the igenic peptide is located in the extracellular domain of a tumor associated polypeptide. In embodiments, the MHC of the MHC-peptide is MHC class I or class II.

Provided herein is a T cell comprising the T cell receptor or chimeric antigen receptor described herein, optionally wherein the T cell is a helper or cytotoxic T cell. In embodiments, the T cell is a T cell of a subject.

Provided herein is a T cell comprising a T cell receptor (TCR) capable of binding at least one neoantigenic peptide described herein or an MHC-peptide complex comprising at least one neoantigenic peptide described herein, wherein the T cell is a T cell isolated from a population ofT cells from a subject that has been incubated with antigen presenting cells and one or more of the at least one neoantigenic peptide described herein for a sufficient time to activate the T cells. In embodiments, the T cell is a CD8+ T cell, a helper T cell or cytotoxic T cell. In embodiments, the tion of T cells from a subject is a population of CD8+ T cells from the subject. In embodiments, the one or more of the at least one neoantigenic e bed herein is a subject-specific neoantigenic peptide. In embodiments. the subject-specific neoantigenic peptide has a different tumor nee-epitope that is an epitope specific to a tumor of the subject. In embodiments, the subject-specific neoantigenic peptide is an expression product of a tumor-specific non-silent mutation that is not present in a non-tumor sample of the subject. In embodiments, the subject-specific neoantigenic peptide binds to a HLA protein of the subject. In embodiments, the subject-specific igenic peptide binds to a HLA protein of the subject with an ICSO less than 500 nM. In embodiments, the activated CD8+ T cells are separated from the antigen ting cells. In embodiments, the antigen presenting cells are dendritic cells or CD40L-expanded B cells. In embodiments, the antigen ting cells are non-transfomied cells. In embodiments, the antigen presenting cells are non-infected cells. In embodiments, the antigen presenting cells are autologous. In ments, the antigen presenting cells have been treated to strip endogenous MHC—associated peptides from their e. In embodiments, the treatment to strip the endogenous MHC-associated peptides ses ing the cells at about 26°C. In embodiments, the treatment to strip the endogenous MHC-associated peptides comprises ng the cells with a mild acid solution. In embodiments. the n presenting cells have been pulsed with at least one neoantigenic e described herein. In embodiments, pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 rig/ml of each of the at least one neoantigenic peptide described herein. In ments, ratio of isolated T cells to antigen presenting cells is between about 30:1 and 300:1. In embodiments, the incubating the isolated population ofT cells is in the presence of IL-2 and IL-7. In embodiments, the MHC of the MHC-peptide is MHC class I or class II. ed herein is a method for activating tumor specific T cells comprising: isolating a population of T cells from a subject; and incubating the isolated population of T cells with antigen presenting cells and at least one neoantigenic peptide described herein for a sufficient time to activate the T cells. In ments, the T cell is a CD8+ T cell, a helper T cell or cytotoxic T cell. In embodiments, the population of T cells from a subject is a population of CD8+ T cells from the subject. In embodiments, the one or more of the at least one igenic peptide described herein is a subject-specific neoantigenic peptide. In embodiments, the subject- specific neoantigenic peptide has a ent tumor nee-epitope that is an epitope specific to a tumor of the subject. In embodiments, the subject-specific neoantigenic peptide is an expression product of a tumor- specific non-silent mutation that is not present in a non-tumor sample of the subject. In embodiments, the subject-specific neoantigenic peptide binds to a HLA protein of the subject. In ments, the subject- specific neoantigenic peptide binds to a HLA n ofthe subject with an IC50 less than 500 nM. In embodiments, the method r comprises separating the activated T cells from the antigen presenting cells.

In embodiments. the method further comprises testing the activated T cells for evidence of reactivity against at least one of neoantigenie peptide of described herein. In ments, the antigen presenting cells are dendritic cells or CD40L-expanded B cells. In embodiments, the antigen presenting cells are non-transformed cells. In embodiments, the antigen presenting cells are non-infected cells. In embodiments, the antigen presenting cells are autologous. In embodiments, the antigen presenting cells have been treated to strip endogenous MHC-associated peptides from their surface. In embodiments, the treatment to strip the endogenous MHC-associated peptides comprises culturing the cells at about 26°C. In ments. the treatment to strip the endogenous MHC-associated es comprises treating the cells with a mild acid solution. In embodiments, the antigen presenting cells have been pulsed with at least one neoantigenie peptide described herein. In embodiments, pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 ug/ml of each of at least one neoantigenie e described herein. In embodiments, ratio of isolated T cells to antigen ting cells is between about 30:1 and 300:1. In ments, the incubating the isolated population of T cells is in the presence of IL—2 and lL-7. In embodiments. the MHC of the MHC- peptide is MHC class I or class II.

Provided herein is a composition comprising activated tumor c T cells produced by a method described .

Provided herein is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of activated tumor c T cell described herein, or produced by a method described herein. In ments, the administering comprises administering from about 10° to 10”, from about 108 to l0l 1. or from about 109 to 1010 of the activated tumor c T cells.

Provided herein is a nucleic acid comprising a promoter ly linked to a polynucleotide encoding the T cell receptor described herein. In embodiments, the TCR is e of binding the at least one neoantigenie peptide in the context of major histoeompatibility complex (MHC) class I or class II.

Provided herein is a nucleic acid comprising a promoter operably lihked to a polynucleotide encoding the chimeric n or described herein. In embodiments, the antigen recognition moiety is capable of binding the at least one neoantigenie peptide in the context of major histocornpatibility complex (MHC) class I or class II. In embodiments, the neoantigenie peptide is located in the extracellular domain of a tumor ated polypeptide. In embodiments, the nucleic acid comprises the CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, 0X40, CD27, CD40L, Tim-3, AZaR, or PD- h embrane region.

Provided herein is an dy or antibody fragment capable of binding at least one neoantigenie peptide described herein or an MHC-peptide complex comprising at least one neoantigenie peptide described herein, optionally wherein the antibody fragment is a bi-specifrc T cell engager (BiTE). In embodiments. the antibody or antibody nt binds to an extracellular portion of the at least one neoantigenie e. In embodiments, the native polypeptide is encoded by a gene selected from the group consisting of: BZM, wherein C2 is RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKIVISNIPAV, or LCSRYSLFLAWRLSSVLQRFRFTHVIQQRMESQIS; EGFR, wherein AxByCz is IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; BTK. wherein AxByCz is MSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFlITEYMANGSLLNYLREMRI-IRFQTQQ LLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND; or ESRI, wherein AxByCz is HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGS'I‘SSHSLQKYYITGEA, NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLPSTLKSLEEKDHIHRVLD KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH.

AGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLCDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLNDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGS'I‘SSHSLQKYYITGE, or IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSI-ISLQKYYITGE; or I-IER2. wherein AxByCz is GSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGLGSPYVSRLLGICLTSTV QLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCM. In ments, the antibody or antibody nt is a bispecific antibody or antibody fragment. In embodiments, one antigen binding domain of the bispecific antibody or antibody fragment is an anti-CD3 binding domain.

Provided herein is a modified cell transfected or transduced with a nucleic acid described herein. In embodiments, the modified cell is a T cell, tumor infiltrating lymphocyte, NK-T cell, TCR-expressing cell, CD4+ T cell. CD8+ T cell, or NK cell. ed herein is a composition comprising the T cell receptor or chimeric antigen receptor described herein.

Provided herein is a composition comprising autologous subject T cells containing the T cell receptor or chimeric antigen receptor described herein. In embodiments, the composition further ses an immune checkpoint inhibitor. In embodiments, the composition further comprises at least two immune oint tors. In embodiments. each of the immune checkpoint inhibitors inhibits a checkpoint n selected from the group ting of CTLA-4, PDLI, PDLZ, PDl, B7-H3, B7-H4, BTLA, HVEM, TlM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CI-IK l, CHKZ, AZaK and B-7 family ligands or a combination thereof. In embodiments, each of the immune checkpoint inhibitors interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDLI, PDL2, PDI, B7-I-I3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIK 2B4, CD160, 5049, CHK l, CHKZ, A2aR, and B-7 family ligands or a combination thereof. In embodiments, the T cells are PD—l and/or CTLA4 ut T cells, optionally,wherein the PD—l and/or CTLA4 knockout T cells are created using a CRISPR system. In embodiments, the composition further comprises an immune modulator or nt. In embodiments, the immune modulator is a co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD4OL, 0X40, CD27, GITR, CD30, DR3, CD69, or 4-IBB. In embodiments, the immune modulator is at least one cancer cell or cancer cell extract. In embodiments, the cancer cell is autologous to the subject in need of the composition. In embodiments. the cancer cell has undergone lysis or been d to UV radiation. In embodiments, the composition further comprises an adjuvant. In ments, the adjuvant is selected from the group consisting of: Poly(I:C), Poly-ICLC, STING agonist, 1018 188, aluminium salts, Amplivax, A815, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, 1C30, 1C3 1, Imiquimod, lmuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvamune, LipoVac, MF59, monophosphoryl lipid A, Montanide [MS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide [SA 51 VG, OK-432, OM-174, OM-l97-MP-EC. ISA-TLRZ agonist. ONTAK. PepTel-‘R‘u vector system. PLG microparticles. resiquimod.

SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, lucan, Pam3Cys, Pam3CSK4, acrylic or methacrylic polymers, copolymers of maleic anhydride, and QSZI stimulon. In embodiments, the composition induces a Immoral response when stered to a subject. In embodiments, the composition induces a T helper cell type 1 when administered to a t. ed herein is a method of inhibiting gromh of a tumor cell expressing a tumor-specific neoepitope. comprising contacting the tumor cell with the peptide. polynucleotide, ry system, vector, composition, antibody, or cells bed herein.

Provided herein is a method of prophylaxis of a subject, comprising contacting a cell of the t with the peptide, polynucleotide, delivery system, vector, composition, antibody, or cells described herein. In embodiments, the native polypeptide is encoded by a gene selected from the group consisting of: 02M, wherein C2 is RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKMSMPAV, or LCSRYSLFLAWRLSSVLQRFRFTHVIQQRMESQIS; EGFR, n AxByCz is IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; BTK, wherein AxByCz is MIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGSLLNYLREMRI-IRFQTQQ LLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND; or ESRl, wherein AxByCz is I-ILMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEA, NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLPSTLKSLEEKDHIHRVLD KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH, IHLMAKAGLTLQQQI-IQRLAQLLLILSI-IIRHMSNKGMEHLYSMKCKNVVPLCDLLLEMLDAHRLHAP SVEETDQSHLATAGSTSSHSLQKYYITGE, AGLTLQQQI-IQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLNDLLLEMLDAI-IRLHAP TSRGGASVEETDQSHLATAGS'I‘SSHSLQKYYITGE, or AGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLLEMLDAHRLI—IAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE; and HERZ, wherein AxByCz is GSGAFGTVYKGIVVIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGLGSPYVSRLLGICLTSTV QLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCM.

Provided herein is a method of treating cancer or initiating, ing, or prolonging an anti-tumor response in a subject in need thereof comprising administering to the subject the peptide. polynucleotide. , composition, antibody, or cells described herein. In embodiments, the subject is a human. In ments, the subject has cancer. In ments, the cancer is selected from the group consisting of urogenital, gynecological, lung, gastrointestinal, head and neck cancer, malignant glioblastoma, malignant mesothelioma, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel Cell Carcinoma or bone and soft tissue sarcomas, hacmatologic neoplasias, multiple mycloma, acute myelogenous leukemia, chronic myelogenous leukemia. myelodysplastic syndrome and acute lymphoblastic leukemia. non—small cell lung cancer (NSCLC), breast cancer, metastatic colorectal cancers, hormone sensitive or hormone refractory prostate , colorectal cancer, ovarian cancer, hepatocellular cancer, renal cell cancer, pancreatic cancer, gastric cancer, oesophageal cancers, hepatocellular cancers, cholangiocellular s, head and neck squamous cell cancer sofi tissue sarcoma, and small cell lung cancer. In embodiments, the peptide, poly-nucleotide, vector, composition, antibody, or cells described herein is for use in treating a ponding cancer according to Table l or Table 2. In embodiments. the peptide. polynucleotide. vector. composition, dy, or cells described herein is for use in treating a subject with an HLA type that is a corresponding HLA type according to Table l or Table 2. In embodiments, the subject has undergone surgical removal of the tumor. In embodiments, the peptide, polynucleotide, vector, composition, or cells is administered via intravenous, intraperitoneal, intratumoral, ermal, or subcutaneous administration. In embodiments, the peptide, polynucleotide, vector, composition, or cells is administered into an anatomic site that drains into a lymph node basin. In embodiments, administration is into multiple lymph node basins. In embodiments, administration is by a subcutaneous or ennal route. In ments. peptide is stered. In embodiments, administration is intratumorally. In embodiments, polynucleotide, optionally RNA, is administered. In embodiments, the polynucleotide is administered intravenously. In embodiments, the cell is a T cell or dendritic cell. In embodiments, the peptide or polynucleotide comprises an n presenting cell ing moiety. In embodiments, the cell is an autologous cell. In embodiments, the method fiirther ses administering at least one immune checkpoint inhibitor to the t. In embodiments, the checkpoint inhibitor is a biologic therapeutic or a small molecule. In embodiments. the oint inhibitor is selected from the group ting of a monoclonal dy, a humanized antibody, a fiilly human antibody and a fusion protein or a combination thereof In embodiments, the checkpoint tors is a PD-l antibody or a PD-Ll antibody. In embodiments, the oint inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, avelumab, durvalumab, atezolizumab, pembrolizumab, and any combination thereof. In embodiments, the checkpoint inhibitor ts a checkpoint protein selected from the group consisting of CTLA-4, PDLI. PDLZ, PDI, B7-I-I3, B7-I-I4, BTLA. HVEM. TIM3. GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN—15049, CHK 1, CHKZ, AZaR, and B-7 family ligands, and any combination thereof. In embodiments, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDLI, PDLZ, PDI, B7-H3, B7-I-I4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, 5049, CHK l, CHK2, A2aR, and B-7 family ligands or a combination thereof. In embodiments, two or more checkpoint tors are stered. In embodiments, at least one of the two or more checkpoint inhibitors is a PD-l antibody or a PD-Ll dy. In embodiments, at least one ofthe two or more checkpoint inhibitors is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, ab, durvalumab, atezolizumab, and lizumab. In embodiments, the oint inhibitor and the composition are administered simultaneously or sequentially in any order. In embodiments, the peptide, polynucleotide, , composition, or cells is administered prior to the checkpoint inhibitor. In embodiments, the e, polynuclcotidc, vector, composition, or cells is administered afler the checkpoint inhibitor. In embodiments. administration of the checkpoint inhibitor is continued throughout neoantigen peptide, polynucleotide, vector, composition, or cell therapy. In embodiments, the neoantigen peptide, polynucleotide, vector, composition, or cell y is administered to subjects that only partially d or do not respond to checkpoint inhibitor therapy. In embodiments, the composition is administered intravenously or subcutaneously. In embodiments, the checkpoint inhibitor is administered intravenously or subcutaneously. In ments, the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the composition. In embodiments, the composition is administered into the same draining lymph node as the checkpoint inhibitor. In ments, the method further ses administering an additional therapeutic agent to the subject either prior to, simultaneously with, or afier treatment with the e, polynucleotide, vector, composition, or cells. In embodiments, the additional agent is a chemotherapeutic agent, an immunomodulatory drug, an immune metabolism modifying drug, a targeted therapy, radiation an anti-angiogenesis agent, or an agent that reduces immune-suppression. In embodiments, the chemotherapeutic agent is an alkylating agent, a omerase inhibitor. an anti-metabolite. or an anti-mitotic agent. In embodiments. the additional agent is an anti- glucocorticoid induced tumor necrosis factor family receptor (GITR) agonistic antibody or dy fragment, ibrutinib, docetaxeol, cisplatin, a CD40 tic antibody or antibody fragment, an IDO inhibitor, or cyclophosphamide. In embodiments, the method s a CD4+ T cell immune response or a CD8+ T cell immune response. In embodiments, the method elicits a CD4+ T cell immune response and a CD8+ T cell immune response.

Provided herein is a method for stimulating an immune response in a subject. comprising administering an effective amount of modified cells or composition described herein. In embodiments, the immune response is cytotoxic and/or humoral immune response. In embodiments, the method stimulates a T cell-mediated immune response in a subject. In ments, the T cell-mediated immune response is directed against a target cell. In embodiments, the target cell is a tumor cell. In embodiments, the modified cells are transfected or transduccd in vivo. In embodiments, the modified cells are transfccted or transduccd ex vivo. In embodiments. the modified cells are autologous subject T cells. In embodiments, the autologous subject T cells are obtained from a subject that has received a neoantigen peptide or nucleic acid vaccine. In embodiments, the neoantigen peptide or nucleic acid vaccine ses at least one personalized igen.

In embodiments, the neoantigen peptide or nucleic acid vaccine comprises at least one additional neoantigenic e listed in Table l or 2. In embodiments, the subject received a chemotherapeutic agent, an immunomodulatory drug, an immune metabolism modifying drug, targeted therapy or radiation prior to and/or during receipt of the neoantigen peptide or nucleic acid vaccine. In embodiments. the t es treatment with at least one checkpoint inhibitor. In embodiments, the autologous T cells are obtained from a subject that has already received at least one round ofT cell therapy containing a neoantigen. In ments, the method r ses adoptive T cell therapy. In embodiments, the adoptive T cell therapy comprises autologous T cells. In embodiments, the autologous T cells are targeted against tumor antigens. In embodiments, the adoptive T cell therapy further comprises allogcnic T cells. In ments, the allogenic T cells are targeted against tiunor ns. In embodiments. the adoptive T cell y is administered before the checkpoint inhibitor, afier the checkpoint inhibitor, or simultaneously eith the checkpoint inhibitor.

Provided herein is a method for evaluating the cy of any of the cells described herein, comprising: (i) measuring the number or concentration of target cells in a first sample obtained from the subject before administering the modified cell, (ii) measuring the number concentration of target cells in a second sample obtained from the subject after administration of the modified cell, and (iii) determining an increase or decrease of the number or concentration of target cells in the second sample compared to the number or concentration of target cells in the first sample. In ments, ent efficacy is detennined by monitoring a clinical e; an increase, enhancement or prolongation ofanti-tumor activity by T cells; an increase in the number of anti-tumor T cells or activated T cells as compared with the number prior to treatment: B cell activity; CD4+ T cell activity; or a combination thereof. In embodiments, treatment efficacy is ined by monitoring a biomarker. In embodiments, the biomarker is selected from the group ting of CEA, Her-Z/neu, bladder tumor antigen, thyroglobulin, alpha-fetoprotein, PSA, CA 125, CA19.9. CA 15.3, leptin. prolactin. osteopontin, IGF-II, CD98, fascin, sPIgR. 143 eta, troponin I. circulating tumor cell RNA or DNA, and b-type natriuretic e. In embodiments, clinical outcome is selected from the group consisting of tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response by the immune system; tumor expansion, recurrence or spread; or a ation thereof. In embodiments, the treatment effect is predicted by presence of T cells or by presence of a gene signature indicating T cell inflammation or a combination thereof.

Provided herein is a method of treating cancer or initiating. enhancing. or prolonging an anti-tumor response in a t in need thereof comprising administering to the subject: the peptide, polynucleotide, , composition, dy, or cells described herein; and at least one checkpoint inhibitor. In embodiments, the method further comprises administration of an immunomodulator or nt. In embodiments, the immunomodulator or adjuvant is selected from the group consisting of Poly(I:C), Poly- ICLC, STING agonist, 1018 [$5, aluminium salts, Amplivax, ASIS, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF. IC30. IC3I, Imiquimod. ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvamune, LipoVac, MF59, monophosphoryl lipid A, Montanide [MS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide [SA 51 VG, OK-432, OM-I 74, OM-l97-MP-EC, ISA-TLRZ agonist, ONTAK, PepTelt’R‘) vector system, PLG microparticles, resiquimod, , virosomes and other virus-like particles, YF-I7D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, acrylic or methacrylic polymers, copolymers of maleic anhydride, and 0821 stimulon. a co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28. CD80. CD86. ICOS. CD40L. 0X40. CD27. GITR. CD30. DR3. CD69. or 4-IBB. In embodiments. the immunomodulator or adjuvant is Poly-1CLC. In embodiments, the checkpoint inhibitor is an anti-PDI antibody or antibody fragment. In embodiments, the anti-PD] antibody or antibody fragment is nivolumab or pembolizumab. In ments, the checkpoint tor is an anti-PD-Ll antibody or antibody fragment. In embodiments, the anti-PD-Ll antibody or antibody fragmentis avelumab, durvalumab or izumab. In embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody or antibody fragment. In embodiments. the anti-CTLA4 dy is ipilimumab or tremelimumab. In embodiments. the method comprises stering both an anti-PDl antibody and an TLA4 antibody. In embodiments, administration of the checkpoint inhibitor is initiated before initiation of stration of the peptide, polynucleotide, vector, composition, antibody, or cell. In embodiments, administration ofthe checkpoint tor is initiated after initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In ments, administration of the checkpoint tor is initiated simultaneously with the initiation of administration of the peptide. polynucleotide, vector. composition. antibody. or cell. In embodiments. the peptide. poly-nucleotide, vector, composition, antibody, or cell is administered intravenously or subcutaneously. In embodiments, the checkpoint inhibitor is administered intravenously or subcutaneously. In embodiments, the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the peptide, polynucleotide, vector, ition, antibody, or cell. In embodiments, the peptide, polynucleotide, vector, composition, antibody, or cell is administered into the same draining lymph node as the checkpoint inhibitor.

Provided herein is a kit comprising the peptide. polynucleotide, vector. composition. antibody, cells. or ition described herein. In embodiments, the cancer is selected from the group consisting of: adrenal, bladder, breast, cervical, colorectal, glioblasoma, head and neck, kidney chromophobe, kidney clear cell, kidney papillary, liver, lung adenocarcinoma, lung squamous, ovarian, pancreatic, melanoma, stomach, uterine corpus endometrial,and uterine osarcoma. In embodiments, the cancer is selected from the group consisting of: prostate cancer, bladder, lung squamous, NSCLC, breast, head and neck, lung adenocarcinoma, GBM. Glioma. CML. AML. supretentorial ependyomas. acute promyelocytic leukemia. solitary fibrous , and crizotinib resistant cancer. In embodiments, the cancer is ed from the group consisting of: CRC, head and neck, stomach, lung squamous, lung adenocarcinoma, te, Bladder. h, renal cell carcinoma, and uterine. In embodiments, the cancer is selected from the group consisting of: melanoma, lung squamous, DLBCL, uterine, head and neck, uterine, liver, and CRC. In embodiments, the cancer is ed from the group consisting of: lymphoid cancer; Burkitt lymphoma, lastoma, prostate adenocarcinoma, colorectal adenocarcinoma; e/Endometrium Adenocarcinoma; MSI+; endometrium serous carcinoma; trium carcinosarcoma—malignant mesodermal mixed tumour; glioma; astrocytoma; GBM, acute myeloid leukaemia ated with MDS: chronic lymphocytic leukaemia-small lymphocytic lymphoma: myelodysplastic syndrome; acute myeloid leukaemia; Iuminal NS carcinoma of breast; chronic myeloid leukaemia; ductal carcinoma of pancreas; c myelomonocytic leukaemia; myelofibrosis; myelodysplastic syndrome; prostate adenocarcinoma; essential thrombocythaemia; and medullomyoblastoma. In embodiments, the cancer is selected from the group consisting of: colorectal, uterine. endometrial, and stomach. In embodiments, the cancer is selected from the group consisting of: cervical, head and neck, anal, stomach, Burkitt‘s lymphoma, and nasopharyngeal carcinoma. In embodiments, the cancer is selected from the group consisting of: bladder, ctal, and stomach. In embodiments, the cancer is selected from the group consisting of: lung, CRC, melanoma, breast, NSCLC, and CLL. In embodiments, the subject is a partial or non-responder to checkpoint inhibitor therapy. In embodiments, the cancer is selected from the group consisting of: bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA). breast cancer. cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), chronic lymphocytic leukaemia (CLL), colorectal cancer (CRC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LlHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma , pancreatic adenocarcinoma (PAAD), te Cancer, skin cutaneous melanoma (SKCM). stomach adenocarcinoma , thyroid adenocarcinoma (TI-ICA), and uterine corpus endometrioid carcinoma . In embodiments. the cancer is ed from the group consisting of: colorectal cancer, uterine cancer, endometrium cancer, stomach cancer, and Lynch syndrome. In embodiments, the cancer is an MSI+ .

The present ion is directed to an ed neoantigenic e comprising a tumor-specific neoepitope defined in Table I, wherein the isolated neoantigenic peptide is not a native ptide. The present invention is also directed to an isolated igenic peptide which comprises a tumor-specific neoepitope and is defined in Table I.

In some embodiments. the isolated neoantigenic peptide is between about 8 to about 50 amino acids in length. In another embodiment, the isolated neoantigenic peptide is between about 15 to about 35 amino acids in length. In another embodiment, the isolated neoantigenic peptide is about 15 amino acids or less in length.

In another embodiment, the isolated neoantigenic peptide is between about 8 and about 11 amino acids in length. In another embodiment, the isolated igenic peptide is 9 or 10 amino acids in length. In another embodiment, the isolated neoantigenic peptide binds major histocompatibility complex (MHC) class I. In another embodiment. the isolated neoantigenic peptide binds MHC class I with a binding affinity of less than about 500 nM.

In some embodiments, the isolated igenic peptide is about 30 amino acids or less in length. In another embodiment, the isolated neoantigenic peptide is between about 6 and about 25 amino acids in .

In another embodiment, the isolated neoantigenic peptide is between about 15 and about 24 amino acids in length. In another embodiment, the isolated igenic peptide is between about 9 and about 15 amino acids in length. In another embodiment. the isolated neoantigenic peptide binds MHC class II. In another embodiment, the isolated igenic peptide binds MHC class II with a binding affinity of less than about 1000 nM.

In some ments, the isolated neoantigenic peptide further ses flanking amino acids. In another embodiment, the flanking amino acids are not native flanking amino acids. In another embodiment, the isolated neoantigenic peptide is linked to at least a second neoantigenic peptide. In another embodiment, the peptides are linked using a poly-glycine or poly-serine linker. In another embodiment. the second neoantigenic peptide binds MHC class I or class II with a binding y of less than about 1000 nM. In r embodiment, the second igenic e binds MHC class I or class II with a binding affinity of less than about 500 nM. In another embodiment, both of the neoepitopes bind to human leukocyte antigen (HLA) -A, -B, -C, -DP, -DQ, or —DR. In another embodiment, the isolated igenic peptide binds a class I HLA and the second neoantigenic peptide binds a class II HLA. In r embodiment, the isolated neoantigenic e binds a class II HLA and the second neoantigenic peptide binds a class I HLA.

In some embodiments, the ed neoantigenic peptide further comprises modifications which increase in vivo half-life, cellular targeting, n uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation. In another embodiment, the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation HESylation, recombinant PEG mimetics, Fe fusion, albumin fusion, nanoparticle attachment, nanoparticulate encapsulation, cholesterol fusion, iron , acylation. arnidation, glycosylation, side chain oxidation. orylation. biotinylation, the addition of a surface active material, the addition of amino acid rnimeties, or the addition of unnatural amino acids. In another ment, the cells that are targeted are antigen ting cells. In another embodiment, the antigen presenting cells are dendritic cells. In another embodiment, the dendritic cells are targeted using DEC205, XCRl, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD1 le, CD83. TSLP receptor, Clec9a or CDla marker. In another embodiment, the dendritic cells are targeted using the CD141, DEC205, or XCRl marker.

In some embodiments. the invention provides an in vivo ry system comprising an isolated neoantigenic peptide described herein. In another ment, the delivery system includes cell-penetrating peptides, rticulate encapsulation, virus like particles, or liposomes. In another embodiment, the cell- penetrating peptide is TAT peptide, herpes simplex virus VP22, transportan, or Antp.

In some embodiments, the invention is directed to a cell comprising an isolated neoantigenic peptide described herein. In another embodiment, the cell is an antigen presenting cell. In another ment, the cell is a dendritic cell.

In some embodiments, the invention is directed to a composition comprising an isolated neoantigenic peptide bed herein. In another embodiment, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 of the isolated neoantigenic peptides comprising a tumor—specific neoepitope defined in Table l or 2. In another ment, the composition comprises between 2 and 20 neoantigenic peptides. In another embodiment, the composition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 l, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 additional neoantigenic peptides. In another embodiment, the composition comprises between about 4 and about 20 additional neoantigenic peptides. In another embodiment. the additional neoantigenic peptide is specific for an individual t's tumor. In another embodiment, the patient specific neoantigenic peptide is ed by identifying ce differences between the genome, exome, and/or transcriptome of the patient's tumor sample and the genome, exome, and/or transeriptome of a non-tumor sample. In another embodiment, the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or circulating tumor cells. In another embodiment, the sequence differences are determined by Next Generation Sequencing.

In some embodiments. the invention is directed to an isolated polynucleotide encoding an isolated neoantigenic peptide described . In another embodiment, the isolated polynucleotide is RNA, optionally a self-amplifying RNA. In r embodiment, the RNA is modified to increase stability, increase cellular targeting, se translation efficiency, adjuvantieity, cytosol accessibility, and/or decrease xicity. In another embodiment, the modification is conjugation to a carrier n, conjugation to a ligand, conjugation to an antibody, eodon optimization, increased GC-content, incorporation of modified nucleosides, incorporation of 5'-eap or cap analog. and/or incorporation of an ed poly-A sequence.

In some embodiments, the invention is directed to a cell comprising a polynucleotide described In some embodiments, the invention is directed to a vector comprising a polynucleotide described herein. In another ment, the polynucleotide is operably linked to a promoter. In another embodiment, the vector comprises a self-amplifying RNA on, plasmid, phage, transposon, cosmid, virus, or virion. In another embodiment, the vector is an adeno-associated virus, herpesvirus, lentivirus, or pseudotypes thereof.

In some embodiments. the invention is directed to an in vivo delivery system comprising an isolated polynucleotide described herein. In another embodiment, the delivery system includes spherical nucleic acids, viruses, virus-like particles, plasmids, bacterial plasmids, or nanoparticles.

In some embodiments, the invention is directed to a cell sing a vector or delivery system described herein. In r embodiment, the cell is an antigen presenting cell. In another embodiment, the cell is a dendritic cell. In another embodiment, the cell is an immature dendritic cell.

In some embodiments. the invention is directed to a composition comprising at least one polynucleotide described herein. In another ment, the ition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 of the isolated polynucleotides. In another ment, the composition comprises between about 2 and about 20 polynucleotides that encode neoantigenic peptides. In another embodiment, the ition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least I l, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least additional neoantigenic polynucleotides encoding for additional neoantigenic peptides. In some embodiments, the composition comprises between about 4 and about 20 additional neoantigenic polynucleotides. In some ments. the isolated poly-“nucleotides and the onal neoantigenic polynucleotides are linked. In some embodiments, the polynucleotides are linked using nucleic acids that encode a pol_x,r-gl_vcine or poly-serine linker. In some embodiments, at least one of the additional neoantigenic peptide is specific for an dual patient's tumor. In some ments, the patient specific neoantigenic peptide is selected by identifying sequence differences between the genome, exome, and/or transcriptome of the patient's tumor sample and the , exome, and/or transcriptomc of a non-tumor . In some embodiments. the samples are fresh or formalin—fixed n embedded tumor tissues. freshly isolated cells. or circulating tumor cells. In some embodiments, the sequence differences are determined by Next Generation Sequencing.

In some embodiments, the invention is directed to a T cell receptor (TCR) capable of binding at least one neoantigenic peptide described herein. In some embodiments, the TCR is capable of binding the isolated neoantigenic peptide in the t of MHC class I or class II.

In some embodiments. the invention is directed to a chimeric n receptor comprising: (i) a T cell tion molecule; (ii) a transmembrane ; and (iii) an antigen recognition moiety e of binding an isolated neoantigenic peptide described herein. In some embodiments, the chimeric antigen receptor contains ta as the T cell activation molecule. In some embodiments, the chimeric antigen receptor further comprises at least one costimulatory signaling domain. In some embodiments, the signaling domain is CD28, 4-1BB, ICOS, 0X40, ITAM, or PC epsilon RI-gamma. In some embodiments, the antigen recognition moiety is capable of binding the isolated neoantigenic peptide in the t of MHC class I or class II. In some embodiments, the chimeric antigen receptor comprises the CD3-zeta. CD28, CTLA-4. ICOS. BTLA.

KIR, LAG3, CD137, 0X40, CD27, CD40L, Tim-3, A2aR, or PD-I embrane region. In some embodiments. the tumor-specific epitope is located in the extracellular domain of a tumor associated polypeptide.

In some embodiments, the invention is ed to a T cell comprising the T cell receptor or chimeric antigen receptor described herein. In some embodiments, the T cell is a helper or cytotoxic T cell.

In some embodiments. the invention is directed to a nucleic acid comprising a promoter operably linked to a polynucleotide encoding a T cell or described herein. In some embodiments, the TCR is e of binding the at least one neoantigenic peptide in the context of major histocompatibility complex (MHC) class I or class II. In another embodiment, the nucleic acid comprises a promoter operably linked to a polynucleotide encoding a chimeric n receptor described herein. In another embodiment, the antigen recognition moiety is capable of binding the at least one igenic peptide in the context of major histocompatibility complex (MHC) class I or class II. In some ments, the tumor—specific epitope is located in the extracellular domain of a tumor associated polypeptide. In some embodiments, the nucleic acid comprises the CD3-zeta, CD28, CTLA-il, ICOS, BTLA, KIR, LAG3, CD137, 0X40, CD27, CD40L, Tim-3, A2aR, or PD-l transmembrane region.

In some embodiments, the invention is directed to an antibody capable of g at least one neoantigenic peptide described herein.

In some ments, the invention is ed to a modified cell transfected or transduced with a nucleic acid described herein. In some ments, the modified cell is a T cell, tumor infiltrating lymphocyte, NK-T cell, TCR-expressing cell, CD4+ T cell, CDS+ T cell, or NK cell.

In some embodiments, the invention is directed to a composition comprising a T cell receptor or chimeric antigen receptor bed herein. In some embodiments, the composition comprises autologous patient T cells containing a T cell receptor or chimeric n receptor. In some ments. the composition further comprises an immune checkpoint inhibitor. In some embodiments, the composition further comprises at least two immune checkpoint inhibitors. In some embodiments, each of the immune checkpoint inhibitors inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDLI, PDL2, PDI, B7-H3, B7-I-I4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK l, CI-IKZ, AZaR, and B-7 family ligands or a combination thereof. In some embodiments, each of the immune checkpoint inhibitors interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDLI, PDLZ, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 284, CD160, 5049, CI-IK l, CHKZ, A2aR, and B-7 family ligands or a combination f.

In some embodiments, the composition further comprises an immune modulator or adjuvant. In another embodiment, the immune tor is a co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, 0X40, CD27, GITR, CD30, DR3. CD69, or 4-lBB. In r embodiment, the immune modulator is at least one cancer cell or cancer cell extract. In another embodiment, the cancer cell is gous to the subject in need of the composition. In another embodiment. the cancer cell has undergone lysis or been exposed to UV radiation. In another embodiment, the adjuvant induces a humoral when administered to a subject. In another embodiment, the adjuvant induces a T helper cell type I when administered to a subject.

In some embodiments, the ion is directed to a method of inhibiting growth of a tumor cell expressing a tumor-specific neoepitope described herein, comprising contacting the tumor cell with the peptide, cleotide. ry system. vector. composition. antibody. or cells of the invention.

In some embodiments, the invention is directed to a method of treating cancer or initiating, enhancing, or prolonging an umor se in a subject in need thereof comprising administering to the subject the peptide, polynucleotide, vector, composition, antibody, or cells bed herein.

In some embodiments, the cancer is selected from adrenal, bladder, breast, cervical, colorectal, glioblasoma, head and neck, kidney chromophobe, kidney clear cell, kidney papillary, liver, lung adeno, lung squamous. ovarian. pancreatic. melanoma, stomach, e corpus endometrial,and uterine carcinosarcoma.

In some ments, the cancer is selected from the group of: prostate cancer, bladder, lung squamous, NSCLC, breast, head and neck, lung adenocarcinoma, GBM, Glioma, CML, AML, supretentorial ependyomas, acute promyelocytic leukemia, solitary fibrous tumors, and crizotinib resistant cancer.

In some embodiments, the cancer is selected from the group consisting of CRC, head and neck, stomach. lung squamous. lung _ Prostate. Bladder. stomach. renal cell carcinoma. and uterine.

In some embodiments, the cancer is selected from the group consisting of melanoma, lung squamous, DLBCL, e, head and neck, uterine, liver, and CRC.

In some embodiments, the cancer is selected from the group consisting of lymphoid cancer; Burkitt lymphoma, lastoma, prostate arcinoma, colorectal arcinoma; Uterine/Endometrium Adenocarcinoma; MSI+; endometrium serous oma; endometrium carcinosarcoma-malignant mesodennal mixed ; glioma; ytoma; GBM. acute myeloid leukaemia associated with MDS; chronic lymphocytic leukaemia-small cytic lymphoma; myelodysplastic syndrome; acute myeloid leukaemia; luminal NS carcinoma of breast; chronic d leukaemia; ductal carcinoma of as; chronic onocytic leukaemia; myelofibrosis; myelodysplastic syndrome; prostate adenocarcinoma; essential thrombocythaemia; and medullomyoblastoma..

In some embodiments, the cancer is selected from the group consisting of colorectal, uterine, endometrial, and stomach.

In some embodiments, the cancer is selected from the group consisting of cervical, head and neck, anal, stomach, Burkitt‘s lymphoma, and nasopharyngeal carcinoma.

In some embodiments, the cancer is selected from the group consisting of bladder, colorectal, and In some embodiments, the cancer is selected from the group consisting of lung, CRC. melanoma, breast, NSCLC, and CLL.

In some embodiments. the cancer is selected from the group consisting of adrenocortical carcinoma (ACC), acute promyelocytic leukemia, acute myeloid leukemia (AML), AML associated with myelodysplastic mes (MDS), anal , astrocytoma, bladder cancer, bladder urothelial oma (BLCA), breast cancer, breast invasive carcinoma (BRCA), Burkitt's Lymphoma, castration-resistant prostate cancer, cervical cancer, cervical squamous cell oma and endocervical adenocarcinoma (CESC), chronic lymphocytic leukaemia-small lymphocytic ma, chronic lymphocytic leukaemia (CLL), chronic myeloid leukaemia (CM L). chronic myelomonocytic leukaemia. colorectal adenocarcinoma, colorectal cancer (CRC), Crizotinib ant non-small cell lung cancer (NSCLC), diffuse large B-cell lymphoma (DLBCL), ductal carcinoma of pancreas, endometrium carcinosarcoma-malignant mesodermal mixed tumour, endometrium serous carcinoma, essential thrombocythaemia, glioblastoma multiforrne (GBM), Glioma, head and neck , head and neck squamous cell carcinoma (I-INSC), invasive lobular carcinoma (ILC) LumA breast cancer, kidney ehromophobc (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal ary cell carcinoma (KIRP), acute myeloid leukemia (LAML). liver hepatocellular carcinoma (LIHC), liver cancer, lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), Luminal NS carcinoma of breast, lung cancer, lymphoid cancer, medullomyoblastoma, melanoma, microsatellite instability (MSI)+ colorectal cancer (CRC), MSI+ endometrioid carcinoma, MSI+ stomach cancer, MSI+ uterine/endometrium cancer, myelodysplastic syndrome, myelofibrosis, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer (NSCLC), ovarian serous cystadenocarcinoma (0V), pancreatic adenocarcinoma , prostate adenocarcinoma (PRAD). prostate cancer. renal cell carcinoma. skin cutaneous melanoma (SKCM). solitary fibrous , stomach adenocareinoma (STAD), h cancer, supretentorial ependyomas, thyroid adenocareinoma (THCA), uterine corpus endometrioid oma (UCEC), or uterine carcinosarcoma (UCS), uterine cancer, and uterine/endometrium adenocarcinoma.

In some embodiments, the cancer is selected from the group consisting of bladder urothelial oma (BLCA), breast invasive carcinoma (BRCA), breast cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). chronic lymphocytic leukaemia (CLL). colorectal cancer (CRC). glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney renal papillary cell carcinoma (KIRP), liver cellular carcinoma (LII-1C), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), Prostate Cancer, skin cutaneous melanoma (SKCM), stomach arcinoma (STAD), thyroid adenoearcinoma , and uterine corpus cndomctrioid carcinoma (UCEC).

In some embodiments. the subject is a human. In another embodiment, the t has cancer. In another embodiment. the cancer is selected from the group consisting of ital, gynecological, lung, gastrointestinal, head and neck cancer, malignant glioblastoma, malignant mesothelioma, non-metastatic or metastatic breast cancer, malignant ma, Merkel Cell oma or bone and soft tissue sarcomas, haematologic neoplasias, multiple myeloma, acute enous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome and acute lymphoblastic ia, non-small cell lung cancer (NSCLC), breast cancer, metastatic ctal cancers, homtone sensitive or hormone refractory prostate cancer, colorectal cancer. ovarian cancer, cellular cancer. renal cell cancer, pancreatic cancer. gastric cancer. ageal cancers, hepatocellular cancers, cholangiocellular s, head and neck squamous cell cancer soft tissue sarcoma, and small cell lung cancer. In another embodiment, the subject has undergone surgical removal of the tumor. In another embodiment, the peptide, polynucleotide, vector, composition, or cells is administered via intravenous, intraperitoneal, intratumoral, intradermal, or subcutaneous administration. In another embodiment, the peptide, polynucleotide, vector, composition, or cells is stered into an anatomic site that drains into a lymph node basin. In another embodiment. the administration is into multiple lymph node basins. In another ment, the administration is by a subcutaneous or intradermal route.

In some embodiments of the method, a peptide is stered. In another embodiment, the administration is intratumorally. In another embodiment of the method, a polynucleotide, optionally RNA, is administered. In another embodiment, the polynucleotide is administered intravenously. In some embodiments of the method, a cell is administered. In another embodiment, the cell is a T cell or dendritic cell. In another embodiment. the e or polynucleotide comprises an n presenting cell targeting moiety.

In another embodiment ofthe method, at least one immune checkpoint inhibitor is also administered to the subject. In another embodiment, the checkpoint inhibitor is a ic therapeutic or a small molecule.

In another embodiment, the checkpoint inhibitor is selected from the group consisting of a monoclonal dy, a humanized antibody, a fully human antibody and a fusion protein or a combination thereof. In another embodiment, the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4. PDLI. PDLZ. PDl. B7-H3. B7-H4. BTLA. HVEM. TIM3. GAL9. LAG3. VISTA. KIR. ZB4.

CD160, CGEN-15049, CHK l, CHKZ, AZaR, and B-7 family s or a combination thereof. In another embodiment, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDLl, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, T1M3, GAL9, LAG3, VISTA, KIR, 284, CD160, CGEN- 15049, CI-IK l, CHKZ, A2aR, and B-7 family ligands or a combination thereof. In r embodiment, two or more checkpoint inhibitors are administered. In another embodiment, the checkpoint inhibitors are: (i) ipilimumab or tremelimumab. and (ii) nivolumab. In another embodiment. the checkpoint inhibitor and the composition are administered simultaneously or sequentially in any order. In another embodiment, a neoantigenic peptide, polynucleotide, vector, composition, or cells is administered prior to the oint inhibitor. In another embodiment, a peptide, polynucleotide, , composition, or cells is administered after the checkpoint inhibitor. In another embodiment, the checkpoint inhibitor is continued throughout neoantigen peptide, polynucleotidc, vector, composition, or cell therapy. In another embodiment, the neoantigen peptide, polynucleotide. vector. composition. or cell therapy is administered to subjects that only partially respond or do not respond to checkpoint inhibitor y. In another embodiment, the composition is administered intravenously or subcutaneously. In r embodiment, the oint inhibitor is administered intravenously or subcutaneously. In another embodiment, the checkpoint inhibitor is administered subcutaneously within about 2 cm ofthe site of administration of the composition. In another embodiment, the composition is administered into the same draining lymph node as the checkpoint inhibitor.

In some embodiments of the method, an additional agent is administered. In another embodiment, the agent is a herapeutic agent, an modulatory drug. an immune metabolism modifying drug, a targeted therapy, radiation an anti-angiogenesis agent, or an agent that reduces -suppression. In another embodiment, the herapeutic agent is an alkylating agent, a topoisomerase inhibitor, an anti- metabolite, or an anti-mitotic agent. In another embodiment, the additional agent is an anti-glucocorticoid induced tumor is factor family receptor (GITR) agonistic antibody or antibody fragment, ibrutinib, docetaxeol, cisplatin, or cyclophosphamide. In another ment, the administration elicits a CD4+ T cell immune response. In another embodiment. the administration elicits a CD4+ T cell immune response and a CD8+ T cell immune response.

In some embodiments, the invention is directed to a method for stimulating an immune response in a t, comprising administering an effective amount of modified cells or composition described herein. In another embodiment, the immune response is cytotoxic and/or humoral immune response. In another embodiment, the method stimulates a T cell-mediated immune se in a subject. In r embodiment, the T cell-mediated immune se is directed against a target cell. In another ment, the target cell is a tumor cell. In another embodiment, the modified cells are transfected or transduced in vivo. In r embodiment, the modified cells are transfected or transduced ex vivo. In another embodiment, the modified cells are autologous patient T cells. In r embodiment, the gous patient T cells are obtained from a patient that has received a neoantigen peptide or nucleic acid vaccine. In another embodiment, the neoantigen peptide or nucleic acid vaccine comprises at least one personalized neoantigen. In another embodiment, the neoantigen e or nucleic acid e comprises at least one additional neoantigenic peptide described herein. In another embodiment, the patient received a chemotherapeutic agent, an immunomodulatory drug, an immune metabolism modifying drug, targeted y or ion prior to and/or during receipt of the neoantigen e or nucleic acid vaccine. In another ment, the patient receives treatment with at least one checkpoint inhibitor. In another embodiment, the autologous T cells are obtained from a patient that has already received at least one round of T cell therapy containing a neoantigen. In another embodiment, the method further comprises adoptive T cell therapy. In another embodiment. the adoptive T cell therapy comprises autologous T cells. In another embodiment, the autologous T cells are targeted against tumor antigens. In another embodiment, the adoptive T cell therapy further comprises allogenic T cells. In another ment, the allogenic T cells are targeted against tumor antigens. In another embodiment, the ve T cell therapy is administered before the checkpoint inhibitor.

In some embodiments, the invention is directed to a method for evaluating the efficacy of treatment comprising; (i) measuring the number or concentration of target cells in a first sample obtained from the subject before administering the modified cell, (ii) ing the number concentration of target cells in a second sample ed from the subject after administration of the modified cell, and (iii) determining an increase or decrease of the number or concentration of target cells in the second sample compared to the number or tration oftarget cells in the first sample. In r embodiment, the treatment efficacy is detemiined by monitoring a clinical outcome; an increase, enhancement or prolongation of anti-tumor activity by T cells; an increase in the number of anti-tumor T cells or activated T cells as compared with the number prior to treatment; B cell activity; CD4 T cell activity; or a combination thereof. In another embodiment. the treatment efficacy is determined by monitoring a biomarker. In r embodiment, the biomarker is selected from the group consisting of CEA, Her-Z/neu, bladder tumor antigen, thyroglobulin, alpha-fetoprotein, PSA, CA 125, CA19.9, CA 15.3, leptin, prolactin, osteopontin, IGF-II, CD98, fascin, sPIgR, 143 eta, troponin I, and b-type natriuretic peptide. In another embodiment, the clinical outcome is selected from the group consisting of tumor regression; tumor shrinkage; tumor is; anti-tumor response by the immune ; tumor expansion. recurrence or spread; or a combination thereof. In another embodiment. the treatment effect is predicted by presence of T cells or by presence of a gene signature indicating T cell inflammation or a combination thereof.

In some embodiments, the ion is directed to a method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need f comprising stering to the subject: (a) the peptide, polynuclcotide, vector, composition, antibody, or cells described herein: and (b) at least one checkpoint inhibitor. In another embodiment. the method further comprises administration of an immunomodulator or adjuvant. In another embodiment. the immunomodulator or adjuvant is selected from the group consisting of Poly-'(IzC), Poly-ICLC, STING agonist, 1018 ISS, aluminium salts, ax, A815, BCG, ,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, 1C31, mod, ImuFact 1MP321, IS Patch, ISS, ISCOMATRIX, Juvamune, c, MF59, monophosphoryl lipid A, Montanide IMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, OK-432, OM-l74, OM-l97-MP-EC, ISA-TLR2 agonist. ONTAK. PepTel‘CEC vector system. PLG microparticles. resiquimod. SRLI72. mes and other like particles, YF-17D, VEGF trap, R848, lucan, Pam3Cys, K4, acrylic or methacrylic polymers, mers of maleic anhydride, and QSZI on. a co-stimulatory ligand, a TNF ligand, an lg amily ligand, CD28, CD80, CD86, ICOS, CD40L, 0X40, CD27, GITK CD30, DR3, CD69, or 4-IBB. In another embodiment, the immunomodulator or adjuvant is Poly-ICLC. In another embodiment, the checkpoint inhibitor is an anti-PD] antibody or antibody fragment. In another ment, the inhibitor of the PD-l pathway is nivolumab. In another embodiment. the checkpoint inhibitor is an anti- CTLA4 antibody or antibody fragment. In another ment, the anti-CTLA4 antibody is umab or tremelimumab. In another embodiment, the method comprises administering both an anti-PD] antibody and an anti-CTLA4 antibody. In another embodiment, the administration of the checkpoint inhibitor is initiated before initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In another embodiment, the administration of the checkpoint inhibitor is initiated aficr initiation of administration of the peptide. polynucleotide, vector, composition. antibody, or cell. In another embodiment, the administration of the checkpoint inhibitor is ted simultaneously with the initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In another embodiment, the peptide, polynucleotide, vector, composition, antibody, or cell is administered intravenously or subcutaneously. In another embodiment, the checkpoint tor is stered intray-renously or subcutaneously. In another embodiment, the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the peptide, polynucleotide, vector, ition, antibody, or cell. In another embodiment, the peptide. polynucleotide, vector. composition. antibody. or cell is administered into the same draining lymph node as the checkpoint inhibitor.

In some embodiments of the therapeutic methods, the additional therapeutic agent is for e, a chemotherapeutic or rapeutic agent, radiation, or immunotherapy. Any suitable therapeutic treatment for a ular cancer may be administered. Examples of chemotherapeutic and biotherapeutic agents include, but are not d to, an angiogenesis inhibitor, such ashydroxy angiostatin K 1-3, DL-a—Difluoromethy!- oroithine. endostatiii. fumagillin. genistein. minocycline. staurosporine. and thalidomide; a DNA intercaitor/cross-linker, such as cin, Carboplatin, Camnistme, Chlorambucil, Cyclophosphamide, cis- Diammineplat num(D) ride (Cispiatin), Melphalan, Mitoxantrone, and Oxaliplatin; a DNA synthesis tor, such as (i)-Amethopterin (Methotrexate), 3-Amino-1,2,4-beiizotnazine 1,4-dioxide, Aminopterin, Cytosine B-D-arabinofiiraiioside, 5-Fmoro-5'~ ridine, 5—Fhsorouracil, Ganciclovir, Hydroxyurea, and Mitomycin C: a DNA-RNA transcription regulator, such as mycin D, Dau orubicin, Doxorubicin, I-Iomoharringtonine. and Idarubiein; an 0T]/.‘{I]K; inhibitor, such as S(-i-)—Camptothecm. Curcumin, (-)- Deguelm, 5,6-Dichiorobenzimidazole 1 -|3-D-ribofiiranoside, Etoposide, Fomiestane, Fostriecin, Hispidin, 2- Immo-l-imidazoli-dineacetic acid (Cyclocreatine), Mevmolin, Trichostatin A, Tyrphostin AG 34, and Tyrphostin AG 879; a gene regulator, such as 5-Aza-2'-deoxyeytidine, S-Azacytidine, aleiferol (Vitamin D3), 4-Hydroxytamoxifen, Melatonin, Mifepristone, Raloxifene, all trans-Retinal (Vitamin A aldehyde), Retinoic acid all trans (Vitamin A acid), 9-cis-Retinoic Acid, l3-cis-Retinoic acid, Retinol (Vitamin A), Tamoxifen. and Troglitazone; a microtubule inhibitor. such as Colchicine. docetaxel. Dolastatirs , Nocodazole, Paclitaxel, yl!otoxin, Rhizoxin, Vinblastine, Vi cristine, Vindesiiie, and Vinorelbine bine); and an unclassified therapeutic agent, such as l7-(All.\,l'iamino)-l 7-demethoxygeldanamycin, 4- Amino-l,8-naphthalimide, in, din A, Cimetidine, Dichioromethylene-diphosphonic acid, lide (Leuprorelin), Luteinizing Hormone-Releasing Hormone, Pifithrin-a, Rapamycin, Sex hormone- binding globulin, Thapsigargin, and Urinary trypsin inhibitor nt (Bikunin). The therapeutic agent may be altretamine. amifostine. asparaginase. capecitabine. cladribine. cisapride. cyiarahirse. dacarbazine (DTlC). dactinomycin, dronabinol, epoetin alpha, “filgrastim, fludarabine, gemcitabine, granisetron, ifosfamide, in'notecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, metoelopramide, mitotane, omeprazole, ondansetron, pilocarpine, prochloroperazine, or topotecan hydrochloride. The therapeutic agent maybe a monoclonal antibody such as rituximab (Rituxan®), alemtuzumab (Campath®), zumab (Avastin®), Cctuximab (Erbitux®), panitumumab (Vectibix®), and zumab (Herceptin®), churafcnib (Zelboraf®) imatinib te (GleeveoE‘). erlotinib (Tarceva‘I'E), gefitinib a®). Vismodegib (ErivedgeTM). 90Y- ibn'tumomab tiuxetan, 1311-tosit.umomab, ado-trastuzumab emtansine, lapatinib (TykerbIE'), pertuzumab (PerjetaTW, ado-trastuzumab emtansine ( adcylaTM), regorafenib (Stivargatfii), sunitinib (Sutentt’Rl), Denosumab ilil), nib (Nexavarl’lii), pazopanib (VotrientiRTI), axitinib (InitaL’RT'I), dasatinib (SpQ‘CellTU), nilotinib (Tasigna‘lkil), bosutinib (Bosulifiii'l), ofatumumab (Arzerra'li‘l), uzumab (Gazy\-’aTM), ibrutinib (ImbruvicaTM), idelalisib (ZydeligiRD), crizotinib (Xalkori®), erlotinib (Tarcevai'lii‘i), afatimb dimaleate rifBD), ceritinib (LDK378/Zykadia), Tositumomab and 131 tumomab (Bexxariii), ibritumomab tiuxetan (Zevalin'ilii'). brentuximab n (Adcetlisflli'), bonezomib (Velcade'i'BZ'). siltuximab (SylvantTM). trametinib ( t'CRZ), dabrafenib (TafinlanE), pembrolizimiab (Kefirudat’jlii‘r), carfilzomib (Kyprolisiftil), rumab (CyramzaTM), Cabozantinib (CometriqTM), vandetanib (CaprelsaIRV), Optionally, the therapeutic agent is a neoantigen. The therapeutic agent may be a cytokine such as interferons (1N Fs), interleukins (lLs), or hematopoietic gromh s. The therapeutic agent may be INF-a, IL—2, Aldesleukin, IL-2, Erythropoietin, Granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocy-“te colony- stimulating factor. The therapeutic agent may be a targeted therapy such as toremifene (Fareston‘CRD). fulvestrant (Faslodexfl'fi), anastrozole dexflii), exemestane (Aromasin’fil'll), letrozole (Femaralfii), ziv- aflibercept (Zaltmp'E), Aiitretinoin (Panretinfii'i), temsirolimus (Tonseli'li‘l), Tretinoin (Vesanoidt’E), ukin diftitox (Ontak'i'BJ), vorinostat (ZoiinzafRJ), romidepsin (lstodax'fi‘l), tene (Targretin-TE‘), pralatrexate _\jn®), !enaliomide (Revlimidt’gi), belinostat (BeleodaqTM), lenaliomide (Revlimid-‘ED, pomalidomide (Pomalyst®), Cabazitaxcl (Jevtana®), enzaluiamidc (Xtandit‘g), abiraterone e (Zfiiga®), radium 223 chloride (Xofigo®), or everolimus (Afmiior'E). nally. the therapeutic agent may be an epigenetic targeted drug such as HDAC inhibitors, kinase inhibitors, DNA methyltransferase inhibitors, histone demethylase inhibitors, or histone ation inhibitors. The epigenetic drugs may be Azacitidine (Vidaza), Decitabine (Dacogen), Vorinostat (Zoiinza), Romidepsin (Istodax), or Ruxolitinib i). For prostate cancer treatment, a preferred chemotherapeutic agent with which anti- CTLA-4 can be combined is paclitaxel (TAXOL).

In some embodiments, the invention is directed to a kit comprising any neoantigen therapeutic described herein.

Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present ion encompasses not only the entire group listed as a whole, but also each member of the group dually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any ofthe group members in the claimed invention.

INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. For example, all publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the kits, compositions, and methodologies that are described in the publications, which might be used in connection with the methods, kits, and compositions described herein. The documents discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be ued as an admission that the inventors bed herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.

BRIEF IPTION OF THE DRAWINGS Figure 1 depicts and exemplary graph of 562 peptides with predicted ty for select HLA Class I molecule between 1 nM and 100,000 nM (plotted on x-axis) that were synthesized. Actual affinity (ICSO (nM)) was measured as described (y-axis). Thick vertical and ntal lines denote 500 nM cutoff between weak and very weak predicted and observed binders, respectively. al dotted line s line-of-best fit (Graphpad Prism) with R2 of 0.45.

Figure 2 depicts and exemplary graph of 275 peptides from Figure 1 that were tested for ity (Tm; hrs) on their tive HLA Class I molecules. Peptides were binned according to observed ty from Figure 1. Median and interquattile range is shown for each bin.

Figure 3A depicts and exemplary graph of HLA-A0210 1+ T cells co-cultured with monocyte-derived dendritie cells loaded with TMPRSS2::ERG fusion neoepitope ALSV; HLA-A02:01) for 10 days.

CD8+ T cells were analyzed for antigen-specificity for 2::ERG fusion neoepitope using multimers al: BV421 and PE).

Figure 3B depicts and exemplary graph of HLA-A0220 1+ T cells tured with monocyte-derived dendritic cells loaded with TMPRSS2::ERG fusion neoepitope (ALNSEALSV; HLA-A02201) for 10 days.

CD8+ T cells were analyzed for antigen-specificity for TMPRSSZ::ERG fusion neoepitope using multimers (validation: APC and BUV396).

La) La) Figure 4A depicts and ary graph of HLA-A02201+ T cells tured with monocyte-derived tic cells loaded with GATA3 frameshift neoepitope (SMLTGPPARV; HLA-A02:01) for 10 days. CD8+ T cells were analyzed for antigen-specificity for GATA3 frameshifi neoepitope using ers (initial: APC and BUV396).

Figure 4B depicts and ary graph of HLA-A02:0 1+ T cells co-cultured with monocyte-derived dendritic cells loaded with GATA3 frameshift neoepitope (SMLTGPPARV; HLA-A02:01) for 10 days. CD8+ T cells were ed for antigen-specificity for GATA3 framcshift neoepitope using multimcrs (validation: PE and BV421).

Figure 5A depicts and exemplary graph of HLA-A0220 l+ T cells co-cultured with monocyte-derived dendritic cells loaded with BZM frameshifi neoepitope (LLCVWVSSI: HLA-A02:01) for 10 days. CD8+ T cells were analyzed for antigen-specificity for [52M frameshift neoepitope using multimers (initial: PE and APC).

Figure SB depicts and ary graph of HLA-A0220 l+ T cells co—culturcd with monocytc-dcrivcd tic cells loaded with [32M frameshift neoepitope (LLCVWVSSI; 2:01) for 10 days. CD8+ T cells were analyzed for n-specificity for [52M frameshift neoepitope using multimcrs (validation: PE and BV42 l).

Figure 6A depicts and exemplary graph of HLA-A02:O l+ T cells co-cultured with monocyte-derived dendritic cells loaded with KRAS G12C neoepitope (KLVVVGACGV; HLA-A02201) for 10 days. CD8' T cells were analyzed for n-specificity for KRAS G 12C frameshifi neoepitope using ers al: BUV396 and BV421), Figure 6B depicts and exemplary graph HLA-A02101+ T cells co-cultured with monocyte-derived dendritic cells loaded with KRAS G12C neoepitope (KLVVVGACGV; HLA-A02:01) for 10 days. CD8’ T cells were analyzed for antigen-specificity for KRAS G 12C frameshifi neoepitope using multimcrs (validation: APC and BUV396).

Figure 7 depicts and exemplary graph ofT cells co-cultured with monocyte-derived dendritic cells loaded [32M frameshift neopeptides for 20 days (restimulation with fresh monocyte-derived dendritic cells on day 20). CD4' T cells were analyzed for antigen-specificity by intracellular cytokine staining afier restimulation with monocyte—derived dendritic cells loaded with BZM frameshifi peptide for 24 hours (right), compared to controls without peptide (lefi).

Figure 8 depicts and exemplary graph ofT cells co-cultured with monocyte-derived dendritic cells loaded BTK C4818 neopeptide for 20 days (restimulation with fresh monoeyte—derived dendritic cells on day ). CD4- T cells were analyzed for antigen-specificity by ellular cydokine staining after restimulation with te-derived dendritic cells loaded with BTK C4818 neopeptide for 24 hours (right), compared to controls wild-type BTK peptide (left).

Figure 9 depicts and exemplary graph ofT cells co-cultured with monocyte-den'ved dendritic cells loaded GATA3 frameshift neopeptides for 20 days (restimulation with fresh monocyte-derived dendritic cells on day 20). CD4+ T cells were analyzed for antigen-specificity by intracellular cytokine staining after restimulation with monocyte-derived dendritic cells loaded with GATA3 frameshift peptide for 24 hours (right). compared to controls without peptide (left).

DETAILED DESCRIPTION OF THE INVENTION Described herein are novel immunotherapeutic agents and uses thereof based on the discovery of neoantigens arising from mutational events unique to an individual's tumor. Accordingly, the invention described herein es peptides, polynucleotides encoding the peptides, and peptide g agents, that can be used, for example, to stimulate an immune response to a tumor associated antigen, to create an genic composition or cancer vaccine for use in treating disease.

I. Definitions The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the ar forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates ise. Furthermore, to the extent that the terms “including", “includes”, “having”, “has”, “with", or variants thereof are used in either the detailed description and/or the claims. such terms are intended to be inclusive in a manner similar to the term “comprising”.

The tenn “about" or “approximately“ can mean within an able error range for the particular value as ined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to %, up to 10%, up to 5%, or up to 1% of a given value. Altematively, particularly with respect to biological systems or processes, the temt can mean within an order of magnitude, within 5-fold, and more preferably within . of a value. Where particular values are described in the ation and claims, unless otherwise stated the term “about“ meaning within an acceptable error range for the particular value should be assumed.

To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

“Neoantigen” means a class of tumor antigens which arise from tumor-specific changes in proteins.

Neoantigens encompass, but are not limited to, tumor antigens which arise from, for example, substitution in the protein sequence. frame shifi mutation. fusion polypeptide. iii-frame deletion. ion. sion of endogenous retroviral polypeptides, and tumor-specific overexpression of polypepitdes.

“Tumor c neoepitope” refers to an epitope that is not present in a reference such as a normal non-cancerous or gennline cell but is found in cancer cells. This es, in particular, situations wherein in a normal non—cancerous or germline cell a ponding epitope is found, however, due to one or more mutations in a cancer cell the sequence of the e is changed so as to result in the neo-epitopc.

A “reference” can be used to correlate and compare the results obtained in the methods of the ion from a tumor specimen. Typically the “reference" may be obtained on the basis of one or more nomial specimens, in particular specimens which are not ed by a cancer disease, either ed from a patient or one or more different individuals, for example, healthy individuals, in particular individuals of the La) ’JI same s. A “reference” can be detemiined empirically by testing a sufficiently large number of normal specimens.

