NEOANTIGENS AND METHODS OF USE THEREOF

Description

[0001] This application 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. Provisional Application No. 62 / 316,547, 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.FIELD OF THE INVENTION

[0002] The field of the present invention relates to immunotherapeutic peptides, nucleic acids encoding the peptides, peptide binding agents, and their use in the immunotherapy of cancer. In one aspect, the invention provides neoantigenic peptides, useful alone or in combination with other tumor-associated peptides, anticancer, or immunomodulatory agents to treat cancer.BACKGROUND OF THE INVENTION

[0003] Tumor vaccines are typically composed of tumor antigens 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 contain 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 peptides 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 patient, 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 proteins are: (a) unique to the tumor cell as the mutation and it's corresponding protein are present only in the tumor; (b) avoid central tolerance and are therefore more likely to be immunogenic; (c) provide an excellent target for immune recognition including by both humoral and cellular immunity. WO 2016 / 187508, WO 2016 / 164833 and WO 2016 / 172722 disclose tumor-specific neoepitopes. Feng Du et al., Medicinal Research Reviews, volume 35, No. 6, pages 1300-1315 (2015) discloses GATA3 mutations in breast cancer patients. Fritsch et al., Cancer Immunology Research, volume 2, No. 6, pages 522-529 (2014) discloses HLA-binding properties of tumor neoepitopes. 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

[0004] The present invention is as set out in the claims. Other disclosures herein not falling within the claims are for the assistance of the skilled person in understanding and practicing the disclosed invention and are not part of the invention. For the avoidance of doubt, it is noted that the present invention does not extend to methods of treatment of the human or animal body. Any references in the description to methods of treatment refer to compounds, pharmaceutical compositions, and medicaments of the present disclosure for use in a method of treatment of the human or animal body by therapy.BRIEF SUMMARY OF THE DISCLOSURE

[0005] 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 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 polypeptide is encoded by a gene selected from the group consisting of: (a) ABL1, wherein AxByCz is (i) VADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGKVYEGVWKKYSLTV AVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVC, (ii) VADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGVVYEGVWKKYSLTV AVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVC, (iii) LLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSATEYLEKKNFIHRDLAARN CLVGENHLVKVADFGLSRLMTGDTYTAHAGAKF, (iv) SLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIIIEFMTYGNLLDYLRECNR QEVNAVVLLYMATQISSAMEYLEKKNFIHRDLA, or (v) STVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQHGEVYEGVWKKYSLT VAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLG; (b) ALK, wherein AxByCz is (i) SSLAMLDLLHVARDIACGCQYLEENHFIHRDIAARNCLLTCPGPGRVAKIADFGMARDIYRASYYRK GGCAMLPVKWMPPEAFMEGIFTSKTDTWSFGVLL, or (ii) QVAVKTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIGVSLQSLPRFILMELMAGGDLKSFLRETRPR PSQPSSLAMLDLLHVARDIACGCQYLEENHFI; (c) BRAF, wherein AxByCz is MIKLIDIARQTAQGMDYLHAKSIIHRDLKSNNIFLHEDLTVKIGDFGLATEKSRWSGSHQFEQLSGSIL WMAPEVIRMQDKNPYSFQSDVYAFGIVLYELM; (d) BTK, wherein AxByCz is MIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGSLLNYLREMRHRFQTQQ LLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND; (e) EEF1B2, wherein AxByCz is MGFGDLKSPAGLQVLNDYLADKSYIEGYVPSQADVAVFEAVSGPPPADLCHALRWYNHIKSYEKEK ASLPGVKKALGKYGPADVEDTTGSGAT; (f) EGFR, wherein AxByCz is (i) SLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIIRNRGENSCKATGQVCHALC SPEGCWGPEPRDCVSCRNVSRGRECVDKCNLL, or (ii) IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; (g) ERBB3, wherein AxByCz is ERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRMVRGTQVYDGKFAIFV MLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDK; (h) ESR1, wherein AxByCz is (i) HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEA, (ii) NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLPSTLKSLEEKDHIHRVLD KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH, (iii) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLCDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, (iv) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLNDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, or (v) IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE; (i) FGFR3, wherein AxByCz is HRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERCPHRPILQAGLPANQTA VLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVG; (j) FRG1B, wherein AxByCz is AVKLSDSRIALKSGYGKYLGINSDELVGHSDAIGPREQWEPVFQNGKMALSASNSCFIRCNEAGDIEA KSKTAGEEEMIKIRSCAEKETKKKDDIPEEDKG; (k) HER2, wherein AxByCz is GSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGLGSPYVSRLLGICLTSTV QLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCM; (1) IDH1, wherein AxByCz is (i) RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGHHAYGDQYRATDFVVPGP GKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, (ii) RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGCHAYGDQYRATDFVVPGPG KVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, (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) LGAGNGGVVFKVSHKPSGLVMARKLIHLEIKPAIRNQIIRELQVLHECNSLYIVGFYGAFYSDGEISIC MEHMDGGSLDQVLKKAGRIPEQILGKVSIAVI; (o) MYC, wherein AxByCz is (i) MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSDLQPPAPSEDIWKKFELLPTPPLSPSRRSG LCSPSYVAVTPFSLRGDNDGG, (ii) FTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLSTPPLSPSRRSGLCSPSY VAVTPFSLRGDNDGGGGSFSTADQLEMVTELLG, or (iii) TNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPIPPLSPSRRSGLCSPSYVA VTPFSLRGDNDGGGGSFSTADQLEMVTELLGG; (p) PDGFRa, wherein AxByCz is VAVKMLKPTARSSEKQALMSELKIMTHLGPHLNIVNLLGACTKSGPIYIIIEYCFYGDLVNYLHKNRD SFLSHHPEKPKKELDIFGLNPADESTRSYVILS; (q) PIK3CA, wherein AxByCz is (i) IEEHANWSVSREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSKITEQEKDFLWSHRHYC VTIPEILPKLLLSVKWNSRDEVAQMYCLVKDWPP, (ii) HANWSVSREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSEITKQEKDFLWSHRHYCVTI PEILPKLLLSVKWNSRDEVAQMYCLVKDWPPIKP, or (iii) LFINLFSMMLGSGMPELQSFDDIAYIRKTLALDKTEQEALEYFMKQMNDARHGGWTTKMDWIFHTI KQHALN; (r) POLE, wherein AxByCz is QRGGVITDEEETSKKIADQLDNIVDMREYDVPYHIRLSIDIETTKLPLKFRDAETDQIMMISYMIDGQG YLITNREIVSEDIEDFEFTPKPEYEGPFCVFN; (s) PTEN, wherein AxByCz is KFNCRVAQYPFEDHNPPQLELIKPFCEDLDQWLSEDDNHVAAIHCKAGKGQTGVMICAYLLHRGKF LKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSY; (t) RAC1, wherein AxByCz is MQAIKCVVVGDGAVGKTCLLISYTTNAFSGEYIPTVFDNYSANVMVDGKPVNLGLWDTAGQEDYD RLRPLSYPQTVGET; and (u) TP53, wherein AxByCz is (i) IRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGSMNRRPILTIITLEDSSG NLLGRNSFEVRVCACPGRDRRTEEENLRKKGEP, (ii) TYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRHCPHHERCSDSDGLAP PQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEV, (iii) EGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNQRPILTIITLEDSSGNLL GRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE, (iv) EGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNWRPILTIITLEDSSGNLL GRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE, or (v) PEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVCVCACPGRDRRTEEENLR KKGEPHHELPPGSTKRALPNNTSSSPQPKKKPL.

[0006] In some cases, the native polypeptide is encoded by the EGFR, ERBB3 or FGFR3 gene and at least one By is expressed extracellularly.

[0007] 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 the native polypeptide; By is absent; each C is an amino acid encoded by a frameshift 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 Cz; and the native polypeptide is encoded by a gene selected from the group consisting of: (a) APC, wherein Cz is (i) AKFQQCHSTLEPNPADCRVLVYLQNQPGTKLLNFLQERNLPPKVVLRHPKVHLNTMFRRPHSCLAD VLLSVHLIVLRVVRLPAPFRVNHAVEW, (ii) APVIFQIALDKPCHQAEVKHLHHLLKQLKPSEKYLKIKHLLLKRERVDLSKLQ, or (iii) MLQFRGSRFFQMLILYYILPRKVLQMDFLVHPA; (b) ARID1A, wherein Cz is (i) ALGPHSRISCLPTQTRGCILLAATPRSSSSSSSNDMIPMAISSPPKAPLLAAPSPASRLQCINSNSRITSGQ WMAHMALLPSGTKGRCTACHTALGRGSLSSSSCPQPSPSLPASNKLPSLPLSKMYTTSMAMPILPLPQ LLLSADQQAAPRTNFHSSLAETVSLHPLAPMPSKTCHHK, (ii) AHQGFPAAKESRVIQLSLLSLLIPPLTCLASEALPRPLLALPPVLLSLAQDHSRLLQCQATRCHLGHPV ASRTASCILP, (iii) PILAATGTSVRTAARTWVPRAAIRVPDPAAVPDDHAGPGAECHGRPLLYTADSSLWTTRPQRVWST GPDSILQPAKSSPSAAAATLLPATTVPDPSCPTFVSAAATVSTTTAPVLSASILPAAIPASTSAVPGSIPL PAVDDTAAPPEPAPLLTATGSVSLPAAATSAASTLDALPAGCVSSAPVSAVPANCLFPAALPSTAGAIS RFIWVSGILSPLNDLQ, (iv) PCRAGRRVPWAASLIHSRFLLMDNKAPAGMVNRARLHITTSKVLTLSSSSHPTPSNHRPRPLMPNLRI SSSHSLNHHSSSPLSLHTPSSHPSLHISSPRLHTPPSSRRHSSTPRASPPTHSHRLSLLTSSSNLSSQHPRR SPSRLRILSPSLSSPSKLPIPSSASLHRRSYLKIHLGLRHPQPPQ, (v) RTNPTVRMRPHCVPFWTGRILLPSAASVCPIPFEACHLCQAMTLRCPNTQGCCSSWAS, or (vi) TNQALPKIEVICRGTPRCPSTVPPSPAQPYLRVSLPEDRYTQAWAPTSRTPWGAMVPRGVSMAHKVA TPGSQTIMPCPMPTTPVQAWLEA; (c) β2M, wherein Cz is (i) RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKMSMPAV, or (ii) LCSRYSLFLAWRLSSVLQRFRFTHVIQQRMESQIS; (d) CDH1, wherein Cz is (i) RSACVTVKGPLASVGRHSLSKQDCKFLPFWGFLEEFLLC, (ii) IQWGTTTAPRPIRPPFLESKQNCSHFPTPLLASEDRRETGLFLPSAAQKMKKAHFLKTWFRSNPTKTK KARFSTASLAKELTHPLLVSLLLKEKQDG, (iii) PTDPFLGLRLGLHLQKVFHQSHAEYSGAPPPPPAPSGLRFWNPSRIAHISQLLSWPQKTEERLGYSSHQ LPRK, (iv) FCCSCCFFGGERWSKSPYCPQRMTPGTTFITMMKKEAEKRTRTLT, or (v) WRRNCKAPVSLRKSVQTPARSSPARPDRTRRLPSLGVPGQPWALGAAASRRCCCCCRSPLGSARSRS PATLALTPRATRSRCPGATWREAASWAE; (e) GATA3, wherein Cz is (i) PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGPPARVPA VPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH; or (ii) PRPRRCTRHPACPLDHTTPPAWSPPWVRALLDAHRAPSESPCSPFRLAFLQEQYHEA; (f) MLL2, wherein Cz is TRRCHCCPHLRSHPCPHHLRNHPRPHHLRHHACHHHLRNCPHPHFLRHCTCPGRWRNRPSLRRLRSL LCLPHLNHHLFLHWRSRPCLHRKSHPHLLHLRRLYPHHLKHRPCPHHLKNLLCPRHLRNCPLPRHLK HLACLHHLRSHPCPLHLKSHPCLHHRRHLVCSHHLKSLLCPLHLRSLPFPHHLRHHACPHHLRTRLCP HHLKNHLCPPHLRYRAYPPCLWCHACLHRLRNLPCPHRLRSLPRPLHLRLHASPHHLRTPPHPHHLR THLLPHHRRTRSCPCRWRSHPCCHYLRSRNSAPGPRGRTCHPGLRSRTCPPGLRSHTYLRRLRSHTCP PSLRSHAYALCLRSHTCPPRLRDHICPLSLRNCTCPPRLRSRTCLLCLRSHACPPNLRNHTCPPSLRSHA CPPGLRNRICPLSLRSHPCPLGLKSPLRSQANALHLRSCPCSLPLGNHPYLPCLESQPCLSLGNHLCPLC PRSCRCPHLGSHPCRLS; (g) PTEN, wherein Cz is (i) SWKGTNWCNDMCIFITSGQIFKGTRGPRFLWGSKDQRQKGSNYSQSEALCVLL, (ii) KRTKCFTFG, (iii) PIFIQTLLLWDFLQKDLKAYTGTILMM, (iv) QKMILTKQIKTKPTDTFLQILR, (v) GFWIQSIKTITRYTIFVLKDIMTPPNLIAELHNILLKTITHHS, (vi) NYSNVQWRNLQSSVCGLPAKGEDIFLQFRTHTTGRQVHVL, or (vii) YQSRVLPQTEQDAKKGQNVSLLGKYILHTRTRGNLRKSRKWKSM; (h) TP53, wherein Cz is (i) SSQNARGCSPRGPCTSSSYTGGPCTSPLLAPVIFCPFPENLPGQLRFPSGLLAFWDSQVCDLHVLPCPQ QDVLPTGQDLPCAAVG, (ii) GAAPTMSAAQIAMVWPLLSILSEWKEICVWSIWMTETLFDIVWWCPMSRLRLALTVPPSTTTTCVTV PAWAA, (iii) TGGPSSPSSHWKTPVVIYWDGTALRCVFVPVLGETGAQRKRISARKGSLTTSCPQGALSEHCPTTPAP LPSQRRNHWMENISPFRSVGVSASRCSES, (iv) FHTPARHPRPRHGHLQAVTAHDGGCEALPPP, (v) CCPRTILNNGSLKTQVQMKLPECQRLLPPWPLHQQLLHRRPLHQPPPGPCHLLSLPRKPTRAATVSV WASCILGQPSL, (vi) VRKHFQTYGNYFLKTTFCPPCRPKQWMI, or (vii) LARTPLPSTRCFANWPRPALCSCGLIPHPRPAPASAPWPSTSSHST; or (i) VHL wherein Cz is (i) ELQETGHRQVALRRSGRPPKCAERPGAADTGAHCTSTDGRLKISVETYTVSSQLLMVLMSLDLDTGL VPSLVSKCLILRVK, (ii) KSDASRLSGA, (iii) RTAYFCQYHTASVYSERAMPPGCPEPSQA, (iv) TRASPPRSSSATAVRASCCPYGSTSTASRSPTQRCRLARAAASTATEVTFGSSEMQGHTMGFWLTKLN YLCHLSMLTDSLFLPISHCQCIL, (v) SSLRITGDWTSSGRSTKIWKTTQMCRKTWSG, or (vi) RRRRGGVGRRGVRPGRVRPGGTGRRGGDGGRAAAARAALGELARALPGHLLQSQSARRAARMAQ LRRRAAALPNAAAWHGPPHPQLPRSPLALQRCRDTRWASG; (j) ACVR2A, wherein AxByCz is (i) GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKRQP, or (ii) GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKTALKYIFVAVRAICVM KSFLIFRRWKSHSPLQIQLHLSHPITTSCSIPWCHLC; (k) C15ORF40, wherein AxByCz is TAEAVNVAIAAPPSEGEANAELCRYLSKVLELRKSDVVLDKVGLALFFFFFETKSCSVAQAGVQWRS LGSLQPPPPGFKLFSCLSFLSSWDYRRMPPCLANFCIFNRDGVSPCWSGWS; (1) CNOT1, wherein AxByCz is (i) LSVIIFFFVYIWHWALPLILNNHHICLMSSIILDCNSVRQSIMSVCFFFFSVIFSTRCLTDSRYPNICWFK, or (ii) LSVIIFFFVYIWHWALPLILNNHHICLMSSIILDCNSVRQSIMSVCFFFFCYILNTMFDR; (m) EIF2B3, wherein AxByCz or Cz is VLVLSCDLITDVALHEVVDLFRAYDASLAMLMRKGQDSIEPVPGQKGKKKQWSSVTSLEWTAQERG CSSWLMKQTWMKSWSLRDPSYRSILEYVSTRVLWMPTSTV; (n) EPHB2, wherein AxByCz or Cz is SIQVMRAQMNQIQSVEGQPLARRPRATGRTKRCQPRDVTKKTCNSNDGKKREWEKRKQILGGGGK YKEYFLKRILIRKAMTVLAGDKKGLGRFMRCVQSETKAVSLQLPLGR; (o) ESRP1, wherein AxByCz is (i) LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKKHEGQIC, or (ii) LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKT; (p) FAM11B, wherein AxByCz is GALCKDGRFRSDIGEFEWKLKEGHKKIYGKQSMVDEVSGKVLEMDISKKKHYNRKISIKKLNRMKV PLMKLITRV; (q) GBP3, wherein AxByCz is RERAQLLEEQEKTLTSKLQEQARVLKERCQGESTQLQNEIQKLQKTLKKKPRDICRIS; (r) JAK1, wherein AxByCz is (i) VNTLKEGKRLPCPPNCPDEVYQLMRKCWEFQPSNRTSFQNLIEGFEALLKTSN or (ii) CRPVTPSCKELADLMTRCMNYDPNQRPFFRAIMRDINKLEEQNPDIVSEKNQQLKWTPHILKSAS; (s) LMAN1, wherein AxByCz is (i) DDHDVLSFLTFQLTEPGKEPPTPDKEISEKEKEKYQEEFEHFQQELDKKKRGIPEGPPRPPRAACGGNI, or (ii) DDHDVLSFLTFQLTEPGKEPPTPDKEISEKEKEKYQEEFEHFQQELDKKKRNSRRATPTSKGSLRRKY LRV; (t) MSH3, wherein AxByCz is (i) TKSTLIGEDVNPLIKLDDAVNVDEIMTDTSTSYLLCISENKENVRDKKKGQHFYWHCGSAACHRRGC V, or (ii) LYTKSTLIGEDVNPLIKLDDAVNVDEIMTDTSTSYLLCISENKENVRDKKRATFLLALWECSLPQARL CLIVSRTLLLVQS; (u) NDUFC2, wherein AxByCz is (i) LPPPKLTDPRLLYIGFLGYCSGLIDNLIRRRPIATAGLHRQLLYITAFFFCWILSCKT, or (ii) SLPPPKLTDPRLLYIGFLGYCSGLIDNLIRRRPIATAGLHRQLLYITAFFLLDIIL; (v) RBM27, wherein AxByCz is NQSGGAGEDCQIFSTPGHPKMIYSSSNLKTPSKLCSGSKSHDVQEVLKKKTGSNEVTTRYEEKKTGSV RKANRMPKDVNIQVRKKQKHETRRKSKYNEDFERAWREDLTIKR; (w) RPL22, wherein AxByCz is (i) MAPVKKLVVKGGKKKEASSEVHS, or (ii) MAPVKKLVVKGGKKRSKF; (x) SEC31A, wherein AxByCz is (i) MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKN, or (ii) MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKLMLLRLNLRKMCGPF; (y) SEC63, wherein AxByCz is (i) AEVFEKEQSICAAEEQPAEDGQGETNKNRTKGGWQQKSKGPKKTAKSKKKETFKKKTYTCAITTVK ATETKAGKWSRWE, or (ii) MAEVFEKEQSICAAEEQPAEDGQGETNKNRTKGGWQQKSKGPKKTAKSKKRNL; (z) SLC35F5, wherein AxByCz is NIMEIRQLPSSHALEAKLSRMSYPVKEQESILKTVGKLTATQVAKISFFFALCGFWQICHIKKHFQTHK LL; (aa) SMAP1, 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) THAP5, wherein AxByCz is VPSKYQFLCSDHFTPDSLDIRWGIRYLKQTAVPTIFSLPEDNQGKDPSKKNPRRKTWKMRKKYAQKP SQKNHLY; (ee) TTK, wherein AxByCz is GTTEEMKYVLGQLVGLNSPNSILKAAKTLYEHYSGGESHNSSSSKTFEKKGEKNDLQLFVMSDTTYK IYWTVILLNPCGNLHLKTTSL; and (ff) XPOT, wherein AxByCz is QQLIRETLISWLQAQMLNPQPEKTFIRNKAAQVFALLFVTEYLTKWPKFFLTFSQ.

[0008] 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 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 Cz, 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 ERAEWRENIREQQKKCFRSFSL TSVELQML TNSCVKLQTVHSIPL TINKEEALQRPV ASDFEPQGLSEA ARWNSKENLLAGPSENDPNLFVALYDFVASG, or (ii) y is 1, and AxByCz is ELQMLTNSCVKLQTVHSIPLTINKEDDESPGLYGFLNVIVHSATGFKQSSKALQRPVASDFEPQGLSE AARWNSKENLLAGPSENDPNLFVALYDFVASGD; (b) the first native polypeptide is encoded by a C11orf95 gene, the second native polypeptide is encoded by an RELA gene, y is 1, and AxByCz is ISNSWDAHLGLGACGEAEGLGVQGAEEEEEEEEEEEEEGAGVPACPPKGPELFPLIFPAEPAQASGPY VEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTD; (c) the first native polypeptide is encoded by a CBFB gene and the second native polypeptide is encoded by an MYH11 gene, y is 0, and AxByCz is LQRLDGMGCLEFDEERAQQEDALAQQAFEEARRRTREFEDRDRSHREEMEVHELEKSKRALETQME EMKTQLEELEDELQATEDAKLRLEVNMQALKGQF; (d) the first native polypeptide is encoded by a CD74 gene and the second native polypeptide is encoded by an ROS1 gene, y is 0, and AxByCz is KGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKAGVPNKPGIPKLLEGS KNSIQWEKAEDNGCRITYYILEIRKSTSNNLQNQ; (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 LPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQLQDKFEHLKMIQQEEIRKL EEEKKQLEGEIIDFYKMKAASEALQTQLSTD; or (ii) an EGFR gene, y is 1, and AxByCz is MRPSGTAGAALLALLAALCPASRALEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGP CRKVCNGIGIGEFKD; (f) the first native polypeptide is encoded by a EML4 gene, the second native polypeptide is encoded by an ALK gene, y is 1, and AxByCz is SWENSDDSRNKLSKIPSTPKLIPKVTKTADKHKDVIINQAKMSTREKNSQVYRRKHQELQAMQMEL QSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDL (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 NDRG1 gene, and AxByCz is MSREMQDVDLAEVKPLVEKGETITGLLQEFDVQEALSVVSEDQSLFECAYGTPHLAKTEMTASSSSD YGQTSKMSPRVPQQDW or (ii) the first native polypeptide is encoded by a TMPRSS2 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) VLDMHGFLRQALCRLRQEEPQSLQAAVRTDGFDEFKVRLQDLSSCITQGKAIETQSSSSEEIVPSPPSP PPLPRIYKPCFVCQDKSSGYHYGVSACEGCKG, or (ii) RSSPEQPRPSTSKAVSPPHLDGPPSPRSPVIGSEVFLPNSNHVASGAGEAAIETQSSSSEEIVPSPPSPPPL PRIYKPCFVCQDKSSGYHYGVSACEGCKG; (k) the first native polypeptide is encoded by a RUNX1 gene, the second native polypeptide is encoded by an CBFA2T1 (RUNX1T1) gene, y is 1, and AxByCz is VARFNDLRFVGRSGRGKSFTLTITVFTNPPQVATYHRAIKITVDGPREPRNRTEKHSTMPDSPVDVKT QSRLTPPTMPPPPTTQGAPRTSSFTPTTLTNGT; (1) 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

[0009] 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 polypeptide, 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 Cz, or y is at least 1 and the at least 8 contiguous amino acids comprises at least one By and / or at least one Cz; and (a) the first native polypeptide is encoded by an AC011997.1 gene, the second native polypeptide is encoded by a LRRC69 gene, y is 1, and AxByCz is MAGAPPPASLPPCSLISDCCASNQRDSVGVGPSEPGNNIKICNESASRK (b) the first native polypeptide is encoded by an EEF1DP3 gene, the second native polypeptide is encoded by a FRY gene, y is 1, and AxByCz is HGWRPFLPVRARSRWNRRLDVTVANGRSWKYGWSLLRVPQVNGIQVLNVSLKSSSNVISY, (c) the first native polypeptide is encoded by a MAD1L1 gene, the second native polypeptide is encoded by a MAFK gene, y is 0, and AxByCz is RLKEVFQTKIQEFRKACYTLTGYQIDITTENQYRLTSLYAEHPGDCLIFKLRVPGSSVLVTVPGL, or (d) the first native polypeptide is encoded by a PPP1R1B gene, the second native polypeptide is encoded by a STARD3 gene, y is 1, and AxByCz is AEVLKVIRQSAGQKTTCGQGLEGPWERPPPLDESERDGGSEDQVEDPALSALLLRPRPPRPEVGAHQ DEQAAQGADPRLGAQPACRGLPGLLTVPQPEPLLAPPSAA.

