Methods of treating pancreatic cancer with PD-1 axis-binding antagonists and RNA vaccines

Abstract

The present disclosure provides methods for treating individuals with pancreatic cancer with personalized cancer vaccines and PD-1 axis antagonists.

Description

[Technical Field]

[0001] The present disclosure relates to methods for treating individuals with pancreatic cancer with personalized cancer vaccines and PD-1 axis antagonists.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 508,248, filed June 14, 2023, and U.S. Provisional Patent Application No. 63 / 476,246, filed December 20, 2022, the entire contents of each of which are incorporated herein by reference.

[0003] Electronic Sequence Listing Reference The contents of the electronic sequence listing (146392064940seqlist.xml; size: 62,168 bytes; creation date: December 7, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0004] Pancreatic cancer is the seventh leading cause of cancer deaths worldwide and the third leading cause of cancer deaths in the United States and Europe (Dalmartello et al., Ann Oncol 2022;33:330-9; and Siegel et al., CA Cancer J Clin 2022;72:7-33). Pancreatic ductal adenocarcinoma (PDAC), which arises in the exocrine tissue of the pancreas, accounts for approximately 90% of pancreatic cancer cases. PDAC has a 5-year survival rate of less than 10% (Haeberle and Esposito. Transl Gastroenterol Hepatol 2019;4:50). Currently, the only potentially curative treatment for PDAC is surgical resection (Rawla et al., World J Oncol 2019;10:10-27; Park et al., JAMA 2021;326:851-62). However, the 5-year survival rate for patients with resected PDAC has been reported to be as low as 12%, depending on the patient population (Bilimoria et al., Cancer 2007;110:1227-34; Ferrone et al., J Gastrointest Surg 2008;12:701-6; Katz et al., Ann Surg Oncol 2009;16:836-47; Ferrone et al., Surgery 2012;152(3Suppl1):S43-9; He et al., HPB (Oxford) 2014;16:83-90; and Conroy et al., JAMA Oncol 2022;e223829.doi:10.1001 / jamaoncol.20223820).

[0005] Immunotherapies, such as immune checkpoint inhibitors, provide clinical benefit to patients with multiple types of solid tumors, including those with mismatch repair-deficient / microsatellite instability-high PDAC (Le et al., Science 2017;357:409-13; and Marabelle et al., J Clin Oncol 2020;38:1-10). However, the majority (>98%) of patients with PDAC have mismatch repair-proficient / microsatellite-stable disease and do not respond to immune checkpoint inhibition (O'Reilly et al., JAMA Oncol 2019;5:1431-8; and Bian and Almhanna. Transl Gastroenterol Hepatol 2021;6:6). The poor immunogenicity of PDAC is attributed to its immunosuppressive tumor microenvironment, paucity of tumor-infiltrating lymphocytes, and low tumor mutational burden leading to the expression of a limited number of immunogenic neoantigens (Lutz et al., Cancer Immunol Res 2014;2:616-31; and Schizas et al., Cancer Treat Rev 2020;86:102016).

[0006] Therapeutic vaccines targeting immunogenic epitopes to activate the immune system against cancer are being developed and investigated, and may be beneficial for the treatment of poorly immunogenic cancers such as pancreatic cancer, including PDAC. However, to date, therapeutic vaccines, while promising, have historically fallen short of expectations. One potential reason is that cancer-specific T cells become functionally exhausted during chronic exposure to cancer cells. Therefore, a combination treatment regimen using two or more targeted cancer immunotherapeutics, such as immune checkpoint inhibitors and therapeutic vaccines targeting immunogenic epitopes, may be required to fully engage the host immune system's antitumor capabilities. For example, a recent phase I study of a personalized RNA vaccine combined with atezolizumab and a chemotherapy regimen in pancreatic ductal adenocarcinoma demonstrated an acceptable safety profile and promising RNA vaccine-induced immune responses (see, e.g., Balachandran et al., Journal of Clinical Oncology 40, no. 16_suppl (June 01, 2022) 2516-2516). However, there remains a need for improved methods for treating pancreatic cancers, such as PDAC.

[0007] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated by reference in their entirety, as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0008] Provided herein is a method for treating a pancreatic cancer tumor in a human patient in need thereof, comprising administering to the patient: (a) a personalized RNA vaccine comprising one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient; (b) a PD-1 axis-binding antagonist; and (c) a chemotherapy treatment; wherein the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment are administered to the patient during a priming phase, during a chemotherapy phase after the priming phase, and during a boost phase after the chemotherapy phase; (i) the priming phase comprises administering to the patient at least one time the RNA vaccine and at least one time the PD-1 axis-binding antagonist; (ii) the chemotherapy phase comprises administering to the patient the chemotherapy treatment; and (iii) the boost phase comprises administering to the patient at least one time the RNA vaccine and at least one time the PD-1 axis-binding antagonist.

[0009] In some embodiments, the pancreatic cancer tumor is a pancreatic ductal adenocarcinoma (PDAC) tumor. In some embodiments, the pancreatic cancer tumor is resectable.

[0010] In some embodiments, the priming phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks after resection of the pancreatic cancer tumor from the patient. In some embodiments, the priming phase begins about 6 weeks and about 12 weeks after resection of the pancreatic cancer tumor from the patient.

[0011] In some embodiments, the priming phase comprises a single administration of the PD-1 axis-binding antagonist, hi some embodiments, the priming phase comprises administration of the PD-1 axis-binding antagonist on day 1 of week 3 of the priming phase.

[0012] In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist at least twice. In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist once every four weeks. In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the priming phase, and every four weeks thereafter. In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist twice. In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 and day 1 of week 5 of the priming phase.

[0013] In some embodiments, the priming phase comprises administering the RNA vaccine two, three, four, five, six, seven, or eight times. In some embodiments, the priming phase comprises administering the RNA vaccine two or three times. In some embodiments, the priming phase comprises administering the RNA vaccine six to eight times, or up to six times. In some embodiments, the priming phase comprises administering the RNA vaccine six times. In some embodiments, the priming phase comprises administering the RNA vaccine once per week. In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of week 1 of the priming phase and once per week thereafter. In some embodiments, the priming phase comprises administering the RNA vaccine six times. In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase.

[0014] In some embodiments, each dose of the PD-1 axis-binding antagonist administered to a patient during the priming phase is administered on the same day as a single dose of the RNA vaccine. In some embodiments, the priming phase consists of 6 weeks. In some embodiments, the RNA vaccine is administered on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase, and the PD-1 axis-binding antagonist is administered on day 1 of week 3 of the priming phase. In some embodiments, the RNA vaccine is administered on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase, and the PD-1 axis-binding antagonist is administered on day 1 of weeks 1 and 5 of the priming phase.

[0015] In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment for at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks, at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, or longer. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment for 23 weeks. In some embodiments, the chemotherapy treatment is administered once every two weeks. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment on day 1 of week 1 of the chemotherapy phase and every two weeks thereafter. In some embodiments, the chemotherapy phase comprises administering 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, or at least 24 or more chemotherapy treatments, hi some embodiments, the chemotherapy phase comprises administering 12 chemotherapy treatments. In some embodiments, the chemotherapy phase consists of 24 weeks, hi some embodiments, the chemotherapy phase comprises administering chemotherapy treatment on day 1 of weeks 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 of the chemotherapy phase.

[0016] In some embodiments, the chemotherapy phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks after the end of the priming phase and after the last administration of the RNA vaccine. In some embodiments, the chemotherapy phase begins within 9 weeks, starting from week 1 of the priming phase. In some embodiments, the priming phase consists of 6 weeks, and the chemotherapy phase begins within 9 weeks, starting from week 1 of the priming phase. In some embodiments, the priming phase consists of 6 weeks, and includes administering chemotherapy treatment starting from day 1 of week 7, starting from week 1 of the priming phase, and every 2 weeks thereafter.

[0017] In some embodiments, the chemotherapy phase comprises 12 administrations of chemotherapy treatment, hi some embodiments, the chemotherapy phase comprises administering chemotherapy treatment on day 1 of weeks 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, beginning with week 1 of the priming phase.

[0018] In some embodiments, the boost phase comprises administering the RNA vaccine two, three, four, five, six, seven, or eight times. In some embodiments, the boost phase comprises administering the PD-1 axis-binding antagonist two, three, four, five, six, seven, or eight times. In some embodiments, the boost phase comprises administering the PD-1 axis-binding antagonist six times and administering the RNA vaccine six times. In some embodiments, the boost phase comprises administering the PD-1 axis-binding antagonist and the RNA vaccine once every four weeks. In some embodiments, the boost phase comprises administering the PD-1 axis-binding antagonist on day 1 of week 1 of the boost phase and every four weeks thereafter. In some embodiments, the boost phase comprises administering the RNA vaccine on day 1 of week 1 of the boost phase and once every four weeks thereafter. In some embodiments, the administration of the RNA vaccine and the PD-1 axis-binding antagonist during the boost phase occurs on the same day. In some embodiments, the boost phase comprises administering the PD-1 axis-binding antagonist and the RNA vaccine on day 1 of week 1 of the boost phase, and every four weeks thereafter. In some embodiments, the boost phase consists of 21 weeks. In some embodiments, the RNA vaccine and the PD-1 axis-binding antagonist are administered on day 1 of weeks 1, 5, 9, 13, 17, and 21 of the boost phase.

[0019] In some embodiments, the boost phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks after the chemotherapy phase has ended. In some embodiments, the boost phase begins up to about 12 weeks after the chemotherapy phase has ended, optionally up to 12 weeks after the last administration of chemotherapy treatment. In some embodiments, the boost phase begins about 3 weeks to about 12 weeks after the chemotherapy phase has ended, optionally about 3 weeks to about 12 weeks after the last administration of chemotherapy treatment; or about 3 weeks or about 4 weeks after the chemotherapy phase has ended, optionally about 3 weeks or about 4 weeks after the last administration of chemotherapy treatment.

[0020] In some embodiments, the boost phase begins at week 27, starting at week 1 of the chemotherapy phase. In some embodiments, the boost phase begins at week 33, starting at week 1 of the priming phase. In some embodiments, the boost phase comprises administering the RNA vaccine and the PD-1 axis-binding antagonist on day 1 of week 33, and every four weeks thereafter, starting at week 1 of the priming phase.

[0021] In some embodiments, the RNA vaccine and the PD-1 axis-binding antagonist are administered six times during the boost phase. In some embodiments, the boost phase comprises administering the RNA vaccine and the PD-1 axis-binding antagonist on day 1 of weeks 33, 37, 41, 45, 49, and 53 of the priming phase, starting at week 1.

[0022] In some embodiments, (a) the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase and the PD-1 axis-binding antagonist on day 1 of week 3 of the priming phase; (b) the chemotherapy phase comprises administering the chemotherapy treatment on day 1 of weeks 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29 of the priming phase, beginning with week 1; and (c) the boost phase comprises administering the RNA vaccine and the PD-1 axis-binding antagonist on day 1 of weeks 33, 37, 41, 45, 49, and 53 of the priming phase, beginning with week 1 of the priming phase.

[0023] In some embodiments, (a) the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase and the PD-1 axis-binding antagonist on day 1 of weeks 1 and 5 of the priming phase; (b) the chemotherapy phase comprises administering the chemotherapy treatment on day 1 of weeks 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29 of the priming phase, beginning with week 1; and (c) the boost phase comprises administering the RNA vaccine and the PD-1 axis-binding antagonist on day 1 of weeks 33, 37, 41, 45, 49, and 53 of the priming phase, beginning with week 1 of the priming phase.

[0024] In some embodiments, the priming phase begins about 6 weeks and about 12 weeks after resection of the pancreatic cancer tumor from the patient.

[0025] In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is nivolumab or pembrolizumab. In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is avelumab or durvalumab. In some embodiments, the anti-PD-L1 antibody comprises (a) a heavy chain variable region (VH) comprising HVR-H1 comprising the amino acid sequence GFTFSDSWIH (SEQ ID NO: 1), HVR-H2 comprising the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 2), and HVR-H3 comprising the amino acid sequence RHWPGGFDY (SEQ ID NO: 3), and (b) a light chain variable region (VL) comprising HVR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO: 4), HVR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO: 5), and HVR-L3 comprising the amino acid sequence QQYLYHPAT (SEQ ID NO: 6). In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-PD-L1 antibody is atezolizumab.

[0026] In some embodiments, the PD-1 axis binding antagonist is administered intravenously to the patient. In some embodiments, the anti-PD-L1 antibody is administered to the patient at a dose of about 1200 mg or about 1680 mg. In some embodiments, the anti-PD-L1 antibody is atezolizumab, and the atezolizumab is administered intravenously to the patient at a dose of about 1680 mg.

[0027] In some embodiments, the chemotherapy treatment comprises one or more of gemcitabine, leucovorin, 5-fluorouracil, capecitabine, irinotecan, liposomal irinotecan, platinum-based chemotherapy agents, taxanes, and any combination thereof. In some embodiments, the platinum-based chemotherapy agent is cisplatin, oxaliplatin, or both. In some embodiments, the taxane is paclitaxel, docetaxel, albumin-bound paclitaxel, or any combination thereof. In some embodiments, the chemotherapy treatment comprises leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin. In some embodiments, the chemotherapy treatment is FOLFIRINOX treatment or mFOLFIRINOX treatment.

[0028] In some embodiments, the chemotherapy treatment is about 85 mg / m 2 Oxaliplatin at a dose of approximately 400 mg / m 2 Leucovorin at a dose of approximately 150 mg / m 2 and / or about 2400 mg / m 2 In some embodiments, the chemotherapy treatment comprises 5-fluorouracil at a dose of 0.05 mg / kg / day. In some embodiments, the chemotherapy treatment is administered intravenously to the patient.

[0029] In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 or 10-20 neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen. In some embodiments, the one or more polynucleotides of the RNA vaccine are formulated with one or more lipids. In some embodiments, the one or more polynucleotides of the RNA vaccine and one or more lipids form lipid nanoparticles. In some embodiments, the one or more polynucleotides of the RNA vaccine and one or more lipids form lipoplexes. In some embodiments, the lipoplex nanoparticles or lipoplexes comprise one or more lipids that form a multi-membrane structure encapsulating the one or more polynucleotides of the RNA vaccine.

[0030] In some embodiments, the one or more lipids comprise at least one cationic lipid and at least one helper lipid. In some embodiments, the one or more lipids comprise (R)N,N,N-trimethyl-2,3-dioleyloxy-1-propanaminium chloride (DOTMA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, at physiological pH, the overall charge ratio of positive to negative charges of the lipid nanoparticle or lipoplex is 1.3:2 (0.65).

[0031] In some embodiments, the one or more polynucleotides of the RNA vaccine are RNA molecules, optionally messenger RNA molecules. In some embodiments, the RNA vaccine is administered to a patient at a dose of about 15 μg, about 21 μg, about 21.3 μg, about 25 μg, about 38 μg, or about 50 μg. In some embodiments, the RNA vaccine is administered to a patient at a dose of about 25 μg. In some embodiments, the RNA vaccine is administered to the patient in two equal halves. In some embodiments, the two equal halves are administered sequentially, optionally with an observation period between the administration of the equal halves. In some embodiments, the approximately 25 μg dose is divided into two equal halves of about 12.5 μg, each administered over one minute, optionally with an observation period of five minutes between the administration of the equal halves. In some embodiments, the RNA vaccine is administered to a patient intravenously.

[0032] In some embodiments, the RNA vaccine comprises an RNA molecule comprising, in a 5' to 3' direction: (1) a 5' cap; (2) a 5' untranslated region (UTR); (3) a polynucleotide sequence encoding a secretory signal peptide; (4) a polynucleotide sequence encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen; (5) a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule; (6) a 3' UTR comprising (a) a 3' untranslated region of an Amino-Terminal Enhancer of Split (AES) mRNA or a fragment thereof, (b) a non-coding RNA of a mitochondrially encoded 12S RNA or a fragment thereof; and (7) a poly(A) sequence.

[0033] In some embodiments, the RNA molecule further comprises a polynucleotide sequence encoding an amino acid linker, wherein the amino acid linker and the polynucleotide sequence encoding a first of the one or more neoepitopes form a first linker-neoepitope module, and the polynucleotide sequence forming the first linker-neoepitope module is located, in a 5' to 3' direction, between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

[0034] In some embodiments, the amino acid linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 39). In some embodiments, the polynucleotide sequence encoding the amino acid linker comprises the sequence GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO: 37).

[0035] In some embodiments, the RNA molecule further comprises, in a 5'→3' direction, at least a second linker-neoepitope module, wherein the at least second linker-neoepitope module comprises a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope, wherein the polynucleotide sequence forming the second linker-neoepitope module is located, in a 5'→3' direction, between the polynucleotide sequence encoding the neoepitope of the first linker-neoepitope module and the polynucleotide sequence encoding at least a portion of a transmembrane domain and a cytoplasmic domain of an MHC molecule, and wherein the neoepitope of the first linker-neoepitope module is different from the neoepitope of the second linker-neoepitope module.

[0036] In some embodiments, the RNA molecule comprises five linker-neoepitope modules, each of which encodes a different neoepitope. In some embodiments, the RNA molecule comprises ten linker-neoepitope modules, each of which encodes a different neoepitope. In some embodiments, the RNA molecule comprises twenty linker-neoepitope modules, each of which encodes a different neoepitope.

[0037] In some embodiments, the RNA molecule further comprises a second polynucleotide sequence encoding an amino acid linker, wherein the second polynucleotide sequence encoding the amino acid linker is between the polynucleotide sequence encoding the 3'-most distal neoepitope and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

[0038] In some embodiments, the 5' cap has the structure: [ka] Contains the D1 diastereomer of

[0039] In some embodiments, the 5'UTR comprises the sequence UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 23). In some embodiments, the 5'UTR comprises the sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 21).

[0040] In some embodiments, the secretory signal peptide comprises the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 27). In some embodiments, the polynucleotide sequence encoding the secretory signal peptide comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 25).

[0041] In some embodiments, at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule comprise the amino acid sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 30). In some embodiments, the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule comprises the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC (SEQ ID NO: 28).

[0042] In some embodiments, the 3' untranslated region of the AES mRNA comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO: 33).

[0043] In some embodiments, the non-coding RNA of the mitochondrially encoded 12S RNA comprises the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO: 35).

[0044] In some embodiments, the 5'UTR comprises the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 31).

[0045] In some embodiments, the poly(A) sequence comprises 120 adenine nucleotides.

[0046] In some embodiments, the RNA comprises, in the 5' to 3' direction, the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 19); a polynucleotide sequence encoding one or more neoepitopes resulting from a cancer-specific somatic mutation present in a tumor specimen; and the polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUA GCGCCCAGGGCAGCGACGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCAC GCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 20).

[0047] In some embodiments, the pancreatic cancer tumor is a resectable PDAC tumor, and is evaluated by preoperative imaging in patients using computed tomography (CT) scans with contrast or magnetic resonance imaging (MRI) before administering the RNA vaccine, PD-1 axis-binding antagonist, and chemotherapy treatment. In some embodiments, the pancreatic cancer tumor is a resectable PDAC tumor that includes one or more characteristics selected from the group consisting of a clear fat plane around the celiac trunk and superior mesenteric artery; patent superior mesenteric vein and portal vein; no superior mesenteric vein or portal vein encirclement; no superior mesenteric artery or hepatic artery encirclement; absence of metastatic disease; and absence of extraregional lymph node disease. In some embodiments, the patient has a histologically confirmed diagnosis of PDAC before administering the RNA vaccine, PD-1 axis-binding antagonist, and chemotherapy treatment.

[0048] In some embodiments, the patient has adenosquamous carcinoma of the pancreas prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0049] In some embodiments, the pancreatic cancer tumor has a tumor, node, metastasis (TNM) pathological staging value of T1-T3, N0-N2, or M0 prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

[0050] In some embodiments, the pancreatic cancer tumor is a resectable PDAC tumor, and the patient has no evidence of PDAC disease after resection of the PDAC tumor, and / or the patient has undergone grossly complete resection of the PDAC tumor before administering the RNA vaccine, the PD-1 axis-binding antagonist, and chemotherapy treatment, optionally, the patient has undergone R0 or R1 resection of the PDAC tumor. In some embodiments, the patient is clearly free of PDAC after resection of the PDAC tumor, and optionally, the absence of PDAC is assessed by CT or MRI scan, one or more biochemical assays, and / or clinical findings. In some embodiments, the pancreatic cancer tumor is a resectable PDAC tumor, and after tumor resection, the patient has no unresolved postoperative complications of grade 3 or higher before administering the RNA vaccine, the PD-1 axis-binding antagonist, and chemotherapy treatment, and optionally, the complications are assessed according to the Clavien Dindo Classification of Surgical Complications.

[0051] In some embodiments, the patient has a CA19-9 level of 180 U / mL or greater prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the patient has a CA19-9 level of less than 180 U / mL prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0052] In some embodiments, at least five neoepitopes resulting from cancer-specific somatic mutations are present in a tumor specimen obtained from the patient prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0053] In some embodiments, the patient has an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 or 1 prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0054] In some embodiments, the patient does not have intraductal papillary mucinous tumor-associated PDAC prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0055] In some embodiments, the patient does not have pancreatic endocrine tumor or acinar cell adenocarcinoma, pancreatic cystadenocarcinoma, or pancreatic malignant squamous cell carcinoma prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

[0056] In some embodiments, the patient has not received adjuvant, neoadjuvant, or induction treatment for pancreatic cancer or systemic anti-cancer treatment for pancreatic cancer before administering the RNA vaccine, the PD-1 axis-binding antagonist, and chemotherapy treatment, and optionally, the pancreatic cancer is PDAC. In some embodiments, the patient has not received cytotoxic chemotherapy, immunotherapy, investigational therapy, or radiation therapy before administering the RNA vaccine, the PD-1 axis-binding antagonist, and chemotherapy.

[0057] In some embodiments, the patient has a spleen before administering the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the patient does not have spleen loss due to splenectomy, spleen injury / infarction, or functional asplenia before administering the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the patient does not have a distal pancreatectomy with splenectomy before administering the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0058] In some embodiments, the patient does not have a pre-existing neuropathy prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

[0059] In some embodiments, the patient does not have an aUGT1A1 genotype associated with a poor metabolizer phenotype prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

[0060] In some embodiments, the patient does not have an autoimmune disease, immunodeficiency, or primary immunodeficiency prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the patient has not been treated with a monoamine oxidase inhibitor (MAOI) within three weeks, a systemic immune stimulant within four weeks, or five drug elimination half-lives, whichever is longer, or a systemic immunosuppressant within two weeks prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0061] In some embodiments, the patient has not undergone an allogeneic stem cell or solid organ transplant prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

[0062] In some embodiments, the method further comprises assessing the disease-free survival (DFS) of said patient after treatment with the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment results in an improvement in the patient's DFS compared to the DFS of a corresponding patient who is not administered the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0063] In some embodiments, the method further comprises assessing the patient's overall survival (OS) after administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment results in improved OS for the patient compared to the OS of a corresponding patient who has not received the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0064] In some embodiments, the method further comprises performing one or more clinical assessments of the patient before, during, and / or after treatment with the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment, wherein the one or more clinical assessments are selected from the group consisting of the European Organisation for Research and Treatment of Cancer QLQ-C30 questionnaire (EORTC QLQ C30), the European Organisation for Research and Treatment of Cancer QLQ-PAN26 questionnaire (EORTC QLQ PAN26), the National Cancer Institute's Patient-Reported Outcomes Common Terminology Criteria for Adverse Events (PRO CTCAE), and the European Organisation for Research and Treatment of Cancer Item Library 46 Questionnaire (EORTC IL46). In some embodiments, administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment results in an improvement in one or more clinical assessments compared to one or more clinical assessments in the patient prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment, and / or compared to one or more clinical assessments in a corresponding patient who has not been administered the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

[0065] In some embodiments, the method further includes assessing antigen-specific and / or tumor-specific T cell responses in the patient before, during, and / or after treatment with the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment results in improved antigen-specific and / or tumor-specific T cell responses in the patient compared to before administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment, and / or compared to a corresponding patient not administered the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0066] In some embodiments, the corresponding patient is a patient with a corresponding pancreatic cancer tumor, and optionally, the pancreatic cancer tumor is a PDAC tumor, and the corresponding patient has a PDAC tumor.In some embodiments, the corresponding patient has been treated with standard therapy for pancreatic cancer, PDAC, or resectable or resected PDAC.In some embodiments, the standard therapy comprises gemcitabine combination therapy or mFOLFIRINOX chemotherapy.

[0067] In some embodiments, corresponding patients were treated with a control treatment comprising mFOLFIRINOX chemotherapy. In some embodiments, the mFOLFIRINOX chemotherapy was about 85 mg / m 2 Oxaliplatin at a dose of approximately 400 mg / m 2 Leucovorin at a dose of approximately 150 mg / m 2 and irinotecan at a dose of approximately 2400 mg / m 2 This drug contains 5-fluorouracil at a dose of 10 mg / kg / day, administered intravenously on day 1 of each 14-day cycle for up to a total of 12 cycles.

[0068] In one aspect, provided is a personalized RNA vaccine for use in a method for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the personalized RNA vaccine is administered in combination with a PD-1 axis-binding antagonist and chemotherapy treatment according to the methods described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient. In another aspect, provided is a PD-1 axis-binding antagonist for use in a method for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the personalized RNA vaccine is administered in combination with a personalized RNA vaccine and chemotherapy treatment according to the methods described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient.

[0069] In one aspect, there is provided a use of a personalized RNA vaccine in the manufacture of a medicament for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the RNA vaccine is administered in combination with a PD-1 axis-binding antagonist and chemotherapy treatment according to the methods described herein, and the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient. In another aspect, there is provided a use of a personalized PD-1 axis-binding antagonist in the manufacture of a medicament for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the PD-1 axis-binding antagonist is administered in combination with a personalized RNA vaccine and chemotherapy treatment according to the methods described herein, and the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient.

[0070] In one aspect, a kit is provided that includes a personalized RNA vaccine for use in a method for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the RNA vaccine is administered in combination with a PD-1 axis-binding antagonist and chemotherapy treatment according to the methods described herein, and the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient. In another aspect, a kit is provided that includes a PD-1 axis-binding antagonist for use in a method for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the PD-1 axis-binding antagonist is administered in combination with a personalized RNA vaccine and chemotherapy treatment according to the methods described herein, and the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient.

[0071] In one aspect, a method for selecting a human patient having a cancer tumor who is likely to respond to a therapy comprising a personalized RNA vaccine is provided, comprising: a) measuring the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from the patient by T cell receptor sequencing; b) comparing the number and / or frequency of the de novo SE TCR clones measured in a) with a reference number and / or reference frequency; and c) selecting the patient who is more likely to respond to a therapy comprising the personalized RNA vaccine if the number and / or frequency of the de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency, wherein the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient, and a number and / or frequency of de novo SE TCR clones that is greater than the reference number and / or reference frequency indicates that the patient is more likely to respond to a therapy comprising the personalized RNA vaccine. In some embodiments, the method further comprises selecting a therapy comprising the personalized RNA vaccine or recommending a therapy comprising the personalized RNA vaccine.In another aspect, a method is provided for selecting a human patient having a cancer tumor who is likely to respond to a therapy comprising a personalized RNA vaccine, the method comprising: a) comparing the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from the patient with a reference number and / or reference frequency; and b) selecting the patient who is more likely to respond to a therapy comprising the personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency, wherein the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient, and the number and / or frequency of de novo SE TCR clones is measured by T cell receptor sequencing, and a number and / or frequency of de novo SE TCR clones greater than the reference number and / or reference frequency indicates that the patient is more likely to respond to a therapy comprising the personalized RNA vaccine.