The term "mutation” refers to a change of or difference in the nucleic acid sequence otide substitution, addition or deletion) compared to a reference. A “somatic mutation” can occur in any of the cells of the body except the germ cells (sperm and egg) and therefore are not passed on to children. These alterations can (but do not always) cause cancer or other diseases. In some embodiments, a mutation is a non- synonymous mutation. The term “non-synonymous mutation” refers to a mutation, for example, a nucleotide substitution. which does result in an amino acid change such as an amino acid substitution in the translation product. A “frameshift” occurs when a on disrupts the normal phase of a gene's codon periodicity (also known as "reading frame”), resulting in the translation of a tive protein sequence. It is possible for different ons in a gene to achieve the same altered reading frame.

As used herein, the term “affinity” refers to a measure of the strength of binding between two members of a binding pair, for example, an HLA-binding peptide and a class I or II HLA. KD is the dissociation constant and has units of molarity. The affinity constant is the inverse of the iation constant. An y constant is sometimes used as a generic temi to describe this chemical entity. It is a direct measure of the energy of binding. Affinity may be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available e SPR units. Affinity may also be expressed as the inhibitory concentration 50 (ICSO), that concentration at which 50% of the peptide is displaced. Likewise, ln(IC50) refers to the natural log of the 1C5”. K0” refers to the off-rate constant, for example, for dissociation of an HLA-binding peptide and a class I or II HLA. hout this disclosure. ng data” results can be sed in terms of “ICsn.” ICsn is the concentration ofthe tested peptide in a binding assay at which 50% inhibition of binding of a labeled reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA n and labeled reference peptide concentrations), these values approximate KD values. Assays for determining binding are well known in the art and are described in , for e, in PCT publications WO 94/20127 and WO 94/03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney. et al.. J. Immunol. 1542247 (1995); and Sette. et al.. Mol. l. 312813 (1994). Altematively. binding can be expressed relative to binding by a reference standard peptide. For example, can be based on its ICSO, relative to the [€50 of a reference standard peptide.

Binding can also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 3392392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill ct al., J. Immunol. 1472189 (1991); do] Gucrcio et al., J. Immunol. 5 (1995)), cell free systems using detergent lysates (e.g., Cerundolo eta1., J. Immunol. 2122069 (1991)). immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 ; Marshall et al., J. Immunol. 15224946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 1 1:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J.

Biol. Chem. 268: 15425 (1993)); high flux e phase assays (Hammer eta1., J. Exp. Med. 18022353 (1994)), and ement of class I MHC ization or assembly (e.g., Ljunggren eta1., Nature 3462476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149: 1896 (1992)). —reactive binding” indicates that a peptide is bound by more than one HLA le; a synonym is degenerate binding.

The term “derived” when used to discuss an epitope is a synonym for red.” A d epitope can be isolated from a natural source, or it can be synthesized according to standard protocols in the art.

Synthetic epitopes can comprise artificial amino acid residues “amino acid mimetics,” such as D isomers of natural occurring L amino acid residues or non-natural amino acid residues such as cyclohexylalanine. A derived or prepared e can be an analog of a native e.

A “diluent” includes sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, l oil, sesame oil and the like. Water is also a diluent for pharmaceutical compositions. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as diluents, for example, in injectable solutions.

An “epitope” is the collective features of a molecule. such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by, for example, an immunoglobulin, T cell receptor, HLA molecule, or chimeric antigen receptor. Altematively, an e can be defined as a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins, chimeric antigen receptors, and/or Major Histocompatibility Complex (MHC) receptors. Epitopes can be prepared by isolation from a natural source, or they can be synthesized according to standard protocols in the art. Synthetic es can comprise artificial amino acid residues. “amino acid mimetics.” such as D isomers of naturally-occurring L amino acid residues or non-naturally-occurring amino acid residues such as cyclohexylalanine. hout this disclosure, es may be referred to in some cases as peptides or peptide epitopes.

It is to be appreciated that proteins or peptides that comprise an epitope or an analog described herein as well as additional amino acid(s) are still within the bounds of the invention. In certain embodiments, the peptide comprises a fragment of an antigen.

In certain embodiments. there is a limitation on the length of a peptide of the invention. The ment that is length-limited occurs when the n or peptide comprising an e described herein ses a region (i.e., a contiguous series of amino acid residues) having 100% identity with a native sequence. In order to avoid the definition of epitope from reading, e.g., on whole natural molecules, there is a limitation on the length of any region that has 100% identity with a native e ce. Thus, for a peptide comprising an epitope described herein and a region with 100% ty with a native e sequence. the region with 100% identity to a native sequence generally has a length of: less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues. In certain ments, an "epitope” described herein is comprised by a peptide having a region with less than 51 amino acid es that has 100% identity to a native peptide sequence. in any increment down to 5 amino acid residues; for example 50. 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28,27, 26, 25, 24, 23, 22, 21, , 19,18, 17, 16,15, l4, 13, 12, ll, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues.

“Human Leukocyte Antigen” or “HLA” is a human class 1 or class 11 Major Histocompatibility Complex (MI-1C) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8TH ED, Lange hing, Los Altos, Calif. (1994).

An "HLA supertype or HLA family". as used herein. describes sets ofHLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding y for peptides bearing certain amino acid motifs are grouped into such HLA supertypes. The terms HLA amily, HLA supertype family, HLA family, and HLA xx-like molecules (where “xx denotes a particular HLA type), are synonyms.

The terms “identical” or t "identity,” in the context of two or more peptide sequences or antigen fragments, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid es that are the same, when compared and aligned for maximum correspondence over a ison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

An ogenic” peptide or an "immunogenic” epitope or de epitope” is a peptide that comprises an allele-specific motif such that the peptide will bind an HLA molecule and induce a cell-mediated or humoral response, for example, cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL) and/or B lymphocyte response. Thus. immunogenic peptides described herein are e of binding to an appropriate HLA molecule and thereafier inducing a CTL (cytotoxic) se, or a HTL (and humoral) response, to the peptide.

As used herein, a ric antigen or” or “CAR” refers to an n binding protein in that includes an immunoglobulin antigen binding domain (e.g., an immunoglobulin variable domain) and a T cell receptor (TCR) constant domain. As used herein, a “constant domain” of a TCR polypeptide es a membrane-proximal TCR constant domain. and may also include a TCR transmembrane domain and/or a TCR cytoplasmic tail. For example, in some embodiments, the CAR is a dimer that includes a first polypeptide comprising a immunoglobulin heavy chain variable domain linked to a TCR.beta. constant domain and a second polypeptide comprising an immunoglobulin light chain variable domain (e.g., a K or 9. variable domain) linked to a TCRa constant domain. In some embodiments, the CAR is a dimer that includes a first polypeptide comprising a immunoglobulin heavy chain variable domain linked to a TCRa constant domain and a second polypeptide comprising an immunoglobulin light chain le domain (e.g., a K or k variable domain) linked to a TCRB constant domain.

The phrases “isolated” or “biologically pure" refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides described herein do not contain some or all of the materials normally ated with the peptides in their in situ environment. An “isolated” epitope refers to an e that does not include the whole sequence of the antigen from which the epitope was derived. Typically the “isolated” epitope does not have attached thereto additional amino acid residues that result in a sequence that has [00% identity over the entire length of a native sequence. The native sequence can be a sequence such as a tumor-associated antigen from which the epitope is derived. Thus, the term “isolated” means that the material is removed from its original environment (e.g., the natural nment if it is naturally occurring). For example, a naturally-occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide, separated from some or all of the coexisting materials in the natural . is isolated. Such a polynucleotide could be part of a vector, and/or such a polynucleotide or peptide could be part of a composition, and still be “isolated” in that such vector or composition is not part of its l environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, and further include such molecules produced synthetically.

“Major Histocompatibility Complex” or “MHC” is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. In . the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed ption of the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3.sup.RD ED., Raven Press, New York (1993).

A “native” or a “wild type” ce refers to a sequence found in nature. Such a sequence can comprise a longer sequence in nature.

A “T cell epitope” is to be understood as meaning a e sequence which can be bound by the MHC molecules of class I or II in the fonn of a e-presenting MHC molecule or MHC complex and then, in this form. be recognized and bound by cytotoxic T-lymphocytes or T-helper cells. respectively.

A “receptor” is to be understood as meaning a ical molecule or a molecule grouping capable of binding a ligand. A receptor may serve, to transmit information in a cell, a cell formation or an organism. The receptor ses at least one receptor unit, for example, where each receptor unit may consist of a protein molecule. The or has a ure which complements that of a ligand and may complex the ligand as a binding partner. The information is itted in particular by conformational changes of the receptor following complexation of the ligand on the surface of a cell. In some embodiments. a receptor is to be understood as meaning in particular proteins of MHC classes I and II capable of g a receptor/ligand complex with a ligand, in ular a peptide or peptide nt of suitable length.

A “ligand” is to be understood as meaning a molecule which has a structure complementary to that of a receptor and is e of forming a complex with this receptor. In some embodiments, a ligand is to be understood as meaning a peptide or peptide fragment which has a suitable length and suitable binding motifs in its amino acid sequence, so that the peptide or peptide fragment is capable of fonning a complex with proteins of MHC class I or MHC class II.

In some embodiments, a “receptor/ligand complex” is also to be understood as meaning a tor/peptide complex” or “receptor/peptide fragment complex”, ing a peptide- or peptide fragment-presenting MHC molecule of class I or of class II.

“Proteins or molecules of the major histocompatibility complex (MHCY’, "MHC molecules”, “MHC proteins” or “HLA ns” are to be tood as meaning proteins capable of g es resulting from the proteolytic cleavage of protein antigens and representing potential cyte epitopes, (e.g., T cell epitope and B cell epitope) transporting them to the cell surface and presenting them there to specific cells, in particular cytotoxic T-lymphocytes, T-helper cells, or B cells. The major histocompatibility complex in the genome ses the c region whose gene products expressed on the cell e are important for binding and presenting endogenous and/or foreign antigens and thus for regulating immunological processes.

The major histocompatibility complex is classified into two gene groups coding for different proteins. namely molecules of MHC class I and molecules of MHC class II. The cellular biology and the expression pattems of the two MHC classes are adapted to these different roles.

The terms “peptide” and "peptide epitope” are used interchangeably with “oligopeptide” in the present specification to designate a series of residues connected one to the other, typically by peptide bonds between the cr-amino and carboxyl groups of adjacent amino acid residues.

“Synthetic peptide” refers to a peptide that is obtained from a non-natural source, e.g.. is man-made.

Such peptides can be produced using such methods as al synthesis or recombinant DNA technology.

“Synthetic peptides” include “fusion proteins.” A “PanDR binding” peptide, a “PanDR binding epitope” is a member of a family of molecules that binds more than one HLA class 11 DR le.

“Phannaceutically acceptable” refers to a generally non-toxic, inert, and/or physiologically compatible composition or component of a composition.

A “phamiaceutical excipient” or “excipient” comprises a material such as an adjuvant. a carrier. pH- ing and ing agents, tonicity adjusting agents, wetting agents, preservatives, and the like. A "pharmaceutical excipient" is an ent which is ceutically acceptable.

The term “motif” refers to a pattern of residues in an amino acid ce of defined length, for example, a peptide of less than about 15 amino acid es in length, or less than about 13 amino acid residues in length, for example, from about 8 to about 13 amino acid residues (e.g., 8, 9, 10, ll, 12, or 13) for a class I HLA motif and from about 6 to about 25 amino acid residues (e.g., 6, 7. 8. 9. 10. ll, 12. l3, 14. 15. l6, l7, l8, 19, 20, 21, 22, 23, 24, or 25) for a class II HLA motif, which is recognized by a particular HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs differ in their pattern of the primary and secondary anchor residues. In some embodiments, an MHC class I motif fies a peptide of 9, 10, or 1 1 amino acid residues in length.

A “supennotifi is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. In some embodiments, a supermotif—bearing e described herein is recognized with high or intermediate affinity (as defined ) by two or more HLA antigens.

The tenn “naturally occurring” as used herein refers to the fact that an object can be found in nature.

For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally According to the invention, the term “vaccine" relates to a pharmaceutical preparation aceutical composition) or t that upon stration induces an immune response, for e, a cellular or humoral immune response, which recognizes and attacks a pathogen or a diseased cell such as a cancer cell. A vaccine may be used for the prevention or treatment of a disease. The term “individualized cancer vaccine" or “personalized cancer vaccine” concerns a particular cancer patient and means that a cancer e is adapted to the needs or special circumstances of an individual cancer patient.

A “protective immune se" or “therapeutic immune response“ refers to a CTL and/or an HTL response to an antigen derived from an pathogenic antigen (e.g., a tumor antigen), which in some way prevents or at least partially arrests disease symptoms, side effects or progression. The immune response can also include an antibody response which has been facilitated by the stimulation of helper T cells. en processing” or “processing” refers to the degradation of a polypeptide or n into procession products, which are fragments of said polypeptide or antigen (e.g.. the degradation of a polypeptide into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, for example, antigen presenting cells, to specific T cells. en presenting cells” (APC) are cells which present peptide fragments of protein antigens in association with MHC molecules on their cell surface. Some APCS may activate antigen specific T cells. sional antigen-presenting cells are very efficient at intemalizing antigen, either by phagocytosis or by receptor-mediated endocytosis, and then displaying a fragment of the antigen, bound to a class II MHC molecule. on their membrane. The T cell recognizes and interacts with the antigen-class II MHC molecule complex on the membrane of the n presenting cell. An additional co-stimulatory signal is then produced by the antigen presenting cell, leading to activation of the T cell. The expression of co-stimulatory molecules is a g feature of professional antigen-presenting cells.

The main types of professional antigen-presenting cells are dendritic cells, which have the broadest range of antigen presentation, and are probably the most important antigen presenting cells, macrophages, B- cells. and certain activated epithelial cells.

Dendritic cells (DCs) are leukocyte populations that present antigens captured in peripheral tissues to T cells via both MHC class II and I antigen presentation pathways. It is well known that tic cells are potent inducers of immune responses and the activation of these cells is a critical step for the induction of antitumoral immunity.

Dendritic cells are conveniently categorized as “immature” and “mature” cells, which can be used as a simple way to discriminate n two well characterized phenotypes. However. this nomenclature should not be construed to exclude all le intermediate stages of differentiation.

Immature dendritic cells are terized as antigen presenting cells with a high capacity for antigen uptake and processing, which correlates with the high expression of Fey receptor and mannose receptor. The mature phenotype is typically terized by a lower expression ofthese markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e. g. CD54 and CD1 1) and costimulatory molecules (e. g.. CD40. CD80, CD86 and 4-1 BB).

The term "residue" refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or nucleic acid (DNA or RNA) that encodes the amino acid or amino acid mimetic.

The lature used to describe es or proteins follows the conventional practice wherein the amino group is presented to the lcfi (the amino- or N-tenninus) and the carboxyl group to the right (the carboxy- or C-terminus) of each amino acid residue. When amino acid residue ons are referred to in a peptide e they are numbered in an amino to carboxyl direction with position one being the residue located at the amino terminal end of the epitope, or the peptide or protein of which it can be a part.

In the formulae enting selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure ae, each residue is generally represented by standard three letter or single letter designations. The L-fomi of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-fonn for those amino acid residues having D-forms is represented by a lower case single letter or a lower case three letter symbol. However, when three letter symbols or full names are used without capitals, they can refer to L amino acid residues. Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or "G. The amino acid sequences of peptides set forth herein are generally designated using the standard single letter .

(A, Alanine; C, Cysteine; D, Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G, Glycine; H, Histidine; I, Isoleucine; K, Lysine; L, Leucine; M. Methionine; N, Asparagine; P, Proline; Q, Glutamine; R, Arginine; S.

; T, Threonine; V, ; W, Tryptophan; and Y, Tyrosine.) The terms “poly-nucleotide” and "nucleic acid” are used interchangeably herein and refer to rs of nucleotides of any , and include DNA and RNA, for example, mRNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, the polynucleotide and nucleic acid can be in vitro transcribed mRNA. In some embodiments. the ucleotide that is stered using the methods of the invention is mRNA.

The terms “identical” or t ity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequenees that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for m correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison re or algorithms or by visual inspection. Various algorithms and software that can be used to obtain aligmnents of amino acid or nucleotide sequences are well-known in the an. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and ions thereof. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity. when compared and aligned for maximum correspondence. as measured using a sequence comparison algorithm or by visual inspection. In some ments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length or any integral value 2between. In some embodiments, identity exists Over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a tide sequence.

A “conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., , arginine, histidine), acidic side chains (e.g., ic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, scrine, ine, tyrosine, cysteine), nonpolar side chains (e.g., e, valinc, leucine, isolcucinc, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.. threonine, yaline, isoleucine) and aromatic side chains (e.g., tyrosine, alanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate peptide function are well-known in the art.

The tenn "vector” as used herein means a construct, which is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors e, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage s, DNA or RNA expression vectors ated with cationic condensing agents. and DNA or RNA expression vectors encapsulated in liposomes.

A polypeptide, antibody, polynueleotide, vector, cell, or composition which is “isolated” is a polypeptide, antibody, polynueleotide, vector. cell, or composition which is in a form not found in nature. ed polypeptides, antibodies, polynueleotides, vectors, cells, or compositions include those which have been ed to a degree that they are no longer in a form in which they are found in nature. In some embodiments. a polypeptide. antibody. polynueleotide. vector. cell. or composition which is ed is substantially pure. In some embodiments, an “isolated eleotide” asses a PCR or quantitative PCR reaction comprising the polynueleotide amplified in the PCR or tative PCR reaction.

The term “substantially pure” as used herein refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

The term “subject” refers to any animal (c.g., a mammal), including, but not limited to, humans, non- human primates, canines, felines, rodents. and the like, which is to be the recipient of a ular treatment.

Typically, the terms “subject" and “patient” are used interchangeably herein in reference to a human subject.

The tenns “effective amount” or “therapeutically effective amount” or “therapeutic effect” refer to an amormt ofa therapeutic effective to ” a disease or disorder in a subject or . The therapeutically effective amount of a drug has a therapeutic effect and as such can prevent the development of a disease or disorder; slow down the development of a disease or disorder; slow down the progression of a disease or disorder; relieve to some extent one or more of the symptoms associated with a disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

The terms “treating" or “treatment" or “to treat" or “alleviating” or “to alleviate” refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt ssion of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that t or slow the development of a targeted pathologic ion or disorder. Thus those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be ted.

As used in the present disclosure and claims, the singular forms “‘a', “an" and “the" include plural forms unless the context y dictates otherwise.

It is understood that terms such as "comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in US. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of" and “consists essentially of ’ have the meaning ascribed to them in US. Patent law, e.g.. they allow for elements not explicitly recited, but e elements that are found in the prior art or that affect a basic or novel characteristic of the invention. Nothing herein is intended as a promise.

The term “and/or” as used in a phrase such as “A and/or B” herein is intended to e both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C ; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). 11. Neoantigens One ofthe critical barriers to developing curative and tumor-specific immunotherapy is the identification and selection of highly specific and restricted tumor antigens to avoid autoimmunity. Tumor neoantigens, which arise as a result of genetic change (e.g., ions, translocations, deletions, missense mutations, splice site mutations, etc.) within malignant cells, represent the most tumor-specific class of antigens. Neoantigens have rarely been used in cancer vaccine or immunogenic compositions due to technical difficulties in fying them. selecting optimized antigens. and producing neoantigens for use in a vaccine or immunogenic composition. These ms may be addressed by: fying mutations in neoplasias/tumors which are present at the DNA level in tumor but not in matched gerrnline samples from a high proportion of subjects having cancer; analyzing the identified mutations with one or more peptide-MHC binding prediction thms to generate a plurality of neoantigen T cell epitopes that are expressed within the neoplasia/tumor and that bind to a high proportion of patient HLA alleles; and sizing the plurality of neoantigenic peptides ed from the sets of all neoantigen peptides and predicted g peptides for use in a cancer vaccine or immunogenic composition suitable for treating a high proportion of ts having cancer.

For example, ating peptide sequencing infonnation into a therapeutic vaccine may include prediction of mutated peptides that can bind to HLA molecules of a high tion of individuals. Efficiently choosing which particular mutations to utilize as immunogen requires the ability to predict which mutated peptides would efficiently bind to a high tion of patient's HLA alleles. Recently. neural network based g approaches with validated binding and non-binding peptides have advanced the accuracy of prediction algorithms for the major HLA-A and -B alleles. However, even using advanced neural network- based algorithms to encode HLA-peptide binding rules, several factors limit the power to predict es presented on HLA alleles.

For example, translating peptide sequencing information into a therapeutic vaccine may e formulating the drug as a multi-epitope vaccine of long peptides. Targeting as many mutated epitopes as practically possible takes advantage of the enormous capacity of the immune system, prevents the opportunity for immunological escape by down-modulation of an immune targeted gene product, and sates for the known inaccuracy of epitope prediction approaches. Synthetic peptides provide a useful means to prepare le gens efficiently and to rapidly translate identification of mutant epitopes to an effective vaccine. Peptides can be readily synthesized ally and easily purified utilizing reagents free of inating bacteria or animal substances. The small size allows a clear focus on the mutated region of the protein and also reduces irrelevant antigenic competition from other components (unmutated protein or viral vector antigens).

For example, translating peptide sequencing information into a therapeutic vaccine may include a combination with a strong vaccine adjuvant. Effective vaccines may require a strong adjuvant to initiate an immune response. For example, poly-ICLC, an agonist ofTLR3 and the RNA helicase-domains ofMDAS and R103, has shown several desirable properties for a vaccine adjuvant. These properties e the induction of local and systemic activation of immune cells in vivo. production of stimulatory chemokines and cytokines, and ation of antigen-presentation by DCs. Funhennore, poly-[CLC can induce e CD4+ and CD8+ responses in humans. Importantly, striking similarities in the upregulation of transcriptional and signal transduction pathways were seen in subjects vaccinated with poly-lCLC and in volunteers who had received the highly ive, replication—competent yellow fever vaccine. Furthermore, >90% of ovarian carcinoma patients immunized with CLC in combination with a NYESO-l peptide e (in addition to Montanide) showed induction of CD4+ and CD8+ T cell. as well as antibody responses to the peptide in a recent phase 1 study. At the same time, poly-ICLC has been extensively tested in more than 25 clinical trials to date and exhibited a relatively benign toxicity profile.

Applicants have ered mutational events in the following genes: ABLI, AC011997, ACVRZA, AFP, AKTl, ALK, , ANAPC 1, APC, ARIDlA, AK AR-v7, ASCLZ, BZM, BRAF, BTK, ClSORF40, CDHl, CLDN6, CNOTI, CT45A5, CTAGlB, DCT, DKK4, .EEF1DP3, EGFR, EIF2B3, env, EPHBZ, ERBB3, ESRl, ESRP1,FAMlllB,FGFR3, FRGIB, GAGEl. , GATA3, GBP3, HERZ, IDH1,JAK1, KIT, KRAS, LMANI, MABEBlé, MAGEAI, MAGEA 10, MAGEA4, MAGEAS, MAGEBI7, MAGEB4, MAGECI, MEK, MLANA, MLL2, MMPI3, MSH3, MSH6, MYC, NDUFCZ, NRAS, PAGEZ, PAGES, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RACl, RBM27, RNF43, RPL22, RUNXl, SEC3lA, SEC63, SF3B1, SLC35F5, SLC45A2, SMAPl, SMAPI, SPOP, TFAM, TGFBRZ, THAPS, TP53, TTK, TYR, UBRS, VHL, XPOT an EEFlDP3zFRY fiision polypeptide. an EGFR:SEPT14 fusion polypeptide. an EGFRVIII deletion polypeptide. an EML41ALK fusion polypeptide, an NDRG12ERG fusion ptide, an AC011997.1:LRRC69 fusion polypeptide, a RUNXl(ex5)-RUNX lTlfusion polypeptide, a TMPRSS2:ERG fusion polypeptide, a NABISTAT6 fusion polypeptide, a NDRGl :ERG fusion polypeptide, a PMLzRARA fusion polypeptide, a PPPlRlB:STARD3 fusion polypeptide, a MADlLl :MAFK fusion polypeptide, a FGFR32TAC fusion polypeptide, a FGFR32TACC3 fusion polypeptide, a BCRzABL fusion polypeptide, a Cl lorf952RELA fusion polypeptide, a CBFBzMYI-Ill fusion polypeptide, a CBFBzMYHl 1 fusion polypeptide, a CD742ROSl fusion polypeptide. a CD742ROS1 fusion polypeptide, ERVE-4: protease, ERVE-4: reverse transcriptase, : reverse transcriptase, ERVE-4: unknown, ERVH-2 matrix protein, ERVH-Z: gag, ERVH-Z: retroviral matrix, ERVH48-1: coat protein, -1: syncytin, ERVl-l envelope protein, ERVK-S gag, ERVK-S env, ERVK-S pol, EBV A73, EBV BALF3, EBV BALF4, EBV BALFS, EBV BARFO, EBV LFZ, EBV RPMSl, HPV-16, HPV-l6 E7, and HPV-16 E6.

In some embodiments, a neoantigen bed herein is due to a onal event in 62M, BTK.

EGFR, GATA3, KRAS, MLLZ, a TMPRSSZ:ERG fusion polypeptide, or TP53.

Neoantigen polypeptides In aspects, the invention provides isolated peptides that comprise a tumor c on from Table l or 2. These peptides and polypeptides are referred to herein as “neoantigenic peptides” or "neoantigenic polypeptides”. The term "peptide” is used interchangeably with “mutant peptide” and “neoantigenic peptide” in the present specification to ate a series of es, typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and yl groups of adjacent amino acids. Similarly, the term "polypeptide" is used interchangeably with “mutant polypeptide" and “neoantigenic polypeptide” in the t specification to designate a series of residues, e.g., L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and yl groups of adjacent amino acids. The polypeptides or peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications. subject to the condition that the modification not y the biological activity of the polypeptides as herein described.

In some embodiments, sequencing methods are used to identify tumor specific mutations. Any le cing method can be used according to the invention, for example, Next Generation Sequencing GIGS) technologies. Third Generation Sequencing methods might substitute for the NGS technology in the future to speed up the sequencing step of the method. For clarification purposes: the terms “Next Generation Sequencing” or "NGS” in the t of the t invention mean all novel high throughput sequencing technologies which, in contrast to the ntional" sequencing methodology known as Sanger chemistry, read nucleic acid templates randomly in parallel along the entire genome by breaking the entire genome into small pieces. Such NGS technologies (also known as massively parallel sequencing technologies) are able to deliver c acid sequence ation of a whole genome, exome, transcriptome (all transcribed sequences of a genome) or methylome (all methylated sequences of a genome) in very short time periods, e.g. within 1-2 weeks. for example. within 1-7 days or within less than 24 hours and allow. in principle. single cell sequencing approaches. Multiple NGS platforms which are cially available or which are mentioned in the literature can be used in the context of the ion e.g. those described in detail in W0 2012/ 159643.

In certain embodiments a neoantigenic peptide described herein le can comprise, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1, about 12, about 13, about 14, about , about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26. about 27. about 28. about 29. about 30. about 31. about 32. about 33. about 34. about 35. about 36. about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about I 10, about 120 or greater amino acid es, and any range derivable therein. In specific embodiments, a neoantigenic peptide molecule is equal to or less than 100 amino acids.

In some ments, neoantigenic peptides and polypeptides described herein for MHC Class I are 13 residues or less in length and usually consist of between about 8 and about 11 residues. particularly 9 or 10 residues. In some embodiments, igenic peptides and polypeptides described herein for MHC Class II are 9-24 residues in length.

A longer neoantigenic peptide can be designed in several ways. In some embodiments, when HLA- binding peptides are ted or known, a longer neoantigenic peptide could consist of ( 1) individual binding peptides with extensions of 2-5 amino acids toward the N- and C-temiinus of each corresponding gene product; or (2) a concatenation of some or all of the binding peptides with extended sequences for each. In other embodiments. when sequencing reveals along (>10 residues) neoepitope sequence present in the tumor (e.g. due to a frameshift, read-through or intron inclusion that leads to a novel peptide ce), a longer igenic peptide could consist of the entire stretch of novel tumor-specific amino acids as either a single longer peptide or several overlapping longer peptides. In some embodiments, use of a longer peptide is presumed to allow for endogenous processing by patient cells and can lead to more ive antigen presentation and induction of T cell responses. In some embodiments, two or more peptides can be used, where the peptides overlap and are tiled over the long igenic peptide.

In some embodiments, the neoantigenic peptides and polypeptides bind an HLA protein (e.g., HLA class I or HLA class II). In specific embodiments the neoantigenic peptides and polypeptides bind an HLA protein with r affinity than the corresponding ype peptide. In specific ments the neoantigenic peptide or polypeptide has an IC50 of at least less than 5000 nM, at least less than 500 nM, at least less than 100 nM, at least less than 50 nM or less.

In some embodiments. the neoantigenic peptides can be from about 8 and about 50 amino acid residues in length, or from about 8 and about 30, from about 8 and about 20. from about 8 and about 18, from about 8 and about 15, or from about 8 and about 12 amino acid residues in length. In some embodiments, the neoantigenic peptides can be from about 8 and about 500 amino acid residues in length, or from about 8 and about 450, from about 8 and about 400, from about 8 and about 350, from about 8 and about 300, from about 8 and about 250, from about 8 and about 200, from about 8 and about 150, from about 8 and about 100, from about 8 and about 50. or from about 8 and about 30 amino acid residues in length.

In some embodiments, the neoantigenic peptides can be at least 8, 9, 10, ll, 12, l3, I4, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid residues in length. In some embodiments, the igenie peptides can be at least8,9,10,ll,12,13,14,15,16,17,18,19,20, 21, 22, 23,24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450. 500 or more amino acid residues in length. In some embodiments. the neoantigenic peptides can be at most 8, 9, 10, 11, 12, 13, 14, 15, l6, 17, 18, 19,20, 21, 22, 23,24, 25, 26,27, 28, 29, 30, 3], 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, or less amino acid residues in length. In some embodiments, the neoantigenic peptides can be at most 8, 9, 10, l 1, 12, l3, 14, 15, l6, l7, l8, 19, 20, 21, 22, 23,24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42,43, 44, 45, 46,47, 48, 49, 50,55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or less amino acid residues in length.

In some embodiments, the neoantigenic es has a total length of at least 8. at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least , at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.

In some embodiments, the neoantigenic peptides has a total length of at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids.

In some embodiments, the neoantigenic peptides can have a pl value of about 0.5 and about 12, about 2 and about 10, or about 4 and about 8. In some embodiments, the neoantigenic peptides can have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or more. In some embodiments, the neoantigenic es can have a pI value ofat most 4.5. 5, 5.5. 6, 6.5, 7. 7.5, or less.

In some embodiments, the neoantigenic peptides can have an HLA binding affinity of between about lpM and about lmM, about lOOpM and about 500pM, about 500pM and about lOttM, about lnM and about luM, or about 10nM and about 1 uMln some embodiments, the neoantigenic peptides can have an HLA binding affinity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 uM, or more. In some embodiments, the neoantigenic peptides can have an HLA binding affinity of at most 2, 3, 4, 5. 6, 7, 8, 9, 10. 15, 20, 25, 30, ,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 pM.

In some embodiments, a igenic peptide bed herein can se carriers such as those well known in the art, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acid residues such as poly L-lysine, poly L-glutamic acid, influenza virus proteins, hepatitis B virus core protein. and the like.

In some embodiments, a neoantigenic peptide described herein can be d by terminal-NHg acylation, e.g., by alkanoyl (C 1-C20) or ycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. in some embodiments these modifications can provide sites for linking to a support or other molecule.

In some embodiments, a neoantigenic peptide described herein can contain modifications such as but not limited to glycosylation. side chain oxidation. biotinylation, phosphorylation, addition of a surface active material, e.g. a lipid, or can be ally modified, e.g., acetylation, etc. Moreover, bonds in the peptide can be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

In some embodiments, a neoantigenic peptide bed herein can contain substitutions to modify a physical property (e.g., stability or solubility) ofthe ing peptide. For example, neoantigenic peptides can be d by the substitution of a cysteine (C) with a—amino butyric acid (“B”). Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substituting a-amino butyric acid for C not only ates this problem, but actually improves binding and crossbinding capability in certain ces. Substitution of cysteine with a-amino butyric acid can occur at any residue of aneoantigenic peptide, e.g., at either anchor or non-anchor positions of an epitope or analog within a peptide, or at other positions of a peptide.

In some embodiments, a neoantigenic peptide described herein can comprise amino acid mimetics or unnatural amino acid residues, e.g. D- or L-naphylalanine; D- or ylglycine; D- or Lthieneylalanine; D- or L-l, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-py-ridinyl)-alanine; D- or L-(3- pyridin_\-'I)-alanine; D- or L—(2-pyrazinyl)-alanine; D- or L-(4—isopropyl)-phenylglycine; D-(trifluoromethyl)- phenylglycine; D-(trifluoro-methyl)-phenylalanine: D-.rho.-fluorophenylalanine; D- or L-.rho.-biphenyl- phenylalanine; D- or L-.rho.-methoxybiphenylphenylalanine; D- or Lindole(allyl)alanines; and, D- or L— alkylalanines, where the alkyl group can be a substituted or unsubstituted methyl, ethyl, , hexyl, butyl, pentyl. isopropyl, iso-butyl, sec-isotyl. iso-pentyl. or a idic amino acid residues. Aromatic rings of a non-natural amino acid e, e.g., lyl, thiophenyl, pyrazolyl, idazolyl, yl, fiiranyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides that have various amino acid mimetics or unnatural amino acid residues are ularly useful, as they tend to manifest increased stability in vivo. Such peptides can also possess improved shelf-life or manufacturing properties.

Peptide stability can be assayed in a number of ways. For instance, peptidases and various ical media. such as human plasma and serum, have been used to test ity. See, e.g., Verhoef, et al., Eur. J.

Drug Metab. Phannacokinetics 11:291 (1986). Half-life of the peptides described herein is iently detemrined using a 25% human serum (v/v) assay. The protocol is as follows: pooled human serum (Type AB, non-heat inactivated) is dilapidated by centrifugation before use. The serum is then diluted to 25% with RPMI-l 640 or another suitable tissue culture medium. At predetennined time intervals, a small amount of reaction on is removed and added to either 6% aqueous trichloroacetic acid (TCA) or ethanol. The cloudy reaction sample is cooled (4°C) for 15 minutes and then sptm to pellet the precipitated serum proteins.

The presence of the peptides is then determined by reversed-phase HPLC using ity-specific chromatography conditions.

In some embodiments, a neoantigenic peptide bed herein can be in solution, lyophylized, or can be in crystal form.

In some embodiments, a neoantigenic peptide described herein can be ed tically, by recombinant DNA technology or al sis. or can be ed from natural sources such as native tumors or enic organisms. Epitopes can be synthesized individually or joined directly or indirectly in a peptide. Although a neoantigenic peptide described herein will be ntially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptide can be synthetically conjugated to be joined to native fragments or particles.

In some embodiments, a neoantigenic peptide described herein can be prepared in a wide variety of ways. In some embodiments. the peptides can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. (See, for example, Stewart & Young, SOLID PHASE PEPTIDE SIS, 2D. ED., Pierce Chemical Co., 1984). Further, individual peptides can be joined using chemical ligation to produce larger peptides that are still within the bounds of the invention.

Altematively, inant DNA technology can be employed wherein a nucleotide sequence which encodes a peptide inserted into an expression vector, ormed or ected into an appropriate host cell and cultivated under conditions suitable for sion. These procedures are generally known in the art, as described generally in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, NY. (1989). Thus, recombinant peptides, which se or consist of one or more epitopes described herein, can be used to present the riate T cell epitope.

In one aspect, the invention described herein also provides compositions comprising one, at least two, or more than two neoantigenic peptides. In some embodiments a composition described herein contains at least two distinct peptides. In some embodiments. the at least two ct peptides are derived from the same polypeptide. By distinct polypeptides is meant that the peptide vary by length, amino acid sequence or both.

The peptides are derived from any polypeptide known to or have been found to contain a tumor specific mutation.

In some embodiments, the isolated neoantigenic peptide is encoded by a gene with a point mutation resulting in an amino acid substitution of the native peptide.

In some embodiments. the isolated neoantigenic peptide is encoded by a ABL gene. In related embodiments, AxByCZ is VADGLITI'LHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGKVYEGVWKKYSLTV AVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVC, VADGLITTLHYPAPKRNKP I V Y GVSPNYDKWEMERTDITMKHKLGGGQYGVVYEGVWKKYSLTV AVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVC, LLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSATEYLEKKNFII-IRDLAARN CLVGENHLVKVADFGLSRLMTGDTYTAHAGAKF SLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYI[IEFMTYGNLLDYLRECNR QEVNAVVLLYMATQISSAMEYLEKKNFIHRDLA, or STVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQHGEVYEGVWKKYSLT VAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLG.

In some embodiments. the isolated neoantigenic peptide is encoded by a ALK gene. In related embodiments, AXB)CZ is SSLAMLDLLHVARDIACGCQYLEENHFIHRDIAARNCLLTCPGPGRVAKIADFGMARDIYRASYYRK PVKWMPPEAFMEGIFI‘SKTDTWSFGVLL or QVAVKTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIGVSLQSLPRFILMELMAGGDLKSFLRETRPR PSQPSSLAMLDLLHVARDIACGCQYLEENHFI.

In some embodiments. the isolated neoantigenic peptide is d by a BRAF gene. In related embodiments, AXB3CZ is MIKLIDIARQTAQGMDYLHAKSIIHRDLKSNNIFLHEDLTVKIGDFGLATEKSRWSGSHQFEQLSGSIL WMAPEVIRMQDKNPYSFQSDVYAFGIVLYELM. In some related embodiments, the neoantigenic peptide is not DFGLATEKSR or FGLATEKSRW.

In some embodiments, the isolated neoantigenic e is encoded by a BTK gene. In related embodiments, AXB,CZ is MIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGSLLNYLREMRHRFQTQQ LLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND.

In some embodiments, the isolated neoantigenic peptide is encoded by a EEFlBZ gene. In d embodiments, AKB,CL is MGFGDLKSPAGLQVLNDYLADKSYIEGYVPSQADVAVFEAVSGPPPADLCHALRWYNHIKSYEKEK ASLPGVKKALGKYGPADVEDTFGSGAT. In some related embodiments, the neoantigenic peptide is not EAVSGPPPA. FEAVSGPPP or FEAVSGPPPA.

In some embodiments, the isolated neoantigenic e is encoded by a EGFR gene. In related embodiments, AXB,CZ is MSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIIRNRGENSCKATGQVCHAL CSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLL or IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA.

In some embodiments, the isolated neoantigenic peptide is encoded by a ERBB3 gene. In related embodiments, AXB,CZ is EFSTLPLPNLRMVRGTQVYDGKF. In some d embodiments, the neoantigenic peptide is not RMVRGTQVY, RNIV, LRMVRGTQV, TLPLPNLRMV, NLRMVRGTQV, or LRMVRGTQVY.

In some embodiments. the ed neoantigenic e is encoded by a ESRl gene. In related embodiments. AXB)CZ is HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEA, NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLPSTLKSLEEKDHIHRVLD KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH, IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLCDLLLEMLDAHRLHAP SVEETDQSHLATAGSTSSHSLQKYYITGE.

IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLNDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, or IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGS'I‘SSHSLQKYYITGE.

In some embodiments. the isolated ncoantigenic peptide is encoded by a FGFR3 gene. In related embodiments. AVByC, is HRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERCPHRPILQAGLPANQTA VLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVG. In some related embodiments, the neoantigenie peptide is not DVLERCPHR, LERCPHRPI, CPHRPILQA, or LERCPI-IRPIL.

In some embodiments, the isolated neoantigenie peptide is d by a FRG 18 gene. In related embodiments, AXB3CZ is AVKLSDSRIALKSGYGKYLGINSDELVGHSDAIGPREQWEPVFQNGKMALSASNSCFIRCNEAGDIEA KSKTAGEEEMIKIRSCAEKETKKKDDIPEEDKG. In some related embodiments. the neoantigenic peptide is not FIR, LSASNSCFI. ALSASNSCF or FQNGKMALSA.

In some embodiments, the isolated neoantigenic peptide is encoded by a HERZ gene. In related embodiments, AXB,CL is GSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGLGSPYVSRLLGICLTSTV QLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCM.

In some ments. the isolated neoantigenic peptide is encoded by a IDHl gene. In related embodiments, AXB)Cz is RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGHHAYGDQYRATDFVVPGP YTPSDGTQKVTYLVHNFEEGGGVAMGM, RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGCHAYGDQYRATDFVVPGPG KVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGGHAYGDQYRATDFVVPGP GKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, or RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGSHAYGDQYRATDFVVPGPG KVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM. In some related embodiments, the neoantigenic peptide is not PIIIGHHAY, GHHAYGDQY, KPIIIGHHAY, IGHHAYGDQY, PIIIGCHAY, GCHAYGDQY, KPIIIGCHAY. IGCHAYGDQY. PIIIGGHAY. GGHAYGDQY. KPIIIGGHAY. or IGGHAYGDQY.

In some embodiments, the isolated neoantigenic peptide is encoded by a KIT gene. In related embodiments, AXBBCZ is GLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIANLLGACTIGGPTLVIT EYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYK or VEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIANLLGACTIGGPTLVIT EYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYK.

In some embodiments, the isolated neoantigenic peptide is MEK gene. In related embodiments, AXB'VCZ is lSELGAGNGGVVFKVSHKPSGLVMARKLIHLEIKPAIRNQIIRELQVLHESNSPYIVGFYGAFYSDGEIS ICMEHMDGGSLDQVLKKAGRIPEQILGKVSI or LGAGNGGVVFKVSHKPSGLVMARKLIHLEIKPAIRNQIIRELQVLHECNSLYIVGFYGAFYSDGEISIC MEHMDGGSLDQVLKKAGRIPEQILGKVSIAVI.

In some embodiments. the isolated neoantigenic peptide is encoded by a MYC gene. In related ments, AXB,CZ is MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSDLQPPAPSEDIWKKFELLPTPPLSPSRRSG LCSPSYVAVTPFSLRGDNDGG, FTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLSTPPLSPSRRSGLCSPSY VAVTPFSLRGDNDGGGGSFSTADQLEMVTELLG, or LDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPIPPLSPSRRSGLCSPSYVA VTPFSLRGDNDGGGGSFSTADQLEMVTELLGG.

In some embodiments, the isolated igenic peptide is encoded by a PDGFRa gene. In related embodiments, AXB,CL is VAVKMLKPTARSSEKQALMSELKIMTHLGPHLNIVNLLGACTKSGPIYIIIEYCFYGDLVNYLHKNRD SFLSHHPEKPKKELDIFGLNPADESTRSYVILS.

In some embodiments. the isolated neoantigenic peptide is encoded by a PIK3CA gene. In related ments, AXB)Cz is IEEHANWSVSREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSKITEQEKDFLWSHRHYC VTIPEILPKLLLSVKWNSRDEVAQMYCLVKDWPP, SREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSEITKQEKDFLWSHRHYCVTI PEILPKLLLSVKWNSRDEVAQMYCLVKDWPPIKP, or LFINLFSMMLGSGMPELQSFDDIAYIRKTLALDKTEQEALEYFMKQMNDARHGGWTTKMDWIFHTI KQHALN. In some related embodiments, the neoantigenic peptide is not ISTRDPLSK, STRDPLSKI, LSKITEQEK, AISTRDPLSK, EKDF, SEITKQEKDF, KQEKDFLWSH, FMKQMNDAR, KQMNDARHG, RHGGWTI‘KM, YFMKQMNDAR, FMKQMNDARH, RI-IGG, QMNDARHGGW, or ARI-IGGW'ITKM.

’JI La) In some embodiments. the ed neoantigenic peptide is encoded by a POLE gene. In related embodiments. AXB)CZ is QRGGVITDEEETSKKIADQLDNIVDMREYDVPYHIRLSIDIETTKLPLKFRDAETDQIMMISYMIDGQG EIVSEDIEDFEFTPKPEYEGPFCVFN. In some related embodiments, the neoantigenic peptide is not ”ITKLPLKFK RDAETDQIM, KFRDAETDQI, ETTKLPLKFR, or RDAETDQIMM.

In some embodiments, the isolated neoantigenic e is encoded by a PTEN gene. In related embodiments, AXB,CZ is KFNCRVAQYPFEDHNPPQLELIKPFCEDLDQWLSEDDNHVAAIHCKAGKGQTGVMICAYLLHRGKF LKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSY. In some related ments, the neoantigenic peptide is not QTGVMICAY, GKGQTGVMI, GQTGVMICAY, or KAGKGQTGVM.

In some embodiments, the ed igenic peptide is encoded by a RACI gene. In related embodiments, AxByCz is MQAIKCVVVGDGAVGKTCLLISY'ITNAFSGEYIPTVFDNYSANVMVDGKPVNLGLWDTAGQEDYD RLRPLSYPQTVGET. In some related embodiments. the neoantigenic peptide is not TTNAFSGEY, FSGEYIPTV, SGEYIPTVF, YTTNAFSGEY, 'ITNAFSGEYI, or FSGEYIPTVF.

In some embodiments. the isolated igenic peptide is encoded by a TP53 gene. In related embodiments, AXB,.CZ is IRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDC'ITIHYNYMCNSSCMGSMNRRPILTIITLEDSSG NLLGRNSFEVRVCACPGRDRRTEEENLRKKGEP, TYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRHCPHHERCSDSDGLAP PQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEV, EGNLRVEYLDDRN'IFRHSVVVPYEPPEVGSDCTI‘IHYNYMCNSSCMGGMNQRPILTIITLEDSSGNLL GRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE, EGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNWRPILTIITLEDSSGNLL GRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE, or PEVGSDC'ITIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVCVCACPGRDRRTEEENLR KKGEPHHELPPGSTKRALPNNTSSSPQPKKKPL. In some related embodiments. the neoantigenic peptide is not SSCMGSMNIL PIL, MGSMNRRPI, CNSSCMGSM, SMNRRPILTL SSCMGSMNRR, NSSCMGSMNK RPIL, MGSM, CMGSIVINRRPI, TEVVRHCPH, VVRHCPHI-IEK SQHMTEVVRH, MNQRPILTI, NQRPILTII, CMGGMNQRPI,GMNQRP1LTI, SSCMGGMNQK NQRPILTIIT, NWRPILTII, SSCMGGMNW, MGGMNWRPI, MNWRPILTI, CMGGMNWRPI, GMNWRPILTI, SSCMGGMNWR, MNWRPILTII, NSSCMGGMNW, NSFEVCVCA, CPGR, or FEVCVCACPG.

In some embodiments, the isolated neoantigenic peptide is d by a gene with a frameshift mutation In some embodiments. the isolated neoantigenic peptide is encoded by a ACVRZA gene. In related embodiments, A‘B)CL is GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKRQP or GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKTALKYIFVAVRAICVM KSFLIFRRWKSHSPLQIQLHLSHPITTSCSIPWCHLC.

In some embodiments, the isolated neoantigenie peptide is C ISORF40 gene. In related embodiments, AxByCL is TAEAVNVAIAAPPSEGEANAELCRYLSKVLELRKSDVVLDKVGLALFFFFFETKSCSVAQAGVQWRS LGSLQPPPPGFKLFSCLSFLSSWDYRRMPPCLANFCIFNRDGVSPCWSGWS.

In some embodiments. the isolated neoantigenie peptide is encoded by a CNOTl gene. In d embodiments, AXB)CZ is LSVIIFFFVYIWHWALPLILNNHHICLMSSIILDCNSVRQSIMSVCFFFFSVIFSTRCLTDSRYPNICWFK or LSVIIFFFVYIWHWALPLILNNHHICLMSSIILDCNSVRQSIMSVCFFFFCYILNTMFDR.

In some embodiments, the isolated neoantigenic peptide is encoded by a EIFZB3 gene. In related embodiments, Ava‘Cz is VLVLSCDLITDVALHEVVDLFRAYDASLAMLMRKGQDSIEPVPGQKGKKKQWSSVTSLEWTAQERG CSSWLMKQTWMKSWSLRDPSYRSILEYVSTRVLWMPTSTV.

In some embodiments. the ed neoantigenic peptide is encoded by a EPHBZ gene. In related embodiments, AXB,.CZ is SIQVMRAQMNQIQSVEGQPLARRPRATGRTKRCQPRDVTKKTCNSNDGKKREWEKRKQILGGGGK YKEYFLKRILIRKAMTVLAGDKKGLGRFMRCVQSETKAVSLQLPLGR.

In some embodiments, the isolated neoantigenie peptide is encoded by a ESRPI gene. In d embodiments. AXB)CZ is LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKKHEGQIC or LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKT.

In some embodiments, the isolated igenie peptide is encoded by a FAMI 13 gene. In related embodiments, AXB)CZ is GALCKDGRFRSDIGEFEWKLKEGHKKIYGKQSMVDEVSGKVLEMDISKKKHYNRKISIKKLNRMKV PLMKLITRV. In some related embodiments. the neoantigenic peptide is not VPL. PLMKLITRV.

RMKVPLMKL, ISKKKHYNR, SKKKHYNRK, KHYNRKISI, SIK, YNRKISIKK, KISIKKLNR, SIKKLNRMK, LNRMKVPLM, ISIKKLNRM, KLI, KLNRMKVPLM, RMKVPLMKLI, ISIKKLNRMK, ISKKKHYNRK, KHYNRKISIK, HYNRKISIKK, KISIKKLNRM, SIKKLNRMKV, LNRMKVPLMK, KVPLMKLITR, DISKKKHYNR, KKHYNRKISI, KKLNRMKVPL, or VPLMKLITRV.

In some embodiments, the isolated igenie peptide is encoded by a GBP3 gene. In d embodiments. AVByC, is RERAQLLEEQEKTLTSKLQEQARVLKERCQGESTQLQNEIQKLQKTLKKKPRDICRIS.

In some embodiments, the isolated neoantigenic peptide is encoded by a JAKl gene. In related embodiments, AXB,CZ is ’JI ’JI VNTLKEGKRLPCPPNCPDEVYQLMRKCWEFQPSNRTSFQNLIEGFEALLKTSN or CRPVTPSCKELADLMTRCMNYDPNQRPFFRAIMRDINKLEEQNPDIVSEKNQQLKWTPHILKSAS.

In some embodiments, the isolated neoantigenic peptide is encoded by a LMANI gene. In related ments, AXBBCz is DDHDVLSFLTFQLTEPGKEPPTPDKEISEKEKEKYQEEFEHFQQELDKKKRGIPEGPPRPPRAACGGN I DDHDVLSFLTFQLTEPGKEPPTPDKEISEKEKEKYQEEFEHFQQELDKKKRNSRRATPTSKGSLRRKY In some embodiments, the isolated neoantigenic peptide is encoded by a MSH3 gene. In d embodiments, AXB,CZ is TKSTLIGEDVNPLIKLDDAVNVDEIMTDTSTSYLLCISENKENVRDKKKGQHFYWHCGSAACHRRGC LYTKSTLIGEDVNPLIKLDDAVNVDEIMTDTSTSYLLCISENKENVRDKKRATFLLALWECSLPQARL CLIVSRTLLLVQS.

In some embodiments, the isolated neoantigenic peptide is encoded by a NDUFCZ gene. In related embodiments, AXB,CZ is LPPPKLTDPRLLYIGFLGYCSGLIDNLIRRRPIATAGLI-IRQLLYITAFFFCWILSCKT, or SLPPPKLTDPRLLYIGFLGYCSGLIDNLIRRRPIATAGLHRQLLYITAFFLLDIIL.

In some embodiments, the isolated igenic peptide is encoded by a RBMZ7 gene. In related embodiments, AXB,CZ is NQSGGAGEDCQIFSTPGHPKMIYSSSNLKTPSKLCSGSKSI—IDVQEVLKKKTGSNEV'ITRYEEKKTGSV RKANRMPKDVNIQVRKKQKHETRRKSKYNEDFERAWREDLTIKR.

In some embodiments, the isolated neoantigenic peptide is encoded by a RPL22 gene. In related embodiments, AXB,CL is MAPVKKLVVKGGKKKEASSEVHS or MAPVKKLVVKGGKKRSKF. In some related embodiments, the neoantigenic peptide is not KRSK or RSK In some embodiments, the isolated neoantigenic peptide is encoded by a SEC3 IA gene. In related embodiments. AXB,.C7, is MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKN MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKLMLLRLNLRKMCGPF.

In some embodiments, the isolated neoantigenic peptide is d by a SEC63 gene. In related ments, AxByCz is AEVFEKEQSICAAEEQPAEDGQGETNKNRTKGGWQQKSKGPKKTAKSKKKETFKKKTYTCAI'ITVK ATETKAGKWSRWE or MAEVFEKEQSICAAEEQPAEDGQGETNKNRTKGGWQQKSKGPKKTAKSKKRNL.

In some embodiments, the isolated neoantigenic peptide is encoded by a SLC35F5 gene. In d embodiments, AXB,CZ is NIMEIRQLPSSHALEAKLSRMSYPVKEQESILKTVGKLTATQVAKISFFFALCGFWQICHIKKHFQTHK In some embodiments, the isolated neoantigenic peptide is d by a SMAPI gene. In related embodiments, AXBBCZ is YDKNAIAITNISSSDAPLQPLVSSPSLQAAVDKNKLEKEKEKKKGREKERKGARKAGKTTY YYDKNAIAITNISSSDAPLQPLVSSPSLQAAVDKNKLEKEKEKKRKRKREKRSQKSRQNHL QLKSCRRKISNWSLKKVPALKKLRSPLWIF. In some related embodiments. the neoantigenic peptide is not ALKKLRSPL, KISNWSLKK, SLKKVPALK, WIF, REK, RKREKRSQK, RSQKSRQNH, SQKSRQNHL, KSRQNHLQL, RRKISNWSL, SLK. KVPALKKLR, HLQLKSCRR, WSLKKVPAL, RQNHLQLKS, KKL, LKKLRSPLW, KKLRSPLWI, KISNWSLKKV, KSRQNHLQLK, SLKKVPALKK, WSLKKVPALK, KRKREKRSQK, RSQKSRQNHL, HLQLKSCRRK, RRKISNWSLK, CRRKISNWSL, NWSLKKVPAL, QKSRQNHLQL, RQNHLQLKSC, LQLKSCRRKI, ALKKLRSPLW, or KKLRSPLWI In some embodiments. the isolated neoantigenic peptide is encoded by a TFAM gene. In related embodiments, AXB,CZ is IYQDAYRAEWQVYKEEISRFKEQLTPSQIMSLEKEIMDKHLKRKAMTKKKRVNTAWKTKKTSFSL or IYQDAYRAEWQVYKEEISRFKEQLTPSQIMSLEKEIMDKHLKRKAMTKKKS.

In some embodiments, the isolated neoantigenic peptide is encoded by a TGFBRZ gene. In related embodiments, AXB,CZ is KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKAW or CVAVWRKNDENITLETVCI-IDPKLPYHDFILEDAASPKCIMKEKKSLVRLSSCVPVALMSAM TTSSSQKNITPAILTCC.

In some embodiments, the isolated neoantigenic peptide is encoded by a THAP5 gene. In related embodiments, AXB)CZ is VPSKYQFLCSDHFTPDSLDIRWGIRYLKQTAVPTIFSLPEDNQGKDPSKKNPRRKTWKMRKKYAQKP In some embodiments, the isolated neoantigenic peptide is encoded by a TTK gene. In related embodiments, AXB,Cz is GTTEEMKYVLGQLVGLNSPNSILKAAKTLYEHYSGGESHNSSSSKTFEKKGEKNDLQLFVMSDTI‘YK IYWTVILLNPCGNLHLKTTSL In some embodiments, the isolated neoantigenic peptide is encoded by a XPOT gene. In related ments. AVByC, is QQLIRETLISWLQAQMLNPQPEKTFIRNKAAQVFALLFVTEYLTKWPKFFLTFSQ.

In some embodiments, the isolated neoantigenic peptide is encoded by 21 APC gene. In related embodiments, C2 is AKFQQCHSTLEPNPADCRVLVYLQNQPGTKLLNFLQERNLPPKVVLRHPKVHLNTMFRRPHSCLAD LIVLRVVRLPAPFRVNHAVEW, APVIFQIALDKPCHQAEVKHLHHLLKQLKPSEKYLKIKHLLLKRERVDLSKLQ. 0r MLQFRGSRFFQMLILYYILPRKVLQMDFLVHPA. In some related embodiments, the igenic peptide is not ADVLLSVl—IL, ADVLLSVHLI, APFRVNHAV, ARHKAVEFL, LSV, CLADVLLSVH, DVLLSVHLI, DVLLSVHLIV, FLQERNLPPK, FRVNHAVEW, VVR, HLIVLRVVRL, HLNTMFRRPH, HPKVHLNTM, HPKVHLNTMF, KAVEFLQER, MFR, KVHLNTMFRR KVVLRHPKV, LLSVHLIVL, LLSVHLIVLR, LPAPFRVNH, LPAPFRVNHA, LQERNLPPKV, LRVVRLPAPF. LSVHLIVLR. LSVl—ILIVLRV. MFRRPHSCL. MFRRPHSCLA. NLPPKVVLR.

NTMFRRPHSC, QERNLPPKV, RHPKVHLNTM, RNLPPKVVL, RNLPPKVVLK RPHSCLADV, RPHSCLADVL, RVVRLPAPF, RVVRLPAPFK SARHKAVEFL, SVHLIVLRV, LRVV, TMFRRPHSC, TMFRRPHSCL, VEFLQERNL, VLLSVHLlV, VLLSVHLlVL, VLRHPKVHL, VLRVVRLPA, VVRLPAPFR, or VVRLPAPFRV.

In some embodiments, the isolated neoantigenic peptide is encoded by a ARIDlA gene. In related embodiments. C7_ is ALGPl-ISRISCLPTQTRGCILLAATPRSSSSSSSNDMIPMAISSPPKAPLLAAPSPASRLQCINSNSRITSGQ WMAHMALLPSGTKGRCTACHTALGRGSLSSSSCPQPSPSLPASNKLPSLPLSKMYTTSMAMPILPLPQ LLLSADQQAAPRTNFHSSLAETVSLHPLAPMPSKTCHHK, AHQGFPAAKESRVIQLSLLSLLIPPLTCLASEALPRPLLALPPVLLSLAQDHSRLLQCQATRCI—ILGHPV ASRTASCILP.

GTSVRTAAR’IXWPRAAIRVPDPAAVPDDHAGPGAECHGRPLLYTADSSLWTFRPQRVWST GPDSILQPAKSSPSAAAATLLPATFVPDPSCPTFVSAAATVSTITAPVLSASILPAAIPASTSAVPGSIPL PAVDDTAAPPEPAPLLTATGSVSLPAAATSAASTLDALPAGCVSSAPVSAVPANCLFPAALPSTAGAIS RFIWVSGILSPLNDLQ, PCRAGRRVPWAASLIHSRFLLMDNKAPAGMVNRARLHlTTSKVLTLSSSSHPTPSNHRPRPLMPN LRl SSSHSLNHHSSSPLSLHTPSSHPSLHISSPRLHTPPSSRRHSSTPRASPPTHSHRLSLLTSSSNLSSQHPRR SPSRLRJLSPSLSSPSKLPIPSSASLHRRSYLKIHLGLRHPQPPQ, RTNPTVRMRPHCVPFWTGRILLPSAASVCPIPFEACHLCQAMTLRCPNTQGCCSSWAS. or TNQALPKIEVICRGTPRCPSTVPPSPAQPYLRVSLPEDRYTQAWAPTSRTPWGAMVPRGVSMAHKVA TPGSQTIMPCPMPTTPVQAWLEA. In some d embodiments, the neoantigenie peptide is not AAWRSCIAL, AAWRSCIALW, AETVSLHPL, AETVSLHPLA, AHMALLPSG, ALWCASSVT, AMPILPLPQL, APLLAAPSPA, APRTNFHSS, APRTNFHSSL, ASNKLPSLPL, AWRSCIALW, CAGRWLWYCW, CIALWCASSV, CPQPSPSLPA, CRRAVSATSW, CTACHTALGK DQQAAPRTNF, ETVSLl-IPLA. FHSSLAETV, GMYSPSRYPR, GQWMAHMAL. GQWMAI-IMALL, GRCTACHTAL, GTAWQLVPL, HMALLPSGT, SGTK, HPLAPMPSK, HSSLAETVSL, HTALGRGSL, IALWCASSV, ILATPPSAA, ILATPPSAAW, IPMAISSPP, IPMAISSPPK, ISSPPKAPL, ISSPPKAPLL, ITSGQWMAHM, CHT, KGRCTACHTA, KLPSLPLSK, LSKM, KMY'ITSMAM, KMYTTSMAMP, ASR, LAAPSPASRL, LAETVSLHPL, LATPPSAAW, LATPPSAAWR, LLAAPSPASR, LLLSADQQAA, LLSADQQAA, LPASNKLPS, LPASNKLPSL, LPLPQLLLS, LLSA. LPSLPLSKM. LPSLPLSKMY. LQCINSNSRI. LQCRRAVSAI LQCRRAVSAT.

LSADQQAAPR, LSKMYTTSM, ’TSMA, LWCASSVTER, LWYCWPTWL, TWLR, MAHMALLPS, MALLPSGTK, MPILPLPQL, MPILPLPQLL, YPR, MYTTSMAMPI, PMAISSPPK, QLVPLQCRR, QQAAPRTNF, QQAAPRTNFH, ALL, RAVSATSWA, RAVSATSWAS, RCAGRWLWY, RCTACHTAL, RGTAWQLVPL, RLQCINSNSR, RRAVSATSW, RTNFHSSLA, RTNFHSSLAE, RTRCAGRWL, RTRCAGRWLW, RWLWYCWPT, RWLWYCWPTW, SAAWRSCIA. CIAL. SGQWMAHMAL. SKMYTTSMA. SKMYTTSMAM. LPL.

SNKLPSLPL, QCI, SPPKAPLLAA, SPSLPASNKL, SRITSGQWM, SSCPQPSPSL, VSL, SSNDMIPMAI, SSSNDMIPM, SSSSNDMIPM, SSSSSSNDM, SSSSSSSNDM, TACHTALGR, TERTRCAGRW, TSMAMPILPL, TTSMAMPIL, TVSLHPLAPM, TWLRGTAWQL, VPLQCRRAV, VSLHPLAPM, VTER’I‘RCAGR, WLRGTAWQL, WLRGTAWQLV, WLWYCWPTW, WLWYCWPTWL, LLPS, WPTWLRGTA, WPTWLRGTAW, WYCWPTWLK SSS, YPRSSSSSSS, Y'ITSMAMPI. or YTI‘SMAMPIL.

In some embodiments. the isolated neoantigenic peptide is encoded by a [32M gene. In related embodiments, C2 is RMERELKKWSIQTCLSARTGLSISCTI‘LNSPPLKKMSMPAV, or LCSRYSLFLAWRLSSVLQRFRFTHVIQQRMESQIS. In some related embodiments, the neoantigenic peptide is not ALAVLALLSF, ALLSFWPGGY, DSGLLTSSSR, ELLCVWVSSI, EWKVKFPEL, FPELLCVWV. FWPGGYPAY, GLLTSSSREW, KFPELLCVW, KFPELLCVWV, KVKFPELLC.

KVKFPELLCV, LAVLALLSF, LAVLALLSFW, LLCVWVSSI, LLCVWVSSIK LLSFWPGGY, LLTSSSREW. LLTSSSREWK. LSFWPGGYPA. LTSSSREWK. LTSSSREWKV. REWKVKFPE.

REWKVKFPEL, SFWPGGYPA, SFWPGGYPAY, SSREWKVKF, SSSREWKVK, SSSREWKVKF, TSSSREWKV, TSSSREWKVK, VKFPELLCV, VKFPELLCVW, WKVKFPELL, or YPAYSKDSGL.

In some ments, the isolated neoantigenic e is encoded by a CDHl gene. In d embodiments, CZ is RSACVTVKGPLASVGRHSLSKQDCKFLPFWGFLEEFLLC, IQWGTITAPRPIRPPFLESKQNCSHFPTPLLASEDRRETGLFLPSAAQKMKKAHFLKTWFRSNPTKTK KARFSTASLAKELTI-IPLLVSLLLKEKQDG.

PTDPFLGLRLGLHLQKVFHQSHAEYSGAPPPPPAPSGLRFWNPSRIAHISQLLSWPQKTEERLGYSSHQ LPRK, FCCSCCFFGGERWSKSPYCPQRMTPGTTFITMMKKEAEKRTRTLT, or WRRNCKAPVSLRKSVQTPARSSPARPDRTRRLPSLGVPGQPWALGAAASRRCCCCCRSPLGSARSRS PATLALTPRATRSRCPGATWREAASWAE.