[0010] In some cases, the isolated neoantigenic peptide comprises a sequence according to Table 1. In cases, x + y + z is at most 500, at most 250, at most 150, at most 125, or at most 100 In cases, x + y + z is at least 8, at least 50, at least 100, at least 200, or at least 300. In cases, z is at most 500, at most 250, at most 150, at most 125, or at most 100. In cases, z is at least 8, at least 50, at least 100, at least 200, or at least 300. In cases, the isolated neoantigenic peptide is from about 8 to about 500 amino acids in length. In cases, the isolated neoantigenic peptide is from about 8 to about 100 amino acids in length. In cases, the isolated neoantigenic peptide is from about 8 to about 50 amino acids in length. In cases, the isolated neoantigenic peptide is from about 15 to about 35 amino acids in length. In cases, the isolated neoantigenic peptide is from about 8 and about 15 amino acids in length. In cases, the isolated neoantigenic peptide is from about 8 and about 11 amino acids in length. In cases, the isolated neoantigenic peptide is 9 or 10 amino acids in length. In cases, the isolated neoantigenic peptide binds major histocompatibility complex (MHC) class I. In cases, the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 500 nM or less. In cases, the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 250 nM or less. In cases, the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 50 nM or less. In cases, the isolated neoantigenic peptide is from about 8 and about 30 amino acids in length. In cases, the isolated neoantigenic peptide is from about 8 to about 25 amino acids in length. In cases, the isolated neoantigenic peptide is from about 15 to about 24 amino acids in length. In cases, the isolated neoantigenic peptide is from about 9 to about 15 amino acids in length. In cases, the isolated neoantigenic peptide binds MHC class II. In cases, the isolated neoantigenic peptide binds MHC class II with a binding affinity of 1000 nM or less. In cases, the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 500 nM or less. In cases, the isolated neoantigenic peptide further comprises flanking amino acids. In cases, the flanking amino acids are not native flanking amino acids In cases, the isolated neoantigenic peptide 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. In cases, the isolated neoantigenic peptide 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 cases, the isolated neoantigenic peptide is a first neoantigenic peptide linked to at least a second neoantigenic peptide. In cases, the isolated neoantigenic peptide is linked to the at least second neoantigenic peptide by a poly-glycine or poly-serine linker. In cases, the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 1000 nM. In cases, the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 500 nM. In cases, isolated neoantigenic peptide and the second neoantigenic peptide bind to human leukocyte antigen (HLA) -A, -B, -C, -DP, -DQ, or -DR. In cases, the isolated neoantigenic peptide binds a class I HLA and the second neoantigenic peptide binds a class II HLA. In cases, the isolated neoantigenic peptide binds a class II HLA and the second neoantigenic peptide binds a class I HLA. In cases, the isolated neoantigenic peptide further comprises a modification which increases in vivo half-life, cellular targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, antigen presentation, or a combination thereof. In cases, 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 fusion, nanoparticle attachment, nanoparticulate 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 addition of unnatural amino acids. In cases, the isolated neoantigenic peptide further comprises a modification which increases cellular targeting to antigen presenting cells. In cases, the antigen presenting cells are dendritic cells. In cases, the dendritic cells are targeted using DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD11c, CD83, TSLP receptor, Clec9a, or CD1a marker. In cases, the dendritic cells are targeted using the CD141, DEC205, Clec9a, or XCR1 marker. In cases, the dendritic cells are autologous cells. In cases, one or more of the dendritic cells are bound to a T cell. In cases, the T cell is an autologous T cell. In cases, the isolated neoantigenic peptide is not a isolated neoantigenic peptide listed in Table 2. In cases, the isolated neoantigenic peptide is linked to at least one additional neoantigenic peptide listed in Table 1 or 2.

[0011] Provided herein is an in vivo delivery system comprising an isolated neoantigenic peptide described herein. In cases, the delivery system includes cell-penetrating peptides, nanoparticulate encapsulation, virus like particles, liposomes, or any combination thereof. In cases, the cell-penetrating peptide is TAT peptide, herpes simplex virus VP22, transportan, Antp, or any combination thereof.

[0012] Provided herein is a cell comprising an isolated neoantigenic peptide described herein. In cases, the cell is an antigen presenting cell. In cases, the cell is a dendritic cell. In cases, the cell is an autologous cell. In cases, the cell is bound to a T cell. In cases, the T cell is an autologous T cell.

[0013] Provided herein is a composition comprising an isolated neoantigenic peptide described herein.

[0014] In cases, 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 1 or 2. In cases, the composition comprises from about 2 to about 20 neoantigenic peptides, or from about 2 to about 30 neoantigenic peptides. In cases, the neoantigen is specific for an individual subject's tumor. In cases, 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, 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 cases, the composition comprises from about 4 to about 20 additional neoantigenic peptides, from about 4 to about 30 additional neoantigenic peptides. In cases, at least on of the additional neoantigenic peptides is specific for an individual subject's tumor. In cases, the subject specific neoantigenic peptide 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 cases, the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or circulating tumor cells. In cases, the sequence differences are determined by Next Generation Sequencing.

[0015] Provided herein is an isolated polynucleotide encoding an isolated neoantigenic peptide described herein. In cases, the polynucleotide is DNA. In cases, the polynucleotide is RNA. In cases, the RNA is a self-amplifying RNA. In cases, the RNA is modified to increase stability, increase cellular targeting, increase translation efficiency, adjuvanticity, cytosol accessibility, and / or decrease cytotoxicity. In cases, 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.

[0016] Provided herein is a cell comprising the polynucleotide described herein.

[0017] Provided herein is a vector comprising the polynucleotide described herein. In cases, the polynucleotide is operably linked to a promoter. In cases, the vector is a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus, or virion. In cases, the vector is derived from an adeno-associated virus, herpesvirus, lentivirus, or a pseudotype thereof.

[0018] Provided herein is an in vivo delivery system comprising the isolated polynucleotide described herein. In cases, the delivery system includes spherical nucleic acids, viruses, virus-like particles, plasmids, bacterial plasmids, or nanoparticles.

[0019] Provided herein is a cell comprising a vector or delivery system described herein. In cases, the cell is an antigen presenting cell. In cases, the cell is a dendritic cell. In cases, the cell is an immature dendritic cell.

[0020] Provided herein is a composition comprising at least one polynucleotide described herein. In cases, 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 polynucleotides. In cases, 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 cases, the neoantigenic peptides are encoded by a vector comprising one or more of the the isolated polynucleotides. In cases, 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 polynucleotides encoding for additional neoantigenic peptides. In cases, one or more of the additional neoantigenic peptides are encoded by a vector comprising one or more of the additional neoantigenic polynucleotides. In cases, the composition comprises from about 4 to about 20 additional neoantigenic polynucleotides, or from about 4 to about 30 additional neoantigenic polynucleotides. In cases, the isolated polynucleotides and the additional neoantigenic polynucleotides are linked. In cases, the polynucleotides are linked using nucleic acids that encode a poly-glycine or poly-serine linker. In cases, at least one of the additional neoantigenic peptide is specific for an individual subject's tumor. In cases, the subject specific neoantigenic peptide 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 cases, the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or circulating tumor cells. In cases, the sequence differences are determined by Next Generation Sequencing.

[0021] 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 cases, the MHC of the MHC-peptide is MHC class I or class II. In cases, TCR is a bispecific TCR further comprising a domain comprising an antibody or antibody fragment capable of binding an antigen. In cases, the antigen is a T cell-specific antigen. In cases, the antigen is CD3. In cases, the antibody or antibody fragment is an anti-CD3 scFv.

[0022] Provided herein is a chimeric antigen receptor comprising: (i) a T cell activation molecule; (ii) a transmembrane region; and (iii) an antigen 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 cases, CD3-zeta is the T cell activation molecule. In cases, the chimeric antigen receptor further comprises at least one costimulatory signaling domain. The In cases, the signaling domain is CD28, 4-1BB, ICOS, OX40, ITAM, or Fc epsilon RI-gamma. In cases, the antigen recognition moiety is capable of binding the isolated neoantigenic peptide in the context of MHC class I or class II. In cases, the CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane region. In cases, the neoantigenic peptide is located in the extracellular domain of a tumor associated polypeptide. In cases, the MHC of the MHC-peptide is MHC class I or class II.

[0023] 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 cases, the T cell is a T cell of a subject.

[0024] 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 of T 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 cases, the T cell is a CD8+ T cell, a helper T cell or cytotoxic T cell. In cases, the population of T cells from a subject is a population of CD8+ T cells from the subject. In cases, the one or more of the at least one neoantigenic peptide described herein is a subject-specific neoantigenic peptide. In cases, the subject-specific neoantigenic peptide has a different tumor neo-epitope that is an epitope specific to a tumor of the subject. In cases, 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 cases, the subject-specific neoantigenic peptide binds to a HLA protein of the subject. In cases, the subject-specific neoantigenic peptide binds to a HLA protein of the subject with an IC50 less than 500 nM. In cases, the activated CD8+ T cells are separated from the antigen presenting cells. In cases, the antigen presenting cells are dendritic cells or CD40L-expanded B cells. In cases, the antigen presenting cells are non-transformed cells. In cases, the antigen presenting cells are non-infected cells. In cases, the antigen presenting cells are autologous. In cases, the antigen presenting cells have been treated to strip endogenous MHC-associated peptides from their surface. In cases, the treatment to strip the endogenous MHC-associated peptides comprises culturing the cells at about 26°C. In cases, the treatment to strip the endogenous MHC-associated peptides comprises treating the cells with a mild acid solution. In cases, the antigen presenting cells have been pulsed with at least one neoantigenic peptide described herein. In cases, pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 µg / ml of each of the at least one neoantigenic peptide described herein. In cases, ratio of isolated T cells to antigen presenting cells is between about 30:1 and 300:1. In cases, the incubating the isolated population of T cells is in the presence of IL-2 and IL-7. In cases, the MHC of the MHC-peptide is MHC class I or class II.

[0025] Provided 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 cases, the T cell is a CD8+ T cell, a helper T cell or cytotoxic T cell. In cases, the population of T cells from a subject is a population of CD8+ T cells from the subject. In cases, the one or more of the at least one neoantigenic peptide described herein is a subject-specific neoantigenic peptide. In cases, the subject-specific neoantigenic peptide has a different tumor neo-epitope that is an epitope specific to a tumor of the subject. In cases, 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 cases, the subject-specific neoantigenic peptide binds to a HLA protein of the subject. In cases, the subject-specific neoantigenic peptide binds to a HLA protein of the subject with an IC50 less than 500 nM. In cases, the method further comprises separating the activated T cells from the antigen presenting cells. In cases, the method further comprises testing the activated T cells for evidence of reactivity against at least one of neoantigenic peptide of described herein. In cases, the antigen presenting cells are dendritic cells or CD40L-expanded B cells. In cases, the antigen presenting cells are non-transformed cells. In cases, the antigen presenting cells are non-infected cells. In cases, the antigen presenting cells are autologous. In cases, the antigen presenting cells have been treated to strip endogenous MHC-associated peptides from their surface. In cases, the treatment to strip the endogenous MHC-associated peptides comprises culturing the cells at about 26°C. In cases, the treatment to strip the endogenous MHC-associated peptides comprises treating the cells with a mild acid solution. In cases, the antigen presenting cells have been pulsed with at least one neoantigenic peptide described herein. In cases, pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 µg / ml of each of at least one neoantigenic peptide described herein. In cases, ratio of isolated T cells to antigen presenting cells is between about 30:1 and 300:1. In cases, the incubating the isolated population of T cells is in the presence of IL-2 and IL-7. In cases, the MHC of the MHC-peptide is MHC class I or class II.

[0026] Provided herein is a composition comprising activated tumor specific T cells produced by a method described herein.

[0027] Provided herein is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of activated tumor specific T cell described herein, or produced by a method described herein. In cases, the administering comprises administering from about 10 6< to 10 12< , from about 10 8< to 10 11< , or from about 10 9< to 10 10< of the activated tumor specific T cells.

[0028] Provided herein is a nucleic acid comprising a promoter operably linked to a polynucleotide encoding the T cell receptor described herein. In cases, 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.

[0029] Provided herein is a nucleic acid comprising a promoter operably linked to a polynucleotide encoding the chimeric antigen receptor described herein. In cases, the antigen recognition moiety is capable of binding the at least one neoantigenic peptide in the context of major histocompatibility complex (MHC) class I or class II. In cases, the neoantigenic peptide is located in the extracellular domain of a tumor associated polypeptide. In cases, the nucleic acid comprises the CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane region.

[0030] Provided herein is an antibody or antibody fragment capable of binding at least one neoantigenic peptide described herein or an MHC-peptide complex comprising at least one neoantigenic peptide described herein, optionally wherein the antibody fragment is a bi-specific T cell engager (BiTE). In cases, the antibody or antibody fragment binds to an extracellular portion of the at least one neoantigenic peptide. In cases, the native polypeptide is encoded by a gene selected from the group consisting of: β2M, wherein Cz is RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKMSMPAV, or LCSRYSLFLAWRLSSVLQRFRFTHVIQQRMESQIS; EGFR, wherein AxByCz is IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; BTK, wherein AxByCz is MIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGSLLNYLREMRHRFQTQQ LLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND; or ESR1, wherein AxByCz is HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEA, NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLPSTLKSLEEKDHIHRVLD KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH, IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLCDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLNDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, or IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE; or HER2, wherein AxByCz is GSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGLGSPYVSRLLGICLTSTV QLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCM. In cases, the antibody or antibody fragment is a bispecific antibody or antibody fragment. In cases, one antigen binding domain of the bispecific antibody or antibody fragment is an anti-CD3 binding domain.

[0031] Provided herein is a modified cell transfected or transduced with a nucleic acid described herein. In cases, 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.

[0032] Provided herein is a composition comprising the T cell receptor or chimeric antigen receptor described herein.

[0033] Provided herein is a composition comprising autologous subject T cells containing the T cell receptor or chimeric antigen receptor described herein. In cases, the composition further comprises an immune checkpoint inhibitor. In cases, the composition further comprises at least two immune checkpoint inhibitors. In cases, each of the immune checkpoint inhibitors inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof. In cases, each of the immune checkpoint inhibitors interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof. In cases, the T cells are PD-1 and / or CTLA4 knockout T cells, optionally, wherein the PD-1 and / or CTLA4 knockout T cells are created using a CRISPR system. In cases, the composition further comprises an immune modulator or adjuvant. In cases, the immune modulator is a co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-1BB. In cases, the immune modulator is at least one cancer cell or cancer cell extract. In cases, the cancer cell is autologous to the subject in need of the composition. In cases, the cancer cell has undergone lysis or been exposed to UV radiation. In cases, the composition further comprises an adjuvant. In cases, the adjuvant is selected from the group consisting of: Poly(I:C), Poly-ICLC, STING agonist, 1018 ISS, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK, PepTel ®< . vector system, PLG microparticles, resiquimod, SRL172, 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 stimulon. In cases, the composition induces a humoral response when administered to a subject. In cases, the composition induces a T helper cell type 1 when administered to a subject.

[0034] Provided herein is a method of inhibiting growth of a tumor cell expressing a tumor-specific neoepitope, comprising contacting the tumor cell with the peptide, polynucleotide, delivery system, vector, composition, antibody, or cells described herein.

[0035] Provided herein is a method of prophylaxis of a subject, comprising contacting a cell of the subject with the peptide, polynucleotide, delivery system, vector, composition, antibody, or cells described herein. In cases, the native polypeptide is encoded by a gene selected from the group consisting of: β2M, wherein Cz is RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKMSMPAV, or LCSRYSLFLAWRLSSVLQRFRFTHVIQQRMESQIS; EGFR, wherein AxByCz is IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; BTK, wherein AxByCz is MIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGSLLNYLREMRHRFQTQQ LLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND; or ESR1, wherein AxByCz is HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEA, NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLPSTLKSLEEKDHIHRVLD KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH, IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLCDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLNDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, or IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE; and HER2, wherein AxByCz is GSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGLGSPYVSRLLGICLTSTV QLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCM.

[0036] Provided herein is a method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof comprising administering to the subject the peptide, polynucleotide, vector, composition, antibody, or cells described herein. In cases, the subject is a human. In cases, the subject has cancer. In cases, 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, haematologic neoplasias, multiple myeloma, 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 cancer, colorectal cancer, ovarian cancer, hepatocellular cancer, renal cell cancer, pancreatic cancer, gastric cancer, oesophageal cancers, hepatocellular cancers, cholangiocellular cancers, head and neck squamous cell cancer soft tissue sarcoma, and small cell lung cancer. In cases, the peptide, polynucleotide, vector, composition, antibody, or cells described herein is for use in treating a corresponding cancer according to Table 1 or Table 2. In cases, the peptide, polynucleotide, vector, composition, antibody, or cells described herein is for use in treating a subject with an HLA type that is a corresponding HLA type according to Table 1 or Table 2. In cases, the subject has undergone surgical removal of the tumor. In cases, the peptide, polynucleotide, vector, composition, or cells is administered via intravenous, intraperitoneal, intratumoral, intradermal, or subcutaneous administration. In cases, the peptide, polynucleotide, vector, composition, or cells is administered into an anatomic site that drains into a lymph node basin. In cases, administration is into multiple lymph node basins. In cases, administration is by a subcutaneous or intradermal route. In cases, peptide is administered. In cases, administration is intratumorally. In cases, polynucleotide, optionally RNA, is administered. In cases, the polynucleotide is administered intravenously. In cases, the cell is a T cell or dendritic cell. In cases, the peptide or polynucleotide comprises an antigen presenting cell targeting moiety. In cases, the cell is an autologous cell. In cases, the method further comprises administering at least one immune checkpoint inhibitor to the subject. In cases, the checkpoint inhibitor is a biologic therapeutic or a small molecule. In cases, 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 In cases, the checkpoint inhibitors is a PD-1 antibody or a PD-L1 antibody. In cases, the checkpoint inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, avelumab, durvalumab, atezolizumab, pembrolizumab, and any combination thereof. In cases, the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands, and any combination thereof. In cases, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof. In cases, two or more checkpoint inhibitors are administered. In cases, at least one of the two or more checkpoint inhibitors is a PD-1 antibody or a PD-L1 antibody. In cases, 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. In cases, the checkpoint inhibitor and the composition are administered simultaneously or sequentially in any order. In cases, the peptide, polynucleotide, vector, composition, or cells is administered prior to the checkpoint inhibitor. In cases, the peptide, polynucleotide, vector, composition, or cells is administered after the checkpoint inhibitor. In cases, administration of the checkpoint inhibitor is continued throughout neoantigen peptide, polynucleotide, vector, composition, or cell therapy. In cases, the neoantigen peptide, polynucleotide, vector, composition, or cell therapy is administered to subjects that only partially respond or do not respond to checkpoint inhibitor therapy. In cases, the composition is administered intravenously or subcutaneously. In cases, the checkpoint inhibitor is administered intravenously or subcutaneously. In cases, the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the composition. In cases, the composition is administered into the same draining lymph node as the checkpoint inhibitor. In cases, the method further comprises administering an additional therapeutic agent to the subject either prior to, simultaneously with, or after treatment with the peptide, polynucleotide, vector, composition, or cells. In cases, 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 cases, the chemotherapeutic agent is an alkylating agent, a topoisomerase inhibitor, an anti-metabolite, or an anti-mitotic agent. In cases, 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 IDO inhibitor, or cyclophosphamide. In cases, the method elicits a CD4+ T cell immune response or a CD8+ T cell immune response. In cases, the method elicits a CD4+ T cell immune response and a CD8+ T cell immune response.

[0037] 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 cases, the immune response is cytotoxic and / or humoral immune response. In cases, the method stimulates a T cell-mediated immune response in a subject. In cases, the T cell-mediated immune response is directed against a target cell. In cases, the target cell is a tumor cell. In cases, the modified cells are transfected or transduced in vivo. In cases, the modified cells are transfected or transduced ex vivo. In cases, the modified cells are autologous subject T cells. In cases, the autologous subject T cells are obtained from a subject that has received a neoantigen peptide or nucleic acid vaccine. In cases, the neoantigen peptide or nucleic acid vaccine comprises at least one personalized neoantigen. In cases, the neoantigen peptide or nucleic acid vaccine comprises at least one additional neoantigenic peptide listed in Table 1 or 2. In cases, 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 cases, the subject receives treatment with at least one checkpoint inhibitor. In cases, the autologous T cells are obtained from a subject that has already received at least one round of T cell therapy containing a neoantigen. In cases, the method further comprises adoptive T cell therapy. In cases, the adoptive T cell therapy comprises autologous T cells. In cases, the autologous T cells are targeted against tumor antigens. In cases, the adoptive T cell therapy further comprises allogenic T cells. In cases, the allogenic T cells are targeted against tumor antigens. In cases, the adoptive T cell therapy is administered before the checkpoint inhibitor, after the checkpoint inhibitor, or simultaneously eith the checkpoint inhibitor.

[0038] Provided herein is a method for evaluating the efficacy 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 cases, treatment efficacy is determined 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 cases, treatment efficacy is determined by monitoring a biomarker. In cases, 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, prolactin, osteopontin, IGF-II, CD98, fascin, sPIgR, 14-3-3 eta, troponin I, circulating tumor cell RNA or DNA, and b-type natriuretic peptide. In cases, 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 thereof. In cases, 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.

[0039] Provided herein is a method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof comprising administering to the subject: the peptide, polynucleotide, vector, composition, antibody, or cells described herein; and at least one checkpoint inhibitor. In cases, the method further comprises administration of an immunomodulator or adjuvant. In cases, the immunomodulator or adjuvant is selected from the group consisting of Poly(I:C), Poly-ICLC, STING agonist, 1018 ISS, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK, PepTel ®< vector system, PLG microparticles, resiquimod, SRL172, 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 stimulon. a co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-1BB. In cases, the immunomodulator or adjuvant is Poly-ICLC. In cases, the checkpoint inhibitor is an anti-PDl antibody or antibody fragment. In cases, the anti-PDl antibody or antibody fragment is nivolumab or pembolizumab. In cases, the checkpoint inhibitor is an anti-PD-L1 antibody or antibody fragment. In cases, the anti-PD-L1 antibody or antibody fragmentis avelumab, durvalumab or atezolizumab. In cases, the checkpoint inhibitor is an anti-CTLA4 antibody or antibody fragment. In cases, the anti-CTLA4 antibody is ipilimumab or tremelimumab. In cases, the method comprises administering both an anti-PD1 antibody and an anti-CTLA4 antibody. In cases, administration of the checkpoint inhibitor is initiated before initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In cases, administration of the checkpoint inhibitor is initiated after initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In cases, administration of the checkpoint inhibitor is initiated simultaneously with the initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In cases, the peptide, polynucleotide, vector, composition, antibody, or cell is administered intravenously or subcutaneously. In cases, the checkpoint inhibitor is administered intravenously or subcutaneously. In cases, the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In cases, the peptide, polynucleotide, vector, composition, antibody, or cell is administered into the same draining lymph node as the checkpoint inhibitor.