[0072] In one aspect, a method of treating a human patient having a cancer tumor is provided, comprising: a) measuring the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from the patient by T cell receptor sequencing; b) comparing the number and / or frequency of de novo SE TCR clones measured in a) with a reference number and / or reference frequency; and c) selecting a patient who is more likely to respond to a therapy comprising a personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency, wherein the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient, and wherein a number and / or frequency of de novo SE TCR clones greater than the reference number and / or reference frequency indicates that the patient is more likely to respond to a therapy comprising the personalized RNA vaccine. In another aspect, a method of treating a human patient having a cancer tumor is provided, comprising: a) comparing the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from the patient with a reference number and / or reference frequency; and b) selecting a patient who is more likely to respond to a therapy comprising a personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient exceeds the reference number and / or reference frequency, thereby treating the cancer tumor, wherein the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient, and the number and / or frequency of de novo SE TCR clones is measured by T cell receptor sequencing, and a number and / or frequency of de novo SE TCR clones that exceeds the reference number and / or reference frequency indicates that the patient is more likely to respond to a therapy comprising the personalized RNA vaccine.

[0073] In some embodiments, the method further comprises administering to the patient a therapy comprising a personalized RNA vaccine, thereby treating the cancer tumor, if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than a reference number and / or reference frequency. In some embodiments, the method further comprises selecting a therapy comprising a personalized RNA vaccine, thereby treating the cancer tumor, if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than a reference number and / or reference frequency. In some embodiments, the number and / or frequency is measured after six administrations of the personalized cancer vaccine. In some embodiments, the reference number is six de novo SE TCR clones. In some embodiments, the reference frequency is 10 -4 It is a de novo SE TCR clone.

[0074] In some embodiments, the cancer tumor is a pancreatic cancer tumor. In some embodiments, the cancer tumor is a pancreatic ductal adenocarcinoma (PDAC) tumor.

[0075] In some embodiments, the therapy comprising the personalized RNA vaccine further comprises a PD-1 axis-binding antagonist. In some embodiments, the therapy further comprises chemotherapy treatment, and the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment are administered to the patient during a priming phase, during a chemotherapy phase after the priming phase, and during a boost phase after the chemotherapy phase, wherein (i) the priming phase comprises administering the RNA vaccine to the patient at least once and administering the PD-1 axis-binding antagonist at least once, (ii) the chemotherapy phase comprises administering the chemotherapy treatment to the patient, and (iii) the boost phase comprises administering the RNA vaccine to the patient at least once and administering the PD-1 axis-binding antagonist at least once. In some embodiments, the PD-1 axis-binding antagonist is atezolizumab. In some embodiments, the chemotherapy treatment is FOLFIRINOX treatment or mFOLFIRINOX treatment.

[0076] In some embodiments, prior to the administering step, the patient is selected by a method comprising: a) measuring the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from the patient by T cell receptor sequencing; b) comparing the number and / or frequency of de novo SE TCR clones measured in a) with a reference number and / or reference frequency; and c) selecting the patient as more likely to respond to a therapy comprising the personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient exceeds the reference number and / or reference frequency, wherein the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient, and wherein a number and / or frequency of de novo SE TCR clones exceeding the reference number and / or reference frequency indicates that the patient is more likely to respond to a therapy comprising the personalized RNA vaccine.

[0077] In one aspect, there is provided an in vitro use of the number and / or frequency of significantly de novo expanded (SE) TCR clones to select patients with cancer tumors who are more likely to respond to a therapy comprising a personalized RNA vaccine, wherein a number and / or frequency of de novo SE TCR clones in a sample from a patient that exceeds a reference number and / or reference frequency selects the patient as more likely to respond to a therapy comprising a personalized RNA vaccine.

[0078] In one aspect, there is provided the use of the number and / or frequency of significantly de novo expanded (SE) TCR clones for the manufacture of a diagnostic agent for assessing the likelihood of a patient with a cancer tumor responding to a therapy comprising a personalized RNA vaccine. [Brief explanation of the drawings]

[0079] [Figure 1]A schematic diagram of the Phase II trial design described in Example 1 is provided. Patients with resectable PDAC undergo screening parts A and B and are then randomized into one of two arms: arm 1, in which patients receive the personalized RNA vaccine in combination with atezolizumab and mFOLFIRINOX, and arm 2, in which patients receive mFOLFIRINOX alone. mFOLFIRINOX: modified leucovorin, 5-fluorouracil (5-FU), irinotecan, oxaliplatin; PDAC: pancreatic ductal adenocarcinoma; Q4W: every 4 weeks.

[0080] [Figure 2A-2B]Figure 2A provides a diagram of the design of the study treatment phase and dosing schedule for arms 1 and 2 of the study described in Example 1. Figure 2A provides a diagram of the design of the study treatment phase and dosing schedule for dosing regimen A for arms 1 and 2. Subjects in arm 1 dosing regimen A of the phase II study will be administered personalized RNA vaccine (at a dose of 25 μg, by IV infusion, once weekly for 6 weeks) in combination with atezolizumab (at a dose of 1680 mg, by IV infusion on the first day of weeks 1 and 5) in the priming phase; mFOLFIRINOX (12 rounds of administration in 14-day cycles starting from week 7) in the chemotherapy phase; and RNA vaccine (at a dose of 25 μg, by IV infusion, 6 rounds of administration in 28-day cycles starting from week 33) in combination with atezolizumab (at a dose of 1680 mg, by IV infusion, 6 rounds of administration in 28-day cycles starting from week 33) in the boost phase. Subjects in arm 2 of the phase II trial will receive mFOLFIRINOX alone in 14-day cycles starting in week 1 for a total of up to 12 rounds. Figure 2B provides a diagram of the study treatment phase and dosing schedule design of dosing regimen B for arms 1 and 2. Subjects in Arm 1 Dosing Regimen B of the Phase II study will receive the personalized RNA vaccine (at a dose of 25 μg, administered IV infusion once weekly for 6 weeks) in combination with atezolizumab (at a dose of 1680 mg administered IV on Day 1 of Week 3) in the priming phase; mFOLFIRINOX (administered for 12 rounds in 14-day cycles starting at Week 7) in the chemotherapy phase; and the RNA vaccine (at a dose of 25 μg, administered IV infusion for 6 rounds in 28-day cycles starting at Week 33) in combination with atezolizumab (administered for 6 rounds in 28-day cycles starting at Week 33) in the boost phase. Subjects in Arm 2 of the Phase II study will receive mFOLFIRINOX alone in 14-day cycles starting at Week 1 for a total of up to 12 rounds. In Figures 2A-2B, B: boost; C: chemotherapy; mFOLFIRINOX: modified leucovorin, 5-fluorouracil (5-FU), irinotecan, oxaliplatin; P: priming.

[0081] [Figure 3] Figure 1 provides a comparative schedule of combined RNA vaccine and chemotherapy treatment in a mouse syngeneic MC38 tumor model. The chemotherapy regimen involved intraperitoneal (ip) injection of modified mouse FOLFIRINOX. The RNA vaccine (referred to as RNA-LPX) involved three doses administered by intravenous injection (iv) on days 0, 7, and 14 (gray arrows). The chemotherapy regimen, as described in Example 2, began at different time points (black arrows) compared to the RNA vaccine. Blood was collected weekly from n = 5 mice per group to analyze T cell responses. Deca: "Decatop"; LPX: lipoplex; FOLFIRINOX: leucovorin, 5-fluorouracil (5-FU), irinotecan, oxaliplatin. n = 10 mice per group.

[0082] [Figure 4]

[0023] Figure 1 provides line graphs showing tumor growth curves for each of the eight treatment groups. LPX: Lipoplex; IV: Intravenous injection; Q14Dx3: 3 rounds, once every 14 days; QWx3: 3 rounds, once weekly; FOLFIRINOX: Leucovorin, 5-fluorouracil (5-FU), irinotecan, oxaliplatin. n = 10 mice per group.

[0083] [Figure 5]Line graphs showing tumor growth curves for each individual mouse in a treatment group are provided, with the group best-fit curve and reference fit overlaid on the graph. Each graph represents results from one of eight treatment groups. Black arrows indicate the time points at which mice received chemotherapy treatment. Gray arrows indicate the time points at which mice received Decatope 1+2 RNA-LPX vaccination. The reference fit and group fit curves are indicated by labeled arrows. LPX: lipoplex; IV: intravenous injection; Q14Dx3: once every 14 days for 3 rounds; QWx3: once every week for 3 rounds; FOLFIRINOX: leucovorin, 5-fluorouracil (5-FU), irinotecan, oxaliplatin. n = 10 mice per group.

[0084] [Figure 6]

[0023] Figure 1 shows the design of the Phase Ia / Ib study described in Example 3. Subjects in the Phase Ia dose-escalation study were administered the RNA vaccine as monotherapy at doses of 25 μg, 38 μg, 50 μg, 75 μg, or 100 μg. During the initial treatment (induction phase), the RNA vaccine was administered on days 1, 8, and 15 of cycle 1, days 1, 8, and 15 of cycle 2, days 1 and 15 of cycle 3, and day 1 of cycle 7 (each cycle was 21 days). During the maintenance phase after initial treatment, the RNA vaccine was administered on day 1 of cycle 13, and then every 8 cycles (i.e., every 24 weeks thereafter, or every 168 days thereafter) until disease progression (PD) (each cycle was 21 days). Subjects in the Phase 1b study received the RNA vaccine at doses of 15 μg (not shown), 25 μg, 38 μg, or 50 μg in combination with 1200 mg of atezolizumab. The Phase 1b study included a dose-escalation phase of the RNA vaccine and an expansion phase in which patients with the indicated checkpoint inhibitor-naive or checkpoint inhibitor-experienced tumor types received the RNA vaccine at doses of 15 μg or 25 μg in combination with atezolizumab. During the initial treatment (induction phase), atezolizumab was administered on day 1 of each of cycles 1 through 12; the RNA vaccine was administered on days 1, 8, and 15 of cycle 1; days 1, 8, and 15 of cycle 2; days 1 and 15 of cycle 3; and day 1 of cycle 7 (each cycle was 21 days long). During the maintenance phase after initial treatment, atezolizumab was administered every 3 weeks starting on day 1 of cycle 13 until disease progression (PD), and the RNA vaccine was administered on day 1 of cycle 13 and every 8 cycles thereafter (i.e., every 24 weeks thereafter or every 168 days thereafter) until disease progression (PD) (each cycle was 21 days).

[0085] [Figure 7] FIG. 1 provides a diagram of the MHC multimer staining assay used to evaluate neoantigen-specific CD8+ T cell immune responses following administration of the RNA vaccine as monotherapy (Phase Ia) or in combination with atezolizumab (Phase Ib), as described in Example 3 herein.

[0086] [Figure 8A-8B]

[0033] Figure 8A shows the results of an MHC multimer staining assay evaluating neoantigen-specific CD8+ T cell immune responses following treatment with a personalized RNA vaccine alone or in combination with atezolizumab from a Phase Ia / Ib study, as described in Example 3 herein. Figure 8A shows the frequency of antigen-specific CD8+ T cells in patients from a Phase Ia study who received a personalized RNA vaccine as monotherapy. The personalized RNA vaccine dosing regimen is indicated below the plot. Figure 8B shows the frequency of antigen-specific CD8+ T cells in patients from a Phase Ib study who received a personalized RNA vaccine in combination with atezolizumab. The dosing regimens for the personalized RNA vaccine and atezolizumab ("Atezo") are indicated below the plot. In Figures 8A-8B, each line represents a unique antigen-specific CD8+ T cell response measured longitudinally. C, cycles.

[0087] [Figure 9]

[0023] Figure 1 shows the results of an MHC multimer staining assay evaluating neoantigen-specific CD8+ T cell immune responses after a boost dose of a personalized RNA vaccine from the Phase Ia / Ib study described in Example 3 herein. The frequencies of antigen-specific CD8+ T cells are shown pre-boost on Day 1 of Cycle 6 (C6D1) or Day 1 of Cycle 12 (C12D1), and post-boost on Day 1 of Cycle 8 (C8D1) or Day 1 of Cycle 14 (C14D1). The timing of the boost dose of personalized RNA vaccine is indicated by the arrow and text below the plot (Day 1 of Cycle 7 [C7D1] and Day 1 of Cycle 13 [C13D1]). Each line represents a unique antigen-specific CD8+ T cell response measured longitudinally.

[0088] [Figure 10]A schematic diagram of significantly expanded T cell receptor (TCR) clones in a single sample is shown to identify clones that expanded upon vaccination. Each dot represents a unique TCR clone identified based on its nucleotide sequence. The frequency of that clone in the pretreatment (i.e., baseline) sample and in the on-treatment sample after six doses of vaccine are plotted on a Log10 scale on the x- and y-axes, respectively. The frequencies of the two time points were compared using a statistical beta-binomial model with an adjusted p-value of 0.01; points are colored based on significance. Significantly expanded (SE) clones are colored black and labeled as de novo if the clone was not detected in the baseline sample, or as pre-existing if the clone was detected at baseline but expanded in frequency after vaccination. The patient's vaccine and Atezo treatment schedule is also plotted.

[0089] [Figure 11] The figures show a higher number of de novo significantly expanded (SE) clones in immune responders to the vaccine (i.e., ELISpot positive). The number of expanded TCR clones at different stages of treatment: 3-Vax (after approximately three vaccine doses), 6-Vax (after approximately six vaccine doses), 8-Vax (after approximately eight vaccine doses), and 9 / 10-Vax (after approximately nine or ten vaccine doses) are plotted for each patient. Each dot represents data from a single patient at the indicated stage of vaccination. Patient immunogenicity was assessed by ELISpot assay. p-values ​​are based on the Wilcoxon Mann-Whitney test. DETAILED DESCRIPTION OF THE INVENTION

[0090] I. Definition Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary widely. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0091] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" includes any combination of two or more such molecules.

[0092] As used herein, the term "about" refers to a normal range of error for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0093] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0094] The term "PD-1 axis-binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis-binding partner with one or more of its binding partners to eliminate T cell dysfunction resulting from signaling on the PD-1 signaling axis, thereby restoring or enhancing T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis-binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0095] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, impairs, or prevents signaling that occurs as a result of the interaction of PD-1 with one or more binding partners, such as PD-L1 and PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In specific aspects, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhexins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, impair, or prevent signaling that results from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that mediated signaling through PD-1, rendering dysfunctional T cells less dysfunctional (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. Specific examples of PD-1 binding antagonists are provided below.

[0096] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L1 with any one or more of its binding partners, e.g., PD-1, B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1, B7-1. In one embodiment, the PD-L1 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that mediated signaling through PD-L1, rendering the dysfunctional T cell less dysfunctional (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. Specific examples of PD-L1 binding antagonists are provided below.

[0097] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L2 with any one or more of its binding partners, e.g., PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-L2 with any one or more of its binding partners, e.g., PD-1. In one embodiment, the PD-L2 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that mediated signaling through PD-L2, rendering dysfunctional T cells less dysfunctional (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0098] "Sustained response" refers to a persistent effect on reducing tumor growth after cessation of treatment. For example, tumor size may remain the same or may be smaller compared to the size at the beginning of the administration phase. In some embodiments, the sustained response has a duration at least equal to the treatment period, at least 1.5, 2.0, 2.5, or 3.0 times the treatment period.

[0099] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient is in a form such that it is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0100] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical disease. Desirable effects of treatment include a reduction in the rate of disease progression, an improvement or palliative of the disease state, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated, including, but not limited to, a reduction in (or destruction of) the proliferation of cancerous cells, a reduction in symptoms caused by the disease, an improvement in the quality of life of those suffering from the disease, a reduction in the dose of other medications required to treat the disease, and / or an increase in the individual's survival time.

[0101] As used herein, "delaying disease progression" means postponing, preventing, slowing, delaying, stabilizing, and / or postponing the onset of a disease (such as cancer). This delay can be of varying lengths of time, depending on the disease being treated and / or the individual's medical history. As will be apparent to those skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the development of metastases, can be delayed.

[0102] An "effective amount" is at least the minimum amount necessary to achieve measurable improvement or prevention of a particular disorder. The effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in an individual. An effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or adverse effects of the treatment. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes manifesting during disease development. For therapeutic use, beneficial or desired results include clinical results such as a reduction in one or more symptoms caused by the disease, improving the quality of life of a person suffering from the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug (e.g., by targeting), delaying disease progression, and / or prolonging survival. In the case of cancer or tumors, an effective amount of a drug may have the effect of reducing the number of cancer cells, reducing tumor size, inhibiting (i.e., slowing or preferably stopping) the infiltration of cancer cells into peripheral organs, inhibiting (i.e., slowing or preferably stopping) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating one or more symptoms associated with the disorder to some extent. An effective amount may be administered in one or more administrations. In the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in the clinical field, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of the administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be achieved or is achieved.

[0103] As used herein, "in conjunction with" or "in combination with" refers to the administration of one treatment modality in addition to another treatment modality. Thus, "in conjunction with" or "in combination with" refers to the administration of one treatment modality to an individual before, during, or after the administration of another treatment modality.

[0104] A "disorder" is any condition for which treatment would be beneficial, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0105] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

[0106] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0107] A "subject" or "individual" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

[0108] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies (such as full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

[0109] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with research, diagnostic, or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95%, and in some embodiments, greater than 99%, by weight of the antibody, as determined, for example, by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, by using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, for example, using Coomassie blue or silver stain. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

[0110] "Native antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the light chain constant domain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0111] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CH1, CH2, and CH3 domains of the heavy chain (collectively, CH), and the CHL (or CL) domain of the light chain.

[0112] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0113] The term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely among antibodies and are used in the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration, connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs within each chain are held in close proximity by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular toxicity.

[0114] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0115] As used herein, the term IgG "isotype" or "subclass" means any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions.

[0116] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known and are described, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000). An antibody may be part of a larger fusion molecule formed by covalent or noncovalent association of the antibody with one or more other proteins or peptides.

[0117] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than the antibody fragments described below. These terms specifically refer to antibodies having heavy chains that include an Fc region.

[0118] A "naked antibody" for purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0119] An "antibody fragment" includes a portion of an intact antibody, preferably including its antigen-binding region. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0120] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, named for its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0121] An "Fv" is the minimum antibody fragment containing a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0122] Fab fragments contain heavy and light chain variable domains, and also contain a light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments in that they have a few additional residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0123] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0124] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with complementary domains on another chain, generating two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described, for example, in EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0125] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for possible minor variations, e.g., naturally occurring variations. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of distinct antibodies. In certain embodiments, such monoclonal antibodies typically comprise a polypeptide sequence that binds to a target, the target-binding polypeptide sequence being obtained by a process that includes selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It is understood that the selected target-binding sequence may be further modified to, for example, improve affinity for the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, create a multispecific antibody, etc., and that an antibody comprising a modified target-binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0126] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be produced using, for example, hybridoma methods (e.g., those described in Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al. al.,J.Mol.Biol.222:581-597(1992);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al. al.,J.Mol.Biol.340(5):1073-1093(2004);Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004);and Lee et al.,J.Immunol.Methods 284(1-2):119-132 (2004); and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0127] The term "monoclonal antibodies" as used herein specifically includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include the PRIMATTZED® antibody, the antigen-binding region of which is derived from, for example, an antibody produced by immunizing macaque monkeys with the antigen of interest.

[0128] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient or donor antibody. These modifications may be made to further refine antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of a human immunoglobulin sequence. Humanized antibodies optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Also see, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0129] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using various techniques known in the art, including phage display libraries. The methods described in Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, such as immunized xenomouse, that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), regarding human antibodies generated by human B cell hybridoma technology.

[0130] A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of the antigen from a second mammalian species. Typically, a species-dependent antibody "specifically binds" to a human antigen (e.g., about 1 x 10 -7 M or less, preferably about 1 × 10 -8 M or less, preferably about 1 × 10 -9The species-dependent antibody has a binding affinity (Kd) value of M or less for a homolog of the antigen from a second non-human mammalian species that is at least about 50-fold, or at least about 500-fold, or at least about 1000-fold weaker than the binding affinity for the human antigen. The species-dependent antibody can be any of the various types of antibodies defined above, but is preferably a humanized or human antibody.

[0131] As used herein, the term "hypervariable region," "HVR," or "HV" refers to a region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, antibodies contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). In native antibodies, H3 and L3 exhibit the highest diversity among the six HVRs, and H3 in particular is thought to play a unique role in conferring superior specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies, consisting only of heavy chains, are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0132] Several HVR delineations are used and encompassed herein. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, instead, refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are shown below. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H30-H35B (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

[0133] HVRs may include the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. The variable domain residues are numbered according to Kabat et al. (see above) for each of these definitions.

[0134] HVRs may include the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. The variable domain residues are numbered according to Kabat et al. (see above) for each of these definitions.

[0135] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0136] The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat-numbered sequence at the regions of homology.

[0137] The Kabat numbering system is commonly used when referring to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is commonly used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0138] The term "linear antibody" refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0139] As used herein, the terms "bind," "specifically bind to," or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, such as biological molecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0140] As used herein, the term "sample" refers to a composition obtained from or derived from a subject and / or individual of interest that contains cellular and / or other molecular entities to be characterized and / or identified, e.g., based on physical, biochemical, chemical, and / or physiological properties. For example, the phrase "disease sample" and variations thereof refer to any sample obtained from a subject of interest that is expected to contain or is known to contain the cellular and / or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts, e.g., homogenized tissue, tumor tissue, cell extracts, and combinations thereof. In some embodiments, the sample is a sample obtained from an individual's cancer (e.g., a tumor sample) containing tumor cells and, optionally, tumor-infiltrating immune cells. For example, the sample may be a tumor specimen embedded in a paraffin block or a tumor specimen comprising freshly cut serial unstained sections. In some embodiments, the sample is from a biopsy and contains 50 or more viable tumor cells (e.g., a core needle biopsy, optionally embedded in a paraffin block; an excision biopsy, an incision biopsy, a punch biopsy, or a forceps biopsy; or a tumor tissue resection).

[0141] A "tissue sample," "tissue biopsy," or "cell sample" refers to a collection of similar cells obtained from a subject's or individual's tissue, e.g., a tumor. The source of the tissue or cell sample may be solid tissue (e.g., a tumor), such as from fresh, frozen, and / or preserved organs, tissue samples, biopsies, and / or aspirates; blood or any blood constituents, such as plasma; bodily fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage in a subject's pregnancy or development. A tissue sample may also be primary or cultured cells or cell lines. Optionally, a tissue or cell sample is obtained from a diseased tissue / organ. A tissue sample may contain compounds not naturally mixed with the native tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0142] As used herein, a "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part (e.g., tissue or cells) of the body of the same subject or individual. For example, a healthy and / or non-diseased cell or tissue adjacent to a diseased cell or tissue (e.g., a cell or tissue adjacent to a tumor). In another embodiment, the reference sample is obtained from an untreated tissue and / or cells of the body of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part (e.g., tissue or cells) of the body of an individual other than the subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and / or cells of the body of an individual other than the subject or individual.

[0143] As used herein, a "reference level," "reference number," or "reference frequency" refers to a number, frequency, amount, etc., having a predetermined value. In this context, "level" encompasses absolute numbers or amounts, relative numbers or amounts, frequencies, proportions, percentages, etc., as well as any value or parameter that can be correlated to or derived therefrom. Those skilled in the art will understand that the reference level is predetermined and set to meet routine requirements, for example, with respect to specificity and / or sensitivity. These requirements may vary, for example, depending on regulatory agencies. For example, the assay sensitivity and / or specificity may be set to a certain limit, for example, 80%, 90%, 95%, 98%, 99%, or 100%. These requirements may also be specified in terms of positive predictive value or negative predictive value. Nevertheless, based on the teachings provided in the present invention, those skilled in the art can arrive at a reference level, reference number, or reference frequency that meets these requirements. For example, the reference level, reference number, or reference frequency can be determined in a reference sample obtained from a patient before treatment administration or in a reference sample obtained from a healthy individual. In one embodiment, the reference level, reference number, or reference frequency is previously determined in the reference sample from the disease entity to which the patient belongs.In certain embodiments, the reference level, reference number, or reference frequency can be statistically calculated or set to be determined from the overall distribution of values ​​in the reference sample from the disease entity being investigated.In one embodiment, the reference level, reference number, or reference frequency is set as a cut-off value determined from the overall distribution of values ​​in the disease entity being investigated, such that the cut-off value indicates, for example, the value at which the predictive rate of immunogenicity and / or immune response to the therapy described herein reaches 100% specificity and 80% sensitivity.The reference level, reference number, or reference frequency can vary from patient to patient, or can vary depending on various physiological parameters of the patient, such as age, sex, or subpopulation, as well as the number of times treated or vaccinated, and (for example) the method used for determination as referred to herein.In one embodiment, the reference sample is derived from essentially the same type of cell, tissue, organ or body fluid source as the sample from the individual or patient subjected to the method of the invention, for example, according to the invention, when blood is used as a sample for determining the level of de novo SE TCR in an individual, the reference level, reference number or reference frequency is also determined in the blood or a part thereof.

[0144] A patient's "effective response" or patient "responsiveness" to treatment with an agent, and similar terms, refers to a clinical or therapeutic benefit conferred on a patient at risk for or suffering from a disease or disorder, such as cancer. In one embodiment, such benefit includes one or more of extending survival (including overall survival and progression-free survival), producing an objective response (including a complete or partial response), or ameliorating the signs or symptoms of cancer.

[0145] A patient who "does not respond effectively" to treatment is one who does not have any of the following: an extension of survival (including overall survival and progression-free survival), an objective response (including a complete or partial response), or an improvement in the signs or symptoms of cancer.

[0146] A patient who is "likely to respond" to a treatment refers to a patient who has been identified based on one or more particular biological characteristics or traits associated with a disease, disorder, or condition (such as cancer) that correlate with treatment responsiveness (or an effective response to treatment). This correlation can be statistically determined, such that a patient identified as "likely to respond" can refer to a patient who has a calculable statistical probability of exhibiting an effective response to treatment.

[0147] In the context of the present invention, the phrase "responsive to" refers to a patient suffering from, suspected of suffering from, prone to suffering from, or diagnosed with a disorder described herein, showing a positive response to a treatment, such as a personalized RNA vaccine described herein. Treatment success can be defined based on progression-free survival (PFS), overall survival (OS), and overall response rate (ORR), including partial or complete response to treatment.