In some embodiments, the isolated igenie peptide is encoded by a GATA3 gene. In related embodiments. C7, is PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLW'ITPPLQHGHRHGLEPCSMLTGPPARVPA VPFDLI—IFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNI—I or PRPRRCTRHPACPLDI—ITI‘PPAWSPPWVRALLDAHRAPSESPCSPFRLAFLQEQYHEA. In some related embodiments, the neoantigenic peptide is not NVL, ADAPAIQPV, ADAPAIQPVL, AESKIMFAT, AESKIMFATL, AlQPVLWTF. PHA, APAIQPVLW, PFDL, ATLQRSSLW, AVPFDLHFCR. CSMLTGPPA. CSMLTGPPAR. DLHFCRSSI. DLHFCRSSIM. EPHLALQPL.

ESKIMFATL, FATLQRSSL, FATLQRSSLW, FCRSSIMKPK, FDLHFCRSSI, FLKAESKIM, FLKAESKIMF, GPPARVPAV, GYMFLKAESK, HADAPAIQPV, HAHADAPAI, HFCRSSIMK, HLALQPLQPH, HMSSLSHISA, HPLQHGHRH, SFW, HRHGLEPCSM, lMFATLQRS, QRSS, IMKPKRDGY, IMKPKRDGYM, MFA, KIMFATLQK KPKRDGYMF, KPKRDGYMFL, LALQPLQPH, LHFCRSSIM, LHFCRSSIMK, LKAESKIMF, LQHGHRHGL, HAH. LQRSSLWCL. LQRSSLWCLC. LSFGPHHPL. LSFGPHPPL. LSFWTTPPL.

LSHISALQPL, MFATLQRSSL, MFLKAESKI, SKIM, MHPPSSLSFW, MKPKRDGYMF, ARV, MSSLSHISA, MSSLSHISAL, NPAALSRHNV, PARVPAVPF, PAVPFDLHF, PEPHLALQPL, PPARVPAVPF, QPVLW'ITPPL, RHGLEPCSM, EPHL, RSSIMKPKR, SALQPLQPH, SHlSALQPL, SIMKPKRDGY, SLSFGPHHPL, SLSFGPHPPL, SLSFW’ITPPL, SMLTGPPAR, SMLTGPPARV, SSLSHISAL, TLQRSSLWCL, TTPPLQHGHR, LAL, VPAVPFDLHF. VPFDLHFCR. YMFLKAESK or YMFLKAESKI.

In some ments, the isolated neoantigenic peptide is encoded by a MLL2 gene. In related ments, C2 is TRRC LCLPHLNI—IHLFLI—IWRSRPCLHRKSHPHLLHLRRLYPHl-ILKHRPCPHHLKNLLCPRHLRNCPLPRHLK HLACLHHLRSHPCPLHLKSHPCLI—IHRRHLVCSHHLKSLLCPLHLRSLPFPHHLRHHACPHHLRTRLCP HHLKNHLCPPHLRYRAYPPCLWCHACLHRLRNLPCPHRLRSLPRPLHLRLHASPHHLRTPPHPHHLR THLLPHHRRTRSCPCRWRSHPCCHYLRSRNSAPGPRGRTCHPGLRSRTCPPGLRSHTYLRRLRSHTCP PSLRSHAYALCLRSHTCPPRLRDHICPLSLRNCTCPPRLRSRTCLLCLRSHACPPNLRNHTCPPSLRSHA CPPGLRNRICPLSLRSHPCPLGLKSPLRSQANALHLRSCPCSLPLGNHPYLPCLESQPCLSLGNHLCPLC PRSCRCPHLGSHPCRLS. In some related embodiments, the igenic peptide is not APGPRGRTC, CHYLRSRNSA, CLRSHTCPPR, CLWCHACLHK CPHLGSHPC, CPHRLRSLPR, SPL, CPPGLRNRI, CPPGLRSHTY, CPPRLRDHI, CPPSLRSHAY, CPRSCRCPH, CPRSCRCPHL, CSLPLGNHPY. CTCPPRLRSR DHICPLSLR. EESPMSPHL. ESPMSPHLR. ESPMSPHLRY.

EVSRLSPCL, GLKSPLRSQA, CPL, GLRNRICPLS, GLRSHTYLR, GLRSHTYLRR, CPPG, HACPPGLRNR, HAYALCLRSH, HHLRTHLLPH, HLGSHPCRL, HLLPHHRRTR, HLRLHASPH, HLRLHASPHH, HLRSCPCSL, HLRTHLLPH, HLRTHLLPHH, HLRTPPHPH, HLRTPPHPHH, HLRYRAYPP, HLRYRAYPPC, HPCCHYLRSK HPHHLRTHL, HPHHLRTHLL, HRLRSLPRPL, HTYLRRLRS, HTYLRRLRSH, HYLRSRNSA, KSPLRSQANA, LESQPCLSL, LHLRLHASPH, PCSL. LLPHHRRTR, LPCPHRLRSL. RRTRS, LPl-IHRRTRSC, LPLGNHPYL, LPRPLHLRL, LRLHASPHHL, LRNCTCPPRL, LRNHTCPPSL, LRNRICPLSL, LRSCPCSLPL, LRSHACPPGL, LRSHACPPNL, LRSHAYALCL, LRSHTCPPRL, LRSHTCPPSL, LRSHTYLRRL, LRSLPRPLHL, LRSQANALHL, LRTPPHPHl-IL, LRYRAYPPCL, LSLGNHLCPL, LSLRSHPCPL, LWCHACLHRL, MSPHLRYRA, MSPHLRYRAY, NLPCPHRLR, NLRNHTCPP, PMSPHLRYR, PPRLRSRTCL, PPSLRSHAY, RAYPPCLWCH, RDHICPLSL, RGRTCHPGL, RGRTCHPGLR. RLHASPHHL. RLHASPHHLR. CPL. RLRDHICPLS. RLRNLPCPH.

RLRNLPCPHR, RLRSI-ITCPP, CPPS, RLRSLPRPL, RLRSLPRPLH, RLRSRTCLL, RLRSRTCLLC, RLSPCLWCHA, RNHTCPPSL, PSLR, RNLPCPHRLR, LSL, RNRICPLSLR, RPLHLRLHA, RPLHLRLHAS, RSCPCRWRSH, RSCPCSLPL, RSHACPPGL, RSHACPPGLR, RSHACPPNL, RSI-IACPPNLK RSHAYALCL, RSHAYALCLR, RSI-IPCCI-IYL, RSHPCCHYLR RSHPCPLGL, RSHPCPLGLK, RSHTCPPRL, RSHTCPPRLR, RSHTCPPSL, RSHTCPPSLR. RSHTYLRRL. RSHTYLRRLR. RSLPRPLHL. RSLPRPLI-ILR RSQANALI-IL.

LHLR, RSRNSAPGP, RSRNSAPGPR, RSRTCLLCL, RSRTCLLCLR, RSRTCPPGL, PGLR, RTCI—IPGLRSR, RTHLLPHHR, RTHLLPHHRR, RTPPHPI-{I-IL, RTPPHPI—IHLR, RTRSCPCRW, CRWK RWRSHPCCH, CCHY, RYRAYPPCL, PCLW, CLR, SLGNHLCPL, SLPLGNHPY, SLPLGNHPYL, SLPRPLHLR, SLPRPLHLRL, CPPR, SLRSHACPPG, SLRSHAYAL, SLRSHAYALC, SLRSHPCPL, SLRSHPCPLG, SPI-IHLRTPP. SPI-II-ILRTPPI-I. SPHLRYRAY, ANAL, SPMSPI—ILRY. SPMSPHLRYK SQANALHLK SQANALHLRS, VSRLSPCLW, WRSHPCCHY, WRSHPCCHYL. YLPCLESQPC, YLRRLRSHTC, YLRSRNSAP, or YLRSRNSAPG.

In some embodiments, the isolated neoantigenic peptide is encoded by a PTEN gene. In related embodiments, CL is SWKGTNWCNDMCIFITSGQIFKGTRGPRFLWGSKDQRQKGSNYSQSEALCVLL, KRTKCFTFG, PIFIQTLLLWDFLQKDLKAYTGTILMM, QKMILTKQIKTKPTDTFLQILR: GFWIQSIKTITRYTIFVLKDIMTPPNLIAELHNILLKTITHHS, NYSNVQWRNLQSSVCGLPAKGEDIFLQFRTI-I'ITGRQVHVL. or YQSRVLPQTEQDAKKGQNVSLLGKYILHTRTRGNLRKSRKWKSM. In some related embodiments, the neoantigenic peptide is not KMLKRTKCF, MLKRTKCFT, LKRTKCFTF, MLKRTKCFTF, KQNKMLKRTK, KMLKRTKCFT, or NKMLKRTKCF.

In some embodiments, the isolated igenic peptide is encoded by a TP53 gene. In related embodiments, CZ is SSQNARGCSPRGPCTSSSYTGGPCTSPLLAPVIFCPFPENLPGQLRFPSGLLAFWDSQVCDLHVLPCPQ QDVLPTGQDLPCAAVG, GAAPTMSAAQIAMVWPLLSILSEWKEICVWSIVVMTETLFDIVVVWCPMSRLRLALTVPPSTTITCVTV PAWAA, TGGPSSPSSHWKTPVVIYWDGTALRCVFVPVLGETGAQRKRISARKGSLTTSCPQGALSEHCPTTPAP LPSQRRNHWMENISPFRSVGVSASRCSES, FHTPARHPRPRHGHLQAVTAHDGGCEALPPP, CCPRTILNNGSLKTQVQMKLPECQRLLPPWPLHQQLLI-IRRPLHQPPPGPCHLLSLPRKPTRAATVSV WASCILGQPSL, VRKHFQTYGNYFLK'ITFCPPCRPKQWMI, or LARTPLPSTRCFANWPRPALCSCGLIPHPRPAPASAPWPSTSSHST. In some related embodiments, the neoantigenic e is not APASAPWPST, APPWPLHQQL, APWPSTSSH, ASCILGQPSL, ATVSVWASCI, CQRLLPPWPL, HQPPPGPCHL, HQQLLHRRPL, IEQWFTEDQV, KLPECQRLL, TVSV, KTYQGSYGFV, KTYQGSYVS, KTYQGSYVSV, LLSLPRKPTR, LPPWPLHQQL, LPRKPTRAA. LPRKPTRAAT. LSLPRKPTR. MKLPECQRL. MKLPECQRLL. MPEAAPPWPL.

WPL, PTRAATVSV, QMKLPECQK QQLLHRRPL. QQLLHRRPLH, QRLLPPWPL, QWFTEDQVQM, RAATVSVWA, RLLPPWPLH, RMPEAAPPW, RPAPASAPW, SLPRKPTRA, SLPRKPTRAA, SQKTYQGSYV, STPRPAPASA, SYGFVWASC, SYGFVWASCI, SYVSVWASC, SYVSVWASCI, TEDQVQMKL, TPRPAPASA, TPRPAPASAP, TRAATVSVW, TVSVWASCI, TVSVWASCIL, GFV, TYQGSYGFVW, TYQGSYVSV, TYQGSYVSVW, VSVWASCIL, WPLHQQLLH. WPSTSSHST. YGFVWASCI. YGFVWASCIL. YQGSYGFVW. YQGSYGFVWA.

YQGSYVSVW, YQGSYVSVWA, YVSVWASCI, or YVSVWASCIL.

In some embodiments, the isolated neoantigenic peptide is encoded by a VHL gene. In related embodiments, C is ELQETGHRQVALRRSGRPPKCAERPGAADTGAHCTSTDGRLKISVETYTVSSQLLMVLMSLDLDTGL VPSLVSKCLILRVK, KSDASRLSGA, RTAYFCQYHTASVYSERAMPPGCPEPSQA, TRASPPRSSSAIAVRASCCPYGSTSTASRSPTQRCRLARAAASTATEVTFGSSEMQGHTMGFWLTKLN YLCI-ILSMLTDSLFLPISHCQCIL, SSLRITGDWTSSGRSTKIWK'ITQMCRKTWSG, or RRRRGGVGRRGVRPGRVRPGGTGRRGGDGGRAAAARAALGELARALPGHLLQSQSARRAARMAQ ALPNAAAWHGPPI—IPQLPRSPLALQRCRDTRWASG. In some related ments, the neoantigenic peptide is not CI-ILSMLTDSL, EMQGHTMGF, FLPISI—ICQC, FLPISHCQCI, FRDAGHTMGF, FWLTKLNYL, GFWLTKLNY, GFWLTKLNYL, GSSEMQGI—ITM, HLSMLTDSL, HLSMLTDSLF, HSYRGHLGSS, HTMGFWLTK, HTMGFWLTKL, KERCLQLSGA, HLSM, HTM, LNYLCHLSM, LNYLCI-ILSML. LPISHCQCI, LPISI-ICQCIL. LSMLTDSLF, LSMLTDSLFL. LTDSLFLPI.

LCHL, MGFWLTKLNY, MLTDSLFLPI, MQGHTMGFW, MQGHTMGFWL, NYLCHLSML, RDAGHTMGF, RDAGHTMGFW, RGHLGSSEM, RIHSYRGHLG, SEMQGHTMG, SEMQGHTMGF, SLFLPISHC, SMLTDSLFL, SSEMQGHTM. TKLNYLCHL, TLKERCLQL, TMGFWLTKL, WLFRDAGHT, WLFRDAGHTM, YLCHLSMLT, or YRGHLGSSEM.

In some ments. the isolated neoantigenic peptide is d by a fusion of a first gene with a second gene. In some ments. the isolated neoantigenic peptide is encoded by an iii-frame fusion ofa first gene with a second gene.

In some embodiments, the isolated neoantigenic peptide is encoded by a BCR gene and an ABL gene.

In related embodiments, AxByCL is ERAEWRENIREQQKKCFRSFSLTSVELQMLTNSCVKLQTVHSIPLTINKEEALQRPVASDFEPQGLSEA ARWNSKENLLAGPSENDPNLFVALYDFVASG or ELQMLTNSCVKLQTVI-ISIPLTINKEDDESPGLYGFLNVIVHSATGFKQSSKALQRPVASDFEPQGLSE AARWNSKENLLAGPSENDPNLFVALYDFVASGD.

In some embodiments, the ed neoantigenic peptide is encoded by a Cl lorf95 gene and a RELA gene. In d embodiments, AxByCZ is ISNSWDAHLGLGACGEAEGLGVQGAEEEEEEEEEEEEEGAGVPACPPKGPELFPLIFPAEPAQASGPY VEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTD.

In some embodiments, the isolated neoantigenie peptide is encoded by a CBFB gene and an MYl-Ill gene.

LQRLDGMGCLEFDEERAQQEDALAQQAFEEARRRTREFEDRDRSHREEMEVHELEKSKRALETQME EMKTQLEELEDELQATEDAKLRLEVNMQALKGQF.

In some embodiments, the ed neoantigenie peptide is encoded by a CD74 gene and an ROSl gene. In related embodiments. AxByC-L is KGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKAGVPNKPGIPKLLEGS KNSIQWEKAEDNGCRITYYILEIRKSTSNNLQNQ.

In some embodiments, the isolated neoantigenie peptide is encoded by a EGFR gene and an SEPT14 gene. In related embodiments, AxByCZ is the first native polypeptide is d by an gene and the second native polypeptide is d by CTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQLQDKFEHLKMIQQEEIRKL EEEKKQLEGEIIDFYKMKAASEALQTQLSTD.

] In some embodiments. the isolated neoantigenie e is encoded by a EGFR gene and an EGFR gene. In related embodiments, AxByCz is MRPSGTAGAALLALLAALCPASRALEEKKGNYVVTDI-IGSCVRACGADSYEMEEDGVRKCKKCEGP CRKVCNGIGIGEFKD.

In some embodiments, the isolated neoantigenie peptide is encoded by a EML4 gene and an ALK gene. In related ments. AxByCz is SWENSDDSRNKLSKIPSTPKLIPKVTKTADKHKDVIINQAKMSTREKNSQVYRRKHQELQAMQMEL QSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDL.

In some embodiments, the isolated neoantigenie peptide is encoded by a FGFR3 gene and an TACC3 gene. In related embodiments, AxByCZ is EGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDRVLTVTSTDVKATQEENRELRSRCE ELHGKNLELGKIMDRFEEVVYQAMEEVQKQKELS.

In some embodiments, the isolated neoantigenie peptide is encoded by a NAB gene and an STAT6 gene. In related embodiments, AKByCZ is RDNTLLLRRVELFSLSRQVARESTYLSSLKGSRLHPEELGGPPLKKLKQEATSKSQIMSLWGLVSKMP PEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCC.

In some embodiments, the isolated neoantigenie peptide is encoded by a NDRGI gene and an ERG gene. In related embodiments, ANByC, is DVDLAEVKPLVEKGETITGLLQEFDVQEALSVVSEDQSLFECAYGTPHLAKTEMTASSSSD YGQTSKMSPRVPQQDW.

In some embodiments. the isolated neoantigenic e is encoded by a TMPRSSZ gene and an ERG gene. In related embodiments. AxByC, is MALNSEALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDW.

In some embodiments, the isolated neoantigenic peptide is d by a PML gene and an RARA gene. In related embodiments, ARByCL is VLDMHGFLRQALCRLRQEEPQSLQAAVRTDGFDEFKVRLQDLSSCITQGKAIETQSSSSEEIVPSPPSP PPLPRIYKPCFVCQDKSSGYHYGVSACEGCKG or RSSPEQPRPSTSKAVSPPI-ILDGPPSPRSPVIGSEVFLPNSNHVASGAGEAAIETQSSSSEEIVPSPPSPPPL CFVCQDKSSGYHYGVSACEGCKG.

In some embodiments, the ed neoantigenic peptide is encoded by a RUNXI gene and an CBFAZTI (RUNXITI) gene. In related embodiments, AxByCL is VARFNDLRFVGRSGRGKSFTLTITVFI'NPPQVATYHRAIKITVDGPREPRNRTEKHSTMPDSPVDVKT QSRLTPPTMPPPPTTQGAPRTSSFTPTTLTNGT In some embodiments. the isolated neoantigenic peptide is encoded by a fusion of a first gene with an exon of a splice variant of the first gene. In some embodiments, the isolated neoantigenic peptide is encoded by a fusion of a first gene with a cryptic exon ofthe first gene.

In some embodiments, the isolated neoantigenic peptide is encoded by a AR-v7 gene and a c exon encoded by the AR—v7 gene. In some embodiments, the isolated neoantigenic peptide is encoded by a AR-v7 gene comprising an exon of a splice t of an AR gene. In related embodiments, AxByCZ is SCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGEKFRVGNCKHLKMTRP In some embodiments, the isolated neoantigenic peptide is encoded by a fusion of a first gene with a second gene, wherein the peptide comprises an amino acid sequence encoded by an out-of frame sequence resulting from the fusion.

In some embodiments, the isolated neoantigenic peptide is encoded by an AC011997.I gene and a LRRC69 gene. In related embodiments, y is I. In related embodiments, AXB,CZ is MAGAPPPASLPPCSLISDCCASNQRDSVGVGPSEPGNNIKICNESASRK.

In some embodiments, the isolated neoantigenic peptide is encoded by an EEFI DP3 gene and a FRY gene. In related embodiments, y is I. In related embodiments, AxBycz is HGWRPFLPVRARSRWNRRLDVTVANGRSWKYGWSLLRVPQVNGIQVLNVSLKSSSNVISY.

In some embodiments, the isolated neoantigenic e is encoded by an MADILI gene and a MAFK gene. In related embodiments, y is 0. In related ments, AxByCZ is QTKIQEFRKACYTLTGYQIDITTENQYRLTSLYAEHPGDCLIFKLRVPGSSVLVTVPGL.

In some embodiments, the isolated neoantigenic peptide is encoded by an PPPIRIB gene and a STARD3 gene. In related ments, y is 1. In related embodiments, AxByCZ is AEVLKVIRQSAGQKTI‘CGQGLEGPWERPPPLDESERDGGSEDQVEDPALSALLLRPRPPRPEVGAHQ DEQAAQGADPRLGAQPACRGLPGLLTVPQPEPLLAPPSAA.

In some ments, the isolated neoantigenic peptide comprises one or more of the e sequences depicted in Table I. In some embodiments. the isolated neoantigenic peptide comprises one or more of the peptide sequences depicted in Table IA, Table 18, Table 1C, Table 1D, Table 1E, and/or Table lF. In some embodiments, the isolated neoantigenic peptide does not comprise one or more of the peptide ces ed in Table 2. In some embodiments, the isolated neoantigenic peptide does not se one or more of the peptide sequences depicted in Table 2A, Table 2B, Table 2C, and/or Table 2D.

Neoantigen polynucleotides Polynucleotides encoding each of the peptides described herein are also part of the invention. As appreciated by one of ordinary skill in the art, various nucleic acid sequences can encode the same e due to the redundancy of the genetic code. Each of these nucleic acids falls within the scope of the present invention. Nucleic acids encoding peptides can be DNA or RNA, for example, mRNA, or a combination of DNA and RNA. In some embodiments, a nucleic acid encoding a peptide is a self—amplifying mRNA. (Brito ct al., Adv. Genet. 2015; 89: 179-233). Any suitable polynueleotide that encodes a peptide described herein falls within the scope of this ion.

The temi RN“A” includes and in some embodiments relates to “mRNA”. The term “mRNA" means “messenger-RNA” and relates to a “transcript” which is generated by using a DNA te and s a peptide or polypeptide. lly, an mRNA ses a 5'-UTR, a protein coding region, and a 3'-UTR. mRNA only possesses limited half-life in cells and in vitro. In some embodiments, the mRNA is self- amplifying mRNA. In the context ofthe present invention, mRNA may be generated by in vitro transcription from a DNA template. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro ription kits commercially ble.

The stability and translation efficiency ofRNA may be modified as required. For example, RNA may be stabilized and its translation increased by one or more modifications having a stabilizing s and/or increasing translation efficiency of RNA. Such modifications are described, for example, in incorporated herein by reference. In order to increase expression of the RNA used according to the present invention, it may be modified within the coding region, i.e. the sequence encoding the expressed peptide or n. without altering the sequence of the expressed peptide or protein. so as to increase the GC—content to increase mRNA stability and to perform a codon optimization and, thus, enhance translation in cells.

The term “modification” in the context of the RNA used in the present invention includes any modification of an RNA which is not naturally present in said RNA. In some embodiments of the invention, the RNA used according to the invention does not have uncapped 5'-triphosphates. Removal of such uncapped phosphates can be achieved by treating RNA with a phosphatase. The RNA ing to the invention may have modified ribonucleotides in order to increase its stability and/or decrease cytotoxicity. For example, In some embodiments, in the RNA used ing to the invention S-methylcytidine is substituted partially or completely, for example, completely, for cytidine. Alternatively or additionally, In some embodiments, in the RNA used according to the invention pseudouridine is substituted partially or completely, for example, completely. for uridine.

In some embodiments the term “modification“ relates to providing an RNA with a 5'-cap or 5‘- cap analog. The term "5'-cap" refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide ted to the mRNA via an unusual 5' to 5' triphosphate linkage. In some embodiments, this guanosine is ated at the 7-position. The term “conventional 5'-cap” refers to a naturally occurring RNA , to the 7-methylguanosine cap (m G). In the context of the present invention, the term “5'-cap” includes a 5'—cap analog that les the RNA cap structure and is modified to possess the ability to stabilize RNA and/or enhance translation of RNA if attached thereto, in vivo and/or in a cell.

In certain embodiments, an mRNA encoding a igen peptide of the invention is administered to a subject in need thereof. In some embodiments, the invention provides RNA, oligoribonucleotide, and polyribonucleotide molecules comprising a modified nucleoside, gene therapy vectors comprising same, gene therapy methods and gene transcription silencing methods comprising same. In some embodiments, the mRNA to be administered comprises at least one modified nucleoside.

The polynucleotides encoding peptides described herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (198]).

Polynucleotides encoding peptides comprising or consisting of an analog can be made simply by tuting the appropriate and desired nucleic acid base(s) for those that encode the native epitope.

A large number of vectors and host systems suitable for producing and administering a igenic peptide described herein are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60. pQE-9 (Qiagen), pBS, leO. phagescript, psiX 174, pBluescript SK, pbsks, pNHSA, pNI-Il6a, le-IISA, pNH46A (Stratagene); ptre99a, pKK223-3, pKK233-3, pDR540, pRITS (Phannacia): pCR (Invitrogen). Eukaryotic: pWLNEO, pSVZCAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); p756 tis); pCEP (lnvitrogen); pCEI une). However, any other d or vector can be used as long as it is replicable and viable in the host.

As representative examples of riate hosts. there can be mentioned: bacterial cells. such as E. coli, Bacillus subtilis. Salmonella urium and various species within the genera Pseudomonas, .S'trepfomyces, and .S‘taphylococcus; fiingal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cells such as COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23: 175 (1981), and other cell lines e of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines or Bowes melanoma: plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings .

Thus, the present invention is also ed to s, and expression s useful for the production and administration of the igenic peptides described herein, and to host cells comprising such vectors.

Host cells are genetically ered (transduced or transfomied or transfected) with the vectors which can be. for example. a cloning vector or an expression vector. The vector can be, for example. in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the polynucleotides. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

For expression of thc ncoantigcnic peptides bed herein, the coding sequence will be provided operably linked start and stop codons. promoter and terminator regions. and in some embodiments. and a ation system to provide an expression vector for expression in the desired cellular host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the d coding sequence. The resulting expression s are transformed into suitable bacterial hosts.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transfonnation of the host cell, e.g., the arnpicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly—expressed gene to direct transcription of a ream structural sequence. Such promoters can be d from operons encoding glycolytic enzymes such as 3- phosphoglycerate kinase (PGK), acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation tion and termination sequences, and in some embodiments, a leader sequence capable of ing ion of translated protein into the periplasmic space or extracellular medium. Optionally, the logous sequence can encode a fiision protein including an N-terminal identification peptide imparting desired characteristics, e.g.. stabilization or simplified purification of expressed recombinant product.

Yeast, insect or ian cell hosts can also be used, employing suitable vectors and control sequences. Examples of ian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23: 175 , and other cell lines capable of expressing a compatible vector, for example, the C 127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will se an origin of replication. a suitable promoter and enhancer, and also any ary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nscribed sequences. Such promoters can also be derived fiom viral sources, such as, e.g., human cytomegalovirus (CMV-IE promoter) or herpes x virus type-l (HSV TK promoter). Nucleic acid sequences derived from the SV40 , and polyadenylation sites can be used to provide the required nontranscribed genetic elements.

Poly-nucleotides encoding ncoantigcnic es described herein can also comprise a ubiquitination signal sequence, and/or a targeting sequence such as an endoplasmic reticulum (ER) signal sequence to facilitate movement of the resulting peptide into the endoplasmic reticulum.

Polynucleotides described herein can be administered and expressed in human cells (e.g., immune cells, including dendritic cells). A human codon usage table can be used to guide the codon choice for each amino acid. Such polynucleotides comprise spacer amino acid es between epitopes and/or analogs, such as those bed above. or can comprise naturally-occurring flanking sequences adjacent to the epitopes and/or s (and/or CTL, I-ITL, and B cell epitopes).

In some ments, a neoantigenic peptide described herein can also be administered/expressed by viral or bacterial s. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. As an example of this ch, vaccinia virus is used as a vector to express nucleotide sequences that encode the neoantigenic peptides described . Vaccinia vectors and methods useful in immunization protocols are described in. e.g.. US. Pat. No. 4.722.848. Another vector is BCG (Bacille Calmette ).

BCG vectors are described by Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the neoantigenie polypeptides described herein, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified x toxin vectors, Sendai virus vectors, poxvirus vectors, canarypox vectors, and x vectors, and the like, will be apparent to those skilled in the art from the description herein. In some embodiments, the vector is Modified Vaccinia Ankara (VA) (e.g. Bavarian Noridic (MVA-BN)).

Standard regulatory sequences well known to those of skill in the art can be included in the vector to ensure expression in the human target cells. Several vector elements are desirable: a promoter with a ream cloning site for polynucleotide, e.g., ne insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human eytomegalovirus (hCMV) promoter. See, eg, US. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences. In some embodiments, the promoter is the CMV-lE promoter.

Polynucleotides described herein can se one or more synthetic or naturally-occurring introns in the transcribed region. The inclusion of mRNA ization sequences and ces for replication in mammalian cells can also be considered for increasing polynucleotide expression.

In addition, a polynucleotide described herein can se immunostimulatory sequences (ISSs or Cst). These sequences can be included in the vector, outside the polynucleotide coding sequence to e immunogenicity.

In some embodiments, the size of at least one antigenic peptide molecule may comprise, but is not limited to, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70. about 80, about 90. about 100. about 110, about 120 or greater amino molecule residues. and any range derivable therein.

In some embodiments, the antigenic peptide molecules are equal to or less than 50 amino acids. In some embodiments, the antigenic peptide molecules are equal to about 20 to about 30 amino acids. A longer peptide may be designed in several ways. For example, when the HLA-binding regions are predicted or known, a longer peptide may consist of : individual binding peptides with an extension of 0-10 amino acids toward the N- and C-terminus of each corresponding gene product. A longer peptide may also consist of a concatenation of some or all ofthe binding peptides with extended sequences for each. In another case, when cing reveals a long (>10 residues) epitope sequence present in the diseased tissue (eg. due to a frameshift, read-through or intron inclusion that leads to a novel peptide sequence), a longer peptide may consist of the entire stretch of novel disease-specific amino acids. In both cases, use of a longer peptide requires endogenous processing by professional n presenting cells such as dcndritie cells and may lead to more effective antigen presentation and induction of T cell responses. In some embodiments. the extended sequence is d to improve the biochemical properties of the polypeptide rties such as solubility or stability) or to improve the likelihood for efficient somal processing of the peptide.

The antigenic peptides and ptides may bind an HLA protein. In some embodiments, the nic peptides may bind an HLA protein with r affinity than a ponding native / wild-type peptide. The antigenic peptide may have an IC50 of about less than 1000 nM, about less than 500 nM, about less than 250 nM. about less than 200 nM. about less than 150 nM. about less than 100 nM. or about less than 50 nM. In some embodiments, the antigenic peptides do not induce an autoimmune response and/or invoke immunological tolerance when administered to a subject.

The invention also provides compositions sing a plurality of antigenic peptides. Reference to antigenic peptides includes any suitable delivery modality that can result in introduction ofthe peptide into a subject‘s cell (e.g., nucleic acid). In some ments, the composition comprises at least 2 or more antigenic peptides. In some ments, the composition contains at least about 3, 4, 5, 6, 7, 8, 9, 10, l l, 12, l3. 14, 15. 16. 17, 18. 19. 20. 21. 22. 23. 24, 25. 26. 27. 28, 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 distinct peptides. In some embodiments, the ition contains at least one peptide from Table I. In some embodiments, the composition contains at least one peptide from Table l and at least one other peptide from Table 1. In some embodiments, the composition contains at least one peptide from Table l and at least one other peptide from Table 2. In some embodiments the composition contains at least 20 distinct peptides. In some ments the composition contains at most 20 distinct peptides. According to the invention. 2 or more of the distinct peptides may be derived from the same polypeptide. For example, if an antigenic mutation s a ptide, two or more of the antigenic peptides may be derived from the polypeptide. In some embodiments, the two or more antigenic peptides derived from the ptide may comprise a tiled array that spans the polypeptide (e.g., the antigenic peptides may comprise a series of overlapping antigenic peptides that spans a portion, or all, of the polypeptide). Antigenic peptides can be derived from any protein coding gene. The antigenic peptides can be derived from mutations in human cancer or from an infectious agent or an autoimmune disease.

The antigenic peptides, polypeptides, and analogs can be further modified to contain additional chemical moieties not normally part of the protein. Those derivatized moieties can improve the solubility, the biological half-life, absorption of the n, or g affinity. The moieties can also reduce or eliminate any ble side s of the proteins and the like. An overview for those moieties can be found in Remington's ceutical Sciences. 20th ed.. Mack Publishing Co.. Easton. PA (2000). For example. nic peptides and polypeptides having the desired activity may be modified as necessary to provide certain desired attributes, eg. improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological ty of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell. For instance, the antigenic e and polypeptides may be subject to various changes, such as substitutions, either conservative or nservative, where such changes might provide for certain advantages in their use. such as improved MHC binding. Such conservative substitutions may encompass ing an amino acid residue with r amino acid residue that is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another. The effect of single amino acid tutions may also be probed using D- amino acids. Such modifications may be made using well known peptide synthesis procedures, as described in e.g., Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Mcicnhofcr, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford. Ill, Pierce). 2d Ed. (1984).

The antigenic peptide may also be modified by extending or decreasing the compounds amino acid sequence, e.g., by the addition or deletion of amino acids. The antigenic peptides, polypeptides, or analogs can also be modified by ng the order or composition of certain residues. It will be appreciated by the skilled artisan that certain amino acid residues ial for biological activity, e.g., those at critical contact sites or conserved residues, may lly not be altered without an adverse effect on biological ty. The non- critical amino acids need not be d to those naturally occurring in proteins, such as L-a— amino acids, or their D-isomers, but may include non-natural amino acids as well. such as [55- amino acids. as well as many derivatives of L-a-amino acids.

An antigen peptide may be optimized by using a series of peptides with single amino acid substitutions to determine the effect of electrostatic charge, hydrophobicity, etc. on MHC binding. For instance, a series of positively d (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions may be made along the length of the peptide revealing different patterns of sensitivity towards various MHC molecules and T cell receptors. In addition. multiple tutions using small. relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be employed. The substitutions may be homo- oligomers or hetero-oligomers. The number and types of residues which are tuted or added depend on the g necessary between essential contact points and certain fiinctional attributes which are sought (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for an MHC molecule or T cell receptor may also be achieved by such substitutions, compared to the affinity ofthe parent peptide. In any event, such substitutions should employ amino acid residues or other molecular fragments chosen to avoid. for example. steric and charge interference which might disrupt binding. Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final peptide.

An antigenic peptide may be modified to provide desired attributes. For instance, the y of the peptides to induce CTL activity can be ed by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. In some embodiments, immunogenic peptides/T helper ates are linked by a spacer molecule. In some embodiments, a spacer comprises relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. Spacers can be selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same es and thus may be a hetero- or homo-oligomer. The -antigenic peptide may be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the peptide. The amino terminus of either the antigenic peptide or the T helper peptide may be acylated.

Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378- 389.

The present invention is based, at least in part, on the ability to present the immune system of the t with one or more e-specific antigens. One of skill in the art from this disclosure and the knowledge in the art will appreciate that there are a variety of ways in which to produce such disease specific ns. In general, such disease specific antigens may be ed either in vitro or in vivo. e specific antigens may be produced in vitro as peptides or polypeptides, which may then be formulated into a vaccine or immunogenic composition and administered to a subject. As described in further detail herein, such in vitro production may occur by a variety of methods known to one of skill in the art such as, for example, peptide synthesis or expression ofa peptide/polypeptide from a DNA or RNA molecule in any ofa variety of bacterial. otic. or viral recombinant expression systems. followed by purification of the expressed peptide/polypeptide. tively, disease specific antigens may be produced in vivo by introducing molecules (e.g., DNA, RNA, viral expression systems, and the like) that encode disease specific ns into a subject, whereupon the encoded disease specific antigens are expressed. The methods of in vitro and in vivo production of antigens is also further described herein as it relates to pharmaceutical compositions and methods of delivery of the therapy.

In some embodiments. the present invention includes modified antigenic peptides. A ation can include a covalent chemical modification that does not alter the primary amino acid sequence of the antigenic peptide itself. Modifications can produce es with desired properties, for example, prolonging the in vivo half-life, increasing the stability, reducing the clearance, altering the immunogenicity or allergenicity, enabling the g of particular antibodies, cellular targeting, antigen , antigen processing, MHC affinity, MHC stability, or antigen presentation. Changes to an antigenic peptide that may be carried out include. but are not limited to, conjugation to a r protein. conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation HESylation, inant PEG cs, Fc fusion, albumin fusion, nanoparticle ment, rticulate encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, the addition of a surface active material, the addition of amino acid mimetics, or the on of unnatural amino acids.

Issues associated with short plasma half- life or susceptibility to protease degradation may be overcome by various modifications. including conjugating or linking the polypeptide sequence to any of a variety of non-proteinaceous rs, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes (see, for example, typically via a linking moiety covalently bound to both the protein and the nonproteinaceous polymer, e.g., a PEG). Such PEG conjugated biomolecules have been shown to possess clinically useful properties, including better physical and l stability, protection against susceptibility to enzymatic degradation, increased solubility, longer in vivo circulating half-life and sed clearance, reduced immunogenicity and antigenicity. and reduced toxicity.

PEGs suitable for conjugation to a polypeptide sequence are generally soluble in water at room temperature, and have the general formula R(0-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a tive group, it generally has from 1 to 8 carbons. The PEG conjugated to the polypeptide sequence can be linear or ed. Branched PEG derivatives, "star-PEGs” and multi-amicd PEGs are contemplated by the present disclosure.

The present sure also contemplates compositions of conjugates wherein the PEGs have different n values and thus the various different PEGs are present in specific ratios. For example, some compositions comprise a mixture of conjugates where n=1, 2, 3 and 4. In some compositions, the tage of conjugates where n=l is 18—25%, the percentage of ates where n=2 is , the percentage of conjugates where n=3 is 12-16%, and the percentage of ates where n=4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods know in the art. For e, cation exchange chromatography may be used to separate conjugates. and a fraction is then identified which contains the conjugate having, for example, the desired number of PEGs attached, purified free from unmodified protein sequences and from conjugates having other numbers of PEGs ed.

PEG may be bound to a polypeptide of the present disclosure via a terminal reactive group (a "spacer”). The spacer is, for example, a terminal ve group which mediates a bond between the free amino or carboxyl groups of one or more of the polypeptide sequences and polyethylene glycol. The PEG having the spacer which may be bound to the free amino group includes N-hydroxysuccinylimide polyethylene glycol which may be prepared by activating succinic acid ester of polyethylene glycol with N— y ylimide. Another activated hylene glycol which may be bound to a free amino group is 2,4-bis(0- methoxypolyethyleneglycol)chloro-s-triazine which may be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride. The activated polyethylene glycol which is bound to the free carboxyl group es polyoxycthylencdiamine.

Conjugation ofone or more of the polypeptide sequences of the present disclosure to PEG having a spacer may be carried out by various conventional s. For example, the conjugation reaction can be d out in solution at a pH of from 5 to 10, at temperature from 4°C to room temperature, for 30 minutes to hours, utilizing a molar ratio of reagent to protein of from 4: l to 30: 1. Reaction conditions may be selected to direct the reaction towards producing predominantly a desired degree of substitution. In general, low temperature, low pH (e.g., pH=5), and short reaction time tend to decrease the number of PEGs ed, whereas high temperature. neutral to high pH (e.g.. pH>7). and longer reaction time tend to se the number of PEGs attached. Various means known in the art may be used to terminate the reaction. In some embodiments the reaction is terminated by acidifying the reaction mixture and freezing at, e.g., -20°C.

The present disclosure also contemplates the use of PEG mimetics. Recombinant PEG mimetics have been developed that retain the utes of PEG (e.g., enhanced serum half— life) while conferring several onal advantageous properties. By way of example, simple polypeptide chains (comprising, for example, Ala. Glu. Gly. Pro. Ser and Thr) capable of g an extended conformation similar to PEG can be produced recombinantly already fused to the peptide or protein drug of interest (e.g., Amunix' XTEN technology; Mountain View, CA). This obviates the need for an additional conjugation step during the manufacturing process. Moreover, established lar biology techniques enable control of the side chain composition of the polypeptide , allowing optimization of genicity and manufacturing properties.

Glycosylation can affect the physical properties of ns and can also be important in protein stability, secretion, and subcellular localization. Proper glycosylation can be important for biological activity.

In fact, some genes from eukaryotic organisms, when expressed in ia (e.g., E. coli) which lack cellular processes for glycosylating proteins, yield proteins that are recovered with little or no activity by virtue of their lack of ylation. Addition of ylation sites can be accomplished by altering the amino acid sequence. The alteration to the polypeptide may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues (for O-linked glycosylation sites) or gine residues (for N- linked glycosylation sites). The structures of ed and O- linked oligosaccharides and the sugar residues found in each type may be different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (hereafier referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O- linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycoprotein.

Embodiments of the present disclosure comprise the generation and use of osylation variants.

The polypeptide sequences of the present disclosure may optionally be altered through s at the DNA level. particularly by mutating the DNA encoding the polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids. Another means of increasing the number of carbohydrate moieties on the polypeptide is by chemical or enzymatic ng of glycosides to the polypeptide. Removal of carbohydrates may be accomplished chemically or enzymatically, or by tution of codons encoding amino acid residues that are glycosylated. al deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a y of endo- and exo-glycosidases.

Additional suitable components and molecules for conjugation include, for example, molecules for targeting to the lymphatic system, thyroglobulin; alburnins such as human serum albumin (HAS); tetanus toxoid; Diphtheria toxoid; polyamino acids such as poly(D-lysine:D-glutamic acid); VP6 polypeptides of ruses; za virus hemaglutinin, influenza virus nucleoprotein'. Keyhole Limpet Hemocyanin (KLH); and hepatitis B virus core protein and surface antigen; or any combination of the foregoing.

Fusion ofalbumin to one or more polypeptides of the present disclosure can, for example, be achieved by genetic manipulation, such that the DNA coding for HSA, or a nt thereof, is joined to the DNA coding for the one or more polypeptide sequences. Thereafter, a suitable host can be transformed or transfected with the fused nucleotide sequences in the form of, for example, a suitable plasmid, so as to express a fiision ptide. The sion may be effected in vitro from, for example, prokaryotie or eukaryotic cells. or in vivo from. for example. a transgenic organism. In some embodiments of the present disclosure, the sion of the fusion protein is performed in mammalian cell lines, for example, CHO cell lines. Transformation is used broadly herein to refer to the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of ous c material nous DNA) from its surroundings and taken up through the cell membrane(s). Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells. Furthermore, albumin itself may be modified to extend its circulating ife. Fusion of the modified albumin to one or more polypeptides can be attained by the genetic manipulation techniques described above or by chemical conjugation; the resulting fusion molecule has a half- life that s that of fusions with non-modified albumin. (See W020] 1/051489). Several albumin-binding strategies have been developed as altematives for direct fusion, including albumin binding through a conjugated fatty acid chain (acylation). Because serum albumin is a transport protein for fatty acids, these natural s with albumin - binding ty have been used for half-life extension of small protein therapeutics. For example, insulin deternir (LEVEMIR), an approved t for diabetes, comprises a myristyl chain conjugated to a genetically-modified insulin. resulting in a long- acting insulin analog.

Another type of modification is to ate (e.g., link) one or more additional components or molecules at the N- and/or inus of a polypeptide sequence, such as another protein (e.g., a protein having an amino acid ce heterologous to the subject protein), or a carrier molecule. Thus, an ary polypeptide sequence can be provided as a conjugate with another component or molecule.

A conjugate modification may result in a polypeptide sequence that retains activity with an additional or complementary function or activity of the second molecule. For example. a polypeptide sequence may be conjugated to a molecule, e.g., to facilitate solubility, storage, in vivo or shelf half-life or stability, reduction in immunogenicity, d or controlled release in vivo, etc. Other functions or activities e a conjugate that reduces toxicity relative to an unconjugated polypeptide sequence, a conjugate that targets a type of cell or organ more efficiently than an unconjugated polypeptide sequence, or a drug to further counter the causes or effects associated with a er or disease as set forth herein (e.g., diabetes).

A polypeptide may also be conjugated to large, slowly metabolized macromolecules such as proteins; ccharides, such as ose, agarose, cellulose, cellulose beads; polymeric amino acids such as poly-'glutamic acid, polylysine; amino acid copolymers; inactivated virus particles; inactivated bacterial toxins such as toxoid from diphtheria, tetanus, cholera, leukotoxin molecules; inactivated bacteria; and dendritic cells.

Additional candidate components and molecules for conjugation include those suitable for isolation or purification. Particular miting examples include binding molecules. such as biotin (biotin-avidin specific binding pair), an dy. a receptor, a ligand, a lectin, or les that comprise a solid t, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test , and membranes.

Purification s such as cation exchange chromatography may be used to separate conjugates by charge difference, which effectively separates conjugates into their various molecular weights. The content of the fractions obtained by cation exchange chromatography may be identified by molecular weight using conventional methods. for example. mass spectroscopy. SDS-PAGE. or other known methods for separating molecular entities by molecular weight.

In some embodiments, the amino- or carboxyl- terminus of a polypeptide ce of the present disclosure can be fused with an immunoglobulin Fc region (e.g., human Fe) to form a fusion conjugate (or fusion molecule). Fe fusion conjugates have been shown to increase the systemic half-life of biophannaceuticals, and thus the biophannaceutical t may require less frequent administration.

Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells that line the blood vessels. and. upon binding, the Fc fusion le is protected from degradation and re- released into the circulation, keeping the molecule in circulation longer. This Fc g is believed to be the mechanism by which endogenous lgG retains its long plasma half-life. More recent Fc-fusion technology links a single copy of a biophannaceutical to the Fc region of an antibody to optimize the pharmacokinetic and phannacodynamic properties ofthe biophamiaceutical as compared to traditional Fc-fusion conjugates.

The present disclosure contemplates the use of other modifications, tly known or developed in the fiJture. of the ptides to e one or more properties. One such method for prolonging the circulation half-life, increasing the stability, reducing the clearance, or altering the immunogenicity or allergenicity of a polypeptide of the present disclosure involves modification of the polypeptide sequences by tion, which utilizes hydroxyethyl starch derivatives linked to other molecules in order to modify the le's characteristics. Various aspects of tion are described in, for example, US. Patent Appln.

Nos. 2007/0134197 and 2006/0258607.

Proteins or peptides may be made by any technique known to those of skill in the art. including the expression of proteins, polypeptides or peptides through standard molecular ical techniques, the ion of proteins or peptides from natural sources, in vitro translation, or the chemical synthesis of proteins or peptides.

Peptides can be readily synthesized chemically utilizing reagents that are free of contaminating bacterial or animal nces (Mcrrificld RB: Solid phase peptide sis. 1. The synthesis ofa tetrapeptide. J. Am. Chem. Soc.85:2149-54, 1963). In some embodiments. antigenic peptides are prepared by (1) parallel solid-phase synthesis on multi-channel instruments using unifomi synthesis and cleavage conditions; (2) ation over a RP-HPLC column with column stripping; and re-washing, but not replacement, between peptides; followed by (3) analysis with a limited set ofthe most infonnative assays. The Good Manufacturing Practices (GMP) footprint can be defined around the set of peptides for an individual patient. thus requiring suite changeover procedures only between syntheses of peptides for different patients. tively, a nucleic acid (e.g., a polynucleotide) encoding an antigenic peptide ofthe invention may be used to produce the antigenic peptide in vitro. The polynucleotide may be, cg, DNA, cDNA, PNA, CNA, RNA, either single- and/or double-stranded, or native or stabilized forms of polynucleotides, such as e.g. polynucleotides with a phosphorothiate backbone, or ations thereof and it may or may not contain introns so long as it codes for the peptide. In some embodiments in vitro translation is used to produce the peptide. An expression vector capable of expressing a polypeptide can also be ed. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation.

Generally, the DNA is inserted into an expression vector, such as a d, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), gh such controls are generally available in the expression vector. The vector is then introduced into the host bacteria for cloning using standard techniques (see, e.g.. Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring , N.Y.).

Expression vectors comprising the isolated polynucleotides, as well as host cells containing the expression vectors, are also contemplated. The antigenic peptides may be provided in the form of RNA or cDNA molecules encoding the desired antigenic peptides. One or more nic es of the ion may be encoded by a single expression vector.

In some embodiments, the polynucleotides may comprise the coding sequence for the disease specific antigenic peptide fused in the same reading frame to a polynucleotide which aids, for example. in expression and/or secretion of a ptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.

In some ments, the polynucleotides can comprise the coding sequence for the e specific antigenic peptide fused in the same reading frame to a marker sequence that allows. for example. for purification of the encoded polypeptide, which may then be orated into a personalized disease vaccine or immunogenic composition. For example, the marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for ation of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin n when a mammalian host (e.g., COS-7 cells) is used. Additional tags include, but are not d to, Calmodulin tags. FLAG tags. Myc tags. S tags, SBP tags. Softag l, Softag 3, V5 tag, Xpress tag.

Isopeptag, SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tags, GST tags, fluorescent protein tags (e.g., green cent protein tags), e binding protein tags, Nus tags, Strep-tag, thioredoxin tag, TC tag, Ty tag, and the like.

In some embodiments, the poly-nucleotides may comprise the coding sequence for one or more of the e specific antigenic peptides fused in the same g frame to create a single concatamerized antigenic peptide construct capable of ing multiple antigenic peptides.

In some embodiments, isolated nucleic acid molecules having a nucleotide sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% cal, at least 90% cal, at least 95% identical, or at least 96%, 9 %, 8% or 99% identical to a cleotide encoding a disease specific nic peptide ofthe present invention, can be provided.

The isolated disease c antigenic es described herein can be produced in vitro (e.g.. in the laboratory) by any suitable method known in the art. Such methods range from direct protein synthetic methods to constructing a DNA ce encoding isolated polypeptide sequences and expressing those sequences in a suitable transformed host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest.

Optionally, the sequence can be mutagenizcd by site-specific mutagenesis to provide fiinctional s thereof. See, e.g. Zoeller et al.. Proc. Natl Acad. Sci. USA 81:5662-5066 (1984) and US. Pat. No.4,588.585.

In some ments, a DNA sequence encoding a polypeptide of interest would be constructed by chemical sis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant ptide of interest is produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid ce can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example. several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5‘ or 3‘ overhangs for complementary assembly Once assembled (e.g., by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequences encoding a particular isolated polypeptide of interest is inserted into an expression vector and optionally operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing. restriction mapping. and expression of a biologically active ptide in a suitable host. As well known in the art, in order to obtain high expression levels of a ected gene in a host, the gene can be operatively linked to transcriptional and translational sion control sequences that are functional in the chosen expression host.

Recombinant expression s may be used to amplify and express DNA encoding the disease specific antigenic peptides. Recombinant expression vectors are replicable DNA constructs which have tic or cDNA-derived DNA fragments ng a disease specific antigenic peptide or a bioequivalent analog ively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (l) a c element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding ce which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and temiination sequences, as described in detail herein. Such regulatory elements can include an operator ce to l transcription.

The y to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of ormants can additionally be incorporated. DNA regions are operatively linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory ) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding ce if it controls the ription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. Generally, operatively linked means contiguous, and in the case of secretory leaders, means contiguous and in g frame. Structural elements intended for use in yeast expression systems e a leader sequence enabling extracellular secretion of translated protein by a host cell. Altematively, where recombinant protein is expressed without a leader or transport sequence, it can e an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

Useful sion vectors for eukaryotic hosts, especially mammals or humans include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, irus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR l, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and tous single-stranded DNA .

Suitable host cells for expression of a polypeptide include prokaryotes, yeast, insect or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive sms, for e E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems could also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian ar hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).

Various mammalian or insect cell culture systems are also advantageously employed to express recombinant protein. Expression of recombinant proteins in mammalian cells can be perfonned because such proteins are generally tly folded, appropriately modified and completely fiinctional. Examples of suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa and BHK cell lines. Mammalian expression vectors can comprise nontranscribed elements such as an origin of replication, a suitable promoter and er linked to the gene to be expressed, and other 5‘ or 3‘ flanking nontranscribed sequences, and 5’ or 3‘ nontranslated sequences, such as ary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Baeulovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and rs, Bio/Technology 6:47 (1988).

The proteins produced by a transformed host can be purified according to any suitable method. Such standard methods include chromatography (e .g.. ion exchange. affinity and sizing column chromatography. and the like), centrifugation, differential lity, or by any other standard technique for protein ation.

Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence, glutathione-S- transferase, and the like can be attached to the protein to allow easy purification by passage over an appropriate affinity . Isolated proteins can also be physically characterized using such techniques as protcolysis, nuclear magnetic resonance and x-ray llography. For e, supematants from systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. ing the concentration step, the concentrate can be applied to a suitable purification matrix. Altematively, an anion exchange resin can be ed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, e, dextran, cellulose or other types commonly employed in protein purification. Altematively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid tography LC) steps employing hydrophobic RP-I-IPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to fiirther purify a cancer stem cell protein-Fe composition. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.

Recombinant protein ed in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by any ient method, including -thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

The present ion also contemplates the use of nucleic acid molecules as vehicles for delivering antigenic peptides/polypeptides to the t in need thereof, in vivo, in the form of, e.g., A vaccines (see. e.g.. W020] 2/ 159643. and W02012/ 159754. hereby incorporated by reference in their entirety).

In some ments antigens may be stered to a patient in need thereof by use of a plasmid.

”Ihese are plasmids which y consist of a strong viral er to drive the in vivo transcription and translation of the gene (or complementary DNA) of interest (Mor, et al., (1995). The Joumal of Immunology 155 (4): 2039—2046). Intron A may sometimes be included to improve mRNA stability and hence increase protein expression er et al. (1997).The Joumal of Immunology 159(12): 6112—6119). Plasmids also include a strong polyadenylation/transcriptional tennination signal, such as bovine growth honnone or rabbit lobulin polyadenylation sequences (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343—410; Robinson et al., (2000). Adv. Virus Res. es in Virus Research 55: l—74; Bohmet al., (1996).

Joumal of Immunological Methods 193 (I): 29—40.). Multieistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein (Lewis et al.. . Advances in Virus Research (Academic Press) 54: 129—88) Plasmids may be introduced into animal tissues by a number of different methods. The two most popular ches are injection of DNA in saline, using a standard hypodermic needle, and gene gun ry. A schematic outline of the construction of a DNA vaccine plasmid and its subsequent delivery by these two methods into a host is illustrated at Scientific an (Weiner et al., (1999) Scientific American 281 (1): 34—41). Injection in saline is nonnally conducted intramuscularly (IM) in skeletal muscle, or intradennally (ID). with DNA being delivered to the extracellular spaces. This can be assisted by electroporation by temporarily damaging muscle fibres with myotoxins such as bupivacaine; or by using hypertonic solutions of saline or sucrose (Alareon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343—410). Immune responses to this method of ry can be affected by many factors, including needle type, needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the animal being injected(Alarcon ct al., . Adv. Parasitol. Advances in Parasitology 42: 343—410).

Gene gun delivery, the other commonly used method of delivery, ballistically accelerates plasmid DNA (pDNA) that has been adsorbed onto gold or tungsten microparticles into the target cells, using compressed helium as an accelerant (Alareon etal., (1999). Adv. tol. Advances in Parasitology 42: 343— 410; Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).

Alternative delivery methods may include aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa, (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129—88) and topical administration of pDNA to the eye and l mucosa (Lewis et al., (1999) es in Virus Research (Academic Press) 54: 129—88). Mueosal surface ry has also been achieved using cationic liposome-DNA preparations, radable microspheres, attenuated Shigella or Listeria vectors for oral stration to the intestinal mucosa, and recombinant adenovirus vectors. DNA or RNA may also be delivered to cells following mild mechanical disruption of the cell membrane, temporarily penneabilizing the cells. Such a mild mechanical disruption of the membrane can be accomplished by gently forcing cells through a small aperture (Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells. Sharei et al, PLOS ONE | DOI:lO.l37l/journal.pone.0118803 April 13, 2015).

In some ments, a disease specific vaccine or immunogenic composition may include separate DNA plasmids encoding, for example, one or more antigenic peptides/polypeptides. As discussed herein, the exact choice of expression vectors can depend upon the peptide/polypeptides to be expressed, and is well within the skill of the ordinary artisan. The expected persistence of the DNA constructs (c.g., in an episomal, non- replicating. tegrated fonn in the muscle cells) is expected to e an increased duration of protection.

One or more antigenic peptides of the invention may be d and expressed in vivo using a viral based system (e.g., an adenovirus system, an adeno associated virus (AAV) vector, a poxvirus, or a lentivirus).

In some embodiments, the disease vaccine or immunogenic ition may include a viral based vector for use in a human patient in need thereof, such as, for example, an adenovirus. ds that can be used for adeno associated virus. adenovirus. and lentivirus delivery have been bed previously (see e.g.. US.

Patent Nos. 6,955,808 and 6,943,019, and US. Patent application No. 20080254008, hereby incorporated by reference).

The peptides and polypeptides of the invention can also be expressed by a vector, e.g., a nucleic acid molecule as herein-discussed, e.g., RNA or a DNA plasmid, a viral vector such as a poxvirus, e.g., orthopox virus, avipox virus, or adenovirus, AAV or irus. This approach involves the use of a vector to s nucleotide sequences that encode the peptide of the ion. Upon introduction into an acutely or chronically infected host or into a noninfected host, the vector expresses the immunogenic peptide, and thereby elicits a host CTL response.

Among vectors that may be used in the practice of the invention, integration in the host genome of a cell is possible with retrovirus gene transfer methods, often resulting in long term expression of the inserted transgcnc. In scom embodiments, the retrovirus is a lentivirus. onally, high transduction efficiencies have been observed in many different cell types and target tissues. The tropism of a retrovirus can be altered by orating n envelope proteins, expanding the potential target population of target cells. A retrovirus can also be engineered to allow for conditional expression of the inserted transgene, such that only certain cell types are infected by the lentivirus. Cell type specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (and hence both lentiviral and retroviral vectors may be used in the practice ofthe invention). Moreover, lentiviral vectors are able to transduce or infect non- dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system may therefore depend on the target tissue. Retroviral s are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for ation and packaging of the vectors, which are then used to integrate the desired nucleic acid into the target cell to provide permanent expression. Widely used retroviral s that may be used in the practice of the invention include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno ncy virus (SIV), human irnmuno deficiency virus (HIV), and ations thereof (see. e.g.. Buchscher et al., (1992) J. Virol. 661273 1-2739; Johann etal.. (1992)]. Virol.66: 1635-1640; Sommnerfelt et al., (1990) Virol.l76:58-59; Wilson et al., (1998) J. Virol.6322374-2378; Miller et al., (1991) J. Virol.65:2220-2224; PCT/US94/05700).

Also usefiil in the practice of the ion is a minimal imate lentiviral vector, such as a iral vector based on the equine infectious anemia virus (EIAV). The vectors may have cytomegalovirus (CMV) promoter driving expression of the target gene. Accordingly, the invention contemplates amongst vector(s) useful in the practice of the ion: viral vectors, including retroviral vectors and lentiviral vectors.

In an embodiment herein the ry is via a lentivirus. Dosages, e.g., 10 u] of a recombinant lentivirus having a titer of l x 109 transducing units (TU)/mL, can be adapted or extrapolated to use of a retroviral or iral vector in the t invention. For transduction in tissues such as the brain, it is necessary to use very small s, so the viral preparation is concentrated by ultracentrifugation. Other methods of concentration such as ultrafiltration or binding to and elution from a matrix may be used. In other embodiments the amount of irus administered may be 1x105 or about 1x105 plaque forming units (PFU), 5x105 or about 5x105 PFU, 1x106 or about 1.x106 PFU, 5x106 or about 5x10“ PFU, 1x107 or about lxlO7PFU, 5x107 or about 5x107 PFU, 1x108 or about 1x108 PFU, 5x108 or about 5x108 PFU, 1x109 or about 1x109PFU, 5x109 or about 5x109 PFU, 1x10lo or about 1x10lo PFU or 5x1010 or about 5x1010 PFU as total single dosage for an average human of 75 kg or adjusted for the weight and size and species of the subject. One of skill in the art can determine suitable dosage. Suitable dosages for a virus can be determined cally.

Also useful in the practice of the invention is an irus . One advantage is the ability of recombinant adenoviruses to efficiently transfer and express inant genes in a variety of mammalian cells and tissues in vitro and in vivo, resulting in the high expression of the transferred nucleic acids. Further, the ability to productively infect quiescent cells, expands the utility of recombinant adenoviral vectors. In addition, high expression levels ensure that the products of the nucleic acids will be expressed to sufficient levels to generate an immune response (see e.g.. US. Patent No.7,029.848. hereby incorporated by reference).

As to adenovirus vectors useful in the practice of the invention, mention is made of US Patent No.6,955,808.

The irus vector used can be selected from the group consisting of the Ad5, Ad35, Ad] 1, C6, and C7 vectors. The sequence of the Adenovirus 5 (“Ad5”) genome has been published. (Chroboczek, 1., Bieber, F., and Jacrot, B. (1992) The Sequence ofthe Genome of Adenovirus Type 5 and Its Comparison with the Genome ofAdenovirus Type 2, Virology 186, 5; the contents if which is hereby incorporated by reference). Ad35 vectors are described in US. Pat. Nos.6,974,695, 6,913,922, and 6,869,794. Adll vectors are described in US. Pat. No. 6.913.922. C6 adenovirus vectors are described in US. Pat. Nos. 6.780.407; 6,537,594; 647; 6,265,189; 6,156,567; 6,090,393; 5,942,235 and 5,833,975. C7 vectors are bed in US. Pat. No. 6,277,558. Adenovirus vectors that are El-defective or deleted, E3- defective or deleted, and/or E4-defective or deleted may also be used. n iruses having ons in the E1 region have improved safety margin because El-defective adenovirus mutants are ation-defective in rmissive cells, or, at the very least, are highly attenuated. iruses having mutations in the E3 region may have enhanced the immunogenieity by ting the mechanism whereby adenovirus down-regulates MHC class I molecules. Adenoviruses having E4 ons may have reduced immunogenicity of the adenovims vector because of suppression of late gene expression. Such vectors may be particularly useful when repeated re- vaccination utilizing the same vector is desired. Adenovirus vectors that are d or mutated in E1, E3, E4, E1 and E3, and El and E4 can be used in accordance with the present ion. Furthennore, "gutless” adenovirus vectors, in which all viral genes are deleted, can also be used in accordance with the present invention. Such vectors require a helper virus for their replication and require a special human 293 cell line expressing both Ela and Cre, a condition that does not exist in natural environment. Such “gutless” vectors are non-immunogenic and thus the vectors may be inoculated multiple times for re-vaceination. The “gutless” adenovirus vectors can be used for insertion of heterologous inserts/genes such as the transgenes of the present invention, and can even be used for co-delivery of a large number of heterologous inserts/genes. In some embodiments, the delivery is via an adenovirus, which may be at a single booster dose. In some embodiments. the adenovirus is delivered via multiple doses. In terms of in vivo delivery. AAV is advantageous over other viral vectors due to low toxicity and low probability of causing insertional mutagenesis because it doesn‘t integrate into the host genome. AAV has a packaging limit of 4.5 or 4.75 Kb.

Constructs larger than 4.5 or 4.75 Kb result in cantly reduced virus production. There are many promoters that can be used to drive nucleic acid molecule expression. AAV ITR can serve as a er and is ageous for eliminating the need for an additional promoter element. For ubiquitous expression, the following promoters can be used: CMV. CAG. CBh. PGK. SV40. Ferritin heavy or light chains. etc. For brain expression, the following promoters can be used: Synapsinl for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Promoters used to drive RNA synthesis can include: P01 111 promoters such as U6 or H1. The use of a Pol 11 promoter and intronie cassettes can be used to express guide RNA (gRNA). With regard to AAV vectors useful in the practice of the invention, mention is made of US Patent Nos. 5658785, 7115391, 3, 6953690, 6, 6924128, 6893865. 6. 653 7540. 6475769 and 6258595. and documents cited therein. As to AAV, the AAV can be AAVl.

AAVZ, AAV5 or any combination thereof. One can select the AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes l, 2, 5 or a hybrid capsid AAV], AAVZ, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for ing cardiac tissue. AAV8 is useful for delivery to the liver. In some embodiments the delivery is via an AAV. The dosage may be adjusted to e the eutic benefit against any side s.

In some embodiments, effectively activating a cellular immune response for a disease vaccine or genic composition can be achieved by expressing the relevant antigens in a vaccine or immunogenic composition in a non-pathogenic microorganism. Well-known examples of such microorganisms are Mycobacterium bovis BCG, Salmonella and Pseudomona (See, US. Patent 91,797, hereby incorporated by reference in its entirety).