[0040] Provided herein is a kit comprising the peptide, polynucleotide, vector, composition, antibody, cells, or composition described herein. In cases, 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. In cases, 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 tumors, and crizotinib resistant cancer. In cases, 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. In cases, the cancer is selected from the group consisting of: melanoma, lung squamous, DLBCL, uterine, head and neck, uterine, liver, and CRC. In cases, the cancer is selected from the group consisting of: lymphoid cancer; Burkitt lymphoma, neuroblastoma, prostate adenocarcinoma, colorectal adenocarcinoma; Uterine / Endometrium Adenocarcinoma; MSI+; endometrium serous carcinoma; endometrium carcinosarcoma-malignant mesodermal mixed tumour; glioma; astrocytoma; GBM, acute myeloid leukaemia associated with MDS; chronic lymphocytic leukaemia-small lymphocytic lymphoma; myelodysplastic syndrome; acute myeloid leukaemia; luminal NS carcinoma of breast; chronic myeloid leukaemia; ductal carcinoma of pancreas; chronic myelomonocytic leukaemia; myelofibrosis; myelodysplastic syndrome; prostate adenocarcinoma; essential thrombocythaemia; and medullomyoblastoma. In cases, the cancer is selected from the group consisting of: colorectal, uterine, endometrial, and stomach. In cases, the cancer is selected from the group consisting of: cervical, head and neck, anal, stomach, Burkitt's lymphoma, and nasopharyngeal carcinoma. In cases, the cancer is selected from the group consisting of: bladder, colorectal, and stomach. In cases, the cancer is selected from the group consisting of: lung, CRC, melanoma, breast, NSCLC, and CLL. In cases, the subject is a partial or non-responder to checkpoint inhibitor therapy. In cases, 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 (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), Prostate Cancer, skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), thyroid adenocarcinoma (THCA), and uterine corpus endometrioid carcinoma (UCEC). In cases, the cancer is selected from the group consisting of: colorectal cancer, uterine cancer, endometrium cancer, stomach cancer, and Lynch syndrome. In cases, the cancer is an MSI+ cancer.

[0041] The present disclosure is directed to an isolated neoantigenic peptide comprising a tumor-specific neoepitope defined in Table 1, wherein the isolated neoantigenic peptide is not a native polypeptide. The present disclosure is also directed to an isolated neoantigenic peptide which comprises a tumor-specific neoepitope and is defined in Table 1.

[0042] In some cases, the isolated neoantigenic peptide is between about 8 to about 50 amino acids in length. In another case, the isolated neoantigenic peptide is between about 15 to about 35 amino acids in length. In another case, the isolated neoantigenic peptide is about 15 amino acids or less in length. In another case, the isolated neoantigenic peptide is between about 8 and about 11 amino acids in length. In another case, the isolated neoantigenic peptide is 9 or 10 amino acids in length. In another case, the isolated neoantigenic peptide binds major histocompatibility complex (MHC) class I. In another case, the isolated neoantigenic peptide binds MHC class I with a binding affinity of less than about 500 nM.

[0043] In some cases, the isolated neoantigenic peptide is about 30 amino acids or less in length. In another case, the isolated neoantigenic peptide is between about 6 and about 25 amino acids in length. In another case, the isolated neoantigenic peptide is between about 15 and about 24 amino acids in length. In another case, the isolated neoantigenic peptide is between about 9 and about 15 amino acids in length. In another case, the isolated neoantigenic peptide binds MHC class II. In another case, the isolated neoantigenic peptide binds MHC class II with a binding affinity of less than about 1000 nM.

[0044] In some cases, the isolated neoantigenic peptide further comprises flanking amino acids. In another case, the flanking amino acids are not native flanking amino acids. In another case, the isolated neoantigenic peptide is linked to at least a second neoantigenic peptide. In another case, the peptides are linked using a poly-glycine or poly-serine linker. In another case, the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 1000 nM. In another case, the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 500 nM. In another case, both of the neoepitopes bind to human leukocyte antigen (HLA) -A, -B, -C, -DP, -DQ, or -DR. In another case, the isolated neoantigenic peptide binds a class I HLA and the second neoantigenic peptide binds a class II HLA. In another case, the isolated neoantigenic peptide binds a class II HLA and the second neoantigenic peptide binds a class I HLA.

[0045] In some cases, the isolated neoantigenic peptide further comprises modifications which increase in vivo half-life, cellular targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation. In another case, 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 fusion, nanoparticle attachment, nanoparticulate 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 addition of unnatural amino acids. In another case, the cells that are targeted are antigen presenting cells. In another case, the antigen presenting cells are dendritic cells. In another case, the dendritic cells are targeted using DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD11c, CD83, TSLP receptor, Clec9a or CD1a marker. In another case, the dendritic cells are targeted using the CD141, DEC205, or XCR1 marker.

[0046] In some cases, the disclosure provides an in vivo delivery system comprising an isolated neoantigenic peptide described herein. In another case, the delivery system includes cell-penetrating peptides, nanoparticulate encapsulation, virus like particles, or liposomes. In another case, the cell-penetrating peptide is TAT peptide, herpes simplex virus VP22, transportan, or Antp.

[0047] In some cases, the disclosure is directed to a cell comprising an isolated neoantigenic peptide described herein. In another case, the cell is an antigen presenting cell. In another case, the cell is a dendritic cell.

[0048] In some cases, the disclosure is directed to a composition comprising an isolated neoantigenic peptide described herein. In another case, 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, or at least 30 of the isolated neoantigenic peptides comprising a tumor-specific neoepitope defined in Table 1 or 2. In another case, the composition comprises between 2 and 20 neoantigenic peptides. In another case, 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, 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 case, the composition comprises between about 4 and about 20 additional neoantigenic peptides. In another case, the additional neoantigenic peptide is specific for an individual patient's tumor. In another case, 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 genome, exome, and / or transcriptome of a non-tumor sample. In another case, the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or circulating tumor cells. In another case, the sequence differences are determined by Next Generation Sequencing.

[0049] In some cases, the disclosure is directed to an isolated polynucleotide encoding an isolated neoantigenic peptide described herein. In another case, the isolated polynucleotide is RNA, optionally a self-amplifying RNA. In another case, the RNA is modified to increase stability, increase cellular targeting, increase translation efficiency, adjuvanticity, cytosol accessibility, and / or decrease cytotoxicity. In another case, 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.

[0050] In some cases, the disclosure is directed to a cell comprising a polynucleotide described herein.

[0051] In some cases, the disclosure is directed to a vector comprising a polynucleotide described herein. In another case, the polynucleotide is operably linked to a promoter. In another case, the vector comprises a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus, or virion. In another case, the vector is an adeno-associated virus, herpesvirus, lentivirus, or pseudotypes thereof.

[0052] In some cases, the disclosure is directed to an in vivo delivery system comprising an isolated polynucleotide described herein. In another case, the delivery system includes spherical nucleic acids, viruses, virus-like particles, plasmids, bacterial plasmids, or nanoparticles.

[0053] In some cases, the disclosure is directed to a cell comprising a vector or delivery system described herein. In another case, the cell is an antigen presenting cell. In another case, the cell is a dendritic cell. In another case, the cell is an immature dendritic cell.

[0054] In some cases, the disclosure is directed to a composition comprising at least one polynucleotide described herein. In another case, 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, or at least 30 of the isolated polynucleotides. In another case, the composition comprises between about 2 and about 20 polynucleotides that encode neoantigenic peptides. In another case, 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, or at least 30 additional neoantigenic polynucleotides encoding for additional neoantigenic peptides. In some cases, the composition comprises between about 4 and about 20 additional neoantigenic polynucleotides. In some cases, the isolated polynucleotides and the additional neoantigenic polynucleotides are linked. In some cases, the polynucleotides are linked using nucleic acids that encode a poly-glycine or poly-serine linker. In some cases, at least one of the additional neoantigenic peptide is specific for an individual patient's tumor. In some cases, 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 genome, exome, and / or transcriptome of a non-tumor sample. In some cases, the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or circulating tumor cells. In some cases, the sequence differences are determined by Next Generation Sequencing.

[0055] In some cases, the disclosure is directed to a T cell receptor (TCR) capable of binding at least one neoantigenic peptide described herein. In some cases, the TCR is capable of binding the isolated neoantigenic peptide in the context of MHC class I or class II.

[0056] In some cases, the disclosure is directed to a chimeric antigen receptor comprising: (i) a T cell activation molecule; (ii) a transmembrane region; and (iii) an antigen recognition moiety capable of binding an isolated neoantigenic peptide described herein. In some cases, the chimeric antigen receptor contains CD3-zeta as the T cell activation molecule. In some cases, the chimeric antigen receptor further comprises at least one costimulatory signaling domain. In some cases, the signaling domain is CD28, 4-1BB, ICOS, OX40, ITAM, or Fc epsilon RI-gamma. In some cases, the antigen recognition moiety is capable of binding the isolated neoantigenic peptide in the context of MHC class I or class II. In some cases, the chimeric antigen receptor comprises the CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane region. In some cases, the tumor-specific epitope is located in the extracellular domain of a tumor associated polypeptide.

[0057] In some cases, the disclosure is directed to a T cell comprising the T cell receptor or chimeric antigen receptor described herein. In some cases, the T cell is a helper or cytotoxic T cell.

[0058] In some cases, the disclosure is directed to a nucleic acid comprising a promoter operably linked to a polynucleotide encoding a T cell receptor described herein. In some cases, 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. In another case, the nucleic acid comprises a promoter operably linked to a polynucleotide encoding a chimeric antigen receptor described herein. In another case, the antigen recognition moiety is capable of binding the at least one neoantigenic peptide in the context of major histocompatibility complex (MHC) class I or class II. In some cases, the tumor-specific epitope is located in the extracellular domain of a tumor associated polypeptide. In some cases, the nucleic acid comprises the CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane region.

[0059] In some cases, the disclosure is directed to an antibody capable of binding at least one neoantigenic peptide described herein.

[0060] In some cases, the disclosure is directed to a modified cell transfected or transduced with a nucleic acid described herein. In some cases, 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.

[0061] In some cases, the disclosure is directed to a composition comprising a T cell receptor or chimeric antigen receptor described herein. In some cases, the composition comprises autologous patient T cells containing a T cell receptor or chimeric antigen receptor. In some cases, the composition further comprises an immune checkpoint inhibitor. In some cases, the composition further comprises at least two immune checkpoint inhibitors. In some cases, each of the immune checkpoint inhibitors inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof. In some cases, each of the immune checkpoint inhibitors interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof.

[0062] In some cases, the composition further comprises an immune modulator or adjuvant. In another case, the immune modulator is a co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-1BB. In another case, the immune modulator is at least one cancer cell or cancer cell extract. In another case, the cancer cell is autologous to the subject in need of the composition. In another case, the cancer cell has undergone lysis or been exposed to UV radiation. In another case, the adjuvant induces a humoral when administered to a subject. In another case, the adjuvant induces a T helper cell type 1 when administered to a subject.

[0063] In some cases, the disclosure 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, polynucleotide, delivery system, vector, composition, antibody, or cells of the disclosure.

[0064] In some cases, the disclosure is directed to a method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof comprising administering to the subject the peptide, polynucleotide, vector, composition, antibody, or cells described herein.

[0065] In some cases, 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, uterine corpus endometrial,and uterine carcinosarcoma.

[0066] In some cases, 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.

[0067] In some cases, the cancer is selected from the group consisting of CRC, head and neck, stomach, lung squamous, lung adeno., Prostate, Bladder. stomach, renal cell carcinoma, and uterine.

[0068] In some cases, the cancer is selected from the group consisting of melanoma, lung squamous, DLBCL, uterine, head and neck, uterine, liver, and CRC.

[0069] In some cases, the cancer is selected from the group consisting of lymphoid cancer; Burkitt lymphoma, neuroblastoma, prostate adenocarcinoma, colorectal adenocarcinoma; Uterine / Endometrium Adenocarcinoma; MSI +< ; endometrium serous carcinoma; endometrium carcinosarcoma-malignant mesodermal mixed tumour; glioma; astrocytoma; GBM, acute myeloid leukaemia associated with MDS; chronic lymphocytic leukaemia-small lymphocytic lymphoma; myelodysplastic syndrome; acute myeloid leukaemia; luminal NS carcinoma of breast; chronic myeloid leukaemia; ductal carcinoma of pancreas; chronic myelomonocytic leukaemia; myelofibrosis; myelodysplastic syndrome; prostate adenocarcinoma; essential thrombocythaemia; and medullomyoblastoma..

[0070] In some cases, the cancer is selected from the group consisting of colorectal, uterine, endometrial, and stomach.

[0071] In some cases, the cancer is selected from the group consisting of cervical, head and neck, anal, stomach, Burkitt's lymphoma, and nasopharyngeal carcinoma.

[0072] In some cases, the cancer is selected from the group consisting of bladder, colorectal, and stomach.

[0073] In some cases, the cancer is selected from the group consisting of lung, CRC, melanoma, breast, NSCLC, and CLL.

[0074] In some cases, the cancer is selected from the group consisting of adrenocortical carcinoma (ACC), acute promyelocytic leukemia, acute myeloid leukemia (AML), AML associated with myelodysplastic syndromes (MDS), anal cancer, astrocytoma, bladder cancer, bladder urothelial carcinoma (BLCA), breast cancer, breast invasive carcinoma (BRCA), Burkitt's Lymphoma, castration-resistant prostate cancer, cervical cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), chronic lymphocytic leukaemia-small lymphocytic lymphoma, chronic lymphocytic leukaemia (CLL), chronic myeloid leukaemia (CML), chronic myelomonocytic leukaemia, colorectal adenocarcinoma, colorectal cancer (CRC), Crizotinib resistant 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 multiforme (GBM), Glioma, head and neck cancer, head and neck squamous cell carcinoma (HNSC), invasive lobular carcinoma (ILC) LumA breast cancer, kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary 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 (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), prostate cancer, renal cell carcinoma, skin cutaneous melanoma (SKCM), solitary fibrous tumors, stomach adenocarcinoma (STAD), stomach cancer, supretentorial ependyomas, thyroid adenocarcinoma (THCA), uterine corpus endometrioid carcinoma (UCEC), or uterine carcinosarcoma (UCS), uterine cancer, and uterine / endometrium adenocarcinoma.

[0075] In some cases, 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 (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), Prostate Cancer, skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), thyroid adenocarcinoma (THCA), and uterine corpus endometrioid carcinoma (UCEC).

[0076] In some cases, the subject is a human. In another case, the subject has cancer. In another case, 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, haematologic neoplasias, multiple myeloma, 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 cancer, colorectal cancer, ovarian cancer, hepatocellular cancer, renal cell cancer, pancreatic cancer, gastric cancer, oesophageal cancers, hepatocellular cancers, cholangiocellular cancers, head and neck squamous cell cancer soft tissue sarcoma, and small cell lung cancer. In another case, the subject has undergone surgical removal of the tumor. In another case, the peptide, polynucleotide, vector, composition, or cells is administered via intravenous, intraperitoneal, intratumoral, intradermal, or subcutaneous administration. In another case, the peptide, polynucleotide, vector, composition, or cells is administered into an anatomic site that drains into a lymph node basin. In another case, the administration is into multiple lymph node basins. In another case, the administration is by a subcutaneous or intradermal route.

[0077] In some cases of the method, a peptide is administered. In another case, the administration is intratumorally. In another case of the method, a polynucleotide, optionally RNA, is administered. In another case, the polynucleotide is administered intravenously. In some cases of the method, a cell is administered. In another case, the cell is a T cell or dendritic cell. In another case, the peptide or polynucleotide comprises an antigen presenting cell targeting moiety.

[0078] In another case of the method, at least one immune checkpoint inhibitor is also administered to the subject. In another case, the checkpoint inhibitor is a biologic therapeutic or a small molecule. In another case, 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. In another case, the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof. In another case, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof. In another case, two or more checkpoint inhibitors are administered. In another case, the checkpoint inhibitors are: (i) ipilimumab or tremelimumab, and (ii) nivolumab. In another case, the checkpoint inhibitor and the composition are administered simultaneously or sequentially in any order. In another case, a neoantigenic peptide, polynucleotide, vector, composition, or cells is administered prior to the checkpoint inhibitor. In another case, a peptide, polynucleotide, vector, composition, or cells is administered after the checkpoint inhibitor. In another case, the checkpoint inhibitor is continued throughout neoantigen peptide, polynucleotide, vector, composition, or cell therapy. In another case, the neoantigen peptide, polynucleotide, vector, composition, or cell therapy is administered to subjects that only partially respond or do not respond to checkpoint inhibitor therapy. In another case, the composition is administered intravenously or subcutaneously. In another case, the checkpoint inhibitor is administered intravenously or subcutaneously. In another case, the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the composition. In another case, the composition is administered into the same draining lymph node as the checkpoint inhibitor.

[0079] In some cases of the method, an additional agent is administered. In another case, the 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 another case, the chemotherapeutic agent is an alkylating agent, a topoisomerase inhibitor, an anti-metabolite, or an anti-mitotic agent. In another case, the additional agent is an anti-glucocorticoid induced tumor necrosis factor family receptor (GITR) agonistic antibody or antibody fragment, ibrutinib, docetaxeol, cisplatin, or cyclophosphamide. In another case, the administration elicits a CD4 +< T cell immune response. In another case, the administration elicits a CD4 +< T cell immune response and a CD8 +< T cell immune response.

[0080] In some cases, the disclosure is directed to a method for stimulating an immune response in a subject, comprising administering an effective amount of modified cells or composition described herein. In another case, the immune response is cytotoxic and / or humoral immune response. In another case, the method stimulates a T cell-mediated immune response in a subject. In another case, the T cell-mediated immune response is directed against a target cell. In another case, the target cell is a tumor cell. In another case, the modified cells are transfected or transduced in vivo. In another case, the modified cells are transfected or transduced ex vivo. In another case, the modified cells are autologous patient T cells. In another case, the autologous patient T cells are obtained from a patient that has received a neoantigen peptide or nucleic acid vaccine. In another case, the neoantigen peptide or nucleic acid vaccine comprises at least one personalized neoantigen. In another case, the neoantigen peptide or nucleic acid vaccine comprises at least one additional neoantigenic peptide described herein. In another case, the patient 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 another case, the patient receives treatment with at least one checkpoint inhibitor. In another case, 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 case, the method further comprises adoptive T cell therapy. In another case, the adoptive T cell therapy comprises autologous T cells. In another case, the autologous T cells are targeted against tumor antigens. In another case, the adoptive T cell therapy further comprises allogenic T cells. In another case, the allogenic T cells are targeted against tumor antigens. In another case, the adoptive T cell therapy is administered before the checkpoint inhibitor.

[0081] In some cases, the disclosure 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) 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 another case, the treatment efficacy is determined 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 case, the treatment efficacy is determined by monitoring a biomarker. In another case, 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, prolactin, osteopontin, IGF-II, CD98, fascin, sPIgR, 14-3-3 eta, troponin I, and b-type natriuretic peptide. In another case, the 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 thereof. In another case, 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.

[0082] In some cases, the disclosure is directed to a method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof comprising administering to the subject: (a) the peptide, polynucleotide, vector, composition, antibody, or cells described herein; and (b) at least one checkpoint inhibitor. In another case, the method further comprises administration of an immunomodulator or adjuvant. In another case, the immunomodulator or adjuvant is selected from the group consisting of Poly(I:C), Poly-ICLC, STING agonist, 1018 ISS, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK, PepTel ®< vector system, PLG microparticles, resiquimod, SRL172, 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 stimulon. a co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-1BB. In another case, the immunomodulator or adjuvant is Poly-ICLC. In another case, the checkpoint inhibitor is an anti-PDl antibody or antibody fragment. In another case, the inhibitor of the PD-1 pathway is nivolumab. In another case, the checkpoint inhibitor is an anti-CTLA4 antibody or antibody fragment. In another case, the anti-CTLA4 antibody is ipilimumab or tremelimumab. In another case, the method comprises administering both an anti-PD1 antibody and an anti-CTLA4 antibody. In another case, the administration of the checkpoint inhibitor is initiated before initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In another case, the administration of the checkpoint inhibitor is initiated after initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In another case, the administration of the checkpoint inhibitor is initiated simultaneously with the initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In another case, the peptide, polynucleotide, vector, composition, antibody, or cell is administered intravenously or subcutaneously. In another case, the checkpoint inhibitor is administered intravenously or subcutaneously. In another case, the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In another case, the peptide, polynucleotide, vector, composition, antibody, or cell is administered into the same draining lymph node as the checkpoint inhibitor.

[0083] In some cases of the therapeutic methods, the additional therapeutic agent is for example, a chemotherapeutic or biotherapeutic agent, radiation, or immunotherapy. Any suitable therapeutic treatment for a particular cancer may be administered. Examples of chemotherapeutic and biotherapeutic agents include, but are not limited 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 Bleomycin, Carboplatin, Carrmistme, Chlorambucil, Cyclophosphamide, cis-Diammineplat num(D) dichloride (Cispiatin), Melphalan, Mitoxantrone, and Oxaliplatin; a DNA synthesis inhibitor, such as (±)-Amethopterin (Methotrexate), 3-Amino-1,2,4-beiizotriazine 1,4-dioxide, Aminopterin, Cytosine β-D-arabinofuraiioside, 5-Fmoro-5'~ deoxyuridine, 5-Fhsorouracil, Ganciclovir, Hydroxyurea, and Mitomycin C; a DNA-RNA transcription regulator, such as Aetinomycin D, Dau orubicin, Doxorubicin, Homoharringtonine, and Idarubicin; an oη / .γηx; inhibitor, such as S(-i-)-Camptothecm, Curcumin, (-)-Deguelm, 5,6-Dichiorobenzimidazole 1 -β-D-ribofuranoside, Etoposide, Formestane, 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'-deoxycytidine, 5-Azacytidine, Cholecalciferol (Vitamin D3), 4-Hydroxytamoxifen, Melatonin, Mifepristone, Raloxifene, all trans-Retinal (Vitamin A aldehyde), Retinoic acid all trans (Vitamin A acid), 9-cis-Retinoic Acid, 13-cis-Retinoic acid, Retinol (Vitamin A), Tamoxifen, and Troglitazone; a microtubule inhibitor, such as Colchicine, docetaxel, Dolastatirs 15, Nocodazole, Paclitaxel, Podophyl!otoxin, Rhizoxin, Vinblastine, Vi cristine, Vindesiiie, and Vinorelbine (Navelbine); and an unclassified therapeutic agent, such as 17-(Allyiamino)-1 7-demethoxygeIdanamycin, 4-Amino-1,8-naphthalimide, Apigenin, Brefeldin A, Cimetidine, Dichioromethylene-diphosphonic acid, Leuprolide (Leuprorelin), Luteinizing Hormone-Releasing Hormone, Pifithrin-a, Rapamycin, Sex hormone-binding globulin, Thapsigargin, and Urinary trypsin inhibitor fragment (Bikunin). The therapeutic agent may be altretamine, amifostine, asparaginase, capecitabine, cladribine, cisapride, cyiarahirse, dacarbazine (DT1C), dactinomycin, dronabinol, epoetin alpha, "filgrastim, fludarabine, gemcitabine, granisetron, ifosfamide, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, metoclopramide, mitotane, omeprazole, ondansetron, pilocarpine, prochloroperazine, or topotecan hydrochloride. The therapeutic agent may be a monoclonal antibody such as rituximab (Rituxan ®< ), alemtuzumab (Campath ®< ), Bevacizumab (Avastin ®< ), Cetuximab (Erbitux ®< ), panitumumab (Vectibix ®< ), and trastuzumab (Herceptin ®< ), Vemurafenib (Zelboraf ®< ) imatinib mesylate (Gleevec ®< ), erlotinib (Tarceva ®< ), gefitinib (Iressa ®< ), Vismodegib (Erivedge ™< ), 90Y-ibritumomab tiuxetan, 1311-tosit.umomab, ado-trastuzumab emtansine, lapatinib (Tykerb ®< ), pertuzumab (Perjeta ™< ), ado-trastuzumab emtansine ( adcyla ™< ), regorafenib (Stivarga ®< ), sunitinib (Sutent ®< ), Denosumab (Xgeva ®< ), sorafenib (Nexavar ®< ), pazopanib (Votrient ®< ), axitinib (Inita ®< ), dasatinib (Sprycel ®< ), nilotinib (Tasigna ®< ), bosutinib (Bosulif ®< ), ofatumumab (Arzerra ®< ), obinutuzumab (Gazyva ™< ), ibrutinib (Imbruvica ™< ), idelalisib (Zydelig ®< ), crizotinib (Xalkori ®< ), erlotinib (Tarceva ®< ), afatimb dimaleate (Giiotrif ®< ), ceritinib (LDK378 / Zykadia), Tositumomab and 1311-tositumomab (Bexxar ®< ), ibritumomab tiuxetan (Zevalin ®< ), brentuximab vedotin (Adcetris ®< ), bortezomib (Velcade ®< ), siltuximab (Sylvant ™< ), trametinib ( ekinist ®< ), dabrafenib (Tafmlar ®< ), pembrolizimiab (Keytruda ®< ), carfilzomib (Kyprolis ®< ), Ramucirumab (Cyramza ™< ), Cabozantinib (Cometriq ™< ), vandetanib (Caprelsa ®< ), Optionally, the therapeutic agent is a neoantigen. The therapeutic agent may be a cytokine such as interferons (INFs), interlcukins (ILs), or hematopoietic growth factors. The therapeutic agent may be INF-α, IL-2, Aldesleukin, IL-2, Erythropoietin, Granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor. The therapeutic agent may be a targeted therapy such as toremifene (Fareston ®< ), fulvestrant (Faslodex ®< ), anastrozole (Arimidex ®< ), exemestane (Aromasin ®< ), letrozole (Femara ®< ), zivaflibercept (Zaltrap ®< ), Aiitretinoin (Panretin ®< ), temsirolimus (Torisel ®< ), Tretinoin (Vesanoid ®< ), denileukin diftitox (Ontak ®< ), vorinostat (Zoiinza ®< ), romidepsin (Istodax ®< ), bexarotene (Targretin ®< ), pralatrexate (Foiotyn ®< ), !enaliomide (Revlimid ®< ), belinostat (Beleodaq ™< ), lenaliomide (Revlimid ®< ), pomalidomide (Pomalyst ®< ), Cabazitaxel (Jevtana ®< ), enzaluiamide (Xtandi ®< ), abiraterone acetate (Zytiga ®< ), radium 223 chloride (Xofigo ®< ), or everolimus (Afiniior ®< ). Aditionally, the therapeutic agent may be an epigenetic targeted drug such as HDAC inhibitors, kinase inhibitors, DNA methyltransferase inhibitors, histone demethylase inhibitors, or histone methylation inhibitors. The epigenetic drugs may be Azacitidine (Vidaza), Decitabine (Dacogen), Vorinostat (Zoiinza), Romidepsin (Istodax), or Ruxolitinib (Jakafi). For prostate cancer treatment, a preferred chemotherapeutic agent with which anti- CTLA-4 can be combined is paclitaxel (TAXOL).