[0148] The term "individualized" in the context of the present invention indicates that the therapy or treatment is specific to each patient, and is designed, constructed, or manufactured based on specifications such as genomic profile, immunological profile, metabolic profile, cancer type, cancer antigen profile, somatic mutation profile, age, gender, etc., or the treatment needs of each individual patient. For example, the RNA vaccines of the present disclosure are personalized for each patient, such that the personalized RNA vaccine targets one or more neoepitopes resulting from cancer-specific somatic mutations present in, for example, a pancreatic cancer tumor specimen from each patient, which may be unique to each patient.

[0149] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; and down-regulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc region to associate with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using various assays disclosed in the definitions herein.

[0150] A cancer or biological sample "having human effector cells" is one that has human effector cells (eg, infiltrating human effector cells) present in the sample in a diagnostic test.

[0151] A cancer or biological sample "having FcR-expressing cells" is one that has FcR expression (e.g., infiltrating FcR-expressing cells) present in the sample in a diagnostic test. In some embodiments, the FcR is an FcγR. In some embodiments, the FcR is an activating FcγR.

[0152] The phrases "selecting a patient" or "identifying a patient," as used herein, refer to using generated information or data related to the number and / or frequency of TCR clones (e.g., significantly expanded (SE) TCR clones, e.g., de novo SE TCR clones) in a patient's sample to identify or select a patient as likely to benefit from a therapy comprising a personalized RNA vaccine. The information or data used or generated can be in any form, e.g., written, oral, or electronic. In some embodiments, using the generated information or data includes communicating, presenting, reporting, storing, transmitting, transferring, providing, disseminating, enforcing, or a combination thereof. In some embodiments, communicating, presenting, reporting, storing, transmitting, transferring, providing, disseminating, enforcing, or a combination thereof is performed by a computer device, an analyzer unit, or a combination thereof. In some further embodiments, communicating, presenting, reporting, storing, transmitting, transferring, providing, disseminating, enforcing, or a combination thereof is performed by a researcher or a medical professional. In some embodiments, the information or data comprises comparing the number and / or frequency of TCR clones (e.g., significantly expanded (SE) TCR clones, e.g., de novo SE TCR clones) to a reference level. In some embodiments, the information or data comprises an indication that a TCR clone (e.g., significantly expanded (SE) TCR, e.g., de novo SE TCR) is present in the sample. In some embodiments, the information or data comprises an indication that the patient is more likely to respond to a therapy comprising a personalized RNA vaccine.

[0153] II. Methods of Treating Pancreatic Cancer Certain aspects of the present disclosure relate to methods for treating pancreatic cancer in a patient, such as a human patient in need thereof, by administering to the patient effective amounts of a personalized RNA vaccine, a PD-1 axis-binding antagonist, and a chemotherapy treatment. In some embodiments, the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen from the patient, e.g., as described in more detail below.

[0154] Any of the personalized RNA vaccines, PD-1 axis binding antagonists, and chemotherapy treatments described herein can be used in the methods of the disclosure.

[0155] The RNA vaccines disclosed herein are designed to induce neoantigen-specific and / or tumor-specific immune responses in patients, such as neoantigen-specific T cell responses, and may increase the size and quality of existing neoantigen-specific T cells. For example, administering a PD-1 axis-binding antagonist in combination with an RNA vaccine can block the patient's PD-L1 / PD-1 pathway, enhancing T cell priming and / or reactivation and / or improving the activity of dysfunctional T cells after tumor antigen exposure, thereby enhancing the RNA vaccine-induced immune response. However, cytotoxic chemotherapy, the standard treatment for pancreatic cancer, can adversely affect RNA vaccine-induced immune responses when administered concomitantly. Therefore, administering an RNA vaccine during a priming phase before chemotherapy (e.g., including up to six priming RNA vaccine administrations, e.g., two or three priming RNA vaccine administrations) can improve a patient's neoantigen-specific and / or tumor-specific immune response (see, e.g., Example 2 herein), which can be maximized and / or maintained when followed up with a boosting phase (e.g., including up to six booster RNA vaccine administrations). RNA vaccine-induced immune responses can be further enhanced by administering the RNA vaccine in combination with a PD-1 axis-binding antagonist during the prime and boost phases, which may result in more robust antitumor immune responses and improved clinical efficacy.

[0156] Thus, in some embodiments, the methods for treating pancreatic cancer provided herein comprise administering a personalized RNA vaccine, a PD-1 axis-binding antagonist, and a chemotherapy treatment during a treatment period comprising three phases: a priming phase, a chemotherapy phase after the priming phase, and a boost phase after the chemotherapy phase, wherein the priming phase comprises administering the RNA vaccine at least once (e.g., up to six administrations of the priming RNA vaccine, e.g., two or three administrations of the priming RNA vaccine) and administering the PD-1 axis-binding antagonist at least once, the chemotherapy phase comprises administering the chemotherapy treatment after administration of the RNA vaccine during the priming phase, and the boost phase comprises administering the RNA vaccine at least once (e.g., up to six administrations of the booster RNA vaccine) and administering at least one dose of the PD-1 axis-binding antagonist.

[0157] In some embodiments, the pancreatic cancer, e.g., pancreatic cancer tumor, treated according to the methods of the present disclosure is exocrine pancreatic cancer or neuroendocrine pancreatic cancer. In some embodiments, the pancreatic cancer is adenocarcinoma (e.g., pancreatic ductal adenocarcinoma), acinar cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma, colloid carcinoma, giant cell tumor, hepatoid carcinoma, mucinous cystic tumor, intraductal papillary mucinous neoplasm, pancreatoblastoma, serous cystadenoma, signet ring cell carcinoma, undifferentiated carcinoma, or solid and pseudopapillary tumor. In some embodiments, the cancer is a pancreatic neuroendocrine tumor. In some embodiments, the pancreatic neuroendocrine tumor is an insulinoma, gastrinoma, glucagonoma, vipoma, somatostatinoma, or PPoma. In some embodiments, the pancreatic cancer, e.g., pancreatic cancer tumor, treated according to the methods of the present disclosure is resectable pancreatic cancer, borderline resectable pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer, or recurrent pancreatic cancer. In some embodiments, the pancreatic cancer, for example, the pancreatic cancer tumor treated according to the method of the present disclosure, is resectable. In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the PDAC is resectable.

[0158] (i) Priming phase In some embodiments, the methods for treating pancreatic cancer provided herein include administering a personalized RNA vaccine and a PD-1 axis-binding antagonist to a patient, such as a human patient in need thereof, during a priming phase of treatment.

[0159] In some embodiments, the priming phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks after resection of a pancreatic cancer tumor, such as a PDAC tumor, from a patient. In some embodiments, the priming phase begins about 6 weeks to about 12 weeks after resection of a pancreatic cancer tumor, such as a PDAC tumor, from a patient.

[0160] In some embodiments, the priming phase comprises from about 1 to about 12 weeks, from about 1 to about 8 weeks, or from about 1 to about 6 weeks, hi some embodiments, the priming phase consists of 6 weeks.

[0161] In some embodiments, the priming phase comprises administering to the patient at least one dose of an RNA vaccine and at least one dose of a PD-1 axis-binding antagonist.

[0162] In some embodiments, the priming phase comprises administering the RNA vaccine to a patient at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or more times. In some embodiments, the RNA vaccine is administered to a patient 1 to 8, 6 to 8, 2 to 6, or 2 or 3 times during the priming phase. In some embodiments, the RNA vaccine is administered to a patient two times during the priming phase. In some embodiments, the RNA vaccine is administered to a patient three times during the priming phase. In some embodiments, the RNA vaccine is administered to a patient six times during the priming phase. In some embodiments, the RNA vaccine is administered to a patient eight times during the priming phase. In other embodiments, the RNA vaccine is administered to a patient up to six times during the priming phase. In some embodiments, the RNA vaccine may be administered as a single composition or in two or more compositions. For example, in some cases, the RNA vaccine is administered as two separate compositions that are administered sequentially.

[0163] In some embodiments, the priming phase comprises administering the RNA vaccine once per week (QW), once per 2 weeks (Q2W), once per 3 weeks (Q3W), once per 4 weeks (Q4W), once per 5 weeks (Q5W), once per 6 weeks (Q6W), once per 7 weeks (Q7W), or once per 8 weeks (Q8W). In some embodiments, the priming phase comprises administering the RNA vaccine once per week (QW), e.g., once every 7 days. In some embodiments, administration of the RNA vaccine during the priming phase begins on day 1 of week 1 of the priming phase. In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of week 1 of the priming phase, and once per week (QW) thereafter, e.g., once every 7 days.

[0164] In some embodiments, the priming phase comprises administering the RNA vaccine six times. For example, in some cases, the RNA vaccine is administered on day 1 of week 1, day 1 of week 2, day 1 of week 3, day 1 of week 4, day 1 of week 5, and day 1 of week 6 of the priming phase.

[0165] In some embodiments, the priming phase comprises administering a single dose of a PD-1 axis-binding antagonist to the patient. In some embodiments, a single dose of a PD-1 axis-binding antagonist is administered to the patient during the priming phase. In some embodiments, the administration of the PD-1 axis-binding antagonist administered to the patient during the priming phase occurs on the same day as administration of the RNA vaccine.

[0166] In some embodiments, the priming phase comprises administering the PD-1 axis-binding antagonist to the patient at least twice. In some embodiments, the priming phase comprises administering the PD-1 axis-binding antagonist to the patient at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, or more times. In some embodiments, the PD-1 axis-binding antagonist is administered to the patient 1 to 8 times, 6 to 8 times, 2 to 6 times, or 2 or 3 times during the priming phase. In some embodiments, the PD-1 axis-binding antagonist is administered to the patient two times during the priming phase. In some embodiments, any administration of the PD-1 axis-binding antagonist administered to the patient during the priming phase occurs on the same day as administration of the RNA vaccine.

[0167] In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist once per week (QW), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every five weeks (Q5W), once every six weeks (Q6W), once every seven weeks (Q7W), or once every eight weeks (Q8W). In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist once per four weeks (Q4W), e.g., once every 28 days. In some embodiments, administration of the PD-1 axis binding antagonist during the priming phase begins on day 1 of week 1 of the priming phase. In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the priming phase, and once every four weeks (Q4W) thereafter, e.g., once every 28 days.

[0168] In some embodiments, the priming phase comprises a single administration of the PD-1 axis-binding antagonist. For example, in some cases, the PD-1 axis-binding antagonist is administered on day 1 of week 3 of the priming phase.

[0169] In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of week 1, day 1 of week 2, day 1 of week 3, day 1 of week 4, day 1 of week 5, and day 1 of week 6 of the priming phase, and administering the PD-1 axis-binding antagonist on day 1 of week 3 of the priming phase.

[0170] In some embodiments, the priming phase comprises two administrations of the PD-1 axis-binding antagonist, for example, in some cases the PD-1 axis-binding antagonist is administered on day 1 of week 1 and day 1 of week 5 of the priming phase.

[0171] In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of week 1, day 1 of week 2, day 1 of week 3, day 1 of week 4, day 1 of week 5, and day 1 of week 6 of the priming phase, and administering the PD-1 axis-binding antagonist on day 1 of week 1 and day 1 of week 5 of the priming phase.

[0172] Exemplary priming phases are provided in Table 1 below. Table 1. Exemplary priming phases A and B. TIFF2026501282000003.tif85170

[0173] In some embodiments, the RNA vaccine is administered to a patient during the priming phase at a dose of about 15 μg to about 50 μg (e.g., about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 38 μg, about 40 μg, about 45 μg, or about 50 μg). In some embodiments, the RNA vaccine is administered to a patient at a dose of about 15 μg, about 21 μg, about 21.3 μg, about 25 μg, about 38 μg, or about 50 μg. In some embodiments, the RNA vaccine is administered to a patient at a dose of 25 μg. In some embodiments, the RNA vaccine is administered to a patient at a dose of about 21 μg. In some embodiments, the RNA vaccine is administered to a patient at a dose of about 21.3 μg. In some embodiments, the RNA vaccine is administered to a patient intravenously. In some embodiments, the total dose of the RNA vaccine may be administered as a single composition or in two or more compositions. For example, in some cases, the RNA vaccine is administered in a total dose of 25 μg, which is split into two compositions administered sequentially. In some embodiments, the RNA vaccine is administered to a patient in two equal halves. In some embodiments, the two equal halves are administered sequentially, optionally with an observation period between the two equal halves. In some embodiments, a dose of approximately 25 μg is split into two equal halves of approximately 12.5 μg, each administered over a one-minute period, optionally with a five-minute observation period between the two equal halves. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 or 10-20 neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen from a patient. In some embodiments, the one or more polynucleotides of the RNA vaccine are formulated with one or more lipids. In some embodiments, the RNA vaccine is formulated as a lipid nanoparticle, where the one or more polynucleotides of the RNA vaccine and one or more lipids form the lipid nanoparticle. In some embodiments, the RNA vaccine is formulated as a lipoplex, wherein one or more polynucleotides and one or more lipids of the RNA vaccine form a lipoplex.

[0174] In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-L1 antibody, for example, as described below. In some embodiments, the anti-PD-L1 antibody is avelumab, durvalumab, or atezolizumab. In one embodiment, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody is administered to the patient at a dose of about 1200 mg or about 1680 mg. In some embodiments, the anti-PD-L1 antibody is administered to the patient at a dose of about 1680 mg. In certain embodiments, the PD-1 axis-binding antagonist is administered to the patient intravenously.

[0175] (ii) Chemotherapy stage In some embodiments, the methods for treating pancreatic cancer provided herein include administering chemotherapy treatment to a patient, such as a human patient in need thereof, e.g., during a chemotherapy phase after a priming phase as described above.

[0176] In some embodiments, the chemotherapy phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks after the end of the priming phase, e.g., after the last administration of an RNA vaccine during the priming phase. In some embodiments, the chemotherapy phase begins between about 1 week and about 9 weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 weeks) after the end of the priming phase, e.g., after the last administration of an RNA vaccine during the priming phase. In some embodiments, the chemotherapy phase begins at week 7, 8, or 9, relative to week 1 of the priming phase. In some embodiments, the chemotherapy phase begins at week 7, relative to week 1 of the priming phase. In some embodiments, the chemotherapy phase begins within 9 weeks of the priming phase, starting from week 1. In some embodiments, the priming phase consists of 6 weeks (e.g., as described above), and the chemotherapy phase begins at either week 7, 8, or 9 of the priming phase, starting from week 1. In some embodiments, the priming phase consists of 6 weeks (e.g., as described above), and the chemotherapy phase begins within 9 weeks of the priming phase, starting from week 1. In some embodiments, the priming phase consists of 6 weeks (e.g., as described above), and the chemotherapy phase begins at week 7 of the priming phase, starting from week 1. In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of week 1, day 1 of week 2, day 1 of week 3, day 1 of week 4, day 1 of week 5, and day 1 of week 6 of the priming phase, and the PD-1 axis-binding antagonist on day 1 of week 1 and day 1 of week 5 of the priming phase, e.g., as described above, and the chemotherapy phase begins on day 1 of week 7, day 1 of week 8, or day 1 of week 9.In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of week 1, day 1 of week 2, day 1 of week 3, day 1 of week 4, day 1 of week 5, and day 1 of week 6 of the priming phase, e.g., as described above, and the PD-1 axis-binding antagonist on day 1 of week 3 of the priming phase, and the chemotherapy phase begins on day 1 of week 7, day 1 of week 8, or day 1 of week 9. In some embodiments, the chemotherapy phase begins on day 1 of week 7.

[0177] In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient once per week (QW), once per two weeks (Q2W), once per three weeks (Q3W), once per four weeks (Q4W), once per five weeks (Q5W), once per six weeks (Q6W), once per seven weeks (Q7W), or once per eight weeks (Q8W). In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient once per two weeks (Q2W), e.g., once every 14 days. In some embodiments, administration of chemotherapy treatment during the chemotherapy phase begins on day 1 of week 1 of the chemotherapy phase. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient on day 1 of week 1 of the chemotherapy phase, and once every two weeks (Q2W) thereafter, e.g., once every 14 days.

[0178] In some embodiments, the chemotherapy period is at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks, at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, or longer. In some embodiments, the chemotherapy period is about 23 weeks. In some embodiments, the chemotherapy period is about 24 weeks.

[0179] In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient 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, or at least 24 or more times. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient at least 12 times. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient 12 times. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient starting on day 1 of week 1 of the chemotherapy phase and every two weeks (Q2W), e.g., every 14 days thereafter, for a total of 12 times. For example, in some cases, the chemotherapy phase includes administering chemotherapy treatment to the patient on week 1, day 1 of week 3, day 1 of week 5, day 1 of week 7, day 1 of week 9, day 1 of week 11, day 1 of week 13, day 1 of week 15, day 1 of week 17, day 1 of week 19, day 1 of week 21, and day 1 of week 23 of the chemotherapy phase.

[0180] In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient in two-week cycles (e.g., 14-day cycles) beginning on day 1 of week 1 of the chemotherapy phase. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient for 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, or at least 24 or more two-week cycles (e.g., 14-day cycles). In some embodiments, the chemotherapy phase comprises at least 12 two-week cycles (e.g., 14-day cycles) of chemotherapy treatment. In some embodiments, the chemotherapy phase comprises 12 two-week cycles (e.g., 14-day cycles) of chemotherapy treatment. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient in two-week cycles (e.g., 14-day cycles) beginning on day 1 of cycle 1 of the chemotherapy phase, for a total of 12 cycles of chemotherapy treatment. For example, in some cases, the chemotherapy phase comprises administering chemotherapy treatment to the patient on day 1 of cycle 1, day 1 of cycle 2, day 1 of cycle 3, day 1 of cycle 4, day 1 of cycle 5, day 1 of cycle 6, day 1 of cycle 7, day 1 of cycle 8, day 1 of cycle 9, day 1 of cycle 10, day 1 of cycle 11, and day 1 of cycle 12 of the chemotherapy phase.

[0181] In some embodiments, the chemotherapy phase begins on week 7 (e.g., day 1 of week 7) from week 1 of the priming phase. In some embodiments, the priming phase comprises 6 weeks (e.g., as described above), and the chemotherapy phase begins on week 7 (e.g., day 1 of week 7) from week 1 of the priming phase. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient on day 1 of week 7, day 1 of week 9, day 1 of week 11, day 1 of week 13, day 1 of week 15, day 1 of week 17, day 1 of week 19, day 1 of week 21, day 1 of week 23, day 1 of week 25, day 1 of week 27, and day 1 of week 29 from week 1 of the priming phase.

[0182] Exemplary chemotherapy phases are shown in Table 2 below. Table 2. Exemplary chemotherapy phases. TIFF2026501282000004.tif254170TIFF2026501282000005.tif80170

[0183] In some embodiments, the chemotherapy treatment administered during the chemotherapy phase is any chemotherapy known in the art or described herein, e.g., chemotherapy for pancreatic cancer, and may be administered during the chemotherapy phase according to any of the chemotherapy phase dosing regimens described herein or according to standard dosing regimens known in the art for such chemotherapy.

[0184] In some specific embodiments, the chemotherapy treatment includes one or more of gemcitabine, leucovorin, 5-fluorouracil, capecitabine, irinotecan, liposomal irinotecan, platinum-based chemotherapy agents, taxanes, and any combination thereof. In some embodiments, the chemotherapy treatment includes leucovorin (e.g., leucovorin calcium, folinic acid, or calcium folate), 5-fluorouracil (e.g., fluorouracil), irinotecan (e.g., irinotecan hydrochloride), and oxaliplatin. In some specific embodiments, the chemotherapy treatment is FOLFIRINOX treatment. FOLFIRINOX is a combination chemotherapy treatment including a combination of leucovorin (e.g., leucovorin calcium, folic acid, or calcium folate), 5-fluorouracil (e.g., fluorouracil), irinotecan (e.g., irinotecan hydrochloride), and oxaliplatin. In some embodiments, the FOLFIRINOX chemotherapy treatment is 85 mg / m 2 oxaliplatin at a dose of 400 mg / m 2 Leucovorin at a dose of 180 mg / m 2 Irinotecan at a dose of 400 mg / m 2 5-fluorouracil bolus at a dose of 2400 mg / m 2 The method comprises administering to the patient a dose of 5-fluorouracil (e.g., administered as an intravenous infusion over about 46 hours). In some embodiments, the chemotherapy treatment is a modified FOLFIRINOX treatment (mFOLFIRINOX). mFOLFIRINOX is a combination chemotherapy treatment that includes a combination of leucovorin (e.g., leucovorin calcium, folic acid, or calcium folate), 5-fluorouracil (e.g., fluorouracil), irinotecan (e.g., irinotecan hydrochloride), and oxaliplatin, but is modified relative to FOLFIRINOX to reduce the dose of one or more individual agents in the combination and / or removal of the 5-fluorouracil bolus. For example, in some cases, the mFOLFIRINOX chemotherapy is administered at a dose of about 85 mg / m 2 Oxaliplatin at a dose of approximately 400 mg / m 2Leucovorin at a dose of approximately 150 mg / m 2 and / or about 2400 mg / m 2 In some embodiments, the chemotherapy phase comprises administering to the patient a dose of 5-fluorouracil of about 85 mg / m (e.g., administered as an intravenous infusion over about 46 hours). 2 Oxaliplatin at a dose of approximately 400 mg / m 2 Leucovorin at a dose of approximately 150 mg / m 2 and irinotecan at a dose of approximately 2400 mg / m 2 In some particular embodiments, the chemotherapy phase comprises administering to the patient 5-fluorouracil at a dose of 85 mg / m over about 2 hours (e.g., ±5 minutes). 2 oxaliplatin at a dose of 400 mg / m2 intravenously over approximately 2 hours (e.g., ±15 minutes) 2 leucovorin at a dose of 150 mg / m over about 90 minutes (e.g., ±5 minutes) starting about 30 minutes after the leucovorin infusion begins 2 Irinotecan at a dose of 2400 mg / m2 administered intravenously and as a continuous infusion over approximately 46 hours (e.g., ±2 hours) 2 This involves administering 5-fluorouracil intravenously at a dose of

[0185] (iii) Boost phase In some embodiments, the methods of treating pancreatic cancer provided herein comprise administering at least one RNA vaccine and at least one PD-1 axis binding antagonist to a patient, such as a human patient in need thereof, during a boost phase following completion of a chemotherapy phase, e.g., as described above.

[0186] In some embodiments, the boost phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks after the end of the chemotherapy phase, e.g., after the last administration of chemotherapy treatment. In some embodiments, the boost phase begins between about 3 weeks and about 12 weeks, or between about 4 weeks and about 12 weeks after the end of the chemotherapy phase, e.g., after the last administration of chemotherapy treatment. In some embodiments, the boost phase begins 3 weeks after the end of the chemotherapy phase, e.g., after the last administration of chemotherapy treatment. In some embodiments, the boost phase begins 4 weeks after the end of the chemotherapy phase, e.g., after the last administration of chemotherapy treatment. In some embodiments, the boost phase begins within 12 weeks after the chemotherapy phase has ended, eg, after the last administration of chemotherapy treatment.

[0187] In some embodiments, the boost phase begins at week 27 (e.g., day 1 of week 27) relative to week 1 of the priming phase. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient on day 1 of week 1, day 1 of week 3, day 1 of week 5, day 1 of week 7, day 1 of week 9, day 1 of week 11, day 1 of week 13, day 1 of week 15, day 1 of week 17, day 1 of week 19, day 1 of week 21, and day 1 of week 23 of the chemotherapy phase, e.g., as described above, and the boost phase begins at week 27 (e.g., day 1 of week 27).

[0188] In some embodiments, the boost phase begins at week 33 (e.g., day 1 of week 33) starting from week 1 of the priming phase. In some embodiments, the chemotherapy phase comprises administering chemotherapy treatment to the patient on day 1 of week 7, day 1 of week 9, day 1 of week 11, day 1 of week 13, day 1 of week 15, day 1 of week 17, day 1 of week 19, day 1 of week 21, day 1 of week 23, day 1 of week 25, day 1 of week 27, and day 1 of week 29 starting from week 1 of the priming phase, e.g., as described above, and the boost phase begins at week 33 (e.g., day 1 of week 33).

[0189] In some embodiments, the boost phase comprises administering the RNA vaccine to a patient once every week (QW), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every five weeks (Q5W), once every six weeks (Q6W), once every seven weeks (Q7W), or once every eight weeks (Q8W). In some embodiments, the boost phase comprises administering the RNA vaccine to a patient once every four weeks (Q4W), e.g., once every 28 days. In some embodiments, the boost phase comprises administering the PD-1 axis-binding antagonist to the patient once per week (QW), once per two weeks (Q2W), once per three weeks (Q3W), once per four weeks (Q4W), once per five weeks (Q5W), once per six weeks (Q6W), once per seven weeks (Q7W), or once per eight weeks (Q8W). In some embodiments, the boost phase comprises administering the PD-1 axis-binding antagonist to the patient once per four weeks (Q4W), e.g., once every 28 days. In some embodiments, the administration of the RNA vaccine and the PD-1 axis-binding antagonist during the boost phase occurs on the same day. In some embodiments, the boost phase comprises administering the RNA vaccine and the PD-1 axis-binding antagonist to the patient once every week (QW), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every five weeks (Q5W), once every six weeks (Q6W), once every seven weeks (Q7W), or once every eight weeks (Q8W). In some embodiments, the boost phase comprises administering the RNA vaccine and the PD-1 axis-binding antagonist to the patient once every four weeks (Q4W), e.g., once every 28 days.

[0190] In some embodiments, administration of the RNA vaccine and / or PD-1 axis-binding antagonist during the boost phase begins on day 1 of week 1 of the boost phase. In some embodiments, the boost phase comprises administering the RNA vaccine and / or PD-1 axis-binding antagonist to a patient on day 1 of the boost phase, and once every four weeks (Q4W) thereafter, for example, once every 28 days. In some embodiments, administration of the RNA vaccine and PD-1 axis-binding antagonist during the boost phase begins on day 1 of week 1 of the boost phase. In some embodiments, the boost phase comprises administering the RNA vaccine and the PD-1 axis-binding antagonist to a patient on day 1 of the boost phase, and once every four weeks (Q4W) thereafter, for example, once every 28 days.

[0191] In some embodiments, the boost phase comprises administering the RNA vaccine to a patient at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or more times. In some embodiments, the RNA vaccine is administered to a patient 1 to 8, 6 to 8, 2 to 6, or 2 or 3 times during the boost phase. In some embodiments, the RNA vaccine is administered to a patient twice during the boost phase. In some embodiments, the RNA vaccine is administered to a patient three times during the boost phase. In some embodiments, the RNA vaccine is administered to a patient six times during the boost phase. In some embodiments, the RNA vaccine is administered to a patient eight times during the boost phase. In other embodiments, the RNA vaccine is administered to a patient at least six times during the boost phase. In other embodiments, the RNA vaccine is administered to a patient up to six times during the boost phase. In some embodiments, the RNA vaccine may be administered as a single composition or in two or more compositions. For example, in some cases, the RNA vaccine is administered as two separate compositions administered sequentially. In some embodiments, the boost phase comprises administering the PD-1 axis binding antagonist to the patient at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, or more times. In some embodiments, the PD-1 axis binding antagonist is administered to the patient 1 to 8 times, 6 to 8 times, 2 to 6 times, or 2 or 3 times during the boost phase. In some embodiments, the PD-1 axis binding antagonist is administered to the patient twice during the boost phase. In some embodiments, the PD-1 axis binding antagonist is administered to the patient three times during the boost phase. In some embodiments, the PD-1 axis binding antagonist is administered to the patient six times during the boost phase. In some embodiments, the PD-1 axis binding antagonist is administered to the patient eight times during the boost phase. In other embodiments, the PD-1 axis-binding antagonist is administered to the patient at least six times during the boost phase.In other embodiments, the PD-1 axis-binding antagonist is administered to the patient up to six times during the boost phase.