In some embodiments, a Poxvirus is used in the disease vaccine or immunogenic composition. These include orthopoxvirus, avipox, vaccinia, MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see e.g.. Verardieta1.. Hum Vaccin ther. 2012 Jul;8(7):96l-70; and Moss. Vaccine. 2013; 31(39): 4220— 4222). Poxvirus expression s were described in 1982 and quickly became widely used for vaccine pment as well as research in numerous fields. Advantages of the vectors include simple construction, ability to accommodate large amounts of foreign DNA and high expression levels. lnforrnation concerning poxviruses that may be used in the practice of the invention, such as Chordopoxvirinae subfamily poxviruses (poxviruses of vertebrates), for instance, orthopoxviruses and avipoxviruscs, c.g., vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g.. ATCC"3 VR-l354). Copenhagen Strain, NYVAC. NYVAC.1. NYVAC.2. MVA, MVA-BN), canarypox vims (e.g., Wheatley C93 Strain, ALVAC), x virus (e.g., FP9 Strain, Webster Strain, ), dovepox, pigeonpox, quailpox, and raccoon pox, inter alia, synthetic or non- naturally ing recombinants thereof, uses thereof, and methods for making and using such recombinants may be found in scientific and patent literature.

In some embodiments, the ia virus is used in the disease vaccine or immunogenic composition to express a antigen. (Rolph et al.. Recombinant viruses as vaccines and immunological tools. Curr Opin Immunol 9:517—524, 1997). The recombinant vaccinia virus is able to replicate within the asm of the infected host cell and the ptide of interest can therefore induce an immune response. Moreover, uses have been widely used as vaccine or immunogenic composition vectors because of their ability to target encoded antigens for processing by the major histocompatibility complex class I pathway by directly infecting immune cells, in particular antigen-presenting cells, but also due to their ability to self-adjuvant.

In some embodiments. ALVAC is used as a vector in a disease vaccine or immunogenic composition.

ALVAC is a canarypox virus that can be modified to s foreign transgenes and has been used as a method for vaccination against both prokaryotic and eukaryotic antigens (Horig H, Lee DS, ght W, et al. Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the 87.1 co-stimulatory molecule. Cancer Immunol Immunother 2000;49:504— 14; von Mchrcn M, Arlen P, Tsang KY, ct al. Pilot study of a dual gene recombinant avipox e containing both carcinoembryonic antigen (CEA) and 37.1 transgenes in patients with recurrent CEA- expressing adenocarcinomas. Clin Cancer Res 2000;6z2219—28; Musey L, Ding Y, Elizaga M, et al. HIV-1 ation administered intramuscularly can induce both systemic and mucosal T cell immunity in HIV-1 - uninfected individuals. J Immunol 2003; 171: 01; Paoletti E. Applications of pox virus vectors to vaccination: an update. Proc Natl Acad Sci U S A 1996;93:11349—53; US. Patent No.7,255,862). In a phase I clinical trial, an ALVAC virus expressing the tumor antigen CEA showed an excellent safety profile and resulted in increased CEA-specific T cell responses in selected patients; objective clinical responses, however, were not observed (Marshall JL. s MJ. Tsang KY. et al. Phase I study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human careinoembryonic n. J Clin Oncol 1999;17:332—7).

In some embodiments, a Modified Vaccinia Ankara (MVA) virus may be used as a viral vector for an antigen vaccine or immunogenic composition. MVA is a member of the Orthopoxvirus family and has been generated by about 570 serial passages on chicken embryo fibroblasts of the Ankara strain of Vaccinia virus (CVA) (for review see Mayr. A.. et al.. ion 3. 6-14. 1975). As a consequence of these passages. the resulting MVA virus contains 31 kilobases less genomic information compared to CVA, and is highly hosT cell cted (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038, 1991). MVA is terized by its extreme attenuation, namely, by a diminished virulence or ious y, but still holds an excellent immunogenicity. When tested in a variety of animal models, MVA was proven to be avirulent, even in immuno-suppressed duals. er, MVA-BN‘E-HERLZ is a candidate immunothcrapy designed for the treatment of HER-Z-positive breast cancer and is currently in clinical trials. (Mandl et al.. Cancer Immunol Immunother. Jan 2012; 61(1): 19—29). s to make and use recombinant MVA has been described (e.g., see US. Patent Nos. 8,309,098 and 5,185,146 hereby incorporated in its entirety).

In some embodiments, recombinant viral particles of the vaccine or immunogenic composition are administered to patients in need thereof.

Neoantigen binding peptides In n embodiments. the present invention provides a g protein (e.g., an antibody or antigen- g fragment thereof), or a T cell receptor (TCR), or a chimeric antigen receptor (CAR) e of binding with a high affinity to a igen peptide:human leukocyte antigen (HLA) complex. In some embodiments, the present invention provides a CAR that is capable of g with a high affinity to a neoantigenic peptide derived from the extracellular domain of a n. In n embodiments, a neoantigen-specific binding protein or TCR or CAR as described herein includes variant polypeptide species that have one or more amino acid substitutions. insertions. or ons in the native amino acid sequence. provided that the binding protein retains or ntially retains its specific binding function. vative substitutions of amino acids are well known and may occur naturally or may be introduced when the binding protein or TCR is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, N Y, 2001). uclcotidc- directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired. tively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis may be used to e immunogen polypeptide ts (see, e.g., Sambrook et al., supra).

A variety of criteria known to persons skilled in the art indicate whether an amino acid that is substituted at a ular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid or a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids may be ed in the following categories: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, , threonine, tyrosine, cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, try-ptophan); amino acids with beta-branched side chains (e.g., threonine. valine. cine). and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine) In certain circumstances, substitution of glutamine for ic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives ofglutamic acid and ic acid, respectively. As understood in the art “similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the ce of a second polypeptide (e.g., using GENEWORKS, Align, the BLAST algorithm, or other algorithms described herein and practiced in the art).

In certain embodiments, a neoantigen specific binding protein, TCR or CAR is capable of (a) specifically binding to a neoantigenzHLA complex on a cell surface independent or in the absence of CD8. In certain embodiments, a neoantigen specific binding protein is a T cell receptor (TCR), a chimeric antigen or or an antigen-binding fragment of a TCR, any ofwhich can be chimeric. humanized or human. In further embodiments, an antigen-binding fragment of the TCR comprises a single chain TCR (scTCR).

In certain embodiments, there is provided a ition comprising a neoantigen-specific binding protein or high affinity recombinant TCR according to any one of the above embodiments and a phannaceutically acceptable carrier, diluent, or excipient.

Methods useful for isolating and purifying recombinantly produced soluble TCR, by way of example, can include obtaining supematants from suitable host cell/vector systems that secrete the recombinant soluble TCR into culture media and then concentrating the media using a commercially available filter. Following tration, the concentrate can be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods can also be employed when ing an gen from its l environment. Methods for large scale production of one or more of the isolated/recombinant soluble TCR described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble TCR may be perfomled according to methods described herein and known in the art.

III. Immunogenic and vaccine itions In some embodiments the present invention is ed to an genic composition, e.g., a vaccine composition capable of g a neoantigen-specific response (e.g., a humeral or cell-mediated immune response). In some embodiments, the immunogenic composition ses neoantigen therapeutics (e.g.. peptides, cleotides. TCR. CAR, cells containing TCR or CAR, tic cell containing polypeptide, dendritic cell containing polynueleotide, antibody, etc.) described herein corresponding to tumor specific neoantigen identified herein.

In some embodiments, immunogenic peptides are identified fiom one or more subjects with a disease or condition. In some embodiments, immunogenic peptides are specific to one or more subjects with a disease or condition. In some embodiments, immunogenic peptides can bind to an HLA that is matched to an HLA haplotype of one or more subjects with a disease or condition.

A person skilled in the art will be able to select neoantigenic therapeutics by g, for example, the generation ofT cells in vitro as well as their efficiency and overall presence, the proliferation, affinity and ion of n T cells for certain peptides, and the functionality of the T cells, e.g. by analyzing the lFN- 7 production or tumor killing by T cells. The most efficient peptides can then combined as an immunogenic ition.

In some embodiments of the present invention the different neoantigenic peptides and/or polypeptides are selected so that one immunogenic composition comprises neoantigenic peptides and/or polypeptides capable of associating with different MHC molecules, such as different MHC class I molecule. In some embodiments, an genic composition comprises neoantigenic peptides and/or polypeptides e of associating with the most ntly occurring MHC class I molecules. Hence immunogenic compositions described herein comprise different peptides capable of associating with at least 2, at least 3, or at least 4 MHC class I or class II les.

In some embodiments, an immunogenic composition described herein is capable of raising a specific cytotoxic T cells response, specific helper T cell response, or a B cell response.

In some embodiments, an immunogenic composition described herein can further comprise an adjuvant and/or a carrier. Examples of useful nts and rs are given herein below. Polypeptides and/or polynuclcotides in the composition can be associated with a carrier such as e.g. a protein or an n- presenting cell such as e.g. a dendritic cell (DC) capable of presenting the e to a T cell or a B cell. In further embodiments, DC-binding peptides are used as carriers to target the neoantigenic peptides and polynucleotides encoding the neoantigen es to dendritic cells (Sioud et al. FASEB J 27: 3272-3283 (2013)).

In embodiments, the neoantigenic ptides or polynueleotides can be provided as n ting cells (c.g., dendritic cells) containing such polypeptides or polynucleotides. In other embodiments, such antigen presenting cells are used to stimulate T cells for use in patients.

In some embodiments, the antigen presenting cells are dendritic cells. In related embodiments, the dendritic cells are autologous tic cells that are pulsed with the neoantigenic peptide or nucleic acid. The neoantigenic peptide can be any suitable peptide that gives rise to an appropriate T cell response. T cell therapy using autologous dendritic cells pulsed with peptides from a tumor associated antigen is disclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278. In some embodiments, the T cell is a CTL. In some embodiments, the T cell is a HTL.

Thus. one embodiment of the present invention an immunogenic composition containing at least one antigen presenting cell (e.g., a tic cell) that is pulsed or loaded with one or more neoantigenic polypeptides or polynucleotides bed herein. In embodiments, such APCs are autologous (e.g., gous dendritic cells). Altematively, peripheral blood mononuclear cells (PBMCs) ed from a patient can be loaded with neoantigenic peptides or polynucleotides ex vivo. In related embodiments, such APCs or PBMCs are injected back into the patient.

The polynucleotide can be any suitable polynucleotide that is capable of transducing the dendritic cell. thus resulting in the presentation of a neoantigenic peptide and induction of immunity. In some embodiments, the polynucleotide can be naked DNA that is taken up by the cells by passive loading. In another embodiment, the polynucleotide is part of a delivery vehicle, for example, a Iiposome, virus like particle, plasmid, or expression vector. In another embodiment, the poly-nucleotide is delivered by a -free delivery system, for example, high performance cleetroporation and high-speed cell deformation). In embodiments, such n presenting cells (APCs) (e.g., dendritic cells) or peripheral blood mononuclear cells ) are used to stimulate a T cell (e.g., an autologous T cell). In related embodiments, the T cell is a CTL. In other d ments, the T cell is an HTL. Such T cells are then injected into the patient. In some embodiments, CTL is injected into the patient. In some embodiments, HTL is injected into the patient. In some embodiments, both CTL and HTL are injected into the patient. Administration of either therapeutic can be performed simultaneously or tially and in any order.

The pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment are intended for parenteral, topical, nasal, oral or local administration. In some embodiments, the pharmaceutical compositions described herein are administered erally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. In embodiments, the composition can be stered intratumorally. The compositions can be administered at the site of surgical excision to induce a local immune response to the tumor. In some embodiments. bed herein are compositions for parenteral administration which comprise a solution of the neoantigenic peptides and immunogenic compositions are dissolved or suspended in an acceptable carrier, for example, an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like. These compositions can be sterilized by conventional, well known sterilization techniques, or can be e filtered. The resulting aqueous ons can be packaged for use as is, or lyophilized, the lyophilizcd preparation being combined with a sterile solution prior to administration. The compositions can contain phamiaceutieally acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering , tonicity adjusting agents, wetting agents and the like, for e, sodium acetate, sodium lactate, sodium chloride, potassium de, calcium chloride, sorbitan urate, triethanolamine oleate, etc.

The tration of neoantigenic peptides and polynucleotides described herein in the phannaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by , and will be selected by fluid volumes, viscosities, etc., ing to the particular mode of administration selected.

The neoantigenic peptides and polynucleotides described herein can also be administered via liposomes, which target the peptides to a particular cells tissue, such as lymphoid tissue. Liposomes are also useful in increasing the half-life of the peptides. Liposomes include emulsions, foams, micelles, ble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to. e.g., a or prevalent among lymphoid cells, such as monoclonal antibodies which bind to the DEC205 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes filled with a desired peptide or polynucleotide described herein can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic polypeptide/polynucleotide compositions.

Liposomes can be formed from standard vesicle-fonning lipids, which generally include neutral and negatively charged phospholipids and a sterol, for e, cholesterol. The selection of lipids is lly guided by consideration of, e.g., liposome size. acid lability and stability of the liposomes in the blood stream.

A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev.

Biophys. . 9; 467 (1980), US. Pat. Nos. 871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

For targeting to the immune cells, a igen polypeptides or polynucleotides to be incorporated into the liposome for cell e detenninants of the desired immune system cells. A liposome suspension containing a peptide can be administered intravenously, locally, topically, etc. in a dose which varies according to. inter alia, the manner of administration. the polypeptide or polynueleotide being delivered. and the stage of the disease being d.

In some embodiments, neoantigen polypeptides and polynucleotides are targeted to dendritic cells. In some embodiments, the neoantigen polypeptides and polynucleotides are target to dendritic cells using the markers DEC205, XCRl, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85], CD85k, CD85d, CD85g, CD85a, TSLP receptor, Clec9a or CDla.

For solid compositions. conventional or nanoparticle nontoxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose. starch, magnesium stearate, sodium saccharin, talcum, cellulose, e, sucrose, magnesium carbonate, and the like. For oral administration, a phannaeeutically acceptable nontoxic composition is formed by incorporating any of the ly employed ents, such as those rs previously listed, and generally 10-95% of active ingredient, that is, one or more neoantigenic polypeptides or cleotides described herein at a concentration of 25%-75%.

For aerosol administration, the neoantigenic polypeptides or polynucleotides can be supplied in finely divided fonn along with a surfactant and lant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as eaproic, octanoie, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic ide. Mixed esters, such as mixed or natural glycerides can be employed. The tant can constitute 0.l%-2 % by weight ofthe composition, or 0.25-5%. The balance of the composition can be propellant. A carrier can also be included as desired, as with, e.g., lecithin for intranasal delivery.

Additional methods for delivering the neoantigenic polynucleotides described herein are also known in the art. For instance, the nucleic acid can be delivered ly, as “naked DNA". This approach is described, for instance, in Wolff et al., Science 247: 468 (1990) as well as US. Pat. Nos. 5,580,859 and ,589,466. The nucleic acids can also be stered using ballistic delivery as described, for instance, in US. Pat. No. 5,204,253. Particles comprised solely of DNA can be administered. tively, DNA can be adhered to particles, such as gold particles.

For therapeutic or immunization purposes. mRNA encoding the neoantigenic peptides. or peptide binding agents can also be administered to the patient. In some ments, the mRNA is selfiamplifying RNA. In a timber ment, the self-amplifying RNA is a part of a tic lipid nanoparticle ation (Geall et al., Proe Natl Acad Sci U S A. 109: 14604—14609 (2012)).

The nucleic acids can also be delivered complexed to cationic nds, such as cationic lipids.

Lipid-mediated gene delivery methods are described, for instance, in W0 96/ 183 72, WO 93/24640; Mannino & Gould—Fogerite, BioTechniques 6(7): 682-691 (1988); US. Pat. No. 5,279,833; WO 91/06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).

The neoantigenic peptides and polypeptides described herein can also be sed by attenuated viruses, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptide described herein. Upon introduction into an acutely or chronically infected host or into a ected host, the recombinant vaccinia virus ses the immunogenic peptide. and thereby elicits a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., US. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides described herein will be apparent to those skilled in the art from the description herein.

Adjuvants are any substance whose admixture into the immunogenie composition increases or otherwise modifies the immune response to the therapeutic agent. rs are scaffold structures. for example a polypeptide or a polysaccharide, to which a neoantigenic polypeptide or polynucleotide, is capable of being associated. Optionally, adjuvants are conjugated ntly or non-covalently to the polypeptides or poly-nucleotides described herein.

The ability of an nt to increase the immune response to an antigen is typically manifested by a significant se in immune-mediated reaction, or reduction in e ms. For example, an increase in humoral immunity can be manifested by a significant increase in the titer of antibodies raised to the antigen. and an se in T cell activity can be manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion. An adjuvant can also alter an immune response, for example, by changing a primarily humoral or T helper 2 response into a primarily cellular, or T helper 1 response.

Suitable adjuvants are known in the art (see, :C), poly-ICLC, STING agonist, 1018 [$8, ium salts, Amplivax, ASlS, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, 1C3 l, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX. Juvamune, LipoVac. MF59, monophosphoryl lipid A, Montanide IMS 1312, ide ISA 206, Montanide ISA 50V, Montanide [SA-51, OK-432, OM- 174, OM-l97-MP-EC, ONTAK, PepTeliK'. vector system, PLG microparticles, resiquimod, , virosomes and other virus-like particles, YF-l7D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, Aquila's QSZl stimulon (Aquila Biotech, ter, Mass, USA) which is derived from saponin, mycobaeterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants also include incomplete Freund's or GM-CSF. Several immunological adjuvants (e.g.. MF59) specific for dendritic cells and their preparation have been described previously (Dupuis M, et al, Cell Immunol. 1998; 186(1): 18-27; Allison A C; Dev Biol Stand. 1998; 92:3-11) (Mosca et al. Frontiers in Bioseience, 2007; -4060) (Gamvrellis et a1. lmmunol & Cell Biol. 2004; 82: 506-516).. Also cytokines can be used. Several cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e .g., TNF—alpha), accelerating the maturation of tic cells into efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, PGEl, PGEZ, IL-l, IL-lb, lL—4, IL-6 and CD40L) (US. Pat. No. 589 incorporated herein by nce in its entirety) and acting as immunoadjuvants (e.g., IL-lZ) lovich D I, et al., J Immunother Emphasis Tumor Immunol. 1996 (6):4l4-4]8).

CpG immunostimulatory oligonucleotides have also been ed to enhance the effects of adjuvants in a vaccine setting. Without being bound by theory, CpG oligonucleotides act by activating the innate (non- ve) immune system via Toll-like receptors (TLR), mainly TLR9. CpG triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens. ing peptide or protein ns, live or killed viruses, dendritic cell immunogenic pharmaceutical compositions, autologous cellular genic pharmaceutical compositions and ccharide conjugates in both prophylactic and therapeutic immunogenic pharmaceutical compositions. lmportantly, it enhances dendritic cell tion and entiation, resulting in enhanced activation of TH] cells and strong cytotoxic T-lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH] bias induced by TLR9 stimulation is maintained even in the presence of adjuvants such as alum or lete Freund's adjuvant (IFA) that normally promote a THZ bias. CpG oligonucleotides show even greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nano particles, lipid ons or similar formulations, which are especially necessary for inducing a strong response when the antigen is relatively weak. They also accelerate the immune response and enabled the antigen doses to be reduced with comparable antibody responses to the full-dose immunogenic pharmaceutical composition without CpG in some experiments (Arthur M. Krieg. Nature Reviews, Drug Discovery. 5, June 2006. 471-484). US. Pat. No. 6,406,705 B1 describes the combined use of CpG oligonucleotides, cleic acid adjuvants and an antigen to induce an antigen-specific immune response. A commercially available CpG TLR9 antagonist is dSLlM e Stem Loop Imrnunomodulator) by Mologen (Berlin, GERMANY), which is a component of the pharmaceutical composition described herein. Other TLR binding molecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 can also be used.

Other examples of useful adjuvants include, but are not limited to, chemically modified Cst (eg.

CpR, Idera). Poly(I:C)(e.g. polyizCIZU). non-CpG bacterial DNA or RNA. ssRNA40 for TLR8, as well as active small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, umab, tremelimumab, and SC58175, which can act therapeutically and/or as an adjuvant. The amounts and concentrations of adjuvants and additives useful in the context of the present ion can readily be detemiined by the skilled artisan without undue experimentation. Additional adjuvants include colony- stirnulating factors. such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF. sargramostirn).

In some embodiments, an immunogenic composition according to the present invention can comprise more than one ent adjuvants. Furthermore, the invention encompasses a therapeutic composition comprising any adjuvant substance including any ofthe above or combinations thereof. It is also plated that the neoantigenic therapeutic (e.g., a humoral or cell-mediated immune se). In some ments, the immunogenic composition comprises ncoantigcn therapeutics (c.g., peptides, clcotides, TCR, CAR, cells containing TCR or CAR. dendritic cell ning ptide, dendritic cell containing polynucleotide. antibody, etc.) and the adjuvant can be administered separately in any appropriate ce.

A carrier can be present independently of an adjuvant. The function of a carrier can for example be to increase the molecular weight of in particular mutant in order to se their activity or genicity, to confer stability, to increase the biological activity, or to increase serum half-life. Furthermore, a carrier can aid presenting peptides to T cells. The carrier can be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell. A carrier protein could be but is not limited to e limpet anin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, globulins, or hormones, such as insulin or palmitic acid. In some embodiments, the carrier comprises a human fibronection type 111 domain (Koide et al. Methods Enzymol. 2012;503:135-56).

For immunization of humans, the carrier must be a logically acceptable cam'er acceptable to humans and safe. However, tetanus toxoid and/or diptheria toxoid are suitable carriers In some embodiments of the invention. Altematively. the r can be ns for example sepharose.

In some embodiments, the polypeptides can be synthesized as multiply linked peptides as an altemative to coupling a polypeptide to a carrier to increase immunogenicity. Such les are also known as multiple antigenic peptides (MAPS).

Neoantigens that induce an immune response can be used as a composition when combined with an acceptable carrier or excipient. Such compositions are useful for in vitro or in vivo analysis or for administration to a subject in vivo or ex vivo for treating a subject with a disease.

Thus phannaceutical compositions can include, in addition to active ingredient, a phannaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the r or other material will depend on the route of stration.

Pharmaceutical formulations comprising a protein of interest, e.g., a neoantigen described herein, can be prepared for storage by mixing the neoantigen having the desired degree of purity with optional physiologically acceptable carriers. excipients or stabilizers (Remington‘s Phannaceutical Sciences 16th edition, Oslo, A. Ed. (1980)), in the form of lyophilized ations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are those that are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride: hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl ns such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- ); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum n, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, harides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; Sugars such as sucrose, mannitol, trchalose or ol; salt-forming counter-ions such as sodium; metal complexes (e. g, Zn- protein complexes); and/or non-ionic surfactants such as E. PLURONICS‘E‘ or polyethylene glycol (PEG). able carriers are physiologically acceptable to the administered patient and retain the therapeutic ties of the compomrds with/in which it is administered. Acceptable rs and their formulations are generally described in, for example, Remington‘ phamiaceutical Sciences (18th Edition, ed.

A. Gennaro, Mack hing Co., Easton, PA 1990). One exemplary carrier is physiological saline. A phannaceutically acceptable carrier is a phannaceutically acceptable material. composition or vehicle. such as a liquid or solid filler, diluent, exeipient, solvent or encapsulating material, involved in ng or transporting the subject compounds from the stration site of one organ, or portion of the body, to another organ, or portion of the body, or in an in vitro assay system. Acceptable carriers are ible with the other ingredients of the formulation and not injurious to a t to whom it is administered. Nor should an acceptable carrier alter the specific activity of the neoantigens.

In one aspect. provided herein are ceutically acceptable or logically acceptable compositions including solvents us or non-aqueous), solutions, ons, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration.

Pharmaceutical compositions or pharmaceutical formulations therefore refer to a composition suitable for pharmaceutical use in a subject. The pharmaceutical compositions and formulations e an amount of a ncoantigcn (or polynucleotidc encoding a ncoantigcn) and a ccutically or physiologically acceptable carrier. Compositions can be fomiulated to be compatible with a ular route of administration (i.e.. systemic or local). Thus, compositions include carriers, ts, or excipients suitable for administration by various routes.

In some embodiments, a composition further comprises an acceptable additive in order to e the stability of the igen in the composition and/or to control the release rate of the composition. Acceptable additives do not alter the specific activity of the neoantigens. Exemplary acceptable additives e, but are not limited to, a sugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran. dextrose. fructose, lactose and mixtures thereof. Acceptable additives can be combined with acceptable rs and/or excipients such as dextrose. Altematively, exemplary acceptable additives include, but are not limited to, a surfactant such as polysorbate 20 or polysorbate 80 to increase stability of the peptide and decrease gelling ofthe solution. The surfactant can be added to the composition in an amount of 0.0 1% to % of the solution. Addition of such acceptable additives increases the stability and half-life of the composition in storage.

The pharmaceutical composition can be administered. for example. by injection. Compositions for injection include aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable cam'ers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). The cam'er can be a t or dispersion medium containing, for example, water, ethanol, polyol (for example, ol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.

Fluidity can be maintained. for example. by the use of a coating such as in. by the maintenance of the required particle size in the ease of sion and by the use of surfactants. Antibacterial and antifungal agents include, for e, ns, chlorobutanol, phenol, ascorbic acid and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be included in the composition. The ing solutions can be packaged for use as is, or lyophilized; the lized preparation can later be combined with a sterile solution prior to administration. For intravenous, injection, or injection at the site of affliction. the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for e. isotonic vehicles such as Sodium Chloride Injection, Ringer’s Injection, ed ‘s Injection. Preservatives, izers, buffers, antioxidants and/or other additives can be included, as needed. Sterile able ons can be prepared by incorporating an active ingredient in the required amount in an riate solvent with one or a combination of ingredients enumerated above. as ed. followed by filtered sterilization. Generally. dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the ease of e powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from a usly sterile- filtered solution thereof.

Compositions can be conventionally administered intravenously, such as by injection of a unit dose, for example. For ion, an active ingredient can be in the form of a parenterally acceptable aqueous solution which is substantially pyrogen-free and has suitable pH, isotonicity and stability. One can prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer‘s Injection, Laetated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required. onally, compositions can be administered via aerosolization.

In some embodiments, the composition is lyophilized, for example, to increase shelf-life in storage.

When the compositions are considered for use in medicaments or any of the methods provided herein, it is plated that the composition can be substantially free of pyrogens such that the composition will not cause an inflammatory reaction or an unsafe allergic reaction when administered to a human patient. Testing compositions for pyrogens and preparing compositions substantially free of pyrogens are well understood to one or ordinary skill of the art and can be accomplished using commercially available kits.

Acceptable carriers can contain a compound that stabilizes, increases or delays absorption, or increases or delays clearance. Such compounds include. for example. carbohydrates. such as glucose. sucrose. or ns; low molecular weight proteins; compositions that reduce the clearance or ysis of peptides; or excipients or other stabilizers and/or buffers. Agents that delay absorption include, for example, aluminum monostearate and n. ents can also be used to ize or to increase or decrease the absorption of the pharmaceutical composition, including mal carriers. To t from digestion the compound can be xed with a composition to render it resistant to acidic and enzymatic hydrolysis, or the compound can be complexed in an appropriately resistant r such as a liposome.

The compositions can be administered in a manner compatible with the dosage fomiulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, ty of the subject‘s immtme system to utilize the active ingredient, and degree of binding capacity desired. e amounts of active ingredient required to be administered depend on the judgment ofthe practitioner and are peculiar to each individual. Suitable regimes for initial administration and booster shots are also variable. but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusions sufficient to maintain concentrations in the blood are contemplated.

Peptide-based immunogenic pharmaceutical compositions can be formulated using any of the well- known techniques, rs, and excipients as suitable and as understood in the art. The polypeptides can be a cocktail of multiple polypeptides containing the same sequence, or a cocktail of multiple copies of ent polypeptides. The peptides can be modified. such as for example by lipidation. or attachment to a r protein. Lipidation can be the covalent attachment of a lipid group to a ptide. Lipidated peptides, or lipidated polypeptides, can stabilize structures and can enhance efficacy of the treatment.

Lipidation can be classified into several different types, such as N-myristoylation, palmitoylation, GPI-anchor addition, prenylation, and several additional types of cations. N-myristoylation is the covalent attachment of myristatc, a C14 ted acid, to a glycine residue. oylation is thioestcr e of long-chain fatty acids (C m) to ne residues. GPI-anchor addition is yl-phosphatidylinositol (GPI) linkage via amide bond. Prenylation is the thioether linkage of an isoprenoid lipid (e.g. famesyl (C-15), geranylgeranyl (C-20)) to cysteine residues. Additional types of modifications can include ment of S- diacylglycerol by a sulfur atom of cysteines, noyl conjugation via serine or threonine residues, S- archaeol conjugation to cysteine residues, and cholesterol attachment.

Fatty acids for generating a lipidated peptides can include C2 to C30 saturated, saturated, or polyunsaturated fatty acyl groups. Exemplary fatty acids can include palmitoyl, myristoyl, stearoyl and decanoyl groups. In some instances, a lipid moiety that has adjuvant property is attached to a polypeptide of interest to elicit or enhance immunogenieity in the absence of an extrinsic adjuvant. A lipidated peptide or lipopeptide can be referred to as a self-adjuvant ptide. Any of the fatty acids described above and elsewhere herein can elicit or enhance immunogenicity of a polypeptide of interest. A fatty acid that can elicit or enhance immunogenicity can include palmitoyl, myristoyl, yl, lauroyl, octanoyl, and decanoyl groups.

Polypeptides such as naked peptides or lipidated peptides can be incorporated into a liposome.

Sometimes, lipidated peptides can be incorporated into a liposome. For example, the lipid portion ofthe lipidated peptide can spontaneously integrate into the lipid bilayer of a liposome. Thus, a lipopeptide can be presented on the “surface” of a liposome.

Exemplary liposomes le for incorporation in the formulations include, and are not limited to, multilamcllar vesicles (MLV), oligolamcllar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium-sized ellar vesicles (MUV). large unilamellar es (LUV), giant unilamellar es (GUV), rnultivesicular vesicles (MVV), single or amcllar vesicles made by reverse- phase evaporation method (REV), multilamcllar vesicles made by the reverse-phase evaporation method (MLV-REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV V), vesicles prepared by extrusion methods (VET), vesicles prepared by French press (FPV), vesicles prepared by fusion (FUV), dehydration-rehydration vesicles (DRV), and bubblesornes (BSV).

Depending on the method of preparation, liposomes can be unilamellar or multilamellar, and can vary in size with diameters ranging from about 0.02 pm to greater than about 10 pm. Liposomes can adsorb many types of cells and then e an incorporated agent (e.g., a e described ). In some cases, the liposomes fuse with the target cell, whereby the contents of the liposome then empty into the target cell. A liposome can be endocytosed by cells that are phagocytic. Endoeytosis can be ed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents.

The liposomes provided herein can also comprise r lipids. In some embodiments the carrier lipids are phospholipids. Carrier lipids capable of forming liposomes include, but are not d to dipalmitoylphosphatidyleholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS). Other suitable phospholipids further include distearoylphosphatidylcholinc (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidyglycerol (DPPG). distearoylphosphatidyglycerol (DSPG). stoylphosphatidylglycerol , dipalmitoylphosphatidic acid (DPPA); dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidyethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE) and the like, or combinations thereof. In some embodiments, the liposomes further comprise a sterol (e.g., cholesterol) which modulates liposome formation. The carrier lipids can be any known non-phosphate polar lipids.

A pharmaceutical composition can be encapsulated within mes using well-known technology.

Biodegradable pheres can also be employed as carriers for the pharmaceutical compositions of this invention.

The pharmaceutical composition can be administered in liposomes or microspheres (or articles). Methods for ing liposomes and microspheres for administration to a patient are well known to those of skill in the art. Essentially. material is dissolved in an aqueous solution. the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonieated, as necessary.

Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Altematively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time g from days to months.

A polypeptide can also be attached to a r protein for delivery. The r n can be an genic r element and can be attached by any recombinant technology. Exemplary carrier proteins include Mariculture keyhole limpet hemocyanin (mcKLH), PEGylated mcKLH, Blue r* Proteins, bovine serum albumin (BSA), cationized BSA, ovalbumin, and bacterial proteins such as tetanus toxoid (1T).

A polypeptide can also be prepared as multiple antigenic peptides (MAPS). Peptides may be attached at the N-tenninus or the C-terminus to small non-immunogenic cores. Peptides built upon this core can offer highly localized peptide density. The core can be a dendritic core residue or matrix composed ofbifunctional units. Suitable core molecules for constructing MAPS can include ammonia, ethylenediamine, aspartic acid, glutamic acid, and lysine. For example, a lysine core molecule can be attached via e bonds through each of its amino groups to two additional lysines.

A polypeptide can be chemically synthesized, or recombinantly sed in a cell system or a cell- free system. A peptide can be synthesized. such as by a liquid-phase sis. a solid-phase synthesis. or by microwave assisted peptide synthesis. A polypeptide can be modified, such as for example, by aeylation, alkylation, amidation, arginylation, polyglutamylation, polyglycylation, butyrylation, gamma-carboxylation, glycosylation, malonylation, hydroxylation, iodination, nucleotide addition (e.g. ADP-ribosylation), oxidation, orylation, adenylylation, propionylation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, glycation, palmitoylation, oylation, isoprenylation or prcnylation (e.g. famcsylation or geranylgeranylation), glypiation. lipoylation. attachement of flavin moiety (e.g. FMN or FAD), attachment of heme C, opantetheinylation, lidene Schiff base formation, amide ion, ethanolamine phosphoglycerol attachment, hypusine fonnuation, biotinylation, tion, lSGylation, SUMUylation, ubiquitination, Neddylation, Pupylation, citrullination, ation, eliminylation, carbamylation, or a combination thereof.

Afier generation ofa polypeptide, the polypeptide can be subjected to one or more rounds of purification steps to remove impurities. The purification step can be a chromatographic step utilizing separation methods such as affinity-based. Size-exclusion based, ion-exchange based. or the like. In some cases, the polypeptide is at most 30%, 40%, 50%, %, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities. In some cases, the polypeptide is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of ties.

A polypeptide can include natural amino acids, unnatural amino acids, or a combination thereof. An amino acid residue can refer to a molecule containing both an amino group and a carboxyl group. Suitable amino acids e. without limitation. both the D- and L-isomers of the naturally-occurring amino acids. as well as turally occum'ng amino acids prepared by organic synthesis or other metabolic routes. The term amino acid, as used herein, includes, t limitation, a-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

The term “(x-amino acid” can refer to a molecule ning both an amino group and a carboxyl group bound to a carbon which is ated the a-carbon.

The tenn “B-amino acid” can refer to a molecule containing both an amino group and a carboxyl group in a [3 configuration.

“Naturally occum'ng amino acid" can refer to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V. A table showing a summary ofthe properties of l amino acids can be found, e.g., in US. Patent Application Publication No. 20130123169. which is herein incorporated by nce.

A peptide provided herein can comprise oneor more hydrophobic, polar, or charged amino acids.

"Hydrophobic amino acids” include small hydrophobic amino acids and large hydrophobic amino acids.

"Small hydrophobic amino acid” can be glycine, alanine, proline, and s thereof. "Large hydrophobic amino acids” can be valine, leucine, isoleucine. phenylalanine, methionine, tryptophan, and analogs thereof.

“Polar amino acids” can be serine, threonine, gine, glutamine, cysteine, tyrosine, and analogs thereof.

"Charged amino acids” can be lysine. ne. histidine. aspartate. glutamate. and analogs thereof.

A peptide provided herein can comprise oneor more amino acid analogs. An “amino acid analog" can be a molecule which is structurally similar to an amino acid and which can be tuted for an amino acid in the formation of a peptidomimetic ycle Amino acid analogs include, without tion, B-amino acids and amino acids where the amino or carboxy group is substituted by a similarly ve group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester).

A peptide provided herein can comprises one or more non-natural amino acids. A “non-natural amino acid” can be an amino acid which is not one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, l, L, K, M, F, P, S, T, W, Y and V. Non-natural amino acids or amino acid analogs include structures disclosed, e.g., in US.

Patent Application Publication No. 23169, which is herein incorporated by reference.

Amino acid analogs can include B-amino acid analogs. Examples of B-amino acid s and analogs of alanine. valine. glycine. leucine. arginine. lysine. aspartic acids. glutamic acids, ne. methionine, phenylalanine, tyrosine, proline, serine, threonine, and tryptophan can include structures disclosed, e.g., in US. Patent Application Publication No. 20130123169, which is herein incorporated by reference.

Amino acid analogs can be racemic. In some instances, the D isomer of the amino acid analog is used.

In some cases, the L isomer of the amino acid analog is used. In some instances, the amino acid analog ses chiral centers that are in the R or S configuration. Sometimes. the amino group(s) of a o acid analog is tuted with a protecting group, e.g., tert-butyloxycarbonyl (BOC group), 9- fluorenylmethyloxyearbonyl (FMOC), tosyl, and the like. Sometimes, the carboxylic acid functional group of a B-amino acid analog is protected, e.g., as its ester derivative. In some cases, the salt of the amino acid analog is used.

A “non-essential” amino acid residue can be a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially altering its essential biological or biochemical activity (e.g., receptor binding or activation). An tial" amino acid residue can be a residue that, when altered from the wild-type ce of the polypeptide, results in abolishing or ntially abolishing the polypeptide's essential biological or biochemical activity.

A “conservative amino acid tution” can be one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families can include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, l, P, F, M, W), beta-branched side chains (e.g., T, V, l) and aromatic side chains (e.g., Y, F, W, H). Thus, a predicted ential amino acid residue in a polypeptide, for example, can be replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions can be substitutions based on ric considerations (e.g. norleucine for methionine) or other ties (e.g. 2- thienylalanine for phenylalanine, or 6-Cl-tryptoplian for phan).

Nucleic acid —based immunogenic pharmaceutical itions can also be administered to a subject.

Nucleic acid —based immunogenic pharmaceutical compositionscan be formulated using any of the well- known techniques, carriers, and excipients as suitable and as understood in the art. The nucleic acid can be DNA, genomic DNA or cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, e, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods. The immunogenic pharmaceutical composition can be a DNA-based immunogenic phannaceutical composition, an RNA-based immunogenic oeutical composition, a hybrid DNA/RNA based immunogenic pharmaceutical composition, or a hybrid nucleic acid/peptide based immunogenic phamiaceutieal composition. The peptide can be a peptide d from a peptide in Table l or 2, a peptide that has a sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more in sequence homology to a peptide in Table l or 2, or a e that has a sequence that is at most 40%, 50%, 60%, 70%, 80%, 90%, 95%, or less in sequence homology to a peptide in Table l or 2.

A nucleic acid described herein can n phosphodiester bonds, although in some cases, as outlined below (for example in the uction of primers and probes such as label probes), c acid analogs are ed that can have alternate backbones, comprising, for example, phosphoramide, phosphorothioate, O- methylphosphoroamidite linkages, and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with bicyclic structures ing locked nucleic acids. positive backbones and non-ribose backbones. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids. Locked nucleic acids (LNAs) are also ed within the definition of nucleic acid analogs.

LNAs are a class of nucleic acid analogues in which the ribose ring is “locked” by a methylene bridge ting the 2-0 atom with the 4’-C atom. These modifications of the ribose-phosphate backbone can be done to increase the stability and half-life of such les in physiological environments. For example, PNAzDNA and LNA-DNA hybrids can exhibit higher stability and thus can be used in some embodiments.

The nucleic acids can be single stranded or double ed, as specified, or contain portions of both double stranded or single stranded sequence. Depending on the application, the nucleic acids can be DNA (including, e.g., genomic DNA, mitochondrial DNA, and cDNA), RNA (including, e.g., mRNA and rRNA) or a hybrid, where the nucleic acid contains any combination of ibo- and ribo-nucleotides, and any combination of bases, including uracil, e, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine. etc.

A c acid —based immunogenic pharmaceutical compositions can be in the form of a vector. A vector can be a circular d or a linear nucleic acid. A circular plasmid or linear nucleic acid can be capable of directing expression of a particular nucleotide ce in an appropriate subject cell. A vector can have a er operably linked to the polypeptide-encoding nucleotide sequence, which can be operably linked to termination signals. A vector can contain sequences required for proper translation of the nucleotide sequence. The vector comprising the nucleotide sequence of interest can be ic. meaning that at least one of its components can be heterologous with respect to at least one of its other components. The expression of the nucleotide sequence in an expression cassette can be under the control of a constitutive promoter or of an inducible promoter, which can initiate transcription only when the host cell is exposed to some particular internal or external stimulus.

The vector can be a plasmid. A plasmid can be useful for transfccting cells with nucleic acid ng the polypeptide, and the transfomied host cells can be cultured and maintained under conditions wherein expression of the polypeptide takes place.

A plasmid can comprise a nucleic acid sequence that encodes one or more of the various polypeptides disclosed herein. A single plasmid can contain coding sequence for a single ptide, or coding sequence for more than one polypeptide. Sometimes, the plasmid can further comprise coding sequence that encodes an adjuvant, such as an immune stimulating molecule, such as a cytokine.

A plasmid can further comprise an initiation codon, which can be upstream of the coding sequence, and a stop codon, which can be downstream of the coding sequence. The initiation and tennination codon can be in frame with the coding sequence. A plasmid can also comprise a promoter that is operably linked to the coding ce, and an enhancer upstream of the coding sequence. The enhancer can be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, FMDV, RSV or EBV.

A plasmid can also comprise a mammalian origin of replication in order to maintain the plasmid extrachromosomally and produce multiple copies of the d in a cell. A plasmid can also comprise a regulatory sequence, which can be well suited for gene sion in a cell into which the plasmid is administered. The coding sequence can comprise a codon that can allow more efficient transcription of the coding sequence in the host cell.

The nucleic acid based genic pharmaceutical compositions can also be a linear nucleic acid immunogenic pharmaceutical composition, or linear expression cassette, that is capable of being efficiently delivered to a subject via oporation and expressing one or more polypeptides disclosed herein.

Cell-based genic ceutical itions can also be administered to a subject. For example, an antigen presenting cell (APC) based immunogenic pharmaceutical composition can be formulated using any of the well-known ques, carriers, and ents as suitable and as understood in the art. APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells. Sometimes, an APC based immunogenic pharmaceutical composition can be a dendritic cell-based immunogenic pharmaceutical composition.

A dendritic cell-based immunogenic pharmaceutical composition can be prepared by any methods well known in the art. In some cases, dendritic cell-based immunogenic pharmaceutical compositions can be prepared through an ex vivo or in vivo method. The ex vivo method can comprise the use of autologous DCs pulsed ex vivo with the polypeptides described herein, to activate or load the DCs prior to stration into the patient. The in vivo method can comprise targeting specific DC receptors using dies coupled with the polypeptides described herein. The DC-based immunogenic pharmaceutical composition can further comprise DC activators such as TLR3, TLR8, and CD40 agonists. The DC-based genic ceutical ition can r comprise adjuvants, and a phannaceutically acceptable r.

An adjuvant can be used to enhance the immune se (humoral and/or cellular) elicited in a patient receiving the immunogenic pharmaceutical composition. Sometimes, adjuvants can elicit a Th I-type se. Other times, adjuvants can elicit a ThZ-typc response. A Thl—typc response can be characterized by the production of cytokines such as IFN-y as opposed to a ThZ-type response which can be characterized by the production of cytokines such as IL-4, IL-5 and IL-10.

In some s, based adjuvants, such as MPLA and MDP, can be used with the immunogenic phamiaceutical compositions disclosed . Monophosphoryl lipid A (MPLA), for example, is an adjuvant that causes increased presentation of liposomal n to specific T Lymphocytes. In addition, a muramyl dipeptide (MDP) can also be used as a suitable adjuvant in ction with the immunogenic pharmaceutical formulations described herein.

Adjuvant can also comprise stimulatory molecules such as cytokines. Non-limiting examples of cytokines include: CCLZO, a-interferon(IFN- a), B-interferon (IFN-B), y- interferon, platelet derived growth factor (PDGF), TNFa, TNFp, GM-CSF, epidermal growth factor (EGF), cutaneous T cell—attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15,, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-l8, MCP-l, MIP-la, MIP-l-, IL-8, L- selectin, P-selectin, E-selectin, CD34, GlyCAM-l, MadCAM-l, LFA-l, VLA-l. Mac-l. p150.95. PECAM. ICAM-l. [CAM-2. [CAM-3. CD2. LFA-3. M-CSF. G-CSF. mutant fonns of IL-18, CD40, CD40L, vascular growth factor. fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth , Fas, TNF receptor, Fit, Apo-l, p55, WSL-l, DR3, TRAMP, Apo-3, AIR LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-l, Ap-l, Ap-2, p38, , MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAIchc, TRAIcheDRCS, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKGZD, MICA. MICB. NKGZA, NKGZB. NKGZC, NKGZE. NKGZF, TAPI, and TAPZ.

Additional adjuvants e: MCP-l, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, ctin, E- selectin, CD34, GlyCAM-l, -l, LFA-l, VLA-l, Mac-l, pl50.95, PECAM, [CAM-l, [CAM-2, [CAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant fomis of IL-l8, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-l, p55, WSL- l, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL- RZ, TRICKZ. DR6. Caspase ICE. Fos. c-jun. Sp-l. Ap-l, Ap-2. p38, p65Rel. MyD88. IRAK. TRAF6. IkB. ve NIK, SAP K, SAP-l, INK, eron response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRCS, TRAIL-R3, TRAIL-R4, RANK, RANK , 0x40, 0x40 LIGAND, NKGZD, MICA, MlCB, NKGZA, NKGZB, NKGZC, NKG2E, NKG2F, TAP], TAPZ and firnctional fragments thereof.

In some aspects, an adjuvant can be a modulator of a toll like receptor. Examples of modulators of toll-like receptors include TLR-9 agonists and are not limited to small molecule modulators of toll-like receptors such as Irniquimod. Other examples of adjuvants that are used in combination with an immunogenic pharmaceutical composition described herein can include and are not d to n, CpG ODN and the like. Sometimes, an adjuvant is selected from bacteria toxoids, polyoxypropylene-polyoxyethylene block polymers, aluminum salts, liposornes, CpG polymers, oil-in-water emulsions, or a ation thereof.

Sometimes, an adjuvant is an oil-in-water emulsion. The oil—in—water emulsion can include at least one oil and at least one tant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocornpatible. The oil ts in the emulsion can be less than 5 um in diameter, and can even have a sub- micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions.

Droplets with a size less than 220 nm can be subjected to filter sterilization.

In some instances, an immunogenic pharmaceutical composition can e carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives), water, oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline solutions, aqueous dextrose and glycerol solutions, flavoring agents, ng agents, detackifiers and other acceptable additives, adjuvants, or binders, other phannaceutically acceptable ary substances as required to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifying agents, wetting agents and the like. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol rnonostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. In another instances. the ceutical preparation is substantially free of vatives. In other instances, the pharmaceutical ation can contain at least one preservative. It will be recognized that, while any suitable carrier known to those of ordinary skill in the art can be employed to administer the pharmaceutical compositions described , the type of carrier will vary depending on the mode of administration.

An immunogenie pharmaceutical composition can include preservatives such as thiomersal or 2- phenoxyethanol. In some instances. the immunogenie ceutical ition is substantially free from (e.g. <10 rig/ml) mercurial material e.g. thiomersal-free. a-Tocopherol succinate may be used as an altemative to mercurial compounds.

For controlling the tonicity, a physiological salt such as sodium salt can be included in the immunogenic phannaceutical composition. Other salts can include potassium chloride. potassium dihydrogen phosphate, disodium phosphate, and/or magnesium chloride, or the like.

An immunogenic pharmaceutical composition can have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, between 240-360 mOSm/kg, or within the range of 290-3 10 mOsm/kg.

An immunogenic pharmaceutical composition can comprise one or more buffers, such as a Tris buffer; a boratc buffer; a ate buffer; a histidinc buffer (particularly with an um hydroxide adjuvant); or a citrate buffer. Buffers. in some cases. are included in the 5-20 mM range.

The pH of the immunogenic pharmaceutical ition can be between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and about 7.8.

An immunogenic pharmaceutical composition can be sterile. The immunogenic pharmaceutical composition can be non-pyrogenic e.g. ning <1 EU (endotoxin unit, a standard measure) per dose, and can be <0.l EU per dose. The composition can be gluten free.

An genic phamiaceutical composition can include detergent e.g. a polyoxyetliylene sorbitan ester surfactant (known as ‘Tweens‘), or an octoxynol (such as octoxynol-9 n X-100) or t- henoxypolyethoxyethanol). The detergent can be present only at trace amounts. The immunogenic phamiaceutical composition can include less than 1 mg/ml of each of octoxynol-IO and polysorbate 80. Other residual ents in trace amounts can be antibiotics (eg. neomycin, kanamycin, polymyxin B).

An immunogenic pharmaceutical composition can be formulated as a sterile solution or suspension, in suitable vehicles, well known in the art. The ceutical compositions can be sterilized by conventional, well-known sterilization techniques. or can be sterile filtered. The resulting s solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.

An immunogenic pharmaceutical composition can be ated with one or more pharmaceutically acceptable salts. Pharmaceutically able salts can include those of the nic ions, such as, for example, sodium, potassium, calcium, magnesium ions, and the like. Such salts can include salts with inorganic or c acids. such as hloric acid. romic acid. phosphoric acid. nitric acid. sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid or maleic acid. In addition, if the agent(s) contain a y group or other acidic group, it can be converted into a pharmaceutically acceptable addition salt with inorganic or organic bases. Examples of suitable bases e sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, cthanolaminc, dicthanolamine, triethanolaminc, and the like.

Phamiaceutical compositions comprising. for example. an active agent such as a peptide, a nucleic acid, an antibody or fragments thereof, and/or an APC described herein, in combination with one or more adjuvants can be formulated to comprise certain molar ratios. For e, molar ratios of about 99:1 to about 1:99 of an active agent such as a peptide, a nucleic acid, an antibody or fragments thereof, and/or an APC described herein, in combination with one or more adjuvants can be used. In some instances, the range of molar ratios of an active agent such as a peptide, a nucleic acid, an antibody or fragments thereof, and/or an APC described herein, in combination with one or more adjuvants can be selected from about 80:20 to about :80; about 75:25 to about 25:75, about 70:30 to about 30:70, about 66:33 to about 33:66, about 60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90. The molar ratio of an active agent such as a peptide, a c acid, an antibody or fragments thereof, and/or an APC bed herein, in combination with one or more adjuvants can be about 1:9, and in some cases can be about 1:1. The active agent such as a peptide, a nucleic acid, an antibody or fragments thereof, and/or an APC described herein, in combination with one or more adjuvants can be formulated together. in the same dosage unit e.g.. in one via]. suppository. tablet, capsule, an aerosol spray; or each agent, form, and/or compound can be formulated in separate units, e.g., two vials, suppositories, tablets, two capsules, a tablet and a vial, an aerosol spray, and the like.

In some instances, an immunogenic pharmaceutical composition can be administered with an additional agent. The choice of the additional agent can depend, at least in part, on the condition being treated.

The additional agent can include, for example, any agents having a therapeutic effect for a en infection (eg. viral infection), including, e.g., drugs used to treat inflammatory conditions such as an NSAID. e.g., ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin. As another example, fomiulations can additionally contain one or more supplements, such as vitamin C, E or other anti-oxidants.

A ceutical composition comprising an active agent such as a peptide, a nucleic acid, an antibody or fragments thereof, and/or an APC described herein, in combination with one or more adjuvants can be formulated in conventional manner using one or more physiologically acceptable carriers, comprising excipients, diluents, and/or auxiliaries, e.g., which facilitate sing of the active agents into preparations that can be administered. Proper formulation can depend at least in part upon the route of administration chosen. The agent(s) bed herein can be delivered to a t using a number of routes or modes of administration, including oral, buecal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, and intramuscular ations, as well as by inhalation.

The active agents can be formulated for parenteral administration (e.g., by injection, for example bolus ion or continuous infusion) and can be presented in unit dose form in ampoules, pre-filled syringes. small volume infusion or in multi-dose containers with an added vative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.

For injectable formulations, the vehicle can be chosen from those known in art to be suitable, including aqueous ons or oil suspensions, or ons, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile s solution, and similar pharmaceutical vehicles. The fomiulation can also comprise polymer itions which are biocompatible, biodegradable, such as actic-eo-glycolic)aeid. These materials can be made into micro or nanospheres, loaded with dmg and fithher coated or derivatized to provide or sustained release performance. Vehicles le for periocular or intraocular injection include, for example, suspensions of therapeutic agent in injection grade water, liposornes and es suitable for lipophilic substances. Other vehicles for periocular or intraocular injection are well known in the art.

In some instances, pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as ine to ease pain at the site of the injection. lly, the ients are supplied either separately or mixed together in unit dosage form. for example. as a dry lyophilized powder or water free concentrate in a hemietically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be ed so that the ients can be mixed prior to administration.

When administration is by injection, the active agent can be formulated in aqueous solutions, specifically in physiologically compatible buffers such as Hanks on, Ringer's solution, or physiological saline buffer. The on can contain fonnulatory agents such as suspending, stabilizing and/or dispersing agents. Altematively, the active nd can be in powder form for constitution with a suitable e, e.g._. sterile pyrogen-free water, before use. In another embodiment, the pharmaceutical composition does not comprise an adjuvant or any other substance added to enhance the immune response stimulated by the peptide.

In another ment, the pharmaceutical composition comprises a substance that inhibits an immune response to the peptide.

In addition to the formulations described previously. the active agents can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation or transcutaneous ry (for example subcutaneously or intramuscularly), intramuscular injection or use of a transderrnal patch. Thus, for example, the agents can be formulated with suitable polymeric or hydrophobic materials (for e as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble tives, for example, as a sparingly soluble salt.

In cases. pharmaceutical compositions sing one or more agents exert local and regional effects when stered topically or injected at or near particular sites of infection. Direct topical application, 6g, of a viscous liquid, solution, suspension, dimethylsulfoxide (DMSO)-based solutions, liposomal formulations, gel, jelly, cream, lotion, ointment, suppository, foam, or aerosol spray, can be used for local administration, to produce for example local and/or regional effects. Pharmaceutically appropriate vehicles for such formulation include, for example, lower aliphatic alcohols, polyglycols (e.g., glycerol or polyethylene glycol), esters of fatty acids, oils. fats. silicones. and the like. Such preparations can also include preservatives (e.g., xybenzoic acid esters) and/or antioxidants (e.g., ascorbic acid and tocopherol). See also Dermatological Formulations: aneous absorption, Barry (Ed), Marcel Dekker Inc], 1983. In another embodiment, local/topical ations comprising a transporter, carrier, or ion channel inhibitor are used to treat epidemral or rnucosal viral infections.

Pharmaceutical compositions can contain a cosmetically or demiatologically acceptable carrier. Such carriers are compatible with skin. nails. mucous membranes. tissues and/or hair. and can include any conventionally used cosmetic or dermatological carrier meeting these requirements. Such carriers can be readily selected by one of ordinary skill in the art. In formulating skin Ointments, an agent or combination of agents can be formulated in an oleaginous hydrocarbon base, an anhydrous absorption base, a water-in-oil absorption base, an oil-in-water water-removable base and/or a water-soluble base. Examples of such carriers and excipients include, but are not limited to, humectants (c.g., urea), glycols (e.g., propylene glycol), alcohols (e.g.. ethanol). fatty acids (e.g.. oleic acid). surfactants (e.g.. isopropyl tate and sodium lauryl sulfate). pyrrolidones, glycerol urate, sulfoxides, terpenes (e.g., menthol), amines, , alkanes, alkanols, water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Ointments and creams can, for example, be formulated with an aqueous or 0in base with the addition of suitable ning and/or gelling agents. Lotions can be formulated with an aqueous or oily base and will in general also containing one or more emulsifying agents, stabilizing agents. dispersing agents, suspending agents, thickening agents, or coloring agents. The construction and use of transdemial patches for the delivery of phamiaceutical agents is well known in the art. Such patches can be constructed for continuous, pulsatile, or on demand delivery of phannaceutical agents.

Lubricants which can be used to form pharmaceutical itions and dosage forms can include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, ol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g.. peanut oil, cottonseed oil, sunflower oil. sesame oil, olive oil. corn oil, and soybean oil). zinc stearate, ethyl oleate, ethyl te, agar, or mixtures thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of tic silica, or mixtures thereof. A lubricant can optionally be added, in an amount of less than about 1 weight percent of the pharmaceutical composition.

The pharmaceutical compositions can be in any form suitable for topical application, including aqueous, s-alcoholic or oily solutions, lotion or serum dispersions, s, anhydrous or oily gels, emulsions obtained by sion of a fatty phase in an aqueous phase (O/W or oil in water) or. conversely.

(W/O or water in oil), microemulsions or alternatively apsules, microparticles or lipid vesicle dispersions of ionic and/or nonionic type. These compositions can be prepared ing to conventional methods. The amounts of the various constituents of the itions are those conventionally used in the art.

These compositions in particular constitute protection, treatment or care creams, milks, s, gels or foams for the face, for the hands, for the body and/or for the mucous nes, or for cleansing the skin. The compositions can also consist of solid preparations tuting soaps or cleansing bars.

Pharmaceutical compositions can n adjuvants such as hydrophilic or ilic gelling agents, hydrophilic or lipophilic active agents, preserving agents, antioxidants, solvents, fragrances, fillers, sunscreens, odor-absorbers and dyestuffs. The amounts of these various adj uvants are those conventionally used in the fields considered and, for example, are from about 0.01% to about 20% of the total weight of the composition. Depending on their nature, these adiuvants can be uced into the fatty phase, into the aqueous phase and/or into the lipid vesicles.

In instances relating to topical/local application, the pharmaceutical compositions can include one or more ation enhancers. For example, the formulations can comprise suitable solid or gel phase rs or exeipients that increase penetration or help delivery of agents or combinations of agents of the invention across a penneability barrier, e.g., the skin. Many of these penetration-enhancing compounds are known in the art of topical formulation, and include, e.g., water, alcohols (e.g., tcrpcncs like methanol, ethanol, 2-propanol), sulfoxides (e.g., dimethyl sulfoxide, decylmethyl sulfoxide. tetradecylmethyl sulfoxide). pyrrolidones (e .g.. 2- pyrrolidone, N-methyl-Z-pyrrolidone. N-(2-hydroxyethyl)pyrrolidone), apram, acetone, dimethylacetamide, dimethylformamide, tetrahydrofurfuryl alcohol, L-a-amino acids, anionic, cationic, amphoteric or nonionic tants (e.g., isopropyl myristate and sodium lauryl sulfate), fatty acids, fatty alcohols (e.g., oleic acid), amines, amides, clofibric acid amides, hexamethylene lauramide, proteolytic enzymes, a-bisabolol, d-limoncne, urea and N,N-dicthyl-m-toluamide, and the like. Additional examples include humeetants (e.g., urea), glycols (e.g., propylene glycol and polyethylene glycol). glycerol monolaurate, s, alkanols, ORGELASE, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and/or other polymers. In r embodiment, the pharmaceutical compositions will e one or more such penetration enhancers.

The ceutical compositions for topical application can include one or more antimicrobial preservatives such as quaternary ammonium compounds, organic mercurials, p-hydroxy benzoates, aromatic alcohols, chlorobutanol, and the like.

The pharmaceutical compositions can be formulated into aerosol ons. suspensions or dry powders. The aerosol can be administered h the respiratory system or nasal passages. For example, one skilled in the art will recognize that a composition of the present invention can be suspended or dissolved in an appropriate carrier, e.g., a ceutically able propellant, and stered directly into the lungs using a nasal spray or inhalant. For example, an aerosol formulation comprising a transporter, carrier, or ion channel inhibitor can be dissolved, suspended or emulsified in a propellant or a mixture of solvent and lant. e.g., for administration as a nasal spray or inhalant. l formulations can n any acceptable propellant under pressure, such as a cosmetically or dennatologically or pharmaceutically acceptable lant, as conventionally used in the art.

An aerosol formulation for nasal administration is generally an s solution designed to be administered to the nasal passages in drops or sprays. Nasal solutions can be similar to nasal secretions in that they are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values outside of this range can additionally be used. Antimicrobial agents or preservatives can also be included in the fonnulation.

An aerosol fomiulation for inhalations and inhalants can be designed so that the agent or combination of agents is carried into the atory tree of the subject when administered by the nasal or oral respiratory route. Inhalation solutions can be administered, for example, by a nebulizer. Inhalations or insufflations, comprising finely ed or liquid drugs, can be delivered to the respiratory system as a pharmaceutical aerosol of a solution or suspension of the agent or combination of agents in a propellant. e.g., to aid in disbursement. Propellants can be liquefied gases, including halocarbons, for example, fluorocarbons such as fluorinated chlorinated arbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.

Halocarbon propellants can include fluorocarbon propellants in which all hydrogens are replaced with fluorine, fluorocarbon propellants in which all ens are replaced with chlorine and at least one fluorine. hydrogen-containing fluorocarbon propellants. and hydrogen-containing chlorofluorocarbon propellants. arbon propellants usefiil in the invention include, for example, propane, isobutane, n- , pentane, isopentane and neopentane. A blend of hydrocarbons can also be used as a propellant. Ether propellants include, for example, dimethyl ether as well as the ethers. An aerosol formulation of the invention can also comprise more than one lant. For example, the aerosol formulation can comprise more than one propellant from the same class, such as two or more fluorocarbons; or more than one, more than two, more than three propellants from different classes, such as a fluorohydrocarbon and a hydrocarbon.

Pharmaceutical compositions of the present invention can also be dispensed with a compressed gas, e.g., an inert gas such as carbon dioxide, s oxide or nitrogen.

Aerosol formulations can also include other components, for example, ethanol, isopropanol, propylene , as well as surfactants or other components such as oils and detergents. These components can serve to stabilize the formulation and/or ate valve components.

The aerosol formulation can be packaged under re and can be ated as an aerosol using solutions, suspensions. emulsions, s and semisolid preparations. For example, a solution aerosol formulation can comprise a solution of an agent of the invention such as a transporter, carrier, or ion channel inhibitor in (substantially) pure propellant or as a mixture of propellant and solvent. The solvent can be used to dissolve the agent and/or retard the evaporation of the propellant. Solvents can include, for example, water, ethanol and glycols. Any combination of suitable solvents can be use, optionally combined with preservatives, antioxidants, and/or other aerosol components.

An aerosol fomiulation can be a dispersion or suspension. A suspension aerosol ation can comprise a suspension of an agent or combination of agents of the t invention, e.g., a orter, carrier, or ion channel inhibitor, and a dispersing agent. Dispersing agents can include, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin and corn oil. A suspension l formulation can also include lubricants, preservatives, antioxidant, and/or other aerosol components.

An aerosol formulation can rly be formulated as an emulsion. An emulsion aerosol formulation can include. for example, an alcohol such as ethanol. a surfactant, water and a propellant. as well as an agent or combination of agents of the ion, e.g., a transporter, carrier, or ion channel. The tant used can be nonionic, anionic or cationic. One example of an emulsion aerosol formulation comprises, for example, ethanol, tant, water and propellant. Another example of an emulsion aerosol fomiulation comprises, for example, vegetable oil, glyceryl monostearate and propane.

The pharmaceutical compounds can be formulated for administration as suppositories. A low melting wax. such as a mixture of triglycerides. fatty acid glycerides. Witepsol $55 (trademark of Dynamite Nobel Chemical, Germany), or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify The pharmaceutical compositions can be ated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

The pharmaceutical compositions can be attached releasably to patible polymers for use in sustained release formulations on, in or attached to inserts for topical, intraocular, ular, or systemic stration. The controlled release from a biocompatible polymer can be utilized with a water soluble polymer to form a instillable tion, as well. The controlled release from a biocompatible polymer, such as for example, PLGA microsphercs or nanospheres, can be utilized in a formulation suitable for intra ocular implantation or injection for sustained release administration, as well. Any suitable biodegradable and biocompatible polymer can be used.

IV. Combinations of CTL peptides and HTL peptides lmmunogenic or e compositions comprising the neoantigenic polypeptides and polynucleotides described herein, or analogs thereof, which have immunostimulatory activity can be modified to provide desired utes, such as improved serum half-life, or to enhance immunogenicity.

For ce, the ability of the neoantigenic peptides to induce CTL activity can be enhanced by linking the e to a sequence which contains at least one epitope that is e of inducing a T helper cell response. In some embodiments, CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of vely small, neutral molecules, such as amino acids or amino acid mimetics, which are ntially uncharged under physiological ions. The spacers are typically selected from. e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or l polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus can be a hetero- or homo-oligomer. When present. the spacer will usually be at least one or two es. more usually three to six residues. Altematively, the CTL peptide can be linked to the T helper peptide without a spacer.