[0084] In some cases, the disclosure is directed to a kit comprising any neoantigen therapeutic described herein.

[0085] Where aspects or cases of the disclosure are described in terms of a Markush group or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but also each member of the group individually 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 of the group members in the claimed invention.

[0086] The documents discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.BRIEF DESDCRIPTION OF THE DRAWINGS

[0087] Figure 1 depicts and exemplary graph of 562 peptides with predicted affinity for select HLA Class I molecule between 1 nM and 100,000 nM (plotted on x-axis) that were synthesized. Actual affinity (IC 50 (nM)) was measured as described (y-axis). Thick vertical and horizontal lines denote 500 nM cutoff between weak and very weak predicted and observed binders, respectively. Diagonal dotted line depicts line-of-best fit (Graphpad Prism) with R 2< of 0.45. Figure 2 depicts and exemplary graph of 275 peptides from Figure 1 that were tested for stability (T 1 / 2 ; hrs) on their respective HLA Class I molecules. Peptides were binned according to observed affinity from Figure 1. Median and interquartile range is shown for each bin. Figure 3A depicts and exemplary graph of HLA-A02:01 +< T cells co-cultured with monocyte-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 neoepitope using multimers (initial: BV421 and PE). Figure 3B depicts and exemplary graph of HLA-A02:01 +< T cells co-cultured with monocyte-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 neoepitope using multimers (validation: APC and BUV396). Figure 4A depicts and exemplary graph of HLA-A02:01 +< 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 analyzed for antigen-specificity for GATA3 frameshift neoepitope using multimers (initial: APC and BUV396). Figure 4B depicts and exemplary graph of HLA-A02:01 +< 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 analyzed for antigen-specificity for GATA3 frameshift neoepitope using multimers (validation: PE and BV421). Figure 5A depicts and exemplary graph of HLA-A02:01 +< T cells co-cultured with monocyte-derived dendritic cells loaded with β2M frameshift neoepitope (LLCVWVSSI; HLA-A02:01) for 10 days. CD8 +< T cells were analyzed for antigen-specificity for β2M frameshift neoepitope using multimers (initial: PE and APC). Figure 5B depicts and exemplary graph of HLA-A02:01 +< T cells co-cultured with monocyte-derived dendritic cells loaded with β2M frameshift neoepitope (LLCVWVSSI; HLA-A02:01) for 10 days. CD8 +< T cells were analyzed for antigen-specificity for β2M frameshift neoepitope using multimers (validation: PE and BV421). Figure 6A depicts and exemplary graph of HLA-A02:01 +< 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 G12C frameshift neoepitope using multimers (initial: BUV396 and BV421). Figure 6B depicts and exemplary graph HLA-A02:01 +< 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 G12C frameshift neoepitope using multimers (validation: APC and BUV396). Figure 7 depicts and exemplary graph of T cells co-cultured with monocyte-derived dendritic cells loaded β2M 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 β2M frameshift peptide for 24 hours (right), compared to controls without peptide (left). Figure 8 depicts and exemplary graph of T cells co-cultured with monocyte-derived dendritic cells loaded BTK C481S neopeptide 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 BTK C481S neopeptide for 24 hours (right), compared to controls wild-type BTK peptide (left). Figure 9 depicts and exemplary graph of T cells co-cultured with monocyte-derived 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

[0088] 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 disclosure described herein provides peptides, polynucleotides encoding the peptides, and peptide binding agents, that can be used, for example, to stimulate an immune response to a tumor associated antigen, to create an immunogenic composition or cancer vaccine for use in treating disease.I. Definitions

[0089] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. 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".

[0090] The term "about" or "approximately" can mean within an acceptable error range for the particular value as determined 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 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0091] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0092] "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 shift mutation, fusion polypeptide, in-frame deletion, insertion, expression of endogenous retroviral polypeptides, and tumor-specific overexpression of polypepitdes.

[0093] "Tumor specific neoepitope" refers to an epitope that is not present in a reference such as a normal non-cancerous or germline cell but is found in cancer cells. This includes, in particular, situations wherein in a normal non-cancerous or germline cell a corresponding epitope is found, however, due to one or more mutations in a cancer cell the sequence of the epitope is changed so as to result in the neo-epitope.

[0094] A "reference" can be used to correlate and compare the results obtained in the methods of the disclosure from a tumor specimen. Typically the "reference" may be obtained on the basis of one or more normal specimens, in particular specimens which are not affected by a cancer disease, either obtained from a patient or one or more different individuals, for example, healthy individuals, in particular individuals of the same species. A "reference" can be determined empirically by testing a sufficiently large number of normal specimens.

[0095] The term "mutation" refers to a change of or difference in the nucleic acid sequence (nucleotide 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 cases, 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 mutation disrupts the normal phase of a gene's codon periodicity (also known as "reading frame"), resulting in the translation of a non-native protein sequence. It is possible for different mutations in a gene to achieve the same altered reading frame.

[0096] 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. K D is the dissociation constant and has units of molarity. The affinity constant is the inverse of the dissociation constant. An affinity constant is sometimes used as a generic term 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 Biacore SPR units. Affinity may also be expressed as the inhibitory concentration 50 (IC 50 ), that concentration at which 50% of the peptide is displaced. Likewise, ln(IC 50 ) refers to the natural log of the IC 50 . K off refers to the off-rate constant, for example, for dissociation of an HLA-binding peptide and a class I or II HLA.

[0097] Throughout this disclosure, "binding data" results can be expressed in terms of "IC 50 ." IC 50 is the concentration of the 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 protein and labeled reference peptide concentrations), these values approximate K D values. Assays for determining binding are well known in the art and are described in detail, for example, 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. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed relative to binding by a reference standard peptide. For example, can be based on its IC 50 , relative to the IC 50 of a reference standard peptide.

[0098] Binding can also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).

[0099] "Cross-reactive binding" indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.

[0100] The term "derived" when used to discuss an epitope is a synonym for "prepared." A derived 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 epitope can be an analog of a native epitope.

[0101] 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, mineral 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.

[0102] 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. Alternatively, an epitope 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 epitopes 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. Throughout this disclosure, epitopes may be referred to in some cases as peptides or peptide epitopes.

[0103] 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 cases, the peptide comprises a fragment of an antigen.

[0104] In certain cases, there is a limitation on the length of a peptide. The case that is length-limited occurs when the protein or peptide comprising an epitope described herein comprises 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 peptide sequence. Thus, for a peptide comprising an epitope described herein and a region with 100% identity with a native peptide 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 cases, an "epitope" described herein is comprised by a peptide having a region with less than 51 amino acid residues 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, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues.

[0105] "Human Leukocyte Antigen" or "HLA" is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, Calif. (1994).

[0106] An "HLA supertype or HLA family", as used herein, describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into such HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecules (where "xx" denotes a particular HLA type), are synonyms.

[0107] The terms "identical" or percent "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 residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0108] An "immunogenic" peptide or an "immunogenic" epitope or "peptide 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 capable of binding to an appropriate HLA molecule and thereafter inducing a CTL (cytotoxic) response, or a HTL (and humoral) response, to the peptide.

[0109] As used herein, a "chimeric antigen receptor" or "CAR" refers to an antigen 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 includes a membrane-proximal TCR constant domain, and may also include a TCR transmembrane domain and / or a TCR cytoplasmic tail. For example, in some cases, 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 κ or λ variable domain) linked to a TCRα constant domain. In some cases, the CAR is a dimer that includes a first polypeptide comprising a immunoglobulin heavy chain variable domain linked to a TCRα constant domain and a second polypeptide comprising an immunoglobulin light chain variable domain (e.g., a κ or λ variable domain) linked to a TCRβ constant domain.

[0110] 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 associated with the peptides in their in situ environment. An "isolated" epitope refers to an epitope 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 100% 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 environment 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 system, 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 natural 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.

[0111] "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 humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed description of the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3.sup.RD ED., Raven Press, New York (1993).

[0112] A "native" or a "wild type" sequence refers to a sequence found in nature. Such a sequence can comprise a longer sequence in nature.

[0113] A "T cell epitope" is to be understood as meaning a peptide sequence which can be bound by the MHC molecules of class I or II in the form of a peptide-presenting MHC molecule or MHC complex and then, in this form, be recognized and bound by cytotoxic T-lymphocytes or T-helper cells, respectively.

[0114] A "receptor" is to be understood as meaning a biological 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 comprises at least one receptor unit, for example, where each receptor unit may consist of a protein molecule. The receptor has a structure which complements that of a ligand and may complex the ligand as a binding partner. The information is transmitted in particular by conformational changes of the receptor following complexation of the ligand on the surface of a cell. In some cases, a receptor is to be understood as meaning in particular proteins of MHC classes I and II capable of forming a receptor / ligand complex with a ligand, in particular a peptide or peptide fragment of suitable length.

[0115] A "ligand" is to be understood as meaning a molecule which has a structure complementary to that of a receptor and is capable of forming a complex with this receptor. In some cases, 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 forming a complex with proteins of MHC class I or MHC class II.

[0116] In some cases, a "receptor / ligand complex" is also to be understood as meaning a "receptor / peptide complex" or "receptor / peptide fragment complex", including a peptide- or peptide fragment-presenting MHC molecule of class I or of class II.

[0117] "Proteins or molecules of the major histocompatibility complex (MHC)", "MHC molecules", "MHC proteins" or "HLA proteins" are to be understood as meaning proteins capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential lymphocyte 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 comprises the genetic region whose gene products expressed on the cell surface 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 patterns of the two MHC classes are adapted to these different roles.

[0118] 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 α-amino and carboxyl groups of adjacent amino acid residues.

[0119] "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 chemical synthesis or recombinant DNA technology. "Synthetic peptides" include "fusion proteins."

[0120] A "PanDR binding" peptide, a "PanDR binding epitope" is a member of a family of molecules that binds more than one HLA class II DR molecule.

[0121] "Pharmaceutically acceptable" refers to a generally non-toxic, inert, and / or physiologically compatible composition or component of a composition.

[0122] A "pharmaceutical excipient" or "excipient" comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like. A "pharmaceutical excipient" is an excipient which is pharmaceutically acceptable.

[0123] The term "motif" refers to a pattern of residues in an amino acid sequence of defined length, for example, a peptide of less than about 15 amino acid residues 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, 11, 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, 11, 12, 13, 14, 15, 16, 17, 18, 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 cases, an MHC class I motif identifies a peptide of 9, 10, or 11 amino acid residues in length.

[0124] A "supermotif" is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. In some cases, a supermotif-bearing peptide described herein is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.

[0125] The term "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 occurring.

[0126] According to the disclosure, the term "vaccine" relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, for example, 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 vaccine is adapted to the needs or special circumstances of an individual cancer patient.

[0127] A "protective immune response" 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.

[0128] "Antigen processing" or "processing" refers to the degradation of a polypeptide or antigen 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.

[0129] "Antigen 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. Professional antigen-presenting cells are very efficient at internalizing 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 antigen 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 defining feature of professional antigen-presenting cells.

[0130] 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.

[0131] 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 dendritic cells are potent inducers of immune responses and the activation of these cells is a critical step for the induction of antitumoral immunity.

[0132] Dendritic cells are conveniently categorized as "immature" and "mature" cells, which can be used as a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation.

[0133] Immature dendritic cells are characterized 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 characterized by a lower expression of these 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).

[0134] 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.

[0135] The nomenclature used to describe peptides or proteins follows the conventional practice wherein the amino group is presented to the left (the amino- or N-terminus) and the carboxyl group to the right (the carboxy- or C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope 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.

[0136] In the formulae representing selected specific cases of the present disclosure, 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 formulae, each residue is generally represented by standard three letter or single letter designations. The L-form 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-form 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 symbol. (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, Serine; T, Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.)

[0137] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length, 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 cases, the polynucleotide and nucleic acid can be in vitro transcribed mRNA. In some cases, the polynucleotide that is administered using the methods of the disclosure is mRNA.

[0138] The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences 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 maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some cases, 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 cases 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 cases, 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 cases, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some cases the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence.

[0139] 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., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, 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.

[0140] The term "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 include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0141] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is "isolated" is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some cases, a polypeptide, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. In some cases, an "isolated polynucleotide" encompasses a PCR or quantitative PCR reaction comprising the polynucleotide amplified in the PCR or quantitative PCR reaction.

[0142] 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.

[0143] The term "subject" refers to any animal (e.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 particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0144] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of a therapeutic effective to "treat" a disease or disorder in a subject or mammal. 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.

[0145] 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 progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition 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 prevented.

[0146] As used in the present disclosure and claims, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise.

[0147] It is understood that terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. 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 U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude 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.

[0148] The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include 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 cases: 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).II. Neoantigens

[0149] One of the 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., inversions, 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 identifying them, selecting optimized antigens, and producing neoantigens for use in a vaccine or immunogenic composition. These problems may be addressed by: identifying mutations in neoplasias / tumors which are present at the DNA level in tumor but not in matched germline samples from a high proportion of subjects having cancer; analyzing the identified mutations with one or more peptide-MHC binding prediction algorithms 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 synthesizing the plurality of neoantigenic peptides selected from the sets of all neoantigen peptides and predicted binding peptides for use in a cancer vaccine or immunogenic composition suitable for treating a high proportion of subjects having cancer.

[0150] For example, translating peptide sequencing information into a therapeutic vaccine may include prediction of mutated peptides that can bind to HLA molecules of a high proportion 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 proportion of patient's HLA alleles. Recently, neural network based learning 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 peptides presented on HLA alleles.

[0151] For example, translating peptide sequencing information into a therapeutic vaccine may include 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 compensates for the known inaccuracy of epitope prediction approaches. Synthetic peptides provide a useful means to prepare multiple immunogens efficiently and to rapidly translate identification of mutant epitopes to an effective vaccine. Peptides can be readily synthesized chemically and easily purified utilizing reagents free of contaminating 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).

[0152] 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 of TLR3 and the RNA helicase-domains of MDA5 and RIG3, has shown several desirable properties for a vaccine adjuvant. These properties include the induction of local and systemic activation of immune cells in vivo, production of stimulatory chemokines and cytokines, and stimulation of antigen-presentation by DCs. Furthermore, poly-ICLC can induce durable 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-ICLC and in volunteers who had received the highly effective, replication-competent yellow fever vaccine. Furthermore, >90% of ovarian carcinoma patients immunized with poly-ICLC in combination with a NYESO-1 peptide vaccine (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.

[0153] Applicants have discovered mutational events in the following genes: ABL1, AC011997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM111B, FGFR3, FRG1B, GAGE1, GAGE10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, XPOT an EEF1DP3:FRY fusion polypeptide, an EGFR:SEPT14 fusion polypeptide, an EGFRVIII deletion polypeptide, an EML4:ALK fusion polypeptide, an NDRG1:ERG fusion polypeptide, an AC011997.1:LRRC69 fusion polypeptide, a RUNX1(ex5)-RUNX1T1 fusion polypeptide, a TMPRSS2:ERG fusion polypeptide, a NAB:STAT6 fusion polypeptide, a NDRG1:ERG fusion polypeptide, a PML:RARA fusion polypeptide, a PPP1R1B:STARD3 fusion polypeptide, a MAD1L1:MAFK fusion polypeptide, a FGFR3:TAC fusion polypeptide, a FGFR3:TACC3 fusion polypeptide, a BCR:ABL fusion polypeptide, a C11orf95:RELA fusion polypeptide, a CBFB:MYH11 fusion polypeptide, a CBFB:MYH11 fusion polypeptide, a CD74:ROS1 fusion polypeptide, a CD74:ROS1 fusion polypeptide, ERVE-4: protease, ERVE-4: reverse transcriptase, ERVE-4: reverse transcriptase, ERVE-4: unknown, ERVH-2 matrix protein, ERVH-2: gag, ERVH-2: retroviral matrix, ERVH48-1: coat protein, ERVH48-1: syncytin, ERVI-1 envelope protein, ERVK-5 gag, ERVK-5 env, ERVK-5 pol, EBV A73, EBV BALF3, EBV BALF4, EBV BALF5, EBV BARF0, EBV LF2, EBV RPMS1, HPV-16, HPV-16 E7, and HPV-16 E6.

[0154] In some cases, a neoantigen described herein is due to a mutational event in β2M, BTK, EGFR, GATA3, KRAS, MLL2, a TMPRSS2:ERG fusion polypeptide, or TP53.Neoantigen polypeptides

[0155] In aspects, the disclosure provides isolated peptides that comprise a tumor specific mutation from Table 1 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 designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. Similarly, the term "polypeptide" is used interchangeably with "mutant polypeptide" and "neoantigenic polypeptide" in the present specification to designate a series of residues, e.g., L-amino acids, connected one to the other, typically by peptide bonds between the α-amino and carboxyl 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 destroy the biological activity of the polypeptides as herein described.

[0156] In some cases, sequencing methods are used to identify tumor specific mutations. Any suitable sequencing method can be used according to the disclosure, for example, Next Generation Sequencing (NGS) 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 context of the present disclosure mean all novel high throughput sequencing technologies which, in contrast to the "conventional" 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 nucleic acid sequence information 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 commercially available or which are mentioned in the literature can be used in the context of the invention e.g. those described in detail in WO 2012 / 159643.

[0157] In certain cases a neoantigenic peptide described herein molecule can comprise, but is not limited to, about 5, about 6, about 7, 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 acid residues, and any range derivable therein. In specific cases, a neoantigenic peptide molecule is equal to or less than 100 amino acids.

[0158] In some cases, 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 cases, neoantigenic peptides and polypeptides described herein for MHC Class II are 9-24 residues in length.

[0159] A longer neoantigenic peptide can be designed in several ways. In some cases, when HLA-binding peptides are predicted 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-terminus of each corresponding gene product; or (2) a concatenation of some or all of the binding peptides with extended sequences for each. In other cases, when sequencing reveals a long (>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 sequence), a longer neoantigenic 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 cases, use of a longer peptide is presumed to allow for endogenous processing by patient cells and can lead to more effective antigen presentation and induction of T cell responses. In some cases, two or more peptides can be used, where the peptides overlap and are tiled over the long neoantigenic peptide.

[0160] In some cases, the neoantigenic peptides and polypeptides bind an HLA protein (e.g., HLA class I or HLA class II). In specific cases the neoantigenic peptides and polypeptides bind an HLA protein with greater affinity than the corresponding wild-type peptide. In specific cases the neoantigenic peptide or polypeptide has an IC 50 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.

[0161] In some cases, 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 cases, 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.

[0162] In some cases, the neoantigenic peptides can be at least 8, 9, 10, 11, 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, or more amino acid residues in length. In some cases, the neoantigenic peptides can be at least 8, 9, 10, 11, 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 cases, the neoantigenic peptides can be at most 8, 9, 10, 11, 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, or less amino acid residues in length. In some cases, the neoantigenic peptides can be at most 8, 9, 10, 11, 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 less amino acid residues in length.

[0163] In some cases, the neoantigenic peptides 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.

[0164] In some cases, 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.

[0165] In some cases, the neoantigenic peptides can have a pI value of about 0.5 and about 12, about 2 and about 10, or about 4 and about 8. In some cases, 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 cases, the neoantigenic peptides can have a pI value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.

[0166] In some cases, the neoantigenic peptides can have an HLA binding affinity of between about 1pM and about 1mM, about 100pM and about 500µM, about 500pM and about 10µM, about 1nM and about 1µM, or about 10nM and about 1µMIn some cases, 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 µM, or more. In some cases, 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, 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 µM.

[0167] In some cases, a neoantigenic peptide described herein can comprise 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.

[0168] In some cases, a neoantigenic peptide described herein can be modified by terminal-NH 2 acylation, e.g., by alkanoyl (C 1 -C 20 ) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some cases these modifications can provide sites for linking to a support or other molecule.

[0169] In some cases, 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 chemically 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.

[0170] In some cases, a neoantigenic peptide described herein can contain substitutions to modify a physical property (e.g., stability or solubility) of the resulting peptide. For example, neoantigenic peptides can be modified by the substitution of a cysteine (C) with α-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 α-amino butyric acid for C not only alleviates this problem, but actually improves binding and crossbinding capability in certain instances. Substitution of cysteine with α-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.

[0171] In some cases, a neoantigenic peptide described herein can comprise amino acid mimetics or unnatural amino acid residues, e.g. D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2-thieneylalanine; D- or L-1, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-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 L-2-indole(allyl)alanines; and, D- or L-alkylalanines, where the alkyl group can be a substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acid residues. Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides that have various amino acid mimetics or unnatural amino acid residues are particularly useful, as they tend to manifest increased stability in vivo. Such peptides can also possess improved shelf-life or manufacturing properties.

[0172] Peptide stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef, et al., Eur. J. Drug Metab. Pharmacokinetics 11:291 (1986). Half-life of the peptides described herein is conveniently determined 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-1640 or another suitable tissue culture medium. At predetermined time intervals, a small amount of reaction solution 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 spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions.

[0173] In some cases, a neoantigenic peptide described herein can be in solution, lyophylized, or can be in crystal form.

[0174] In some cases, a neoantigenic peptide described herein can be prepared synthetically, by recombinant DNA technology or chemical synthesis, or can be isolated from natural sources such as native tumors or pathogenic organisms. Epitopes can be synthesized individually or joined directly or indirectly in a peptide. Although a neoantigenic peptide described herein will be substantially free of other naturally occurring host cell proteins and fragments thereof, in some cases the peptide can be synthetically conjugated to be joined to native fragments or particles.