[0192] In some embodiments, the boost phase involves administering the RNA vaccine to the patient every four weeks (Q4W), e.g., every 28 days, beginning on day 1 of week 1 of the boost phase, e.g., for a total of six administrations of the RNA vaccine. For example, in some cases, the boost phase involves administering the RNA vaccine to the patient on day 1 of week 1, day 1 of week 5, day 1 of week 9, day 1 of week 13, day 1 of week 17, and day 1 of week 21 of the boost phase. In some embodiments, the boost phase involves administering the PD-1 axis-binding antagonist to the patient every four weeks (Q4W), e.g., every 28 days, beginning on day 1 of week 1 of the boost phase, e.g., for a total of six administrations of the PD-1 axis-binding antagonist. For example, in some cases, the boost phase involves administering the PD-1 axis-binding antagonist to the patient on day 1 of week 1, day 1 of week 5, day 1 of week 9, day 1 of week 13, day 1 of week 17, and day 1 of week 21 of the boost phase. In some embodiments, the boost phase involves administering the RNA vaccine and the PD-1 axis-binding antagonist to the patient every four weeks (Q4W), e.g., every 28 days, beginning on day 1 of week 1 of the boost phase, e.g., for a total of six administrations of the RNA vaccine and the PD-1 axis-binding antagonist. For example, in some cases, the boost phase involves administering the RNA vaccine and the PD-1 axis-binding antagonist to the patient on day 1 of week 1, day 1 of week 5, day 1 of week 9, day 1 of week 13, day 1 of week 17, and day 1 of week 21 of the boost phase. In some embodiments, the boost phase consists of 21 weeks.

[0193] In some embodiments, the boost phase comprises administering the RNA vaccine and / or PD-1 axis-binding antagonist to the patient in a 4-week cycle (e.g., a 28-day cycle), e.g., beginning on day 1 of week 1 of the boost phase. In some embodiments, the boost phase comprises administering the RNA vaccine to the patient at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least 11 times, at least 12 times, or more times over a 4-week cycle (e.g., a 28-day cycle). In some embodiments, the RNA vaccine is administered for 1 to 8 cycles, 6 to 8 cycles, 2 to 6 cycles, or 2 or 3 cycles during the boost phase in a 4-week cycle (e.g., a 28-day cycle). In some embodiments, the RNA vaccine is administered for 2 cycles in a 4-week cycle (e.g., a 28-day cycle) during the boost phase. In some embodiments, the RNA vaccine is administered for 3 cycles in a 4-week cycle (e.g., a 28-day cycle) during the boost phase. In some embodiments, the RNA vaccine is administered for six cycles in four-week cycles (e.g., 28-day cycles) during the boost phase. In some embodiments, the RNA vaccine is administered for eight cycles in four-week cycles (e.g., 28-day cycles) during the boost phase. In other embodiments, the RNA vaccine is administered for at least six cycles in four-week cycles (e.g., 28-day cycles) during the boost phase. In some embodiments, the RNA vaccine is administered for six or fewer cycles in four-week cycles (e.g., 28-day cycles) during the boost phase.

[0194] In some embodiments, the boost phase comprises administering the PD-1 axis binding antagonist to the patient for 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, or more 4-week cycles (e.g., 28-day cycles). In some embodiments, the PD-1 axis binding antagonist is administered for 1 to 8, 6 to 8, 2 to 6, or 2 or 3 cycles during the boost phase in a 4-week cycle (e.g., 28-day cycle). In some embodiments, the PD-1 axis binding antagonist is administered for 2 cycles during the boost phase in a 4-week cycle (e.g., 28-day cycle). In some embodiments, the PD-1 axis binding antagonist is administered for 3 cycles during the boost phase in a 4-week cycle (e.g., 28-day cycle). In some embodiments, the PD-1 axis binding antagonist is administered for 6 cycles during the boost phase in a 4-week cycle (e.g., a 28-day cycle). In some embodiments, the PD-1 axis binding antagonist is administered for 8 cycles during the boost phase in a 4-week cycle (e.g., a 28-day cycle). In some embodiments, the PD-1 axis binding antagonist is administered for at least 6 cycles during the boost phase in a 4-week cycle (e.g., a 28-day cycle). In some embodiments, the PD-1 axis binding antagonist is administered for no more than 6 cycles during the boost phase in a 4-week cycle (e.g., a 28-day cycle).

[0195] In some embodiments, the boost phase involves administering the RNA vaccine to the patient in 4-week cycles (e.g., 28-day cycles) beginning on day 1 of week 1 of the boost phase, for a total of six cycles, for example. For example, in some cases, the boost phase involves administering the RNA vaccine to the patient on day 1 of cycle 1, day 1 of cycle 2, day 1 of cycle 3, day 1 of cycle 4, day 1 of cycle 5, and day 1 of cycle 6 of the boost phase.

[0196] In some embodiments, the boost phase involves administering the PD-1 axis-binding antagonist to the patient in 4-week cycles (e.g., 28-day cycles) beginning on day 1 of week 1 of the boost phase, e.g., for a total of 6 cycles. For example, in some cases, the boost phase involves administering the PD-1 axis-binding antagonist to the patient on day 1 of cycle 1, day 1 of cycle 2, day 1 of cycle 3, day 1 of cycle 4, day 1 of cycle 5, and day 1 of cycle 6 of the boost phase.

[0197] In some embodiments, the boost phase involves administering the RNA vaccine and the PD-1 axis-binding antagonist to the patient in 4-week cycles (e.g., 28-day cycles) beginning on day 1 of week 1 of the boost phase, e.g., for a total of 6 cycles. For example, in some cases, the boost phase involves administering the RNA vaccine and the PD-1 axis-binding antagonist to the patient on day 1 of cycle 1, day 1 of cycle 2, day 1 of cycle 3, day 1 of cycle 4, day 1 of cycle 5, and day 1 of cycle 6 of the boost phase.

[0198] In some embodiments, the boost phase begins on week 33 (e.g., day 1 of week 33), e.g., as described above, starting from week 1 of the priming phase. In some embodiments, the boost phase involves administering the RNA vaccine to a patient starting on day 1 of week 33, e.g., as described above, starting from week 1 of the priming phase, and every four weeks thereafter (Q4W; e.g., every 28 days), e.g., for a total of six doses of the RNA vaccine. For example, in some cases, the boost phase involves administering the RNA vaccine to a patient on day 1 of week 33, day 1 of week 37, day 1 of week 41, day 1 of week 45, day 1 of week 49, and day 1 of week 53 of the boost phase, e.g., as described above, starting from week 1 of the priming phase. In some embodiments, the boost phase involves administering the PD-1 axis-binding antagonist to the patient starting on day 1 of week 33 of the priming phase, e.g., as described above, and every four weeks (Q4W; e.g., every 28 days) thereafter, e.g., for a total of six doses of the PD-1 axis-binding antagonist. For example, in some cases, the boost phase involves administering the PD-1 axis-binding antagonist to the patient on day 1 of week 33, day 1 of week 37, day 1 of week 41, day 1 of week 45, day 1 of week 49, and day 1 of week 53, starting on week 1 of the priming phase. In some embodiments, the boost phase involves administering the RNA vaccine and the PD-1 axis-binding antagonist to the patient starting on day 1 of week 33 of the priming phase, e.g., as described above, and every four weeks (Q4W; e.g., every 28 days) thereafter, e.g., for a total of six doses of the RNA vaccine and the PD-1 axis-binding antagonist. For example, in some cases, the boost phase involves administering the RNA vaccine and the PD-1 axis-binding antagonist to the patient on day 1 of week 33, day 1 of week 37, day 1 of week 41, day 1 of week 45, day 1 of week 49, and day 1 of week 53 of the boost phase, starting on week 1 of the priming phase.

[0199] An exemplary boost phase is provided in Table 3 below. Table 3. Exemplary boost phase. TIFF2026501282000006.tif162170

[0200] In some embodiments, the RNA vaccine is administered to a patient during the boost phase at a dose of about 15 μg to about 50 μg (e.g., about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 38 μg, about 40 μg, about 45 μg, or about 50 μg). In some embodiments, the RNA vaccine is administered to a patient at a dose of about 15 μg, about 21 μg, about 21.3 μg, about 25 μg, about 38 μg, or about 50 μg. In some embodiments, the RNA vaccine is administered to a patient at a dose of 25 μg. In some embodiments, the RNA vaccine is administered to a patient at a dose of about 21 μg. In some embodiments, the RNA vaccine is administered to a patient at a dose of about 21.3 μg. In some embodiments, the RNA vaccine is administered to a patient intravenously. In some embodiments, the total dose of the RNA vaccine may be administered as a single composition or in two or more compositions. For example, in some cases, the RNA vaccine is administered in a total dose of 25 μg, which is split into two compositions administered sequentially. In some embodiments, the RNA vaccine dose is administered to a patient in two equal halves. In some embodiments, the two equal halves are administered sequentially, optionally with an observation period between the administration of the equal halves. In some embodiments, a dose of approximately 25 μg is split into two equal halves of approximately 12.5 μg, each administered over a one-minute period, optionally with a five-minute observation period between the administration of the equal halves. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 or 10-20 neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen from a patient. In some embodiments, the one or more polynucleotides of the RNA vaccine are formulated with one or more lipids. In some embodiments, the RNA vaccine is formulated as a lipid nanoparticle, where the one or more polynucleotides of the RNA vaccine and one or more lipids form the lipid nanoparticle. In some embodiments, the RNA vaccine is formulated as a lipoplex, wherein one or more polynucleotides and one or more lipids of the RNA vaccine form a lipoplex.

[0201] In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-L1 antibody, for example, as described below. In some embodiments, the anti-PD-L1 antibody is avelumab, durvalumab, or atezolizumab. In one embodiment, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody is administered to the patient at a dose of about 1200 mg or about 1680 mg. In some embodiments, the anti-PD-L1 antibody is administered to the patient at a dose of about 1680 mg. In certain embodiments, the PD-1 axis-binding antagonist is administered to the patient intravenously.

[0202] Any of the priming, chemotherapy, and boost phases described herein can be used in any combination in the methods for treating pancreatic cancer provided herein.

[0203] For example, in some embodiments, the methods of treating pancreatic cancer provided herein comprise administering to a patient a personalized RNA vaccine, a PD-1 axis-binding antagonist, and a chemotherapy treatment during a treatment period comprising a priming phase, a chemotherapy phase following the priming phase, and a boost phase following the chemotherapy phase, wherein the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase, and the PD-1 axis-binding antagonist on day 1 of week 3 of the priming phase; the chemotherapy phase comprises administering the chemotherapy treatment on day 1 of weeks 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29 of the priming phase, starting from week 1; and the boost phase comprises administering the RNA vaccine and the PD-1 axis-binding antagonist on day 1 of weeks 33, 37, 41, 45, 49, and 53 of the priming phase, starting from week 1. In another example, the method of treating pancreatic cancer provided herein comprises administering to a patient a personalized RNA vaccine, a PD-1 axis-binding antagonist, and a chemotherapy treatment during a treatment period comprising a priming phase, a chemotherapy phase following the priming phase, and a boost phase following the chemotherapy phase, wherein the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase, and the PD-1 axis-binding antagonist on day 1 of weeks 1 and 5 of the priming phase; the chemotherapy phase comprises administering the chemotherapy treatment on day 1 of weeks 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29 of the priming phase, starting from week 1; and the boost phase comprises administering the RNA vaccine and the PD-1 axis-binding antagonist on day 1 of weeks 33, 37, 41, 45, 49, and 53 of the priming phase, starting from week 1.In some embodiments, the priming phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks after resection of a pancreatic cancer tumor, such as a pancreatic ductal adenocarcinoma (PDAC) tumor, from a patient. In some embodiments, the priming phase begins about 6 weeks to about 12 weeks after resection of a pancreatic cancer tumor, such as a pancreatic ductal adenocarcinoma (PDAC) tumor, from a patient.

[0204] Tumor-bearing individuals In some embodiments, the pancreatic cancer tumor is a resectable PDAC tumor, which is evaluated by preoperative imaging in human patients using computed tomography (CT) scans with contrast or magnetic resonance imaging (MRI) before administering the RNA vaccine, PD-1 axis-binding antagonist, and chemotherapy treatment. In some embodiments, CT scans with contrast may be contraindicated. In some embodiments where CT scans with contrast are contraindicated, CT scans without contrast are used in conjunction with MRI scans before administering the RNA vaccine, PD-1 axis-binding antagonist, and chemotherapy treatment. In some embodiments, other imaging techniques are performed if clinically indicated. Other imaging techniques include, but are not limited to, positron emission tomography (PET), ultrasound, 3D ultrasound, radiography, etc. Contrast agents include, for example, gadolinium, iron oxide, manganese(II), iodine (e.g., Iohexol), barium sulfate, etc. In some embodiments, the pancreatic cancer tumor is a resectable PDAC tumor comprising one or more features selected from the group consisting of: a distinct fat plane around the celiac trunk and superior mesenteric artery; patent superior mesenteric vein and portal vein; no superior mesenteric vein or portal vein encasement; no superior mesenteric artery or hepatic artery encasement; absence of metastatic disease; and absence of extraregional lymph node disease.

[0205] In some embodiments, the human patient has a histologically confirmed diagnosis of PDAC before administering the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the human patient has adenosquamous carcinoma of the pancreas before administering the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the human patient does not have intraductal papillary mucinous neoplasia-associated PDAC before administering the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the human patient does not have pancreatic endocrine tumor or pancreatic acinar cell adenocarcinoma, pancreatic cystadenocarcinoma, or pancreatic malignancy before administering the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0206] In some embodiments, the pancreatic cancer tumor has a tumor, node, metastasis (TNM) pathological staging value of T1-T3, N0-N2, or M0 prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the staging value is assessed according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual, 8th edition (Amin, MB et al., eds., American Joint Committee on Cancer (AJCC) Cancer Staging Manual; 8th ed. New York: Springer 2017).

[0207] In some embodiments, the pancreatic cancer tumor is a resectable PDAC tumor, and the human patient has no evidence of PDAC disease after resection of the PDAC tumor, and / or the human patient underwent macroscopically complete resection of the PDAC tumor before administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the human patient further underwent R0 or R1 resection of the PDAC tumor. In some embodiments, the human patient demonstrated a clear absence of PDAC after resection of the PDAC tumor. In some embodiments, the absence of PDAC is assessed by CT or MRI scan, one or more biochemical assays, and / or clinical findings. In some embodiments, one of the multiple biochemical assays includes, but is not limited to, a carcinoembryonic antigen (CEA) and a CA19-9 assay.

[0208] In some embodiments, the pancreatic cancer tumor is a resectable PDAC tumor, and after tumor resection, the human patient had no unresolved postoperative complications of grade 3 or higher prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment. In some embodiments, complications are evaluated according to the Clavien-Dindo Classification of Surgical Complications. The Clavien-Dindo Classification of Surgical Complications identifies five stages: Grade 1: deviation from the normal postoperative course does not require pharmacological treatment or surgical, endoscopic, and radiological intervention; Grade 2: pharmacological treatment with drugs may be required; Grade 3: surgical, endoscopic, and radiological intervention may be required, with or without general anesthesia; Grade 4: occurrence of life-threatening complications that may require IC / ICU management and may include single or multiple organ dysfunction; Grade 5: patient death.

[0209] In some embodiments, the human patient has a CA19-9 level of 180 U / mL or greater before administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment. In some embodiments, the patient has a CA19-9 level of less than 180 U / mL before administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment. CA19-9, also known as carbohydrate antigen 19-9 or cancer antigen 19-9, is a pancreatic cancer antigen that can be present in blood samples taken from human pancreatic cancer patients or humans suspected of having pancreatic cancer.

[0210] In some embodiments, at least five neoepitopes resulting from cancer-specific somatic mutations are present in a tumor specimen obtained from a human patient prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0211] In some embodiments, the human patient, prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment, has an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 or 1. The ECOG Performance Status assesses a patient's ability to care for themselves, their daily activities, and their physical abilities (e.g., walking, working, etc.).

[0212] In some embodiments, the human patient has not received adjuvant, neoadjuvant, or induction treatment for pancreatic cancer, or systemic anti-cancer treatment for pancreatic cancer, before administering the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment. In some embodiments, the pancreatic cancer is PDAC.

[0213] In some embodiments, the human patient has not received cytotoxic chemotherapy, immunotherapy, investigational therapy, or radiation therapy prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and chemotherapy.

[0214] In some embodiments, the human patient has a spleen before administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the human patient does not have spleen loss due to splenectomy, spleen injury / infarction, or functional asplenia before administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the human patient does not have a distal pancreatectomy with splenectomy before administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0215] In some embodiments, the human patient does not have a pre-existing neuropathy prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

[0216] In some embodiments, the human patient does not have the aUGT1A1 genotype, which is associated with a poor metabolism phenotype, before administration of the RNA vaccine, the PD-1 axis binding antagonist, and chemotherapy treatment. The UGT1A1 gene encodes the UDP-glucuronosyltransferase enzyme, which aids in the metabolism of drugs such as irinotecan (SN-38), acetaminophen (paracetamol), carvedilol, etoposide, lamotrigine, and simvastatin. Human patients with the aUGT1A1 genotype may be at increased risk of irinotecan toxicity after chemotherapy treatment with mFOLFIRINOX (see, for example, Correia Marques, S. and Ikediobi, ON (2010), Hum Genomics; 4(4):238-249).

[0217] In some embodiments, the human patient does not have an autoimmune disease, immunodeficiency, or primary immunodeficiency prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the human patient has not been treated with a monoamine oxidase inhibitor (MAOI) within three weeks, a systemic immune stimulant within four weeks, or a systemic immunosuppressant within five drug elimination half-lives, whichever is longer, or within two weeks prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, the human patient has not undergone an allogeneic stem cell or solid organ transplant prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. Response to administration

[0218] In some embodiments, the methods described herein further comprise assessing disease-free survival (DFS) of a human patient after administration of the RNA vaccine, the PD-1 axis-binding antagonist, and chemotherapy treatment. DFS is measured as the time from patient randomization to either the first recurrence of PDAC or the first occurrence of a new cancer, or death from any cause, as determined by the investigator. In some embodiments, new cancer does not include malignancies with a negligible risk of metastasis or death (e.g., 5-year OS rate >90%), including, but not limited to, adequately treated in situ carcinoma of the cervix, non-melanoma skin cancer, localized prostate cancer, in situ ductal carcinoma, or stage I uterine cancer. In some embodiments, administration of the RNA vaccine, the PD-1 axis-binding antagonist, and chemotherapy treatment results in improved DFS of the human patient compared to the DFS of a corresponding human patient not administered the RNA vaccine, the PD-1 axis-binding antagonist, and chemotherapy treatment.

[0219] In some embodiments, the methods described herein further comprise assessing the overall survival (OS) of a human patient after treatment with the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. OS is defined as the time from patient randomization to death from any cause. In some embodiments, administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment results in improved OS of the human patient compared to the OS of a corresponding human patient who has not received the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0220] In some embodiments, the methods described herein further comprise conducting one or more clinical assessments of the human patient before, during, and / or after treatment with the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment, wherein the one or more clinical assessments are selected from the group consisting of the European Organisation for Research and Treatment of Cancer QLQ-C30 questionnaire (EORTC QLQ C30), the European Organisation for Research and Treatment of Cancer QLQ-PAN26 questionnaire (EORTC QLQ PAN26), the National Cancer Institute's Patient-Reported Outcomes Common Terminology Criteria for Adverse Events (PRO CTCAE), and the European Organisation for Research and Treatment of Cancer Item Library 46 Questionnaire (EORTC IL46). In some embodiments, clinical assessments are administered in the following order: EORTC QLQ-C30, EORTC QLQ-PAN26, PRO CTCAE, and EORTC IL46. The QLQ-C30 consists of 30 questions assessing five aspects of patient functioning (physical, emotional, role, cognitive, and social), three symptom scales (fatigue, nausea and vomiting, pain), overall health / quality of life, and six single items with a recall period of the previous week (dyspnea, insomnia, loss of appetite, constipation, diarrhea, and financial difficulties). Scale scores can be obtained for multi-item scales. The QLQ-PAN26 consists of 26 questions assessing nine pancreatic cancer-related and treatment-related symptoms (pain, eating-related items, cachexia, liver symptoms, side effects, altered bowel habits, ascites, dyspepsia, and flatulence) and five emotional domains specific to pancreatic cancer (body image, satisfaction with medical care, sexuality, fears about future health, and ability to plan for the future) (see, e.g., Mackay et al., HPB (Oxford); 24: 443-451 (2022)).The PRO-CTCAE is used to characterize the presence, frequency, severity, and / or degree of interference with daily functioning of 78 patient-reportable symptomatic treatment toxicities (see, e.g., Basch et al., J Natl Cancer Inst;106:dju244 (2014); and Dueck et al., JAMA Oncol;1:1051-1059 (2015)). The PRO-CTCAE contains 124 questions rated either dichotomously (to determine presence vs. absence) or on a 5-point Likert scale (to determine frequency, severity, and interference with daily functioning). Treatment toxicities can occur with observable signs (e.g., vomiting) or non-observable symptoms (e.g., nausea). The standard PRO-CTCAE recall period is the previous 7 days. The IL46 is a validated single-item question used to assess the overall impact of side effects and is used in conjunction with the PRO-CTCAE to assess treatment tolerability. Symptomatic adverse events from the PRO-CTCAE item bank include, but are not limited to, sore mouth / throat, nausea, vomiting, diarrhea, shortness of breath, cough, rash, hair loss, hand-foot syndrome, neuropathy, dizziness, headache, joint pain, fatigue, bruising, chills, nosebleeds, injection site or IV site pain. In some embodiments, administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment results in an improvement in one or more clinical assessments compared to one or more clinical assessments in the human patient prior to administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment, and / or compared to one or more clinical assessments in a corresponding human patient who has not been administered the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment.

[0221] In some embodiments, the methods described herein further comprise assessing antigen-specific and / or tumor-specific T cell responses in the human patient before, during, and / or after treatment with the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment results in improved antigen-specific and / or tumor-specific T cell responses in the human patient compared to before administration of the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment, and / or compared to a corresponding human patient not administered the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment. In some embodiments, antigen-specific and / or tumor-specific T cell responses in human patients can be assessed by immune monitoring assays, including, but not limited to, IFN-γ release assays (e.g., ELISpot); tumor or immune biomarker (e.g., PDL1, CD8) assays; whole exome sequencing, whole genome sequencing, or RNA sequencing and TCR sequencing to analyze mutational changes; monitoring immune cell infiltration and tracking antigen-specific T cells; analysis of circulating tumor DNA (ctDNA); analysis of cytokines, phenotype, and function of antigen-specific T cells, T cell repertoire, immune cell subset numbers, percentages, and functional status (including T cell subsets and myeloid-derived suppressor cells); titers of antibodies against reference antigens; single-cell transcriptional genetic analysis; and detection of T cells specific for neoantigens encoded in personalized cancer vaccines.

[0222] In some embodiments, the corresponding human patient is a human patient with a corresponding pancreatic cancer tumor. In some embodiments, the pancreatic cancer tumor is a PDAC tumor, and the corresponding human patient has a PDAC tumor. In some embodiments, the corresponding human patient has been treated with standard therapy for pancreatic cancer, PDAC, or resectable or resected PDAC. In some embodiments, the standard therapy comprises gemcitabine combination therapy or mFOLFIRINOX chemotherapy. mFOLFIRINOX has a favorable benefit-risk profile and has been established as the current standard of care after resection of PDAC in eligible patients (Conroy et al., N Engl J Med;379:2395-2406(2018); and Conroy et al., JAMA Oncol 2022;e223829(2022),doi:10.1001 / jamaoncol.20223820 online ahead of print). The mFOLFIRINOX therapy includes the following individual agents: leucovorin, 5FU, irinotecan, and oxaliplatin. In some embodiments, corresponding human patients are treated with a control treatment that includes mFOLFIRINOX chemotherapy. In some embodiments, the mFOLFIRINOX chemotherapy is administered at a dose of about 85 mg / m 2 Oxaliplatin at a dose of approximately 400 mg / m 2 Leucovorin at a dose of approximately 150 mg / m 2 and irinotecan at a dose of approximately 2400 mg / m 2 This drug contains 5-fluorouracil at a dose of 10 mg / kg / day, administered intravenously on day 1 of each 14-day cycle for up to a total of 12 cycles.

[0223] Compositions for use in treating pancreatic cancer In one aspect, a personalized RNA vaccine is provided for use in a method for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the RNA vaccine is administered in combination with a PD-1 axis-binding antagonist and chemotherapy treatment according to the methods described herein, and the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the human patient. In another aspect, a use of a personalized RNA vaccine is provided in the manufacture of a medicament for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the RNA vaccine is administered in combination with a PD-1 axis-binding antagonist and chemotherapy treatment according to the methods described herein, and the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the human patient.

[0224] In one aspect, provided is a PD-1 axis-binding antagonist for use in a method for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the PD-1 axis-binding antagonist is administered in combination with a personalized RNA vaccine and chemotherapy treatment according to the methods described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the human patient. In another aspect, provided is use of a personalized PD-1 axis-binding antagonist in the manufacture of a medicament for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the PD-1 axis-binding antagonist is administered in combination with a personalized RNA vaccine and chemotherapy treatment according to the methods described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the human patient.

[0225] Administration Methods and Additional Therapies The PD-1 axis binding antagonist, RNA vaccine, and chemotherapy treatment can be administered by the same or different routes of administration. In some embodiments, the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the RNA vaccine is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally (e.g., in a lipoplex or lipid nanoparticle). In some embodiments, the chemotherapy treatment is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the RNA vaccine is administered intravenously (e.g., in a lipoplex). In some embodiments, the PD-1 axis binding antagonist, the RNA vaccine, and the chemotherapy treatment are administered intravenously.In some cases, the chemotherapy treatment can include combination chemotherapy.In such cases, each individual agent in the combination can be administered by the same administration route or by different administration routes, for example, as described above.

[0226] When the PD-1 axis binding antagonist and the RNA vaccine are administered on the same day, for example, during the priming and / or boosting phase of treatment, they can be administered in any order. For example, the PD-1 axis binding antagonist and the RNA vaccine can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the RNA vaccine is administered before the PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are in separate compositions. In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are in the same composition.