Although the CTL peptide epitope can be linked directly to the T helper peptide e, CTL epitope/HTL epitope conjugates can be linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are lly selected from, e.g., Ala, Gly, or other l spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus can be a hetero- or ligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues. The CTL peptide epitope can be linked to the T helper peptide e either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic e or the T helper e can be ed.

HTL peptide epitopes can also be modified to alter their biological properties. For example, peptides comprising HTL epitopes can contain D-amino acids to increase their resistance to proteases and thus extend their serum half-life. Also, the epitope peptides can be conjugated to other molecules such as lipids, proteins or sugars, or any other synthetic compounds, to increase their biological activity. For example, the T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.

In certain embodiments. the T helper peptide is one that is recognized by T helper cells present in the majority of the population. This can be accomplished by selecting amino acid sequences that bind to many, most, or all of the HLA class II molecules. These are known as “loosely l-ILA-restricted” or “promiscuous" T helper ces. Examples of amino acid sequences that are promiscuous include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE), Plasmodiumfalcipamm CS protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS), and Streptococcus 18kD protein at positions 116 (GAVDSILGGVATYGAA). Other examples include peptides bearing a DR 17 supemrotif, or either of the DR3 motifs.

Altematively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely I-lLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT ation WO 95/07707). These synthetic compounds called Pan-DR—binding epitopes (e.g., PADRE, Epimmune, Inc, San Diego, CA) are designed to bind most HLA-DR (human HLA class II) molecules. For instance, a —binding epitope peptide having the formula: aKXVWANTLKAAa, where "X” is either cyclohexylalanine, phenylalanine. or tyrosine. and a is either D-alanine or L-alanine. has been found to bind to most HLA-DR alleles, and to stimulate the se ofT helper lymphocytes from most individuals, regardless of their HLA type. An altemative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.

In some embodiments it can be desirable to include in a neoantigen eutic (e.g., peptides. polynucleotides, TCR, CAR, cells ning TCR or CAR, dendritic cell containing polypeptide, dendritic cell containing polynucleotide. dy. etc.) in phamraceutieal itions (e.g.. immunogenic compositions) at least one component of which primes xic T lymphocytes. Lipids have been fied as agents capable of g CTL in vivo against viral antigens. For example, palmitic acid residues can be ed to the a-and a- amino groups of a lysine residue and then linked, e.g., via one or more g residues such as Gly, Gly-Gly-, Ser, Ser—Ser, or the like, to an immunogenic neoantigenic peptide. The lipidated peptide can then be administered either directly in a micclle or particle, incorporated into a liposome, or fied in an adjuvant. In some embodiments, a particularly effective immunogenic construct ses palmitic acid attached to 8- and a- amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. coli oteins, such as tripalmitoyl-S- glycerylcysteinlyseryl- serine (P3CSS) can be used to prime virus specific CTL when ntly attached to an appropriate peptide. (See, e.g., Deres, et al., Nature 3422561, 1989). Neoantigenic peptides described herein can be d to P3CSS. for example. and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. er, because the induction of neutralizing dies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses to infection.

As noted herein, onal amino acids can be added to the termini of a neoantigenic peptide to provide for ease of linking es one to r, for coupling to a carrier support or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide. or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-tenninus of the peptide or oligopeptide. However, it is to be noted that modification at the carboxyl terminus of a T cell epitope can, in some cases, alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural ce by being modified by terminal-NHZ acylation, e.g., by alkanoyl (Cl-C20) or thioglycolyl acctylation, terminal-carboxyl amidation, c.g., a, mcthylamine, etc.

In some instances these cations can provide sites for g to a support or other molecule.

An embodiment of an immunogenic composition described herein comprises ex vivo administration of a cocktail of epitope-bearing neoantigenic polypeptide or polynucleotides to PBMC, or isolated DC therefrom, from the patient's blood. A ceutical to facilitate harvesting of dendritic cells (DCs) can be used, including GM—CSF, IL-4, IL-6, IL-IB, and TNFa. After pulsing the DCs with peptides or polynucleotides encoding the peptides, and prior to reinfusion into patients, the DC are washed to remove unbound peptides.

In this embodiment, a vaccine or genic composition comprises peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces. The ition is then administered to the patient. In other embodiments, such pulsed DCs are used to stimulate T cells suitable for use in T cell therapy.

V. Multi-epitope immunogenic compositions A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the neoantigenic peptides described herein are a particularly useful embodiment of the invention. In some embodiments. the nucleic acid is RNA. In some embodiments. minigene constructs encoding a neoantigenic peptide comprising one or multiple epitopes described herein are used to administer nucleic acids encoding the neoantigenic peptides described herein uses.

The use of multi-epitope minigenes is described An, L. and Whitton, J. L., J. Virol. 7122292, 1997; n, S. A. et al., J. Immunol. 1572822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitopc DNA plasmid encoding supermotif- and/or motif- g neoantigen peptides. a universal helper T cell epitope (or multiple tumor associated n HTL es), and an endoplasmic reticulum-translocating signal sequence can be engineered.

The immunogenicity ofa multi-epitopic minigene can be tested in transgenic mice to evaluate the magnitude of immune se induced t the es tested. Further, the immunogenicity of DNA- encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells I l l transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: I.) generate a cell mediated and/or humoral se and 2.) that the induced immune cells recognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected neoepitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes can be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These neoepitope-encoding DNA ces can be directly adjoined, so that when translated, a continuous polypeptide sequence is d. To optimize expression and/or immunogenicity. additional elements can be orated into the minigene design.

Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, a ubiquitination signal ce, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes can be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.

The minigene sequence can be converted to DNA by ling oligonueleotides that encode the plus and minus strands of the minigene. Overlapping oligonueleotides (30-100 bases long) can be synthesized, phosphorylated, d and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA . This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.

Standard regulatory sequences well known to those of skill in the art can be included in the vector to ensure expression in the target cells. For example, a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (eg. ampicillin or kanamycin resistance). Numerous promoters can be used for this e, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., US. Patent Nos. 5,580,859 and ,5 89,466 for other suitable promoter sequences.

Additional vector modifications can be used to ze minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally- occurring introns could be incorporated into the transcribed region of the minigene. The inclusion ofmRNA ization sequences and sequences for replication in mammalian cells can also be considered for increasing minigene expression.

Once an expression vector is selected, the minigene can be cloned into the polylinker region downstream of the er. This plasmid is transformed into an appropriate E. coli , and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, can be confirmed using restriction mapping and DNA sequence analysis.

Bacterial cells harboring the t d can be stored as a master cell bank and a working cell bank.

In on, immunomodulatory sequences appear to play a role in the immunogenicity of DNA es. These sequences can be ed in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity. In some ments, the sequences are immunostimulatory. In another embodiment. the sequences are [$55 or Cst.

In some embodiments, a bi-cistronic expression vector which allows production of both the minigene- encoded epitopes and a second protein (included to e or decrease immunogenicity) can be used.

Examples of ns or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), ulatory molecules, or for HTL responses, pan-DR binding ns. Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, y improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co- expression of imrnunosuppressive molecules (eg. TGF-B) can be beneficial in certain diseases.

Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium. and grown to saturation in shaker flasks or a bioreaetor ing to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, rnia). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA.” is currently being used for intramuscular (1M) stration in al trials. To maximize the immunotherapeutie effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA can be used. A variety of methods have been described, and new techniques can become available. Cationic lipids can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); US. Pat No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 3 . In addition, glycolipids, fusogenic liposomes, peptides and compounds ed to collectively as tive. interactive. non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

In another ment, the nucleic acid is introduced into cells by use of high-speed cell deformation. During high-speed deformation, cells are ed such that temporary disruptions occur in the cell membrane, thus allowing the nucleic acid to enter the cell. Altemativcly, protein can be ed from expression vectors — in a bacterial expression vector. for example, and the proteins can then be delivered to the cell.

Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for rd CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green cent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (5 lCr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 5'Cr e, indicates both production of, and HLA tation of, minigene-encoded CTL epitopes. Expression of HTL epitopes can be evaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations.

Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (IP) for lipid- complexed DNA). An exemplary protocol is twenty-one days after immunization, splenocytes are ted and restimulated for 1 week in the presence of peptides ng each epitope being tested. Thereafter, for CTL or cells, assays are conducted for cytolysis of e-loaded, SlCr-labeled target cells using rd techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes. demonstrates DNA vaccine function for in vivo induction of CTLs. lmmunogenicity of HTL epitopes is evaluated in enic mice in an analogous manner. tively, the nucleic acids can be administered using ballistic delivery as bed, for instance, in US. Patent No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to les, such as gold particles.

VI. Cells for use in the invention In one aspect, the present invention also provides cells expressing a neoantigen-recognizing receptor that activates an immunoresponsive cell (e.g.. T cell receptor (TCR) or chimeric antigen receptor (CAR)). and methods of using such cells for the treatment of a disease that requires an enhanced immune response.

Such cells e genetically modified immunoresponsive cells (e.g., T cells, Natural Killer (NK) cells, cytotoxic T cytes (CTL) cells, helper T lymphocyte (HTL) cells) expressing an antigen- recognizing receptor (e.g., TCR or CAR) that binds one of the neoantigenic peptides described herein, and methods of use therefore for the treatment of neoplasia and other pathologies where an increase in an n- specific immune se is desired. T cell activation is mediated by a TCR or 21 CAR targeted to an antigen.

The present invention es cells expressing a combination of an antigen-recognizing receptor that activates an immunoresponsive cell (e.g., TCR, CAR) and a ic co-stimulating receptor (CCR), and methods of using such cells for the treatment of a disease that requires an enhanced immune response. In some embodiments, tumor antigen-specific T cells, NK cells, CTL cells or other immunoresponsive cells are used as shuttles for the selective enrichment of one or more co-stimulatory s for the treatment or prevention of neoplasia. Such cells are administered to a human subject in need thereof for the treatment or prevention of a particular cancer.

In some embodimentsthe tumor antigen-specific human lymphocytes that can be used in the methods of the invention e, without limitation, peripheral donor lymphocytes genetically modified to express chimeric antigen receptors (CARS) (Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45), peripheral donor cytes genetically modified to express a fiill-Iength tumor antigen-recognizing T cell receptor complex comprising the a and p heterodimer (Morgan. R. A.. et al. 2006 Science 314:126-129). lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies (Panelli, M. C., et al. 2000] Immunol [64:495-504; Panelli, M. C ., et al. 2000 J Immunol 82-4392), and selectively in vitro-expanded antigen-specific peripheral bloodleukocytes employing artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells (Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, G. A., et al. 2003 Blood 10222498-2505). The T cells may be autologous. allogcncic, or derived in vitro from engineered progenitor or stem cells.

In some embodiments, the immunotherapeutic is an engineered receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a B-cell receptor (BCR), an adoptive T cell therapy (ACT), or a tive thereof. In other aspects, the engineered receptor is a chimeric n receptor (CAR). In some aspects, the CAR is a first generation CAR. In other aspects, the CAR is a second generation CAR. In still other s, the CAR is a third generation CAR.

In some aspects, the CAR comprises an extracellular portion. a transrnernbrane n. and an intracellular portion. In some aspects, the intracellular portion comprises at least one T cell co-stimulatory domain. In some s, the T cell co-stimulatory' domain is selected from the group consisting of CD27, CD28, TNFRS9 (4-lBB), TNFRSF4 (0X40), TNFRSF8 , CD40LG (CD40L), ICOS, ITGBZ (LFA-l), CD2, CD7, KLRC2 (NKGZC), TNFRSIS (GITR), TNFRSF l4 (HVEM), or any combination thereof.

In some aspects, the engineered receptor binds a . In some aspects. the binding is specific to a peptide specific to one or more subjects suffering from a disease or condition.

In some aspects. the immunotherapeutic is a cell as described in detail herein. In some aspects. the therapeutic is a cell comprising a receptor that specifically binds a peptide. In some aspects, the immunotherapeutic is a cell used in ation with the peptides/nucleic acids of this invention. In some embodiments, the cell is a patient cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is tumor infiltrating lymphocyte.

In some aspects, a subject with a ion or disease is treated based on a T cell or repertoire of the subject. In some embodiments. an antigen e is selected based on a T cell receptor repertoire of the subject. In some embodiments, a subject is treated with T cells expressing TCRs specific to an antigen or peptide. In some embodiments, a subject is treated with an antigen or peptide specific to TCRs, e.g., subject specific TCRs. In some ments, a subject is treated with an antigen or peptide specific to T cells expressing TCRs, e.g., t specific TCRs. In some ments, a subject is treated with an antigen or peptide specific to t specific TCRs.

In some embodiments. an irnrnunogenic antigen composition or vaccine is selected based on TCRs identified in one or more subjects. In some embodiments identification of a T cell repertoire and g in Functional assays is used to ine an imrnunogenic composition or vaccine to be administered to one or more subjects with a condition or disease. In some embodiments, the imrnunogenic composition is an antigen vaccine. In some embodiments, the antigen vaccine comprises subject specific antigen peptides. In some embodiments, antigen peptides to be included in an antigen vaccine are selected based on a quantification of subject specific TCRs that bind to the antigens. In some embodiments. n peptides are selected based on a binding affinity of the peptide to a TCR. In some embodiments, the selecting is based on a combination of both the quantity and the binding affinity. For example, a TCR that binds ly to an antigen in a functional assay, but that is not highly represented in a TCR repertoire may be a good candidate for an antigen vaccine because T cells expressing the TCR would be advantageously amplified.

In some embodiments, antigens are selected for administering to one or more ts based on g to TCRs. In some ments. T cells. such as T cells from a subject with a disease or condition. can be expanded. Expanded T cells that express TCRs specific to an immunogenic antigen peptide, can be administered back to a subject. In some embodiments, suitable cells, e.g., PBMCs, are transduced or transfected with eleotides for expression of TCRs specific to an immunogenic antigen peptide and administered to a subject. T cells expressing TCRs specific to an immunogenic antigen peptide can be expanded and administered back to a subject. In some embodiments, T cells that express TCRs specific to an immunogenic antigen peptide that result in cytolytic activity when incubated with autologous diseased tissue can be expanded and administered to a subject. In some embodiments, T cells used in functional assays result in binding to an immunogenic antigen peptide can be expanded and stered to a t. In some embodiments, TCRs that have been determined to bind to subject specific immunogenic antigen es can be expressed in T cells and administered to a subject.

Co-Stimulatory Ligands In some embodiments, the cells of the invention are provided with at least one co-stimulatory ligand which is a non-antigenspecific signal important for full activation of an immune cell. Co-stimulatory ligands include, without tion, tumor necrosis factor (TNF) ligands, cytokines (such as IL-2, IL- 12, IL-15 or ILZ I), and immunoglobulin (Ig) superfamily ligands.

Tumor necrosis factor (TNF) is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its primary role is in the tion of immune cells. Tumor is factor (TNF) ligands share a number of common es. The majority of the ligands are synthesized as type II transmembrane proteins containing a short cytoplasmic segment and a vely long extracellular region. TNF ligands e, without limitation, nerve growth factor (NGF), CD4OL (CD4OL)/CD154, CD137L/4-lBBL, tinnor necrosis factor alpha (TNFa), CD l34L/OX4OL/CD252, CD27L/CD70, Fas ligand (FasL), CD3OL/CD153, tumor is factor [3 (TNF(3)/lymphotoxin-alpha (LTa), lymphotoxin-beta (ur(3), CD257/B cell-activating factor (BAFF)/Blys/THANK/Tal l-l, glueocortieoid-induced TNF Receptor ligand (GITRL), and TNF- relatcd apoptosis-inducing ligand (TRAIL), LIGHT (TNFSFI4). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble ns that are involved in the ition, binding, or adhesion processes of cells. These ns share structural features with immunoglobulins--they possess an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, without limitation, CD80 and CD86, both ligands for CD28.

Compositions comprising genetically modified immunoresponsive cells of the invention can be provided ically or directly to a subject for the treatment of a neoplasia. In some embodiments. cells of the invention are directly injected into an organ of interest (e.g., an organ affected by a tumor). Altematively, compositions comprising genetically modified immunoresponsive cells are ed indirectly to the organ of st, for example, by administration into the atory system (e.g., the tumor vasculature). Expansion and differentiation agents can be provided prior to, during or after stration of the cells to increase production ofT cells, NK cells, or CTL cells in vitro or in vivo.

The modified cells can be administered in any physiologically acceptable vehicle. normally intravascularly, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). Genetically modified immunoresponsive cells of the invention can comprise a purified population of cells. Those skilled in the art can readily determine the tage of genetically d immunoresponsive cells in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, or the like. If desired, factors can also be included, including, but not limited to, interleukins, e.g. IL-Z, lL-3, lL-6, and lL-l l, as well as the other interleukins, the colony stimulating factors, such as G-, M- and GM-CSF, interferons, e.g. y-interferon and erythropoietin.

Compositions of the invention include pharmaceutical compositions comprising genetically modified immunoresponsive cells or their progenitors and a phannaceutieally acceptable carrier. Administration can be autologous or heterologous. For example, immunoresponsive cells, or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells of the ion or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present invention (e.g., a pharmaceutical composition containing a genetically d immunoresponsive cell), it will generally be formulated in a unit dosage injectable fonn ion, suspension, on).

VII. Methods of use and pharmaceutical itions The neoantigen therapeutics (e.g., es, ucleotides, TCR CAR, cells containing TCR or CAR dendritic cell containing polypeptide, dendritie cell containing polynucleotide, antibody, etc.) described herein are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as the treatment of . In some embodiments, the eutic treatment methods comprise thcrapy. In certain embodiments, a neoantigen peptide is useful for activating, promoting, increasing, and/or enhancing an immune response. redirecting an existing immune response to a new target, increasing the immunogenicity of a tumor, inhibiting tumor , reducing tumor volume, increasing tumor cell sis, and/or reducing the tumorigenicity of a tumor. The methods of use can be in vitro. ex vivo, or in vivo methods.

In some aspects, the present invention provides methods for ting an immune response in a subject using a neoantigen therapeutic described herein. In some embodiments. the invention provides methods for promoting an immune se in a subject using a neoantigen therapeutic described herein. In some embodiments, the invention provides methods for increasing an immune response in a subject using a neoantigen peptide described herein. In some embodiments, the invention provides methods for enhancing an immune response using a neoantigen peptide. In some embodiments, the activating, promoting, increasing, and/or ing of an immune se comprises increasing cell-mediated immunity. In some ments. the activating. ing. increasing. and/or ing of an immune response comprises increasing T cell activity or humoral ty. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune se comprises increasing CTL or HTL activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune se comprises increasing NK cell ty. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T cell activity and increasing NK cell activity. In some ments, the activating. promoting, increasing, and/or enhancing of an immune response ses increasing CTL ty and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of Tregs. In some embodiments, the immune response is a result of antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor cell. In some embodiments, the antigenic stimulation is cancer.

In some embodiments, the invention provides methods of ting, promoting, sing, and/or enhancing of an immune response using a neoantigen therapeutic bed herein. In some embodiments, a method comprises administering to a subject in need thereof a therapeutically effective amount of a igen therapeutic that delivers a neoantigen polypeptide or polynucleotide to a tumor cell. In some embodiments, a method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigen therapeutic that binds the tumor associated antigen and is internalized by the tumor cell. In some embodiments, a method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigen ptide that is internalized by a tumor cell, and the neoantigen peptide is processed by the cell. In some embodiments. a method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigen polypeptide that is internalized by a tumor cell, and an nic peptide is presented on the surface of the tumor cell. In some embodiments, a method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigen polypeptide that is lized by the tumor cell, is processed by the cell, and an antigenic peptide is presented on the surface of the tumor cell.

In some embodiments. a method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigen polypeptide or polynucleotide described herein that delivers an ous polypeptide comprising at least one nic peptide to a tumor cell, wherein the antigenic peptide is presented on the surface of the tumor cell. In some embodiments, the antigenic peptide is presented on the e of the tumor cell in complex with a MHC class I le. In some embodiments, the antigenic peptide is presented on the surface of the tumor cell in complex with a MHC class II molecule.

In some embodiments, a method comprises contacting a tumor cell with a neoantigen polypeptide or polynucleotide described herein that delivers an exogenous polypeptide comprising at least one antigenic peptide to the tumor cell, wherein the antigenic peptide is presented on the e of the tumor cell. In some embodiments, the antigenic peptide is presented on the surface of the tumor cell in complex with a MHC class I molecule. In some embodiments, the antigenic peptide is presented on the surface of the tumor cell in complex with a MHC class II molecule.

In some embodiments, a method ses administering to a subject in need thereofa therapeutically effective amount of a neoantigen polypeptide or polynucleotide described herein that delivers an ous polypeptide comprising at least one antigenic peptide to a tumor cell, wherein the antigenic peptide is presented on the surface of the tumor cell, and an immune response against the tumor cell is induced. In some embodiments, the immune response against the tumor cell is increased. In some embodiments, the neoantigen polypeptide or poly-nucleotide delivers an exogenous polypeptide comprising at least one antigenic peptide to a tumor cell, n the nic peptide is presented on the surface of the tumor cell, and tumor growth is inhibited.

In some embodiments, a method comprises administering to a subject in need thereof a therapeutically effective amount of a igen polypeptide or polynucleotide described herein that delivers an exogenous polypeptide sing at least one antigenic peptide to a tumor cell, wherein the antigenic peptide is presented on the e of the tumor cell, and T cell killing directed against the tumor cell is induced. In some embodiments, T cell killing directed against the tumor cell is enhanced. In some ments, T cell killing directed against the tumor cell is sed.

In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a neoantigen therapeutic described herein, wherein the agent is an antibody that specifically binds the neoantigen described herein. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of the antibody.

The present invention provides methods of redirecting an existing immune response to a tumor. In some embodiments, a method of redirecting an existing immune response to a tumor ses administering to a subject a therapeutically ive amount of a neoantigen therapeutic described herein. In some embodiments, the ng immune response is against a virus. In some embodiments, the virus is selected from the group consisting of: measles virus, varicclla-zoster virus (VZV; chickenpox virus), za virus, mumps virus, poliovirus, rubella virus, rotavirus, hepatitis A virus (I-IAV), hepatitis B virus (HBV). Epstein Barr virus (EBV), and cytomegalovirus (CMV). In some ments, the virus is varicella—zoster virus. In some embodiments, the virus is cytomegalovirus. In some embodiments, the virus is measles virus. In some ments, the existing immune response has been acquired after a l viral infection. In some embodiments, the existing immune response has been acquired after vaccination against a virus. In some embodiments, the existing immune response is a cell-mediated response. In some embodiments, the existing immune response comprises cytotoxic T cells (CTLs) or HTLs.

In some embodiments, a method of redirecting an existing immune response to a tumor in a subject comprises administering a fusion n comprising (i) an antibody that specifically binds a neoantigen and (ii) at least one igenic peptide described herein, wherein (a) the fusion protein is internalized by a tumor cell afier binding to the tumor-associated antigen; (b) the neoantigenic peptide is processed and presented on the surface of the tumor cell associated with a MHC class I molecule; and (c) the neoantigenic e/MHC Class I complex is recognized by xic T cells. In some embodiments. the xic T cells are memory T cells. In some embodiments, the memory T cells are the result of a vaccination with the neoantigenic peptide.

The present invention provides methods of increasing the immunogenicity of a tumor. In some embodiments, a method of increasing the immunogenicity of a tumor ses contacting a tumor or tumor cells with an effective amount of a neoantigen therapeutic described herein. In some embodiments, a method of sing the genicity of a tumor comprises administering to a subject a therapeutically effective amount ofa neoantigen therapeutic described .

The present invention also provides methods for inhibiting growth of a tumor using a neoantigen therapeutic described herein. In certain embodiments, a method of inhibiting growth of a tumor comprises contacting a cell mixture with a neoantigen therapeutic in vitro. For example, an immortalized cell line or a cancer cell line mixed with immune cells (e.g., T cells) is cultured in medium to which a neoantigenic peptide is added. In some ments, tumor cells are isolated from a patient sample, for example, a tissue biopsy, pleural effusion, or blood sample, mixed with immune cells (e.g., T cells), and cultured in medium to which a neoantigen therapeutic is added. In some embodiments. a neoantigen therapeutic increases. promotes, and/or enhances the activity of the immune cells. In some embodiments, a neoantigen therapeutic inhibits tumor cell . In some ments, a neoantigen therapeutic activates killing of the tumor cells.

In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or the t had a tumor which was at least partially d.

In some embodiments, a method of inhibiting growth of a tumor comprises redirecting an existing immune response to anew target. comprising administering to a subject a therapeutically effective amount of a neoantigen therapeutic, wherein the existing immune response is against an antigenic peptide red to the tumor cell by the neoantigenic peptide.

In certain embodiments, the tumor comprises cancer stem cells. In n embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of the neoantigen therapeutic. In some embodiments, a method of reducing the frequency of cancer stem cells in a tumor in a subject, sing administering to the subject a therapeutically effective amount of a neoantigen therapeutic is provided.

In addition, in some aspects the invention provides a method of reducing the tumorigenicity of a tumor in a t, sing administering to the subject a therapeutically effective amount of a neoantigen therapeutic bed herein. In certain embodiments, the tumor comprises cancer stem cells. In some ments, the tumorigenicity of a tumor is reduced by reducing the frequency of cancer stem cells in the tumor. In some embodiments. the methods se using the neoantigen therapeutic described herein. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by stration of a igen therapeutic described herein.

In some embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a tumor selected from the group consisting of: colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, ndocrinc tumor, gastrointestinal tumor, melanoma, al tumor. bladder tumor. glioblastoma and head and neck tumor. In certain embodiments. the tumor is a colorectal tumor. In certain embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a breast tumor. In some embodiments, the tumor is a lung tumor. In certain embodiments, the tumor is a pancreatic tumor. In certain embodiments, the tumor is a melanoma tumor. In some embodiments, the tumor is a solid tumor.

The present invention r provides methods for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a neoantigen therapeutic described herein.

In some embodiments, a method of treating cancer comprises redirecting an existing immune response to a new target, the method comprising administering to a subject a therapeutically ive amount of neoantigen therapeutic, wherein the existing immune response is against an nic peptide red to the cancer cell by the neoantigenic peptide.

The present invention provides for methods of treating cancer comprising administering to a subject a therapeutically effective amount of a neoantigen therapeutic described herein (e.g., a subject in need of treatment). In certain embodiments, the subject is a human. In certain embodiments. the subject has a cancerous tumor. In certain embodiments, the subject has had a tumor at least partially removed.

In n embodiments, the cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung , ovarian cancer, liver cancer, breast , kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, ncurocndocrinc cancer, bladder cancer, glioblastoma, and head and neck cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is ovarian . In certain embodiments. the cancer is colorectal cancer. In certain embodiments. the cancer is breast cancer. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is ma. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer comprises a solid tumor.

In some embodiments, the cancer is a hematologic . In some ment, the cancer is selected from the group ting of: acute myelogcnous leukemia (AML), Hodgkin lymphoma, multiple myeloma, T cell acute lyrnphoblastic leukemia (T—ALL). chronic lymphocytic leukemia (CLL), hairy cell leukemia. chronic myelogcnous leukemia (CML), non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and cutaneous T cell lymphoma (CTCL).

In some embodiements, the neoantigen eutic is administered as a combination therapy.

Combination therapy with two or more therapeutic agents uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action can result in additive or etic effects. Combination therapy can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s). Combination therapy can decrease the likelihood that resistant cancer cells will develop. In some embodiments, combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells.

Cancers include. but are not limited to. B cell . e.g. multiple myeloma. Waldenstrom's macroglobulinemia the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and cytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer (e. g. , metastatic, hormone refractory prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, al cancer, uterine or endometrial cancer. cancer of the oral cavity or pharynx, liver , kidney cancer, testicular . biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present invention e human sarcomas and carcinomas, e. g. , fibrosarcoma, myxosarcoma, liposarcoma, chondrosarooma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, ngiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer. pancreatic cancer. breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma. adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary arcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, carcinoma, ma, nal carcinoma, Wilms' tumor, al cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma. pinealoma. hemangioblastoma acoustic neuroma. oligodendroglioma, meningioma. melanoma, neuroblastoma, blastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and themia vera, lymphoma (Hodgkin's disease and non-Hodgkin's e), multiple myeloma, Waldenstrom's lobulinemia, and heavy chain disease. In some embodiments, the cancer whose phenotype is determined by the method of the invention is an epithelial cancer such as. but not limited to. bladder cancer. breast cancer. cervical cancer. colon cancer, logic cancers, renal cancer, eal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin . In other embodiments, the cancer is breast cancer, prostate cancer, lung , or colon cancer. In still other ments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial s may be characterized in various other ways including. but not limited to. serous, endometrioid. mucinous, clear cell. brenner. or undifferentiated. In some embodiments, the present invention is used in the treatment, diagnosis, and/or prognosis of lymphoma or its subtypes, including, but not d to, mantle cell lymphoma. Lymphoproliferative disorders are also ered to be proliferative diseases.

In some embodiments, the combination of an agent described herein and at least one additional therapeutic agent results in additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the agent. In some ments, the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s).

In certain embodiments, in addition to administering a neoantigen therapeutic described herein, the method or treatment further comprises stering at least one additional therapeutic agent. An additional therapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the agent. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

Therapeutic agents that can be administered in combination with the neoantigen therapeutic described herein include chemotherapeutic agents. Thus, in some embodiments, the method or treatment es the administration of an agent bed herein in combination with a chemotherapeutic agent or in ation with a cocktail of chemotherapeutic agents. Treatment with an agent can occur prior to. rently with, or subsequent to stration of chemotherapies. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive stration in either order but generally within a time period such that all active agents can exert their biological activities aneously. Preparation and dosing schedules for such chemotherapeutic agents can be used according to cturers' instructions or as detemiined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in The Chemotherapy Source Book. 4th Edition. 2008. M.

C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.

Useful classes of chemotherapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and clear platinum complexes and carboplatin), anthracyelines, otics, anti-folates, antimetabolites, chemotherapy sensitizers, duocarmycins, ides, fluorinatcd dines, ionophores. lexitropsins. nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, omerase inhibitors, vinca alkaloids, or the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an totic, a topoisomerase inhibitor, or an angiogenesis inhibitor.

Chemotherapeutic agents usefiil in the instant invention e, but are not limited to, ting agents such as thiotepa and cyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan. ulfan and piposulfan: aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trieQ-rlenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, rethamine, mechlorethamine oxide hloride, mclphalan, ichin, phcncstcrinc, prcdnimustinc, trofosfamidc, uracil d; nitrosureas such as cannustine. chlorozotocin. fotemustine. lomustine. nimustine. ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azasen'ne, bleomycins. cactinomycin, calicheamicin, carabicin, mycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazooxo-L- norleucine, doxorubicin, epirubicin, esorubicin, icin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonign'n, strcptozocin, tubercidin, ubcnimcx, zinostatin, zorubicin; anti-mctabolitcs such as rcxatc and 5- fluorouracil (S-FU); folic acid ues such as denopterin, methotrexate. pteropterin, rexate; purine analogs such as fludarabine, aptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, cannofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, S-FU: androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfonnithine; elliptinium acetate; cid; gallium nitrate; yurea; an; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; Z-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogemianium; tenuazonic acid; tnaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; onitol; mitolactol; pipobroman; gacflosine; arabinoside (Ara- C); taxoids, e.g. paclitaxel (TAXOL) and docetax'el (TAXOTERE); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; cin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPTl l; topoisomerase inhibitor RES 2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA): and pharmaceutically acceptable salts, acids or derivatives of any of the above. Chemotherapeutic agents also e anti-hormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, fcnc, aromatasc inhibiting 4(5)-imidazolcs, 4 hydroxfiamoxifcn, trioxifene, keoxifene, 18. onapristone, and toremifene (FARESTON); and anti-androgens such as flutamide. nilutamide, bicalutamide, leuprolide, and lin; and pharmaceutically able salts, acids or derivatives of any of the above. In certain embodiments, the onal therapeutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin.

In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g.. topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin e, mitoxantrone HCI, actinomyein D, etoposide, can HCI, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or tives of any of these. In some embodiments, the additional therapeutic agent is irinotecan.

In certain embodiments, the chemotherapeutic agent is an anti-metabolite. An anti-metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions. yet different enough to interfere with one or more normal functions of cells, such as cell on. Anti- metabolites e, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, S-azacytidine, 6 mercaptopurine, azathioprine, 6-thioguanine, pentostatin, bine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any ofthese. In certain embodiments, the additional therapeutic agent is abine.

In certain embodiments, the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin. In some embodiments, the agent is a taxane. In certain ments, the agent is paclitaxel or xel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitavel (TAXOL), xel (TAXOTERE), albumin-bound paclitaxel (ABRAXANE), DHA-paclitaxel, or PG-paclitavel. In certain altemative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or phannaceutically acceptable salts. acids. or derivatives thereof. In some embodiments. the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plkl. In certain embodiments, the additional therapeutic agent is axel. In some embodiments, the additional therapeutic agent is n- bound paclitaxel.

In some embodiments, an additional therapeutic agent comprises an agent such as a small molecule.

For example, treatment can involve the ed administration of an agent of the present invention with a small molecule that acts as an inhibitor against tumor-associated antigens ing. but not limited to. EGFR.

HERZ (ErbB2), and/or VEGF. In some embodiments, an agent of the present invention is administered in combination with a n kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), nib T), lapatanib, vandetanib (ZACTIMA), AEE788, Cl-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In some embodiments, an additional therapeutic agent comprises an mTOR inhibitor. In another embodiment, the onal therapeutic agent is chemotherapy or other inhibitors that reduce the number of Tng cells. In certain embodiments. the therapeutic agent is cyclophosphamide or an anti-CTLA4 antibody. In another embodiment, the additional therapeutic reduces the presence of d-derived suppressor cells. In a further embodiment, the additional therapeutic is carbotaxol. In another ment, the additional therapeutic agent shifis cells to a T helper 1 response. In a further embodiment, the onal eutic agent is ibrutinib.

In some embodiments, an additional therapeutic agent comprises a biological molecule, such as an antibody. For example. treatment can involve the combined administration of an agent of the present invention with antibodies against tumor-associated antigens including, but not limited to, antibodies that bind EGFR, HERZ/ErbBZ, and/or VEGF. In certain embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In certain ments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor dy). In certain embodiments, the onal therapeutic agent is bevacizumab (AVASTIN), rumab, trastuzumab (HERCEPTIN), pertuzumab ARG). panitumumab (VECTIBIX). nimotuzumab. zalutumumab. or cetuximab (ERBITUX).

The agents and compositions provided herein may be used alone or in ation with tional therapeutic regimens such as surgery, ation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or ted). A set of tumor antigens can be useful, e.g., in a large fraction of cancer ts.

In some embodiments. at least one or more chemotherapeutic agents may be administered in addition to the ition comprising an immunogenic vaccine. In some embodiments, the one or more chemotherapeutic agents may belong to different s of chemotherapeutic agents.

Examples of chemotherapy agents include, but are not limited to, alkylating agents such as nitrogen mustards (e.g. inechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxant’li‘r), ifosfamide, and melphalan); nitrosoureas (e.g. oso-N-methylurea, streptozocin, carmustine (BCNU), lomustine, and semustine); alkyl sulfonates (e.g. busulfan); tetrazines (e.g. dacarbazine (DTIC), mitozolomide and temozolomide (Temodarilii)); aziridines (e.g. thiotepa, mytomycin and diaziquone); and platinum drugs (e.g. cisplatin, carboplatin, and oxaliplatin); non-classical alkylating agents such as procarbazine and altretamine (hexamethylmelamine); anti-metabolite agents such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine E), bine, floxuridine, fludarabine, nelarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin, thioguanine, Vidaza; anti-mierotubule agents such as vinca alkaloids (e.g. vincristine, stine, vinorelbine. vindesine and vinflunine); taxanes (e.g. paclitaxel (Tami-11)). docetaxel (Tax'oteretlii)); podophyllotoxin (e.g. etoposide and teniposide); epothilones (e.g. ixabepilone (lxempral’LRDD; estramustine (Emcytfll‘l); anti-tumor antibiotics such as anthracyclines (e.g. ubicin, doxorubicin (Adriamycint‘liTI, epirubicin, idarubicin); actinomycin-D; and bleomycin; topoisomerase 1 inhibitors such as topotecan and irinotecan (CPT—l 1); topoisomerase II tors such as etoposide (VP-16), teniposide, ntrone, novobiocin, mcrbarone and aclarubiein; osteroids such as prcdnisonc, mcthylprcdnisolonc (Solumedrole‘). and dexamethasonc (Decadron®); L-asparaginase; bortezomib (Velcade®); immunotherapeutic agents such as rituximab (Rituxan®), alemtuzumab (Campath®), thalidomide, lenalidomide (Revlimid®), BCG, eukin-2, interferon-alfa and cancer vaccines such as ProvengeaE‘; homione therapeutic agents such as fiilvestrant (FaslodexfE‘i), tamoxifen, toremifene (FarestontRi), anastrozole (ArimidexGKD), exemestan (Aromasin-i'liil), letrozole (Femara'li‘), megestrol acetate (Megacenlifi), estrogens, tamide (Casodex®), flutamide (Eulexin-I'E'), nilutamide (Nilandront’fili‘l), leuprolide ntR‘) and goserelin (Zoladex-‘BD; differentiating agents such as retinoids. tretinoin (ATRA or AtralinCET'i). bexarotene (Targretint’ii'?) and c trioxide (Arsenox'IRD); and targeted therapeutic agents such as imatinib (GleeveoR‘I), gefitinib (Iressaa'li') and nib (Sutent'R'I). In some embodiments, the chemotherapy is a cocktail therapy.

Examples of a cocktail therapy includes, but is not limited to, CHOP/R-CHOP (rituxan, eyclophosphamide, hydroxydoxorubicin, vincristine, and prednisone), EPOCH (etoposide, prednisone, vincristine, cyclophosphamidc, hydroxydoxorubicin), CVAD (cyclophosphamide, vincristinc, ydoxorubicin. dexamethasone), FOLFOX (fluorouraeil (S-FU), leucovorin. oxaliplatin). ICE (ifosfamide, carboplatin, etoposide), DHAP (high-dose bine [ara-C], dexamethasone, cisplatin), ESHAP side, methylprednisolone, cytarabine [ara-C], cisplatin) and CMF (cyclophosphamide, methotrexate, fluouracil).

In certain embodiments, an additional therapeutic agent comprises a second immunotherapeutic agent.

In some embodiments, the additional immunothcrapcutic agent es, but is not limited to, a colony stimulating factor. an interleukin. an antibody that blocks immunosuppressive functions (e.g.. an anti-CTLA4 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody), an antibody that enhances immune cell functions (e.g., an anti-GITR antibody, an X-40 antibody, an anti-CD40 antibody, or an antil BB antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), a soluble ligand (e.g., GITRL, GITRL-Fc, OX-40L, OX-40L-Fc, CD40L, CD40L—Fc, 4-lBB ligand, or 4-lBB ligand-Fe), or a member of the B7 family (e.g., CD80, CD86). In some embodiments, the additional therapeutic agent targets CTLA-4, CD28, CD3, PD-l, PD-Ll, TIGIT, GITR, OX-40, CD-40, or 4- In some embodiments, the additional therapeutic agent is an immune checkpoint tor. In some embodiments, the immune checkpoint tor is an anti-PD-l antibody, an anti-PD-Ll antibody, an anti- CTLA-4 antibody, an anti-CD28 antibody, an anti-TIGIT antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, an anti-GITR dy, an anti1BB antibody, or an anti-OX-40 antibody. In some ments, the additional therapeutic agent is an anti-TIGIT dy. In some embodiments, the additional therapeutic agent is an anti-PD-I antibody selected from the group consisting of: mab (OPDIVO). pembrolizumab (KEYTRUDA), pidilzumab, MEDIO680, REGN2810. BGB-A317, and PDROOl. In some embodiments, the additional therapeutic agent is an anti-PD-LI antibody selected from the group consisting of: BMS935559 (MDX-l 105), izumab (MPDL3280A), durvalumab (MEDI4736), and avelumab (MSBOOIO718C). In some embodiments, the onal therapeutic agent is an anti-CTLA-4 antibody ed from the group consisting of: ipilimumab Y) and trcmelimumab. In some embodiments, the additional therapeutic agent is an anti-LAG-3 antibody selected from the group consisting of: EMS-986016 and LAGSZS. In some embodiments, the additional therapeutic agent is an anti-OX-40 antibody ed from the group consisting of: MEDI6469, MEDIOS62, and MOXR09I6. In some embodiments, the additional therapeutic agent is an antiIBB antibody selected from the group consisting of: PF-05082566.

In some embodiments, the neoantigen therapeutic can be administered in ation with a biologic le selected from the group consisting of: adrenomedullin (AM). angiopoietin (Ang). BMPs. BDNF.

EGF, opoietin (EPO), FGF, GDNF. granulocyte colony stimulating factor (G-CSF), granuloeyte- macrophage colony stimulating factor (GM-C SF), macrophage colony stimulating factor (M-CSF), stem cell factor (SCF), GDP9, HGF, HDGF, IGF, migration-stimulating , myostatin (GDP-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-a, TGF-B, TNF-a, VEGF, PlGF, gamma-IFN, IL-1, IL-2, IL—3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and lL-18.

In some embodiments. treatment with a neoantigen therapeutic described herein can be accompanied by surgical removal of tumors, removal of cancer cells, or any other surgical therapy deemed necessary by a treating physician.

In certain ments, treatment involves the administration of a neoantigen therapeutic described herein in combination with ion therapy. Treatment with an agent can occur prior to, concurrently with, or subsequent to administration of radiation therapy. Dosing schedules for such radiation therapy can be determined by the skilled medical tioner.

Combined administration can include eo-administration, either in a single phannaeeutical formulation or using separate formulations, or consecutive stration in either order but generally within a time period such that all active agents can exert their ical activities simultzuieously.

It will be appreciated that the combination of a neoantigen therapeutic described herein and at least one additional therapeutic agent can be administered in any order or concurrently. In some embodiments, the agent will be administered to patients that have previously undergone treatment with a second therapeutic agent. In certain other embodiments. the neoantigen therapeutic and a second therapeutic agent will be administered substantially simultaneously or concurrently. For example, a subject can be given an agent while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy). In certain embodiments, a neoantigen therapeutic will be administered within 1 year of the treatment with a second therapeutic agent. It will fiirther be appreciated that the two (or more) agents or treatments can be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously).

For the treatment of a disease. the appropriate dosage of a neoantigen therapeutic bed herein s on the type of e to be treated, the severity and course of the disease, the responsiveness of the disease, whether the agent is administered for eutic or preventative purposes, previous therapy, the patient's clinical y, and so on, all at the discretion ofthe treating physician. The neoantigen therapeutic can be stered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the e state is achieved (c.g., reduction in tumor size). Optimal dosing schedules can be ated from measurements of drug accumulation in the body of the patient and will vary depending on the ve potency of an individual agent. The administering physician can determine Optimum dosages, dosing methodologies, and repetition rates.

In some ments, a neoantigen therapeutic can be administered at an initial higher "loading” dose, followed by one or more lower doses. In some embodiments, the frequency of administration can also change. In some embodiments, a dosing n can comprise administering an initial dose, followed by onal doses (or “maintenance” doses) once a week. once every two weeks. once every three weeks. or once every month. For example, a dosing regimen can comprise administering an l loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose. Or a dosing regimen can comprise administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week. Or a dosing regimen can comprise administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.

As is known to those of skill in the art. administration of any therapeutic agent can lead to side effects and/or toxicities. In some cases, the side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose. In some cases, therapy must be discontinued, and other agents can be tried. However, many agents in the same therapeutic class display r side s and/or toxicities, meaning that the patient either has to stop therapy, or if possible, suffer from the unpleasant side effects associated with the therapeutic agent.

In some embodiments. the dosing schedule can be limited to a specific number of administrations or “cycles”. In some embodiments, the agent is administered for 3, 4, 5, 6, 7, 8, or more cycles. For example, the agent is administered every 2 weeks for 6 cycles, the agent is administered every 3 weeks for 6 , the agent is administered every 2 weeks for 4 cycles, the agent is administered every 3 weeks for 4 , etc.

Dosing schedules can be decided upon and subsequently modified by those skilled in the art.

The present invention provides methods of administering to a subject a igen therapeutic described herein sing using an intermittent dosing gy for administering one or more agents, which can reduce side effects and/or toxicities associated with administration of an agent, herapeutic agent. etc. In some embodiments, a method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of a neoantigen therapeutic in combination with a therapeutically effective dose of a herapeutic agent, wherein one or both of the agents are administered according to an ittent dosing strategy. In some embodiments, a method for treating cancer in a human subject comprises stering to the subject a therapeutically effective dose of a neoantigen therapeutic in combination with a therapeutically effective dose of a second immunotherapeutic agent. wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises stering an initial dose of a neoantigen eutic to the t, and administering subsequent doses of the agent about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a neoantigen therapeutic to the subject, and administering subsequent doses of the agent about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a neoantigen eutic to the subject, and administering subsequent doses of the agent about once every 4 weeks. In some embodiments, the agent is stered using an intemiittent dosing strategy and the additional therapeutic agent is administered weekly.

The present invention provides compositions comprising the neoantigen therapeutic described herein.

The present invention also provides pharmaceutical compositions comprising a neoantigen therapeutic described herein and a phannaceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions find use in immunotherapy. In some embodiments. the compositions find use in inhibiting tumor gromh. In some embodiments, the pharmaceutical compositions find use in inhibiting tumor groyyth in a subject (e.g., a human patient). In some embodiments. the compositions find use in treating cancer. In some embodiments, the pharmaceutical compositions find use in treating cancer in a subject (e.g., a human patient).

Formulations are prepared for storage and use by combining a igen therapeutic of the present invention with a phamiaceutically acceptable vehicle (c.g., a carrier or excipicnt). Those of skill in the art generally consider phannaceutically acceptable carriers. excipients, and/or stabilizers to be ve ingredients of a ation or pharmaceutical composition. Exemplary formulations are listed in W0 2015/09581 l. le pharrnaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other c acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadccyldimcthylbcnzyl ammonium chloride, hexamethonium chloride, konium chloride. benzethonium chloride, phenol. butyl or benzyl alcohol, alkyl parabens. such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins: hilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, gine, histidine, arginine, or lysine; carbohydrates such as monosaecharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, ose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as tein complexes; and non- ionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 225t Edition, 2012, Pharmaceutical Press. London.) In some embodiments, the vehicle is 5% dextrose in water.

The pharmaceutical compositions described herein can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdennal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or ls. including by nebulizer. racheal, and intranasal; oral; or parenteral ing intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).

The eutic formulation can be in unit dosage form. Such fonnulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories.

The ncoantigcnic peptides described herein can also be ped in microcapsules. Such microcapsules are prepared. for example, by coacervation ques or by acial polymerization, for example, hydroxymethyIceIIquse or gelatin-microcapsules and methylmethacylate) microcapsules, respectively, in dal drug delivery s (for example, Iiposornes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22st Edition, 2012, Pharmaceutical Press, London.

In certain embodiments, phannaceutical fomiulations e a neoantigen therapeutic described herein complexed with liposomes. Methods to produce liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter.

In certain embodiments, ned-release preparations comprising the neoantigenic peptides described herein can be produced. Suitable examples of sustained-release preparations include semi- permeable matrices of solid hydrophobic polymers containing an agent. where the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L- glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, able lactic acid-glycolic acid mers such as the LUPRON DEPOTTM (injectable pheres composed of lactic acid-glycolic acid copolymcr and leuprolidc acetate), sucrose acetate isobutyratc, and -(-)hydroxybutyric acid.

The present sure provides s of treatment comprising an immunogenic vaccine. Methods of treatment for a disease (such as cancer or a viral ion) are provided. A method can comprise administering to a subject an effective amount of a composition comprising an immunogenic antigen. In some embodiments, the antigen ses a viral antigen. In some embodiments, the antigen ses a tumor antigen.

Non-limiting examples of vaccines that can be prepared include a peptide-based vaccine, a nucleic ased vaccine, an antibody based vaccine, a T cell based vaccine, and an antigen-presenting cell based vaccine.

Vaccine itions can be formulated using one or more physiologically acceptable rs including excipients and auxiliaries which tate processing of the active agents into preparations which can be used pharmaceutically. Proper formulation can be dependent upon the route of stration .

Any of the well-known techniques, carriers, and excipients can be used as suitable and as understood in the In some cases. the vaccine composition is formulated as a peptide-based e. a nucleic acid-based vaccine, an antibody based vaccine, or a cell based vaccine. For example, a vaccine composition can e naked cDNA in cationic lipid formulations; lipopeptides (e.g, Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), naked cDNA or peptides, encapsulated e.g., in poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g, Eldridge, et al., Molec. l. -294, 1991: Alonso et al, Vaccine 12:299-306, 1994; Jones et al, Vaccine 13:675-681, 1995); peptide composition contained in immune stimulating complexes (ISCOMS) (e.g., Takahashi et al, Nature 344:873-875, 1990; Hu et al. Clin Exp Immunol. 113:235—243, 1998); or multiple antigen peptide systems (MAPS) (see e.g., Tam, J. P., Proc. Natl Acad. Sci. USA. 85:5409-5413, 1988; Tam, J.P., J. Immunol. Methods 196217-32, 1996). Sometimes, a vaccine is fomiulated as a peptide- based vaccine, or nucleic acid based vaccine in which the nucleic acid encodes the polypeptides. Sometimes, a vaccine is formulated as an dy based vaccine. Sometimes, a vaccine is fonnulated as a cell based vaccine.

The amino acid ce of an identified disease-specific immunogenic neoantigen peptide can be used develop a phannaceutically acceptable composition. The source of antigen can be, but is not limited to, natural or synthetic proteins, including glycoproteins, peptides, and ntigens; antibody/antigen complexes; lipoproteins; RNA or a translation product thereof; and DNA or a polypeptide encoded by the DNA. The source of antigen may also comprise non-transformed, transformed. transfected, or transdueed cells or cell lines. Cells may be transformed. transfected. or uced using any of a variety of expression or retroviral vectors known to those of ordinary skill in the art that may be employed to express recombinant antigens. Expression may also be achieved in any appropriate host cell that has been transformed, transfected, or transduced with an expression or retroviral vector containing a DNA molecule encoding recombinant n(s). Any number of transfection, transformation, and transduction protocols known to those in the art may be used. Recombinant vaccinia vectors and cells infected with the vaccina vector, may be used as a source of antigen.

A composition can comprise a synthetic disease-specific immunogenic neoantigen peptide. A composition can comprise two or more disease-specific immunogenic neoantigen peptides. A composition may comprise a precursor to a disease-specific immunogenic peptide (such as a n, peptide, DNA and RNA). A precursor to a disease-specific immunogenic peptide can generate or be generated to the identified disease-specific genic neoantigen peptide. In some embodiments, a therapeutic composition ses a precursor of an immunogenic peptide. The precursor to a disease-specific immunogenic peptide can be a ug. In some embodiments. the composition comprising a disease-specific immunogenic neoantigen peptide may further se an adjuvant. For example, the neoantigen e can be utilized as a vaccine. In some embodiments, an immunogenic vaccine may comprise a phannaceutically acceptable immunogenic neoantigen peptide. In some embodiments, an immunogenic vaccine may se a pharmaceutically acceptable precursor to an genic neoantigen peptide (such as a protein, peptide, DNA and RNA). In some embodiments, a method of ent comprises administering to a subject an effective amount of an antibody specifically recognizing an immunogenic neoantigen peptide. In some embodiments, a method of treatment comprises administering to a subject an effective amount of a soluble TCR or TCR analog specifically recognizing an immunogenic neoantigen peptide.

The methods described herein are ularly usefiil in the personalized medicine context, where immunogenic neoantigen peptides are used to develop therapeutics (such as vaccines or therapeutic antibodies) for the same individual. Thus, a method of treating a disease in a subject can comprise identifying an immunogenic neoantigen peptide in a subject ing to the methods described herein; and synthesizing the peptide (or a precursor thereof); and administering the peptide or an antibody cally recognizing the peptide to the subject. In some embodiments, an sion pattern of an immunogenic igen can serve as the ial basis for the generation of patient specific vaccines. In some embodiments, an expression n of an immunogenic neoantigen can serve as the essential basis for the generation of a vaccine for a group of ts with a particular disease. Thus, particular diseases, 6. g. , particular types of , can be selectively treated in a t group.

In some embodiments, the peptides described herein are structurally normal ("shared“) ns that can be recognized by autologous anti-disease T cells in a large patient group. In some embodiments, an antigen-expression pattern of a group of diseased subjects whose disease expresses structurally normal ("shared”) neoantigens is determined.

There are a variety of ways in which to produce immunogenic neoantigens. Proteins or peptides may be made by any technique known to those of skill in the art. including the sion of proteins. polypeptides or peptides through standard molecular biological techniques, the isolation of ns or es from natural sources, in vitro ation, or the chemical synthesis of proteins or peptides. In general, such disease specific igens may be produced either in vifro or in vivo. lmmunogenic neoantigens may be produced in vitro as peptides or polypeptides, which may then be ated into a personalized vaccine or genic composition and administered to a subject. In vitro production of immunogenic igens can comprise peptide sis or expression of a peptide/polypeptide from a DNA or RNA molecule in any of a variety of bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptide/polypeptide. Altematively, immunogenic neoantigens can be produced in vivo by introducing molecules (e.g. and viral expression systems) that encode an immunogenic igen into a , DNA, RNA, subject, whereupon the encoded immunogenic neoantigens are expressed. In some embodiments, a poly-nucleotide encoding an immunogenic igen peptide can be used to e the neoantigen peptide in vitro.

In some embodiments. a polynucleotide comprises a sequence with at least 60%, 65%. 70%]. 75%. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%. or 100% identity to a polynucleotide encoding an immunogenic neoantigen.

The polynucleotide may be, e. g., DNA, cDNA, PNA, CNA, RNA, - and/or double-stranded, native or stabilized forms of polynucleotides, or combinations thereof. A nucleic acid encoding an immunogenic neoantigen peptide may or may not contain introns so long as it codes for the peptide. In some embodiments in vitro translation is used to e the peptide.

Expression vectors comprising sequences encoding the neoantigen, as well as host cells containing the expression vectors, are also contemplated. Expression vectors suitable for use in the present invention can comprise at least one expression control element operationally linked to the nucleic acid sequence. The expression control elements are inserted in the vector to control and regulate the expression of the nucleic acid sequence. Examples of expression control elements are well known in the art and include, for example, the lac system. operator and promoter regions of phage lambda, yeast promoters and promoters derived from a, adenovirus, retrovirus or SV40. Additional operational elements include, but are not limited to, leader ces, termination codons, enylation signals and any other sequences necessary or red for the appropriate transcription and subsequent translation of the nucleic acid sequence in the host system. It will be understood by one skilled in the art the correct combination of expression control elements will depend on the host system chosen. It will further be understood that the sion vector should contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers.

The neoantigen peptides may be provided in the form of RNA or cDNA molecules encoding the desired neoantigen peptides. One or more neoantigen peptides of the invention may be encoded by a single expression vector. Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. if necessary. the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), although such controls are generally available in the sion vector. The vector is then introduced into the host bacteria for cloning using standard techniques. Useful expression vectors for eukaryotic hosts, especially mammals or humans include, for example, vectors comprising sion l sequences from SV40, bovine papilloma virus, adenovirus and cytomcgalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR l. pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and tous single-stranded DNA phages.

In embodiments, a DNA sequence encoding a ptide of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such ucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those eodons that are favored in the host cell in which the recombinant polypeptide of st is produced. Standard methods can be applied to size an isolated polynucleotide sequence encoding an isolated polypeptide of interest.

Suitable host cells for expression of a polypeptide include prokaryotes, yeast, insect or higher eukaryotic cells under the control of appropriate ers. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells e established cell lines of mammalian origin. Cell-free translation systems can also be employed. Appropriate cloning and expression vectors for use with ial, fungal, yeast, and mammalian cellular hosts are well known in the art. Various mammalian or insect cell culture systems can be ed to s recombinant protein. Exemplary mammalian host cell lines include, but are not limited to COS-7, L cells, C 127, 3T3, e hamster ovary (CHO), 293, HeLa and BHK cell lines. Mammalian expression vectors can comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other ’ or 3~ flanking nscribed sequences, and S~ or 3‘ nontranslated sequences, such as ary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.

The proteins produced by a transformed host can be d according to any suitable . Such standard methods include chromatography (e.g., ion ge, affinity and sizing column chromatography, and the like), centrifugation, differential solubility, or by any other standard technique for protein purification.

Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence, glutathione-S- transferase, and the like can be attached to the protein to allow easy purification by e over an appropriate y coltunn. Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.

A vaccine can comprise an entity that binds a polypeptide sequence described herein. The entity can be an antibody. Antibody-based vaccine can be formulated using any of the well-known techniques, carriers, and excipients as suitable and as understood in the art. In some embodiments, the peptides described herein can be used for making neoantigen specific therapeutics such as antibody therapeutics. For example, neoantigens can be used to raise and/or identify dies specifically recognizing the neoantigens. These antibodies can be used as therapeutics. The antibody can be a natural dy, a chimeric antibody, a humanized antibody, or can be an dy fragment. The antibody may recognize one or more of the polypeptides described herein. The antibody can recognize a polypeptide that has a sequence that is at most 40%, 50%, 60%, 70%, 80%, 90%, 95%, or less in sequence homology to a polypeptide described herein. The antibody can recognize a ptide that has a ce that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more in sequence homology to a polypeptide described herein. The antibody can also recognize a polypeptide with sequence length is at least 30%, 40%, 50%, 6 %, 70%, 80%, 90%, 95%, or more in sequence length compared to a polypeptide described herein. The antibody can recognize a polypeptide with a sequence length at most 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or less in sequence length compared to a polypeptide bed herein.

The present invention also contemplates the use of nucleic acid molecules as vehicles for delivering igen peptides/polypeptides to the subject in need f, in vivo, in the form of, e. g., DNA/RNA vaccines.

In some ments, the vaccine is a nucleic acid vaccine. In some embodiments, neoantigens can be administered to a subject by use of a plasmid. Plasmids may be introduced into animal tissues by a number of different methods, e. g. or aerosol instillation of naked DNA on mucosal es, such as the , injection nasal and lung mucosa. In some embodiments, physical delivery, such as with a "gene—gun" may be used. The exact choice of expression vectors can depend upon the e/polypeptides to be sed, and is well within the skill of the ordinary artisan.

In some embodiments, the c acid encodes an immunogenic peptide or peptide precursor. In some embodiments, the nucleic acid vaccine comprises sequences flanking the sequence coding the immunogenic peptide or peptide precursor. In some embodiments, the nucleic acid vaccine comprises more than one immunogenic e. In some embodiments, the nucleic acid vaccine is a sed e. In some embodiments, the nucleic acid vaccine is a RNA-based vaccine. In some embodiments, the RNA-based vaccine comprises mRNA. In some embodiments. the RNA-based e comprises naked mRNA. In some embodiments, the RNA-based vaccine comprises modified mRNA (e. g., mRNA protected from degradation using protamine. mRNA containing modified 5‘ CAP structure or mRNA containing modified nucleotides). In some embodiments, the RNA-based vaccine comprises single-stranded mRNA.

The polynucleotide may be substantially pure, or contained in a suitable vector or delivery system.

Suitable vectors and delivery systems e viral. such as systems based on adenovirus. vaccinia virus. retroviruses, herpes virus, adeno-associated virus or hybrids containing elements of more than one virus. Non- viral delivery systems include cationic lipids and cationic polymers (e.g. cationic liposomes).

One or more neoantigen es can be encoded and expressed in vivo using a viral based system.

Viral vectors may be used as recombinant vectors in the present ion, n a portion of the viral genome is deleted to uce new genes without destroying infectivity of the virus. The viral vector of the present invention is a nonpathogenic virus. In some embodiments the viral vector has a tropism for a specific cell type in the mammal. In another embodiment, the viral vector of the present invention is able to infect professional antigen presenting cells such as dendritic cells and macrophages. In yet another embodiment of the present invention, the viral vector is able to infect any cell in the mammal. The viral vector may also infect tumor cells. Viral vectors used in the present invention include but is not limited to Poxvirus such as vaccinia virus, avipox virus, fowlpox virus and a highly attenuated vaccinia virus (Ankara or MVA), retrovirus, adenovirus, baculovirus and the like.

A vaccine can be delivered via a variety of . Delivery routes can include oral (including buccal and sub-lingual), rectal, nasal, topical, transdennal patch, ary, vaginal, suppository, or parenteral (including intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous) administration or in a form suitable for administration by aerosolization, inhalation or insufflation. General information on drug delivery systems can be found in Ansel et al., Pharmaceutical Dosage Femis and Drug Delivery Systems ncott Williams & Wilkins, Baltimore Md. (1999). The vaccine described herein can be administered to muscle. or can be administered via intradennal or subcutaneous injections. or transdermally, such as by iontophoresis. Epidermal stration of the vaccine can be employed.

In some instances, the e can also be formulated for administration via the nasal passages. ations suitable for nasal stration, wherein the carrier is a solid, can include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is stered in the manner in which snuff is taken, i.e., by rapid inhalation h the nasal passage from a container of the powder held close up to the nose. The formulation can be a nasal spray. nasal drops. or by aerosol administration by nebulizer. The formulation can include aqueous or oily ons of the vaccine.

The vaccine can be a liquid preparation such as a suspension, syrup or elixir. The vaccine can also be a preparation for parenteral, subcutaneous, intradennal, intramuscular or intravenous administration (e.g., injectable stration), such as a sterile suspension or on.

The vaccine can include material for a single immunization, or may include material for multiple immunizations (i.e. a ‘multidose' kit). The inclusion of a preservative is preferred in multidose anangements.

As an tive (or in addition) to including a preservative in multidose compositions, the compositions can be contained in a container having an aseptic r for l of material.

The vaccine can be stered in a dosage volume of about 0.5 mL, gh a half dose (i.e. about 0.25 mL) can be stered to children. mes the vaccine can be administered in a higher dose e.g. about 1 ml.

The vaccine can be administered as a l, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dose-course regimen.

Sometimes, the e is administered as a l, 2, 3, or 4 dose-course regimen. Sometimes the vaccine is administered as a l dose-course regimen. Sometimes the vaccine is stered as a 2 dose-course regimen.

The administration of the first dose and second dose can be separated by about 0 day, 1 day, 2 days, 5 days. 7 days. 14 days. 21 days. 30 days. 2 months. 4 months. 6 months. 9 months. 1 year. 1.5 years. 2 years. 3 years. 4 years, or more.

The vaccine described herein can be stered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years.

Sometimes, the vaccine described herein is administered every 2, 3, 4, 5, 6, 7, or more years. Sometimes, the vaccine described herein is administered every 4, 5, 6, 7, or more years. Sometimes, the vaccine described herein is administered once.

The dosage examples are not limiting and are only used to ify particular dosing regiments for administering a vaccine described herein. The effective amount for use in humans can be determined from animal models. For example, a dose for humans can be formulated to achieve circulating, liver, topical and/or gastrointestinal concentrations that have been found to be effective in s. Based on animal data, and other types of r data, those skilled in the art can determine the effective amounts of a vaccine ition appropriate for humans.

The effective amount when referring to an agent or ation of agents will generally mean the dose ranges. modes of administration. fonnulations, etc. that have been ended or approved by any of the various regulatory or advisory zations in the medical or phannaoeutical arts (e.g., FDA, AMA) or by the manufacturer or supplier.

In some aspects, the e and kit described herein can be stored at between 2°C and 8°C. In some instances, the vaccine is not stored frozen. In some instances, the vaccine is stored in temperatures of such as at -20°C or -80°C. In some instances, the vaccine is stored away from sunlight.

VIII. Kits The neoantigen therapeutic described herein can be provided in kit form together with instructions for administration. Typically the kit would include the desired neoantigen therapeutic in a container, in unit dosage form and instructions for administration. Additional therapeutics, for example, cytokines, lymphokines, checkpoint inhibitors, antibodies, can also be included in the kit. Other kit components that can also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipicnts.

Kits and articles of manufacture are also provided herein for use with one or more methods described herein. The kits can contain one or more neoantigen polypeptides. The kits can also contain nucleic acids that encode one or more of the polypeptides described herein, antibodies that ize one or more of the polypeptides described herein, or APC-based cells activated with one or more of the polypeptides described herein. The kits can further n adjuvants, reagents, and buffers necessary for the makeup and ry of the vaccines.

The kits can also include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the ner(s) comprising one of the separate elements, such as the polypeptides and adjuvants, to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.