[0175] In some cases, a neoantigenic peptide described herein can be prepared in a wide variety of ways. In some cases, 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 SYNTHESIS, 2D. ED., Pierce Chemical Co., 1984). Further, individual peptides can be joined using chemical ligation to produce larger peptides.

[0176] Alternatively, recombinant DNA technology can be employed wherein a nucleotide sequence which encodes a peptide inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. 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, N.Y. (1989). Thus, recombinant peptides, which comprise or consist of one or more epitopes described herein, can be used to present the appropriate T cell epitope.

[0177] In one aspect, the disclosure described herein also provides compositions comprising one, at least two, or more than two neoantigenic peptides. In some cases a composition described herein contains at least two distinct peptides. In some cases, the at least two distinct 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.

[0178] In some cases, the isolated neoantigenic peptide is encoded by a gene with a point mutation resulting in an amino acid substitution of the native peptide.

[0179] In some cases, the isolated neoantigenic peptide is encoded by a ABL gene. In related cases, A x B y C z is VADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGKVYEGVWKKYSLTV AVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVC, VADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGVVYEGVWKKYSLTV AVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVC, LLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSATEYLEKKNFIHRDLAARN CLVGENHLVKVADFGLSRLMTGDTYTAHAGAKF, SLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIIIEFMTYGNLLDYLRECNR QEVNAVVLLYMATQISSAMEYLEKKNFIHRDLA, or STVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQHGEVYEGVWKKYSLT VAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLG.

[0180] In some cases, the isolated neoantigenic peptide is encoded by a ALK gene. In related cases, A x B y C z is SSLAMLDLLHVARDIACGCQYLEENHFIHRDIAARNCLLTCPGPGRVAKIADFGMARDIYRASYYRK GGCAMLPVKWMPPEAFMEGIFTSKTDTWSFGVLL or QVAVKTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIGVSLQSLPRFILMELMAGGDLKSFLRETRPR PSQPSSLAMLDLLHVARDIACGCQYLEENHFI.

[0181] In some cases, the isolated neoantigenic peptide is encoded by a BRAF gene. In related cases, A x B y C z is MIKLIDIARQTAQGMDYLHAKSIIHRDLKSNNIFLHEDLTVKIGDFGLATEKSRWSGSHQFEQLSGSIL WMAPEVIRMQDKNPYSFQSDVYAFGIVLYELM. In some related cases, the neoantigenic peptide is not DFGLATEKSR or FGLATEKSRW.

[0182] In some cases, the isolated neoantigenic peptide is encoded by a BTK gene. In related cases, A x B y C z is MIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGSLLNYLREMRHRFQTQQ LLEMCKDVCEAMEYLESKQFLHRDLAARNCLVND.

[0183] In some cases, the isolated neoantigenic peptide is encoded by a EEF1B2 gene. In related cases, A x B y C z is MGFGDLKSPAGLQVLNDYLADKSYIEGYVPSQADVAVFEAVSGPPPADLCHALRWYNHIKSYEKEK ASLPGVKKALGKYGPADVEDTTGSGAT. In some related cases, the neoantigenic peptide is not EAVSGPPPA, FEAVSGPPP or FEAVSGPPPA.

[0184] In some cases, the isolated neoantigenic peptide is encoded by a EGFR gene. In related cases, A x B y C z is MSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIIRNRGENSCKATGQVCHAL CSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLL or IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA.

[0185] In some cases, the isolated neoantigenic peptide is encoded by a ERBB3 gene. In related cases, A x B y C z is EFSTLPLPNLRMVRGTQVYDGKF. In some related cases, the neoantigenic peptide is not RMVRGTQVY, LPLPNLRMV, LRMVRGTQV, TLPLPNLRMV, NLRMVRGTQV, or LRMVRGTQVY.

[0186] In some cases, the isolated neoantigenic peptide is encoded by a ESR1 gene. In related cases, A x B y C z is HLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYGLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEA, NQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLPSTLKSLEEKDHIHRVLD KITDTLIHLMAKAGLTLQQQHQRLAQLLLILSH, IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLCDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLNDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE, or IHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLSDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGE.

[0187] In some cases, the isolated neoantigenic peptide is encoded by a FGFR3 gene. In related cases, A x B y C z is HRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERCPHRPILQAGLPANQTA VLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVG. In some related cases, the neoantigenic peptide is not DVLERCPHR, LERCPHRPI, CPHRPILQA, or LERCPHRPIL.

[0188] In some cases, the isolated neoantigenic peptide is encoded by a FRG1B gene. In related cases, A x B y C z is AVKLSDSRIALKSGYGKYLGINSDELVGHSDAIGPREQWEPVFQNGKMALSASNSCFIRCNEAGDIEA KSKTAGEEEMIKIRSCAEKETKKKDDIPEEDKG. In some related cases, the neoantigenic peptide is not SASNSCFIR, LSASNSCFI, ALSASNSCF or FQNGKMALSA.

[0189] In some cases, the isolated neoantigenic peptide is encoded by a HER2 gene. In related cases, A x B y C z is GSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGLGSPYVSRLLGICLTSTV QLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCM.

[0190] In some cases, the isolated neoantigenic peptide is encoded by a IDH1 gene. In related cases, A x B y C z is RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGHHAYGDQYRATDFVVPGP GKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGCHAYGDQYRATDFVVPGPG KVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGGHAYGDQYRATDFVVPGP GKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM, or RVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGSHAYGDQYRATDFVVPGPG KVEITYTPSDGTQKVTYLVHNFEEGGGVAMGM. In some related cases, the neoantigenic peptide is not PIIIGHHAY, GHHAYGDQY, KPIIIGHHAY, IGHHAYGDQY, PIIIGCHAY, GCHAYGDQY, KPIIIGCHAY, IGCHAYGDQY, PIIIGGHAY, GGHAYGDQY, KPIIIGGHAY, or IGGHAYGDQY.

[0191] In some cases, the isolated neoantigenic peptide is encoded by a KIT gene. In related cases, A x B y C z is VEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIANLLGACTIGGPTLVIT EYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYK or VEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIANLLGACTIGGPTLVIT EYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYK.

[0192] In some cases, the isolated neoantigenic peptide is MEK gene. In related cases, A x B y C z is ISELGAGNGGVVFKVSHKPSGLVMARKLIHLEIKPAIRNQIIRELQVLHESNSPYIVGFYGAFYSDGEIS ICMEHMDGGSLDQVLKKAGRIPEQILGKVSI or LGAGNGGVVFKVSHKPSGLVMARKLIHLEIKPAIRNQIIRELQVLHECNSLYIVGFYGAFYSDGEISIC MEHMDGGSLDQVLKKAGRIPEQILGKVSIAVI.

[0193] In some cases, the isolated neoantigenic peptide is encoded by a MYC gene. In related cases, A x B y C z is MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSDLQPPAPSEDIWKKFELLPTPPLSPSRRSG LCSPSYVAVTPFSLRGDNDGG, FTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLSTPPLSPSRRSGLCSPSY VAVTPFSLRGDNDGGGGSFSTADQLEMVTELLG, or TNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPIPPLSPSRRSGLCSPSYVA VTPFSLRGDNDGGGGSFSTADQLEMVTELLGG.

[0194] In some cases, the isolated neoantigenic peptide is encoded by a PDGFRa gene. In related cases, A x B y C z is VAVKMLKPTARSSEKQALMSELKIMTHLGPHLNIVNLLGACTKSGPIYIIIEYCFYGDLVNYLHKNRD SFLSHHPEKPKKELDIFGLNPADESTRSYVILS.

[0195] In some cases, the isolated neoantigenic peptide is encoded by a PIK3CA gene. In related cases, A x B y C z is IEEHANWSVSREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSKITEQEKDFLWSHRHYC VTIPEILPKLLLSVKWNSRDEVAQMYCLVKDWPP, HANWSVSREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSEITKQEKDFLWSHRHYCVTI PEILPKLLLSVKWNSRDEVAQMYCLVKDWPPIKP, or LFINLFSMMLGSGMPELQSFDDIAYIRKTLALDKTEQEALEYFMKQMNDARHGGWTTKMDWIFHTI KQHALN. In some related cases, the neoantigenic peptide is not ISTRDPLSK, STRDPLSKI, LSKITEQEK, AISTRDPLSK, SKITEQEKDF, SEITKQEKDF, KQEKDFLWSH, FMKQMNDAR, KQMNDARHG, RHGGWTTKM, YFMKQMNDAR, FMKQMNDARH, KQMNDARHGG, QMNDARHGGW, or ARHGGWTTKM.

[0196] In some cases, the isolated neoantigenic peptide is encoded by a POLE gene. In related cases, A x B y C z is QRGGVITDEEETSKKIADQLDNIVDMREYDVPYHIRLSIDIETTKLPLKFRDAETDQIMMISYMIDGQG YLITNREIVSEDIEDFEFTPKPEYEGPFCVFN. In some related cases, the neoantigenic peptide is not TTKLPLKFR, RDAETDQIM, KFRDAETDQI, ETTKLPLKFR, or RDAETDQIMM.

[0197] In some cases, the isolated neoantigenic peptide is encoded by a PTEN gene. In related cases, A x B y C z is KFNCRVAQYPFEDHNPPQLELIKPFCEDLDQWLSEDDNHVAAIHCKAGKGQTGVMICAYLLHRGKF LKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSY. In some related cases, the neoantigenic peptide is not QTGVMICAY, GKGQTGVMI, GQTGVMICAY, or KAGKGQTGVM.

[0198] In some cases, the isolated neoantigenic peptide is encoded by a RAC1 gene. In related cases, A x B y C z is MQAIKCVVVGDGAVGKTCLLISYTTNAFSGEYIPTVFDNYSANVMVDGKPVNLGLWDTAGQEDYD RLRPLSYPQTVGET. In some related cases, the neoantigenic peptide is not TTNAFSGEY, FSGEYIPTV, SGEYIPTVF, YTTNAFSGEY, TTNAFSGEYI, or FSGEYIPTVF.

[0199] In some cases, the isolated neoantigenic peptide is encoded by a TP53 gene. In related cases, A x B y C z is IRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGSMNRRPILTIITLEDSSG NLLGRNSFEVRVCACPGRDRRTEEENLRKKGEP, TYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRHCPHHERCSDSDGLAP PQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEV, EGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNQRPILTIITLEDSSGNLL GRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE, EGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNWRPILTIITLEDSSGNLL GRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE, or PEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVCVCACPGRDRRTEEENLR KKGEPHHELPPGSTKRALPNNTSSSPQPKKKPL. In some related cases, the neoantigenic peptide is not SSCMGSMNR, GSMNRRPIL, MGSMNRRPI, CNSSCMGSM, SMNRRPILTI, SSCMGSMNRR, NSSCMGSMNR, MGSMNRRPIL, MCNSSCMGSM, CMGSMNRRPI, TEVVRHCPH, VVRHCPHHER, SQHMTEVVRH, MNQRPILTI, NQRPILTII, CMGGMNQRPI, GMNQRPILTI, SSCMGGMNQR, NQRPILTIIT, NWRPILTII, SSCMGGMNW, MGGMNWRPI, MNWRPILTI, CMGGMNWRPI, GMNWRPILTI, SSCMGGMNWR, MNWRPILTII, NSSCMGGMNW, NSFEVCVCA, EVCVCACPGR, or FEVCVCACPG.

[0200] In some cases, the isolated neoantigenic peptide is encoded by a gene with a frameshift mutation In some cases, the isolated neoantigenic peptide is encoded by a ACVR2A gene. In related cases, A x B y C z is GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKRQP or GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKTALKYIFVAVRAICVM KSFLIFRRWKSHSPLQIQLHLSHPITTSCSIPWCHLC.

[0201] In some cases, the isolated neoantigenic peptide is C15ORF40 gene. In related cases, A x B y C z is TAEAVNVAIAAPPSEGEANAELCRYLSKVLELRKSDVVLDKVGLALFFFFFETKSCSVAQAGVQWRS LGSLQPPPPGFKLFSCLSFLSSWDYRRMPPCLANFCIFNRDGVSPCWSGWS.

[0202] In some cases, the isolated neoantigenic peptide is encoded by a CNOT1 gene. In related cases, A x B y C z is LSVIIFFFVYIWHWALPLILNNHHICLMSSIILDCNSVRQSIMSVCFFFFSVIFSTRCLTDSRYPNICWFK or LSVIIFFFVYIWHWALPLILNNHHICLMSSIILDCNSVRQSIMSVCFFFFCYILNTMFDR.

[0203] In some cases, the isolated neoantigenic peptide is encoded by a EIF2B3 gene. In related cases, A x B y C z is VLVLSCDLITDVALHEVVDLFRAYDASLAMLMRKGQDSIEPVPGQKGKKKQWSSVTSLEWTAQERG CSSWLMKQTWMKSWSLRDPSYRSILEYVSTRVLWMPTSTV.

[0204] In some cases, the isolated neoantigenic peptide is encoded by a EPHB2 gene. In related cases, A x B y C z is SIQVMRAQMNQIQSVEGQPLARRPRATGRTKRCQPRDVTKKTCNSNDGKKREWEKRKQILGGGGK YKEYFLKRILIRKAMTVLAGDKKGLGRFMRCVQSETKAVSLQLPLGR.

[0205] In some cases, the isolated neoantigenic peptide is encoded by a ESRP1 gene. In related cases, A x B y C z is LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKKHEGQIC or LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKT.

[0206] In some cases, the isolated neoantigenic peptide is encoded by a FAM11B gene. In related cases, A x B y C z is GALCKDGRFRSDIGEFEWKLKEGHKKIYGKQSMVDEVSGKVLEMDISKKKHYNRKISIKKLNRMKV PLMKLITRV. In some related cases, the neoantigenic peptide is not KLNRMKVPL, PLMKLITRV, RMKVPLMKL, ISKKKHYNR, SKKKHYNRK, KHYNRKISI, HYNRKISIK, YNRKISIKK, KISIKKLNR, SIKKLNRMK, LNRMKVPLM, ISIKKLNRM, MKVPLMKLI, KLNRMKVPLM, RMKVPLMKLI, ISIKKLNRMK, ISKKKHYNRK, KHYNRKISIK, HYNRKISIKK, KISIKKLNRM, SIKKLNRMKV, LNRMKVPLMK, KVPLMKLITR, DISKKKHYNR, KKHYNRKISI, KKLNRMKVPL, or VPLMKLITRV.

[0207] In some cases, the isolated neoantigenic peptide is encoded by a GBP3 gene. In related cases, A x B y C z is RERAQLLEEQEKTLTSKLQEQARVLKERCQGESTQLQNEIQKLQKTLKKKPRDICRIS.

[0208] In some cases, the isolated neoantigenic peptide is encoded by a JAK1 gene. In related cases, A x B y C z is VNTLKEGKRLPCPPNCPDEVYQLMRKCWEFQPSNRTSFQNLIEGFEALLKTSN or CRPVTPSCKELADLMTRCMNYDPNQRPFFRAIMRDINKLEEQNPDIVSEKNQQLKWTPHILKSAS.

[0209] In some cases, the isolated neoantigenic peptide is encoded by a LMAN1 gene. In related cases, A x B y C z is DDHDVLSFLTFQLTEPGKEPPTPDKEISEKEKEKYQEEFEHFQQELDKKKRGIPEGPPRPPRAACGGNI or DDHDVLSFLTFQLTEPGKEPPTPDKEISEKEKEKYQEEFEHFQQELDKKKRNSRRATPTSKGSLRRKY LRV.

[0210] In some cases, the isolated neoantigenic peptide is encoded by a MSH3 gene. In related cases, A x B y C z is TKSTLIGEDVNPLIKLDDAVNVDEIMTDTSTSYLLCISENKENVRDKKKGQHFYWHCGSAACHRRGC V or LYTKSTLIGEDVNPLIKLDDAVNVDEIMTDTSTSYLLCISENKENVRDKKRATFLLALWECSLPQARL CLIVSRTLLLVQS.

[0211] In some cases, the isolated neoantigenic peptide is encoded by a NDUFC2 gene. In related cases, A x B y C z is LPPPKLTDPRLLYIGFLGYCSGLIDNLIRRRPIATAGLHRQLLYITAFFFCWILSCKT, or SLPPPKLTDPRLLYIGFLGYCSGLIDNLIRRRPIATAGLHRQLLYITAFFLLDIIL.

[0212] In some cases, the isolated neoantigenic peptide is encoded by a RBM27 gene. In related cases, A x B y C z is NQSGGAGEDCQIFSTPGHPKMIYSSSNLKTPSKLCSGSKSHDVQEVLKKKTGSNEVTTRYEEKKTGSV RKANRMPKDVNIQVRKKQKHETRRKSKYNEDFERAWREDLTIKR.

[0213] In some cases, the isolated neoantigenic peptide is encoded by a RPL22 gene. In related cases, A x B y C z is MAPVKKLVVKGGKKKEASSEVHS or MAPVKKLVVKGGKKRSKF. In some related cases, the neoantigenic peptide is not VVKGGKKRSK or VKGGKKRSK

[0214] In some cases, the isolated neoantigenic peptide is encoded by a SEC31A gene. In related cases, A x B y C z is MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKN or MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKLMLLRLNLRKMCGPF.

[0215] In some cases, the isolated neoantigenic peptide is encoded by a SEC63 gene. In related cases, A x B y C z is AEVFEKEQSICAAEEQPAEDGQGETNKNRTKGGWQQKSKGPKKTAKSKKKETFKKKTYTCAITTVK ATETKAGKWSRWE or MAEVFEKEQSICAAEEQPAEDGQGETNKNRTKGGWQQKSKGPKKTAKSKKRNL.

[0216] In some cases, the isolated neoantigenic peptide is encoded by a SLC35F5 gene. In related cases, A x B y C z is NIMEIRQLPSSHALEAKLSRMSYPVKEQESILKTVGKLTATQVAKISFFFALCGFWQICHIKKHFQTHK LL.

[0217] In some cases, the isolated neoantigenic peptide is encoded by a SMAP1 gene. In related cases, A x B y C z is YEKKKYYDKNAIAITNISSSDAPLQPLVSSPSLQAAVDKNKLEKEKEKKKGREKERKGARKAGKTTY S or KYEKKKYYDKNAIAITNISSSDAPLQPLVSSPSLQAAVDKNKLEKEKEKKRKRKREKRSQKSRQNHL QLKSCRRKISNWSLKKVPALKKLRSPLWIF. In some related cases, the neoantigenic peptide is not ALKKLRSPL, KISNWSLKK, SLKKVPALK, KLRSPLWIF, KKRKRKREK, RKREKRSQK, RSQKSRQNH, SQKSRQNHL, KSRQNHLQL, RRKISNWSL, RKISNWSLK, KVPALKKLR, HLQLKSCRR, WSLKKVPAL, RQNHLQLKS, KKVPALKKL, LKKLRSPLW, KKLRSPLWI, KISNWSLKKV, KSRQNHLQLK, SLKKVPALKK, WSLKKVPALK, KRKREKRSQK, RSQKSRQNHL, HLQLKSCRRK, RRKISNWSLK, CRRKISNWSL, NWSLKKVPAL, QKSRQNHLQL, RQNHLQLKSC, LQLKSCRRKI, ALKKLRSPLW, or KKLRSPLWI

[0218] In some cases, the isolated neoantigenic peptide is encoded by a TFAM gene. In related cases, A x B y C z is IYQDAYRAEWQVYKEEISRFKEQLTPSQIMSLEKEIMDKHLKRKAMTKKKRVNTAWKTKKTSFSL or IYQDAYRAEWQVYKEEISRFKEQLTPSQIMSLEKEIMDKHLKRKAMTKKKS.

[0219] In some cases, the isolated neoantigenic peptide is encoded by a TGFBR2 gene. In related cases, A x B y C z is KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKAW or EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKSLVRLSSCVPVALMSAM TTSSSQKNITPAILTCC.

[0220] In some cases, the isolated neoantigenic peptide is encoded by a THAP5 gene. In related cases, A x B y C z is VPSKYQFLCSDHFTPDSLDIRWGIRYLKQTAVPTIFSLPEDNQGKDPSKKNPRRKTWKMRKKYAQKP SQKNHLY.

[0221] In some cases, the isolated neoantigenic peptide is encoded by a TTK gene. In related cases, A x B y C z is GTTEEMKYVLGQLVGLNSPNSILKAAKTLYEHYSGGESHNSSSSKTFEKKGEKNDLQLFVMSDTTYK IYWTVILLNPCGNLHLKTTSL

[0222] In some cases, the isolated neoantigenic peptide is encoded by a XPOT gene. In related cases, A x B y C z is QQLIRETLISWLQAQMLNPQPEKTFIRNKAAQVFALLFVTEYLTKWPKFFLTFSQ.

[0223] In some cases, the isolated neoantigenic peptide is encoded by a APC gene. In related cases, C z is AKFQQCHSTLEPNPADCRVLVYLQNQPGTKLLNFLQERNLPPKVVLRHPKVHLNTMFRRPHSCLAD VLLSVHLIVLRVVRLPAPFRVNHAVEW, APVIFQIALDKPCHQAEVKHLHHLLKQLKPSEKYLKIKHLLLKRERVDLSKLQ, or MLQFRGSRFFQMLILYYILPRKVLQMDFLVHPA. In some related cases, the neoantigenic peptide is not ADVLLSVHL, ADVLLSVHLI, APFRVNHAV, ARHKAVEFL, CLADVLLSV, CLADVLLSVH, DVLLSVHLI, DVLLSVHLIV, FLQERNLPPK, FRVNHAVEW, HLIVLRVVR, HLIVLRVVRL, HLNTMFRRPH, HPKVHLNTM, HPKVHLNTMF, KAVEFLQER, KVHLNTMFR, KVHLNTMFRR, KVVLRHPKV, LLSVHLIVL, LLSVHLIVLR, LPAPFRVNH, LPAPFRVNHA, LQERNLPPKV, LRVVRLPAPF, LSVHLIVLR, LSVHLIVLRV, MFRRPHSCL, MFRRPHSCLA, NLPPKVVLR, NTMFRRPHSC, QERNLPPKV, RHPKVHLNTM, RNLPPKVVL, RNLPPKVVLR, RPHSCLADV, RPHSCLADVL, RVVRLPAPF, RVVRLPAPFR, SARHKAVEFL, SVHLIVLRV, SVHLIVLRVV, TMFRRPHSC, TMFRRPHSCL, VEFLQERNL, VLLSVHLIV, VLLSVHLIVL, VLRHPKVHL, VLRVVRLPA, VVRLPAPFR, or VVRLPAPFRV.