[0227] More than one type of RNA may be administered to a patient, e.g., a patient may receive one RNA vaccine with a combination of neoepitopes and another RNA vaccine with a different combination of neoepitopes. In some embodiments, a first RNA vaccine having, e.g., 5-20 or 10-20 neoepitopes is administered in combination with a second RNA vaccine having, e.g., 5-20 or 10-20 different or alternative neoepitopes.

[0228] In some embodiments, the methods for treating pancreatic cancer provided herein may further include administering an additional therapy to the patient. The additional therapy may be radiation therapy, surgery (e.g., resection of a pancreatic cancer tumor), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an antiemetic). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery, e.g., resection of a pancreatic cancer tumor. In some embodiments, the additional therapy is a combination of radiation therapy and surgery, e.g., resection of a pancreatic cancer tumor. In some embodiments, the additional therapy is gamma irradiation.

[0229] III. RNA vaccines Certain aspects of the present disclosure relate to personalized cancer vaccines (ICVs). In some embodiments, the personalized cancer vaccine is an RNA vaccine. Exemplary RNA vaccine features are described below. In some embodiments, the present disclosure provides an RNA polynucleotide comprising one or more features / sequences of the RNA vaccines described below. In some embodiments, the RNA polynucleotide is a single-stranded mRNA polynucleotide. In other embodiments, the present disclosure provides a DNA polynucleotide encoding an RNA comprising one or more features / sequences of the RNA vaccines described below.

[0230] The personalized cancer vaccine comprises a personalized neoantigen (i.e., a tumor-associated antigen (TAA) specifically expressed in a patient's cancer) that has been identified as having potential immunostimulatory activity. In the embodiments described herein, the personalized cancer vaccine is a nucleic acid, e.g., messenger RNA. Thus, without wishing to be bound by theory, it is believed that upon administration, the personalized cancer vaccine is taken up and translated by antigen-presenting cells (APCs), and the expressed protein is presented via major histocompatibility complex (MHC) molecules on the surface of the APCs. This results in the induction of both cytotoxic T lymphocyte (CTL) and memory T cell-dependent immune responses against cancer cells expressing the TAA.

[0231] A personalized cancer vaccine (e.g., an RNA vaccine) typically includes multiple neoantigenic epitopes ("neoepitopes"), e.g., 2, 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, 28, 29, or 30 neoepitopes, or at least 2, 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, 28, 29, or 30 neoepitopes, optionally with linker sequences between individual neoepitopes. In some embodiments, neoepitope, as used herein, refers to a novel epitope that is specific to a patient's cancer but not found in the patient's normal cells. In some embodiments, the neoepitope is presented to T cells when bound to MHC. In some embodiments, the personalized cancer vaccine also includes a 5' mRNA cap analog, a 5' UTR, a signal sequence, a domain that promotes antigen expression, a 3' UTR, and / or a polyA tail. In some embodiments, the RNA vaccine includes one or more polynucleotides encoding 5-20 or 10-20 neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen. In some embodiments, the RNA vaccine includes one or more polynucleotides encoding at least five neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen. In some embodiments, the RNA vaccine includes one or more polynucleotides encoding 5-20 neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen. In some embodiments, the RNA vaccine includes one or more polynucleotides encoding 5-10 neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen.

[0232] In some embodiments, the production of the RNA vaccines of the present disclosure is a multi-step process whereby somatic mutations in a patient's tumor are identified by next-generation sequencing (NGS) and immunogenic neoantigenic epitopes (or "neoepitopes") are predicted. RNA cancer vaccines targeting the selected neoepitopes are produced for each patient. In some embodiments, the vaccine is an RNA-based cancer vaccine consisting of up to two messenger RNA molecules, each encoding up to 10 neoepitopes (up to 20 neoepitopes total) specific to the patient's tumor.

[0233] In some embodiments, expressed nonsynonymous mutations are identified by whole exome sequencing (WES) of tumor DNA and peripheral blood mononuclear cell (PBMC) DNA (as a source of patient-derived healthy tissue) and tumor RNA sequencing (to assess expression). From the resulting list of mutant proteins, potential neoantigens are predicted using a bioinformatics workflow that ranks their potential immunogenicity based on multiple factors, including the binding affinity of predicted epitopes to individual major histocompatibility complex (MHC) molecules and the expression level of associated RNA. The mutation discovery, prioritization, and confirmation process is complemented by a database that provides comprehensive information on the expression level of each wild-type gene in healthy tissue. This information enables the development of personalized risk mitigation strategies by eliminating candidate subjects with unfavorable risk profiles. Mutations occurring in proteins with potentially higher autoimmune risk in vital organs are excluded and not considered for vaccine production. In some embodiments, CD8 for each individual patient is used. + T cells and / or CD4 +Up to 20 MHC1 and MHCII neoepitopes predicted to elicit T cell responses are selected for inclusion in the vaccine. Vaccination against multiple neoepitopes is expected to increase the breadth and magnitude of the overall immune response to personalized cancer vaccines and may help mitigate the risk of immune escape that can occur when tumors are exposed to the selective pressure of an effective immune response (Tran E, Robbins PF, Lu YC, et al. N Engl J Med 2016;375:2255-62; Verdegaal EM, de Miranda NF, Visser M, et al. Nature 2016;536:91-5).

[0234] In some embodiments, the RNA vaccine comprises one or more polynucleotide sequences encoding an amino acid linker. For example, an amino acid linker can be used between two tumor-specific neoepitope sequences, between a tumor-specific neoepitope sequence and a fusion protein tag (e.g., comprising a sequence derived from an MHC complex polypeptide), or between a secretory signal peptide and a tumor-specific neoepitope sequence. In some embodiments, the RNA vaccine encodes multiple linkers. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5 to 20 neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen, wherein the polynucleotides encoding each epitope are separated by polynucleotides encoding linker sequences. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5 to 10 neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen, wherein the polynucleotides encoding each epitope are separated by polynucleotides encoding linker sequences. In some embodiments, a polynucleotide encoding a linker sequence is also present between the polynucleotide encoding the N-terminal fusion tag (e.g., a secretory signal peptide) and the polynucleotide encoding one of the neoepitopes, and / or between the polynucleotide encoding one of the neoepitopes and the polynucleotide encoding the C-terminal fusion tag (e.g., comprising a portion of an MHC polypeptide). In some embodiments, two or more linkers encoded by an RNA vaccine comprise different sequences. In some embodiments, an RNA vaccine encodes multiple linkers, all of which share the same amino acid sequence.

[0235] In some embodiments, the RNA molecule further comprises a polynucleotide sequence encoding an amino acid linker, wherein the amino acid linker and the polynucleotide sequence encoding a first of the one or more neoepitopes form a first linker-neoepitope module, and the polynucleotide sequence forming the first linker-neoepitope module is located, in a 5' to 3' direction, between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule. In some embodiments, the RNA molecule further comprises, in a 5' to 3' direction, at least a second linker-neoepitope module, the at least second linker-neoepitope module comprising a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope, the polynucleotide sequence forming the second linker-neoepitope module being between, in a 5' to 3' direction, the polynucleotide sequence encoding the neoepitope of the first linker-neoepitope module and the polynucleotide sequence encoding at least a portion of a transmembrane domain and a cytoplasmic domain of an MHC molecule, and the neoepitope of the first linker-neoepitope module is different from the neoepitope of the second linker-neoepitope module. In some embodiments, the RNA molecule comprises five linker-neoepitope modules, each of which encodes a different neoepitope. In some embodiments, the RNA molecule comprises 10 linker-neoepitope modules, each of which encodes a different neoepitope, hi some embodiments, the RNA molecule comprises 20 linker-neoepitope modules, each of which encodes a different neoepitope.In some embodiments, the RNA molecule further comprises a second polynucleotide sequence encoding an amino acid linker, wherein the second polynucleotide sequence encoding the amino acid linker is between the polynucleotide sequence encoding the 3'-most distal neoepitope and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

[0236] Various linker sequences are known in the art. In some embodiments, the linker is a flexible linker. In some embodiments, the linker comprises G, S, A, and / or T residues. In some embodiments, the linker consists of glycine and serine residues. In some embodiments, the linker is about 5 to about 20 amino acids in length or about 5 to about 12 amino acids in length, e.g., 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, or about 20 amino acids in length. In some embodiments, the linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 39). In some embodiments, the linker for an RNA vaccine comprises the sequence GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO: 37). In some embodiments, the linker for an RNA vaccine is encoded by DNA comprising the sequence GGCGGCTCTGGAGGAGGCGGCTCCGGAGGC (SEQ ID NO: 38).

[0237] In some embodiments, the RNA vaccine comprises a 5' cap. The basic mRNA cap structure consists of two nucleosides (e.g., two guanines) and a 7-methyl group on the distal guanine, i.e., m 7 It is known that the 5' cap contains a 5'-5' triphosphate bond between GpppG and the nucleotide. Exemplary cap structures can be found, for example, in U.S. Patent Nos. 8,153,773 and 9,295,717, and Kuhn, A. et al. (2010) Gene Ther. 17:961-971. In some embodiments, the 5' cap has the structure m2 7,2’-O Gpp sIn some embodiments, the 5' cap is a beta-S-ARCA cap. The S-ARCA cap structure can be (e.g., m 7 At the C2' position of G, the 5' cap comprises a 2'-O methyl substitution and an S substitution at one or more of the phosphate groups. In some embodiments, the 5' cap comprises the following structure: [ka]

[0238] In some embodiments, the 5'-cap is the D1 diastereoisomer of beta-S-ARCA (see, e.g., U.S. Pat. No. 9,295,717). The * in the above structure indicates a stereogenic P center that can exist in two diastereoisomers (designated D1 and D2). Beta-S-ARCA or the D1 diastereoisomer of beta-S-ARCA (D1) is the diastereoisomer of beta-S-ARCA that elutes first on an HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereoisomer of beta-S-ARCA (beta-S-ARCA (D2)). The HPLC is preferably analytical HPLC. In one embodiment, a Supelcosil LC-18-T RP column of the following format is preferably used for separation: 5 μm, 4.6 × 250 mm, and a flow rate of 1.3 ml / min can be applied. In one embodiment, a gradient of methanol in ammonium acetate within 15 minutes is used, e.g., a linear gradient of 0-25% methanol in 0.05 M ammonium acetate, pH 5.9. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0239] In some embodiments, the RNA vaccine comprises a 5' UTR. Certain untranslated sequences found 5' to the protein-coding sequence in mRNA have been shown to increase translation efficiency. See, e.g., Kozak, M. (1987) J. Mol. Biol. 196:947-950. In some embodiments, the 5' UTR comprises a sequence from human alpha globin mRNA. In some embodiments, the RNA vaccine comprises a 5' UTR sequence of UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 23). In some embodiments, the 5' UTR sequence of the RNA vaccine is encoded by DNA comprising the sequence TTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 24). In some embodiments, the 5' UTR of the RNA vaccine comprises the sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 21). In some embodiments, the 5'UTR sequence of the RNA vaccine is encoded by DNA comprising the sequence GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 22).

[0240] In some embodiments of the methods provided herein, the constant region of an exemplary RNA vaccine comprises the ribonucleotide sequence (5'→3') of SEQ ID NO: 42. The linkage between the first two G residues is an aberrant linkage (5'→5')-pp, e.g., as shown in Table 4. s p-. "N" refers to the position of the polynucleotide sequence encoding one or more (e.g., 1-20) neoepitopes (separated by optional linkers). The insertion site of the tumor-specific sequence (C131-A132; marked in bold) is shown in bold. See Table 4 for modified bases and unusual linkages in exemplary RNA sequences. Table 4 TIFF2026501282000008.tif48170

[0241] In some embodiments, the RNA vaccine comprises a polynucleotide sequence encoding a secretory signal peptide. As known in the art, a secretory signal peptide is an amino acid sequence that, upon translation, directs transport of a polypeptide from the endoplasmic reticulum to the secretory pathway. In some embodiments, the signal peptide is derived from a human polypeptide, such as an MHC polypeptide. See, for example, Kreiter, S. et al. (2008) J. Immunol. 180:309-318, which describes exemplary secretory signal peptides that improve processing and presentation of MHC class I and II epitopes in human dendritic cells. In some embodiments, upon translation, the signal peptide is N-terminal to one or more neoepitope sequences encoded by the RNA vaccine. In some embodiments, the secretory signal peptide comprises the sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 27). In some embodiments, the secretory signal peptide of the RNA vaccine comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 25). In some embodiments, the secretory signal peptide of the RNA vaccine is encoded by DNA comprising the sequence ATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC (SEQ ID NO: 26).

[0242] In some embodiments, the RNA vaccine comprises a polynucleotide sequence encoding at least a portion of the transmembrane and / or cytoplasmic domain. In some embodiments, the transmembrane and / or cytoplasmic domain is derived from the transmembrane / cytoplasmic domain of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes occurring in all vertebrates. The function of MHC proteins or molecules in signaling between lymphocytes and antigen-presenting cells in a normal immune response involves their binding to peptides and presenting them for potential recognition by T cell receptors (TCRs). MHC molecules bind peptides within intracellular processing compartments and present these peptides on the surface of antigen-presenting cells to T cells. The human MHC region, also known as HLA, is located on chromosome 6 and includes class I and class II regions. The class I alpha chain is a glycoprotein with a molecular weight of approximately 44 kDa. This polypeptide chain is somewhat longer than 350 amino acid residues. It can be divided into three functional regions: the outer, transmembrane, and cytoplasmic regions. The ectodomain is 283 amino acid residues long and is divided into three domains: alpha1, alpha2, and alpha3. These domains and regions are usually encoded by separate exons of class I genes. The transmembrane domain spans the lipid bilayer of the plasma membrane. It usually consists of 23 hydrophobic amino acid residues arranged in an alpha helix. The cytoplasmic domain, i.e., the portion facing the cytoplasm and connected to the transmembrane domain, is typically 32 amino acid residues long and can interact with elements of the cytoskeleton. The alpha chain interacts with beta2-microglobulin, thus forming an alpha-beta2 dimer on the cell surface. The term "MHC class II" or "class II" refers to major histocompatibility complex class II proteins or genes. Within the human MHC class II domain are the DP, DQ, and DR subregions of the class II alpha and beta chain genes (i.e., DP alpha, DP beta, DQ alpha, DQ beta, DR alpha, and DR beta). Class II molecules are heterodimers consisting of an alpha chain and a beta chain, respectively.Both chains are glycoproteins with molecular weights of 31-34 kDa (α) or 26-29 kDa (β). The total length of the alpha chain varies from 229 to 233 amino acid residues, while the total length of the beta chain varies from 225 to 238 residues. Both the alpha and beta chains consist of an ectodomain, a connecting peptide, a transmembrane domain, and a cytoplasmic tail. The ectodomain consists of two domains, alpha1 and alpha2, or beta1 and beta2. The connecting peptides are beta and 9 residues long in the alpha and beta chains, respectively. The connecting peptide connects the two domains to a transmembrane domain consisting of 23 amino acid residues in both the alpha and beta chains. The length of the cytoplasmic domain, i.e., the portion facing the cytoplasm and connected to the transmembrane domain, varies from 3 to 16 residues in the alpha chain and 8 to 20 residues in the beta chain. Exemplary transmembrane / cytoplasmic domain sequences are described in U.S. Patent Nos. 8,178,653 and 8,637,006. In some embodiments, upon translation, the transmembrane and / or cytoplasmic domain is at the C-terminus of one or more neoepitope sequences encoded by the RNA vaccine. In some embodiments, the transmembrane and / or cytoplasmic domain of the MHC molecule encoded by the RNA vaccine comprises the sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 30). In some embodiments, the transmembrane and / or cytoplasmic domain of the MHC molecule comprises the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCAUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGC (SEQ ID NO: 28).In some embodiments, the transmembrane and / or cytoplasmic domain of the MHC molecule is encoded by DNA comprising the sequence ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACAGCC (SEQ ID NO: 29).

[0243] In some embodiments, the RNA vaccine comprises both a polynucleotide sequence encoding a secretory signal peptide N-terminal to one or more neoepitope sequences and a polynucleotide sequence encoding a transmembrane domain and / or a cytoplasmic domain C-terminal to one or more neoepitope sequences. The combination of such sequences has been shown to improve the processing and presentation of MHC class I and II epitopes in human dendritic cells. See, for example, Kreiter, S. et al. (2008) J. Immunol. 180:309-318.

[0244] In bone marrow DCs, the RNA is released into the cytosol and translated into poly-neoepitope peptides. The polypeptide contains an additional sequence to enhance antigen presentation. In some embodiments, a signal sequence (sec) from the MHCII heavy chain at the N-terminus of the polypeptide is used to target the nascent molecule to the endoplasmic reticulum, which has been shown to enhance MHCII presentation efficiency. Without wishing to be bound by theory, it is believed that the transmembrane and cytoplasmic domains of the MHCII heavy chain direct the polypeptide to the endosomal / lysosomal compartment, which has been shown to improve MHCII presentation.

[0245] In some embodiments, the RNA vaccine comprises a 3' UTR. Specific untranslated sequences found in the 3' pair of protein-coding sequences in mRNA have been shown to improve RNA stability, translation, and protein expression. Polynucleotide sequences suitable for use as 3' UTRs are described, for example, in PG Publication No. U.S. Patent Application Publication No. 20190071682. In some embodiments, the 3' UTR comprises the 3' untranslated region of AES or a fragment thereof and / or a non-coding RNA of mitochondrially encoded 12S RNA. The term "AES" refers to Amino-Terminal Enhancer of Split and includes the AES gene (see, e.g., NCBI Gene ID: 166). The protein encoded by this gene belongs to the Groucho / TLE family of proteins and can function as a homo-oligomer or hetero-oligomer with other family members to dominantly suppress the expression of other family member genes. An exemplary AES mRNA sequence is provided in NCBI Reference Sequence Accession No. NM_198969. The term "MT_RNR1" refers to mitochondrially encoded 12S RNA and includes the MT_RNR1 gene (see, e.g., NCBI gene ID: 4549). This RNA gene belongs to the Mt_rRNA class. Diseases associated with MT-RNR1 include restrictive cardiomyopathy and auditory neuropathy. Among its associated pathways are ribosome biogenesis in eukaryotes and CFTR translation fidelity (Class I mutations). An exemplary MT_RNR1 RNA sequence is found within NCBI Reference Sequence Accession Number NC_012920. In some embodiments, the 3'UTR of the RNA vaccine comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO: 33).In some embodiments, the 3'UTR of the RNA vaccine comprises the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO: 35). In some embodiments, the 3'UTR of the RNA vaccine comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO: 33) and the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO: 35). In some embodiments, the 3'UTR of the RNA vaccine comprises the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 31).In some embodiments, the 3'UTR of the RNA vaccine is encoded by DNA comprising the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC (SEQ ID NO: 34). In some embodiments, the 3'UTR of the RNA vaccine is encoded by DNA comprising the sequence CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG (SEQ ID NO: 36). In some embodiments, the 3'UTR of the RNA vaccine is encoded by DNA comprising the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC (SEQ ID NO: 34) and the sequence CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG (SEQ ID NO: 36).In some embodiments, the 3'UTR of the RNA vaccine is encoded by DNA comprising the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (SEQ ID NO: 32).

[0246] In some embodiments, the RNA vaccine comprises a poly(A) tail at its 3' end. In some embodiments, the poly(A) tail comprises more than 50 or more than 100 adenine nucleotides. For example, in some embodiments, the poly(A) tail comprises 120 adenine nucleotides. This poly(A) tail has been demonstrated to increase RNA stability and translation efficiency (Holtkamp, ​​S et al. (2006) Blood 108:4009-4017). In some embodiments, RNA comprising a poly(A) tail is produced by transcribing a DNA molecule comprising a polynucleotide sequence encoding at least 50, 100, or 120 consecutive adenine nucleotides and a recognition sequence for a type IIS restriction endonuclease in the 5' to 3' direction of transcription. Exemplary poly(A) tails and 3' UTR sequences that improve translation can be found, for example, in U.S. Patent No. 9,476,055.

[0247] In some embodiments, an RNA vaccine or RNA molecule of the present disclosure comprises the following general structure (5' to 3' direction): (1) a 5' cap; (2) a 5' untranslated region (UTR); (3) a polynucleotide sequence encoding a secretory signal peptide; (4) a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule; (5) a 3' UTR comprising (a) a 3' untranslated region of an Amino-Terminal Enhancer of Split (AES) mRNA or a fragment thereof, and (b) a non-coding RNA of a mitochondrially encoded 12S RNA or a fragment thereof; and (6) a poly(A) sequence. In some embodiments, the RNA vaccine or RNA molecule of the disclosure comprises, in 5' to 3' direction, the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 19); and the polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAG CUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGC UCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUU (SEQ ID NO: 20).Advantageously, RNA vaccines comprising this combination and orientation of structures or sequences are characterized by one or more of improved RNA stability, increased translation efficiency, improved antigen presentation and / or processing (e.g., by DCs), and increased protein expression.

[0248] In some embodiments, an RNA vaccine or RNA molecule of the present disclosure comprises the sequence (5' to 3' direction) of SEQ ID NO: 42. In some embodiments, N refers to a polynucleotide sequence encoding 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 30 different neoepitopes. In some embodiments, N refers to a polynucleotide sequence encoding one or more linker-epitope modules (e.g., 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 30 different linker-epitope modules). In some embodiments, N refers to a polynucleotide sequence encoding one or more linker-epitope modules (e.g., 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 30 different linker-epitope modules) and an additional amino acid linker at the 3' end.

[0249] In some embodiments, the RNA vaccine or RNA molecule further comprises a polynucleotide sequence encoding at least one neoepitope, wherein the polynucleotide sequence encoding the at least one neoepitope is located, in a 5' to 3' direction, between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule. In some embodiments, the RNA molecule comprises a polynucleotide sequence encoding 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, or 20 different neoepitopes.

[0250] In some embodiments, the RNA vaccine or RNA molecule further comprises, in the 5' to 3' direction, a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope. In some embodiments, the polynucleotide sequences encoding the amino acid linker and the neoepitope form a linker-neoepitope module (e.g., contiguous sequences in the 5' to 3' direction within the same open reading frame). In some embodiments, the polynucleotide sequence forming the linker-neoepitope module is located, in the 5' to 3' direction, between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule, or between the sequence of SEQ ID NO: 19 and the sequence of SEQ ID NO: 20. In some embodiments, the RNA vaccine or RNA molecule comprises 2, 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, 28, 29, or 30 linker-epitope modules. In some embodiments, each of the linker-epitope modules encodes a different neoepitope. In some embodiments, the RNA vaccine or RNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 linker-epitope modules, and the RNA vaccine or RNA molecule comprises polynucleotides encoding 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, or 20 different neoepitopes. In some embodiments, the RNA vaccine or RNA molecule comprises 5, 10, or 20 linker-epitope modules. In some embodiments, each of the linker-epitope modules encodes a different neoepitope.In some embodiments, the linker-epitope modules form a contiguous sequence in the same open reading frame in a 5' to 3' direction. In some embodiments, the polynucleotide sequence encoding the linker of the first linker-epitope module is 3' to the polynucleotide sequence encoding the secretory signal peptide. In some embodiments, the polynucleotide sequence encoding the neoepitope of the last linker-epitope module is 5' to the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule.

[0251] In some embodiments, the RNA vaccine is at least 800 nucleotides, at least 1000 nucleotides, or at least 1200 nucleotides in length. In some embodiments, the RNA vaccine is less than 2000 nucleotides in length. In some embodiments, the RNA vaccine is at least 800 nucleotides but less than 2000 nucleotides in length, at least 1000 nucleotides but less than 2000 nucleotides in length, at least 1200 nucleotides but less than 2000 nucleotides in length, at least 1400 nucleotides but less than 2000 nucleotides in length, at least 800 nucleotides but less than 1400 nucleotides in length, or at least 800 nucleotides but less than 2000 nucleotides in length. For example, the constant region of an RNA vaccine comprising the above elements is approximately 800 nucleotides in length. In some embodiments, an RNA vaccine comprising five tumor-specific neoepitopes (e.g., each encoding 27 amino acids) is greater than 1300 nucleotides in length. In some embodiments, an RNA vaccine comprising ten tumor-specific neoepitopes (e.g., each encoding 27 amino acids) is greater than 1800 nucleotides in length.

[0252] In some embodiments, one or more polynucleotides of an RNA vaccine are formulated with one or more lipids. In some embodiments, the RNA vaccine is formulated as a lipid nanoparticle, where one or more polynucleotides of the RNA vaccine and one or more lipids form a lipid nanoparticle. In some embodiments, the RNA vaccine is formulated as a lipoplex, where one or more polynucleotides of the RNA vaccine and one or more lipids form a lipoplex. In some embodiments, a lipoplex nanoparticle formulation for RNA (RNA-lipoplex) is used to enable IV delivery of the RNA vaccine of the present disclosure. In some embodiments, a lipoplex nanoparticle formulation for RNA cancer vaccines containing the synthetic cationic lipid (R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propanaminium chloride (DOTMA) and the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is used to enable, for example, IV delivery. The DOTMA / DOPE liposome components are optimized for IV delivery and targeting of antigen-presenting cells in the spleen and other lymphoid organs.

[0253] In some embodiments, the lipid nanoparticles or lipoplexes comprise at least one cationic lipid. The cationic lipid may be monocationic or polycationic. Any cationic amphiphilic molecule, for example, a molecule comprising at least one hydrophilic and lipophilic portion, is a cationic lipid within the meaning of the present invention. In one embodiment, the positive charge is carried by at least one cationic lipid, and the negative charge is carried by RNA. In one embodiment, the lipid nanoparticles or lipoplexes comprise at least one helper lipid. The helper lipid may be a neutral or anionic lipid. The helper lipid may be a natural lipid, such as a phospholipid or a natural lipid analog, or a completely synthetic lipid, or a lipid-like molecule dissimilar to a natural lipid. In one embodiment, the cationic lipid and / or the helper lipid are bilayer-forming lipids.

[0254] In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or an analog or derivative thereof, and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) or an analog or derivative thereof.

[0255] In one embodiment, the at least one helper lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or an analog or derivative thereof, cholesterol (Chol) or an analog or derivative thereof, and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or an analog or derivative thereof.

[0256] In one embodiment, the molar ratio of the at least one cationic lipid to the at least one helper lipid is 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1: 1. In one embodiment, in this ratio, the molar amount of cationic lipid results from the molar amount of cationic lipid multiplied by the number of positive charges on the cationic lipid.

[0257] In one embodiment, lipids are contained in the vesicles that encapsulate the RNA.The vesicles can be multilamellar vesicles, unilamellar vesicles, or a mixture thereof.The vesicles can be lipoplexes or lipid nanoparticles.

[0258] One or more RNA vaccine formulations described herein can be formed by mixing RNA and cationic lipids, adjusting the positive charge to negative charge ratio of the cationic lipid to RNA according to the (+ / -) charge ratio of the cationic lipid to RNA. The + / - charge ratio of the cationic lipid to RNA in the lipid nanoparticles or lipoplexes described herein can be calculated by the following formula: (+ / - charge ratio) = [(amount of cationic lipid (mol)) * (total number of positive charges in cationic lipid)]: [(amount of RNA (mol)) * (total number of negative charges in RNA)]. The amount of RNA and the amount of cationic lipid can be easily determined by those skilled in the art, taking into account the loading amount during the preparation of nanoparticles or lipoplexes. For further description of exemplary nanoparticles, see, for example, U.S. Patent Application Publication No. 20150086612 to PG.