The es of manufacture provided herein n packaging materials. Examples of pharmaceutical ing materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging al suitable for a selected fonnulation and intended mode of stration and treatment.

A kit typically includes labels listing contents and/or ctions for use, and package inserts with instructions for use. A set of instructions will also lly be included.

The ion will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes. and are not intended to limit the invention in any manner.

Those of skill in the art will readily ize a variety of non-critical parameters that can be changed or modified to yield altemative embodiments according to the invention. All patents, patent applications, and printed publications listed herein are incorporated herein by reference in their ty.

EXAMPLES Example 1: Identification of mutant sequences with genic potential ants have discovered mutations that are recurrent in cancer patients. See, Tables 1 and 2.

Neoantigenic peptides described herein can be derived from the mutations listed in Tables 1 and 2.

For example, a neoantigenic peptide can be derived from a tution mutation, e.g., the KRAS G12V mutation. The neonatigenie peptide can be a peptide comprising 5, 6, 7, 8, 9, 10, ll, 12, l3, 14, 15, l6, l7, l8, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acid residues of the protein, e.g., KRAS including the substitution (G 12V). The substitution may be positioned anywhere along the length of the neoantigenic e. For example, it can be located in the N terminal third of the neoantigenic peptide, the central third of the neoantigenic peptide or the C terminal third of the neoantigenic peptide. In another e. the substituted residue is located 2-5 residues away from the N terminal end or 2-5 residues away from the C terminal end. Neoantigenic peptides can similarly derived from tumor specifc insertion mutations where the neonatigenie peptide comprises one or more, or all of the inserted residues.

Neoantigenic peptides can also be derived from frame shift mutation, fusion polypeptides, in-frame deletions, and splice variants. The tumor specific polypeptides resulting from these mutations can be characterized by the existence of a tion point between a polypeptide sequence encoded by the germline and the tumor specific mutant ptide d by the tumor specific mutation. The neonatigenie peptide can be a peptide ofabout 5, 6, 7, 8, 9, 10, ll, 12, l3, l4, l5, l6, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, or 100 amino acid residues that encompasses the transition point between the germline encoded native sequence and the tumor specific polypeptide sequence. The transition point can be positioned anywhere along I 3 8 the length of the neoantigenic peptide. For example, it can be d in the N temlinal third of the neoantigenic peptide. the l third of the neoantigenic peptide or the C terminal third of the neoantigenic peptide. In another example, the transition point is d 2-5 residues away from the N terminal end or 2-5 residues away from the C terminal end. A neoantigenic peptide derived from frame shift mutation and splice variants can also be a e consisting of tumor specific mutant residues.

For each mutation event listed in Tables 1 and 2, the full-length amino acid sequence of the mutant protein was derived. Any constituent 9mer or lOmcr not found in the gemlline protein sequence was flagged as a nee-peptide and scored for binding potential on six common HLA alleles (HLA-A01 :01. HLA-A0201.

HLA-A0301, HLA-A1 1:01, HLA-A2402, HLA-80702, and HLA-80801) using available algorithms. Any peptide scoring better than 1000 nM was nominated. The nominated peptides are listed ill Tables 1 and 2 followed by the high scoring HLA s in parenthesis.

Table 1 :;:;:; Exemplary; on Sequence Brotein Ghan . Context TABLE 1\ POINT MlTATIONl VADGLITTLHYPAPKRNK ggfigfimgfifig 10)” PTVYGVSPNYDKWEMER Chronic myeloid leukemia KLGGGQYGK (A03 0'1) TDITMKHKLGGGQYGEV (CML). Acute lymphocytic KLGGGQYGKV (A112 01 ) YEGVWKKYSLTVAVKTL ' leukemia (ALL).

K ' ‘EGVWKK (AOZ'OL A03“) KEDTMEVEEFLKEAAVM Gastrointestinal stromal KVYEGVWKKY (A0301) KEIKHPNLVQLLGVC tumors (GIST) EGV ( 1424.02) QYGKVYEGVW (A2402) VADGLITTLHYPAPKRNK ggfing'”$3320? 10)” PTVYGVSPNYDKWEMER Chronic myeloid leukemia KLGGGQYGV (A02 6'1) HKLGGGQYGVV (CML). Acute lympllocytic KLGGGQYGVV (A020 l) E255V YEGVWKK) SLTVAVKV'i/IL QYGVVYEGV ( A2402) leukemia (AILL).

Nee WW W ' VVYEGVWKK (A02.01 A0301) VVYEGVWKKY ) LLGVCT PPFYHTEFMT ATEY (A0101) Chronic mveloid leukemia YGNLLDYLRECNRQEVN - ISSATEYLEK (A0301), .

, (CML). Acute lymphocytlc AVVLL‘ MATQISSAIEYL SSATEYLEK (A0“ 01)"' 1e“ ek mi (ALL) EKKNFIHRDLAARNCLVG .3 ~ TQISSI’J'EYL (A0201) Gastronltestlnal stromal.

ENHLVKVADFGLSRLMT YMATQISSAT ) tumoys (GIST) GDTYTAHAGAKF SLTVAVKTLKEDTMEVEE FYIIIEFMTY (A2402) Chronic myeloid leukemia FLKEAAVMKEIKHPNLVQ llEFMTYGNL (A0201) .

, (CML). Acute lympllocytlc LLGVCTREPPFYHLEFM“ T3151 IIIEFMTYG (A02 01) leukemia (ALL) GNLLDYLRECNRQEVNA ' ‘ “IEFMTYGN (A0101) Gastromtestlnal stromal. .

VVLLYMATQISSANIEYLE YIHEFMTYG (A0201) tumors (GIST) KKNFIHRDLA STV GL1 HYP KR Chr°nic in 1el NKPTVYGVSPNYDKWEM -‘ °idle lkemia1 (CML), Acute lympllocytlc.

MKHKLGGGQflG GQHGEVYEGV (A0201) leukemia (ALL) EVYEGVWKKYSLTVAVK KLGGGQHGEV (A0201) ” . .

TLKEDTMEVEEFLKEAAV [Guia’fs'i‘gfg‘g‘l 5mm“ MKEIKHPNLVQLLG Exemplary: Brutein €11an-_ c SSLAMLDLLHVARDIACG NHFIHRDIAARN 01269A MLPVKWMPPEAFMEGIF SKTDTWSFGVLL L1196M FILM (A02.0l. B08.01) SLPRFILMEL (A0201, A2402.

BO702,B0&0I) NHKLHNARQTAQGMDYL HAKSHHRDLKSNNELHE DLTVKIGDFGLATEKSRW LATEKSRWS (A0201. 308.01) CRC. GBM. KIRP. LUAD.

SGSHQFEQLSGSILWMAP LATEKSRWSG (A0201. 1308.01) SKCM. THCA EVUUMQDKNPYSFQSDVY AFGHHXELM EYTAANGSLL(A2+OD MANGSLLNY (A0101, A03.0l.

MIKEGSMSEDEFIEEAKV All.01) MMNLSHEKLVQLYGVCT MANGSLLNYL , 307.02.

IlTEYMANGgLLN 808.01) YLREMRHRFQTQQLLEM SLLNYLREM(A020L30202 CKDVCEAMEYLESKQFL Boxon HRDLAARNCLVND YMANGSLLN (A0201) YMANGSLLNY (A01.01. A03.0l.

AlLOl MGFGDLKSPAGLQVLND YLADKSYIEGYVPSQADV AVFEAVSQPPPADLCHAL EEF1B2 GPPPADLCHAL (B0702) RWYNHIKSYEKEKASLPG VKKALGKYGPADVEDTT GSGAT SLNHSLGLRSLKEEDGD VIISGNKNLCYANTINWK KLFGTSGQKTKHRNRGE ENSCK (A0301) SCKATGQVCHALCSPEGC WGPEPRDCVSCRNVSRG RECVDKCNLL Exemplary: ' Mmafian§e§uenec .. ..3 , .. . fireteinGhan- z (.ry :5 v 55; CLTSTVQLIM (A01.01. A IMQLMPFGC (A0201) IMQLMPFGCL (A0201. A24.02, 808.01) LIMQLMPFG (A0201) IPVAIKELREATSPKANKE LIMQLMPFGC (A0201) ILDEAYVMASVDNPHVC LTSTVQLIM (A0101) RLLGICLTSTVQLIMQL :- MQLMPFGCL (A0201, 30702, EGFR T790M FGCLLDYVREHKDNIGSQ 1308.01) NSCLC,PRAD YLLNWCVQIAKGMNYLE MQLMPFGCLL (A0201, A2402, DRRLVHRDLAA 808.01) QLIMQLMPF (A0201. A2402 1308.01) QLIMQLMPFG (A0201) MQL (A0201) VQLIMQLMPF (A0201. A2402, 808.01) ERCEWMGNLEIVLTGHN ADLSFLQWIREVTGYVLV AMNEFSTLPLPNLRMVRG , ERBB3 V104M CRC, Stomach Cancer TQVYDGKFAIFVMLNYN TNSSHALRQLRLTQLTEIL SGGVYIEKNDK GLLLEMLDA ) HLMAKAGLTLQQQHQRL LYGLLLEML (A2402) LSHIRHMSNKGM NVVPL‘),GLL ) EHLYSMKCKNVVPLYQL PLYGLLLEM (A02.0 1) Breast Cancer AHRLHAPTSRG PLYGLLLEML (A0201. A2402) ASVEETDQSHLATAGSTS VPLY LLLEM (130G 7 .02) SHSLQKWITGEA VVPLYGLLL (A0201, A2402) NQGKCVEGMVEIFDWL gtgijgdKSL (A0201. A2402.

SEEISILLNscmgiEEE‘XGWTFLPST (A0101) S463P — GVYTFLPSTL (A0201, A2402) Breast Cancer SLEEKDHIHRVLDKITDTL TFLPSTLKSL (A2402), IHLMAKAGLTLQQQHQR VYTFLPSTL (A2402) LAQLLLILSH YTFLPSTLK (A0301) IHLMAKAGLTLQQQHQR NVVPLCDLL (A0201) LAQLLLILSHIRHMSNKG NVVPLCDLLL(A02.01) MEHLYSMKCKNVVPLQD PLCDLLLEM ) ESRl Yi37C' Breast Cancer LLLEMLDAHRLHAPTSRG PLCDLLLEML (A0201) GASVEETDQSHLATAGST VPLCDLLLEM (130702) KYYITGE LLL (A0201, A2402) AGLTLQQQHQR DLL (A02.01) LAQLLLILSHIRHMSNKG NVVPLNDLLL ) MEHLYSMKCKNWPLED ESRl H 537N, PLNDLLLEM (A02.0 1) Breast Cancer LLLEMLDAHRLHAPTSRG PLNDLLLEML (A0201) GASVEETDQSHLATAGST VPLNDLLLEM (307 02) SSHSLOKYYITGE ' IHLMAKAGLTLQQQHQR NVVPLSDLL (A0201) LAQLLLILSHIRHMSNKG NVVPLSDLLL (A02.01) MEHLYSMKCKNVVPL§D PLSDLLLEM (A0201) ESR] Y’37S. Breas‘ cancer LLLEMLDAHRLHAPTSRG PLSDLLLEML (A0201) GASVEETDQSHLATAGST VPLSDLLLEM (1307.02) SSHSLQKYYITGE VVPLSDLLL (A0201, A2402) Exemplary; MmafianSequenec. ’ Reptideszl LA allele: .

Brute") (Elma. ; . 3., _.; .gzgsContexgtgz .3. ,zlefis) 55; ,3. 1.1: 3.; :1. 1.1: .

HRIGGIKLRHQQWSLVME SWPSDRGNYTCVVENKF YTLDVLERQPH ' ' HRPI (A0201. B0801) BLCA, HNSC, KIRP, ILQAGLPANQTAVLGSDV YTLDVLERC (A0201) EFHCKVYSDAQPHIQWL HVEVNGSKVG AVKLSDSRIALKSGYGKY LGINSDELVGHSDAIGPRE QWEPVFQNGKMALgASN GBM, KIRP, PRAD. SKCM SCFIRCNEAGDIEAKSKTA GEEEMIKIRSCAEKETKK KDDIPEEDKG GSGAFGTVYKGIWIPDGE NVKIPVAIKVLRENTSPK‘ V777L NKEILDEAYVMAGLGSPY VMAGLGSPYV (A0201. A0301) (Resistance) ICLTSTVQLVTQL MPYGCLLDHVRENRGRL GSIDLLNWCM LKQMWKSPNGTI RNILGGTVFREAIICKNIPR LVSGWVKPIIIGflI-[AYGD KPIIIGHHA (807.02) BLCA, GBM, PRAD QYRATDFVVPGPGKVEIT YTPSDGTQKVTYLVHNFE EGGGVAMGM RVEEFKLKQMWKSPNGTI TVFREAIICKNIPR LVSGWVKPIIIGQHAYGD KPIIIGCHA (B07.02) BLCA. GBM. PRAD QYRATDFVVPGPGKVEIT YTPSDGTQKVTYLVHNFE EGGGVAMGM RVEEFKLKQMWKSPNGTI RNILGGTVFREAIICKNIPR BLCA, BRCA. CRC, GBM, LVSGWVKPIIIGQHAYGD KPIIIGGHA (B07.02) HNSC, LUAD, PAAD, QYRATDFVVPGPGKVEIT PRAD, UCEC YTPSDGTQKVTYLVHNFE EGGGVAMGM RVEEFKLKQMWKSPNGTI RNILGGTVFREAIICKNIPR KPIIIG§HAYGD KPIIIGSHA (B07.02) QYRATDFWPGPGKVEIT YTPSDGTQKVTYLVHNFE EGGGVAMGM VAVKMLKPSAHLTEREA LMSELKVLSYLGNHMNI IIEYCCYGDL ) NLLGACTIGGPTLVILEYC Gastrointestinal stromal TIGGPTLVII (A0201) CYGDLLNFLRRKRDSFIC tumors (GIST) VIIEYCCYG ) KQEDHAEAALYKNLLHS KESSCSDSTNE VEATAYGLIKSDAAMTV HMNIANLLGA (A0201) AVKMLKPSAHLTEREAL IANLLGACTI (A0201) MSELKVLSYLGNHMNIA LGA (A0201) NLLGACTIGGPTLVITEYC YLGNHMNIA (A0201. 808.01) CYGDLLNFLRRKRDSFIC YLGNHMNIAN (A0201) KQEDHAEAALYK Exemplary; ' MmafianSequenec. ’ Bentideszl LA : . firestein Chan ~2555C0illteXst25 . .3. 16(25):; ,3. .3.) 3.; 3. {4.1.

ISELGAGNGGWFKVSHK PSGLVMARKLIHLEIKPAI RNQIIRELQVLHEgNSPYI VLHESNSPY (A0301) MEK Melanoma VGFYGAFYSDGEISICME VLHESNSPYI (A0201) HMDGGSLDQVLKKAGRI LQVLHECNSL (A020 1‘ 808.01) LGAGNGGVVFKVSHKPS YGAF (A2402) GLVMARKLIHLEIKPAIRN NSLYIVGFY (A0101) QIIRELQVLHECNSLYIVG QVLHECNSL , 308.01) Melanoma FYGAFYSDGEISICMEHM SLYIVGFYG (A0201) DGGSLDQVLKKAGRIPEQ SLYIVGFYGA (A0201) ILGKVSIAVI VLHECNSLY (A0301) VLHECNSLY] (A0201. A0301) FTNRNYDLDYD FYQQQQQSDL ) SVQPYFYCDEEENFYQQQ QQQSDLQPPA ) QQSQLQPPAPSEDIWKKF PPA (A0201) ELLPTPPLSPSRRSGLCSPS YQQQQQSDL (A0201, B0801) YVAVTPFSLRGDNDGG FTNRNYDLDYDSVQP CDEEENFYQQQQQSELQP PAPSEDIWKKFELL§TPPL FELLST’PPL (A0201, 808.01) SPSRRSGLCSPSYVAVTPF LLSTPPLSPS ) SLRGDNDGGGGSFSTAD 0‘ LEMVTELLG TNRNYDLDYDSVQPYFY CDEEENFYQQQQQSELQP FELLPIPPL (A0201) IWKKFELLPLPPLS IWKKFELLPI (A2402) PSRRSGLCSPSYVAVTPFS LLPIPPLSPS (A0201. 307.02) LRGDNDGGGGSFSTADQ LPIPPLSPS(B07.02) LEMVTELLGG I VAVKMLKPTARSSEKQA IIEYCFYGDL (A0201) LMSELKIMTHLGPHLNIV IIIEYCFYG (A0201) NLLGACTKSGPIYIILEYCF Chronic Eosinophilic IYIIIEYCF (A2402) YGDLVNYLHKNRDSFLS Leukemia YCFY (A2402) HHPEKPKKELDIFGLNPA YIIIEYCFYG (A0201) DESTRSYVILS I IEEHANWSVSREAGFSYS HAGLSNRLARDNELREN BLCA. BRCA, CESC, CRC, DKEQLKAISTRDPLSEITE GBM, HNSC, KIRC, KIRP, PIK3CA KITEQEKDFL (A0201) QEKDFLWSHRHYCVTIPE LIHC. LUAD. LUSC.

LSVKWNSRDEVA PRAD. UCEC MYCLVKDWPP I HANWSVSREAGFSYSHA GLSNRLARDNELRENDKE BLCA, BRCA, CESC, CRC, QLKAISTRDPLSEITEQEK STRDPLSEITK (A0301) GBM, HNSC, KIRC, KIRP, PIK3CA DFLWSHRHYCVTIPEILPK DPLSEITK (A0301) LIHC, LUAD, LUSC, LLLSVKWNSRDEVAQMY PRAD, SKCM, UCEC CLVKDWPPIKP I LFINLFSMMLGSGMPELQ SFDDIAYIRKTLALDKTEQ BRCA, CESC, CRC. GBM, PIK3CA H1047R EALEYFMKQMNDARHG HNSC, LII-1C, LUAD.

WTTKMDWIFHTIKQHAL LUSC, PRAD, UCEC :1:3: Exemplary 1:3: 1.-:' :Mntatiqn Sequence ; Beptides.(HL-A allele Brotein Chan lets) ,.

C0 orecta a enocarcmomd.

Ulerine/Endonlelrium Adenocarcinoma; Coloreclal QRGGVITDEEETSKKIAD adenocarcinoma, MSI+; QLDNIVDMREYDVPYHIR Uterine/Endomctrium LSIDIETTKLPLKFBDAET LPLKFRDAET (307.02) Adenocarcinonm, MSI+1 DQIMMISYMIDGQGYLIT Endometrioid carcinoma; NREIVSEDIEDFEFTPKPE Endometrium Scmus VFN carcinoma; Endomctrium Carcinosarcoma-malignanl mesodcnnal mixed tumour; Glioma: Astrocvtoma: GBM AQYPFEDHNPPQ LELIKPFCEDLDQWLSED DNHVAAIHCKAGKGQTG QTGVMICAYL (A02.01) BRCA. CESC, CRC. GBM.

VMICAYLLHRGKFLKAQ :I KIRC, LUSC, UCEC ALDFYGEVRTRDKKGVTI PSQRRYVYYYSY MQAIKCWVGDGAVGKT CLLlSYTTNAFgGEYIPTV IPTV (A0201. A2402) FDNYSANVMVDGKPVNL Melanoma GLWDTAGQEDYDRLRPL SYPQTVGET lRVEGNLRVEYLDDRNTF RHSWVPYEPPEVGSDCT SMNRRPILT . B0801) BLCA. BRCA. CRC. GBM.

TIHYNYMCNSSCMG§MN YMCNSSCMGS (A0201) HNSC. LUSC. PAAD.

RRPlLTIITLEDSSGNLLGR NSFEVRVCACPGRDRRTE EENLRKKGEP TYSPALNKMFCQLAKTCP VQLWVDSTPPPGTRVRA BLCA. BRCA. CRC. GBM.

MAIYKQSQHMTEVVRflC W \l U- :1: HNSC. LUAD. PAAD.

PHHERCSDSDGLAPPQHL| PRAD. UCEC RVEGNLRVEYLDDRNTF - HSVWPYEPPEV EGNLRVEYLDDRNTFRHS VWPYEPPEVGSDCTTIH BLCA, BRCA, CRC, GBM, YNYMCNSSCMGGMNQR GMNQRPILT (A0201 ) HNSC,K1RC, LIHC, PlLTllTLEDSSGNLLGRNS LUSC, PAAD. PRAD, FEVRVCACPGRDRRTEEE UCEC NLRKKGEPHHE EGNLRVEYLDDRNTFRHS WVPYEPPEVGSDCTTIH BLCA. BRCA. CRC. GBM.

YNYMCNSSCMGGMNER GMNWRPILT (A0201) HNSC. LIHC. LUSC.

PILTIITLEDSSGNLLGRNS PAAD. SKCM. UCEC FEVRVCACPGRDRRTEEE NLRKKGEPHHE PEVGSDCTTIHYNYMCNS SCMGGMNRRPILTIITLED BLCA. BRCA. CRC. GBM.

GRNSFEVQVCAC FEVC (A0201) HNSC. LUSC. PAAD.

PGRDRRTEEENLRKKGEP HHELPPGSTKRALPNNTS SSPQPKKKPL \"lSl-ASSOCIA'I‘EI) TABLE 18 FMMESHIFTS ' Proéiein Chan. ; CDKDKRRHCFA I U 3 H MS1+ CRC”MSI+ ‘ TWKNISGSIEIVKQGCWL Uterine/Endomelrium ACVR2A D96fs +1 DRTDCVEKKR 0 P Cancer, MSI+ Stomach Cancer, Lvnch syndrome ALKYIFVAV (A0201, 808.01) ALKYIFVAVR (A0301) AVRAICVMK (A0301) AVRAICVMKS (A0301) CVEKKTALK (A0301) CVEKKTALKY (A0101) LLF (A2402. 308.01) LIFR (A030 I) GVEPCYGDKDKRRHCFA FLIFRRWKS (A0201. B0801) TWKNISGSIEIVKQGCWL FRRWKSHSPL (B0801) MSI+ CRC. MSI+ DDINCYDRTDCVEKKmFVAVRAICV (A0201. 808.01) e/Endometrium ACVR2A D96f5' _1‘ KYIFVAVRAICVMKSFLIF FVAVRAICVM (B0801) Cancer, MSI+ Stomach RRWKSHSPLO 10 LHLSHPI IQLHLSHPI (A0201) Cancer, Lynch syndrome KSFLIFRRWK (A0301) KTALKYIFV ) KYIFVAVRAI (A2402) PLQI (A2402) TALKYIFVAV (A0201, 808.01) VAVRAICVMK ) VMKSFLIFR (A0301) VMKSFLIFRR (A0301) YIFVAVRAI (A0201) ALFFFFFET (A0201) ALFFFFFETK (A0301) AQAGVQWRSL (A0201) CLANFCIFNR (A0301) CLSFLSSWDY (A0101. A0301) FFETKSCSV (B0801) FFFETKSCSV (A0201) TAEAVNVAIAAPPSEGEA FKLFSCLSFL (A0101) NAELCRYLSKVLELRKSD FLSSWDYRRM (A0201) MSI+ CRC, MSI+ VVLDKVGLALFFFFFETK GFKLFSCLSF (A2402) . .

‘ Utenne/Endometnum C I 50RF40 ”32% +1‘ SCSVA . AGVWRSL—GSL KLFSCLSFL (A0201. A0301) Cancer. MSI+ Stomach KLFSCLSFLS (A0201. A0301) Cancer. Lynch syndrome DYRRMPPCLANFCIFNRD LALFFFFFET (A0201) GVSPCWSGWS* LFFFFFETK (A0301) LSFLSSWDY ) LSFLSSWDYR (A0301) RMPPCLANF (A2402) LANF (A2402) SLQPPPPGFK (A03 .0 l) SL (A0201) LSVIIFFFVYIWHWALPLI FFFSVIFST ) MSI+ CRC, MSI+ NNHHICLMSSIILDCNSVR MSVCFFFFSV (A0201) Uterine/Endometrium L 1544fs; +1 QSIMSVCFFFFSVIFSTRCL SVCFFFFSV (A0201. 808.01) Cancer. MSI+ Stomach SVCFFFFSVI (A0201) Cancer. Lvnch svndrome LSVIIFFFVYIWHWALPLI MSI+ CRC. MSI+ FFCYILNTMF (A2402) NNHHICLMSSHLDCNSVR . .

L1544fs; -1 MSVCFFFFCY (A0101) ngg’e‘f/a‘gff‘seggéh FCYI (A0201) Cancer Lvnch syndrome Exemplary; MmafinnSequencc. ’ Reptideszl ,LA : firemen! (Sham ; .21ecs) f DLITDVALHEW DLFRAYDASLAMLMRKG MSI+ CRC, MSI+ QDSIEPVPGQKGKKK KQWSSVTSL<A02.01) Uterine/Endometn'um EIF2B3 AlSlfs;-l VLWMPTSTV (A0201) MKOTWMKSWSLRDPS - Cancer, MSI+ Stomach Cancer, Lynch syndrome SILEYVSTRVLWMPTSTV SIQVMRAQMNQIQSVEGO PLARRPRATGRTKRCQPR MSI+ CRC. MSI+ DVTKKTCNSNDGKKRfl Uterine/Endometrium KlOZOfS; -1 EKRKI ILGGGGKYKEYFL ILIRKAMTV (A0201) Cancer, MSI+ Stomach KRILIRKAMTVLAGDKKG Cancer. Lynch syndrome LDFLGEFATDIRTHGVHM MSI+ CRC, MSI+ N51215: +1 VLNHQGRPSGDAFIQMKS Uterine/Endometrium , MSI+ Stomach , Lynch syndrome MSI+ CRC, MSI+ N512fs; —l LDFLGEFATDIRTHGVHM Utcrinc/Endometrium VLNHQGRPSGDAFIQMKS Cancer, MSI+ Stomach ADRAFMAAQKCHKKE Cancer, Lynch syndrome GALCKDGRFRSDIGEFEW MSI+ CRC. MSI+ KLKEGHKKIYGKQSMVD Uterine/Endometrium FAMlllB ; -l LEMDISKKKM RMKVPLMK (A0301) Cancer, MSI+ Stomach NRKISIKKLNRMKVPLMK Cancer, Lynch me RERAQLLEEQEKTLTSKL MSI+ CRC, MSI+ QEQARVLKERCQGESTQL Uterine/Endometrium TLKKKPRDI (808.01) QNEIQKLQKTLKKKPRDI Cancer, MSI+ h CRIS* Cancer. Lynch syndrome MSI+ CRC, MSI+ VNTLKEGKRLPCPPNCPD P86lfs; +1 LIEGFEALLK (A03.01) Uterine/Endometrium E KCWEFQPSNR Cancer. MSI+ Stomach TSFQNLIEGFEALLKTSN* Cancer, Lynch syndrome QQLKWTPHI (A0201) QLKWTPHILK (A0301) IVSEKNQQLK (A0301) CRPVTPSCKELADLMTRC MSI+ CRC, MSI+ K860fs; -l HILK (A0301) MNYDPNQRPFFRAIMRDI Uterine/Endometrium QQLKWTPHI (A2402) Cancer, MSI+ Stomach NQQLKWTPHIL (1308.01) Cancer, Lynch syndrome TPHI (B08.01) QLKWTPHIL (808.01) DDHDVLSFLTFQLTEPGK MSI+ CRC, MSI+ RAAC (B0702) EPPTPDKEISEKEKEKYQE Uterine/Endometrium E305fs; +1 PPRPPRAAC (B0702) EFEHFQQELDKKKRGIPE Cancer, MSI+ Stomach GPPRPPRAACGGNI* Cancer, Lynch me DDHDVLSFLTFQLTEPGK MSI+ CRC, MSI+ EPPTPDKEISEKEKEKYQE Uterine/Endometrium E305fs; -l SLRRKYLRV (B08.01) EFEHFQQELDKKKRNSRR Cancer, MSI+ Stomach ATPTSKGSLRRKYLRV* Cancer, Lynch syndrome TKSTLIGEDVNPLIKLDDA MSI+ CRC. MSI+ N385fs; +1 VNVDEIMTDTSTSYLLCIS Uterine/Endomem'um ENKENVRDKKKG O HFY SAACHRRGCV (B0801) Cancer, MSI+ Stomach CHRRGCV* Cancer. Lynch syndrome Exemplary; ' Mmafianfiequenee. ’ Bentideszl LA allele: .

Einstein Chan; ; .. . .,,.,exam 116(5) , LPQA (A0201 CLIVSRTLL (308.01) CLIVSRTLLL (A0201, B0801) FLLALWECS (A0201) LIGEDVNPLIKLD FLLALWECSL (A0201. B0801) DAVNVDEIMTDTSTSYLL MSI+CRC, MSI+ IVSRTLLLV (A0201) CISENKENVRDKKRATFL Uterine/Endometn'um LIVSRTLLL (A0201, B0801) Cancer, MSI+ Stomach LIVSRTLLLV (A0201) Cancer, Lynch syndrome LLALWECSL . B0801) LPQARLCLI (B0801, 1307.02) CLIV (808.01) NVRDKKRATF (B0801) SLPQARLCLI (A0201. B0801) LPPPKLTDPRLLYIGFLGY FFCWILSCK (A0301) MSI+ CRC. MSI+ CSGLIDNLIRRRPIATAGL FFFCWILSCK (A0301) e/Endometrium HRQLLYITAFFFCWILSCK ITAFFFCWI ) Cancer, MSI+ Stomach LYITAFFFCW (A2402) Cancer, Lynch syndrome YITAFFFCWI (A0201) ITAFFLLDI (A0201) SLPPPKLTDPRLLYIGFLG LLYITAFFL (A0201, 808.01) MSI+ CRC, MSI+ F69fs; -1 YCSGLIDNLIRRRPIATAG LLYITAFFLL (A0201, A2402) e/Endometrium LHRQLLYITAFFLLDIIU‘ LYITAFFLL ) Cancer, MSI+ Stomach LYITAFFLLD ) , Lynch syndrome YITAFFLLDI (A0201) NQSGGAGEDCQIFSTPGH PKMIYSSSNLKTPSKLCSG MSI+ CRC. MSI+ SKSHDVQEVLKKKTGS TRY (A0101) Uterine/Endometrium VTTRYEEKKTGSVRKAN MPKDVNIQV (1307.02) Cancer, MSI+ Stomach RMPKDVNIOOVRKKKHE TGSNEVTI'RY (A0101) Cancer, Lynch syndrome TRRKSKYNEDFERAWRE MSI+ CRC, MSI+ MAPVKKLVVKGGKKKE- Uterine/Endometrium Cancer. MSI+ Stomach Cancer, Lynch syndrome MSI+ CRC, MSI+ Kles; -1 MAPVKKLVVKGGKKM Uterine/Endometrium F* Cancer, MSI+ Stomach Cancer, Lynch syndrome MSI+ CRC, MSI+ I462fs; +1 MPSHQGAEQQQQQHHVF Uterine/Endometrium SEC3 1A ISQVVTEKEFLSRSDQL O 0‘ Cancer. MSI+ Stomach AVQSQGFINYCQKKE , Lynch syndrome KKLMLLRLNL (A0201) KLMLLRLNL (A0201. A0301, B0702, B0801) KLMLLRLNLR (A0301) LLRLNLRKM (B0801) AEQQQQQHHVF LMLLRLNL (308.01) MSI+ CRC, MSI+ I462fs; -l KEFLSRSDQLO OLMLLRLNLRK (A0301) Uterine/Endometrium SEC31A AVQSQGFINYCQKKLML LNLRKMCGPF (808.01) Cancer. MSI+ Stomach RLNLRKMCGPF* MLLRLNLRK (A0301) Cancer, Lynch syndrome MLLRLNLRKM . A0301.

B0801) NLRKMCGPF (B0801) NYCQKKLMLL (A2402) YCOKKLMLL (B0801) ary: ' Mmafianfiequenec. ’ Peptndes(HLA al1e1e5 .. fitment Chem 5.5;. . .22 FKKKTYTCAI (B08.0 1) lTTVKATETK (A0301) AEVFEKEQSICAAEEQPA KSKKKETT’K (A0301) MSI+ CRC. MSI+ EDGQGETNKNRTKGGWQ KSKKKETFKK (A0301) K530fs; +1 Uterine/Endometrium KKTAKSKKKETF KTYTCAITTV (A0201, A2402) Cancer, MSI+ Stomach KKKTYTCAITTVKATETK TFKKKTYTC 1) Cancer, Lynch syndrome AGKWSRWE* TYTCAITTV (A2402) TYTCAITTVK (A0301) YTCAITTVK (A0301) MAEVFEKEQSICAAEEQP MSI+ CRC, MSI+ AEDGQGETNKNRTKGGW Uterine/Endometrium K529fs; -l TAKSKKRNL (3081”) QQKSKGPKKTAKSKKM Cancer. MSI+ Stomach L* Cancer. Lynch syndrome QLPSSHALEAKLS MSI+ CRC. MSI+ RMSYPVKEQESILKTVGK Uterine/Endometrium C248fs; -l FALCGFWQI (A0201) LTATQVAKISFFFALCGF Cancer. MSI+ Stomach W 0 ICHIKKHF O THKLL* Cancer Lynch syndrome YEKKKYYDKNAIAITNISS MSI+ CRC. MSI+ SDAPLQPLVSSPSLQAAV Uterine/Endometrium DKNKLEKEKEKKKGREK Cancer, MSI+ Stomach ERKGARKAGKTTYS* Cancer. Lynch syndrome KYEKKKYYDKNAIAITNI SSSDAPLQPLVSSPSLQAA LKKLRSPL (B0801) MSI+ CRC. MSI+ VDKNKLEKEKEKKRKRK SLKKVPAL (BO8.01) Uterine/Endometrium Kl7lfs;-l S O KSR 0 NHL I LKSC RKISNWSLKK (A0301) Cancer, MSI+ h RRKISNWSLKKVPALKKL VPALKKLRSPL (307.02) Cancer, Lynch syndrome RSPLWIF* KRVNTAWKTK (A0301) MSI+ CRC. MSI+ RFKEQLTPSQIMSLEKEIM RVNTAWKTK (A0301) Uterine/Endometrium E14815; +1 DKHLKRKAMTKKKRVN KTKK ) Cancer. MSI+ h AWKTKKTSFSL* TKKKRVNTA (B0801) Cancer, Lynch syndrome WKTKKTSFSL 808.01 MSI+ CRC. MSI+ E l48fs; -l IYQDAYRAEWQVYKEEIS Uterine/Endometrium TPSQIMSLEKEIM Cancer. MSI+ h KAMTKKKg Cancer. Lynch syndrome MSI+ CRC. MSI+ KPQEVCVAVWRKNDENI Uterine/Endometrium TLETVCHDPKLPYHDFILE Cancer, MSI+ Stomach DAASPKCIMKEKKKAW* Cancer. Lynch syndrome ALMSAMTTS (A0201) AMTTSSSQK(A03.01.A1101) AMTTSSSQKN (A0301) CIMKEKKSL (808.01) CIMKEKKSLV (808.01) EKPQEVCVAVWRKNDEN IMKEKKSL (808.01) MSI+ CRC, MSI+ ITLETVCHDPKLPYHDFIL IMKEKKSLV (B0801) Uterine/Endomctrium K128fs: -1 KCIMKEKKSLVR KSLVRLSSCV (A0201) , MSI+ Stomach LSSCVPVALMSAMTTSSS LVRLSSCVPV (A0201) Cancer, Lynch syndrome OKNITPAILTCC* RLSSCVPVA , A0301) RLSSCVPVAL (A0201) SAMTTSSSQK (A0301, A1101) SLVRLSSCV (A0201) VPVALMSAM (B0702) VRLSSCVPVA A0201 Exemplary;- an Sequence : es.:(HLA allele: ..mtem (Elma .. sizicontexlzi .. .. .. .. . E‘Esi exam ..1e(zs)).sa :222; VPSKYQFLCSDHFTPDSL MSI+ CRC. MSI+ RYLKQTAVPTIFS Ulcrine/Endomelrium K99fs; -l GKDPSKKNPRRK KMRKKYAQK (A03 .0 1) . MSI+ Stomach Cancer. Lynch syndrome FVMSDTTYK (A0301) GTTEEMKYVLGQLVGLN FVMSDTTYKI (A0201) MSI+ CRC. MSI+ SPNSILKAAKTLYEI-{YSG KTFEKKGEK (A0301) R854fs: -l Uterine/Endometrium GESHNSSSSKTFEKKfl LFVMSD'ITYK (A0301) Cancer. MSI+ Stomach NDLOLFVMSD'ITYKIYW MSDTTYKIY (A0101) Cancer. Lynch syndrome TVILLNPCGNLHLKTTSL* VMSDTTYKI (A0201) VMSDTTYKIY (A0101) QQLIRETLISWLQAQMLN MSI+ CRC. MSI+ F126fs: -l PQPEKTFIRNKAAQVFAL Uterine/Endometrium XPOT YLTKWPKFFL (A0201) LTKWPKFFLTFS Cancer. MSI+ Stomach Cancer. Lvnch sv 11drome TABLE I(‘ F RAM ES 11 1 FT V1352fs 35W MEEEEPNIKEE FLQERNLPP (A0201)”1L FRRPHSCLA (308.01) Fl354fs LIVLRVVRL (808.01) CRC. LUAD. UCEC. STAD Q1378fs LLSVHLIVL (A0201. 808.01) Sl398fs EVKHLHHLL (308.01) HLHHLLKQLK (A0301) $142 lfs ERV (B0801) R1435fs KIKHLLLKR (A0301) T1438fs KPSEKYLKI (307.02) Pl442fs APVIFO CHOAEV KYLKIKHLL (A2402) Pl443fs LKILKPSEKYLK HLLL (A2402) CRC. LUAD. UCEC, STAD Vl452fs IKHLLLKRERVDLSKLO * LLKQLKPSEK (A0301) P1453fs LLKRERVDL (308.01) K1462fs RVDL (808.01) El464fs KYLK (A0301) YLKIKHLLL (A0201. 308.01) YLKIKHLLLK (A0301) [LPRKVLQM (B0801) KVLQMDFLV (A0201. A2402) T148715 ML 0 FRGSRFF O MLILYYI QMDF (B0702. 808.01) H1490fs CRC. LUAD. UCEC. STAD PRKVL O MDFLVHPA* LQMDFLVHPA ) L148815 QMDFLVHPA (A0201) YILPRKVLQM (A0201. B0801) ALGPHSRISCLPTOTRGCI APSPASRLQC (307.02) LLAATPRSSSSSSSNDMIP HPLAPMPSKT (307.02) Q1306fs MAISSPPKAPLLAAPSPAS LLL (A0201) Sl3l6fs RLOOCINSNSRITSGWMA LLLSADQQA (A0201) Y1324fs STAD. UCEC. BLCA.

HMALLPSGTKGRCTACH LPTQTRGCI (307.02) ARIDIA "1134er BRCA. LUSC. CESC. (313513 KIRC. UCS LPASNKLPSLPLSKMYTT RISCLPTQTR (A0301) 0137st MAMPILPLPQLLLSADQQ SLAETVSLH (A0301) P146715 AAPRI NFHSSLAETVSLH TPRSSSSSS (807.02) PLAPMPSKTCHHK* TPRSSSSSSS B0702 Exemplatyg: " n§e¢ju¢n¢¢ oteinGhan._ é;=; inexam lees) ALPPVLLSL (A0201 ALPPVLLSLA (A0201) LAL (A0201) ASRTASCIL (B07.02) EALPRPLLAL (B0801) HLGHPVASR (A0301) TAS (B07.02) HPVASRTASC (807.02) IIQLSLLSLL (A0201) IQLSLLSLL (A0201) IQLSLLSLLI (A0201, A2402) AH O GFPAAKESRVI 0 LSL $674fs LLALPPVLL (A0201) STAD. UCEC. BLCA. 725fs LLIPPLTCL ) BRCA. LUSC. CESC.

R727fs LLIPPLTCLA (A0201) 0 O ATRCHLGHPVASRT KIRC. UCS I736fs LLSLLIPPL (A0201) LLSLLIPPLT (A0201) LPRPLLALPP (307.02) QLSLLSLLI ) RLLQCQATR (A0301) RPLLALPPV (307.02) RPLLALPPVL (B07.02) SLAQDHSRL (A0201) SLAQDHSRLL (A0201) SLLIPPLTCL (A0201) SLLSLLIPP (A0201) SLLSLLIPPL (A0201, B0801) Exemplary; Mmafian§e§uenec.

Bratein €11an-_ ' ; .cxam ,2le(2$) AAATSAASTL (307.02) AAIPASTSAV (307.02) AIPASTSAV (A0201) ALPAGCVSSA (A0201) APLLTATGSV (307.02) SIL (307.02) ATLLPATTV (A0201) ATVSTTTAPV (A0201) LFPA (A0201) CLFPAALPST (110201) AAA (307.02) FPAALPSTA (307.02) FPAALPSTAG (307.02) GAECHGRPL (307.02) GAISRFIWV (A0201) ILPAAIPAST (A0201) IWVSGILSPL (A24.02) LLTATGSVSL (A0201) LLYTADSSL (A0201) PILAATGTSVRTAARTWV LPAAATSAA (307.02) PRAAIRVPDPAAVPDDHA LPAAATSAAS (307.02) GPGAECHGRPLLYTADSS G-‘l l4fs AST (307.02 ) LWTTRPIRVWSTGPDSIL Q473fs LPAGCVSSA (807.02) OPAK P AAAATLLPAT H477fs SSAP (B0702) TVPDPSCPTFVSAAATVS ‘ STAD. UCEC. BLCA.

S499fs BRCA. LUSC. CESC.

P504fs =532235111107123)AVPGSIPLPAVDDTAAPPE KIRC, UCS Q548fs P549fs —RFIWVSGILQPAKSSPSAA (B67 4 Oi) AASTLDALPAGCVSSAPV ' RPQRVWSTG (1307.02) SAVPANCLFPAALPSTAG RVWSTGPDSI (A0201) AISRFIWVSGILSPLNDLI SAVPGSIPL (307.02) SILPAAIPA (A0201) SLPAAATSA ) SLPAAATSAA (A0201) SLWTTRPQR (AO3.01) SLWTTRPQRV (A02.0l) SPSAAAATL (307.02) ATLL (307.02) TLDALPAGCV ) TVSTTTAPV (A0201) VLSASILPA (A0201) VLSASILPAA (A0201) VPANCLFPA (307.02) VPANCLFPAA (307.02) VPDPSCPTF (307.02 ) VPGSIPLPA (307.02) VPGSIPLPAV (307.02) WVSGILSPL (A0201) YTADSSLWTT (A0201) Exemplary; ' Mutafianfieémencc. . ._ _. . atein (Elma-g Essa inexam ,: 2 APAGMVNRA (BO702) ASLHRRSYL (B0801) ASLHRRSYLK (A0301) FLLMDNKAPA (A0201) HPRRSPSRL 2. B0801) HPSLHISSP (BO702) HRRSYLKIHL 1) HSRFLLMDNK (A0301) KLPIPSSASL (A0201) KVLTLSSSSH (A0301) LIHSRFLLM (B0801) LLMDNKAPA (A0201) LMDNKAPAGM (A0201) LPIPSSASL (B0702) MPNLRISSS (B0702. 808.01) MPNLRISSSH (BO702) NLRISSSHSL (B0702, 808.01) PPTHSHRLSL (BO702) T433fs PCRAGRRVPWAASLIHSR RAGRRVPWAA (B0801) A441fs FLLMDNKAPAGMVNRAR RARLHITTSK (A0301) Y447fs LHITTSKVLTLSSSSHPTPS SLNH (A0301) P483fs LMPNLRISSSHSL RLHTPPSSR (A0301) P484fs STAD. UCEC. BLCA, NHHSSSPLSLHTPSSHPSL SSRR (A0301) P504fs BRCA. LUSC. CESC.

HISSPRLHTPPSSRRHSSTP RLRILSPSL (A0201. B0702.

SS 191‘s KIRC. UCS RASPPTHSHRLSLLTSSSN B0801) H544fs P549fs P554fs SASLHRRSYL (B0702, 808.01) Q563fs SLHISSPRL (A0201) SLHRRSYLK (A0301) SLHRRSYLKI (808.01) FLL (A0201) SLlHSRFLLM (A0201. B0801) SLLTSSSNL (A0201) SLNHHSSSPL (A0201. 807.02.

B0801) SLSSPSKLPI (A0201) TPS ) SPLSLHTPSS (B0702) SPPTHSHRL (BO702) SPRLHTPPS (BO702) SPRLHTPPSS (B0702) SPSLSSPSKL (B0702) SYLKIHLGL (A2402) IRPRPL (B0702, B0801) TPSSHPSLHI (BO702) Exemplary; Mmafianfiequenec ’ PeptidesKHLA allele: . . firetein (Sham ; . .,...exam defis) , CVPFWTGRH. (B0702) HCVPFWTGRIL (80702) ILLPSAASV (A02.01) ILLPSAASVC (A02.01) LLPSAASVCPI (A02.01) LPSAASVCPI (30702) MRPHCVPF (B0801) RILLPSAASV (A02.01) A2137fs RTNPTVRMRPHCVPFWT RMRPHCVPF (A2402. B0702. STAD, UCEC. BLCA, P2139fs GRILLPSAASVCPLPFEAC 308.01) BRCA. LUSC. CESC.

L l 970fs HLC I AMTLRCPNTI GCC VPFW (A2402) KIRC, UCS V l 994fs RMR (A0301) SVCPIPFEA (A02.01) TVRMRPHCV (B0801) TVRMRPHCVPF (308.01) VPFWTGRIL (807.02) VPFWTGRILL (B0702) VRMRPHCVPF (308.01) AMVPRGVSM (30702, ) AMVPRGVSMA (A02.01) RTPW ) CPMPITPVQA (307.02) CPSTVPPSPA 2) GVSM (307.02. 308.01) TN I ALPKIEVICRGTPRCP MPCPMPITPV (307.02) STVPPSPA IPYLRVSLPED MPTI'PVQAW (307.02) STAD, UCEC. BLCA, RYT I AWAPTSRTPWGAM MPTTPVQAWL (307.02) BRCA. LUSC. CESC.

MAHKVATPGS ' SLPEDRYTQA (A02.01) KIRC, ucs SPAQPYLRV (307.02) SPAQPYLRVS 2) TIMPCPMPT (A02.01) TPVQAWLEA (30702) TSRTPWGAM (307.02) VPPSPAQPYL 2) VPRGVSMAH (307.02) CLSARTGLSI (308.01) CTTLNSPPLK (A0301) KKWSIITCLSAR GLSISCTTL (A02.01) TGLSISCTI'LNSPPLKKMS SPPLKKMSM (B0702. B0801) CRC. STAD. SKCM, HNSC TLNSPPLKK (A0301) TI'LNSPPLK (A0301) TI'LNSPPLKK (A0301) LQRFRFTHV (308.01) THVI (308.01) LCSRYSLFLAWRLSSVL ' RLSSVLQRF (A2402) CRC, STAD. SKCM, HNSC RLSSVLQRFR (A0301) VLORFRFTHV (A0201. 308.01) ASVGRHSLSK (A0301) KFLPFWGFL (A2402) A69 lfs LASVGRHSL (307.02) P708fs ILC LumA Breast Cancer LPFWGFLEEF (307.02) L71 lfs PFWGFLEEF (A2402) SVGRHSLSK A0301 Exemplary: ' Mmafinn§e§uenee Brutein Elma; ; APRPIRPPF 2) APRPIRPPFL (B0702) AQKMKKAHFL (BO801) FLPSAAQKM (A0201) H121fs GLFLPSAAQK (A0301) P126fs HPLLVSLLL (BO702) H128fs KAHFLKTWFR (A0301) WG'I'I‘TAPRPIRPPFLES N144fs ASL ) KNCSHFPTPLLASEDRR V157fs KMKKAHFLK (A0301) P159fs KTWFRSNPTK (A0301) ILC LumA Breast Cancer N166fs LAKELTHPL (807.02. 80801) N18 lfs LAKELTHPLL (308.01) F189fs KARF ) P201[s QKMKKAHFL (B0801) F205fs RFSTASLAK (A0301) RPIRPPFLES (B0702) RSNPIKTKK (A0301) SLAKELTHPL (A0201, 80801) TKKARFSTA (B0801) GLRFWNPSR (A0301) [SQLLSWPQK (A0301) PTDPFLGLRLGLHL 0KVF V114fs, RIAHISQLL (A0201) HOSHAEYSGAPPPPPAPS P127fs RLGYSSHQL ) GLRFWNPSRIAHIS . LLS ILC LumA Breast Cancer V132fs SQLLSWPQK (A0301) P160fs SQL (B0801) WPQKTEERL (B0702) EAEKRTRTL (B0801) GTI'FITMMK (A0301) GTTFITMMKK (A0301) L731fs FCCSCCFFGGERWSKSPY 11\/1KKEAEK(AO3.01) R749fs CPIRMTPGTFFITMMKKE RMTPGTIFI (A0201) ILC LumA Breast Cancer E757fs SPYCPQRMT (307.02) 759fs TMMKKEAEK (1103.01) TPGTTFITM (307.02) TPGTTFITMM (307.02) TTFITMMKK (A0301) WRRNCKAPVSLRKSVO T PARSSPARPDRTRRLPSLG CPGATWREA (807.02) VPGIPWALGAAASRRCC CPGATWREAA (807.02) ILC LumA Breast Cancer CCCRSPLGSARSRSPATL ‘ RSRCPGATWR (A0301) LTPRATRSRCPGATWREA TPRATRSRC (B0702) Exemplary; MmafianSequenec. ’ Peptides(HLA allele firestein (Sham ; P394fs P387fs S398fs H400fs M401fs S408fs P409fs S408fs PGRPLOTHVLPEPHLAL 0 P409fs PL 0 HADAPAI I P T419fs VLWTTPPL O HGHRHGLEP HVLPEPHLAL (807.02) H424fs CSMLTGPPARVPAVPFDL RPLQTHVLPE (807.02) Breast Cancer P425fs HFCRSSIMKPKRDGYMFL VLWTTPPLQH ) S427fs KAESKIMFATL ORSSLWC F43 lfs S430fs H434fs H435fs S438fs M443fs G444fs *445fs CSPF (807.02) CPLDHTTPPA (807.02) P426fs PRPRRCTRHPACPLDHTT FLQEQYHEA (A0201. ) H434fs PPAW PPWVRALLDAHR RLAFLQEQYH (A03 .01) Breast Cancer P4338 APSESPCSPFRLAFL O EOY SPCSPFRLAF (807.02) T44lfs SPPWVRALL (807.02) YPACPLDHTT (807.02) ALHLRSCPC (808.01) TRRCHCCPHLRSHPCPHH CLHHRRHLV (808.01) LRNHPRPHHLRHHACHH CLHHRRHLVC(80801) HLRNCPHPHFLRHCTCPG CLHRKSHPHL 1) RWRNRPSLRRLRSLLCLP CPP (808.01) HLNHHLFLHWRSRPCLH CLRSHTCPP (808.01) KSHPHLLHLRRLYPHHLK CLWCHACLH (A0301) HRPCPHHLKNLLCPRHLR CPHHLKNHL (807.02) NCPLPRHLKHLACLHHLR CPHHLKNLL (807.02) SHPCPLHLKSHPCLHHRR CPHHLRTRL (807.02. 808.01) HLKSLLCPLHLR CPLHLRSLPF (807.02. 808.01) P519fs SLPFPHHLRHHACPHHLR CPLPRHLKHL (807.02, 808.01) E524fs TRLCPHHLKNHLCPPHLR CPLSLRSHPC (807.02) P647fs YRAYPPCLWCHACLHRL CPRHLRNCPL (807.02, ) $654fs RNLPCPHRLRSLPRPLHLR FPHHLRHHA (807.02. 808.01) STAD, BLCA. CRC.

L656fs LHASPHHLRT‘PPHPHHLR FPHHLRHHAC(BO702.BO8.01) HNSC,BRCA 755fs THLLPHHRRTRSCPCRWR CPP (808.01) L761fs YLRSRNSAPGPR HACLHRLRNL (808.01) Q773fs GRTCHPGLRSRTCPPGLR HLACLHHLR (A0301) SHTYLRRLRSHTCPPSLRS HLCPPHLRY (A0301) HAYALCLRSHTCPPRLRD HLCPPHLRYR (A0301) HICPLSLRNCTCPPRLRSR HLKHLACLH (A0301) Exemplary: ' Mmafian§e§uenee Brutein €11an-_ “ ' ; exam 3) HLRSHPCPL (1307.02. 08.01) HLRSHPCPLH (A03.01) HLRSLPFPH (A0301) HLRTRLCPH (A03.01, ) HLVCSHHLK ) HPCLHHRRHL (307.02. 308.01) HPGLRSRTC (307.02) HPHLLHLRRL (307.02, 808.01) HRKSHPHLL (B08.01) HRRTRSCPC (808.01) KSHPHLLHLR (A0301) KSLLCPLHLR (A0301) LLCPLHLRSL (A0201. ) LLHLRRLYPH (1308.01) LPRHLKHLA (1307.02) LPRHLKHLAC (30702. B0801) HTC (B08.01) LRRLYPHHL 1) LVCSHHLKSL (B08.01) NLRNHTCPPS (30801) PLHLRSLPF (B08.01) RLCPHHLKNH (A0301) RLYPHHLKH (A03 .0 1) RLYPHHLKHR ) RPCPHHLKNL (130702) RSHPCPLHLK (A0301) RSLPFPHHLR (A0301) RTRLCPHHL (307.02) RTRLCPHHLK (A03.0l) SLLCPLHLR (A03.01) SLRSHACPP (808.01) SPLRSQANA (307.02) YLRRLRSHT (808.01) YPHHLKHRPC (130702, 130801) 1122fs 1135fs FITSGQIFK (A0301) WK TNW NDM 1m UCEC.PRAD.SKCM.

A148“ '1:ITSGQIF (A2402) OOIFKGTRGPRFLWGSKD STADBRCA-LUSC- L152fs SQSEALCVL (A0201) ROOKGSNYSSEALCVLU K1RC,LIHC, K1RP,GBM 1316sz CVLL (A0201) 116st 1.26er UCEC, PRAD, SKCM, PTEN K266fs KRTKCFTFG* RCA,LUSC, KIRC, LIHC, KIRP, GBM PIFI o TLLLWDFL o KDLKA AYTGTILMM (A2402) Egggig’ 18.5324 YTGTILMM* DLKAYTGTIL (B08.01) KIRC*LIHC km, 63M T319fs ILTKQIKTK (A03.0l) T321fs KMILTKQIK ) UCEC, PRAD. SKCM, "KMILTK IKTKPTDTFL K327fs KPTDTFLQI (30702) STAD, BRCA. LUSC, A328fs KPTDTFLQIL (30702) KIRC, LII-1C, KIRP, GBM A333fs MILTKQIKTK (A0301) Exemplary: Brutein €11an-_ ; .-.-.,exam:1e(ES) :2: ITRY'I‘IFVLK (A0301) LIAELHNIL (A0201) LIAELHNILL (A0201) UCEC, PRAD. SKCM, STAD, BRCA. LUSC, NLIAELHNIL ) KIRC, LIHC, KlRP, GBM RYTIFVLKDI (A2402) TITRYTIFVL (A020 I) TPPNLIAEL (1307.02) T202fs G209fs FLQFRTH'IT (A0201, B0801) C21 lfs DIFL (307.02) UCEC, PRAD. SKCM, I224fs LQFRTH'I'TGR (A0301) STAD, BRCA. LUSC, G230fs NLQSSVCGL (A0201) KIRC, LII-1C, KIRP, GBM P23 lfs PAK (A0301) R233fs VQWRNLQSSV (A0201) D236fs 625 lfs GQNVSLLGK (A0301) E256fs HTRTRGNLRK (A0301) K260fs ILHTRTRGNL (B0801) Q26 lfs UCEC, PRAD, SKCM, KGQNVSLLGK (A0301) L265fs NVSLLGKYILHTRTRGNL STAD. BRCA, LUSC, LLGKYILHT (A0201) M270fs RKSRKWKSM* KIRC, LII-1C. KIRP. GBM LRKSRKWKSM (808.01) H272fs SLLGKYILH (A0301) T286fs SLLGKYILHT (A0201) E288fs A70fs CTSPLLAPV (A0201) P72fs FPENLPGQL (807.02) A76fs GLLAFWDSQV ) A79fs TFCPFPENL (A2402) P89fs LLAFWDSQV (A0201) W9lfs BRCA. CRC. LUAD.

SS 0 NARGCSPRGPCTSSS LLAPVIFCP (A0201) S9615 PRAD, HNSC, LUSC, TGGPCTSPLLAPVIFCPFP aLLAPVIFCPF (A0201. A2402) V97fs PAAD, STAD, BLCA, 0V, NLPG ILRFPSGLLAFWDS LPCPQQDVL (B0702) V97fs LIHC. SKCM, UCEC.

O VCDLHVLPC ' O ODVLPT LAF 02) G108fs LAML, UCS. KICH. GBM, ODLPCAAVG* RFPSGLLAFW (A2402) G1 l7fs ACC SPLLAPVIF 2) S 12 1 fs SPRGPCTSS (30702) V122fs SPRGPCTSSS (1307.02) C 124fs HVL (A0201) K139fs VIFCPFPENL (A0201) V l 43fs Exemplary: ' Mmafian§e§uenee Brutein €11an-_ ; cxam,21e(es) :2: AMVWPLLSI (A02. 1) AMVWPLLSIL (A0201) AQIAMVWPL , A2402) WPLL (A0201) CPMSRLRLA 2, B0801) V173fs CPMSRLRLAL (307.02. B0801) H178fs IAMVWPLLSI , A2402, D186fs 308.01) H193fs ILSEWKEICV (A0201) L194fs 1VWWCPMSR (A0301) BRCA. CRC. LUAD, El98fs IVWWCPMSRL (A0201) PRAD= HNSC, LUSC, V203fs IWMTETLFDI (A2402) PAAD. STAD. BLCA, 0V.

E204fs LLSILSEWK (A0301) LIHC. SKCM. UCEC.

L206fs MSAAQIAMV (A0201) LAML, UCS. KICH. GBM.

D207fs MSRLRLALT (B0801) ACC N210fs MSRLRLALTV (308.01) T211fs MVWPLLSIL ) F212fs RLALTVPPST (A0201) V225fs WWC (A0201) S24lfs TLFDIVWWCP (A0201) TMSAAQIAMV (A0201) VWSIWMTETL (A2402) WMTETLFDI (A0201, A2402) WMTETLFDIV (A0101. A0201) R248fs P250fs SZGOfs N263fs G266fs N268fs V272fs ALRCVFVPV (A0201, 808.01) V274fs ALRCVFVPVL (A0201, 808.01) P278fs PTI‘ (A0201) D281fs AQRKRISARK (A0301) R282fs GAQRKRISA (808.01) T284fs T P P HWKTPVVIY HWMENISPF(A24.02) BRCA.CRC.LUAD.

E285fs WDGTALRCVFVPVLGET LPSQRRNHW (1307.02) PRAD. HNSC. LUSC.

L289fs GAORKRISARKGSLTTSC LPSQRRNHWM (30702, B0801) PAAD. STAD. BLCA, 0v, K292fs POOGALSEHCP'I'I‘PAPLPS NISPFRSVGV (A0201) LIHC, SKCM. UCEC. 301fs RRNHWMENISPFRSVGVS RISARKGSL (130702. B0801) LAML, UCS, KICH. GBM, S303fs ASRCSES* SPFRSVGVSA (30702) ACC T3 12fs SPSSHWKTPV (1307.02, 808.01) S3 l4fs TALRCVFVPV (A0201) K3 19fs VIYWDGTAL (A0201) K320fs VlYWDGTALR ) 322fs VLGETGAQRK (A0301) Y327fs F328fs L330fs R3 3315 R335fs R337fs E3 39fs Exemplary; Mmafian§e§uenec.

Bratein €11an-_ ; S l49fs P15 lfs BRCA. CRC. LUAD, P152fs HPRPRHGHL 2, 808.01) PRAD, HNSC, LUSC, V157fs FHTPARHPRPRHGHL 0AV HPRPRHGHLQ ) PAAD. STAD, BLCA. 0V, Q165fs TAHDGGCEALPPP* LQA (BO702) LIHC, SKCM, UCEC.

S 166fs RPRHGHLQAV (B0702, B0801) LAML. UCS, KICH. GBM, H168fs ACC V173fs P47fs D48fs D49fs Q52fs F54fs E56fs P5815 P60fs E62fs GSLKTQVQMK (A0301) M66fs BRCA. CRC, LUAD, CCPRTILNNGSLKTO V OM PPGPCHLLSL (B0702) P72fs PRAD, HNSC, LUSC, V73fs PAAD. STAD, BLCA. OV.

P75fs LIHC, SKCM, UCEC.

A78fs LAML. UCS, KICH. GBM, P82fs ACC P85fs S96fs P98fs T102fs Y103fs GlOst F109fs R1 lOfs Gl l7fs BRCA. CRC, LUAD, PRAD, HNSC, LUSC, VRKHFITYGNYFLKTTFC CPPCRPKQWM (B0702) PAAD. STAD, BLCA. 0V.

TTFCPPCRPK (A0301) LIHC. SKCM. UCEC.

LAML. UCS. KICH. GBM.

C124fs L130fs N131fs CFANWPRPAL (A2402) C 135fs FANWPRPAL (B0702. B0801) Kl39fs GLIPHPRPA (A0201) Al38fs HPRPAPASA (B0702, B0801) BRCA. CRC, LUAD, Tl40fs HPRPAPASAP (B0702) PRAD, HNSC. LUSC.

V143fs LARTPLPSTRCFANWPRP IPHPRPAPA 2, 308.01) PAAD. STAD. BLCA, OV, Q144fs ALCSCGLIPHPRPAPASAP IPHPRPAPAS (BO702) , SKCM, UCEC, V147fs WPSTSSHST* RPALCSCGL (BO702) LAML. UCS, KICH. GBM, T150fs RPALCSCGLI (BO702) P151fs TPLPSTRCF (B0702) P152fs WPRPALCSC (BO702) G154fs WPRPALCSCG (BO702) R156fs R158fs A161fs ary: ' Mmafian§e§uenee Brutein Guam ; .:.:.,examagle(zs) 52;.

ALRRSGRPPK (A0301) GLVPSLVSK (A0301) KISVETYTV ) SLDL (A0201, ) LMSLDLDTGL (A02.0l) LMVLMSLDL (A0201) LVSKCLILRV (A0201) Ll78fs MSL (A0201, 308.01) D179fs ELOOETGHRVALRRSGRP RPGAADTGA (307.02) LIB-lfs PKCAERPGAADTGAHCTS RPGAADTGAH (307.02) T202fs _-SLDLDTGLV(A02.0]) K1RC,K1RP R205fs —SLVSKCLIL(A0201. 308.01) 321315 KCLILRVK* SQLLMVLMSL(A02.01) 021215 TVSSQLLMV (A0201) TYTVSSQLL (A2402) TYTVSSQLLM (A2402) VLMSLDLDT (A0201) VPSLVSKCL (307.02) VSKCLILRVK (A0301) YTVSSQLLM(A01.01) LLMV (A0201) L158fs K159fs KSDASRLSGA* KIRC. KIRP Rl6lfs Ql64fs RTAYFCOYHTASVYSER- FCQYHTASV (B0801), , KIRC. KIRP MPPGCPEPS . A* 868fs S72fs I75fs CPYGSTSTA (307.02) SSOfs CPYGSTSTAS (307.02) P86fs LARAAASTAT (307.02) P97fs TRASPPRSSSAIAVRASCC FLP (A0201) 1109fs PYGSTSTASRSPTIRCRL . PPRSSSAIAV (307.02) H] les RAAASTATEVTFGSSEMO RAAASTATEV (307.02) L] l6fs KIRC. KIRP HTM FWLTKLNYL HL SPPRSSSA] (307.02) GlZ3fs SMLTDSLFLPISHCOCIL“ SPPRSSSAIA (307.02) T124fs SPTQRCRLA (307.02) N131fs TQRCRLARA (308.01) Ll35fs ARAA (308.01) V137fs Gl44fs Dl43fs Il47fs K17 lfs P l 72fs N174fs SSLRITGDWTSSGRSTKIW KIWKTTQMCR (A0301) KIRC KIRP L l 78fs KTTOMCRKTWSG* WTSSGRSTK (A03 .0 l) ‘ L l 88fs ._ , ary, :Mutation Sequence _ Peptides.(HLA allele " Protein Chan_ “Context v.examle(s))m 33"?“th 55 7 ,- -. , .- .. - ALGELARAL (A020 l) AQLRRRAAA (808.01) AQLRRRAAAL (808.01) veers ARRAARMAQL (B0801) Q7315 RRRRGGVGRRGVRPGRV SPL (307.02. B0801) vsus RPGGTGRRGGDGGRAAA HPQLPRSPLA (B0702) F9115 ARAALGELARALPGHLL 04LARALPGHL (1307.02) T100fs _Q-—Q_S SARRAARMA LRRRA LARALPGHLL (BO/.02)a KlRC KIRP.