[0224] In some cases, the isolated neoantigenic peptide is encoded by a ARID1A gene. In related cases, C z is ALGPHSRISCLPTQTRGCILLAATPRSSSSSSSNDMIPMAISSPPKAPLLAAPSPASRLQCINSNSRITSGQ WMAHMALLPSGTKGRCTACHTALGRGSLSSSSCPQPSPSLPASNKLPSLPLSKMYTTSMAMPILPLPQ LLLSADQQAAPRTNFHSSLAETVSLHPLAPMPSKTCHHK, AHQGFPAAKESRVIQLSLLSLLIPPLTCLASEALPRPLLALPPVLLSLAQDHSRLLQCQATRCHLGHPV ASRTASCILP, PILAATGTSVRTAARTWVPRAAIRVPDPAAVPDDHAGPGAECHGRPLLYTADSSLWTTRPQRVWST GPDSILQPAKSSPSAAAATLLPATTVPDPSCPTFVSAAATVSTTTAPVLSASILPAAIPASTSAVPGSIPL PAVDDTAAPPEPAPLLTATGSVSLPAAATSAASTLDALPAGCVSSAPVSAVPANCLFPAALPSTAGAIS RFIWVSGILSPLNDLQ, PCRAGRRVPWAASLIHSRFLLMDNKAPAGMVNRARLHITTSKVLTLSSSSHPTPSNHRPRPLMPNLRI SSSHSLNHHSSSPLSLHTPSSHPSLHISSPRLHTPPSSRRHSSTPRASPPTHSHRLSLLTSSSNLSSQHPRR SPSRLRILSPSLSSPSKLPIPSSASLHRRSYLKIHLGLRHPQPPQ, RTNPTVRMRPHCVPFWTGRILLPSAASVCPIPFEACHLCQAMTLRCPNTQGCCSSWAS, or TNQALPKIEVICRGTPRCPSTVPPSPAQPYLRVSLPEDRYTQAWAPTSRTPWGAMVPRGVSMAHKVA TPGSQTIMPCPMPTTPVQAWLEA. In some related cases, the neoantigenic peptide is not AAWRSCIAL, AAWRSCIALW, AETVSLHPL, AETVSLHPLA, AHMALLPSG, ALWCASSVT, AMPILPLPQL, APLLAAPSPA, APRTNFHSS, APRTNFHSSL, ASNKLPSLPL, AWRSCIALW, CAGRWLWYCW, CIALWCASSV, CPQPSPSLPA, CRRAVSATSW, CTACHTALGR, DQQAAPRTNF, ETVSLHPLA, FHSSLAETV, GMYSPSRYPR, GQWMAHMAL, GQWMAHMALL, GRCTACHTAL, GTAWQLVPL, HMALLPSGT, HMALLPSGTK, HPLAPMPSK, HSSLAETVSL, HTALGRGSL, IALWCASSV, ILATPPSAA, ILATPPSAAW, IPMAISSPP, IPMAISSPPK, ISSPPKAPL, ISSPPKAPLL, ITSGQWMAHM, KGRCTACHT, KGRCTACHTA, KLPSLPLSK, KLPSLPLSKM, KMYTTSMAM, KMYTTSMAMP, LAAPSPASR, LAAPSPASRL, LAETVSLHPL, LATPPSAAW, LATPPSAAWR, LLAAPSPASR, LLLSADQQAA, LLSADQQAA, LPASNKLPS, LPASNKLPSL, LPLPQLLLS, LPLPQLLLSA, LPSLPLSKM, LPSLPLSKMY, LQCINSNSRI, LQCRRAVSA, LQCRRAVSAT, LSADQQAAPR, LSKMYTTSM, LSKMYTTSMA, LWCASSVTER, LWYCWPTWL, LWYCWPTWLR, MAHMALLPS, MALLPSGTK, MPILPLPQL, MPILPLPQLL, MYSPSRYPR, MYTTSMAMPI, PMAISSPPK, QLVPLQCRR, QQAAPRTNF, QQAAPRTNFH, QWMAHMALL, RAVSATSWA, RAVSATSWAS, RCAGRWLWY, RCTACHTAL, RGTAWQLVPL, RLQCINSNSR, RRAVSATSW, RTNFHSSLA, RTNFHSSLAE, RTRCAGRWL, RTRCAGRWLW, RWLWYCWPT, RWLWYCWPTW, SAAWRSCIA, SAAWRSCIAL, SGQWMAHMAL, SKMYTTSMA, SKMYTTSMAM, SMAMPILPL, SNKLPSLPL, SPASRLQCI, SPPKAPLLAA, SPSLPASNKL, SRITSGQWM, SSCPQPSPSL, SSLAETVSL, SSNDMIPMAI, SSSNDMIPM, SSSSNDMIPM, SSSSSSNDM, SSSSSSSNDM, TACHTALGR, TERTRCAGRW, TSMAMPILPL, TTSMAMPIL, TVSLHPLAPM, TWLRGTAWQL, VPLQCRRAV, VSLHPLAPM, VTERTRCAGR, WLRGTAWQL, WLRGTAWQLV, WLWYCWPTW, WLWYCWPTWL, WMAHMALLPS, WPTWLRGTA, WPTWLRGTAW, WYCWPTWLR, YPRSSSSSS, YPRSSSSSSS, YTTSMAMPI, or YTTSMAMPIL.

[0225] In some cases, the isolated neoantigenic peptide is encoded by a β2M gene. In related cases, C z is RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKMSMPAV, or LCSRYSLFLAWRLSSVLQRFRFTHVIQQRMESQIS. In some related cases, the neoantigenic peptide is not ALAVLALLSF, ALLSFWPGGY, DSGLLTSSSR, ELLCVWVSSI, EWKVKFPEL, FPELLCVWV, FWPGGYPAY, GLLTSSSREW, KFPELLCVW, KFPELLCVWV, KVKFPELLC, KVKFPELLCV, LAVLALLSF, LAVLALLSFW, LLCVWVSSI, LLCVWVSSIR, LLSFWPGGY, LLTSSSREW, LLTSSSREWK, LSFWPGGYPA, LTSSSREWK, LTSSSREWKV, REWKVKFPE, REWKVKFPEL, SFWPGGYPA, SFWPGGYPAY, SSREWKVKF, SSSREWKVK, SSSREWKVKF, TSSSREWKV, TSSSREWKVK, VKFPELLCV, VKFPELLCVW, WKVKFPELL, or YPAYSKDSGL.

[0226] In some cases, the isolated neoantigenic peptide is encoded by a CDH1 gene. In related cases, C z is RSACVTVKGPLASVGRHSLSKQDCKFLPFWGFLEEFLLC, IQWGTTTAPRPIRPPFLESKQNCSHFPTPLLASEDRRETGLFLPSAAQKMKKAHFLKTWFRSNPTKTK KARFSTASLAKELTHPLLVSLLLKEKQDG, PTDPFLGLRLGLHLQKVFHQSHAEYSGAPPPPPAPSGLRFWNPSRIAHISQLLSWPQKTEERLGYSSHQ LPRK, FCCSCCFFGGERWSKSPYCPQRMTPGTTFITMMKKEAEKRTRTLT, or WRRNCKAPVSLRKSVQTPARSSPARPDRTRRLPSLGVPGQPWALGAAASRRCCCCCRSPLGSARSRS PATLALTPRATRSRCPGATWREAASWAE.

[0227] In some cases, the isolated neoantigenic peptide is encoded by a GATA3 gene. In related cases, C z is PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGPPARVPA VPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH or PRPRRCTRHPACPLDHTTPPAWSPPWVRALLDAHRAPSESPCSPFRLAFLQEQYHEA. In some related cases, the neoantigenic peptide is not AALSRHNVL, ADAPAIQPV, ADAPAIQPVL, AESKIMFAT, AESKIMFATL, AIQPVLWTT, ALQPLQPHA, APAIQPVLW, ARVPAVPFDL, ATLQRSSLW, AVPFDLHFCR, CSMLTGPPA, CSMLTGPPAR, DLHFCRSSI, DLHFCRSSIM, EPHLALQPL, ESKIMFATL, FATLQRSSL, FATLQRSSLW, FCRSSIMKPK, FDLHFCRSSI, FLKAESKIM, FLKAESKIMF, GPPARVPAV, GYMFLKAESK, HADAPAIQPV, HAHADAPAI, HFCRSSIMK, HLALQPLQPH, HMSSLSHISA, HPLQHGHRH, HPPSSLSFW, HRHGLEPCSM, IMFATLQRS, IMFATLQRSS, IMKPKRDGY, IMKPKRDGYM, KAESKIMFA, KIMFATLQR, KPKRDGYMF, KPKRDGYMFL, LALQPLQPH, LHFCRSSIM, LHFCRSSIMK, LKAESKIMF, LQHGHRHGL, LQPHADHAH, LQRSSLWCL, LQRSSLWCLC, LSFGPHHPL, LSFGPHPPL, LSFWTTPPL, LSHISALQPL, MFATLQRSSL, MFLKAESKI, MFLKAESKIM, MHPPSSLSFW, MKPKRDGYMF, MLTGPPARV, MSSLSHISA, MSSLSHISAL, NPAALSRHNV, PARVPAVPF, PAVPFDLHF, PEPHLALQPL, PPARVPAVPF, QPVLWTTPPL, RHGLEPCSM, RHNVLPEPHL, RSSIMKPKR, SALQPLQPH, SHISALQPL, SIMKPKRDGY, SLSFGPHHPL, SLSFGPHPPL, SLSFWTTPPL, SMLTGPPAR, SMLTGPPARV, SSLSHISAL, TLQRSSLWCL, TTPPLQHGHR, VLPEPHLAL, VPAVPFDLHF, VPFDLHFCR, YMFLKAESK or YMFLKAESKI.

[0228] In some cases, the isolated neoantigenic peptide is encoded by a MLL2 gene. In related cases, C z is TRRCHCCPHLRSHPCPHHLRNHPRPHHLRHHACHHHLRNCPHPHFLRHCTCPGRWRNRPSLRRLRSL LCLPHLNHHLFLHWRSRPCLHRKSHPHLLHLRRLYPHHLKHRPCPHHLKNLLCPRHLRNCPLPRHLK HLACLHHLRSHPCPLHLKSHPCLHHRRHLVCSHHLKSLLCPLHLRSLPFPHHLRHHACPHHLRTRLCP HHLKNHLCPPHLRYRAYPPCLWCHACLHRLRNLPCPHRLRSLPRPLHLRLHASPHHLRTPPHPHHLR THLLPHHRRTRSCPCRWRSHPCCHYLRSRNSAPGPRGRTCHPGLRSRTCPPGLRSHTYLRRLRSHTCP PSLRSHAYALCLRSHTCPPRLRDHICPLSLRNCTCPPRLRSRTCLLCLRSHACPPNLRNHTCPPSLRSHA CPPGLRNRICPLSLRSHPCPLGLKSPLRSQANALHLRSCPCSLPLGNHPYLPCLESQPCLSLGNHLCPLC PRSCRCPHLGSHPCRLS. In some related cases, the neoantigenic peptide is not APGPRGRTC, CHYLRSRNSA, CLRSHTCPPR, CLWCHACLHR, CPHLGSHPC, CPHRLRSLPR, CPLGLKSPL, CPPGLRNRI, CPPGLRSHTY, CPPRLRDHI, CPPSLRSHAY, CPRSCRCPH, CPRSCRCPHL, CSLPLGNHPY, CTCPPRLRSR, DHICPLSLR, EESPMSPHL, ESPMSPHLR, ESPMSPHLRY, EVSRLSPCL, GLKSPLRSQA, GLRNRICPL, GLRNRICPLS, GLRSHTYLR, GLRSHTYLRR, GLRSRTCPPG, HACPPGLRNR, HAYALCLRSH, HHLRTHLLPH, HLGSHPCRL, HLLPHHRRTR, HLRLHASPH, HLRLHASPHH, HLRSCPCSL, HLRTHLLPH, HLRTHLLPHH, HLRTPPHPH, HLRTPPHPHH, HLRYRAYPP, HLRYRAYPPC, HPCCHYLRSR, HPHHLRTHL, HPHHLRTHLL, HRLRSLPRPL, HTYLRRLRS, HTYLRRLRSH, HYLRSRNSA, KSPLRSQANA, LESQPCLSL, LHLRLHASPH, LHLRSCPCSL, LLPHHRRTR, LPCPHRLRSL, LPHHRRTRS, LPHHRRTRSC, LPLGNHPYL, LPRPLHLRL, LRLHASPHHL, LRNCTCPPRL, LRNHTCPPSL, LRNRICPLSL, LRSCPCSLPL, LRSHACPPGL, LRSHACPPNL, LRSHAYALCL, LRSHTCPPRL, LRSHTCPPSL, LRSHTYLRRL, LRSLPRPLHL, LRSQANALHL, LRTPPHPHHL, LRYRAYPPCL, LSLGNHLCPL, LSLRSHPCPL, LWCHACLHRL, MSPHLRYRA, MSPHLRYRAY, NLPCPHRLR, NLRNHTCPP, PMSPHLRYR, PPRLRSRTCL, PPSLRSHAY, RAYPPCLWCH, RDHICPLSL, RGRTCHPGL, RGRTCHPGLR, RLHASPHHL, RLHASPHHLR, RLRDHICPL, RLRDHICPLS, RLRNLPCPH, RLRNLPCPHR, RLRSHTCPP, RLRSHTCPPS, RLRSLPRPL, RLRSLPRPLH, RLRSRTCLL, RLRSRTCLLC, RLSPCLWCHA, RNHTCPPSL, RNHTCPPSLR, RNLPCPHRLR, RNRICPLSL, RNRICPLSLR, RPLHLRLHA, RPLHLRLHAS, RSCPCRWRSH, RSCPCSLPL, RSHACPPGL, RSHACPPGLR, RSHACPPNL, RSHACPPNLR, RSHAYALCL, RSHAYALCLR, RSHPCCHYL, RSHPCCHYLR, RSHPCPLGL, RSHPCPLGLK, RSHTCPPRL, RSHTCPPRLR, RSHTCPPSL, RSHTCPPSLR, RSHTYLRRL, RSHTYLRRLR, RSLPRPLHL, RSLPRPLHLR, RSQANALHL, RSQANALHLR, RSRNSAPGP, RSRNSAPGPR, RSRTCLLCL, RSRTCLLCLR, RSRTCPPGL, RSRTCPPGLR, RTCHPGLRSR, RTHLLPHHR, RTHLLPHHRR, RTPPHPHHL, RTPPHPHHLR, RTRSCPCRW, RTRSCPCRWR, RWRSHPCCH, RWRSHPCCHY, RYRAYPPCL, RYRAYPPCLW, SHAYALCLR, SLGNHLCPL, SLPLGNHPY, SLPLGNHPYL, SLPRPLHLR, SLPRPLHLRL, SLRNCTCPPR, SLRSHACPPG, SLRSHAYAL, SLRSHAYALC, SLRSHPCPL, SLRSHPCPLG, SPHHLRTPP, SPHHLRTPPH, SPHLRYRAY, SPLRSQANAL, SPMSPHLRY, SPMSPHLRYR, SQANALHLR, SQANALHLRS, VSRLSPCLW, WRSHPCCHY, WRSHPCCHYL, YLPCLESQPC, YLRRLRSHTC, YLRSRNSAP, or YLRSRNSAPG.

[0229] In some cases, the isolated neoantigenic peptide is encoded by a PTEN gene. In related cases, C z is SWKGTNWCNDMCIFITSGQIFKGTRGPRFLWGSKDQRQKGSNYSQSEALCVLL, KRTKCFTFG, PIFIQTLLLWDFLQKDLKAYTGTILMM, QKMILTKQIKTKPTDTFLQILR, GFWIQSIKTITRYTIFVLKDIMTPPNLIAELHNILLKTITHHS, NYSNVQWRNLQSSVCGLPAKGEDIFLQFRTHTTGRQVHVL, or YQSRVLPQTEQDAKKGQNVSLLGKYILHTRTRGNLRKSRKWKSM. In some related cases, the neoantigenic peptide is not KMLKRTKCF, MLKRTKCFT, LKRTKCFTF, MLKRTKCFTF, KQNKMLKRTK, KMLKRTKCFT, or NKMLKRTKCF.

[0230] In some cases, the isolated neoantigenic peptide is encoded by a TP53 gene. In related cases, C z is SSQNARGCSPRGPCTSSSYTGGPCTSPLLAPVIFCPFPENLPGQLRFPSGLLAFWDSQVCDLHVLPCPQ QDVLPTGQDLPCAAVG, GAAPTMSAAQIAMVWPLLSILSEWKEICVWSIWMTETLFDIVWWCPMSRLRLALTVPPSTTTTCVTV PAWAA, TGGPSSPSSHWKTPVVIYWDGTALRCVFVPVLGETGAQRKRISARKGSLTTSCPQGALSEHCPTTPAP LPSQRRNHWMENISPFRSVGVSASRCSES, FHTPARHPRPRHGHLQAVTAHDGGCEALPPP, CCPRTILNNGSLKTQVQMKLPECQRLLPPWPLHQQLLHRRPLHQPPPGPCHLLSLPRKPTRAATVSV WASCILGQPSL, VRKHFQTYGNYFLKTTFCPPCRPKQWMI, or LARTPLPSTRCFANWPRPALCSCGLIPHPRPAPASAPWPSTSSHST. In some related cases, the neoantigenic peptide is not APASAPWPST, APPWPLHQQL, APWPSTSSH, ASCILGQPSL, ATVSVWASCI, CQRLLPPWPL, HQPPPGPCHL, HQQLLHRRPL, IEQWFTEDQV, KLPECQRLL, KPTRAATVSV, KTYQGSYGFV, KTYQGSYVS, KTYQGSYVSV, LLSLPRKPTR, LPPWPLHQQL, LPRKPTRAA, LPRKPTRAAT, LSLPRKPTR, MKLPECQRL, MKLPECQRLL, MPEAAPPWPL, PEAAPPWPL, PTRAATVSV, QMKLPECQR, QQLLHRRPL, QQLLHRRPLH, QRLLPPWPL, QWFTEDQVQM, RAATVSVWA, RLLPPWPLH, RMPEAAPPW, RPAPASAPW, SLPRKPTRA, SLPRKPTRAA, SQKTYQGSYV, STPRPAPASA, SYGFVWASC, SYGFVWASCI, SYVSVWASC, SYVSVWASCI, TEDQVQMKL, TPRPAPASA, TPRPAPASAP, TRAATVSVW, TVSVWASCI, TVSVWASCIL, TYQGSYGFV, TYQGSYGFVW, TYQGSYVSV, TYQGSYVSVW, VSVWASCIL, WPLHQQLLH, WPSTSSHST, YGFVWASCI, YGFVWASCIL, YQGSYGFVW, YQGSYGFVWA, YQGSYVSVW, YQGSYVSVWA, YVSVWASCI, or YVSVWASCIL.

[0231] In some cases, the isolated neoantigenic peptide is encoded by a VHL gene. In related cases, C z is ELQETGHRQVALRRSGRPPKCAERPGAADTGAHCTSTDGRLKISVETYTVSSQLLMVLMSLDLDTGL VPSLVSKCLILRVK, KSDASRLSGA, RTAYFCQYHTASVYSERAMPPGCPEPSQA, TRASPPRSSSAIAVRASCCPYGSTSTASRSPTQRCRLARAAASTATEVTFGSSEMQGHTMGFWLTKLN YLCHLSMLTDSLFLPISHCQCIL, SSLRITGDWTSSGRSTKIWKTTQMCRKTWSG, or RRRRGGVGRRGVRPGRVRPGGTGRRGGDGGRAAAARAALGELARALPGHLLQSQSARRAARMAQ LRRRAAALPNAAAWHGPPHPQLPRSPLALQRCRDTRWASG. In some related cases, the neoantigenic peptide is not CHLSMLTDSL, EMQGHTMGF, FLPISHCQC, FLPISHCQCI, FRDAGHTMGF, FWLTKLNYL, GFWLTKLNY, GFWLTKLNYL, GSSEMQGHTM, HLSMLTDSL, HLSMLTDSLF, HSYRGHLGSS, HTMGFWLTK, HTMGFWLTKL, KERCLQLSGA, KLNYLCHLSM, LFRDAGHTM, LNYLCHLSM, LNYLCHLSML, LPISHCQCI, LPISHCQCIL, LSMLTDSLF, LSMLTDSLFL, LTDSLFLPI, LTKLNYLCHL, MGFWLTKLNY, MLTDSLFLPI, MQGHTMGFW, MQGHTMGFWL, NYLCHLSML, RDAGHTMGF, RDAGHTMGFW, RGHLGSSEM, RIHSYRGHLG, SEMQGHTMG, SEMQGHTMGF, SLFLPISHC, SMLTDSLFL, SSEMQGHTM, TKLNYLCHL, TLKERCLQL, TMGFWLTKL, WLFRDAGHT, WLFRDAGHTM, YLCHLSMLT, or YRGHLGSSEM.

[0232] In some cases, the isolated neoantigenic peptide is encoded by a fusion of a first gene with a second gene. In some cases, the isolated neoantigenic peptide is encoded by an in-frame fusion of a first gene with a second gene.

[0233] In some cases, the isolated neoantigenic peptide is encoded by a BCR gene and an ABL gene. In related cases, A x B y C z is ERAEWRENIREQQKKCFRSFSLTSVELQMLTNSCVKLQTVHSIPLTINKEEALQRPVASDFEPQGLSEA ARWNSKENLLAGPSENDPNLFVALYDFVASG or ELQMLTNSCVKLQTVHSIPLTINKEDDESPGLYGFLNVIVHSATGFKQSSKALQRPVASDFEPQGLSE AARWNSKENLLAGPSENDPNLFVALYDFVASGD.

[0234] In some cases, the isolated neoantigenic peptide is encoded by a C11orf95 gene and a RELA gene. In related cases, A x B y C z is ISNSWDAHLGLGACGEAEGLGVQGAEEEEEEEEEEEEEGAGVPACPPKGPELFPLIFPAEPAQASGPY VEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTD.

[0235] In some cases, the isolated neoantigenic peptide is encoded by a CBFB gene and an MYH11 gene. LQRLDGMGCLEFDEERAQQEDALAQQAFEEARRRTREFEDRDRSHREEMEVHELEKSKRALETQME EMKTQLEELEDELQATEDAKLRLEVNMQALKGQF.

[0236] In some cases, the isolated neoantigenic peptide is encoded by a CD74 gene and an ROS1 gene. In related cases, A x B y C z is KGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKAGVPNKPGIPKLLEGS KNSIQWEKAEDNGCRITYYILEIRKSTSNNLQNQ.

[0237] In some cases, the isolated neoantigenic peptide is encoded by a EGFR gene and an SEPT14 gene. In related cases, A x B y C z is the first native polypeptide is encoded by an gene and the second native polypeptide is encoded by LPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQLQDKFEHLKMIQQEEIRKL EEEKKQLEGEIIDFYKMKAASEALQTQLSTD.

[0238] In some cases, the isolated neoantigenic peptide is encoded by a EGFR gene and an EGFR gene. In related cases, A x B y C z is MRPSGTAGAALLALLAALCPASRALEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGP CRKVCNGIGIGEFKD.

[0239] In some cases, the isolated neoantigenic peptide is encoded by a EML4 gene and an ALK gene. In related cases, A x B y C z is SWENSDDSRNKLSKIPSTPKLIPKVTKTADKHKDVIINQAKMSTREKNSQVYRRKHQELQAMQMEL QSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDL.

[0240] In some cases, the isolated neoantigenic peptide is encoded by a FGFR3 gene and an TACC3 gene. In related cases, A x B y C z is EGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDRVLTVTSTDVKATQEENRELRSRCE ELHGKNLELGKIMDRFEEVVYQAMEEVQKQKELS,

[0241] In some cases, the isolated neoantigenic peptide is encoded by a NAB gene and an STAT6 gene. In related cases, A x B y C z is RDNTLLLRRVELFSLSRQVARESTYLSSLKGSRLHPEELGGPPLKKLKQEATSKSQIMSLWGLVSKMP PEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCC.

[0242] In some cases, the isolated neoantigenic peptide is encoded by a NDRG1 gene and an ERG gene. In related cases, A x B y C z is MSREMQDVDLAEVKPLVEKGETITGLLQEFDVQEALSVVSEDQSLFECAYGTPHLAKTEMTASSSSD YGQTSKMSPRVPQQDW.

[0243] In some cases, the isolated neoantigenic peptide is encoded by a TMPRSS2 gene and an ERG gene. In related cases, A x B y C z is MALNSEALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDW.

[0244] In some cases, the isolated neoantigenic peptide is encoded by a PML gene and an RARA gene. In related cases, A x B y C z is VLDMHGFLRQALCRLRQEEPQSLQAAVRTDGFDEFKVRLQDLSSCITQGKAIETQSSSSEEIVPSPPSP PPLPRIYKPCFVCQDKSSGYHYGVSACEGCKG or RSSPEQPRPSTSKAVSPPHLDGPPSPRSPVIGSEVFLPNSNHVASGAGEAAIETQSSSSEEIVPSPPSPPPL PRIYKPCFVCQDKSSGYHYGVSACEGCKG.

[0245] In some cases, the isolated neoantigenic peptide is encoded by a RUNX1 gene and an CBFA2T1 (RUNX1T1) gene. In related cases, A x B y C z is VARFNDLRFVGRSGRGKSFTLTITVFTNPPQVATYHRAIKITVDGPREPRNRTEKHSTMPDSPVDVKT QSRLTPPTMPPPPTTQGAPRTSSFTPTTLTNGT

[0246] In some cases, 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 cases, the isolated neoantigenic peptide is encoded by a fusion of a first gene with a cryptic exon of the first gene.