[0259] In one embodiment, the charge ratio of positive to negative charges across the lipid nanoparticles (e.g., at physiological pH) is 1.4:1 to 1:8, preferably 1.2:1 to 1:4, such as 1:1 to 1:3, for example 1:1.2 to 1:2, 1:1.2 to 1:1.8, 1:1.3 to 1:1.7, particularly 1:1.4 to 1:1.6, for example about 1:1.5. In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipid nanoparticles is In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipid nanoparticles is between 1.6:2 (0.8) and 1:2 (0.5), or between 1.6:2 (0.8) and 1.1:2 (0.55). In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipid nanoparticles is 1.3:2 (0.65). In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipid nanoparticles is 1.0:2.0 or greater. In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipid nanoparticles is 1.9:2.0 or less. In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipid nanoparticles is 1.0:2.0 or greater and 1.9:2.0 or less.

[0260] In another embodiment, the charge ratio of positive to negative charges across the lipoplex (e.g., at physiological pH) is 1.4:1 to 1:8, preferably 1.2:1 to 1:4, such as 1:1 to 1:3, for example 1:1.2 to 1:2, 1:1.2 to 1:1.8, 1:1.3 to 1:1.7, particularly 1:1.4 to 1:1.6, such as about 1:1.5. In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipoplex is In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipoplex is between 1.6:2 (0.8) and 1:2 (0.5), or between 1.6:2 (0.8) and 1.1:2 (0.55). In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipoplex is 1.3:2 (0.65). In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipoplex is 1.0:2.0 or greater. In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipoplex is 1.9:2.0 or less. In some embodiments, at physiological pH, the charge ratio of positive to negative charges across the lipoplex is 1.0:2.0 or greater and 1.9:2.0 or less.

[0261] In one embodiment, the lipoplex or lipid nanoparticle comprises DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of positive charges in DOTMA to negative charges in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the lipoplex or lipid nanoparticle comprises DOTMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, and the charge ratio of positive charges in DOTMA to negative charges in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the lipoplex or lipid nanoparticle comprises DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, with a charge ratio of positive charges in DOTMA to negative charges in RNA of 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the lipoplex or lipid nanoparticle comprises DOTMA and DOPE in a molar ratio of 2:1 to 1:2, preferably 2:1 to 1:1, with a charge ratio of positive charges in DOTMA to negative charges in RNA of 1.4:1 or less. In one embodiment, the lipoplex or lipid nanoparticle comprises DOTMA and cholesterol in a molar ratio of 2:1 to 1:2, preferably 2:1 to 1:1, with a charge ratio of positive charges in DOTMA to negative charges in RNA of 1.4:1 or less. In one embodiment, the lipoplex or lipid nanoparticle comprises DOTAP and DOPE in a molar ratio of 2:1 to 1:2, preferably 2:1 to 1:1, and the charge ratio of positive charges in DOTAP to negative charges in RNA is 1.4:1 or less.

[0262] In one embodiment, the zeta potential of the lipoplexes or lipid nanoparticles is -5 or less, -10 or less, -15 or less, -20 or less, or -25 or less. In various embodiments, the zeta potential of the lipoplexes or lipid nanoparticles is -35 or more, -30 or more, or -25 or more. In one embodiment, the lipoplexes or lipid nanoparticles have a zeta potential of 0 mV to -50 mV, preferably 0 mV to -40 mV or -10 mV to -30 mV.

[0263] In some embodiments, the polydispersity index of the lipoplexes or lipid nanoparticles is 0.5 or less, 0.4 or less, or 0.3 or less, as measured by dynamic light scattering.

[0264] In some embodiments, the liposomes, lipoplexes, or lipid nanoparticles have an average diameter in the range of about 50 nm to about 1000 nm, about 100 nm to about 800 nm, about 200 nm to about 600 nm, about 250 nm to about 700 nm, or about 250 nm to about 550 nm, as measured by dynamic light scattering.

[0265] In some embodiments, the personalized cancer vaccine is administered intravenously, for example, the RNA vaccine is administered to a human patient at a dose of 15 μg, 21 μg, 21.3 μg, 25 μg, 38 μg, or 50 μg. In some embodiments, 15 μg, 21 μg, 21.3 μg, 25 μg, 38 μg, or 50 μg of RNA is delivered per administration (i.e., the dose weight reflects the weight of the administered RNA, not the total weight of the administered formulation or lipoplex). In some embodiments, the RNA vaccine is administered to a human patient at a dose of about 25 μg. In some embodiments, the RNA vaccine is administered to a human patient at a dose of about 21 μg. In some embodiments, the RNA vaccine is administered to a human patient at a dose of about 21.3 μg. More than one personalized cancer vaccine may be administered to a subject, for example, a subject may be administered one personalized cancer vaccine having a combination of neoepitopes and a separate personalized cancer vaccine having a different combination of neoepitopes. In some embodiments, a first personalized cancer vaccine having five neoepitopes is administered in combination with a second personalized cancer vaccine having five alternative epitopes. In some embodiments, a first personalized cancer vaccine having ten neoepitopes is administered in combination with a second personalized cancer vaccine having ten alternative epitopes.

[0266] In some embodiments, the personalized cancer vaccine is administered to be delivered to the spleen. For example, the personalized cancer vaccine can be administered to deliver one or more antigens (e.g., tumor-specific neoantigens) to antigen-presenting cells (e.g., in the spleen).

[0267] Either the personalized cancer vaccines or RNA vaccines of the present disclosure may find use in the methods described herein. For example, in some embodiments, the PD-1 axis binding antagonists of the present disclosure are administered in combination with personalized cancer vaccines (ICV), such as the RNA vaccines described herein.

[0268] Further provided herein are DNA molecules encoding any of the RNA vaccines of the present disclosure. For example, in some embodiments, the DNA molecules of the present disclosure include the following general structure (5' to 3' direction): (1) a polynucleotide sequence encoding a 5' untranslated region (UTR); (2) a polynucleotide sequence encoding a secretory signal peptide; (3) a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule; (4) a polynucleotide sequence encoding (a) a 3' untranslated region of an Amino-Terminal Enhancer of Split (AES) mRNA or a fragment thereof; and (b) a polynucleotide sequence encoding a 3' UTR comprising a non-coding RNA of a mitochondrially encoded 12S RNA or a fragment thereof; and (5) a polynucleotide sequence encoding a poly(A) sequence. In some embodiments, a DNA molecule of the disclosure comprises, in the 5' to 3' direction, the polynucleotide sequence GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC (SEQ ID NO: 40) and the polynucleotide sequence TACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (SEQ ID NO: 41).

[0269] In some embodiments, the DNA molecule further comprises, in the 5' to 3' direction, a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope. In some embodiments, the polynucleotide sequences encoding the amino acid linker and the neoepitope form a linker-neoepitope module (e.g., contiguous sequences in the 5' to 3' direction within the same open reading frame). In some embodiments, the polynucleotide sequence forming the linker-neoepitope module is located, in the 5' to 3' direction, between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule, or between the sequence of SEQ ID NO:40 and the sequence of SEQ ID NO:41. In some embodiments, the DNA molecule comprises 2, 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, 28, 29, or 30 linker-epitope modules, each linker-epitope module encoding a different neoepitope. In some embodiments, the DNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 linker-epitope modules, and the DNA molecule comprises polynucleotides encoding 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, or 20 different neoepitopes. In some embodiments, the DNA molecule comprises 5, 10, or 20 linker-epitope modules. In some embodiments, each of the linker-epitope modules encodes a different neoepitope. In some embodiments, the linker-epitope modules form a contiguous sequence in the 5' to 3' direction within the same open reading frame.In some embodiments, the polynucleotide sequence encoding the linker of the first linker-epitope module is 3' to the polynucleotide sequence encoding the secretory signal peptide, hi some embodiments, the polynucleotide sequence encoding the neoepitope of the last linker-epitope module is 5' to the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule.

[0270] Also provided herein are methods of producing any of the RNA vaccines of the present disclosure, comprising transcribing a DNA molecule of the present disclosure (e.g., by transcription of linear double-stranded DNA or plasmid DNA, e.g., by in vitro transcription). In some embodiments, the method further comprises isolating and / or purifying the transcribed RNA molecule from the DNA molecule.

[0271] In some embodiments, the RNA or DNA molecules of the present disclosure allow the RNA to be transcribed under the control of a 5' RNA polymerase promoter and contain a type IIS restriction cleavage site containing a polyadenylation cassette (poly(A) sequence), with the recognition sequence located 3' to the poly(A) sequence but the cleavage site located upstream, and therefore within the poly(A) sequence. Restriction cleavage at the type IIS restriction cleavage site allows the plasmid to be linearized within the poly(A) sequence, as described in U.S. Patent Nos. 9,476,055 and 10,106,800. The linearized plasmid can then be used as a template for in vitro transcription, with the resulting transcript terminating in an unmasked poly(A) sequence. Any of the type IIS restriction cleavage sites described in U.S. Patent Nos. 9,476,055 and 10,106,800 may be used.

[0272] In some embodiments of the methods provided herein, the RNA vaccine comprises one or more polynucleotides encoding 5 to 20 (any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen. In certain embodiments, the RNA vaccine is formulated with one or more lipids. In certain embodiments, the one or more polynucleotides and one or more lipids of the RNA vaccine form a lipoplex. In certain embodiments, the lipoplex comprises one or more lipids that form a multilayer structure encapsulating the one or more polynucleotides of the RNA vaccine. In certain embodiments, the one or more lipids comprise at least one cationic lipid and at least one helper lipid. In certain embodiments, the one or more lipids comprise (R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propanaminium chloride (DOTMA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In certain embodiments, at physiological pH, the charge ratio of positive to negative charges across the liposome is 1.3:2 (0.65).

[0273] In certain embodiments, the RNA vaccine comprises an RNA molecule comprising, in a 5' to 3' direction: (1) a 5' cap; (2) a 5' untranslated region (UTR); (3) a polynucleotide sequence encoding a secretory signal peptide; (4) a polynucleotide sequence encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen; (5) a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule; (6) a 3' untranslated region of (a) an Amino-Terminal Enhancer of Split (AES) mRNA or a fragment thereof; and (b) a 3' UTR comprising a non-coding RNA of mitochondrially encoded 12S RNA or a fragment thereof; and (7) a poly(A) sequence.

[0274] In certain embodiments, the RNA molecule further comprises a polynucleotide sequence encoding an amino acid linker, wherein the amino acid linker and the polynucleotide sequence encoding a first of the one or more neoepitopes form a first linker-neoepitope module, and the polynucleotide sequence forming the first linker-neoepitope module is located, in a 5' to 3' direction, between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule. In certain embodiments, the amino acid linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 39). In certain embodiments, the polynucleotide sequence encoding the amino acid linker comprises the sequence GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO: 37).

[0275] In certain embodiments, the RNA molecule further comprises, in the 5'→3' direction, at least a second linker-epitope module, the at least second linker-epitope module comprising a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope, wherein the polynucleotide sequence forming the second linker-neoepitope module is located, in the 5'→3' direction, between the polynucleotide sequence encoding the neoepitope of the first linker-neoepitope module and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule, and the neoepitope of the first linker-epitope module is different from the neoepitope of the second linker-epitope module. In certain embodiments, the RNA molecule comprises five linker-epitope modules, each of the five linker-epitope modules encoding a different neoepitope. In certain embodiments, the RNA molecule comprises 5 linker-epitope modules, each of the 5 linker-epitope modules encoding a different neoepitope. In certain embodiments, the RNA molecule comprises 10 linker-epitope modules, each of the 10 linker-epitope modules encoding a different neoepitope. In certain embodiments, the RNA molecule comprises 20 linker-epitope modules, each of the 20 linker-epitope modules encoding a different neoepitope.

[0276] In certain embodiments, the RNA molecule further comprises a second polynucleotide sequence encoding an amino acid linker, wherein the second polynucleotide sequence encoding the amino acid linker is between the polynucleotide sequence encoding the distal-most neoepitope in the 3' direction and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

[0277] In certain embodiments, the 5' cap comprises a D1 diastereoisomer of the following structure: [ka]

[0278] In certain embodiments, the 5'UTR comprises the sequence UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 23). In certain embodiments, the 5'UTR comprises the sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 21).

[0279] In certain embodiments, the secretory signal peptide comprises the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 27). In certain embodiments, the polynucleotide sequence encoding the secretory signal peptide comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 25).

[0280] In certain embodiments, at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule comprise the amino acid sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 30). In certain embodiments, the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule comprises the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC (SEQ ID NO: 28).

[0281] In certain embodiments, the 3' untranslated region of the AES mRNA comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO: 33). In certain embodiments, the non-coding RNA of the mitochondrially encoded 12S RNA comprises the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO: 35). In certain embodiments, the 3'UTR comprises the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 31).

[0282] In certain embodiments, the poly(A) sequence comprises 120 adenine nucleotides.

[0283] In certain embodiments, the RNA vaccine comprises, in 5' to 3' direction, the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 19); a polynucleotide sequence encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen; and the polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGA UAGCGCCCAGGGCAGCGACGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCA CGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 20).

[0284] IV. PD-1 axis binding antagonists In some embodiments, a personalized cancer vaccine (e.g., an RNA vaccine) of the present disclosure is administered in combination with a PD-1 axis binding antagonist.

[0285] For example, PD-1 axis-binding antagonists include PD-1-binding antagonists, PDL1-binding antagonists, and PDL2-binding antagonists. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

[0286] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner(s). In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the binding partner of PDL1 is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the binding partner of PDL2 is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0287] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody).

[0288] In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number 946414-94-4). Nivolumab (Bristol-Myers Squibb / Ono), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168. In some embodiments, the anti-PD-1 antibody comprises heavy and light chain sequences: (a) The heavy chain has the amino acid sequence: QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWY DGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 11); (b) The light chain comprises the amino acid sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

[0289] In some embodiments, the anti-PD-1 antibody comprises six HVR sequences from SEQ ID NO:11 and SEQ ID NO:12 (e.g., three heavy chain HVRs of SEQ ID NO:11 and three light chain HVRs of SEQ ID NO:12). In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable domain from SEQ ID NO:11 and a light chain variable domain from SEQ ID NO:12.

[0290] In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number 1374853-91-4). Pembrolizumab (Merck), also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO 2009 / 114335. In some embodiments, the anti-PD-1 antibody comprises heavy and light chain sequences: (a) The heavy chain has the amino acid sequence: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGG INPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 13), (b) the light chain has the amino acid sequence: Contains EIVLTQSPAT LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14).

[0291] In some embodiments, the anti-PD-1 antibody comprises six HVR sequences from SEQ ID NO: 13 and SEQ ID NO: 14 (e.g., three heavy chain HVRs of SEQ ID NO: 13 and three light chain HVRs of SEQ ID NO: 14). In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable domain from SEQ ID NO: 13 and a light chain variable domain from SEQ ID NO: 14.

[0292] In some embodiments, the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.

[0293] In some embodiments, the anti-PD-1 antibody is PDR001 (CAS Registry Number 1859072-53-9; Novartis). PDR001 is a humanized IgG4 anti-PD1 antibody that blocks the binding of PDL1 and PDL2 to PD-1.

[0294] In some embodiments, the anti-PD-1 antibody is REGN2810 (Regeneron). REGN2810 is a human anti-PD1 antibody, also known as LIBTAYO® and cemiplimab-rwlc.

[0295] In some embodiments, the anti-PD-1 antibody is BGB-108 (BeiGene). In some embodiments, the anti-PD-1 antibody is BGB-A317 (BeiGene).

[0296] In some embodiments, the anti-PD-1 antibody is JS-001 (Shanghai Junshi). JS-001 is a humanized anti-PD1 antibody.

[0297] In some embodiments, the anti-PD-1 antibody is STI-A1110. STI-A1110 is a human anti-PD1 antibody.

[0298] In some embodiments, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR-1210 is a human IgG4 anti-PD1 antibody.

[0299] In some embodiments, the anti-PD-1 antibody is PF-06801591 (Pfizer).

[0300] In some embodiments, the anti-PD-1 antibody is TSR-042 (also known as ANB011; Tesaro / AnaptysBio).

[0301] In some embodiments, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).

[0302] In some embodiments, the anti-PD-1 antibody is ENUM 244C8 (Enumeral Biomedical Holdings). ENUM 244C8 is an anti-PD-1 antibody that inhibits PD-1 function without blocking the binding of PDL1 to PD-1.

[0303] In some embodiments, the anti-PD-1 antibody is ENUM 388D4 (Enumeral Biomedical Holdings). ENUM 388D4 is an anti-PD-1 antibody that competitively inhibits the binding of PDL1 to PD-1.

[0304] In some embodiments, the PD-1 antibody comprises six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and / or heavy chain variable domains and light chain variable domains from the PD-1 antibodies set forth in the following: WO 2015 / 112800 (applicant: Regeneron), WO 2015 / 112805 (applicant: Regeneron), WO 2015 / 112900 (applicant: Novartis), U.S. Patent Application Publication No. 20150210769 (assigned to Novartis), WO 2016 / 089873 (applicant: Celgene), WO 2015 / 035606 (applicant: Beigene), WO 2015 / 085847 (applicant: Shanghai Hengrui Pharmaceutical / Jiangsu Hengrui), and the like. Medicine), WO 2014 / 206107 (applicant: Shanghai Junshi Biosciences / Junmeng Biosciences), WO 2012 / 145493 (applicant: Amplimmune), U.S. Patent No. 9,205,148 (assigned to MediImmune), WO 2015 / 119930 (applicant: Pfizer / Merck), WO 2015 / 119923 (applicant: Pfizer / Merck), WO 2016 / 032927 (applicant: Pfizer / Merck), WO 2014 / 179664 (applicant: AnaptysBio), WO 2016 / 106160 (applicant: Enumeral), and WO 2014 / 194302 (applicant: Sorrento).

[0305] In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. AMP-224 (CAS Registry Number 1422184-00-6; GlaxoSmithKline / MedImmune), also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342.

[0306] In some embodiments, the PD-1 binding antagonist is a peptide or a small molecule compound. In some embodiments, the PD-1 binding antagonist is AUNP-12 (PierreFabre / Aurigene). See, e.g., WO 2012 / 168944, WO 2015 / 036927, WO 2015 / 044900, WO 2015 / 033303, WO 2013 / 144704, WO 2013 / 132317, and WO 2011 / 161699.

[0307] In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PD-1. In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL1. In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL1 and VISTA. In some embodiments, the PDL1 binding antagonist is CA-170 (also known as AUPM-170). In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL1 and TIM3. In some embodiments, the small molecule is a compound described in WO 2015 / 033301 and WO 2015 / 033299.

[0308] In some embodiments, the PD-1 axis binding antagonist is an anti-PDL1 antibody. Various anti-PDL1 antibodies are contemplated and described herein. In any of the cases herein, the isolated anti-PDL1 antibody can bind to human PDL1, e.g., human PDL1 as set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof. In some embodiments, the anti-PDL1 antibody can inhibit the binding between PDL1 and PD-1 and / or the binding between PDL1 and B7-1. In some embodiments, the anti-PDL1 antibody is a monoclonal antibody. In some embodiments, the anti-PDL1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the anti-PDL1 antibody is a humanized antibody. In some embodiments, the anti-PDL1 antibody is a human antibody. Examples of anti-PDL1 antibodies useful in the methods of the present invention, and methods for making them, are described in PCT Patent Application WO 2010 / 077634 and U.S. Patent No. 8,217,149, which are incorporated herein by reference.

[0309] In some embodiments, the anti-PDL1 antibody comprises a heavy chain variable region sequence and a light chain variable region sequence: (a) the heavy chain variable region comprises the HVR-H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 1), AWISPYGGSTYYADSVKG (SEQ ID NO: 2), and RHWPGGFDY (SEQ ID NO: 3), respectively; (b) the light chain variable region comprises the HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 4), SASFLYS (SEQ ID NO: 5), and QQYLYHPAT (SEQ ID NO: 6), respectively.

[0310] In some embodiments, the anti-PDL1 antibody is MPDL3280A, also known as atezolizumab and TECENTRIQ® (CAS Registry Number 1422185-06-5), and is described in WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Proposed INN:List 112, Vol. 28, No. 4, published January 16, 2015 (see page 485). In some embodiments, the anti-PDL1 antibody comprises heavy and light chain sequences: (a) the heavy chain variable region sequence comprises the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7); (b) The light chain variable region sequence comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 8).

[0311] In some embodiments, the anti-PDL1 antibody comprises a heavy chain and a light chain sequence: (a) The heavy chain has the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 9), (b) The light chain comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

[0312] In some embodiments, the anti-PDL1 antibody is avelumab (CAS Registry Number: 1537032-82-8). Avelumab, also known as MSB0010718C, is a human monoclonal IgG1 anti-PDL1 antibody (Merck KGaA, Pfizer). In some embodiments, the anti-PDL1 antibody comprises a heavy chain and a light chain sequence: (a) The heavy chain has the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 15), (b) The light chain comprises the amino acid sequence: QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 16).

[0313] In some embodiments, the anti-PDL1 antibody comprises six HVR sequences from SEQ ID NO: 15 and SEQ ID NO: 16 (e.g., three heavy chain HVRs of SEQ ID NO: 15 and three light chain HVRs of SEQ ID NO: 16). In some embodiments, the anti-PDL1 antibody comprises a heavy chain variable domain from SEQ ID NO: 15 and a light chain variable domain from SEQ ID NO: 16.

[0314] In some embodiments, the anti-PDL1 antibody is durvalumab (CAS Registry Number: 1428935-60-7). Durvalumab, also known as MEDI4736, is an Fc-optimized human monoclonal IgG1 kappa anti-PDL1 antibody (MedImmune, AstraZeneca) described in WO 2011 / 066389 and U.S. Patent Application Publication No. 2013 / 034559. In some embodiments, the anti-PDL1 antibody comprises heavy and light chain sequences: (a) The heavy chain has the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 17), (b) the light chain comprises the amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18).

[0315] In some embodiments, the anti-PDL1 antibody comprises six HVR sequences from SEQ ID NO: 17 and SEQ ID NO: 18 (e.g., three heavy chain HVRs of SEQ ID NO: 17 and three light chain HVRs of SEQ ID NO: 18). In some embodiments, the anti-PDL1 antibody comprises a heavy chain variable domain from SEQ ID NO: 17 and a light chain variable domain from SEQ ID NO: 18.

[0316] In some embodiments, the anti-PDL1 antibody is MDX-1105 (Bristol Myers Squibb). MDX-1105, also known as BMS-936559, is an anti-PDL1 antibody described in WO 2007 / 005874.

[0317] In some embodiments, the anti-PDL1 antibody is LY3300054 (Eli Lilly).

[0318] In some embodiments, the anti-PDL1 antibody is STI-A1014 (Sorrento). STI-A1014 is a human anti-PDL1 antibody.

[0319] In some embodiments, the anti-PDL1 antibody is KN035 (Suzhou Alphamab), a single domain antibody (dAB) generated from a camel phage display library.

[0320] In some embodiments, the anti-PDL1 antibody is comprised of a cleavable moiety or linker that, when cleaved (e.g., by proteases in the tumor microenvironment), activates the antibody antigen-binding domain, rendering it capable of binding its antigen, e.g., by removing a non-binding steric moiety. In some embodiments, the anti-PDL1 antibody is CX-072 (CytomX Therapeutics).

[0321] In some embodiments, the PDL1 antibody comprises six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and / or heavy chain variable domains and light chain variable domains derived from the PDL1 antibodies described in: U.S. Patent Application Publication No. 20160108123 (assigned to Novartis), WO 2016 / 000619 (applicant: Beigene), WO 2015 / 145493 (applicant: Amplimmune), U.S. Patent No. 9205148 (assigned to Medimmune), WO 2015 / 181634 (applicant: Sorrento), and WO 2016 / 061142 (applicant: Novartis).

[0322] In still further specific embodiments, the antibody further comprises a human or mouse constant region. In still further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In still further specific embodiments, the human constant region is IgG1. In still further embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In still further embodiments, the mouse constant region is IgG2A.

[0323] In further specific aspects, the antibody has reduced or minimal effector function. In yet further specific aspects, the minimal effector function results from an "effector-less Fc mutation" or an aglycosylation mutation. In yet further embodiments, the effector-less Fc mutation is an N297A or D265A / N297A substitution in the constant region. In some embodiments, the isolated anti-PDL1 antibody is aglycosylated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of a glycosylation site from an antibody is conveniently accomplished by altering the amino acid sequence to remove one of the tripeptide sequences described above (for N-linked glycosylation sites). This alteration can be made by substituting an asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

[0324] In yet a further embodiment, the present disclosure provides a composition comprising any of the above-described anti-PDL1 antibodies in combination with at least one pharmaceutically acceptable carrier.

[0325] In yet a further embodiment, the present disclosure provides a composition comprising an anti-PDL1, anti-PD-1, or anti-PDL2 antibody, or antigen-binding fragment thereof, provided herein and at least one pharmaceutically acceptable carrier. In some embodiments, the anti-PDL1, anti-PD-1, or anti-PDL2 antibody, or antigen-binding fragment thereof, administered to an individual is a composition comprising one or more pharmaceutically acceptable carriers. Any of the pharmaceutically acceptable carriers described herein or known in the art may be used.

[0326] In some embodiments, the PD-1 axis-binding antagonist is administered intravenously to a human patient. In some embodiments, the anti-PD-L1 antibody is administered to a human patient at a dose of about 1200 mg or about 1680 mg, for example, about 1100 mg, 1150 mg, 1200 mg, 1250 mg, or 1300 mg, or about 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 mg, 1680 mg, 1690 mg, 1700 mg, or more. In some embodiments, the anti-PD-L1 antibody is atezolizumab, and atezolizumab is administered intravenously to a human patient at a dose of about 1680 mg.

[0327] V. Chemotherapy Treatment In some embodiments, a personalized cancer vaccine (e.g., an RNA vaccine) of the present disclosure is administered in combination with a PD-1 axis binding antagonist and a chemotherapy treatment.

[0328] For example, the chemotherapy treatment may include a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, gemcitabine, leucovorin, 5-fluorouracil, capecitabine, irinotecan, liposomal irinotecan, platinum-based chemotherapy agents, auristatins, vinca alkaloids, podophyllotoxins, taxanes, baccatin derivatives, cryptophycins, maytansinoids, combretastatins, and dolastatins. In some embodiments, the chemotherapy treatment includes one or more of gemcitabine, leucovorin, 5-fluorouracil, capecitabine, irinotecan, liposomal irinotecan, platinum-based chemotherapy agents, taxanes, and any combination thereof. Further examples of chemotherapeutic agents include auristatins, DNA minor groove binders, DNA minor groove alkylating agents, enedienes, lexitropsins, duocarmycins, taxanes, puromycins, dolastatins, maytansinoids, and vinca alkaloids.