P103fs AALPNAAAWHGPPHPQL MAQLRRRAA (B0102. 808.01). _, . $11115 PRSPLAL ; RCRDTRWASG * MAQLRRRAAA (807.02. 808.01) " '12:: - QLRRRAAAL (807.02. B0801) [>12be RAAALPNAAA (B0702) RMAQLRRRAA (B0702. B0801) SOSARRAARM (B0801) TABLE 11) (‘RYPTK‘ EXON ' SCKVFFKRAAEGKQKYL GMTLGEKFRV (A02:() 1) CASRNDCTIDKFRRKNCP Prostate Cancer. Castmtton- cn‘ lic final exon RVGNCKHLK (A03.01) . - p SCRLRKCYEAGMTLGEK res1stant Prostate Cancer RVGNCKHLKMTRP* OUT OF FRAME TABLE IE FUSIONS "3 ACOI I997. IILR " MAGAPPPASLPPCSLISDC AC01 [997.1 C69 GPSEPGNNI (B0702) LUSC. Breast . Head CASNQRDSVGVGPSEPQ: :LRRC69 KlCNESASRK (A0301) and Neck Cancer. LUAD NNIKICNESASRK* *out-of-frame GlQVLNVSLK (A0301) IQVLNVSLK ) KSSSNVISY (A0101. A0301) KYGWSLLRV (A2402) HGWRPFLPVRARSRWNR RSWKYGWSL (A0201) EEFIDP32FRY RLDVTVANGR2§2WKYG SLKSSSNVI(B()8.01) EEF 1DP3 Breast Cancer *out-of—framc WSLLRVPQVNGIQVLNV SWKYGWSLL (A2402) SLKSSSNVISYE* TVANGRSWK (A0301) VPQVNGIQV (807.02) IQVL (80702) VTVANGRSWK ) WSLLRVPOV (808.01) HPGDCLIFKL (807.02) RLKEVFQTKIQEFRKACY KLRVPGSSV (307.02) MADlLl:M . IDI'ITENQYRLTS KLRVPGSSVL 2) MAD ”“1 'MAFKLYAEHPGDCLIFK::LRVP‘ AFK RVPGSSVLV (15102.01) GSSVLVTVPGU- PGL (A02 .0 1) VPGSSVLV’TV (B0702) AEVLKVIRQSAGQKTTCG QGLEGPWERPPPLDESER DGGSEDQVEDPALS:A:Q PPPlRlB:S PPPlRlB:STAR RPPR (A0301) . LRPRPPRPEVGAHQDEQ Breast Cancer TARD3 D.) ALSALLLRPR (A0301) AA ; GADPRLGA ;PACR TVPQPEPLLAP PSAA" 1N FRAME DELETIONS Table 1F and FUSIONS ‘3 Exemplary Mutation Sequence Pratein Chan. exam jets»; ERAEWRENIREQQKKCFR SFSLTSVELQMLTNSCVK LQTVHSIPLTINKEzzEALQ L BCR.ABL LTINKEEAL (A0201. B0801) RPVASDFEPQGLSEAAR WNSKENLLAGPSENDPN LFVALYDFVASG ELQMLTNSCVKLQTVHSI SE53JSKEATDGEESCEEEI:31:31 IVHSATGFK (A0301) BCRzABL BCRzABL ATGFKQSSK (A0301) RPVASDFEPQCLSEAAR LLAGPSENDPN LFVALYDFVASGD ISNSWDAHLGLGACGEAE . GLGVQGAEEEEEEEEEEE ELFPLIFPA . B0801) -. EEGAGVPACPPKGPELF C1 lorf93.RELA KGPELFPLI (A0201. A2402) Supretentonal ependyomas.

PLIFPAEPAQASGPYVEII KGPELFPLIF (A24 02) EQPKQRGMRFRYKCEG ‘ RSAGSIPGERSTD LQRLDGMGCLEFDEERA AQQAFEEARRR T'REFEDRDRSHREEMExV (variant "type a") HELEKSKRALETQMEE MKTQLEELEDELQATE DAKLRLEVNMQALKGQ KGSFPENLRHLKNTMETI DWKVFESWMHHWLLFE MSRHSLEQKPTDAPPK::A . . NSCLC. Crimtinib CD74:ROSl (exon6sexon32) KPTDAPPKAGV ) GVPNKPGIPKLLEGSKN resistance SIQWEKAEDNGCRITYY MRPSGTAGAALLALLAA LCPASRALEEKKzgzNYW EGFvaIl VRACGADSYEM GNYV (A0201) (internal deletion EEDGVRKCKKCEGPCRK VCNGIGIGEFKD LPQPPICTIDVYMIMVKC WMIDADSRPKFRELIIEFS IQLQDKFEHL (A0201, 308.01) EGFR:SEP KMARDPQRYLVIQ::LQD QLQDKFEHL (A0201, B0801) GBM, Glioma. Head and EGFR'SEPTH 14 ' MIQQEEIRKLE QLQDKFEHLK (A0301) Neck Cancer EEKKQLEGEIIDFYKMK YLVIQLQDKF (A0201. A2402) AASEALQTQLSTD SWENSDDSRNKLSKIPSTP EML-leLK EML-kALK KHQELQAMQMEEQSPE STREKNSQV (B08.01) VYRRKHQEL (A24.02 ) YKLSKLRTSTIMTDYNP NYCFAGKTSSISDL EGHRMDKPANCTHDLYM IMRECWHAAPSQRPTFKQ FGFR3.TA LVEDLDRVLTVTSTD::VK VLTVTSTDV (A0201) FGFR3_TACC3' Bladder Cancer, LUSC ATQEENRELRSRCEELH VLTVTSTDVK (A0301) GKNLELGKIMDRFEEW YQAMEEVQKQKELS Exemplaryz;:s- Mutatiun Sequence Peptides:(HLA alleleta ; g; Bratein ; 4.; : ,4 22;; exam 16(5)): 3;; 32:5; 3.; 3 35;; 3.. 3.; 2:2; 3, 3.. 25;; 5., ;.,.

RDNTLLLRRVELFSLSRQ %§mg£¥:fi’z%28n VARESTYLSSLKGSRLHP TSK (A0301) NAB:STA'I‘6 EELGGPPLKKLKQE::ATS NAB:STA’I‘t ("variant 1" of KSIIMSLWGLVSKMPPE (szclgll/Inilszvvgél/ $822811)A24 02 Solitary fibrous tumors Chmielccki ct a1. KVQRLYVDFPQHLRHL Bog 01) LGDWLESQPWEFLVGS sQIMSLWGLV(A02.01) DAFCC TSKSQIMSL (308.01) MSREMQDVDLAEVKPLV . EKGETITGLLQEFDVQ::E NDRSI'ER NDRGl:ERG ALSVVSEDQSLFECAYG taggggégimgigl) Prostate Cancer ' ) TPHLAKTEMTASSSSDY SPRVPQQDW VLDMHGFLRQALCRLRQ QAAVRTDGFDEF PML:RARA KVRLQDLSSCITQGKAJE Acute promyelocytic (exon32ex018) TQSSSSEEIVPSPPSPPPL leukemia PRIYKPCFVCQDKSSGY HYGVSACEGCKG RSSPEQPRPSTSKAVSPPH LDGPPSPRSPVIGSEVFLP PMLzRARA NSNl-IVASGAGEAzAzlET 0 Acute promyelocytic (exonézexorfi) SSSSEEIVPSPPSPPPLPRI leukemia YKPCFVCQDKSSGYHY VSACEGCKG VARFNDLRFVGRSGRGKS FTLTITVFTNPPQVATYHR RUNXl(ex5)- AIKITVDGPREPRzflzRTE GPREPRNRT (807.02) RUNXlTl(ex2) HSTMPDSPVDVKTQSRL RNRTEKHSTM (B0801) TPPTMPPPPTTQGAPRT SSFTPTTLTNGT MALNS::EALSVVSEDQSL ALNSEALSV (A0201) TMPRSS2: 3 FECAYGTPHLAKTEMTAS TMPRSSZ'ERG ALNSEALSVV (A0201) Prostate Cancer QTSKMSPRVPQQ MALNSEALSV (A0201. B0801) Underlined AAs ent non-native AAs ZBolded AAs represent native AAs of the amino acid sequence encoded by the second of the two fused genes KBolded and underlined AAs represent non-native AAs of the amino acid ce d by the second of the two fused genes due to a framshift.

Table 2 ExemplaniziDiseases 22.2; V H H .. ‘ " ' ~ ~ ~ 'l‘ahch. POINT ATIO'H MSDVAIVKEGWLHKRG KYIKTWRPRY (A2402) BRCA. CESC, HNSC, KYIKTWRPRYFLLKNDG WLHKRGKYI (A0201. 307.02. LUSC. PRAD. SKCM.

TFIGYKERPQDVDQREAPB0801) THCA AQCQLMKTER WLHKRGKYIK (A0301) Exemplary: ' Mutatinn Sequence. fireteinChan .ssCantextsa ~.

TMLVLEGSGNLVLYTGV APKPLSKLL (B0702) GBM. LUSC. PAAD.

VRVGKVFIPGLPAPSLTM GVSAPKPLSK (A0301) SNTMPRPSTPLDGVSAPK VSAPKPLSK (A03.01) PLSKLLGSLDEVVLLSPV PELRDSSKLHDSLYNED TFO OLGTYIHSI HRIGGIKLRHQQWSLVM CPHRPILQA (307.02) ESWPSDRGNYTCWEN KFGSIRQTYTLDVLERQP HRPILQAGLPANQTAVL GSDVEFHCKVYSDAQPH Io WLKHVEVNGSKVG MREPIYMHSTMVFLPWE KLSDSRTAL , 807.02. KIRP. PRAD, SKCM LH'IKKGPSPPEQFMAVK 308.01) LSDSRIALKSGYGKYLGI KLSDSRTALK (A0301) NSDELVGHSDAIGPREQ LSDSRTALK (AOLOL A0301) WEPVFQNGKMALLASNS RTALKSGYGK (A03.01) TALKSGYGK ) AVKLSDSRIALKSGYGK ALSASNSCF (A0201, A2402, GBM. KIRP, PRAD.

YLGINSDELVGHSDAIGP 307.02) SKCM REQWEPVFQNGKMAL§ ALSASNSCF] (A0201) ASNSCFIRCNEAGDIEAK FQNGKMALSA . B0801) SKTAGEEEMIKIRSCAEK ETKKKDDIPEEDKG AMPNQAQMRILKETELR KVSRENTSPK (A0301) KVKVLGSGAFGTVYKGI L755$ WIPDGENVKIPVAIKV§R (Resistance) ENTSPKANKEILDEAYV MAGVGSPYVSRLLGICL RVEEFKLKQMWKSPNG KPIIIGGHAY(BO7.02) BLCA, BRCA, CRC, GBM, IRNILGGTVFREAIICKN 1- HNSC, LUAD, PAAD, RLVSGWVKPIIIGQHAYG PRAD, UCEC DQYRATDFWPGPGKVE ITYTPSDGTQKVTYLVH .

FEEGGGVAMGM MTEYKLVWGAQGVGK KLWVGACGV (A0201) SALTIQLIQNHFVDEYDP LWVGACGV (A0201) TIEDSYRKQVVIDGETCL VVGACGVGK (A0301. Al 1.01) VWGACGVGK (A0301) MTEYKLWVGAQGVGK WGADGVGK (A1 1.01) BLCA. BRCA, CESC, SALTIQLIQNHFVDEYDP VGK (A1101) CRC. GBM. HNSC, KIRP.

TIEDSYRKQVVIDGETCL KLWVGADGV (A0201) LIHC. LUAD. PAAD.

LDILDTAGIE LVWGADGV A0201 SKCM, UCEC MTEYKLVWGAXGVGK KLWVGAVGV (A0201) BRCA. CESC, CRC.

SALTIQLIQNHFVDEYDP GV (A0201) LUAD, PAAD, THCA, TIEDSYRKQVVIDGETCL WGAVGVGK (A03.01, A1 1.01) UCEC LDILDTAGQE GVGK , Al 1.01) AGGVGKSALTIQLIQNHF HEEY (A0101) CRC. LUSC, PAAD.

TIEDSYRKQVVI SKCM, UCEC LDILDTAGflEEY SAMRDQYMRTGEGFLC VFAINNTKSFEDIHHYRE QIKRVKDSEDVPM Exemplary; mm,fiequcnee. (item (Elma; ;:Cante,x ~ AGGVGKSALTIQLIQNHF ILDTAGLEEY (A01.01) CRC. GBM. HNSC. LUAD.

VDEYDPTIEDSYRKQVVI LLDILDTAGL (A02.01) SKCM, UCEC DGETCLLDILDTAGLEEY SAMRDQYMRTGEGFLC VFAINNTKSFEDIHHYRE O IKRVKDSEDVPM AGGVGKSALTIQLIQNHF ILDTAGKEEY (A0 1.0 l) BLCA, CRC, LIHC. LUAD.

VDEYDPTIEDSYRKQVVI LUSC, SKCM, TI-ICA, DGETCLLDILDTAGfiEEY UCEC SAMRDQYMRTGEGFLC VFAINNSKSFADINLYRE o IKRVKDSDDVPM AGGVGKSALTIQLIQNHF ILDTAGREEY (A0 1 .0 I ) BLCA, CRC. LUSC.

VDEYDPTIEDSYRKQVVI PAAD. PRAD. SKCM.

DGETCLLDILDTAGgEEY THCA. UCEC SAMRDQYMRTGEGFLC SKSFADINLYRE o SDDVPM IEEHANWSVSREAGFSYS AISTRDPLSK 1) BLCA. BRCA, CESC, CRC. GBM, HNSC, KIRC, DKEQLKAISTRDPLSEI a KIRP, LIHC. LUAD, PIK3CA QEKDFLWSHRHYCVTIP LUSC, PRAD, UCEC EILPKLLLSVKWNSRDEV VKDWPP KFNCRVAQYPFEDHNPP QTGVMICAY(A01.01) BRCA. CESC, CRC. GBM, QLELIKPFCEDLDQWLSE KIRC, LUSC, UCEC DDNHVAAIHCKAGKGQ GVMICAYLLHRGKFLKA QEALDFYGEVRTRDKKG Melanoma AVCKSKKSWQARHTGIK QEV (A0201) AML associated with MDS; IVQQIAILMGCAILPHLRS Chronic 1ymphocylic LVEIIEHGLVDEQQEVRT| leukaemia-small SALAIAALAEAATPYGIE lymphocytic lymphoma; SFDSVLKPLWKGIRQHR Myelodysplastic syndrome; GKGLAAFLKAI AML: Luminal NS carcinoma of breast: Chronic myeloid leukaemia; Ductal carcinoma of pancreas; Chronic myclomonocylic leukaemia; Chronic lymphocytic mia-small lymphocytic lymphoma; Myelofibrosis; Myelodysplastic syndrome; PRAD; ial thrombocythaemia; Exemplary11 Mutation ce ..; Brotein Chan EmplrvD . .. 1Context11 , azexamldsfng ..

YLSL LLLVSCPKSEVRA FVQGKDWGL (A02.01. B0801) PRAD KFKFSILNAKGEETKAME SQRAYRFVQGKDWGLK KFIRRDFLLDEANGLLPD DKLTLFCEVSVVQDSVNI SGQNTMNMVKVPE LLVSCPKSEVRA FVQGKDWGV (A0201) KFKFSILNAKGEETKAME SQRAYRFVQGKDWGXK KFIRRDFLLDEANGLLPD DKLTLFCEVSVVQDSVNI SGQNTMNMVKVPE IRVEGNLRVEYLDDRNT CMGSMNRRPI (A0201. B0801) BLCA. BRCA, CRC, GBM, YEPPEVGSDCTGSMNRRPIL (308.01) HNSC. LUSC. PAAD.

TIHYNYMCNSSCMG§MN RPI (B0801) PRAD RRPILTIITLEDSSGNLLG MGSMNRRPIL (B0801) RNSFEVRVCACPGRDRR SMNRRPILTI (A0201, A2402, TEEENLRKKGEP 308.01) EGNLRVEYLDDRNTFRH CMGGMNQRPI (A0201, 808.01) BLCA. BRCA, CRC, GBM, SVVVPYEPPEVGSDC'ITI GMNQRPILTI (A0201. B0801) HNSC. KIRC. LIHC.

HYNYMCNSSCMGGMNQNQRPILTII (A(12.01. 308.01) LUSC. PAAD. PRAD.

RPILTIITLEDSSGNLLGR UCEC VCACPGRDRRT EEENLRKKGEPHHE EGNLRVEYLDDRNTFRH CMGGMNWRPI (A0201. A2402, BLCA. BRCA., CRC, GBM.

SVVVPYEPPEVGSDCTFI B11801) HNSC, LIHC, LUSC.

HYNYMCNSSCMGGMN GMNWRPILTI (A0201. 808.01) PAAD. SKCM. UCEC —1'0 'JI LI) flRPILTIITLEDSSGNLLG MNWRPILTI (A0201, A2402, RNSFEVRVCACPGRDRR B0801) TEEENLRKKGEPHHE MNWRPILTII (A0201. A2402) PEVGSDCTTIHYNYMCN NSFEVCVCA (A0201) BLCA‘ BRCA, CRC, GBM, SSCMGGMNRRPILTIITLE HNSC, LUSC. PAAD, DSSGNLLGRNSFEVQVC UCEC ACPGRDRRTEEENLRKK GEPHHELPPGSTKRALPN NTSSSPQPKKKPL PEVGSDCTTIHYNYMCN NSFEVHVCA (A0201) BRCA. CRC. GBM. HNSC., GMNRRPILTIITLE USC, PAAD.

DSSGNLLGRNSFEVflVC UCEC —1'U 'JI 'vJ ACPGRDRRTEEENLRKK LPPGSTKRALPN NTSSSP o PKKKPL TEWRRCPHHERCSDSD WPCEPPEV (A0201) BLCA. BRCA. GBM.

GLAPPQHLIRVEGNLRVE VVVPCEPPEV (A0201) HNSC. LIHC. LUAD.

YLDDRNTFRHSVWPQE LUSC. PAAD. SKCM.

DCTFIHYNYMC UCEC NSSCMGGMNRRPILTIIT LEDSSGNLLGRNSF \‘ISl-ASSOCIATED Table 28 FRAMESHIFTS ' YNFDKNYKDWQSAVECI TPPL (A0201) MSI+ CRC, MSI+ AVLDVLLCLANYSRGGD LLPEDTPPL (A0201) c/Endomcm'um MSl-16 F1088fs; +1 GPMCRPVILLPEDTPPQ Cancer, MSI+ Stomach RA Cancer. Lvnch svndromc Table 2C FRAMESHIFT Exemplary utatmn Sequence atein Chan-1: AKFCHSTLEPNPADC APFRVNHAV (30702) CRC.LUAD.UCEC.

RVLVYLOONPGTKLLNF CLADVLLSV (A0201) STAD LOERNLPPKVVLRHPKV FLQERNLPPK (A03.01) HLNTMFRRPHSCLADVL HLIVLRVVRL . ) LSVHLIVLRWRLPAPFR HPKVHLNTM (307.02, B08.01) NTMF (307.02, B08.01) KVHLNTMFR (A0301) KVHLNTMFRR (A0301) LPAPFRVNHA (307.02) MFRRPHSCL (307.02, 308.01) MFRRPHSCLA (308.01) NTMFRRPHSC (B08.01) RPHSCLADV (307.02) RPHSCLADVL (307.02) RWRLPAPFR (A0301) SVHLIVLRV (A02.01) TMFRRPHSC (B08.01) TMFRRPHSCL (A0201. 308.01) VLLSVHLIV (A0201) VLLSVHLIVL 1) VLRWRLPA (308.01) VVRLPAPFR (A03.01) x Chan. (nation Sequence ALGPHSRISCLFTOTRGCI AMPILPLPQL (A02.01) STAD. UCEC. BLCA.

LLAATPRSSSSSSSNDMIP PSPA (BO7.02) BRCA. LUSC. CESC.

MAISSPPKAPLLAAPSPA APRTNFHSS (BO702) KIRC, UCS SRL O CINSNSRITSG 0WM APRTNFHSSL (B0702, BO801) AHMALLPSGTKGRCTAC CPQPSPSLPA (B0702) HTALGRGSLSSSSCPOPS GQWMAHMAL ) PSLPASNKLPSLPLSKMY GQWMAHMALL ) PILPLPOLLLSA HMALLPSGTK (A0301) DOA AAPRTNFHSSLAET HTALGRGSL (B0702) VSLHPLAPMPSKTCHHK IPMAISSPP (BO702) IPMAISSPPK (1307.02) KLPSLPLSK (A0301) KLPSLPLSKM (A0201) KMYTTSMAM (A0201. A0301) LLAAPSPASR (A0301) LLLSADQQAA ) LLSADQQAA (A0201) LPASNKLPS (BO702) Y1324fs LPASNKLPSL (B0702, B0801) ARID lA LPLPQLLLSA (BO702) LPSLPLSKM (BO702) LSKMYTTSM (808.01) MALLPSGTK ) MPILPLPQL ) MPILPLPQLL (130702) MYTTSMAMPI (A2402) PMAISSPPK (A0301) QWMAHMALL (A2402) SKMYTTSMAM (1307.02) SMAMPILPL (A0201, 130702, SNKLPSLPL (808.01) SPASRLQCI (30702, 130801) SPPKAPLLAA (1307.02) SPSLPASNKL (B0702) YTTSMAMPI (A0201) YTTSMAMPIL (A0201) RSYRRMIHLWWTAO ISL HPA (A0201) STAD, UCEC. BLCA, GVCRSLTVACCTGGLVG GLTHPAHQPL (A0201) BRCA. LUSC, CESC, TPLSISRPTSRAR 0 SCCL HPAHQPLGSM (B0702) KIRC, UCS ARID lA PGLTHPAH I PLGSM* LTHPAHQPL (BO702) RPTSRARQSC (1307.02) RQSCCLPGL (A0201) TSRAROSCCL (130801) VSLRGLSCARA ELLCVWVSSI (A0201) CRC, STAD, SKCM, GGYPAYSKDSG EWKVKFPEL (808.01) HNSC LLTSSSREWKVKFPELLC KFPELLCVW (A2402) LLCVWVSSI (A0201) LLTSSSREWK (A0301) LTSSSREWK (A0301) YPAYSKDSGL BO702 AIAKAVCS 0ESRDVLCE CLQCLWALL (A0201) Breast Cancer L328fs LSDHHNHTLEEEC 0WGP CQWGPCLQCL (A0201) GATA3 N3 34fs CL 0 CLWALL 0 AS 0 Y* OOWGPCLCLW (A2402) 32%;“ Chan 2 PGRPLTHVLPEPHLAL AIQPVLWTT (A02.01) 8132151 Cancer PL 0 PHADHAHADAPAI o ALQPLQPHA (A0201) LoHGHRHGL DLHFCRSSIM (308.01) TGPPARVPAVPF EPHLALQPL (307.02, 308.01) DLHFCRSSIMKPKRDGY ESKIMFATL (308.01) MFLKAESKIMFATLORSS FATLQRSSL (307.02, 308.01) LWCLCSNH* FLKAESKIM (308.01) FLKAESKIMF 1) GPPARVPAV (307.02) DGYM (308.01) KIMFATLQR (A03.0l) S408fs KPKRDGYMF (30702) S408fs KPKRDGYMFL (307.02) S430fs LHFCRSSIM (308.01) H434fs LQHGHRHGL (30801) H435fs MFATLQRSSL (307.02. B08.01) SKI (A2402) MLTGPPARV (A0201) QPVLWTTPPL 2) SMLTGPPARV (A0201) TLQRSSLWCL (A0201) LAL (A02.01) VPAVPFDLHF (307.02) ESK (A03.01) YMFLKAESKI (A0201. A0301, A2402. B08.01 _APGPRGRTC(307.02) STAD, BLCA. CRC, =CLRSHTCPPR(A03.01)HLRNCPHPHFLRHCTCPCCLWCHACLHR (A0301) HNSC, BRCA SLRRLRSLLCLP CPHLGSHPC 2) FLHWRSRPCLH CPLGLKSPL (307.02) RKSHPHLLHLRRLYPHH CPRSCRCPH (307.02) LKHRPCPHHLKNLLCPR CPRSCRCPHL (307.02, ) HLRNCPLPRHLKHLACL CSLPLGNHPY (A0101) HHLRSHPCPLHLKSHPCL GLRNRICPL (A0201, 307.02, VCSHHLKSLLC 308.01) PLHLRSLPFPHHLRHHAC GLRSHTYLR ) PHHLRTRLCPHHLKNHL GLRSHTYLRR (A0301) CPPHLRYRAYPPCLWCH GPRGRTCHPG (307.02) ACLHRLRNLPCPHRLRSL HLGSHPCRL (308.01) HLRLHASPHHLRTP HLRLHASPH (A0301) PHPHHLRTHLLPHHRR HLRSCPCSL (307.02, B08.0l) =HLRTHLLPH (A0301)RNSAPGPRGRTCHPGLRSHLRTHLLPHH (A0301) RTCPPGLRSHTYLRRLRS HLRYRAYPP (308.01) HTCPPSLRSHAYALCLRS HLRYRAYPPC (308.01) HTCPPRLRDHICPLSLRN HPHHLRTHL (307.02) LRSRTCLLCLRS HPHHLRTHLL (307.02. 308.01) HACPPNLRNHTCPPSLRS HTYLRRLRSH (A0301) HACPPGLRNRICPLSLRS LPCPHRLRSL (307.02, 308.01) HPCPLGLKSPLRS 0 ANAL LPHHRRTRSC (307.02. 308.01) HLRSCPCSLPLGNHPYLP LPLGNHPYL (307.02) CLSLGNHLCPLC LPRPLHLRL (B07.02,B08.01) PRSCRCPHLGSHPCRLS* NLRNHTCPP (308.01) PPRLRSRTCL (307.02, 308.01) RLHASPHHL (A0201) RLHASPHHLR (A0301) RLRDHICPL (A0201. B()7.()2.

Exemplary utatmn Sequence atein Chan- ' 1308.01) RLRNLPCPH (A03.01) RLRNLPCPHR (A0301) RLRSHTCPP (130801) RLRSLPRPL (30702, B08.01) RLRSLPRPLH (A0301) CLL (30702, BO8.01) RNRICPLSL (B0702, 808.01) RPLHLRLHA (307.02) RPLHLRLHAS (1307.02) RSHACPPGLR (A0301) RSHACPPNLR (A0301) RSHAYALCLR (A0301) RSHPCCHYLR (A0301) RSHPCPLGLK (A0301) RSHTCPPSLR (A03.01) RSLPRPLHLR 1) RSRTCLLCL (30702) RSRTCLLCLR 1) RSRTCPPGL 02) RSRTCPPGLR (A03.01) HHRR (A0301 ) RTRSCPCRWR (A0301 ) RYRAYPPCL (A2402) RYRAYPPCLW (A2402) SLGNHLCPL (A0201,130702, 308.01) HPYL (A0201) SLPRPLHLRL (A0201) SLRNCTCPPR (A03.01) SLRSHAYAL (A0201, 30702. 808.01) SLRSHPCPL (A0201, , SPHHLRTPP (307.02) SPHHLRTPPH (1307.02) SPLRSQANAL (30702. B08.01) YLRRLRSHTC ) YLRSRNSAP (308.01) YLRSRNSAPG (30301) GPRSHPLPRLWHLLLOV ALAPTLTHM (A0201) STAD, BLCA, CRC.

TOTSFALAPTLTHMLSPH ALAPTLTHML (A02.0l) HNsc, BRCA -_ LLQVTQTSFA (A02.01) ML” ”334% LQVTQTSFAL (A0201) RLWHLLLQV (A0201) Exemplary: Brutein (Shan- ; MIQLCTQLARF (A2402) RGKGGGVPPSPPLALGP' RMQLCTQLA (A0201) RMQLCTQLAR (A03 .0 1) SPPLALGPRM (130702) TQLARFFPI . A2402.

B08.01) KYEKKKYYDKNAIAITNI KSRQNHLQL (307.02) MSI+ CRC, MSI+ SSSDAPLQPLVSSPSLQA ALKKLRSPL 01, 1307.02) Uterine/Endometrium - CRRK(A03.01) Cancer, MSI+ Stomach KREKRSOOOKSRNHLLK KISNWSLKK (A0301, A1101) Cancer SNWSLKKVPAL LKKV(A03.01) KKLRSPLWIF KLRSPLWIF (A2402) KSRQNHLQLK (A0301) NWSLKKVPAL (308.01) SLKKVPALK (A0301, Al 1.01) SLKKVPALKK (A03.0l) SQKSRQNHL (30801) WSLKKVPAL (B08.0l) WSLKKVPALK (A0301) CCPRTILNNGSLKTIOV RLL (A0201) BRCA. CRC. LUAD, ORLLPPWPLH o KPTRAATVSV (1307.02) PRAD, HNSC, LUSC, P58fs IOLLHRRPLHPPPGPCHL LPPWPLHQQL (30702) PAAD. STAD, BLCA. OV.

P72fs LSLPRKPTRAATVSVWA LPRKPTRAA (1307.02, 1308.01) LIHC, SKCM. UCEC. 610st LPRKPTRAAT (1307.02) LAML, UCS. KICH. GBM.

R1 lOfs QQLLHRRPL (1308.01) ACC RLLPPWPLH (A0301) LARTPLPSTRCFANWPRP APASAPWPST (807.02) BRCA. CRC, LUAD, ALCSCGLIPHPRPAPASA APWPSTSSH (B0702) PRAD= HNSC, LUSC, P152fs RPAPASAPW (807.02) PAAD. STAD. BLCA. OV.

WPSTSSHST (B0702) LIHC. SKCM. UCEC.

LAML, UCS. KICH‘ GBM, SQGLYSSSASSGKCLME HLL (1307.02) VTVDRNCLEVLPTKMSY K2120fs AANLKNVMNMQNRQK KGKNSPCCOOOKKLRVN Exemplary22-Mutation ce Peptides(HLA allele 2;; Bratein Chang : 2;s;; .23; 2;; ;.;s; ‘ ‘ ;.;.; ;.;.; -_ ;.;.;. ;.;.; ;.;.; ;.; ;.;.; ;.;.. 2;: 2 TRASPPRSSSAIAVRASC FLPISHCQCI (A02.01) KIRC KIRP CPYGSTSTASRSPTQRCR FWLTKLNYL (A24.02 308.01) LARAAASTATEVTFGSSE HLSMLTDSL (A0201) MOGHTMGFWLTKLNYL HTMGFWLTK (A0301) CHLSMLTDSLFLPISHC O HTMGFWLTKL (A0201) KLNYLCHLSM (A0201) LPISl-ICQCI (B0702, B0801) QCIL (307.02, 1308.01) L116fs LTDSLFLPI (A0101, A0201) G123fs LTKLNYLCHL (808.01) MLTDSLFLPI (A0101. A0201. 808.01) MQGHTMGFWL ) NYLCHLSML (A2402) SMLTDSLFL (A0201) TMGFWLTKL (A0201) MLT (A0201) TABLEZD INSER’I“ LGSGAFGTVYKGIWIPD CILDEAYVMAY (A0101) Lung Cancer ENVKIPVAIKVLRENTSP VMAYVMAGV (A020 I) LDEAYVMAfl YVMAYVMAG (A0201. 307.02.

HERZ G776insYVMA MGVGSPYVSRLLGICL 1308.01) STVQLVTQLMPYGCLLD YVMAYVMAGV (A0201, 1307.02, HVRENRGRLGSQDLLN 1308.01) lUnderlined AAs represent non-native AAs ZBolded AAs represent native AAs of the amino acid sequence d by the second of the two fused genes 3Bolded and underlined AAs represent non-native AAs of the amino acid sequence encoded by the second of the two fused genes due to a framshift.

In the Tables above, for one or more of the exemplary fusions; a sequence that comes before the first “z” belongs to an exon sequence of a polypeptide encoded by a first gene, a ce that comes afier the second belongs to an exon sequence of a polypeptide encoded by a second gene. and an amino acid that appears between symbols is encoded by a codon that is split between the exon sequence of a polypeptide encoded by a first gene and the exon sequence of a polypeptide encoded by a second gene.

However, in some ments, for example, NAB:STAT6, the NAB exon is linked to the 5‘ UTR of STAT6 and the first amino acid that appears after the junction is the normal start codon of STAT6 (there is no frame present at this site (as it is not normally translated».

AR-V7 in the tables above can also be ered. in some embodiments. a splice variant ofthe AR gene that encodes a protein that lacks the ligand binding domain found in full length AR.

Example 2: HLA Class I and Class 11 Binding Assays The following e of peptide binding to HLA molecules demonstrates quantification of binding affinities of HLA class I and class 11 peptides. Binding assays can be performed with peptides that are either motif-bearing or not bearing.

Epstein-Barr virus (EBV)-transfomied gous cell lines, fibroblasts, CIR, or 721.22 transfectants are used as sources of HLA class 1 molecules. Cell lysates are prepared and HLA molecules purified in accordance with disclosed protocols (Sidney et 211., Current Protocols in logy 18.3.1 (1998); Sidney. ct 211.. J. Immunol. 154:247 (1995); Sette. et 211.. Mol. Immunol. 311813 (1994)). HLA molecules are purified from lysates by affinity chromatography. The lysates are passed over a column of Sepharose CL-4B beads coupled to an appropriate antibody. The anti-HLA column is then washed with 10 mM Tris-HCL, pH 8.0, in 1% NP-40, PBS, and PBS containing 0.4% n-oetylglueoside and HLA molecules are eluted with 50 mM diethylamine in 0.15 M NaCl containing 0.4% n-oetylglucoside, pH 1 1.5. A 1/25 volume of 2.0 M Tris, pH 6.8, is added to the eluate to reduce the pH to ~80. Eluates are then concentrated by centrifugation in Centriprep 30 concentrators (Amicon. Beverly. MA). Protein content is evaluated by a BCA n assay (Pierce Chemical Co., Rockford, IL) and confirmed by SDS-PAGE.

A detailed description of the protocol utilized to measure the binding affinity of peptides to Class I and Class 11 MHC has been published (Sette et a1, Mol. Immunol. 312813, 1994; Sidney et al., in t Protocols in Immunology, ies, Ed, John Wiley & Sons, New York, Section 18.3, 1998). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled e inhibitors and 1-10 nM ”SI-radiolabeled probe peptides for 48 n in PBS containing 0.05% Nonidet P-40 (NP40) (or 200/o w/v digitonin for H-2 IA assays) in the presence of a protease inhibitor cocktail. All assays are at pH 7.0 with the exception of DRBl*0301, which was perfonned at pH 4.5, and DRB1*1601 (DRZleBl) and DRB4*0101 (DRw53), which were performed at pH 5.0.

Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration on 7.8 mm x 15 cm TSK200 columns (TosoHaas 16215, Montgomeryville, PA). Because the large size of the radiolabeled peptide used for the DRB1* 1501 (DR2w2BI) assay makes separation ofbound from d peaks more difficult under these conditions. all DRB1*1501 (DR2w2Bl) assays were performed using a 7.8 mm x 30 cm TSK2000 column eluted at 0.6 mLs/min. The eluate from the TSK columns is passed through a Beekman 170 radioisotope detector, and radioactivity is plotted and ated using a t—Packard 3396A integrator, and the fraction of peptide bound is determined.

Radiolabeled peptides are iodinated using the chloramine-T method. Typically, in preliminary experiments, each MHC preparation is d in the ce of fixed amounts of radiolabeled es to ine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent tion and direct binding assays are performed using these HLA concentrations.

Since under these ions [label] < [HLA] and [€502 [HLA], the measured IC50 values are reasonable approximations of the true Kn values. Peptide inhibitors are typically tested at concentrations ranging from 120 ug/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different ments, a relative binding figure is calculated for each peptide by dividing the ICsn of a positive control for inhibition by the 1C5" for each tested peptide (typically unlabeled versions of the abeled probe peptide). For database es, and inter-experiment comparisons, relative binding values are compiled. These values can uently be ted back into leo nM values by dividing the ICso nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation has proven to be the most accurate and tent for comparing peptides that have been tested on different days. or with different lots of purified MHC.

Because the antibody used for HLA-DR purification (LB3.1) is n specific, [51 molecules are not separated from [53 (and/or [34 and [35) molecules. The [31 specificity of the binding assay is obvious in the cases of DRBl*010] (DRl), DRBl*0802 (DR8w2), and 803 (DR8w3), where no [33 is expressed. It has also been demonstrated for DRB1*0301 (DR3) and DRB3*0101 (DR52a), DRBl*O401 (DR4w4), DRB1*O404 (DR4w14), 405 (DR4w15), DRB1*1101 (DRS), DRB1*1201 2), DRB1* 1302 (DR6w19) and DRB1*0701 (DR7). The m of B chain city for DRB1* 1501 (DR2w2Bl). 101 (DRZw'ZBZ), DRB1*1601 (DRZWZIBI), DRBS*0201 (DR51Dw21), and DRB4*0101 ) assays is circumvented by the use of asts. Development and validation of assays with regard to DRB molecule specificity have been described previously (see, e.g., Southwood et al., J. l. 160:3363-3373, 1998).

A detailed description of the protocol utilized to measure the binding stability of peptides to Class I MHC has been published (Hamdahl et al. J Immunol Methods. 374:5-12, 2011). Briefly. synthetic genes encoding biotinylated MHC-I heavy and light chains are expressed in E. coli and purified from inclusion bodies using rd s. The light chain ([32m) is radio-labeled with iodine ( ”51), and combined with the purified MHC-1 heavy chain and peptide of interest at 18°C to initiate pMHC-I complex formation. These reactions are carried out in streptavidin coated microplates to bind th biotinylatcd MHC-I heavy chains to the surface and allow measurement of radiolabeled light chain to monitor complex fonnation. Dissociation is initiated by addition of higher coneentations of unlabled light-chain and incubation at 37°C. Stability is defined as the length of time in hours it takes for half of the complexes to dissociate, as measured by scintillation counts Live cell/flow cytometry-based assays can also be used, e.g., an assay utilizing a TAP-deficient hybridoma cell line T2 (American Type Culture Collection (ATCC ion No. CRL-1992), Manassas, Va). TAP deficiency in this cell line leads to inefficient loading of MHCI in the ER and an excess of empty MHCIs. Salter and Cresswell. EMBO J. 52943-49 (1986); Salter, Immunogenetics 212235-46 (1985). Empty MHCls are highly unstable and short—lived. When T2 cells are cultured at reduced temperatures, empty MHCIs appear transiently on the cell surface, where they can be stabilized by ous addition of MHCI- binding peptides. To i this binding assay, peptide-receptive MHCIs were induced by culturing aliquots of 107 T2 cells overnight at 26 °C in serum free AIM-V medium alone, or in medium containing escalating concentrations (0.1 to 100 uM) of peptide. Cells were then washed twice with PBS, and subsequently incubated with a fluorescent tagged HLA-AOZOl-speeific monoclonal antibody. 337.2. to quantify cell surface expression. s were acquired on a FACS Calibur instrument (Becton Dickinson) and the mean fluorescence intensity (MFI) determined using the accompanying Cellquest software.

Example 3: HLA Class 1 Binding Affinity HLA g assays were used to evaluate either the affinity of interaction of a given epitope with a selected HLA molecule or the stability. Each of these measurements was. e.g.. tive of the ability of an epitope to elicit a CD8+ T cell se.

A subset of peptides from Table l (n=562) were synthesized and their affinity for their given HLA class 1 molecule was measured as described. The values are shown in Table 3. The measurements are plotted against the predicted ty in Figure 1. These data show a strong correlation between prediction and measurement (dotted line represents best fit, R2 = 0.45), demonstrating the value of the predictions. However, the outliers demonstrate the importance of these measurements. Thick vertical and horizontal lines are shown at 500 nM for the predicted affinity and observed affinity, respectively. 500nM is ly accepted in the field as the maximum affinity for an epitope that is a “weak binder” to HLA class 1. Therefore, the points in the lower right quadrant (prediction greater than 500 nM, measurement less than 500 nM) are epitopes that were considered very weak binders but were observed to bind within an acceptable range. Epitopes in this quadrant (n=75) represent 30.5% of epitopes not considered to be binders by tion (combination of bottom right and top right quadrants, n=246).

Table 3: HLA Class I Binding Affinity and Stability redicted fl 1.»: .. 3.. : . , .5 .z, . -:: .3. . 3,. ..(nM) ,.. . .3 . .3: —_——_I- —_——_-_ —_—_-El_.- —_——_-m —_——_.- —_——_.- —_——_-I- —————-n. —————m- —————-m- BCRzABL 308-01 LTINKEEAL , 395.0 (e13a2, akab2a2) BCRzABL A02.01 LTINKEEAL 12671.0 44130 — (c13a2, akab2a2) —————-mu —_——_-I. —_———-E_ C l (+ 1) A03 .01 LFFFFFETK 658.4 413.3 not measured —————-m 1455.0 —————-_ —————- —————-_ —————- . EGFR""1. A0201 ALEEKKGNYV 2445.0 141.0 l deletlon) —————-_ —————.— —————m- —————.— —————.— —————-_ —————-— —————-_ —————-— ————m.- —————.— —————- —————-m- —————-m- —————-m- —————-m —————m- —————m- —————-n. —_———n- HERZ, - G776insYVMA ‘ - . . _ G776insYVMA ‘ - . _-__-—G776insYVMA ' - .

G776insYVMA ‘ ~ - . , G776insYVMA ' - . .

HERZ, _ _ stsss ————-z_ —————-_ 553.0 ————-m_ as —————- —————-m —————-m —————-— —————-m —_———-m —————-m —————m_ —————-_ —————-n- —————-m- —————m_ —————-_ —————- —————m- —————.— NAB:STAT6 ("variant 1” of A0201 SQIMSLWGL 14.0 62.0 1 Chmielecki et a1.

NAB:STAT6 (“variant 1” of A0201 IMSLWGLVS 3630.0 7321.0 Chmielecki et a1.) NAB:STAT6 ("variant 1” of A2402 SQIMSLWGL 1604.0 8516.0 0 ecki et a1.) NAB:STAT6 (“variant 1” of 808.01 SQIMSLWGL 4587.0 15997.0 Chmiclccki ct a1.) NDRGlERG A0201 LLQEFDVQEA 200.0 NDRGI:ERG A0201 QEAL 2229.0 502800 2“ 11:13:12.: 1:31:53; K1399: 9:39:31 K222121151, -——-- 1112:2121, K1929: Kain-1mm NDUFC2(- A0201 LLYITAFFLL 323.2 KCDT14)(_1) NDUFC2(- “”2 _ ‘ LLY‘TAFFLL Kcvm -1 NDUFC2(- A2402 LYITAFFLLD 414.3 KCDT14 _1 —————-a_ —————-— —————- —————.— —————m —————m- —————.— ——_——-n_ —————m- ————m:_m- —————m_ —————.— ————-m-_ —————- ———-::-—— —————-_ —————-_ —————- Example 3: HLA Class I Binding Stability A subset of the peptides used for affinity measurements were also used for stability measurements using the assay described (n=275). These data are shown in Table 3. The data are d according to 4 bins in Figure 2, with the median and interquartile intervals overlaid. Less than 50 nM was considered by the field as a strong binder, 50-150 nM was considered an intermediate binder, 150—500 nM was considered a weak , and greater than 500 nM was considered a very weak binder. The connection between the ed stability and observed affinity was evident by the sing median stability across these binned stability intervals. r, there is considerable overlap between the bins, and antly there are epitopes in all bins with observed stability in the multiple hour range, including the very weak binders.

Example 4: Immunogencity Assays Immunogenicity assays are used to test the ability of each test peptide to expand T cells. Mature professional APCs are prepared for these assays in the following way. Monocytes are enriched from healthy human donor PBMCs using a bead-based kit (Miltenyi). Enriched cells are plated in GM-CSF and IL-4 to induce re DCs. After 5 days, immature DCs are incubated at 37°C with each peptide for 1 hour before addition of a cytokine tion cocktail (GM-CSF. IL-lB. IL-4. IL-6. TNFa. PGEl B). Cells are incubated at 37°C to mature DCs.

After maturation of DCs, PBMCs (either bulk or enriched for T cells) are added to mature dendritic cells with proliferation cytokines. Cultures are red for peptide-specific T cells using a combination of functional assays and/or tetramer ng. Parallel genicity assays with the modified and parent peptides allowed for comparisons of the relative efficiency with which the peptides expanded peptide-specific T cells.

Tetramer Staining. MHC tetramers are purchased or ctured on-site, and are used to e peptide-specific T cell ion in the immunogenicity assays. For the assessment, tetramer is added to 1x105 cells in PBS containing 1% FCS and 0.1% sodium azide (FACS buffer) according to manufacturer's instructions. Cells are incubated in the dark for 20 s at room temperature. dies specific for T cell markers, such as CD8, are then added to a final tration suggested by the manufacturer, and the cells are incubated in the dark at 4 0C for 20 minutes. Cells are washed with cold FACS buffer and resuspended in buffer containing 1% fonnaldehyde. Cells are acquired on a FACS Calibur (Becton Dickinson) instrument, and are analyzed by use of Cellquest software (Becton son). For analysis of tetramer positive cells, the lymphocyte gate is taken from the forward and side-scatter plots. Data are ed as the percentage of cells that were CDSVTetramerf Intracellular cytokine staining. In the absence of well-established tetramer staining to identify antigen-specific T cell populations, antigen-specificity can be estimated using assessment of cytokine production using well-established flow cytometry assays. Briefly. T cells are stimulated with the peptide of interest and compared to a control. Afier stimulation, production of cytokines by CD4+ T cells (e.g., IFN*{ and TNFOL) are assessed by intracellular staining. These cytokines, especially IFNy, used to identify stimulated cells.

ELISPOT. Peptide-specific T cells are functionally enumerated using the T assay (BD Biosciences), which measures the release of lFNgamma from T cells on a single cell basis. Target cells (T2 or HLA-A0201 transfected C le) were pulsed with 10 uM peptide for 1 hour at 37 °C, and washed three times. 1x105 peptide-pulsed targets are co-cultured in the ELISPOT plate wells with varying concentrations of T cells (5x102 to 2x103) taken from the immunogenicity culture. Plates are developed according to the cturer's protocol, and analyzed on an ELISPOT reader (Cellular Technology Ltd.) with accompanying software. Spots corresponding to the number of lFNgamma-producing T cells are reported as the absolute number of spots per number ofT cells . T cells expanded on modified peptides are tested not only for their ability to recognize s pulsed with the modified peptide, but also for their ability to recognize targets pulsed with the parent peptide.

C0107 staining. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide. The lytic granules of T cells have a lipid bilaycr that contains lysosomal-associated membrane roteins ("LAMPS”), which include the molecules CD 107a and b. When cytotoxic T cells are activated through the T cell receptor. the membranes of these lytic granules mobilize and fuse with the plasma membrane of the T cell. The granule contents are released, and this leads to the death of the target cell. As the granule membrane fiises with the plasma membrane, C107a and b are exposed on the cell surface, and therefore are markers of degranulation. Because degranulation as measured by CD107 a and b staining is reported on a single cell basis, the assay is used to functionally enumerate peptide-specific T cells. To perform the assay, peptide is added to HLA-AOZOl-transfectcd cells C IR to a final concentration of 20 uM, the cells were incubated for 1 hour at 37 °C. and washed three times. 1x105 of the peptide-pulsed CIR cells were aliquoted into tubes, and antibodies specific for CD 107 a and b are added to a final concentration suggested by the manufacturer (Becton Dickinson). Antibodies are added prior to the addition ofT cells in order to “capture” the CD 107 molecules as they transiently appear on the surface during the course of the assay. 1x105 T cells from the immunogenicity culture are added next, and the samples were incubated for 4 hours at 37 0C.

The T cells are further stained for additional cell surface molecules such as CD8 and acquired on a FACS Calibur instrument (Becton Dickinson). Data is analyzed using the accompanying est software. and results were reported as the percentage of CD8+ CD107 a and b+ cells.

] Cytotoxicity assays. Cytotoxic activity is measured using a chromium release assay. Target T2 cells are labeled for 1 hour at 37 °C with NaSICr and washed 5x103 target T2 cells were then added to varying numbers of T cells from the immunogenicity culture. Chromium e is measured in supematant harvested after 4 hours of incubation at 37°C. The percentage of specific lysis is ated as: Experimental release-spontaneous release/Total release-spontaneous release x 100 Immunogenicity assays were d out to assess whether each peptide can elicit a T cell response by antigen-specific ion. Though current methods are imperfect, and ore negative results do not imply a peptide is incapable of inducing a response, a positive result demonstrates that a peptide can induce a T cell response. l peptides from Table 3 were tested for their capacity to elicit CD8+ T cell responses with multimer ts as described. Each positive result was measured with a second multimer preparation to avoid any preparation biases. Several examples of positive multimer staining are shown in Figures 3-6, with multimer negative cells (lefi) and antigen-specific imer—double-positive) cells (right) shown.

HLA-A02201+ T cells were co—eultured with monoeyte-derived dendritic cells loaded with TMPRSS2::ERG fusion neoepitope (ALNSEALSV; HLA-A02:01) for 10 days. CD8° T cells were analyzed for antigen-specificity for TMPRSS2::ERG fusion tope using multimers (initial: BV421 and PE; validation: APC and BUV396) (Figure 3).

HLA-A02:Ol+ T cells were co-cultured with monocytc-derivcd dendritic cells loaded with GATA3 frameshift neoepitope (SMLTGPPARV; 2201) for 10 days. CD8+ T cells were analyzed for antigen- specificity for GATA3 hift tope using multimers (initial: APC and ; validation: PE and BV421) (Figure 4).

HLA-AOIZ:01+ T cells were tured with monocyte-derived dendritic cells loaded with [32M frameshift neoepitope (LLCVWVSSI; HLA-A02201) for 10 days. CD8+ T cells were analyzed for antigen- specificity for [52M frameshift neoepitope using multimers (initial: PE and APC; validation: PE and BV421) (Figure 5).

HLA-A02201+ T cells were co—cultured with monocyte—derived dendritic cells loaded with KRAS G 12C neoepitope (KLVVVGACGV; HLA-A02101) for 10 days. CD8+ T cells were analyzed for antigen- city for KRAS G12C frameshift neoepitope using multimers (initial: BU V396 and BV42]; tion: APC and BUV396) (Figure 6).

While antigen-specific CD8+ T cell responses are readily assessed using well-established HLA Class I multimer technology. CD4+ T cell responses require a te assay to evaluate because HLA Class II multimer technology is not well-established. In order to assess CD4+ T cell responses, T cells were re- stimulated with the peptide of interest and compared to a control. In the case of a completely novel sequence (e.g., arising from a frame-shift or fusion), the control was no peptide. 1n the case of a point-mutation, the control was the WT peptide. After stimulation, production of cytokincs by CD4- T cells (c.g., IFNy and TNFa) were assessed by intracellular staining. These cytokines, especially lFNy, used to identify stimulated cells. Antigen-specific CD4+ T cell responses showed increased ne production relative to control.

Examples of antigen-specific CD4+ T cell responses generated against neopeptides are shown in s 7-9.

T cells were co-cultured with te-derived dendritic cells loaded B2M frameshift neopeptides for days (restimulation with fresh 1nonocy10-derived dendritic cells on day 20). CD4' T cells were analyzed for antigen-specificity by intracellular cytokine staining after restimulation with monocyte-derived dendritic cells loaded with [52M frameshift peptide for 24 hours, compared to controls without e (Figure 7).

Naive T cells were co-cultured with monocyte-derived dendritic cells loaded BTK C4818 neopeptide for 20 days mulation with fresh monocyte-denved dendritic cells on day 20). CD4+ T cells were analyzed for n-specificity by intracellular ne staining afier restimulation with monocyte-derived dendritic cells loaded with BTK C481 S neopeptide for 24 hours, compared to controls ype BTK peptide e Naive T cells were co-cultured with monocyte-derived dendritic cells loaded GATA3 frameshifi neopeptides for 20 days (restimulation with fresh monocyte-derived dendritic cells on day 20). CD4+ T cells were analyzed for antigen-specificity by intracellular cytokine staining afier restimulation with te- derived dendritic cells loaded with GATA3 frameshift peptide for 24 hours, compared to controls without peptide (Figure 9).

Example 5: Selection of CTL and HTL epitopes for inclusion in an tumor-specific vaccine.

This example illustrates the procedure for the selection of peptide es for vaccine itions of the invention. The peptides in the composition can be in the form of a c acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or may be single and/or polyepitopic peptides.

Epitopes are ed which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For example, vaccine can include 1-2 epitopes that come from at least one tumor antigen . Epitopes from one region can be used in combination with epitopes from one or more additional tumor antigen regions.

Epitopes can be selected, for example, that have a binding y of an ICso of 500 nM or less for an HLA class 1 molecule, or for class 11, an ICSO of 1000 nM or less.

When creating a polyepitopic compositions, e.g. a minigene, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as those employed when ing a peptide comprising nested epitopes. Additionally, however, upon determination of the nucleic acid sequence to be provided as a minigene. the peptide sequence encoded thereby is analyzed to determine whether any ‘junctional epitopes" have been created. A j unctional epitope is a potential HLA binding epitope, e.g., as predictedby motif analysis. Junctional epitopes are generally to be avoided e the recipient may bind to an HLA molecule and te an immune response to that epitope, which is not present in a native protein sequence.

Peptide epitopes for inclusion in vaccine compositions are, for example, selected from those listed in the Tables. A vaccine composition sed of selected peptides. when administered. is safe. cious. and elicits an immune response similar in magnitude of an immune response that inhibits tumor growth.

Example 6: e Composition for Prophylactic or Therapeutic Uses lrnmunogenic or vaccine compositions of the invention are used to inhibit tumor growth. For example, a polyepitopic composition (or a c acid comprising the same) containing multiple CTL and HTL epitopes is administered to individuals having tumors. The dose of peptide for the immunization is from about 1 to about 50,000 pg, generally loo-5,000 ug, for a 70 kg t. The initial administration may be followed by booster dosages at 4 weeks followed by evaluation of the ude of the immune se in the patient, by techniques that determine the presence of epitope-specific CTL tions in a PBMC sample.

Additional booster doses are administered as ed. The composition is found to be both safe and efficacious to inhibit tumor growth.

Altematively, the polyepitopic composition can be administered as a nucleic acid, for example as RNA, in accordance with methodologies known in the art and disclosed herein.

Neoantigen binding agents. such as TCR or CARS can be can be administered in accordance with methodologies known in the art and disclosed herein. The binding agents can be administered as polynucleotides, for example DNA or RNA, ng the binding agents as part of cellular therapy.

Altematively, the binding agents can be prepared as antibodies or fragments thereof capable of recognizing the specific peptidele-IC complex coupled to cytotoxic agents or T cell binding agents e of re-directing patient T cells to tumor cells expressing the epitopes listed in the Tables.

Neoantigen peptides. ucleotides. binding agents. or cells expressing these molecules can be delivered to the same patient via le methodologies known in the art, and can further be combined with other cancer therapies (e.g., herapy, surgery, radiation, checkpoint inhibitors, etc).

Example 7: Administration of Compositions Using Dendritic Cells Vaccines sing epitopes of the invention may be administered using dendritic cells. In this example. peptide-pulsed or nucleic acid-pulsed dendritic cells can be administered to a patient to stimulate a CTL response in viva. In this method dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide or encoding DNA or RNA CTL and HTL epitopes of the ion. The dendritic cells are d back into the patient to elicit CTL and HTL responses in viva. The induced CTL and HTL then destroy (CTL) or facilitate destruction (HTL) of the specific target tumor cells that bear the proteins from which the epitopes in the vaccine are derived.

Altematively. ex viva CTL or HTL responses to a particular tumor-associated antigen can be induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells, such as dendritic cells, and the appropriate immunogenic peptides or nucleic acids. After an appropriate incubation time ally about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate ction (HTL) of their specific target cells, i.e., tumor cells. e 8: Identification of Neoepitopes Cancer cells containing ct genetic changes that alter amino acid sequence could generate ial novel T cell epitopes, such as those shown in the Tables. Identifying which patients tumors contain tumor-specific neoepitopes as identified in the Tables can comprise identification of DNA mutations. As one approach, whole genome or whole exome sequencing using well—established techniques oftumor versus matched ne samples from patients can be carried out. As an additional approach, RNA sequencing of tumor and appropriately matched normal samples may also be conducted. As an additional approach, specific assays based on targeted sequencing, either next generation sequencing techniques or well-established Sanger sequencing can also be conducted. Additionally, highly specific Polymerase-chain reaction based assays may be developed.

Example 9: Specificity of neoantigen peptide recognition by CD8+ or CD4+ cells In various embodiments, the present teachings include sure of discrimination between d and ype sequences by vaccine-induced CD8+ cells. To determine whether e-induced T cells could ize naturally processed antigen. a tumor cell line can be transduced with a multi-mini-gene construct encoding mutated (MUT) or ype (WT) sequences of peptides incorporated into a vaccine. Each minigene can consists of 21 aa encoding either the MUT or WT sequences. Vaccine-induced cells, specific for a protein containing a particular mutation can be incubated with MUT or WT expressing cancer cells, supematants can be ted afier 24 h of incubation, and IFN-y produced by cells can be measured in tants by ELISA.

Neoantigen-specific cells recognition of mutated and wild type es can also be detemiined in a standard 4 h 5|Cr—release assay.