[0247] In some cases, the isolated neoantigenic peptide is encoded by a AR-v7 gene and a cryptic exon encoded by the AR-v7 gene. In some cases, the isolated neoantigenic peptide is encoded by a AR-v7 gene comprising an exon of a splice variant of an AR gene. In related cases, A x B y C z is SCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGEKFRVGNCKHLKMTRP

[0248] In some cases, 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.

[0249] In some cases, the isolated neoantigenic peptide is encoded by an AC011997.1 gene and a LRRC69 gene. In related cases, y is 1. In related cases, A x B y C z is MAGAPPPASLPPCSLISDCCASNQRDSVGVGPSEPGNNIKICNESASRK.

[0250] In some cases, the isolated neoantigenic peptide is encoded by an EEF1DP3 gene and a FRY gene. In related cases, y is 1. In related cases, A x B y C z is HGWRPFLPVRARSRWNRRLDVTVANGRSWKYGWSLLRVPQVNGIQVLNVSLKSSSNVISY.

[0251] In some cases, the isolated neoantigenic peptide is encoded by an MAD1L1 gene and a MAFK gene. In related cases, y is 0. In related cases, A x B y C z is RLKEVFQTKIQEFRKACYTLTGYQIDITTENQYRLTSLYAEHPGDCLIFKLRVPGSSVLVTVPGL.

[0252] In some cases, the isolated neoantigenic peptide is encoded by an PPP1R1B gene and a STARD3 gene. In related cases, y is 1. In related cases, A x B y C z is AEVLKVIRQSAGQKTTCGQGLEGPWERPPPLDESERDGGSEDQVEDPALSALLLRPRPPRPEVGAHQ DEQAAQGADPRLGAQPACRGLPGLLTVPQPEPLLAPPSAA.

[0253] In some cases, the isolated neoantigenic peptide comprises one or more of the peptide sequences depicted in Table 1. In some cases, the isolated neoantigenic peptide comprises one or more of the peptide sequences depicted in Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, and / or Table 1F. In some cases, the isolated neoantigenic peptide does not comprise one or more of the peptide sequences depicted in Table 2. In some cases, the isolated neoantigenic peptide does not comprise one or more of the peptide sequences depicted in Table 2A, Table 2B, Table 2C, and / or Table 2D.Neoantigen polynucleotides

[0254] Polynucleotides encoding each of the peptides described herein are also part of the disclosure. As appreciated by one of ordinary skill in the art, various nucleic acid sequences can encode the same peptide due to the redundancy of the genetic code. Each of these nucleic acids falls within the scope of the present disclosure. Nucleic acids encoding peptides can be DNA or RNA, for example, mRNA, or a combination of DNA and RNA. In some cases, a nucleic acid encoding a peptide is a self-amplifying mRNA. (Brito et al., Adv. Genet. 2015; 89:179-233). Any suitable polynucleotide that encodes a peptide described herein falls within the scope of this disclosure.

[0255] The term "RNA" includes and in some cases relates to "mRNA". The term "mRNA" means "messenger-RNA" and relates to a "transcript" which is generated by using a DNA template and encodes a peptide or polypeptide. Typically, an mRNA comprises a 5'-UTR, a protein coding region, and a 3'-UTR. mRNA only possesses limited half-life in cells and in vitro. In some cases, the mRNA is self-amplifying mRNA. In the context of the present disclosure, 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 transcription kits commercially available.

[0256] The stability and translation efficiency of RNA may be modified as required. For example, RNA may be stabilized and its translation increased by one or more modifications having a stabilizing effects and / or increasing translation efficiency of RNA. Such modifications are described, for example, in PCT / EP2006 / 009448. In order to increase expression of the RNA used according to the present disclosure, it may be modified within the coding region, i.e. the sequence encoding the expressed peptide or protein, 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.

[0257] The term "modification" in the context of the RNA used in the present disclosure includes any modification of an RNA which is not naturally present in said RNA. In some cases of the disclosure, the RNA used according to the disclosure does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating RNA with a phosphatase. The RNA according to the disclosure may have modified ribonucleotides in order to increase its stability and / or decrease cytotoxicity. For example, In some cases, in the RNA used according to the disclosure 5-methylcytidine is substituted partially or completely, for example, completely, for cytidine. Alternatively or additionally, In some cases, in the RNA used according to the disclosure pseudouridine is substituted partially or completely, for example, completely, for uridine.

[0258] In some cases 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 connected to the mRNA via an unusual 5' to 5' triphosphate linkage. In some cases, this guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, to the 7-methylguanosine cap (m G). In the context of the present disclosure, the term "5'-cap" includes a 5'-cap analog that resembles 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.

[0259] In certain cases, an mRNA encoding a neoantigen peptide of the disclosure is administered to a subject in need thereof. In some cases, the disclosure 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 cases, the mRNA to be administered comprises at least one modified nucleoside.

[0260] 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 (1981). Polynucleotides encoding peptides comprising or consisting of an analog can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native epitope.

[0261] A large number of vectors and host systems suitable for producing and administering a neoantigenic 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, pD 10, phage script, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pCR (Invitrogen). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); p75.6 (Valentis); pCEP (Invitrogen); pCEI (Epimmune). However, any other plasmid or vector can be used as long as it is replicable and viable in the host.

[0262] As representative examples of appropriate hosts, there can be mentioned: bacterial cells, such as E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus; fungal 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 capable 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 herein.

[0263] Thus, the present disclosure is also directed to vectors, and expression vectors useful for the production and administration of the neoantigenic peptides described herein, and to host cells comprising such vectors.

[0264] Host cells are genetically engineered (transduced or transformed 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.

[0265] For expression of the neoantigenic peptides described herein, the coding sequence will be provided operably linked start and stop codons, promoter and terminator regions, and in some cases, and a replication 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 desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts.

[0266] Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived 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 initiation and termination sequences, and in some cases, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

[0267] Yeast, insect or mammalian cell hosts can also be used, employing suitable vectors and control sequences. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. Such promoters can also be derived from viral sources, such as, e.g., human cytomegalovirus (CMV-IE promoter) or herpes simplex virus type-1 (HSV TK promoter). Nucleic acid sequences derived from the SV40 splice, and polyadenylation sites can be used to provide the required nontranscribed genetic elements.

[0268] Polynucleotides encoding neoantigenic peptides 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.

[0269] 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 residues between epitopes and / or analogs, such as those described above, or can comprise naturally-occurring flanking sequences adjacent to the epitopes and / or analogs (and / or CTL, HTL, and B cell epitopes).

[0270] In some cases, a neoantigenic peptide described herein can also be administered / expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. As an example of this approach, vaccinia virus is used as a vector to express nucleotide sequences that encode the neoantigenic peptides described herein. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). 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 neoantigenic polypeptides described herein, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, Sendai virus vectors, poxvirus vectors, canarypox vectors, and fowlpox vectors, and the like, will be apparent to those skilled in the art from the description herein. In some cases, the vector is Modified Vaccinia Ankara (VA) (e.g. Bavarian Noridic (MVA-BN)).

[0271] 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 downstream cloning site for polynucleotide, e.g., minigene 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 cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences. In some cases, the promoter is the CMV-IE promoter.

[0272] Polynucleotides described herein can comprise one or more synthetic or naturally-occurring introns in the transcribed region. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells can also be considered for increasing polynucleotide expression.

[0273] In addition, a polynucleotide described herein can comprise immunostimulatory sequences (ISSs or CpGs). These sequences can be included in the vector, outside the polynucleotide coding sequence to enhance immunogenicity.

[0274] In some cases, 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.

[0275] In some cases, the antigenic peptide molecules are equal to or less than 50 amino acids. In some cases, 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 either: 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 of the binding peptides with extended sequences for each. In another case, when sequencing reveals a long (>10 residues) epitope sequence present in the diseased tissue (e.g. 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 antigen presenting cells such as dendritic cells and may lead to more effective antigen presentation and induction of T cell responses. In some cases, the extended sequence is altered to improve the biochemical properties of the polypeptide (properties such as solubility or stability) or to improve the likelihood for efficient proteasomal processing of the peptide.

[0276] The antigenic peptides and polypeptides may bind an HLA protein. In some cases, the antigenic peptides may bind an HLA protein with greater affinity than a corresponding 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 cases, the antigenic peptides do not induce an autoimmune response and / or invoke immunological tolerance when administered to a subject.

[0277] The disclosure also provides compositions comprising a plurality of antigenic peptides. Reference to antigenic peptides includes any suitable delivery modality that can result in introduction of the peptide into a subject's cell (e.g., nucleic acid). In some cases, the composition comprises at least 2 or more antigenic peptides. In some cases, the composition contains at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 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, or 500 distinct peptides. In some cases, the composition contains at least one peptide from Table 1. In some cases, the composition contains at least one peptide from Table 1 and at least one other peptide from Table 1. In some cases, the composition contains at least one peptide from Table 1 and at least one other peptide from Table 2. In some cases the composition contains at least 20 distinct peptides.. In some cases the composition contains at most 20 distinct peptides. According to the disclosure, 2 or more of the distinct peptides may be derived from the same polypeptide. For example, if an antigenic mutation encodes a polypeptide, two or more of the antigenic peptides may be derived from the polypeptide. In some cases, the two or more antigenic peptides derived from the polypeptide 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.

[0278] 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 protein, or binding affinity. The moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000). For example, antigenic peptides and polypeptides having the desired activity may be modified as necessary to provide certain desired attributes, e.g. improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell. For instance, the antigenic peptide and polypeptides may be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding. Such conservative substitutions may encompass replacing an amino acid residue with another 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 substitutions 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 & Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2d Ed. (1984).

[0279] The antigenic peptide may also be modified by extending or decreasing the compound's amino acid sequence, e.g., by the addition or deletion of amino acids. The antigenic peptides, polypeptides, or analogs can also be modified by altering the order or composition of certain residues. It will be appreciated by the skilled artisan that certain amino acid residues essential for biological activity, e.g., those at critical contact sites or conserved residues, may generally not be altered without an adverse effect on biological activity. The noncritical amino acids need not be limited 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 β-γ-δ- amino acids, as well as many derivatives of L-a-amino acids.

[0280] 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 charged (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 substitutions 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 substituted or added depend on the spacing necessary between essential contact points and certain functional 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 of the 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.

[0281] An antigenic peptide may be modified to provide desired attributes. For instance, the ability of the peptides to induce CTL activity can be enhanced by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. In some cases, immunogenic peptides / T helper conjugates are linked by a spacer molecule. In some cases, 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 residues 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.

[0282] The present disclosure is based, at least in part, on the ability to present the immune system of the patient with one or more disease-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 antigens. In general, such disease specific antigens may be produced either in vitro or in vivo. Disease 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 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. Alternatively, 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 antigens 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.

[0283] In some cases, the present disclosure includes modified antigenic peptides. A modification can include a covalent chemical modification that does not alter the primary amino acid sequence of the antigenic peptide itself. Modifications can produce peptides with desired properties, for example, prolonging the in vivo half-life, increasing the stability, reducing the clearance, altering the immunogenicity or allergenicity, enabling the raising of particular antibodies, cellular targeting, antigen uptake, 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 carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticulate 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 addition of unnatural amino acids.

[0284] 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 polymers, 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 thermal stability, protection against susceptibility to enzymatic degradation, increased solubility, longer in vivo circulating half-life and decreased clearance, reduced immunogenicity and antigenicity, and reduced toxicity.

[0285] 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 protective group, it generally has from 1 to 8 carbons. The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star-PEGs" and multi-armed PEGs are contemplated by the present disclosure.

[0286] The present disclosure 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 percentage of conjugates where n=1 is 18-25%, the percentage of conjugates where n=2 is 50-66%, the percentage of conjugates where n=3 is 12-16%, and the percentage of conjugates where n=4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods know in the art. For example, 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 attached.

[0287] 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 reactive 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-hydroxy succinylimide. Another activated polyethylene glycol which may be bound to a free amino group is 2,4-bis(0- methoxypolyethyleneglycol)-6-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 includes polyoxyethylenediamine.

[0288] Conjugation of one or more of the polypeptide sequences of the present disclosure to PEG having a spacer may be carried out by various conventional methods. For example, the conjugation reaction can be carried out in solution at a pH of from 5 to 10, at temperature from 4°C to room temperature, for 30 minutes to 20 hours, utilizing a molar ratio of reagent to protein of from 4: 1 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 attached, whereas high temperature, neutral to high pH (e.g., pH>7), and longer reaction time tend to increase the number of PEGs attached. Various means known in the art may be used to terminate the reaction. In some cases the reaction is terminated by acidifying the reaction mixture and freezing at, e.g., -20°C.

[0289] The present disclosure also contemplates the use of PEG mimetics. Recombinant PEG mimetics have been developed that retain the attributes of PEG (e.g., enhanced serum half- life) while conferring several additional advantageous properties. By way of example, simple polypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser and Thr) capable of forming 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 molecular biology techniques enable control of the side chain composition of the polypeptide chains, allowing optimization of immunogenicity and manufacturing properties.

[0290] Glycosylation can affect the physical properties of proteins 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 bacteria (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 glycosylation. Addition of glycosylation 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 asparagine residues (for N-linked glycosylation sites). The structures of N-linked 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 (hereafter 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. Some cases of the present disclosure comprise the generation and use of N-glycosylation variants.

[0291] The polypeptide sequences of the present disclosure may optionally be altered through changes 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 coupling of glycosides to the polypeptide. Removal of carbohydrates may be accomplished chemically or enzymatically, or by substitution of codons encoding amino acid residues that are glycosylated. Chemical deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases.

[0292] Additional suitable components and molecules for conjugation include, for example, molecules for targeting to the lymphatic system, thyroglobulin; albumins such as human serum albumin (HAS); tetanus toxoid; Diphtheria toxoid; polyamino acids such as poly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses; influenza virus hemaglutinin, influenza virus nucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B virus core protein and surface antigen; or any combination of the foregoing.

[0293] Fusion of albumin 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 fragment 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 fusion polypeptide. The expression may be effected in vitro from, for example, prokaryotic or eukaryotic cells. In some cases of the present disclosure, the expression 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 exogenous genetic material (exogenous 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 half-life. 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 exceeds that of fusions with non-modified albumin. (See WO2011 / 051489). Several albumin-binding strategies have been developed as alternatives 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 ligands with albumin - binding activity have been used for half-life extension of small protein therapeutics. For example, insulin detemir (LEVEMIR), an approved product for diabetes, comprises a myristyl chain conjugated to a genetically-modified insulin, resulting in a long- acting insulin analog.

[0294] Another type of modification is to conjugate (e.g., link) one or more additional components or molecules at the N- and / or C-terminus of a polypeptide sequence, such as another protein (e.g., a protein having an amino acid sequence heterologous to the subject protein), or a carrier molecule. Thus, an exemplary polypeptide sequence can be provided as a conjugate with another component or molecule.

[0295] 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, delayed or controlled release in vivo, etc. Other functions or activities include 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 disorder or disease as set forth herein (e.g., diabetes).

[0296] A polypeptide may also be conjugated to large, slowly metabolized macromolecules such as proteins; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads; polymeric amino acids such as polyglutamic 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.

[0297] Additional candidate components and molecules for conjugation include those suitable for isolation or purification. Particular non-limiting examples include binding molecules, such as biotin (biotin-avidin specific binding pair), an antibody, a receptor, a ligand, a lectin, or molecules that comprise a solid support, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes. Purification methods 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.

[0298] In some cases, the amino- or carboxyl- terminus of a polypeptide sequence of the present disclosure can be fused with an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to increase the systemic half-life of biopharmaceuticals, and thus the biopharmaceutical product may require less frequent administration.

[0299] Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells that line the blood vessels, and, upon binding, the Fc fusion molecule is protected from degradation and re- released into the circulation, keeping the molecule in circulation longer. This Fc binding is believed to be the mechanism by which endogenous IgG retains its long plasma half-life. More recent Fc-fusion technology links a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical as compared to traditional Fc-fusion conjugates.

[0300] The present disclosure contemplates the use of other modifications, currently known or developed in the future, of the polypeptides to improve 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 hesylation, which utilizes hydroxyethyl starch derivatives linked to other molecules in order to modify the molecule's characteristics. Various aspects of hesylation are described in, for example, U.S. Patent Appln. Nos. 2007 / 0134197 and 2006 / 0258607.

[0301] 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 biological techniques, the isolation of proteins or peptides from natural sources, in vitro translation, or the chemical synthesis of proteins or peptides.

[0302] Peptides can be readily synthesized chemically utilizing reagents that are free of contaminating bacterial or animal substances (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc.85:2149-54, 1963). In some cases, antigenic peptides are prepared by (1) parallel solid-phase synthesis on multi-channel instruments using uniform synthesis and cleavage conditions; (2) purification over a RP-HPLC column with column stripping; and re-washing, but not replacement, between peptides; followed by (3) analysis with a limited set of the most informative 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.

[0303] Alternatively, a nucleic acid (e.g., a polynucleotide) encoding an antigenic peptide of the disclosure may be used to produce the antigenic peptide in vitro. The polynucleotide may be, e.g., 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 combinations thereof and it may or may not contain introns so long as it codes for the peptide. In some cases in vitro translation is used to produce the peptide. An expression vector capable of expressing a polypeptide can also be prepared. 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 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 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 Harbor, N.Y.).

[0304] 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 antigenic peptides of the invention may be encoded by a single expression vector.

[0305] In some cases, 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 polypeptide 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.

[0306] In some cases, the polynucleotides can comprise the coding sequence for the disease 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 incorporated 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 purification 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 protein when a mammalian host (e.g., COS-7 cells) is used. Additional tags include, but are not limited to, Calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag, Isopeptag, SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tags, GST tags, fluorescent protein tags (e.g., green fluorescent protein tags), maltose binding protein tags, Nus tags, Strep-tag, thioredoxin tag, TC tag, Ty tag, and the like.

[0307] In some cases, the polynucleotides may comprise the coding sequence for one or more of the disease specific antigenic peptides fused in the same reading frame to create a single concatamerized antigenic peptide construct capable of producing multiple antigenic peptides.

[0308] In some cases, 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% identical, at least 90% identical, at least 95% identical, or at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding a disease specific antigenic peptide of the present invention, can be provided.

[0309] The isolated disease specific antigenic peptides 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 sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable transformed host. In some cases, 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 mutagenized by site-specific mutagenesis to provide functional analogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Pat. No.4,588,585.

[0310] In some cases, a DNA sequence encoding a polypeptide of interest would be constructed by chemical synthesis 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 polypeptide 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 sequence 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

[0311] 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 polypeptide in a suitable host. As well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene can be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0312] Recombinant expression vectors may be used to amplify and express DNA encoding the disease specific antigenic peptides. Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a disease specific antigenic peptide or a bioequivalent analog operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail herein. Such regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants 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 leader) 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 sequence if it controls the transcription 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 reading frame. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0313] Useful expression vectors for eukaryotic hosts, especially mammals or humans include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR 1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and filamentous single-stranded DNA phages.

[0314] 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 organisms, for example 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 cellular hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).

[0315] Various mammalian or insect cell culture systems are also advantageously employed to express recombinant protein. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified and completely functional. 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 enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0316] 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 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 passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography. For example, supernatants 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. Following the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, 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 chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a cancer stem cell protein-Fc composition. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.

[0317] Recombinant protein produced 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 convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

[0318] The present disclosure also contemplates the use of nucleic acid molecules as vehicles for delivering antigenic peptides / polypeptides to the subject in need thereof, in vivo, in the form of, e.g., DNA / RNA vaccines (see, e.g., WO2012 / 159643, and WO2012 / 159754).

[0319] In some cases antigens may be administered to a patient in need thereof by use of a plasmid. These are plasmids which usually consist of a strong viral promoter to drive the in vivo transcription and translation of the gene (or complementary DNA) of interest (Mor, et al., (1995). The Journal of Immunology 155 (4): 2039-2046). Intron A may sometimes be included to improve mRNA stability and hence increase protein expression (Leitner et al. (1997).The Journal of Immunology 159 (12): 6112-6119). Plasmids also include a strong polyadenylation / transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410; Robinson et al., (2000). Adv. Virus Res. Advances in Virus Research 55: 1-74; Böhmet al., (1996). Journal of Immunological Methods 193 (1): 29-40.). Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88)

[0320] Plasmids may be introduced into animal tissues by a number of different methods. The two most popular approaches are injection of DNA in saline, using a standard hypodermic needle, and gene gun delivery. 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 American (Weiner et al., (1999) Scientific American 281 (1): 34-41). Injection in saline is normally conducted intramuscularly (IM) in skeletal muscle, or intradermally (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 (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410). Immune responses to this method of delivery 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 et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410).

[0321] 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 (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410; Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).

[0322] 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 vaginal mucosa (Lewis et al., (1999) Advances in Virus Research (Academic Press) 54: 129-88). Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration 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 permeabilizing 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:10.1371 / journal.pone.0118803 April 13, 2015).

[0323] In some cases, 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 (e.g., in an episomal, non-replicating, non-integrated form in the muscle cells) is expected to provide an increased duration of protection.

[0324] One or more antigenic peptides of the disclosure may be encoded 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 cases, the disease vaccine or immunogenic composition may include a viral based vector for use in a human patient in need thereof, such as, for example, an adenovirus. Plasmids that can be used for adeno associated virus, adenovirus, and lentivirus delivery have been described previously (see e.g., U.S. Patent Nos. 6,955,808 and 6,943,019, and U.S. Patent application No. 20080254008).

[0325] 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 lentivirus. This approach involves the use of a vector to express nucleotide sequences that encode the peptide of the invention. 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.

[0326] Among vectors that may be used in the practice of the disclosure, integration in the host genome of a cell is possible with retrovirus gene transfer methods, often resulting in long term expression of the inserted transgene. In some cases, the retrovirus is a lentivirus. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. The tropism of a retrovirus can be altered by incorporating foreign 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 of the disclosure). 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 vectors 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 replication 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 vectors that may be used in the practice of the disclosure include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., (1992) J. Virol. 66:2731-2739; Johann et al., (1992) J. Virol.66:1635-1640; Sommnerfelt et al., (1990) Virol.176:58-59; Wilson et al., (1998) J. Virol.63:2374-2378; Miller et al., (1991) J. Virol.65:2220-2224; PCT / US94 / 05700).

[0327] Also useful in the practice of the disclosure is a minimal non-primate lentiviral vector, such as a lentiviral vector based on the equine infectious anemia virus (EIAV). The vectors may have cytomegalovirus (CMV) promoter driving expression of the target gene. Accordingly, the disclosure contemplates amongst vector(s) useful in the practice of the disclosure: viral vectors, including retroviral vectors and lentiviral vectors.

[0328] In a case herein the delivery is via a lentivirus. Dosages, e.g., 10 µl of a recombinant lentivirus having a titer of 1 x 10 9< transducing units (TU) / mL, can be adapted or extrapolated to use of a retroviral or lentiviral vector in the present disclosure. For transduction in tissues such as the brain, it is necessary to use very small volumes, 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 cases the amount of lentivirus administered may be 1x10 5< or about 1x10 5< plaque forming units (PFU), 5x10 5< or about 5x10 5< PFU, 1x10 6< or about 1.x10 6< PFU, 5x10 6< or about 5x10 6< PFU, 1x10 7< or about 1x107PFU, 5x10 7< or about 5x10 7< PFU, 1x10 8< or about 1x10 8< PFU, 5x10 8< or about 5x10 8< PFU, 1x10 9< or about 1x10 9< PFU, 5x10 9< or about 5x10 9< PFU, 1x10 10< or about 1x10 10< PFU or 5x10 10< or about 5x10 10< 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 empirically.