[0329] In some embodiments, the platinum-based chemotherapeutic agent is cisplatin, oxaliplatin, or both. Cisplatin, also known as Platinol® and Platinol®-AQ, is an antineoplastic alkylating agent. Oxaliplatin, also known as eloxatin, is also an antineoplastic alkylating agent.

[0330] In some embodiments, the taxane is paclitaxel, docetaxel, albumin-bound paclitaxel, or any combination thereof. Taxanes are anti-microtubule agents that act to halt the process of mitosis, thereby preventing cancer cells from dividing and growing.

[0331] Standard chemotherapy treatment for patients after PDAC resection involves adjuvant therapy with gemcitabine combination therapy or mFOLFIRINOX. Gemcitabine monotherapy has been the standard first-line treatment for advanced pancreatic cancer for over 20 years. However, recently, combination chemotherapy regimens (e.g., FOLFIRINOX or gemcitabine / nab-paclitaxel) have been shown to achieve higher response rates and better overall survival rates than gemcitabine monotherapy. These combination therapies have become the standard first-line treatment for advanced pancreatic cancer and are also available as treatment options for borderline resectable and locally advanced pancreatic cancer (see, e.g., Saung, MT, and Zheng, L., Clin Ther;39(11):2125-2134(2017)).

[0332] In some embodiments, the chemotherapy treatment includes leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin. Combination treatment with leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin is also known as FOLFIRINOX. In some embodiments, the chemotherapy treatment is FOLFIRINOX treatment or modified FOLFIRINOX (mFOLFIRINOX) treatment. Modified treatment regimens in mFOLFIRINOX can be selected to reduce the incidence and severity of hematologic toxic effects and diarrhea without reducing treatment efficacy. In some embodiments, the chemotherapy treatment is about 85 mg / m 2 Oxaliplatin at a dose of approximately 400 mg / m 2 Leucovorin at a dose of approximately 150 mg / m 2 and / or about 2400 mg / m 2 In some embodiments, the chemotherapy treatment comprises 5-fluorouracil at a dose of 0.05 mg / kg / day. In some embodiments, the chemotherapy treatment is administered intravenously to the human patient. In some embodiments, the chemotherapy treatment is administered as described herein.

[0333] VI. Medicines and Formulations Also provided herein are pharmaceutical compositions and formulations, for example, for the treatment of pancreatic cancer. In some embodiments, the pharmaceutical compositions and formulations further comprise a pharmaceutically acceptable carrier.

[0334] After preparing the antibody of interest (e.g., techniques for producing antibodies that can be formulated as disclosed herein are detailed herein and known in the art), a pharmaceutical formulation containing the antibody is prepared. In certain embodiments, the formulated antibody has not been subjected to prior lyophilization, and the formulation of interest herein is an aqueous formulation. In certain embodiments, the antibody is a full-length antibody. In one embodiment, the antibody in the formulation is an antibody fragment, such as F(ab')2. The therapeutically effective amount of the antibody present in the formulation is determined, for example, by considering the desired dose volume and mode of administration. Exemplary antibody concentrations in formulations are about 25 mg / mL to about 150 mg / mL, or about 30 mg / mL to about 140 mg / mL, or about 35 mg / mL to about 130 mg / mL, or about 40 mg / mL to about 120 mg / mL, or about 50 mg / mL to about 130 mg / mL, or about 50 mg / mL to about 125 mg / mL, or about 50 mg / mL to about 120 mg / mL, or about 50 mg / mL to about 110 mg / mL, or about 50 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 90 mg / mL, or about 50 mg / mL to about 80 mg / mL, or about 54 mg / mL to about 66 mg / mL. In some embodiments, the anti-PDL1 antibody described herein (e.g., atezolizumab) is administered at a dose of about 1200 mg. In some embodiments, an anti-PD1 antibody described herein (such as pembrolizumab) is administered at a dose of about 200 mg. In some embodiments, an anti-PD1 antibody described herein (such as nivolumab) is administered at a dose of about 240 mg (e.g., every 2 weeks) or 480 mg (e.g., every 4 weeks).

[0335] In some embodiments, the RNA vaccines described herein are administered at a dose of about 15 μg, about 21 μg, about 21.3 μg, about 25 μg, about 38 μg, or about 50 μg. For example, in some embodiments, the RNA vaccines are administered to human patients at a dose of about 21 μg, about 21.3 μg, or about 25 μg.

[0336] The pharmaceutical compositions and formulations described herein can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing an active ingredient (such as an antibody or polypeptide) having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, proteins such as albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases (eg, chondroitinases).

[0337] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation including a histidine-acetate buffer.

[0338] The compositions and formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other, and such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0339] The active ingredient can also be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions, for example, by microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0340] Sustained-release preparations may also be prepared.Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, these matrices being in the form of shaped articles, for example, films or microcapsules.Preparations used for in vivo administration are generally sterilized.Sterilization can be easily achieved, for example, by filtering through a sterile filtration membrane.

[0341] Pharmaceutical formulations of atezolizumab and pembrolizumab are commercially available. For example, atezolizumab is known under the trade name TECENTRIQ® (as described elsewhere herein). Pembrolizumab is known under the trade name KEYTRUDA® (as described elsewhere herein). In some embodiments, atezolizumab and the RNA vaccine, or pembrolizumab and the RNA vaccine, are provided in separate containers. In some embodiments, atezolizumab and pembrolizumab are used and / or prepared for administration to an individual as described in the prescribing information available with the commercially available products.

[0342] VII. Manufactured Articles or Kits Further provided herein is an article of manufacture or kit comprising the RNA vaccine of the present disclosure. Further provided herein is an article of manufacture or kit comprising a PD-1 axis binding antagonist (e.g., atezolizumab or pembrolizumab). In some embodiments, the article of manufacture or kit further comprises a package insert containing instructions for using the RNA vaccine and / or the PD-1 axis binding antagonist (e.g., in conjunction with the RNA vaccine) to treat or delay the progression of pancreatic cancer in an individual. Also provided herein is an article of manufacture or kit comprising a PD-1 axis binding antagonist (e.g., atezolizumab or pembrolizumab) and an RNA vaccine.

[0343] In some embodiments, a kit is provided that includes a personalized RNA vaccine for use in a method for treating a pancreatic cancer tumor in a human in need thereof, wherein the RNA vaccine is administered in combination with a PD-1 axis-binding antagonist and chemotherapy treatment according to the methods described herein, and the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the human. In some embodiments, the kit includes a PD-1 axis-binding antagonist for use in a method for treating a pancreatic cancer tumor in a human in need thereof, wherein the PD-1 axis-binding antagonist is administered in combination with a personalized RNA vaccine and chemotherapy treatment according to the methods described herein, and the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the human.

[0344] In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers can be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on or associated with the container can provide instructions for use. The article of manufacture or kit can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the product further includes one or more additional agents (e.g., chemotherapeutic agents and anti-neoplastic agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0345] VIII. Patient Selection Methods Further provided herein is a method for selecting a patient (e.g., a human patient) having a cancer tumor who is likely to respond to a therapy comprising a personalized RNA vaccine, the method comprising: (a) measuring the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from the patient by T cell receptor sequencing; (b) comparing the number and / or frequency of de novo SE TCR clones measured in (a) with a reference number and / or reference frequency; and (c) selecting the patient who is more likely to respond to a therapy comprising the personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency, wherein the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient, and a higher number and / or frequency of de novo SE TCR clones than the reference number and / or reference frequency indicates that the patient is more likely to respond to a therapy comprising the personalized RNA vaccine. In some embodiments, the method further comprises selecting a therapy comprising the personalized RNA vaccine or recommending a therapy comprising the personalized RNA vaccine.

[0346] Also provided herein is a method for selecting a human patient having a cancer tumor who is likely to respond to a therapy comprising a personalized RNA vaccine, comprising: a) comparing the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from the patient with a reference number and / or reference frequency; and b) selecting the patient who is more likely to respond to a therapy comprising the personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency, wherein the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor sample obtained from the patient, and the number and / or frequency of de novo SE TCR clones is measured by cell receptor sequencing, and a number and / or frequency of de novo SE TCR clones greater than the reference number and / or reference frequency indicates that the patient is more likely to respond to a therapy comprising the personalized RNA vaccine.

[0347] Extensively expanded de novo (SE) TCR clones The present disclosure describes methods for quantifying, analyzing, and using de novo SE TCR clones to determine the potential for therapeutic efficacy and immunogenic response to the personalized RNA vaccines described herein.

[0348] T cell receptor (TCR) clones are generated during TCR gene rearrangement, and clonality can be identified and characterized, for example, by identifying clonal expansion of T cells after antigen exposure, to identify the presence of a specific antigen in an individual. During T cell maturation, CD4+ and CD8+ T cells undergo T cell receptor gene rearrangement at the TCR-α, -β, -γ, and -δ loci. TCR gene rearrangement involves somatic splicing of the variable (V), joining (J), and diversity (D) regions of the genome of immature T cells, including the β but not the α chain. This process is responsible for the diversity of the antigen-binding region of the T cell receptor. Up to 10 15(see, e.g., Ndifon et al. 2012) to 10 61 It is estimated that there are potentially 100 TCR clones (see, e.g., Mora & Walczak 2019). If successful in surviving and maturing, each of these T cells has the capacity to further expand, giving rise to a population of T cell clones, each containing the same TCR sequence derived from the same single T cell. If the TCR of a naive T cell binds with sufficient affinity to a specific antigen (e.g., a cancer neoantigen)-MHC complex, the T cell will expand clonally. This expansion significantly increases the abundance of T cells that recognize a specific pathogen (e.g., a cancer tumor neoepitope), thereby enabling an effective immune response. As a result of the unique V(D)J recombination occurring in each mature T cell, the nucleotide and / or amino acid sequence of the TCR present on each T cell can serve as a natural molecular barcode for tracking the presence, number, and abundance (i.e., frequency) of T cell clones at various stages of treatment.

[0349] As used herein, a significantly expanded (SE) TCR clone refers to a TCR clone that expands in frequency (i.e., increases in percentage or proportion compared to the total number of TCR clones) during and / or after treatment compared to baseline (i.e., compared to pre-treatment), as determined by methods such as TCR sequencing ("TCR-seq"). Statistical methods, such as a binomial model based on Fisher's exact test (DeWitt et al., J Virol 2015;89(8):4517-4526) or a beta-binomial model (Rytlewski et al., PLoS One 2019;14(3):e0213684), can be applied to the data to identify which TCR clones exhibit a statistically significant increase in clonal frequency during and / or after administration of a personalized RNA vaccine. In some embodiments, the frequency of a TCR clone measured during or after treatment administration is compared to the frequency of a TCR clone measured at pre-treatment baseline using a beta-binomial model. In some embodiments, the frequency of TCR clones measured during or after treatment administration is compared to the frequency of TCR clones measured at baseline before treatment using Fisher's exact test. In some embodiments, a statistical correction, such as a post-hoc correction, for example, a Benjamini-Hochberg correction, is performed to control the false discovery rate. See, for example, Figure 10.

[0350] In some embodiments, the SE TCR clone is present in the sample at baseline. In some embodiments, the SE TCR clone is present in the sample at baseline. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8or lower frequencies. In some embodiments, the SE TCR clone frequency is significantly higher after administration of a personalized RNA vaccine treatment compared to the baseline before treatment. Without wishing to be bound by theory, it is believed that these SE TCR clones respond to the personalized RNA vaccine but do not respond to the cancer tumor being treated, as the SE TCR clones did not (or only minimally) proliferate clonally before administration of a personalized RNA vaccine targeting the cancer tumor.

[0351] In other embodiments, the SE TCR clone is a de novo SE TCR clone and was not detected in the pre-treatment sample, i.e., the TCR clone arose after the start of administration of the personalized RNA vaccine, thereby indicating that the TCR clone specifically arose as a result of the treatment.

[0352] Determining the number and / or frequency of de novo SE TCR clones In the present disclosure, measurements of de novo SE TCR clones include the number and / or frequency of de novo SE TCR clones. The number of TCR clones is a quantification of the number of unique TCR clones present in a sample. The frequency of a TCR clone is a quantification of the abundance of T cells expressing a particular TCR clone.

[0353] As described in the section above, the frequency of each TCR clone can be compared to the TCR clones at the pre-treatment baseline to identify which TCR clones have significantly expanded.TCR clones that have significantly expanded but were not present before treatment can then be identified and quantified to identify the number of de novo SE TCR clones.The number of de novo SE TCR clones can then be compared to a reference level, as described in more detail below.

[0354] The method for quantifying the frequency of a TCR clone depends on the technique used to measure the TCR clone, such as bulk TCR sequencing (bulkTCRseq) or single-cell TCR sequencing (scTCR-seq), as described below. In some cases, the frequency of a TCR clone is calculated as the percentage or proportion of T cells expressing a particular TCR clone divided by the total number of T cells in the sample. In other examples, the frequency of a TCR clone is quantified as the number of mRNA reads for a particular TCR clone divided by the total number of TCR read counts in the sample. TCR clones with higher frequencies represent clonally expanded T cells, as described above.

[0355] The presence, number, and frequency of each TCR clone can be assessed using high-throughput sequencing, such as T cell receptor sequencing (TCR-seq), including bulk TCR-seq or single-cell TCR-seq (scTCR-seq), including, but not limited to, 10X Chromium Single Cell 5' Sequencing with V(D)J Enrichment or Single-Cell Immune Profiling. TCR-seq can identify the presence and sequence of each TCR clone present in a sample, e.g., a patient sample, such as a cancer patient sample. Single-cell TCR-seq provides both TCR messenger RNA (mRNA) expression and TCR clone frequency in the same assay. In some embodiments, TCR clones are identified by nucleic acid sequencing, e.g., transcriptional gene analysis. In some embodiments, TCR clones are identified by proteomic immune profiling analysis.

[0356] A sample containing T cells is collected from an individual, e.g., a patient, such as a cancer patient. The sample may be obtained from any tissue, organ, biopsy, or bodily fluid in which T cells are present. For example, the sample may be obtained from blood, lymph, bone marrow, spleen, lymph nodes (such as peripheral lymph nodes of a tumor), cerebrospinal fluid, tonsils, tumor biopsy, etc. In some embodiments, the sample is obtained from blood. In some embodiments, peripheral blood mononuclear cells (PBMCs) are isolated from a blood sample. In some embodiments, T cells are enriched from the isolated PBMCs. In some embodiments, the enriched T cells are CD4+ T cells. In some embodiments, the enriched T cells are CD8+ T cells. In some embodiments, the enriched T cells are CD3+ T cells, and the CD3+ T cell population includes both CD4+ T cells and CD8+ T cells.

[0357] In some embodiments, the individual is a cancer patient, such as a human cancer patient. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), melanoma, renal cell carcinoma (RCC), breast cancer, colorectal cancer (CRC), ovarian cancer, prostate cancer, urinary bladder cancer (UBC), cervical cancer, bone cancer, head and neck squamous cell carcinoma (HNSCC), and pancreatic cancer. In some embodiments, the cancer tumor is a pancreatic cancer tumor. In some embodiments, the cancer tumor is a pancreatic ductal adenocarcinoma (PDAC) tumor. In some embodiments, at least five neoepitopes resulting from cancer-specific somatic mutations are present in a tumor specimen obtained from a human cancer patient before administration of the personalized RNA vaccine.

[0358] A sample can be obtained from an individual (e.g., a cancer patient) at any time during and / or after treatment. A reference sample is obtained from the individual before administration of treatment to establish a baseline value, e.g., a baseline number and / or frequency of TCR clones. The sample is then analyzed for the number and / or frequency of TCR clones, e.g., SE TCR clones, particularly de novo SE TCR clones, as described herein. In some embodiments, the sample is obtained after one, two, three, four, five, six, seven, eight, nine, ten, or more administrations of a personalized RNA vaccine described herein. In some embodiments, the sample is obtained after two, three, six, eight, nine, and / or ten administrations of a personalized RNA vaccine. In some embodiments, the sample is obtained after six administrations of the personalized RNA vaccine.

[0359] In some embodiments, the sample is obtained after the end of treatment. In some embodiments, the sample is obtained about 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks after the end of treatment. , obtained either 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or later.

[0360] In some embodiments, the number and / or frequency of TCR clones is measured before initiation of treatment with a personalized cancer vaccine. In some embodiments, the number and / or frequency of de novo SE TCR clones is measured after one, two, three, four, five, six, seven, eight, nine, ten, or more doses of a personalized cancer vaccine. In some embodiments, the number and / or frequency of de novo SE TCR clones is measured after six doses of a personalized cancer vaccine.

[0361] In some embodiments, the number and / or frequency of de novo SE TCR clones is measured after completion of treatment with a personalized cancer vaccine. In some embodiments, the number and / or frequency of de novo SE TCR clones is measured 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or 14 weeks after completion of treatment. Measured at any of the following times: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or later.

[0362] Comparison of de novo SE TCR clone measurements with reference levels The number of T cell clones or clones and the frequency of each T cell clone or clone in a patient treated with a personalized RNA vaccine of the present disclosure are compared with a reference value or level (e.g., a reference number or reference frequency). This comparison is performed to detect the patient's immune response or immunogenicity to the personalized RNA vaccine. In some embodiments, the reference level, e.g., the reference number or reference frequency, can be determined in a reference sample obtained from the patient before administration of the personalized RNA vaccine. In certain embodiments, the reference sample obtained from the patient before administration of the personalized RNA vaccine is a baseline sample. In certain embodiments, the reference level can be statistically calculated or set so as to be determined from the overall distribution of values ​​in reference samples from cancer patients, for example, pancreatic cancer patients, for example, pancreatic ductal adenocarcinoma patients.

[0363] The reference number and / or reference frequency of de novo SE TCR clones can define a cutoff whereby a number or frequency of de novo SE TCR clones above that cutoff correlates with an increased likelihood that a patient will exhibit an immune response to the personalized RNA vaccine as a single agent or in combination with checkpoint blockade, as defined in this disclosure, indicative of improved or otherwise successful cancer treatment, as determined by computational analysis.

[0364] In one embodiment, the reference level is set as a cutoff value, e.g., such that the cutoff value indicates a value at which the predictive rate of immunogenicity and / or immune response to a therapy described herein reaches about 100% specificity and about 80% sensitivity. In some embodiments, the specificity may reach any of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the sensitivity may reach any of about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. In some embodiments, the sensitivity may reach about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% or higher.

[0365] In some embodiments, the reference number is about 2 to about 15 SE TCR clones. In some embodiments, the reference number is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 SE TCR clones. In some embodiments, the reference number is 6 SE TCR clones.

[0366] In some embodiments, the reference number is about 2 to about 15 de novo SE TCR clones. In some embodiments, the reference number is about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 de novo SE TCR clones. In some embodiments, the reference number is 6 de novo SE TCR clones.

[0367] In some embodiments, the reference frequency is about 10 -2 ~about 10 -6 In some embodiments, the reference frequency is about 10 -2 , 10 -3 , 10 -4 , 10 -5 or 10 -6In some embodiments, the reference frequency is about 10 -4 These are de novo SE TCR clones.

[0368] Selecting patients more likely to respond to therapy, including personalized RNA vaccines In some embodiments, the number and / or frequency of de novo SE TCR clones in a patient sample that exceeds the reference number and / or reference frequency indicates that the patient is more likely to respond to a therapy that includes a personalized RNA vaccine. The number and / or frequency of de novo SE TCR clones correlates with the immunogenic response or immunogenicity to the personalized RNA vaccine. An increased immunogenic response or immunogenicity to the personalized RNA vaccine correlates with and indicates an increased treatment responsiveness.

[0369] With access to the TCR repertoire (e.g., number and / or frequency of de novo SE TCR clones) in peripheral blood at baseline (i.e., before treatment) and after initiation of treatment administration, a predictive model can detect an immune response to a personalized RNA vaccine based on the number of de novo SE TCR clones. Machine learning approaches can further improve the predictive algorithm. In some embodiments, patients are selected as likely to respond to a therapy comprising a personalized RNA vaccine using a predictive model. In some embodiments, patients are selected as likely to respond to a therapy comprising a personalized RNA vaccine using a computer-based predictive model. In some embodiments, patients are selected as likely to respond to a therapy comprising a personalized RNA vaccine using machine learning. In some embodiments, patients are selected as likely to respond to a therapy comprising a personalized RNA vaccine based on the number and / or frequency of SE TCR clones. In some embodiments, patients are selected as likely to respond to a therapy comprising a personalized RNA vaccine based on the number and / or frequency of de novo SE TCR clones. In some embodiments, patients are selected as likely to respond to a therapy comprising a personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones is greater than a reference number and / or reference frequency.

[0370] In some embodiments, patients are selected as likely to respond to a therapy comprising a personalized RNA vaccine if the number of SE TCR clones is greater than about any of 3, 4, 5, 6, 7, 8, 9, 10 or more SE TCR clones. In some embodiments, patients are selected as likely to respond to a therapy comprising a personalized RNA vaccine if the number of de novo SE TCR clones is greater than about any of 3, 4, 5, 6, 7, 8, 9, 10 or more de novo SE TCR clones. In some embodiments, patients are selected as likely to respond to a therapy comprising a personalized RNA vaccine if the number of de novo SE TCR clones is greater than 6 de novo SE TCR clones.

[0371] In some embodiments, the patient has a frequency of de novo SE TCR clones of about 10 -6 , 10 -5 , 10 -4 , 10 -3 or 10 -2 In some embodiments, a patient is selected as likely to respond to a therapy comprising a personalized RNA vaccine if the frequency of the de novo SE TCR clone is higher than 10 -4 De novo SE TCR clones are selected as more likely to respond to therapy, including personalized RNA vaccines, if they are more likely to respond to therapies.

[0372] Other embodiments Also provided herein is a method of treating a patient (such as a human patient) having a cancer tumor, the method comprising: a) measuring the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from the patient by T cell receptor sequencing; b) comparing the number and / or frequency of de novo SE TCR clones measured in a) with a reference number and / or reference frequency; and c) selecting a patient who is more likely to respond to a therapy comprising a personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient exceeds the reference number and / or reference frequency, wherein the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient, and wherein a number and / or frequency of de novo SE TCR clones that exceeds the reference number and / or reference frequency indicates that the patient is more likely to respond to a therapy comprising the personalized RNA vaccine.

[0373] In another aspect, a method of treating a human patient having a cancer tumor is provided, comprising: a) comparing the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from the patient with a reference number and / or reference frequency; and b) selecting a patient who is more likely to respond to a therapy comprising a personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient exceeds the reference number and / or reference frequency, thereby treating the cancer tumor, wherein the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient, and the number and / or frequency of de novo SE TCR clones is measured by T cell receptor sequencing, and a number and / or frequency of de novo SE TCR clones that exceeds the reference number and / or reference frequency indicates that the patient is more likely to respond to a therapy comprising the personalized RNA vaccine.

[0374] In some embodiments, the method further comprises administering to the patient a therapy comprising a personalized RNA vaccine, thereby treating the cancer tumor, when the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency. In some embodiments, the method further comprises selecting a therapy comprising a personalized RNA vaccine, thereby treating the cancer tumor, when the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency.

[0375] In some embodiments, the cancer tumor is a pancreatic cancer tumor. In some embodiments, the cancer tumor is a pancreatic ductal adenocarcinoma (PDAC) tumor.

[0376] In some embodiments, the therapy comprising the personalized RNA vaccine further comprises a PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist is atezolizumab.

[0377] In some embodiments, the therapy further comprises chemotherapy treatment, wherein the RNA vaccine, the PD-1 axis-binding antagonist, and the chemotherapy treatment are administered to the patient during a priming phase, during a chemotherapy phase after the priming phase, and during a boost phase after the chemotherapy phase, wherein (i) the priming phase comprises administering the RNA vaccine at least once and administering the PD-1 axis-binding antagonist at least once to the patient, (ii) the chemotherapy phase comprises administering the chemotherapy treatment to the patient, and (iii) the boost phase comprises administering the RNA vaccine at least once and administering the PD-1 axis-binding antagonist at least once to the patient. In some embodiments, the chemotherapy treatment is FOLFIRINOX treatment or mFOLFIRINOX treatment.

[0378] In some embodiments of the treatment methods described in the section above, prior to the administering step, the patient is selected by the patient selection method described above.

[0379] In some embodiments, the steps of the method are performed by a single entity. In other embodiments, individual steps may be performed by different entities. For example, the step of determining the number and / or frequency of de novo SE TCR clones may be performed by an entity different from the entity that performs the comparison and selection steps described above.

[0380] The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description, and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. [Example]

[0381] The present disclosure will be more fully understood by reference to the following examples, which, however, should not be construed as limiting the scope of the present invention. The examples and embodiments described herein are for illustrative purposes only, and it is understood that various modifications or changes thereto will be suggested to those skilled in the art and will be included within the spirit and scope of this application and the appended claims.

[0382] Example 1: A Phase II, Open-Label, Multicenter, Randomized Study of the Efficacy and Safety of a Personalized Cancer Vaccine Plus Atezolizumab and mFOLFIRINOX Versus mFOLFIRINOX Alone in Patients with Resected Pancreatic Ductal Adenocarcinoma This example describes a phase II, open-label, multicenter, randomized trial evaluating the efficacy and safety of a personalized cancer vaccine plus atezolizumab and modified leucovorin, 5-fluorouracil (5-FU), irinotecan, and oxaliplatin (mFOLFIRINOX) compared with mFOLFIRINOX in patients with resected PDAC who had not received prior systemic anticancer treatment for PDAC and had no evidence of disease after surgery. This study aims to identify a more effective adjuvant therapy for PDAC, because the majority of patients who receive the current standard of care adjuvant therapy, gemcitabine combination therapy or mFOLFIRINOX, after PDAC resection experience disease recurrence and death.

[0383] Test Purpose The objective of this study was to evaluate the efficacy and safety of a personalized cancer vaccine plus atezolizumab and mFOLFIRINOX versus mFOLFIRINOX in patients with resected PDAC. Test Design

[0384] The study will enroll approximately 260 patients at approximately 80 sites worldwide. As shown in Figure 1, the Phase II trial includes: i) a two-part screening period (Part A and Part B); ii) a treatment period consisting of one or three phases (priming, chemotherapy, and boost), depending on the treatment arm; and iii) a follow-up period. The total duration of study participation for each patient is expected to range from 1 day to more than 6 years.

[0385] Screening will be conducted in two parts, referred to as Part A and Part B. During Part A, blood and tumor tissue specimens will be tested to determine the presence of at least five neoepitopes, enabling the creation of a personalized cancer vaccine for each patient. Patients will further undergo limited screening and medical history review for eligibility. During Screening Part B, patient eligibility will be confirmed, including assessments collected within 28 days and 14 days of enrollment. Patients will be randomized to Arm 1 or Arm 2 using a stratified permuted block randomization scheme to achieve an approximately 1:1 ratio between the two treatment arms. Randomization will be stratified by surgical margin status (R0 vs. R1) and lymph node involvement (N0 vs. N+). Study treatment will begin within 7 days of randomization.

[0386] As shown in Figures 2A-2B, two alternative dosing regimens, Dosing Regimen A and Dosing Regimen B, are used in this study.