Claims (1)

1. CLAIMS 1. An isolated neoantigenic peptide comprising a tumor-specific neoepitope, wherein the isolated neoantigenic e is not a native polypeptide, wherein the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: AxByCl, each A and C represents an amino acid corresponding to the native polypeptide, y is at least 1 and B ents an amino acid substitution or insert of the native polypeptide, satleast8, the at least 8 contiguous amino acids comprises By, and the native polypeptide is encoded by a gene selected from the group consisting of: (a) ABLl, wherein AxByCZ is (i) VADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGKVYEGV WKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVC, (ii) VADGLITFLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGVVYEGV WKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVC, (iii) REPPFYIlTEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSATEYLEKKNFI HRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAKF, (iv) SLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIIIEFMTYGNL LDYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLA, or (v) STVADGLITFLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQHGEVYEG VWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLG; (b) ALK, wherein AXBVVCZ is (i) SSLAMLDLLHVARDIACGCQYLEENHFIHRDIAARNCLLTCPGPGRVAKIADFGMARDI YRASYYRKGGCAMLPVKWMPPEAFMEGIFTSKTDTWSFGVLL, or (ii) QVAVKTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIGVSLQSLPRFILMELMAGGDLK SFLRETRPRPSQPSSLAMLDLLHVARDIACGCQYLEENHFI; (c) BRAF, wherein AXB,.Cz is MIKLIDIARQTAQGMDYLHAKSIIHRDLKSNNIFLHEDLTVKIGDFGLATEKSRWSGSHQFEQ LSGSILWMAPEVIRMQDKNPYSFQSDVYAFGIVLYELM; (d) BTK, wherein AxByCZ is MIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGSLLNYLREMRHR FQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND; (e) EEFl B2, wherein A_.‘B,-CZ is MGFGDLKSPAGLQVLNDYLADKSYIEGYVPSQADVAVFEAVSGPPPADLCHALRWYNHIK SYEKEKASLPGVKKALGKYGPADVEDTTGSGAT; (f) EGFR, wherein AxByCZ is (i) SLNITSLGLRSLKEISDGDVIISGNKNLCYANT]NWKKLFGTSGQKTKIIRNRGENSCKAT GQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLL, or (ii) lPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLL DYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; (g) ERBB3, wherein AXB,CZ is ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRMVRGTQVYDG KFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDK; (h) ESRl, wherein AxByCZ is (i) HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLLEML DAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEA, (ii) NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLPSTLKSLEEK DHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH, (iii) AGLTLQQQHQRLAQLLLILSHIRHMSNKGMEI—ILYSMKCKNVVPLCDLLLEML DAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, (iv) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEI—ILYSMKCKNVVPLNDLLLEML DAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, or (v) lHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLLEML DAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE; (i) FGFR3, wherein AxByCz is HRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGS[RQTYTLDVLERCPHRPILQAGLP ANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVG; (i) FRGIB, wherein AXB,CZ is SRIALKSGYGKYLGINSDELVGHSDAIGPREQWEPVFQNGKMALSASNSCFIRCNE AGDIEAKSKTAGEEEMIKIRSCAEKETKKKDDIPEEDKG; (k) HERZ, n AxByCZ is GSGAFGTVYKGlWlPDGENVKlPVAlKVLRENTSPKANKEILDEAYVMAGLGSPYVSRLLGI CLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCM; (I) lDHl, whcrcin AxByCZ is (i) RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGHHAYGDQYRA TDFVVPGPGKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, (ii) RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWKPIIIGCHAYGDQYRA TDFVVPGPGKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, (iii) RVEEFKLKQMWKSPNGTIRNILGGTVFREAI[CKNIPRLVSGWVKPIIIGGHAYGDQYRA GPGKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM. or (iv) RVEEFKLKQMWKSPNGTIRNILGGTVFREAI[CKNIPRLVSGWVKPIIIGSHAYGDQYRAT DFVVPGPGKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM; (m) KIT, wherein AAB,.C,_ is (i) VEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIANLLGACT IGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYK, or (ii) VEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIANLLGACT IGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYK; (n) MEK, wherein AXB,CZ is (i) lSELGAGNGGVVFKVSHKPSGLVMARKLIHLEIKPAlRNQllRELQVLHESNSPYIVGFYG EISICMEHMDGGSLDQVLKKAGRIPEQILGKVSI, or (ii) LGAGNGGVVFKVSHKPSGLVMARKLIHLEIKPAIRNQIIRELQVLHECNSLYIVGFYGAF YSDGEISICMEHMDGGSLDQVLKKAGRIPEQILGKVSIAVI; (o) MYC, wherein AxByCZ is (i) MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSDLQPPAPSEDIWKKFELLPTP PLSPSRRSGLCSPSYVAVTPFSLRGDNDGG, (ii) FTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLSTPPLSPSRR SGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLG, or (iii) TNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPIPPLSPSRRS GLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGG; (p) PDGFRa, wherein AxByCz is KPTARSSEKQALMSELKIMTHLGPHLNIVNLLGACTKSGPIYIIIEYCFYGDLVNYL HKNRDSFLSHHPEKPKKELDIFGLNPADESTRSYVILS; (q) PIK3CA, wherein A,‘ByCZ is (i) IEEHANVVSVSREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSKITEQEKDFL WSHRHYCVTIPEILPKLLLSVKWNSRDEVAQMYCLVKDWPP. (ii) HANWSVSREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSEITKQEKDFLWS HRHYCVTIPEILPKLLLSVKWNSRDEVAQMYCLVKDVVPPIKP, or (iii) LF1N LFSMMLGSGMPELQSFDDlAYlRKTLALDKTEQEALEYFMKQMNDARHGGWTTK MDWIFHTIKQHALN; (r) POLE, whcrcin AxByCz is QRGGVITDEEETSKKIADQLDNIVDMREYDVPYI-IIRLSIDIETTKLPLKFRDAETDQIMMISY MIDGQGYLITNREIVSEDIEDFEFTPKPEYEGPFCVFN; (s) PTEN, wherein AxByCZ is AQYPFEDHNPPQLELIKPFCEDLDQWLSEDDNHVAAIHCKAGKGQTGVMICAYLL HRGKFLKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSY; (t) RAC 1, wherein AxByCZ is MQAIKCVVVGDGAVGKTCLLISYTTNAFSGEYIPTVFDNYSANVMVDGKPVNLGLWDTAG QEDYDRLRPLSYPQTVGET; and (u) TP53, wherein AXB,,CZ is (i) lRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTFIHYNYMCNSSCMGSMNRRPIL TIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEP, (ii) TYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRHCPHHE RCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEV. (iii) EYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNQRPILTII GNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE, (iv) EGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTI‘IHYNYMCNSSCMGGMNWRPILTII TLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE, or (v) PEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVCVCACPGRD RRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPL. The isolated neoantigenic e of claim 1, wherein the native polypeptide is encoded by the EGFR, ERBB3 or FGFR3 gene and at least one B, is expressed ellularly. D) An isolated neoantigenic peptide comprising a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native ptide, wherein the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: AxByCZ, wherein each A is an amino acid corresponding to the native polypeptide; B, is absent; each C is an amino acid encoded by a frameshifi of a sequence encoding the native polypeptide: x + y + z is at least 8; the at least 8 contiguous amino acids comprises at least one Cl; and the native polypeptide is encoded by a gene selected from the group consisting of: (a) APC, wherein C2 is (i) HSTLEPNPADCRVLVYLQNQPGTKLLNFLQERNLPPKVVLRHPKVHLNTM FRRPHSCLADVLLSVHLIVLRVVRLPAPFRVNHAVEW, (ii) APVIFQIALDKPCHQAEVKHLHHLLKQLKPSEKYLKIKHLLLKRERVDLSKLQ, or (iii) MLQFRGSRFFQMLILYYILPRKVLQMDFLVHPA; (b) ARIDlA, wherein C, is (i) ALGPHSRISCLPTQTRGCILLAATPRSSSSSSSNDMIPMAISSPPKAPLLAAPSPASRLQC lNSNSRlTSGQWMAHMALLPSGTKGRCTACHTALGRGSLSSSSCPQPSPSLPASNKLP SLPLSKMY'I'I'SMAMPILPLPQLLLSADQQAAPRTNFHSSLAETVSLHPLAPMPSKTCH (ii) AHQGFPAAKESRVIQLSLLSLLIPPLTCLASEALPRPLLALPPVLLSLAQDHSRLLQCQ ATRCHLGHPVASRTASCILP. (iii) PILAATGTSVRTAARTWVPRAAIRVPDPAAVPDDHAGPGAECHGRPLLYTADSSLWT TRPQRVWSTGPDSILQPAKSSPSAAAATLLPATTVPDPSCPTFVSAAATVSTTTAPVLS ASlLPAAlPASTSAVPGSIPLPAVDDTAAPPEPAPLLTATGSVSLPAAATSAASTLDALP AGCVSSAPVSAVPANCLFPAALPSTAGAISRFIWVSGILSPLNDLQ, (iv) PCRAGRRVPWAASLIHSRFLLMDNKAPAGMVNRARLHITTSKVLTLSSSSHPTPSNH RPRPLMPNLRISSSHSLNHHSSSPLSLHTPSSHPSLHISSPRLHTPPSSRRHSSTPRASPPT HSHRLSLLTSSSNLSSQHPRRSPSRLRILSPSLSSPSKLPIPSSASLHRRSYLKIHLGLRHP QPPQ, (v) RTNPTVRMRPHCVPFWTGRILLPSAASVCPIPFEACHLCQAMTLRCPNTQGCCSSWA S, or (vi) TNQALPKIEVICRGTPRCPSTVPPSPAQPYLRVSLPEDRYTQAWAPTSRTPWGAMVPR GVSMAHKVATPGSQTIMPCPMPTTPVQAWLEA; (e) 62M. wherein C2 is (i) KKWSIQTCLSARTGLSISC'ITLNSPPLKKMSMPAV, or (ii) LCSRYSLFLAWRLSSVLQRFRFTI—IVIQQRMESQISz (d) CDHl, wherein CA is (i) RSACVTVKGPLASVGRHSLSKQDCKFLPFWGFLEEFLLC, (ii) IQWGTTTAPRPIRPPFLESKQNCSHFPTPLLASEDRRETGLFLPSAAQKMKKAHFLKT WFRSNPTKTKKARFSTASLAKELTHPLLVSLLLKEKQDG. (iii) PTDPFLGLRLGLHLQKVFHQSHAEYSGAPPPPPAPSGLRFWNPSRIAHISQLLSWPQKT EERLGYSSHQLPRK, (iv) FCCSCCFFGGERWSKSPYCPQRMTPGTTFITMMKKEAEKRTRTLT, or (v) WRRNCKAPVSLRKSVQTPARSSPARPDRTRRLPSLGVPGQPWALGAAASRRCCCCC RSPLGSARSRSPATLALTPRATRSRCPGATWREAASWAE; (6) GATA3. wherein C1 is (i) THVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSM LTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH; (ii) PRPRRCTRHPACPLDH'ITPPAWSPPWVRALLDAHRAPSESPCSPFRLAFLQEQYHEA; MLLZ, wherein Cz is TRRCHCCPHLRSHPCPI-H-ILRNHPRPI-II-lLRI-Il—lACI—ll-Il-ILRNCPHPHFLRHCTCPGRWRNRPS LRRLRSLLCLPHLNHHLFLHWRSRPCLHRKSHPHLLHLRRLYPHHLKHRPCPHI-ILKNLLCP RHLRNCPLPRHLKHLACLHHLRSHPCPLHLKSHPCLHHRRHLVCSHHLKSLLCPLHLRSLPF PHI-ILRHHACPHHLRTRLCPHHLKNHLCPPHLRYRAYPPCLWCHACLHRLRNLPCPHRLRS LPRPLHLRLHASPHHLRTPPHPHHLRTHLLPHHRRTRSCPCRWRSHPCCHYLRSRNSAPGPR GRTCHPGLRSRTCPPGLRSHTYLRRLRSHTCPPSLRSHAYALCLRSHTCPPRLRDHICPLSLR RLRSRTCLLCLRSHACPPNLRNHTCPPSLRSHACPPGLRNRICPLSLRSHPCPLGLK SPLRSQANALHLRSCPCSLPLGNHPYLPCLESQPCLSLGNHLCPLCPRSCRCPHLGSHPCRLS (g) PTEN, wherein CLis (i) SWKGTNWCNDMCIFITSGQIFKGTRGPRFLWGSKDQRQKGSNYSQSEALCVLL, (ii) KRTKCFTFG, (iii) PIFIQTLLLWDFLQKDLKAYTGTILMM. (W) QKMILTKQIKTKPTDTFLQILR, (V) GFWIQSIKTITRY'HFVLKDIMTPPNLIAELHNILLKTITHHS, (vi) NYSNVQWRNLQSSVCGLPAKGEDIFLQFRTHTTGRQVHVL, or (vii) YQSRVLPQTEQDAKKGQNVSLLGKYILHTRTRGNLRKSRKWKSM; (h) TP53, whcrcin CZ is (i) SSQNARGCSPRGPCTSSSYTGGPCTSPLLAPVIFCPFPENLPGQLRFPSGLLAFWDSQV CDLHVLPCPQQDVLPTGQDLPCAAVG, (ii) GAAPTMSAAQIAMVWPLLSILSEWKEICVWSIWMTETLFDIVWWCPMSRLRLALTV FCVTVPAWAA, (iii) TGGPSSPSSHWKTPVVIYWDGTALRCVFVPVLGETGAQRKRISARKGSLTTSCPQGA LSEHCPTTPAPLPSQRRNHWMENISPFRSVGVSASRCSES, (M FHTPARHPRPRHGHLQAVTAHDGGCEALPPP, (V) CCPRTILNNGSLKTQVQMKLPECQRLLPPWPLHQQLLHRRPLHQPPPGPCHLLSLPRK PTRAATVSVWASCILGQPSL, (vi) VRKHFQTYGNYFLKTTFCPPCRPKQWMI, or (vii) LARTPLPSTRCFANWPRPALCSCGLIPHPRPAPASAPWPSTSSHST; or (i) VHL wherein CZ is (i) ELQETGHRQVALRRSGRPPKCAERPGAADTGAHCTSTDGRLKISVETYTVSSQLLMV LMSLDLDTGLVPSLVSKCLILRVK. (ii) KSDASRLSGA, (iii) RTAYFCQYHTASVYSERAMPPGCPEPSQA, (W) TRASPPRSSSAIAVRASCCPYGSTSTASRSPTQRCRLARAAASTATEVTFGSSEMQGH TMGFWLTKLNYLCHLSMLTDSLFLPISHCQCIL, (V) GDWTSSGRSTKIWKTTQMCRKTWSG, or (vi) RRRRGGVGRRGVRPGRVRPGGTGRRGGDGGRAAAARAALGELARALPGHLLQSQS ARRAARMAQLRRRAAALPNAAAWHGPPHPQLPRSPLALQRCRDTRWASG; (i) ACVRZA, wherein AXBYCZ is (i) GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKRQP, or (ii) GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKTALKYIF VAVRAICVMKSFLIFRRWKSHSPLQIQLHLSHPITFSCSIPWCHLC; (k) C 0, wherein AxByCZ is VAIAAPPSEGEANAELCRYLSKVLELRKSDVVLDKVGLALFFFFFETKSCSVAQA GVQWRSLGSLQPPPPGFKLFSCLSFLSSWDYRRMPPCLANFClFNRDGVSPCWSGWS; (1) CNOTl, wherein AxByCZ is (i) LSVIIFFFVYIWHWALPLILNNHHICLMSSIILDCNSVRQSIMSVCFFFFSVIFSTRCLTDS RYPNICWFK. or (ii) LSV]IFFFVYIWHWALPLILNNHH]CLMSSIILDCNSVRQSIMSVCFFFFCYILNTMFDR; (m) , wherein AKB,CZ or C2 is VLVLSCDLITDVALHEVVDLFRAYDASLAMLMRKGQDSIEPVPGQKGKKKQWSSVTSLE WTAQERGCSSWLMKQTWMKSWSLRDPSYRSILEYVSTRVLWMPTSTV; (n) EPHBZ, wherein AxByCZ or CZ is SIQVMRAQMNQIQSVEGQPLARRPRATGRTKRCQPRDVTKKTCNSNDGKKREWEKRKQIL GGGGKYKEYFLKRILIRKAMTVLAGDKKGLGRFMRCVQSETKAVSLQLPLGR; ESRP] wherein AxByCZ is (i) LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKKHEGQI C, or (ii) LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKT; (p) FAMl 1B, wherein AXB,CZ is GALCKDGRFRSDlGEFEWKLKEGHKKIYGKQSMVDEVSGKVLEMDISKKKHYNRKISIKK LNRMKVPLMKLITRV; (q) GBP3, wherein A.\.B),CZ is RERAQLLEEQEKTLTSKLQEQARVLKERCQGESTQLQNEIQKLQKTLKKKPRDICRJS; JAKl, wherein AXB).C,_ is (i) VNTLKEGKRLPCPPNCPDEVYQLMRKCWEFQPSNRTSFQNLIEGFEALLKTSN or (ii) CRPVTPSCKELADLMTRCMNYDPNQRPFFRAIMRDINKLEEQNPDIVSEKNQQLKWT PHILKSAS; (s) LMAN 1, wherein AXB,CZ is (i) DDHDVLSFLTFQLTEPGKEPPTPDKEISEKEKEKYQEEFEHFQQELDKKKRGIPEGPPR PPRAACGGNI, or (ii) DDHDVLSFLTFQLTEPGKEPPTPDKEISEKEKEKYQEEFEHFQQELDKKKRNSRRATP TSKGSLRRKYLRV; (0 MSH3, wherein AxByCz is (i) TKSTLIGEDVNPLIKLDDAVNVDEIMTDTSTSYLLCISENKENVRDKKKGQHFYWHC GSAACHRRGCV, or (ii) LYTKSTLIGEDVNPLIKLDDAVNVDEIMTDTSTSYLLCISENKENVRDKKRATFLLAL WECSLPQARLCLIVSRTLLLVQS; (n) NDUFCZ, wherein AxByCzis (i) LPPPKLTDPRLLYIGFLGYCSGLIDNLIRRRPIATAGLHRQLLYITAFFFCW'ILSCKT, or (ii) SLPPPKLTDPRLLYIGFLGYCSGLIDNLlRRRPlATAGLHRQLLYlTAFFLLDllL; (V) RBM27, wherein AxByCZ is NQSGGAGEDCQIFSTPGHPKMIYSSSNLKTPSKLCSGSKSHDVQEVLKKKTGSNEVTTRYE EKKTGSVRKANRMPKDVNIQVRKKQKHETRRKSKYNEDFERAWREDLTIKR; (W) RPL22, wherein AxBycZ is (i) MAPVKKLVVKGGKKKEASSEVHS, or (ii) LVVKGGKKRSKF; (X) SEC31A, wherein AxByCZ is (i) MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKN, or (ii) MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKLMLLRL NLRKMCGPF; SEC63, wherein AXB,.CZis (i) AEVFEKEQSICAAEEQPAEDGQGETNKNRTKGGWQQKSKGPKKTAKSKKKETFKKK TYTCAITTVKATETKAGKWSRWE, or (ii) MAEVFEKEQSICAAEEQPAEDGQGETNKNRTKGGWQQKSKGPKKTAKSKKRNL; (Z) 5, wherein AxByCz is NIMEIRQLPSSHALEAKLSRMSYPVKEQESILKTVGKLTATQVAKISFFFALCGFWQICHIKK HFQTHKLL; (aa) SMAPl, wherein AxByCzis (i) YEKKKYYDKNAlAlTNlSSSDAPLQPLVSSPSLQAAVDKNKLEKEKEKKKGREKERK GARKAGKTTYS, or (ii) KYEKKKYYDKNAIAITNISSSDAPLQPLVSSPSLQAAVDKNKLEKEKEKKRKRKREK RSQKSRQNHLQLKSCRRKISNWSLKKVPALKKLRSPLWIF; (bb) TFAM, wherein AXB)CZ is (i) IYQDAYRAEWQVYKEEISRFKEQLTPSQIMSLEKEIMDKHLKRKAMTKKKRVNTAW KTKKTSFSL, or (ii) IYQDAYRAEWQVYKEEISRFKEQLTPSQIMSLEKEIMDKHLKRKAMTKKKS: (CC) TGFBRZ, wherein AxByCZ is (i) VAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKAW. or (ii) EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKSLVRLSS CVPVALMSAMTTSSSQKNITPAILTCC; (dd) THAPS, wherein AxByCZ is VPSKYQFLCSDHFTPDSLDIRWGIRYLKQTAVPTIFSLPEDNQGKDPSKKNPRRKTWKMRK KYAQKPSQKNHLY; (ee) TTK, wherein AXB).CZ is GTI‘EEMKYVLGQLVGLNSPNSILKAAKTLYEHYSGGESHNSSSSKTFEKKGEKNDLQLFV MSD'ITYKIYWTVILLNPCGNLHLKTI‘SL; and (fl) XPOT, n AXB,CZ is QQLIRETLISWLQAQMLNPQPEKTFIRNKAAQVFALLFVTEYLTKWPKFFLTFSQ. An isolated neoantigenic e comprising a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native polypeptide, wherein the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: AxByCZ, wherein each A is an amino acid ponding to a first native polypeptide; each C is an amino acid corresponding to a second native polypeptide, or a cryptic exon or exon of a splice variant of the first native polypeptide, each B is an amino acid that is not an amino acid corresponding to the first native polypeptide, the second native polypeptide, or the c exon of the first native polypeptide, and x + y + z is at least 8, wherein y is absent and the at least 8 contiguous amino acids comprises at least one AK and at least one C1, or y is at least 1 and the at least 8 contiguous amino acids comprises at least one 3,. wherein: (a) the first native polypeptide is encoded by a BCR gene, the second native ptide is encoded by an ABL gene, and (i) y is 0, and AxByCZ is ENIREQQKKCFRSFSLTSVELQMLTNSCVKLQTVHSIPLTINKEEALQRPVAS DFEPQGLSEAARWNSKENLLAGPSENDPNLFVALYDFVASG. or (ii) y is l, and AxByCZ is ELQMLTNSCVKLQTVHSIPLTINKEDDESPGLYGFLNVIVHSATGFKQSSKALQRPVAS DFEPQGLSEAARWNSKENLLAGPSENDPNLFVALYDFVASGD: (b) the first native polypeptide is encoded by a C l lorf95 gene, the second native polypeptide is encoded by an RELA gene, y is l, and AXB‘VCZ is ISNSWDAHLGLGACGEAEGLGVQGAEEEEEEEEEEEEEGAGVPACPPKGPELFPLIFPAEPA QASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTD: (c) the first native polypeptide is encoded by a CBFB gene and the second native polypeptide is encoded by an MYHJ 1 gene, y is 0, and AxByCZ is LQRLDGMGCLEFDEERAQQEDALAQQAFEEARRRTREFEDRDRSHREEMEVHELEKSKRA LETQMEEMKTQLEELEDELQATEDAKLRLEVNMQALKGQF; (d) the first native polypeptide is encoded by a CD74 gene and the second native polypeptide is encoded by an ROSl gene, y is 0, and A,B,C, is KGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKAGVPNKPGIP KLLEGSKNSIQWEKAEDNGCRITYYILEIRKSTSNNLQNQ1 the first native polypeptide is encoded by an EGFR gene and the second native polypeptide is encoded by (i) an SEPT14 gene, y is 0, and AXB’VCZ is LPQPPICTIDVYMIMVKCWMlDADSRPKFRELIIEFSKMARDPQRYLVIQLQDKFEHLK MlQQEElRKLEEEKKQLEGEIIDFYKMKAASEALQTQLSTD; or (ii) an EGFR gene, y is l, and AxByCZ is AGAALLALLAALCPASRALEEKKGNYVVTDHGSCVRACGADSYEMEEDGV RKCKKCEGPCRKVCNGIGIGEFKD; (f) the first native polypeptide is encoded by a EML4 gene, the second native polypeptide is encoded by an ALK gene, y is l, and AxByCZ is SWENSDDSRNKLSKIPSTPKLIPKVTKTADKHKDVIINQAKMSTREKNSQVYRRKHQELQA SPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDL (g) the first native polypeptide is encoded by a FGFR3 gene, the second native ptide is encoded by an TACC3 gene, y is 0, and AxByCZ is KPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDRVLTVTSTDVKATQEENR ELRSRCEELHGKNLELGKIMDRFEEVVYQAMEEVQKQKELS, (h) the first native polypeptide is encoded by a NAB gene, the second native polypeptide is encoded by an STAT6 gene, y is at least 1, and ARB,CL is RDNTLLLRRVELFSLSRQVARESTYLSSLKGSRLHPEELGGPPLKKLKQEATSKSQlMSLWG LVSKMPPEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCC; the second native polypeptide is encoded by an ERG. y is 0. and (i) the first native polypeptide is encoded by a NDRGl gene, and AXB)CZ is MSREMQDVDLAEVKPLVEKGETITGLLQEFDVQEALSVVSEDQSLFECAYGTPHLAK TEMTASSSSDYGQTSKMSPRVPQQDW or (ii) the first native polypeptide is d by a TMPRSSZ gene, and AxByCZ is MALNSEALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDW; (j) the first native polypeptide is encoded by a PML gene, the second native polypeptide is encoded by an RARA gene, y is 1, and AxByCz is (i) VLDMHGFLRQALCRLRQEEPQSLQAAVRTDGFDEFKVRLQDLSSCITQGKAIETQSSS SEEIVPSPPSPPPLPRIYKPCFVCQDKSSGYHYGVSACEGCKG, or (ii) RSSPEQPRPSTSKAVSPPHLDGPPSPRSPVIGSEVFLPNSNHVASGAGEAAIETQSSSSEE IVPSPPSPPPLPRIYKPCFVCQDKSSGYHYGVSACEGCKG; (k) the first native polypeptide is encoded by a RUNXl gene, the second native polypeptide is encoded by an CBFAZTl (RUNXlTl) gene, y is l, and A,‘B,.Cz is VARFNDLRFVGRSGRGKSFTLTITVFTNPPQVATYHRAIKITVDGPREPRNRTEKHSTMPDS PVDVKTQSRLTPPTMPPPPTFQGAPRTSSFTPTTLTNGT; (l) the first native polypeptide is encoded by a AR—v7 gene, the cryptic exon or the exon of a splice variant is encoded by the AR-v7 gene. y is 0. and AXB,C2 is SCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGEKFRVGNCKH LKMTRP. 'JI An isolated neoantigenic peptide comprising a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native ptide, wherein the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: ANByC, wherein each A is an amino acid corresponding to a first native polypeptide; each B is an amino acid that is not an amino acid ponding to the first native polypeptide or the second native ptide, each C is an amino acid encoded by a frameshifi of a sequence encoding a second native polypeptide; x + y + z is at least 8, wherein y is absent and the at least 8 contiguous amino acids comprises at least one C7,. or y is at least 1 and the at least 8 uous amino acids comprises at least one By and/or at least one Cl; and (a) the first native ptide is encoded by an AC011997.1 gene, the second native polypeptide is encoded by a LRRC69 gene, y is l, and AxB,CZ is MAGAPPPASLPPCSLlSDCCASNQRDSVGVGPSEPGNNIKICNESASRK (b) the first native polypeptide is d by an EEFIDPB gene. the second native polypeptide is encoded by a FRY gene, y is l, and AXB_\CZ is HGWRPFLPVRARSRWNRRLDVTVANGRSWKYGWSLLRVPQVNGIQVLNVSLKSSSNVIS (e) the first native polypeptide is encoded by a MADlLl gene, the second native polypeptide is encoded by a MAFK gene, y is 0, and AxByCZ is RLKEVFQTKIQEFRKACYTLTGYQIDITTENQYRLTSLYAEHPGDCLIFKLRVPGSSVLVTVP GL, or (d) the first native polypeptide is encoded by a PPPl R] B gene, the second native polypeptide is encoded by a STARD3 gene, y is l, and AxByCZ is AEVLKVIRQSAGQKTTCGQGLEGPWERPPPLDESERDGGSEDQVEDPALSALLLRPRPPRPE VGAHQDEQAAQGADPRLGAQPACRGLPGLLTVPQPEPLLAPPSAA. The isolated neoantigenic peptide of any one of claims 1-5, wherein the isolated neoantigenic peptide comprises a sequence according to Table l. The isolated neoantigenic peptide of any one of claims 1-6, n x + y + z is at most 500, at most 250, at most 150, at most 125, or at most 100 The isolated neoantigenic peptide of any one of claims 1-7, wherein x + y + z is at least 8, at least 50, at least 100. at least 200. or at least 300. The isolated neoantigenic peptide of any one of claims 1-8, wherein z is at most 500, at most 250, at most 150, at most 125, or at most 100. 10. The isolated neoantigenic peptide of any one of claims 1-9, wherein z is at least 8, at least 50, at least 100, at least 200, or at least 300. ll. The isolated neoantigenic peptide of any one of claims 1-10, wherein the isolated neoantigenic e is from about 8 to about 500 amino acids in . 12. The isolated neoantigenic peptide of claim 1 1, n the isolated neoantigenic peptide is from about 8 to about 100 amino acids in length. l3. The isolated neoantigenic peptide of claim 12, wherein the isolated neoantigenic peptide is from about 8 to about 50 amino acids in . 14. The isolated neoantigenic peptide of claim 13, wherein the isolated neoantigenic peptide is from about 15 to about 35 amino acids in . . The ed neoantigenic peptide of claim 13. n the isolated neoantigenic peptide is from about 8 and about 15 amino acids in . 16. The ed neoantigenic peptide of claim 13, wherein the isolated neoantigenic peptide is from about 8 and about 11 amino acids in length. 17. The isolated neoantigenic peptide of claim 13, wherein the isolated neoantigenic peptide is 9 or 10 amino acids in length 18. The ed neoantigenic peptide of any one of claims 1-17. wherein the isolated neoantigenic peptide binds major histocompatibility complex (MHC) class I. 19. The isolated neoantigenic peptide of claim 18, wherein the isolated neoantigenic peptide binds MHC class I with a binding afilnity of about 500 nM or less. 20. The isolated neoantigenic peptide of claim 19, n the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 250 nM or less. 21. The isolated neoantigenic peptide of claim 19. wherein the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 50 nM or less. 22. The isolated neoantigenic peptide of any one of claims 1-2], wherein the isolated neoantigenic peptide is from about 8 and about 30 amino acids in length. 23. The isolated iféOahtigemg peptide of claim 22, wherein the isolated neoantigenic peptide is from about 8 to about 25 amino acids in length. . The isolated neoantigenic peptide of claim 23, wherein the isolated neoantigenic peptide is from about 15 to about 24 amino acids in length. . The isolated igenic peptide of claim 23, n the isolated neoantigenic peptide is from about 9 to about 15 amino acids in . . The isolated neoantigenic peptide of any one of claims l-l7 and 22-25, wherein the isolated neoantigenic peptide binds MHC class II. 27. The isolated neoantigenic peptide of claim 26, wherein the isolated neoantigenic peptide binds MHC class II with a g affinity of 1000 nM or less. 28. The isolated neoantigenic peptide of claim 27, wherein the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 500 nM or less. 29. The isolated neoantigenic peptide of any one of claims 1-28, wherein the isolated igenic peptide further comprises flanking amino acids. 30. The isolated igenic peptide of claim 29, wherein the flanking amino acids are not native flanking amino acids 31. The isolated igenic peptide of any one of claims 1-30, wherein the isolated neoantigenic e has a total length of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70. at least 80, at least 90, at least 100. at least 150, at least 200. at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids. 32. The ed neoantigenic peptide of any one of claims 1-31, wherein the isolated neoantigenic peptide has a total length of at most 8, at most 9, at most 10, at most 1 l, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40. at most 50. at most 60. at most 70. at most 80, at most 90, at most 100. at most 150. at most 200. at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids. 33. The ed neoantigenic peptide of any one of claims 1-32, wherein the isolated neoantigenic peptide is a first neoantigenic peptide linked to at least a second neoantigenic peptide. 34. The isolated neoantigenic peptide of claim 33, wherein the isolated neoantigenic peptide is linked to the at least second igenic peptide by a poly-glycine or poly-serine linker. . The isolated neoantigenic peptide of claim 33 or 34, wherein the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 1000 nM. 36. The isolated neoantigenic peptide of claim 35, wherein the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 500 nM. 37. The isolated neoantigenic peptide of claim 35 or 36, wherein ed neoantigenic peptide and the second neoantigenic peptide bind to human yte antigen (HLA) -A. -B. -C. -DP. -DQ. or -DR. 38. The isolated neoantigenic peptide of any one of claims 35-37, n the isolated neoantigenic peptide binds a class I HLA and the second neoantigenic peptide binds a class II HLA. 39. The isolated neoantigenic peptide of any one of claims 35-37, wherein the isolated igenic peptide binds a class II HLA and the second neoantigenic peptide binds a class I HLA. 40. The isolated neoantigenic peptide of any one of claims 35-37, wherein the isolated neoantigenic peptide further comprises a modification which increases in viva half-life. cellular ing. antigen uptake, antigen processing, MHC affinity, MHC stability, antigen presentation, or a ation thereof. 41. The isolated neoantigenic peptide of claim 40, wherein the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation HESylation, recombinant PEG mimetics, Fc fusion, albumin fiision, nanoparticlc attachment, nanoparticulatc encapsulation. terol fusion, iron fusion, acylation. ion. ylation. side chain oxidation, phosphorylation, biotinylation, the addition of a surface active material, the addition of amino acid mimetics, or the addition of unnatural amino acids. 42. The ed neoantigenic peptide of claim 40 or 41, wherein the isolated neoantigenic peptide further comprises a modification which increases cellular targeting to antigen ting cells. 43. The isolated neoantigenic peptide of claim 42, wherein the antigen presenting cells are dendritic cells. 44. The isolated neoantigenic peptide of claim 43, wherein the dendritic cells are ed using DEC205, XCRl. CD197, CD80. CD86. CD123. CD209, CD273. CD283. CD289, CD184, CD85h, CD85j, CD85k, CDSSd, CD85g, CD85a, CD141, CD1 1c, CD83, TSLP receptor, Clec9a, or CDla marker. .. . The isolated neoantigenic peptide of claim 44, wherein the dendritic cells are targeted using the CD141, DEC205, Clec9a, or XCRl marker. 46. The isolated neoantigenic e of any one of claims 42-45, wherein the dendritic cells are autologous cells. 47. The isolated neoantigenic e of any one of claims 42-46, wherein one or more of the dendritic cells are bound to a T cell. 48. The isolated neoantigenic peptide of claim 47, wherein the T cell is an autologous T cell. 49. The isolated neoantigenic peptide of any one of claims 1-49, wherein the isolated neoantigenic peptide is not 21 isolated neoantigenic peptide listed in Table 2. U. 0 . The isolated neoantigenic peptide of any one of claims 1-48, wherein the isolated neoantigenic peptide is linked to at least one onal neoantigenic peptide listed in Table l or 2. UI .— . An in vivo delivery system comprising the isolated neoantigenic peptide of any of claims 1-50. LII [\J . The delivery system of claim 51, wherein the delivery system includes cell-penetrating peptides, nanoparticulate encapsulation, virus like les, liposomes, or any combination thereof. . The delivery system of claim 52, wherein the cell-penetrating peptide is TAT peptide, herpes simplex virus VP22. transportan. Antp. or any combination thereof. Ul 4:- . A cell comprising the isolated neoantigenic peptide of any of claims 1-50. LII IJ.. . The cell of claim 54, wherein the cell is an antigen presenting cell. UI O\ The cell of claim 55, wherein the cell is a dendritic cell. 'JI \l . The cell of any one of claims 54-56, wherein the cell is an autologous cell. 'Jl 00 . The cell of any one of claims 54-5 7, wherein the cell is bound to a T cell. . The cell of claim 58. wherein the T cell is an autologous T cell. 60. A ition comprising the isolated neoantigenic peptide of any of claims 1-50. 61. The composition of claim 60, wherein the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70. at least 80, at least 90, or at least 100 of the isolated neoantigenic peptides comprising a tumor-specific neoepitope according to Table l or 2. 62. The composition of claim 61, wherein the composition comprises from about 2 to about 20 igenic peptides, or from about 2 to about 30 neoantigenic peptides. 63. The composition of claim 61 or 62, wherein the neoantigen is specific for an individual subject's tumor. 64. The composition of any one of claims 60-63, wherein the composition fiirther ses at least 1, at least 2. at least 3. at least 4, at least 5. at least 6. at least 7, at least 8. at least 9, at least 10, at least 1 1. at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 additional igenic peptides. . The composition of claim 64, wherein the composition comprises from about 4 to about 20 additional neoantigenic es. from about 4 to about 30 additional neoantigenic peptides. 66. The composition of any of claims 60-65, wherein at least onof the additional neoantigenic peptides is specific for an individual t's tumor. 67. The composition of claim 66, wherein the subject specific neoantigenic peptide is selected by identifying ce differences between the genome, exome, and/or transcriptome of the subject's tumor sample and the genome, cxomc, and/or transcriptome of a non-tumor sample. 68. The ition of claim 67. wherein the samples are fresh or fomialin-fixed paraffin embedded tumor tissues, y isolated cells, or circulating tumor cells. 69. The composition of claim 67 or 68, wherein the sequence differences are detemiined by Next Generation Sequencing. 70. An ed polynucleotide encoding the isolated igenic peptide of any one of claims 1-50. 71. The isolated polynucleotide of claim 70, wherein the polynucleotide is DNA. 72. The isolated polynucleotide of claim 70. wherein the polynucleotide is RNA. 73. The isolated polynucleotide of claim 72, wherein the RNA is a self-amplifying RNA. 74. The isolated polynucleotide of claim 72 or 73, wherein the RNA is modified to increase stability, increase cellular ing, increase translation efficiency, adiuvanticiw, cytosol accessibility, and/or decrease cytotoxicity. . The isolated polynucleotidc of claim 74, n the modification is conjugation to a carrier protein, conjugation to a ligand. conjugation to an antibody. codon optimization. increased GC-content. oration ofmodified nucleosides, incorporation of 5'-cap or cap analog, and/or incorporation of an unmasked poly-A sequence. 76. A cell sing the polynucleotide of any of claims 70-75. 77. A vector comprising the polynucleotide of any one of claims 70—75. 78. The vector of claim 77, wherein the polynucleotide is operably linked to a promoter. 79. The vector of claim 77 or 78. wherein the vector is a self-amplifying RNA replicon, d. phage. transposon, cosmid, virus, or virion. 80. The vector of claim 79, wherein the vector is derived from an adeno-associated virus, herpesvirus, lentivirus, or a pseudotype thereof. 81. An in vivo delivery system comprising the isolated polynucleotide of any of claims 70-75. 82. The delivery system of claim 81, n the delivery system includes spherical nucleic acids, viruses, virus-like particles, plasmids, bacterial plasmids, or nanoparticles. 83. A cell comprising the vector or ry system of any of claims 77-82. 84. The cell of claim 83, wherein the cell is an antigen presenting cell. .. . The cell ofclaim 84, wherein the cell is a tic cell. 86. The cell of claim 85, wherein the cell is an immature dendritic cell. 87. A composition comprising at least one polynucleotide of any of claims 70-75. 88. The composition of claim 87, wherein the composition comprises at least 2, at least 3, at least 4, at least 5. at least 6. at least 7. at least 8. at least 9. at least 10. at least 1 l. at least 12. at least 13. at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 of the isolated cleotides. 89. The composition of claim 88, n the composition comprises from about 2 and about 20 of the isolated polynucleotidcs, or from about 2 to about 30 of the isolated polynucleotides. 90. The composition of any one of claims 87—89, wherein the neoantigenic peptides are encoded by a vector comprising one or more of the the isolated cleotides. 91. The composition of any one of claims 87-90, wherein the composition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 l, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30. at least 40, at least 50. at least 60. at least 70. at least 80. at least 90. or at least 100 additional neoantigenic polynucleotides encoding for additional neoantigenic es. 92. The composition of any one of claims 88-91, wherein one or more of the additional neoantigenic peptides are encoded by a vector comprising one or more of the additional neoantigenic polynucleotides. 93. The composition of claim 91 or 92, wherein the composition comprises from about 4 to about 20 additional neoantigenic polynucleotides. or from about 4 to about 30 additional neoantigenic polynucleotides. . The ition of claim 91 or 92, wherein the isolated polynucleotides and the additional neoantigenic poly-nucleotides are linked. . The composition of claim 94, wherein the polynucleotides are linked using nucleic acids that encode a poly-glycine or poly-scrinc linker. . The ition of any of claims 87-95, wherein at least one of the additional neoantigenic peptide is ic for an individual t 's tumor. 97. The composition of claim 96, wherein the subject specific igenic peptide is selected by identifying sequence differences between the genome, exome, and/or transcriptome of the t's tumor sample and the genome, exome, and/or transcriptome of a non-tumor sample. 98. The composition of claim 97, wherein the samples are fresh or fomialin-fixed paraffin embedded tumor tissues, y isolated cells, or circulating tumor cells. 99. The composition of claims 97 or 98. wherein the sequence differences are determined by Next Generation Sequencing. 100 . A T cell receptor (TCR) capable of binding at least one neoantigenic e listed in any of claims 1-50 or an MHC-peptide complex comprising at least one igenic peptide listed in any of claims 1-50. 101 . The TCR of claim 100, wherein the MHC of the MHC-peptide is MHC class I or class II. 102 . The TCR of claim 100 or 101. wherein TCR is a bispecific TCR further comprising a domain comprising an antibody or antibody fragment capable of binding an antigen. 103. The TCR of claim 102, wherein the antigen is a T cell-specific n. 104. The TCR of claim 102 or 103, wherein the antigen is CD3. 105 . The TCR of claim 104, wherein the antibody or antibody fragment is an anti-CD3 scFv. 106 . A chimeric antigen receptor comprising: (i) a T cell activation molecule; (ii) a transmembrane region; and (iii) an antigen recognition moiety e of g at least one neoantigenic peptide listed in any of claims 1-50 or an MHC-peptide complex comprising at least one neoantigenic peptide listed in any of claims 1-50. 107. The chimeric antigen receptor of claim 106, wherein CD3-zeta is the T cell activation molecule. 108. The chimeric antigen receptor of claim 106 or 107, further comprising at least one costimulatory signaling . 109. The chimeric antigen receptor of any of claims 106-108, wherein the signaling domain is CD28, 4- 138, ICOS, 0X40, ITAM, or Fc n RI-gamma. 110. The chimeric antigen receptor of any of claims 106-109, wherein the n recognition moiety is capable of binding the isolated neoantigenic peptide in the context of MHC class I or class 11. 111. The chimeric antigen or of any of claims 106-110, comprising the CD3-zeta, CD28, CTLA-4, ICOS. BTLA. KIR. LAG3. CD137. 0X40. CD27. CD40L. Tim-3. AZaK or PD-l transmembrane region. 112. The chimeric antigen receptor of any of claims 106-111, wherein the neoantigenic peptide is located in the extracellular domain of a tumor associated polypeptide. 113. The chimeric antigen receptor of any of claims 106-112, wherein the MHC of the MHC-peptide is MHC class 1 or class II. . A T cell comprising the T cell receptor or chimeric antigen receptor of any of claims 100-1 13, optionally wherein the T cell is a helper or cytotoxic T cell. . The T cell of claim I 14, wherein the T cell is a T cell ofa t. A T cell comprising a T cell receptor (TCR) capable of binding at least one neoantigenic peptide of any of claims 1-50 or an MHC-peptide complex sing at least one neoantigenic peptide of any of claims [-50, wherein the T cell is a T cell isolated from a population of T cells from a t that has been incubated with antigen presenting cells and one or more of the at least one neoantigenic e of any of claims 1-50 for a sufficient time to activate the T cells. 117. The T cell of claim 1 16, wherein the T cell is a CD8+ T cell. a helper T cell or cytotoxic T cell. 118. The T cell of claim 1 16 or 117. wherein the population of T cells from a t is a population of CD8+ T cells from the subject. 119. The T cell of any one of claims 116-118, n the one or more of the at least one igenic peptide of any of claims 1-50 is a subject-specific neoantigenic peptide. 120. The T cell of any one of claims 1 16-119. wherein the t-specific neoantigenic peptide has a ent tumor neo—epitope that is an e specific to a tumor of the subject. 121. The T cell of any one of claims 116-120. wherein the subject-specific neoantigenic peptide is an expression product of a tumor-specific non-silent mutation that is not present in a non-tumor sample of the subject. 122. The T cell of any one of claims 1 16-121. wherein the subject-specific neoantigenic peptide binds to a HLA protein of the subject. 123. The T cell of any one of claims 116-122, wherein the subject—specific neoantigenic peptide binds to a HLA protein of the subject with an IC50 less than 500 nM. 124. The T cell of any one of claims 1 16-123, wherein the activated CD8+ T cells are separated from the antigen presenting cells. 125. The T cell of any one of claims 1 16-124, wherein the antigen presenting cells are dendritic cells or CD40L-expanded B cells. 126. The T cell of any one of claims 116-125. wherein the antigen presenting cells are non-transformed cells. 127. The T cell of any one of claims 116-126, wherein the antigen presenting cells are non-infected cells. 128. The T cell of any one of claims 116-127. wherein the antigen presenting cells are autologous. 129. The T cell of any one of claims 1 . wherein the antigen presenting cells have been treated to strip endogenous sociated peptides from their surface. 130. The T cell of claim 129, wherein the treatment to strip the endogenous MHC-associated peptides ses culturing the cells at about 26°C. 131. The T cell of claim 129, wherein the treatment to strip the endogenous MHC-associated peptides comprises treating the cells with a mild acid solution. 132. The T cell of any one of claims 116-131. wherein the antigen presenting cells have been pulsed with the at least one neoantigenic peptide of any of claims 1-50. 133. The T cell of claim 132, wherein pulsing comprises incubating the n presenting cells in the presence of at least about 2 ug/ml of each of the at least one neoantigenic peptide of any of claims 1- 134. The T cell of any one of claims 3. n ratio of isolated T cells to antigen presenting cells is between about 30:1 and 300:1. 135. The T cell of any one of claims 116-134. wherein the incubating the isolated population of T cells is in the presence of lL-2 and lL-7. 136. The T cell of any one of claims 116-135, wherein the MHC of the MHC-peptide is MHC class I or class II. 137. A method for activating tumor c T cells sing: (a) isolating a tion of T cells from a subject; and (b) incubating the isolated population of T cells with antigen presenting cells and at least one neoantigenic peptide of any of claims l-50 for a sufficient time to activate the T cells. 138. The method of claim 137, wherein the T cell is a CD8+ T cell, a helper T cell or cflotoxic T cell. 139. The method of claim 137 or 138, n the population of T cells from a subject is a population of CD8+ T cells from the t. 140. The method of any one of claims 137-139, wherein the one or more of the at least one neoantigenic peptide of any of claitns 1-50 is a subject-specific neoantigenic peptide. 141. The method of any one of claims 137-140, wherein the subject-specific neoantigenic peptide has a different tumor nee-epitope that is an epitope specific to a tumor of the subject. 142. The method of any one of claims 137-141, wherein the subject-specific neoantigenic peptide is an expression product of a tumor-specific non-silent mutation that is not t in a non-tumor sample of the subject. 2 l 0 143. The method of any one of claims 137- 142, wherein the subject-specific igenic peptide binds to a l-ILA protein of the subject. 144. The method of any one of claims 137-143, wherein the subject-specific neoantigenic peptide binds to a HLA protein of the subject with an leu less than 500 nM. 145. The method of any one of claims 137-144, wherein the method further comprises separating the activated T cells from the antigen presenting cells. 146. The method of any one of claims 137-145, wherein the method further comprises testing the ted T cells for evidence of reactivity against at least one of igenic peptide of any of claims 1-50. 147. The method of any one of claims 137-146, wherein the antigen presenting cells are dendritic cells or expanded B cells. 148. The method of any one of claims 137-147, wherein the antigen presenting cells are non-transfonned cells. 149. The method of any one of claims 137-148. wherein the antigen presenting cells are non-infected cells. 150. The method of any one of claims 137-149, wherein the antigen presenting cells are autologous. 151. The method of any one of claims 137-150, wherein the antigen presenting cells have been treated to strip endogenous MHC-associated peptides from their e. 152. The method of claim 151, wherein the treatment to strip the endogenous MI-lC-associated peptides comprises culturing the cells at about 26°C. 153. The method of claim 151. wherein the treatment to strip the endogenous MHC-associated peptides comprises ng the cells with a mild acid solution. 154. The method of any one of claims 137-153, wherein the antigen presenting cells have been pulsed with the at least one igenic peptide of any of claims 1-50. . The method of claim 154, wherein pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 ug/ml of each of the at least one neoantigenic peptide of any of claims 1- 156. The method of any one of claims 137-155, wherein ratio of isolated T cells to antigen presenting cells is between about 30:1 and 300:1. 157. The method of any one of claims 137-156, wherein the ting the isolated population of T cells is in the presence of IL-2 and IL-7. 158. The method of any one of claims 137-157. wherein the MHC of the MHC-peptide is MHC class I or class II. 159. A ition sing activated tumor specific T cells produced by the method of any one of claims 137-158. 160. A method oftreating cancer in a subject comprising administering to the subject a eutically effective amount of activated tumor Specific T cell of any one of claims 1 16-136. or produced by the method ofany one of claims 137-158. 161. The method of claim 160, wherein the administering comprises administering from about 10" to 10”, from about 108 to 10”, or from about 109 to 10'0 of the activated tumor specific T cells. 162. A nucleic acid comprising a promoter operably linked to a polynucleotide encoding the T cell receptor of any one of claims 1 14-161. 163. The nucleic acid of claim 162. wherein the TCR is capable of binding the at least one neoantigenic peptide in the context of major histocompatibility complex (MHC) class I or class II. 164. A nucleic acid comprising a promoter operably linked to a polynucleotide encoding the chimeric antigen receptor of any one of claims 106-113. 165. The nucleic acid of claim 164, wherein the antigen recognition moiety is capable of binding the at least one igenic peptide in the context of major histocompatibility complex (MHC) class 1 or class II. 166. The nucleic acid of claim 164, wherein the neoantigenic peptide is located in the extracellular domain of a tumor associated polypeptide. 167. The nucleic acid of any of claims 6, comprising the CD3-zeta, CD28, , ICOS, BTLA, KIR, LAG3, CD137, 0X40, CD27, CD40L, Tim-3, AZaK or PD-l transmembrane region. 168. An antibody or antibody nt capable of binding at least one neoantigenic peptide of any of claims 1-50 or an MHC-peptide complex comprising at least one neoantigenic peptide of any of claims 1-50, ally wherein the antibody fragment is a bi-specific T cell engager (BiTE). 169. The antibody or antibody fragment of claim 168, wherein the antibody or dy fragment binds to an ellular portion of the at least one igenic peptide, 170. The dy or antibody fragment of claim 168 or 169, wherein (a) the native polypeptide is encoded by a [32M gene, wherein CZ is (i) RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKMSMPAV, or (ii) LCSRYSLFLAWRLSSVLQRFRFTHVIQQRMESQIS; (b) the native polypeptide is d by a EGFR gene, wherein AxByCZ is IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLL DYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; (c) the native polypeptide is encoded by a BTK gene, wherein AxByCZ is MIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGSLLNYLREM RHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND; or (d) the native polypeptide is encoded by an ESRI gene, wherein AxByCz is (i) GLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLL EMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEA, (ii) NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSlILLNSGVYTFLPSTLKSL EEKDHII-lRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH. (iii) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLCDLLL EMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, (iv) lHLMAKAGLTLQQQHQRLAQLLLlLSHlRHMSNKGMEHLYSMKCKNVVPLNDLLL EMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, or (v) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLL EMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE; and (e) the native polypeptide is encoded by an HERZ gene, wherein AxByCZ is GSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGLGSPYVSRL LGlCLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCM; (f) the native polypeptide is d by a GATA3 gene, wherein C2 is (i) PGRPLQTHVLPEPHLALQPLQPHADl-IAHADAPAIQPVLWTTPPLQHGHRHGLEPCS MLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSN H, or (ii) PRPRRCTRHPACPLDHTTPPAWSPPWVRALLDAHRAPSESPCSPFRLAFLQEQYHE A; or (g) the first native polypeptide is d by a TMPRSSZ gene, the second native ptide is encoded by an ERG, y is 0, and AKBYCZ is ALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDW. 171. The antibody or antibody fragment of any one of claims 0. wherein the antibody or antibody fragment is a bispecific antibody or antibody fragment. 172. The antibody or dy nt of claim 171, wherein one antigen binding domain of the bispecific antibody or antibody fragment is an anti-CD3 binding domain. 173. A modified cell transfected or transduced with the nucleic acid of any one of claims 2. 174. The modified cell of claim 173, wherein the modified cell is a T cell, tumor infiltrating lymphocyte, NK-T cell. TCR-expressing cell. CD4+ T cell. CD8+ T cell. or NK cell. 175. A composition comprising the T cell receptor or chimeric antigen receptor of any of claims 100-] 13. 176. A composition comprising autologous subject T cells containing the T cell receptor or chimeric antigen receptor of any of claims 100-1 13. 177. The composition of claim 175 or 176, wherein the composition r comprises an immune checkpoint inhibitor. 178. The composition of claim 177, wherein the composition further comprises at least two immune checkpoint inhibitors. 179. The composition of claim 177 or 178, wherein each of the immune checkpoint inhibitors inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDLl, PDLZ, PDl, B7-H3, B7- H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 234, CD160, CGEN-15049, CHK l, Cl-IK2. AZaR. and B-7 famin ligands or a combination thereof. 180. The composition of claim 177 or 178, wherein each of the immune checkpoint inhibitors interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDLl, PDLZ, PDl, B7-H3, B7-H4, BTLA, HVEM, T1M3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN- 15049, CHK l, CHKZ, A2aR, and B-7 family ligands or a combination thereof. 181. The composition of any one of claims 176-180, whcrcin the T cells are PD-l and/or CTLA4 knockout T cells. ally.wherein the PD-l and/or CTLA4 knockout T cells are created using a CRISPR system. 182. The composition of any one of the ing claims, wherein the ition further ses an immune modulator or adjuvant. 183. The composition of claim 182, wherein the immune modulator is a co—stimulatory ligand, a TNF ligand, an lg supcrfamily , CD28, CD80, CD86, ICOS, CD40L, 0X40, CD27, GITR, CD30, DR3, CD69, or 4-1BB. . The composition of claim 182, wherein the immune modulator is at least one cancer cell or cancer cell extract. . The composition of claim 184, wherein the cancer cell is autologous to the subject in need of the composition. 186. The composition of claim 185, wherein the cancer cell has undergone lysis or been exposed to UV radiation. 187. The composition of claim 182, wherein the ition further comprises an nt. 188. The composition of claim 187, wherein the adjuvant is selected from the group consisting of: Poly(l:C), Poly-ICLC, STING agonist, 1018 ISS, aluminium salts, Amplivax, AS 15, BCG, CP- 870,893, CpG7909, CyaA, dSLlM, GM-CSF, 1C30, 1C31, lmiquimod, lmuFact , 18 Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide [SA 51 VG, OK-432, OM-174, OM-l97-MP-EC. RZ agonist. ONTAK. PepTell’R'Ti. vector system. PLG articles. resiquimod, , virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta- glucan, Pam3Cys, Pam3CSK4, acrylic or methacrylic rs, copolymers of maleic anhydride, and QS21 stimulon. 189. The composition of claim 187 or 188, wherein the composition induces a humoral response when administered to a subjcct. 190. The composition of claim 189, wherein the composition induces a T helper cell type 1 when administered to a subject. 191. A method of inhibiting growth of a tumor cell sing a tumor-specific neoepitope, comprising contacting the tumor cell with the peptide, polynucleotide, delivery system, vector, composition, antibody, or cells of any of claims 1- 190. 192. A method of laxis of a subject, comprising contacting a cell of the subject with the peptide, poly-nucleotide. delivery system. vector. composition. antibody. or cells of any of claims 1-190. 193. The method of claim 192, wherein (a) the native polypeptide is encoded by a [52M gene. wherein CZ is (i) RMERELKKWSlQTCLSARTGLSlSCTI‘LNSPPLKKMSMPAV, or (ii) LCSRYSLFLAWRLSSVLQRFRFTHVIQQRMESQIS; (b) the native polypeptide is encoded by a EGFR gene, wherein AxByCz is IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLL DYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; (C) the native polypeptide is d by a BTK gene, wherein AXB'VCZ is MIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGSLLNYLREM RHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND; or (d) the native ptide is encoded by an ESRI gene, wherein AxByCZ is (i) HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLL EMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEA, (ii) NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLPSTLKSL EEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH, (iii) AGLTLQQQHQRLAQLLLILSI-IIRHMSNKGMEHLYSMKCKNVVPLCDLLL EMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE. (iv) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLNDLLL EMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE. or (v) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLL EMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE; and the native polypeptide is encoded by an HERZ gene, wherein L is GSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGLGSPYVSRL LGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMz (0 the native polypeptide is encoded by a GATA3 gene. wherein CZ is (i) PGRPLQTHVLPEPHLALQPLQPHADl-IAHADAPAIQPVLWTTPPLQHGHRHGLEPCS MLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSN H, or (ii) PRPRRCTRHPACPLDHTTPPAWSPPWVRALLDAHRAPSESPCSPFRLAFLQEQYHE A; or (g) the first native polypeptide is encoded by a TMPRSSZ gene. the second native polypeptide is encoded by an ERG, y is 0, and AxByCZ is MALNSEALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDW. 194. A method oftreating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof comprising stering to the subject the peptide. polynucleotide, vector. composition, antibody, or cells of any of claims 1-190. 195. The method of any one of claims 191-194, wherein the subject is a human. 196. The method of claim 195, wherein the subject has cancer. 197. The method of claim 196, wherein the cancer is selected from the group consisting of urogenital, logical, lung, gastrointestinal, head and neck cancer, malignant glioblastoma, malignant mesothelioma, non—metastatic or metastatic breast cancer. malignant melanoma. Merkel Cell Carcinoma or bone and soft tissue sarcomas, haematologic neoplasias, le myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastie syndrome and acute lymphoblastic leukemia, non-small cell lung cancer (N SCLC), breast cancer, metastatic colorectal s, hormone sensitive or hormone refractory prostate cancer, colorectal cancer, ovarian cancer, ccllular cancer, renal cell cancer. pancreatic cancer, gastric cancer, ocsophagcal cancers, hepatocellular cancers. eholangioeellular cancers, head and neck squamous cell cancer soft tissue sarcoma, and small cell lung . 198. The method of any one of claims 191-197, wherein the peptide, polynucleotide, vector, composition, antibody, or cells of any of claims 1- 190 is for use in treating a corresponding cancer according to Table l or Table 2. 199. The method of any one of claims 191-198, wherein the peptide, cleotide, vector, composition, antibody, or cells of any of claims 1-190 is for use in treating a subject with an HLA type that is a corresponding I-ILA type according to Table 1 or Table 2. 200. The method of any of claims 191- 199, n the subject has one surgical removal of the tumor. 201. The method of any one of claims 191-200, wherein the peptide, polynucleotide, vector, composition, or cells is administered via intravenous, intraperitoneal, intratumoral, intradermal, or aneous administration. 202. The method of claim 201. wherein the peptide. poly-nucleotide. vector. composition. or cells is administered into an anatomic site that drains into a lymph node basin. 203. The method of claim 202, wherein administration is into multiple lymph node basins. 204. The method of any one of claims 3, wherein administration is by a subcutaneous or intradennal route. 205. The method of claim 201, n peptide is administered. 206. The method of claim 205, n administration is intratumorally. 207. The method of claim 201, wherein polynucleotide, optionally RNA, is stered. 208. The method of claim 20] or 207, wherein the polynucleotide is administered intravenously. 209. The method of any claim 201, wherein the cell is a T cell or dendritic cell. 210. The method of claim 201 or 209, wherein the peptide or polynucleotide comprises an antigen ting cell targeting moiety. 211. .The method ofclaim 201 or 209 or 210, n the cell is an autologous cell. 212. The method of any of claims 191-211, further comprising administering at least one immune checkpoint inhibitor to the subject. 213. The method of claim 212, wherein the checkpoint inhibitor is a biologic therapeutic or a small molecule. 214. The method of claim 212 or 213. wherein the checkpoint inhibitor is selected from the group consisting of a monoclonal antibody, a humanized antibody, a fully human antibody and a fusion protein or a combination thereof 215. The method of any of claims 212-214, wherein the checkpoint inhibitors is a PD-l dy or a PD- Ll antibody. 216. The method of any one of claims 212-214, wherein the checkpoint inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, avelumab. durvalumab, izumab, pembrolizumab, and any ation thereof. 217. The method of any one of claims 212-216, wherein the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA—4, PDLl, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN—15049, CHK l, CHKZ, A2aR, and B-7 family ligands, and any ation thereof. 218. The method of any of claims 212-217, n the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDLl. PDL2. PDl. B7-H3, B7- H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 284, CD160, 5049, CHK l, CHK2, A2aR, and B-7 family ligands or a combination thereof. 219. The method of any one of claims 8, wherein two or more checkpoint tors are administered. 220. The method of claim 219, wherein at least one ofthe two or more oint inhibitors is a PD-l antibody or a PD-Ll antibody. 221. The method of claim 219, wherein at least one of the two or more checkpoint inhibitors is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, avelumab, durvalumab, atezolizumab, and pembrolizumab. 222. The method of any of claims 212-221, wherein the checkpoint inhibitor and the composition are administered simultaneously or sequentially in any order. 223. The method of claim 222, wherein the peptide, polynucleotide. vector, composition, or cells is administered prior to the checkpoint tor. 224. The method of claim 222, wherein the peptide, poly-nucleotide, vector, composition, or cells is administered afier the oint inhibitor. . The method of claim 222, wherein stration of the checkpoint inhibitor is continued throughout igen peptide. polynucleotide. vector. composition. or cell therapy. . The method of any one of claims 212-225, n the neoantigen peptide, poly-nucleotide, vector, composition, or cell therapy is administered to subjects that only partially respond or do not respond to checkpoint inhibitor therapy. 227. The method of any one of claims 191-226, wherein the composition is administered intravenously or subcutaneously. 228. The method of any one of claims 212-227. n the checkpoint inhibitor is administered intravenously or subcutaneously. 229. The method of any one of claims 212-228, wherein the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration ofthe composition. 230. The method of claim 229, wherein the composition is administered into the same ng lymph node as the checkpoint inhibitor. 231. The method of any of claims 191-230, fithher sing administering an additional therapeutic agent to the subject either prior to, simultaneously with, or after treatment with the peptide, polynucleotide, vector, composition, or cells. 232. The method of claim 231, wherein the additional agent is a chemotherapeutic agent, an immunomodulatory drug, an immune metabolism modifying drug, a targeted therapy, radiation an anti-angiogenesis agent, or an agent that reduces immtme-suppression. 233. The method of claim 232, wherein the chemotherapeutic agent is an alkylating agent, a topoisomerase inhibitor. an anti-metabolite. or an anti-mitotic agent. 234. The method of claim 231, n the additional agent is an anti-glucocorticoid induced tumor necrosis factor family receptor (GITR) agonistic antibody or antibody fragment, ibrutinib, docetaxeol, cisplatin, a CD40 agonistic antibody or antibody fragment, an lDO tor, or cyclophosphamide. . The method of any of claims l9l-234, which elicits a CD4+ T cell immune response or a CD8- T cell immune response. 236. The method of any of claims 191-235, which elicits a CD4+ T cell immune response and a CD8+ T cell immune response. 237. A method for stimulating an immune response in a t, comprising administering an effective amount of modified cells or composition of any of claims 173-190. 238. The method of claim 237, wherein the immune response is xic and/or l immune response. 239. The method of claim 237, wherein the method ates a T cell-mediated immune response in a 240. The method of claim 239, wherein the T cell-mediated immune response is directed against a target cell. 241 . The method of claim 240, wherein the target cell is a tumor cell. 242. The method of any of claims 1. n the modified cells are transfected or transduced in vivo. 243. The method of any of claims 237-242, wherein the d cells are transfected or transduced ex vivo. 244. The method of any of claims 237-243, wherein the modified cells are autologous subject T cells. 245. The method of claim 244, wherein the autologous subject T cells are obtained from a subject that has received a neoantigen peptide or nucleic acid vaccine. 246. The method of claim 245, wherein the neoantigen peptide or nucleic acid vaccine comprises at least one alized neoantigen. 247. The method of claim 246, wherein the neoantigen peptide or c acid vaccine comprises at least one additional neoantigenic peptide listed in Table l or 2. 248. The method of claim 247, wherein the subject received a chemotherapeutic agent, an immunomodulatory drug. an immune metabolism modifying drug, targeted y or radiation prior to and/or during receipt of the igen peptide or nucleic acid vaccine. 249. The method of any of claims 237-248, wherein the subject receives treatment with at least one checkpoint inhibitor. . The method of any of claims 237-249, n the autologous T cells are obtained from a subject that has already received at least one round of T cell therapy containing a neoantigen. . The method of any of claims 237-250, wherein the method fithher comprises adoptive T cell therapy. . The method of claim 251, wherein the adoptive T cell y comprises autologous T cells. . The method of claim 252, wherein the autologous T cells are targeted against tumor antigens. . The method of claim 251 or 252, wherein the adoptive T cell therapy fiirther comprises allogenic T cells. . The method of claim 254, wherein the allogenic T cells are targeted against tumor antigens. . The method of any of claims 25 l-255, wherein the adoptive T cell therapy is administered before the checkpoint inhibitor. after the checkpoint inhibitor. or simultaneously with the checkpoint inhibitor. . A method for evaluating the efficacy of any of claims 173-190, comprising: (i) measuring the number or concentration of target cells in a first sample obtained from the t before stering the modified cell, (ii) measuring the number tration of target cells in a second sample obtained from the subject afier administration of the modified cell, and (iii) determining an increase or decrease of the number or concentration of target cells in the second sample compared to the number or concentration of target cells in the first sample. 258. The method of claim 257, wherein treatment efficacy is determined by monitoring a clinical outcome; an increase, enhancement or prolongation of umor activity by T cells; an increase in the number of anti-tumor T cells or activated T cells as compared with the number prior to treatment; B cell activity; CD4+ T cell activity; or a combination thereof. 259. The method of claim 258, wherein treatment efficacy is determined by monitoring a biomarker. 260. The method of claim 259. wherein the biomarker is selected from the group consisting of CEA. Her- 2/neu, bladder tumor antigen, thyroglobulin, alpha-fetoprotein, PSA, CA 125, CA19.9, CA 15.3, leptin, tin, osteopontin, IGF-lI, CD98, fascin, sPIgK 143 eta, troponin I, ating tumor cell RNA or DNA, and b-type natriuretic peptide. 261. The method of claim 258, wherein clinical outcome is selected from the group consisting of tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response by the immune system: tumor expansion. recurrence or spread; or a combination f. 262. The method of claim 258, wherein the treatment effect is predicted by presence ofT cells or by presence of a gene ure indicating T cell inflammation or a combination thereof. 263. A method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof comprising stering to the subject: (a) the peptide, polynuclcotide, vector, ition, antibody, or cells of any of claims 1-106; and (b) at least one checkpoint inhibitor. 264. The method of claim 263, further comprising administration of an immunomodulator or adjuvant. 265. The method of claim 264, wherein the immunomodulator or adjuvant is selected from the group ting of Poly-'(IzC), CLC , STING agonist, 1018 155, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM—CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvamune, LipoVac, MF59, osphoryl lipid A, Montanide lMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, , OM-l74, OM-l97-MP-EC. lSA-TLRZ agonist. ONTAK. PepTel® vector system. PLG mieroparticles, resiquimod, SRL 172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, acrylic or methacrylic polymers, copolymers of maleic anhydride, and QS21 on. a co-stimulatory ligand, a TNF ligand, an lg superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, 0X40, CD27, GITR, CD30, DR3, CD69, or 4-1BB. 266. The method of claim 265, n the modulator or adjuvant is Poly-ICLC. 267. The method of any one of claims 263-266. wherein the checkpoint inhibitor is an anti-PD] antibody or antibody fragment. 268. The method of claim 267, wherein the anti-PD] antibody or antibody fragment is mab or pembolizumab. 269. The method of any one of claims 263-266, wherein the checkpoint inhibitor is an anti-PD-Ll antibody or dy fragment. 270. The method of claim 267, wherein the anti-PD-Ll antibody or antibody fragmentis avelumab. durvalumab or izumab. 271. The method of any one of claims 263-266, wherein the checkpoint inhibitor is an anti-CTLA4 antibody or antibody fragment. 272. The method of claim 271, wherein the anti-CTLA4 antibody is ipilimumab or tremelimumab. 273. The method of any one of claims 263-272, wherein the method comprises administering both an anti- PDl antibody and an anti-CTLA4 antibody. 274. The method of any one of claims 263-272, wherein administration of the checkpoint inhibitor is initiated before initiation of administration of the e, polynucleotide, vector, composition, antibody, or cell. 275. The method of any one of claims 263-272, wherein administration of the checkpoint inhibitor is initiated after initiation of administration of the peptide, polynucleotide, vector. composition, antibody. or cell. 276. The method of any one of claims 263-272, wherein administration of the checkpoint inhibitor is initiated simultaneously with the initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. 277. The method of any one of claims 263-276, wherein the peptide, polynucleotide, vector, composition, antibody, or cell is stered intravenously or subcutaneously. 278. The method of any one of claims 263-276, wherein the checkpoint inhibitor is administered enously or subcutaneously. 279. The method of any one of claims 263-278, wherein the checkpoint inhibitor is stered subcutaneously within about 2 cm of the site of administration ofthe peptide, polynucleotide, , composition, antibody, or cell. 280. The method of claim 279, wherein the peptide, polynucleotide, vector, composition, antibody, or cell is administered into the same ng lymph node as the checkpoint inhibitor. 281. A kit comprising the peptide. polynucleotide, vector. composition, dy. cells, or composition of any one ofclaims 1-136, 159, or 168-190. 282. The method of any one of claims 191-256, wherein the cancer is selected from the group consisting of: adrenal, bladder, breast, cervical, colorectal, glioblasoma, head and neck, kidney chromophobe, kidney clear cell, kidney papillary, liver, lung adenocarcinoma, lung squamous, ovarian, pancreatic, melanoma, stomach, uterine corpus endometrial,and uterine carcinosarcoma. 283. The method of any one of claims 6. wherein the cancer is selected from the group ting of: prostate cancer, bladder, lung squamous, NSCLC, breast, head and neck, lung adenocarcinoma, GBM, Glioma, CML, AML, supretentorial ependyomas, acute promyelocytic leukemia, solitary fibrous , and crizotinib resistant cancer. 284. The method of claim 108, wherein the cancer is selected from the group consisting of: CRC, head and neck, stomach, lung squamous, lung adenocarcinoma, Prostate, Bladder. stomach, renal cell carcinoma, and uterine. . The method of any one of claims 6, n the cancer is selected from the group consisting of: melanoma, lung us, DLBCL, uterine, head and neck, uterine, liver, and CRC. 286. The method of any one of claims 191-256, wherein the cancer is selected from the group consisting of: lymphoid cancer; Burkitt lymphoma, neuroblastoma, te adenocarcinoma, colorectal BioNTech US Inc. 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