[0329] Also useful in the practice of the disclosure is an adenovirus vector. One advantage is the ability of recombinant adenoviruses to efficiently transfer and express recombinant 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., U.S. Patent No.7,029,848). As to adenovirus vectors useful in the practice of the disclosure, mention is made of US Patent No.6,955,808. The adenovirus vector used can be selected from the group consisting of the Ad5, Ad35, Ad11, C6, and C7 vectors. The sequence of the Adenovirus 5 ("Ad5") genome has been published. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285). Ad35 vectors are described in U.S. Pat. Nos.6,974,695, 6,913,922, and 6,869,794. Ad11 vectors are described in U.S. Pat. No. 6,913,922. C6 adenovirus vectors are described in U.S. Pat. Nos. 6,780,407; 6,537,594; 6,309,647; 6,265,189; 6,156,567; 6,090,393; 5,942,235 and 5,833,975. C7 vectors are described in U.S. Pat. No. 6,277,558. Adenovirus vectors that are E1-defective or deleted, E3- defective or deleted, and / or E4-defective or deleted may also be used. Certain adenoviruses having mutations in the E1 region have improved safety margin because E1-defective adenovirus mutants are replication-defective in non-permissive cells, or, at the very least, are highly attenuated. Adenoviruses having mutations in the E3 region may have enhanced the immunogenicity by disrupting the mechanism whereby adenovirus down-regulates MHC class I molecules. Adenoviruses having E4 mutations may have reduced immunogenicity of the adenovirus 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 deleted or mutated in E1, E3, E4, E1 and E3, and E1 and E4 can be used in accordance with the present disclosure. Furthermore, "gutless" adenovirus vectors, in which all viral genes are deleted, can also be used in accordance with the present disclosure. Such vectors require a helper virus for their replication and require a special human 293 cell line expressing both E1a 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-vaccination. The "gutless" adenovirus vectors can be used for insertion of heterologous inserts / genes such as the transgenes of the present disclosure, and can even be used for co-delivery of a large number of heterologous inserts / genes. In some cases, the delivery is via an adenovirus, which may be at a single booster dose. In some cases, 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 significantly reduced virus production. There are many promoters that can be used to drive nucleic acid molecule expression. AAV ITR can serve as a promoter and is advantageous 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: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Promoters used to drive RNA synthesis can include: Pol III promoters such as U6 or H1. The use of a Pol II promoter and intronic cassettes can be used to express guide RNA (gRNA). With regard to AAV vectors useful in the practice of the disclosure, mention is made of US Patent Nos. 5658785, 7115391, 7172893, 6953690, 6936466, 6924128, 6893865, 6793926, 6537540, 6475769 and 6258595, and documents cited therein. As to AAV, the AAV can be AAV1, AAV2, 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 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAV8 is useful for delivery to the liver. In some cases the delivery is via an AAV. The dosage may be adjusted to balance the therapeutic benefit against any side effects.

[0330] In some cases, effectively activating a cellular immune response for a disease vaccine or immunogenic 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, U.S. Patent No.6,991,797).

[0331] In some cases, 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., Verardiet al., Hum Vaccin Immunother. 2012 Jul;8(7):961-70; and Moss, Vaccine. 2013; 31(39): 4220-4222). Poxvirus expression vectors were described in 1982 and quickly became widely used for vaccine development 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. Information concerning poxviruses that may be used in the practice of the disclosure, such as Chordopoxvirinae subfamily poxviruses (poxviruses of vertebrates), for instance, orthopoxviruses and avipoxviruses, e.g., vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g., ATCC ®< VR-1354), Copenhagen Strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox virus (e.g., Wheatley C93 Strain, ALVAC), fowlpox virus (e.g., FP9 Strain, Webster Strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, inter alia, synthetic or non- naturally occurring recombinants thereof, uses thereof, and methods for making and using such recombinants may be found in scientific and patent literature.

[0332] In some cases, the vaccinia 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 cytoplasm of the infected host cell and the polypeptide of interest can therefore induce an immune response. Moreover, Poxviruses 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.

[0333] In some cases, ALVAC is used as a vector in a disease vaccine or immunogenic composition. ALVAC is a canarypox virus that can be modified to express foreign transgenes and has been used as a method for vaccination against both prokaryotic and eukaryotic antigens (Horig H, Lee DS, Conkright W, et al. Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule. Cancer Immunol Immunother 2000;49:504-14; von Mehren M, Arlen P, Tsang KY, et al. Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas. Clin Cancer Res 2000;6:2219-28; Musey L, Ding Y, Elizaga M, et al. HIV-1 vaccination administered intramuscularly can induce both systemic and mucosal T cell immunity in HIV-1-uninfected individuals. J Immunol 2003;171:1094-101; Paoletti E. Applications of pox virus vectors to vaccination: an update. Proc Natl Acad Sci U S A 1996;93:11349-53; U.S. 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, Hawkins MJ, Tsang KY, et al. Phase I study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen. J Clin Oncol 1999;17:332-7).

[0334] In some cases, 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., Infection 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 restricted (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038, 1991). MVA is characterized by its extreme attenuation, namely, by a diminished virulence or infectious ability, but still holds an excellent immunogenicity. When tested in a variety of animal models, MVA was proven to be avirulent, even in immuno-suppressed individuals. Moreover, MVA-BN ®< -HER2 is a candidate immunotherapy designed for the treatment of HER-2-positive breast cancer and is currently in clinical trials. (Mandl et al., Cancer Immunol Immunother. Jan 2012; 61(1): 19-29). Methods to make and use recombinant MVA has been described (e.g., see U.S. Patent Nos. 8,309,098 and 5,185,146 hereby incorporated in its entirety).

[0335] In some cases, recombinant viral particles of the vaccine or immunogenic composition are administered to patients in need thereof.Neoantigen binding peptides

[0336] In certain cases, the present disclosure provides a binding protein (e.g., an antibody or antigen-binding fragment thereof), or a T cell receptor (TCR), or a chimeric antigen receptor (CAR) capable of binding with a high affinity to a neoantigen peptide:human leukocyte antigen (HLA) complex. In some cases, the present disclosure provides a CAR that is capable of binding with a high affinity to a neoantigenic peptide derived from the extracellular domain of a protein. In certain cases, 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 deletions in the native amino acid sequence, provided that the binding protein retains or substantially retains its specific binding function. Conservative 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). Oligonucleotide-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. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis may be used to prepare immunogen polypeptide variants (see, e.g., Sambrook et al., supra).

[0337] A variety of criteria known to persons skilled in the art indicate whether an amino acid that is substituted at a particular 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 included 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, serine, threonine, tyrosine, cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), 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 glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic 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 sequence of a second polypeptide (e.g., using GENEWORKS, Align, the BLAST algorithm, or other algorithms described herein and practiced in the art).

[0338] In certain cases, a neoantigen specific binding protein, TCR or CAR is capable of (a) specifically binding to a neoantigen:HLA complex on a cell surface independent or in the absence of CD8. In certain cases, a neoantigen specific binding protein is a T cell receptor (TCR), a chimeric antigen receptor or an antigen-binding fragment of a TCR, any of which can be chimeric, humanized or human. In further cases, an antigen-binding fragment of the TCR comprises a single chain TCR (scTCR).

[0339] In certain cases, there is provided a composition comprising a neoantigen-specific binding protein or high affinity recombinant TCR according to any one of the above cases and a pharmaceutically acceptable carrier, diluent, or excipient.

[0340] Methods useful for isolating and purifying recombinantly produced soluble TCR, by way of example, can include obtaining supernatants 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 concentration, 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 isolating an immunogen from its natural 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 performed according to methods described herein and known in the art.III. Immunogenic and vaccine compositions

[0341] In some cases the present disclosure is directed to an immunogenic composition, e.g., a vaccine composition capable of raising a neoantigen-specific response (e.g., a humoral or cell-mediated immune response). In some cases, the immunogenic composition comprises neoantigen therapeutics (e.g., peptides, polynucleotides, TCR, CAR, cells containing TCR or CAR, dendritic cell containing polypeptide, dendritic cell containing polynucleotide, antibody, etc.) described herein corresponding to tumor specific neoantigen identified herein.

[0342] In some cases, immunogenic peptides are identified from one or more subjects with a disease or condition. In some cases, immunogenic peptides are specific to one or more subjects with a disease or condition. In some cases, immunogenic peptides can bind to an HLA that is matched to an HLA haplotype of one or more subjects with a disease or condition.

[0343] A person skilled in the art will be able to select neoantigenic therapeutics by testing, for example, the generation of T cells in vitro as well as their efficiency and overall presence, the proliferation, affinity and expansion of certain T cells for certain peptides, and the functionality of the T cells, e.g. by analyzing the IFN-γ production or tumor killing by T cells. The most efficient peptides can then combined as an immunogenic composition.

[0344] In some cases of the present disclosure 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 cases, an immunogenic composition comprises neoantigenic peptides and / or polypeptides capable of associating with the most frequently 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 molecules.

[0345] In some cases, 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.

[0346] In some cases, an immunogenic composition described herein can further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are given herein below. Polypeptides and / or polynucleotides in the composition can be associated with a carrier such as e.g. a protein or an antigen-presenting cell such as e.g. a dendritic cell (DC) capable of presenting the peptide to a T cell or a B cell. In further cases, DC-binding peptides are used as carriers to target the neoantigenic peptides and polynucleotides encoding the neoantigen peptides to dendritic cells (Sioud et al. FASEB J 27: 3272-3283 (2013)).

[0347] In cases, the neoantigenic polypeptides or polynucleotides can be provided as antigen presenting cells (e.g., dendritic cells) containing such polypeptides or polynucleotides. In other cases, such antigen presenting cells are used to stimulate T cells for use in patients.

[0348] In some cases, the antigen presenting cells are dendritic cells. In related cases, the dendritic cells are autologous dendritic 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 cases, the T cell is a CTL. In some cases, the T cell is a HTL.

[0349] Thus, one case of the present disclosure an immunogenic composition containing at least one antigen presenting cell (e.g., a dendritic cell) that is pulsed or loaded with one or more neoantigenic polypeptides or polynucleotides described herein. In cases, such APCs are autologous (e.g., autologous dendritic cells). Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient can be loaded with neoantigenic peptides or polynucleotides ex vivo. In related cases, such APCs or PBMCs are injected back into the patient.

[0350] 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 cases, the polynucleotide can be naked DNA that is taken up by the cells by passive loading. In another case, the polynucleotide is part of a delivery vehicle, for example, a liposome, virus like particle, plasmid, or expression vector. In another case, the polynucleotide is delivered by a vector-free delivery system, for example, high performance electroporation and high-speed cell deformation). In cases, such antigen presenting cells (APCs) (e.g., dendritic cells) or peripheral blood mononuclear cells (PBMCs) are used to stimulate a T cell (e.g., an autologous T cell). In related cases, the T cell is a CTL. In other related cases, the T cell is an HTL. Such T cells are then injected into the patient. In some cases, CTL is injected into the patient. In some cases, HTL is injected into the patient. In some cases, both CTL and HTL are injected into the patient. Administration of either therapeutic can be performed simultaneously or sequentially and in any order.

[0351] The pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment are intended for parenteral, topical, nasal, oral or local administration. In some cases, the pharmaceutical compositions described herein are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. In cases, the composition can be administered intratumorally. The compositions can be administered at the site of surgical excision to induce a local immune response to the tumor. In some cases, described 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 sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0352] The concentration of neoantigenic peptides and polynucleotides described herein in the pharmaceutical 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 weight, and will be selected by fluid volumes, viscosities, etc., according to the particular mode of administration selected.

[0353] 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, insoluble 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 receptor 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-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, for example, cholesterol. The selection of lipids is generally 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. Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

[0354] For targeting to the immune cells, a neoantigen polypeptides or polynucleotides to be incorporated into the liposome for cell surface determinants 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 polynucleotide being delivered, and the stage of the disease being treated.

[0355] In some cases, neoantigen polypeptides and polynucleotides are targeted to dendritic cells. In some cases, the neoantigen polypeptides and polynucleotides are target to dendritic cells using the markers DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, TSLP receptor, Clec9a or CD1a.

[0356] 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, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more neoantigenic polypeptides or polynucleotides described herein at a concentration of 25%-75%.

[0357] For aerosol administration, the neoantigenic polypeptides or polynucleotides can be supplied in finely divided form along with a surfactant and propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides can be employed. The surfactant can constitute 0.1%-20% by weight of the 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.

[0358] Additional methods for delivering the neoantigenic polynucleotides described herein are also known in the art. For instance, the nucleic acid can be delivered directly, as "naked DNA". This approach is described, for instance, in Wolff et al., Science 247: 1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466. The nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles.

[0359] For therapeutic or immunization purposes, mRNA encoding the neoantigenic peptides, or peptide binding agents can also be administered to the patient. In some cases, the mRNA is self-amplifying RNA. In a further case, the self-amplifying RNA is a part of a synthetic lipid nanoparticle formulation (Geall et al., Proc Natl Acad Sci U S A. 109: 14604-14609 (2012)).

[0360] The nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for instance, in WO 96 / 18372, WO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833; WO 91 / 06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).

[0361] The neoantigenic peptides and polypeptides described herein can also be expressed 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 noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. 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.

[0362] Adjuvants are any substance whose admixture into the immunogenic composition increases or otherwise modifies the immune response to the therapeutic agent. Carriers 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 covalently or non-covalently to the polypeptides or polynucleotides described herein.

[0363] The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reaction, or reduction in disease symptoms. 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 increase 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.

[0364] Suitable adjuvants are known in the art (see, WO 2015 / 095811) and include, but are not limited to poly(I:C), poly-ICLC, STING agonist, 1018 ISS, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel ®< . vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) which is derived from saponin, mycobacterial 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 Bioscience, 2007; 12:4050-4060) (Gamvrellis et al. Immunol & 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 dendritic cells into efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-1b, IL-4, IL-6 and CD40L) (U.S. Pat. No. 5,849,589 incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J Immunother Emphasis Tumor Immunol. 1996 (6):414-418).

[0365] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) 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, including peptide or protein antigens, live or killed viruses, dendritic cell immunogenic pharmaceutical compositions, autologous cellular immunogenic pharmaceutical compositions and polysaccharide conjugates in both prophylactic and therapeutic immunogenic pharmaceutical compositions. Importantly, it enhances dendritic cell maturation and differentiation, resulting in enhanced activation of TH1 cells and strong cytotoxic T-lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of adjuvants such as alum or incomplete Freund's adjuvant (IFA) that normally promote a TH2 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 emulsions 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). U.S. Pat. No. 6,406,705 B1 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and an antigen to induce an antigen-specific immune response. A commercially available CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) 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.

[0366] Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g. CpR, Idera), Poly(I:C)(e.g. polyi:CI2U), non-CpG bacterial DNA or RNA, ssRNA40 for TLR8, as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, 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 disclosure can readily be determined by the skilled artisan without undue experimentation. Additional adjuvants include colony-stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim).

[0367] In some cases, an immunogenic composition according to the present disclosure can comprise more than one different adjuvants. Furthermore, the disclosure encompasses a therapeutic composition comprising any adjuvant substance including any of the above or combinations thereof. It is also contemplated that the neoantigenic therapeutic (e.g., a humoral or cell-mediated immune response). In some cases, the immunogenic composition comprises neoantigen therapeutics (e.g., peptides, polynucleotides, TCR, CAR, cells containing TCR or CAR, dendritic cell containing polypeptide, dendritic cell containing polynucleotide, antibody, etc.) and the adjuvant can be administered separately in any appropriate sequence.

[0368] 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 increase their activity or immunogenicity, 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 keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. In some cases, the carrier comprises a human fibronection type III domain (Koide et al. Methods Enzymol. 2012;503:135-56). For immunization of humans, the carrier must be a physiologically acceptable carrier acceptable to humans and safe. However, tetanus toxoid and / or diptheria toxoid are suitable carriers In some cases of the disclosure. Alternatively, the carrier can be dextrans for example sepharose.

[0369] In some cases, the polypeptides can be synthesized as multiply linked peptides as an alternative to coupling a polypeptide to a carrier to increase immunogenicity. Such molecules are also known as multiple antigenic peptides (MAPS).

[0370] 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.

[0371] Thus pharmaceutical compositions can include, in addition to active ingredient, a pharmaceutically 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 carrier or other material will depend on the route of administration.

[0372] 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 Pharmaceutical Sciences 16t...

Claims

1. A pharmaceutical composition comprising: a recombinant or synthetic neoantigenic peptide or a polynucleotide encoding the recombinant neoantigenic peptide for use in treatment of cancer in a subject in need thereof; wherein the neoantigenic peptide comprises a tumor-specific neoepitope, wherein the tumor-specific neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by a formula:         AxByCz, wherein: each A is an amino acid corresponding to a native polypeptide encoded by a GATA3 gene; By is absent; each C is an amino acid encoded by a frameshift of a sequence encoding the native polypeptide; the at least 8 contiguous amino acids comprises at least one C; x + y + z is at least 8; and wherein Cz is (i) PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPL QHGHRHGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGY MFLKAESKIMFATLQRSSLWCLCSNH; and (a) the tumor-specific neoepitope is FLKAESKIMF (SEQ ID NO: 536) and the subject express an MHC molecule encoded by a B08:01 HLA allele, (b) the tumor-specific neoepitope is IMKPKRDGYM (SEQ ID NO: 550) and the subject express an MHC molecule encoded by a B08:01 HLA allele, (c) the tumor-specific neoepitope is LHFCRSSIM (SEQ ID NO: 556) and the subject express an MHC molecule encoded by a B08:01 HLA allele, (d) the tumor-specific neoepitope is MFATLQRSSL (SEQ ID NO: 567) and the subject express an MHC molecule encoded by a B07:02 HLA allele or a B08:01 HLA allele, (e) the tumor-specific neoepitope is MFLKAESKI (SEQ ID NO: 568) and the subject express an MHC molecule encoded by a A24:02 HLA allele, (f) the tumor-specific neoepitope is YMFLKAESKI (SEQ ID NO: 598) and the subject express an MHC molecule encoded by a A24:02 HLA allele or a B08:01 HLA allele, (g) the tumor-specific neoepitope is HVLPEPHLAL (SEQ ID NO: 1428), (h) the tumor-specific neoepitope is RPLQTHVLPE (SEQ ID NO: 1429), (i) the tumor-specific neoepitope is VLWTTPPLQH (SEQ ID NO: 1430) and optionally the subject expresses an MHC molecule encoded by a A03:01 HLA allele, (j) the tumor-specific neoepitope is EPHLALQPL (SEQ ID NO: 529) and the subject express an MHC molecule encoded by a B07:02 or B08:01 HLA allele, or (k) the tumor-specific neoepitope is VLPEPHLAL (SEQ ID NO: 594) and the subject express an MHC molecule encoded by a A02.01 HLA allele; or wherein Cz is (ii) PRPRRCTRHPACPLDHTTPPAWSPPWVRALLDAHRAPSESPCSP FRLAFLQEQYHEA and (a) the tumor-specific neoepitope sequence is APSESPCSPF (SEQ ID NO: 1431), (b) the tumor-specific neoepitope sequence is CPLDHTTPPA (SEQ ID NO: 1432), (c) the tumor-specific neoepitope sequence is FLQEQYHEA (SEQ ID NO: 1433), (d) the tumor-specific neoepitope sequence is RLAFLQEQYH (SEQ ID NO: 1434), (e) the tumor-specific neoepitope sequence is SPCSPFRLAF (SEQ ID NO: 1435), or (f) the tumor-specific neoepitope sequence is SPPWVRALL (SEQ ID NO: 1436); wherein the cancer is breast cancer.

2. The pharmaceutical composition for use according to claim 1, wherein Cz is PRPRRCTRHPACPLDHTTPPAWSPPWVRALLDAHRAPSESPCSPFRLAFL QEQYHEA and (a) the tumor-specific neoepitope is APSESPCSPF (SEQ ID NO: 1431), (b) the tumor-specific neoepitope is CPLDHTTPPA (SEQ ID NO: 1432), (c) the tumor-specific neoepitope is FLQEQYHEA (SEQ ID NO: 1433), (d) the tumor-specific neoepitope is RLAFLQEQYH (SEQ ID NO: 1434), (e) the tumor-specific neoepitope is SPCSPFRLAF (SEQ ID NO: 1435), or (f) the tumor-specific neoepitope is SPPWVRALL (SEQ ID NO: 1436).

3. The pharmaceutical composition for use according to claim 1, wherein Cz is: PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHR HGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFA TLQRSSLWCLCSNH and (a) the tumor-specific neoepitope is FLKAESKIMF (SEQ ID NO: 536) and the subject express an MHC molecule encoded by a B08:01 HLA allele, (b) the tumor-specific neoepitope is IMKPKRDGYM (SEQ ID NO: 550) and the subject express an MHC molecule encoded by a B08:01 HLA allele, (c) the tumor-specific neoepitope is LHFCRSSIM (SEQ ID NO: 556) and the subject express an MHC molecule encoded by a B08:01 HLA allele, (d) the tumor-specific neoepitope is MFATLQRSSL (SEQ ID NO: 567) and the subject express an MHC molecule encoded by a B07:02 HLA allele or a B08:01 HLA allele, (e) the tumor-specific neoepitope is MFLKAESKI (SEQ ID NO: 568) and the subject express an MHC molecule encoded by a A24:02 HLA allele, (f) the tumor-specific neoepitope is YMFLKAESKI (SEQ ID NO: 598) and the subject express an MHC molecule encoded by a A24:02 HLA allele or a B08:01 HLA allele, (g) the tumor-specific neoepitope is HVLPEPHLAL (SEQ ID NO: 1428), (h) the tumor-specific neoepitope is RPLQTHVLPE (SEQ ID NO: 1429), (i) the tumor-specific neoepitope is VLWTTPPLQH (SEQ ID NO: 1430) and optionally the subject expresses an MHC molecule encoded by a A03:01 HLA allele, (j) the tumor-specific neoepitope is EPHLALQPL (SEQ ID NO: 529) and the subject express an MHC molecule encoded by a B07:02 or B08:01 HLA allele, or (k) the tumor-specific neoepitope is VLPEPHLAL (SEQ ID NO: 594) and the subject express an MHC molecule encoded by a A02.01 HLA allele.

4. The pharmaceutical composition for use according to any one of claims 1-3, wherein the neoantigenic peptide is at least 9 and at most 500 amino acids in length.

5. The pharmaceutical composition for use according to any one of claims 1-4, wherein the tumor-specific neoepitope binds to a MHC class I molecule with a binding affinity of 500 nM or less.

6. The pharmaceutical composition for use according to any one of claims 1-5, wherein the pharmaceutical composition comprises at least two different neoantigenic peptides, wherein the gene encoding the tumor-specific neoepitope of each of the at least two different neoantigenic peptides is GATA3.

7. The pharmaceutical composition for use according to any one of claims 1 and 3-6, wherein the tumor-specific neoepitope is FLKAESKIMF (SEQ ID NO: 536) and the subject express an MHC molecule encoded by a B08:01 HLA allele.

8. The pharmaceutical composition for use according to any one of claims 1 and 3-6, wherein the tumor-specific neoepitope is IMKPKRDGYM (SEQ ID NO: 550) and the subject express an MHC molecule encoded by a B08:01 HLA allele.

9. The pharmaceutical composition for use according to any one of claims 1 and 3-6, wherein the tumor-specific neoepitope is LHFCRSSIM (SEQ ID NO: 556) and the subject express an MHC molecule encoded by a B08:01 HLA allele.

10. The pharmaceutical composition for use according to one of claims 1, and 3-6, wherein the tumor-specific neoepitope is MFATLQRSSL (SEQ ID NO: 567) and the subject express an MHC molecule encoded by a B07:02 HLA allele or a B08:01 HLA allele.

11. The pharmaceutical composition for use according to any one of claims 1 and 3-6, wherein the tumor-specific neoepitope is MFLKAESKI (SEQ ID NO: 568) and the subject express an MHC molecule encoded by a A24:02 HLA allele.

12. The pharmaceutical composition for use according to any one of claims 1 and 3-6, wherein the tumor-specific neoepitope is YMFLKAESKI (SEQ ID NO: 598) and the subject express an MHC molecule encoded by a A24:02 HLA allele or a B08:01 HLA allele.

13. The pharmaceutical composition for use according to any one of claims 1 and 3-6, wherein the tumor-specific neoepitope is VLWTTPPLQH (SEQ ID NO: 1430) and optionally the subject expresses an MHC molecule encoded by a A03:01 HLA allele.