[0387] An overview of dosing regimen A for arms 1 and 2 is shown in Figure 2A. Patients in the experimental arm (arm 1) will receive personalized cancer vaccine, atezolizumab, and mFOLFIRINOX over three treatment phases, with the following approximate timing: Priming period (weeks 1-6) Individualized cancer vaccine 25 μg IV in 7-day cycles starting on day 1 of week 1 for a total of 6 cycles (6 doses). Atezolizumab 1680 mg IV on day 1 of week 1 and day 1 of week 5 for a total of two doses. Chemotherapy period (weeks 7-29) mFOLFIRINOX (oxaliplatin 85mg / m 2 , leucovorin 400 mg / m 2 , irinotecan 150 mg / m 2 , 5-FU 2400 mg / m 2 ) IV in 14-day cycles starting on Day 1 of Week 7 for a maximum of 12 cycles (12 doses). Boost period (weeks 33-53) Individualized cancer vaccine 25 μg IV in 28-day cycles starting on day 1 of week 33 for a total of 6 cycles (6 doses). Atezolizumab 1680 mg IV in 28-day cycles starting on day 1 of week 33 for a total of 6 cycles (6 doses).

[0388] An overview of dosing regimen B for arms 1 and 2 is shown in Figure 2B. The chemotherapy and boost phases of regimen B are the same as the corresponding phases in regimen A. The priming phase of regimen B is modified as follows: Priming period (weeks 1-6) Individualized cancer vaccine 25 μg IV administered in 7-day cycles (i.e., once weekly, QW) starting on day 1 of week 1 for a total of 6 doses. Atezolizumab 1680 mg IV given once on day 1 of week 3.

[0389] The priming phase of Regimen B was designed to increase the immunogenicity and clinical activity of the personalized cancer vaccine when administered in combination with atezolizumab. Biomarker samples will be collected from patients who have received cevumeran but have not yet received atezolizumab.

[0390] ...

Claims

1. 1. A method for treating a pancreatic cancer tumor in a human patient in need thereof, comprising: (a) a personalized RNA vaccine comprising one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from said patient; (b) a PD-1 axis binding antagonist, and (c) chemotherapy treatment to said patient, the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment are administered to the patient during a priming phase, during a chemotherapy phase after the priming phase, and during a boost phase after the chemotherapy phase; (i) the priming phase comprises at least one administration of the RNA vaccine and at least one administration of the PD-1 axis binding antagonist to the patient; (ii) the chemotherapy phase comprises administering the chemotherapy treatment to the patient; and (iii) the boost phase comprises at least one administration of the RNA vaccine and at least one administration of the PD-1 axis binding antagonist to the patient.

2. 10. The method of claim 1, wherein the pancreatic cancer tumor is a pancreatic ductal adenocarcinoma (PDAC) tumor.

3. 3. The method of claim 1 or claim 2, wherein the pancreatic cancer tumor is resectable.

4. 4. The method of claim 3, wherein the priming phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks after resection of the pancreatic cancer tumor from the patient.

5. 4. The method of claim 3, wherein the priming phase begins about 6 weeks to about 12 weeks after resection of the pancreatic cancer tumor from the patient.

6. 6. The method of any one of claims 1 to 5, wherein the priming phase comprises a single administration of the PD-1 axis binding antagonist.

7. 7. The method of claim 6, wherein the priming phase comprises administering the PD-1 axis binding antagonist on day 1 of week 3 of the priming phase.

8. 6. The method of any one of claims 1 to 5, wherein the priming phase comprises at least two administrations of the PD-1 axis binding antagonist.

9. 9. The method of any one of claims 1-5 and 8, wherein the priming phase comprises administering the PD-1 axis binding antagonist once every four weeks.

10. 10. The method of claim 9, wherein the priming phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the priming phase, and every four weeks thereafter.

11. 11. The method of any one of claims 1-5 and 8-10, wherein the priming phase comprises two administrations of the PD-1 axis binding antagonist.

12. 12. The method of claim 11, wherein the priming phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 and day 1 of week 5 of the priming phase.

13. 13. The method of any one of claims 1 to 12, wherein the priming phase comprises 2, 3, 4, 5, 6, 7, or 8 administrations of the RNA vaccine.

14. 14. The method of claim 13, wherein the priming phase comprises two or three administrations of the RNA vaccine.

15. 14. The method of any one of claims 1 to 13, wherein the priming phase comprises 6 to 8 administrations of the RNA vaccine, or up to 6 doses of the RNA vaccine.

16. 16. The method of claim 15, wherein the priming phase comprises six administrations of the RNA vaccine.

17. The method of any one of claims 1 to 16, wherein the priming phase comprises administering the RNA vaccine once a week.

18. 18. The method of claim 17, wherein the priming phase comprises administering the RNA vaccine on day 1 of week 1 of the priming phase, and once weekly thereafter.

19. The method of any one of claims 1 to 18, wherein the priming phase comprises six administrations of the RNA vaccine.

20. 20. The method of claim 19, wherein the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase.

21. 21. The method of any one of claims 1-20, wherein each dose of the PD-1 axis binding antagonist administered to the patient during the priming phase occurs on the same day as administration of the RNA vaccine.

22. 22. The method of any one of claims 1 to 21, wherein the priming phase consists of 6 weeks.

23. 23. The method of claim 22, wherein the RNA vaccine is administered on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase, and the PD-1 axis binding antagonist is administered on day 1 of week 3 of the priming phase.

24. 23. The method of claim 22, wherein the RNA vaccine is administered on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase, and the PD-1 axis binding antagonist is administered on day 1 of weeks 1 and 5 of the priming phase.

25. 25. The method of any one of claims 1-24, wherein the chemotherapy phase comprises administering the chemotherapy treatment for at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks, at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, or more.

26. 26. The method of any one of claims 1 to 25, wherein the chemotherapy phase comprises administering the chemotherapy treatment for 23 weeks.

27. The method of any one of claims 1 to 26, wherein said chemotherapy treatment is administered once every two weeks.

28. 28. The method of claim 27, wherein the chemotherapy phase comprises administering the chemotherapy treatment on day 1 of week 1 of the chemotherapy phase, and every two weeks thereafter.

29. 29. The method of any one of claims 1-28, wherein the chemotherapy phase comprises administering the chemotherapy treatment 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, or at least 24 or more times.

30. 30. The method of claim 29, wherein the chemotherapy phase comprises 12 administrations of the chemotherapy treatment.

31. The method of any one of claims 1 to 30, wherein the chemotherapy phase consists of 24 weeks.

32. 32. The method of any one of claims 1-31, wherein the chemotherapy phase comprises administering the chemotherapy treatment on day 1 of weeks 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 of the chemotherapy phase.

33. 33. The method of any one of claims 1-32, wherein the chemotherapy phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks after the priming phase ends and after the last administration of the RNA vaccine.

34. 33. The method of any one of claims 1 to 32, wherein the chemotherapy phase begins within 9 weeks from the first week of the priming phase.

35. 35. The method of claim 34, wherein the priming phase consists of 6 weeks and the chemotherapy phase begins within 9 weeks of the priming phase.

36. 36. The method of any one of claims 1-35, wherein the priming phase consists of 6 weeks and the chemotherapy phase comprises administering the chemotherapy treatment starting on day 1 of week 7 of the priming phase, starting on week 1, and every two weeks thereafter.

37. 37. The method of claim 36, wherein the chemotherapy phase comprises 12 administrations of the chemotherapy treatment.

38. 38. The method of claim 37, wherein said chemotherapy phase comprises administering said chemotherapy treatment on day 1 of weeks 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, beginning with week 1 of said priming phase.

39. 39. The method of any one of claims 1 to 38, wherein the boost phase comprises 2, 3, 4, 5, 6, 7 or 8 administrations of the RNA vaccine.

40. 40. The method of any one of claims 1-39, wherein the boost phase comprises 2, 3, 4, 5, 6, 7, or 8 administrations of the PD-1 axis binding antagonist.

41. 41. The method of any one of claims 1-40, wherein the boost phase comprises six administrations of the PD-1 axis binding antagonist and six administrations of the RNA vaccine.

42. 42. The method of any one of claims 1-41, wherein the boost phase comprises administering the PD-1 axis binding antagonist and the RNA vaccine once every four weeks.

43. 43. The method of any one of claims 1-42, wherein the boost phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the boost phase, and every four weeks thereafter.

44. 44. The method of any one of claims 1-43, wherein the boost phase comprises administering the RNA vaccine on day 1 of week 1 of the boost phase and every four weeks thereafter.

45. 45. The method of any one of claims 1-44, wherein administration of the RNA vaccine and the PD-1 axis binding antagonist during the boost phase occurs on the same day.

46. 46. ​​The method of claim 45, wherein the boost phase comprises administering the PD-1 axis binding antagonist and the RNA vaccine on day 1 of week 1 of the boost phase, and every four weeks thereafter.

47. 47. The method of any one of claims 1 to 46, wherein the boost phase consists of 21 weeks.

48. 48. The method of any one of claims 1-47, wherein the RNA vaccine and the PD-1 axis binding antagonist are administered on day 1 of weeks 1, 5, 9, 13, 17, and 21 of the boost phase.

49. 49. The method of any one of claims 1-48, wherein the boost phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks after the chemotherapy phase has ended.

50. 49. The method of any one of claims 1-48, wherein the boost phase begins up to about 12 weeks after the chemotherapy phase ends, optionally up to about 12 weeks after the last administration of the chemotherapy treatment.

51. The boost period is about 3 weeks to about 12 weeks after the chemotherapy phase has ended, optionally about 3 weeks to about 12 weeks after the last administration of the chemotherapy treatment; or 49. The method of any one of claims 1 to 48, wherein the method begins about 3 weeks or about 4 weeks after the chemotherapy phase has ended, optionally about 3 weeks or about 4 weeks after the last administration of the chemotherapy treatment.

52. 49. The method of any one of claims 1 to 48, wherein the boost phase begins at week 27 of the chemotherapy phase, starting at week 1.

53. 49. The method of any one of claims 1 to 48, wherein the boost phase begins at week 33 from week 1 of the priming phase.

54. 54. The method of claim 53, wherein the boost phase comprises administering the RNA vaccine and the PD-1 axis binding antagonist on day 1 of week 33 of the priming phase, and every four weeks thereafter.

55. 55. The method of claim 54, wherein the RNA vaccine and the PD-1 axis binding antagonist are administered six times during the boost phase.

56. 56. The method of claim 55, wherein the boost phase comprises administering the RNA vaccine and the PD-1 axis binding antagonist on day 1 of weeks 33, 37, 41, 45, 49, and 53 starting from week 1 of the priming phase.

57. (a) the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase, and administering the PD-1 axis binding antagonist on day 1 of week 3 of the priming phase; (b) the chemotherapy phase comprises administering the chemotherapy treatment on day 1 of weeks 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, beginning with week 1 of the priming phase; (c) the boost phase comprises administering the RNA vaccine and the PD-1 axis binding antagonist on day 1 of weeks 33, 37, 41, 45, 49, and 53 starting from week 1 of the priming phase; The method of any one of claims 1 to 7, 13 to 23, and 25 to 56.

58. (a) the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, and 6 of the priming phase, and administering the PD-1 axis binding antagonist on day 1 of weeks 1 and 5 of the priming phase; (b) the chemotherapy phase comprises administering the chemotherapy treatment on day 1 of weeks 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, beginning with week 1 of the priming phase; (c) the boost phase comprises administering the RNA vaccine and the PD-1 axis binding antagonist on day 1 of weeks 33, 37, 41, 45, 49, and 53 starting from week 1 of the priming phase; The method of any one of claims 1 to 5, 8 to 22, and 24 to 56.

59. 59. The method of claim 57 or claim 58, wherein the priming phase begins about 6 weeks to about 12 weeks after resection of the pancreatic cancer tumor from the patient.

60. 60. The method of any one of claims 1 to 59, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

61. 61. The method of claim 60, wherein the PD-1 binding antagonist is an anti-PD-1 antibody.

62. 62. The method of claim 61, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.

63. 60. The method of any one of claims 1 to 59, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

64. 64. The method of claim 63, wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.

65. 65. The method of claim 64, wherein the anti-PD-L1 antibody is avelumab or durvalumab.

66. the anti-PD-L1 antibody, (a) a heavy chain variable region (VH) comprising an HVR-H1 comprising the amino acid sequence GFTFSDSWIH (SEQ ID NO: 1), an HVR-H2 comprising the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 2), and an HVR-H3 comprising the amino acid sequence RHWPGGFDY (SEQ ID NO: 3); and (b) a light chain variable region (VL) comprising an HVR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO: 4), an HVR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO: 5), and an HVR-L3 comprising the amino acid sequence QQYLYHPAT (SEQ ID NO: 6); 65. The method of claim 64, comprising:

67. The anti-PD-L1 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:

7. Ｈ ) and a light chain variable region (V Ｌ 65. The method of claim 64, comprising:

68. 65. The method of claim 64, wherein the anti-PD-L1 antibody is atezolizumab.

69. 69. The method of any one of claims 1-68, wherein the PD-1 axis binding antagonist is administered to the patient intravenously.

70. 70. The method of any one of claims 64-69, wherein the anti-PD-L1 antibody is administered to the patient at a dose of about 1200 mg or about 1680 mg.

71. 71. The method of claim 70, wherein the anti-PD-L1 antibody is atezolizumab, and the atezolizumab is administered intravenously to the patient at a dose of about 1680 mg.

72. 72. The method of any one of claims 1-71, wherein the chemotherapy treatment comprises one or more of gemcitabine, leucovorin, 5-fluorouracil, capecitabine, irinotecan, liposomal irinotecan, platinum-based chemotherapy agents, taxanes, and any combination thereof.

73. 73. The method of claim 72, wherein the platinum-based chemotherapeutic agent is cisplatin, oxaliplatin, or both.

74. 74. The method of claim 72 or claim 73, wherein the taxane is paclitaxel, docetaxel, albumin-bound paclitaxel, or any combination thereof.

75. 73. The method of any one of claims 1 to 72, wherein the chemotherapy treatment comprises leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin.

76. 73. The method of any one of claims 1 to 72, wherein the chemotherapy treatment is a FOLFIRINOX treatment or mFOLFIRINOX treatment.

77. the chemotherapy treatment comprising: Approximately 85mg / m ２ oxaliplatin at a dose of; Approximately 400mg / m ２ Leucovorin at a dose of; Approximately 150mg / m ２ and / or irinotecan at a dose of Approximately 2400mg / m ２ 5-fluorouracil at a dose of 73. The method of any one of claims 1 to 72, comprising:

78. 78. The method of any one of claims 1 to 77, wherein said chemotherapy treatment is administered to said patient intravenously.

79. 79. The method of any one of claims 1-78, wherein the RNA vaccine comprises one or more polynucleotides encoding 5 to 20 or 10 to 20 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen.

80. 80. The method of any one of claims 1 to 79, wherein the one or more polynucleotides of the RNA vaccine are formulated with one or more lipids.

81. 81. The method of claim 80, wherein the one or more polynucleotides and the one or more lipids of the RNA vaccine form lipid nanoparticles.

82. 81. The method of claim 80, wherein the one or more polynucleotides and the one or more lipids of the RNA vaccine form a lipoplex.

83. 83. The method of claim 81 or 82, wherein the lipid nanoparticle or lipoplex comprises one or more lipids that form a multi-membrane structure that encapsulates the one or more polynucleotides of the RNA vaccine.

84. 84. The method of claim 83, wherein the one or more lipids comprise at least one cationic lipid and at least one helper lipid.

85. 84. The method of claim 83, wherein the one or more lipids comprise (R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propanaminium chloride (DOTMA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

86. 86. The method of claim 85, wherein the lipid nanoparticle or lipoplex has an overall positive to negative charge ratio of 1.3:2 (0.65) at physiological pH.

87. 87. The method of any one of claims 1 to 86, wherein the one or more polynucleotides of the RNA vaccine are RNA molecules, optionally messenger RNA molecules.

88. 88. The method of any one of claims 1-87, wherein the RNA vaccine is administered to the patient at a dose of about 15 μg, about 21 μg, about 21.3 μg, about 25 μg, about 38 μg, or about 50 μg.

89. 89. The method of claim 88, wherein the RNA vaccine is administered to the patient at a dose of about 25 μg.

90. 90. The method of any one of claims 1 to 89, wherein the RNA vaccine is administered to the patient intravenously.

91. The RNA vaccine is (1) 5' cap, (2) 5' untranslated region (UTR), (3) a polynucleotide sequence encoding a secretory signal peptide; (4) a polynucleotide sequence encoding the one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen; (5) a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule; （６） (a) the 3' untranslated region of an Amino-Terminal Enhancer of Split (AES) mRNA or a fragment thereof; and (b) a non-coding RNA of mitochondrially encoded 12S RNA or a fragment thereof and a 3'UTR comprising (7) poly(A) sequence 91. The method of any one of claims 1 to 90, comprising an RNA molecule comprising:

92. 92. The method of claim 91, wherein the RNA molecule further comprises a polynucleotide sequence encoding an amino acid linker, wherein the polynucleotide sequence encoding the amino acid linker and a first of the one or more neoepitopes form a first linker-neoepitope module, and wherein the polynucleotide sequence forming the first linker-neoepitope module is located in a 5'3' direction between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

93. 93. The method of claim 92, wherein the amino acid linker comprises the sequence GGSGGGGGSGG (SEQ ID NO: 39).

94. 93. The method of claim 92, wherein the polynucleotide sequence encoding the amino acid linker comprises the sequence GGCGGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO: 37).

95. 95. The method of any one of claims 92 to 94, wherein the RNA molecule further comprises, in a 5' to 3' direction, at least a second linker-neoepitope module, the at least second linker-neoepitope module comprising a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope, the polynucleotide sequence forming the second linker-neoepitope module being located, in a 5' to 3' direction, between the polynucleotide sequence encoding the neoepitope of the first linker-neoepitope module and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule, and the neoepitope of the first linker-neoepitope module is different from the neoepitope of the second linker-neoepitope module.

96. 96. The method of claim 95, wherein the RNA molecule comprises five linker-neoepitope modules, each of the five linker-neoepitope modules encoding a different neoepitope.

97. 96. The method of claim 95, wherein the RNA molecule comprises 10 linker-neoepitope modules, each of the 10 linker-neoepitope modules encoding a different neoepitope.

98. 96. The method of claim 95, wherein the RNA molecule comprises 20 linker-neoepitope modules, each of the 20 linker-neoepitope modules encoding a different neoepitope.

99. 99. The method of any one of claims 91-98, wherein the RNA molecule further comprises a second polynucleotide sequence encoding an amino acid linker, the second polynucleotide sequence encoding the amino acid linker being between the polynucleotide sequence encoding the 3'-most distal neoepitope and the polynucleotide sequence encoding the at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

100. The 5' cap has the structure: 【Chemistry 1】 The method of any one of claims 91 to 99, comprising the D1 diastereoisomer of

101. 101. The method of any one of claims 91 to 100, wherein the 5'UTR comprises the sequence UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 23).

102. 101. The method of any one of claims 91 to 100, wherein the 5'UTR comprises the sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 21).

103. 103. The method of any one of claims 91 to 102, wherein the secretory signal peptide comprises the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 27).

104. 103. The method of any one of claims 91 to 102, wherein the polynucleotide sequence encoding the secretory signal peptide comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 25).

105. 105. The method of any one of claims 91-104, wherein the at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule comprises the amino acid sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 30).

106. 105. The method of any one of claims 91-104, wherein the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule comprises the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC (SEQ ID NO: 28).

107. 107. The method of any one of claims 91 to 106, wherein the 3' untranslated region of the AES mRNA comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCCUGGGUACCCCGAGUCUCCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO: 33).

108. 108. The method of any one of claims 91-107, wherein the non-coding RNA of the mitochondrially encoded 12S RNA comprises the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO: 35).

109. The 3'UTR has the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGGUACCCCGAGUCUCCCCCGACCUCGGG UCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCUCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAACG CUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 31).

110. 110. The method of any one of claims 91 to 109, wherein the poly(A) sequence comprises 120 adenine nucleotides.

111. The RNA vaccine is Polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCACAGACUCAGAGAGAGAACCCGCCACCAUGAGAGUGAUGGGCCCCC AGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCCGGAAGC (SEQ ID NO: 19), a polynucleotide sequence encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in said tumor specimen; and Polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGC UACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAAGGGCGGCAGCUACAGCCAGGCCGCCA GCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUG CAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGGUACCCCGAGUCUCCCCCGACCUCCG GGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACC UCCCAAGCACGCAGCAAUGCAGCUCAAACGCUUAGCCUAGCCACACCCCCCAGGGAAACAG CAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGU CAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 20) 91. The method of any one of claims 1 to 90, comprising an RNA molecule comprising:

112. 112. The method of any one of claims 1-111, wherein the pancreatic cancer tumor is a resectable PDAC tumor and is assessed by preoperative imaging in the patient using a computed tomography (CT) scan with contrast or magnetic resonance imaging (MRI) prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

113. the pancreatic cancer tumor is A distinct fat plane around the celiac trunk and superior mesenteric artery, Patent superior mesenteric vein and portal vein, No envelopment of the superior mesenteric vein or portal vein, No encirclement of the superior mesenteric artery or hepatic artery, absence of metastatic disease, and Absence of extraregional lymph node disease 113. The method of any one of claims 1 to 112, wherein the tumor is a resectable PDAC tumor comprising one or more characteristics selected from the group consisting of:

114. The method of any one of claims 1-113, wherein the patient has a histologically confirmed diagnosis of PDAC prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

115. The method of any one of claims 1 to 114, wherein the patient has adenosquamous carcinoma of the pancreas prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

116. 116. The method of any one of claims 1-115, wherein the pancreatic cancer tumor has a tumor, node, metastasis (TNM) pathological staging value of T1-T3, N0-N2, or M0 prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

117. the pancreatic cancer tumor is a resectable PDAC tumor; the patient had no evidence of PDAC disease after resection of the PDAC tumor, and / or the patient undergoes macroscopically complete resection of the PDAC tumor prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment; Optionally, the patient has undergone R0 or R1 resection of the PDAC tumor.

117. The method of any one of claims 1 to 116.

118. The method of claim 117, wherein the patient is demonstrably free of PDAC following resection of the PDAC tumor, optionally wherein the absence of PDAC is assessed by CT or MRI scan, one or more biochemical assays, and / or clinical findings.

119. The method of any one of claims 1-118, wherein the pancreatic cancer tumor is a resectable PDAC tumor, and after resection of the tumor, the patient has no unresolved post-operative complications of >= Grade 3 prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment, optionally wherein the complications are assessed according to the Clavien-Dindo Classification of Surgical Complications.

120. The method of any one of claims 1-119, wherein the patient has a CA19-9 level of 180 U / mL or greater prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

121. The method of any one of claims 1-119, wherein the patient has a CA19-9 level of less than 180 U / mL prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

122. 122. The method of any one of claims 1-121, wherein at least five neoepitopes resulting from cancer-specific somatic mutations are present in the tumor specimen obtained from the patient prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

123. The method of any one of claims 1-122, wherein the patient has an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 or 1 prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

124. The method of any one of claims 1-123, wherein the patient does not have intraductal papillary mucinous tumor-associated PDAC prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

125. The method of any one of claims 1 to 124, wherein the patient does not have pancreatic endocrine tumor or acinar cell adenocarcinoma, pancreatic cystadenocarcinoma, or pancreatic malignant squamous cell carcinoma prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

126. The method of any one of claims 1-125, wherein the patient has not received adjuvant, neoadjuvant, or induction treatment for pancreatic cancer, or systemic anti-cancer treatment for pancreatic cancer, prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment, and optionally, the pancreatic cancer is PDAC.

127. The method of any one of claims 1-126, wherein the patient has not received cytotoxic chemotherapy, immunotherapy, investigational therapy, or radiation therapy prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

128. The method of any one of claims 1 to 127, wherein the patient has a spleen prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

129. The method of any one of claims 1 to 128, wherein the patient does not have loss of a spleen due to splenectomy, splenic injury / infarction, or functional asplenia prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

130. The method of any one of claims 1 to 129, wherein the patient has not undergone distal pancreatectomy with splenectomy prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

131. The method of any one of claims 1-130, wherein the patient does not have a pre-existing neuropathy prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

132. The method of any one of claims 1-131, wherein the patient does not have an aUGT1A1 genotype associated with a poor metabolizer phenotype prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

133. The method of any one of claims 1-132, wherein the patient does not have an autoimmune disease, immunodeficiency, or primary immunodeficiency prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

134. the patient, prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment, Monoamine oxidase inhibitors (MAOIs) within 3 weeks, systemic immunostimulants within 4 weeks or 5 drug elimination half-lives, whichever is longer; or Systemic immunosuppressants within the last 2 weeks The method of any one of claims 1 to 133, wherein the patient has not been treated with

135. The method of any one of claims 1-134, wherein the patient has not undergone an allogeneic stem cell or solid organ transplant prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

136. The method of any one of claims 1-135, further comprising assessing the disease-free survival (DFS) of the patient after treatment with the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

137. 137. The method of claim 136, wherein administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment results in an improvement in DFS in the patient compared to a DFS in a corresponding patient who is not administered the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

138. The method of any one of claims 1-137, further comprising assessing the overall survival (OS) of the patient after treatment with the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

139. 139. The method of claim 138, wherein administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment results in an improvement in OS for the patient compared to the OS of a corresponding patient who is not administered the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

140. and performing one or more clinical assessments of the patient before, during, and / or after treatment with the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment, wherein the one or more clinical assessments are based on a clinical score determined using a European Organization for Research and Treatment of Cancer QLQ-C30 questionnaire (EORTC QLQ C30), a European Organization for Research and Treatment of Cancer QLQ-PAN26 questionnaire (EORTC QLQ PAN26), a National Cancer Institute's Common Terminology Criteria for Patient-Reported Outcomes Adverse Events (PRO CTCAE), a National Cancer Institute's Common Terminology Criteria for Adverse Events (NCCI ...

140. The method of any one of claims 1 to 139, wherein the questionnaire is selected from the group consisting of: European Organisation for Research and Treatment of Cancer Questionnaire Item Library 46 Questionnaire (EORTC IL46, European Organization for Research and Treatment of Cancer Item Library 46 Questionnaire).

141. The method of claim 140, wherein administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment results in an improvement in the one or more clinical assessments compared to the one or more clinical assessments in the patient prior to administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment, and / or compared to the one or more clinical assessments in a corresponding patient who has not been administered the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

142. The method of any one of claims 1-141, further comprising assessing antigen-specific and / or tumor-specific T cell responses in the patient before, during, and / or after treatment with the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

143. The method of claim 142, wherein administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment results in an improved antigen-specific and / or tumor-specific T cell response in the patient compared to before administration of the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment, and / or compared to a corresponding patient who has not been administered the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment.

144. The method of any one of claims 137, 139, 141 and 143, wherein the corresponding patient is a patient having a corresponding pancreatic cancer tumor, and optionally, the pancreatic cancer tumor is a PDAC tumor, and the corresponding patient has a PDAC tumor.

145. The method of any one of claims 137, 139, 141 and 143-144, wherein the corresponding patient has been treated with standard of care for pancreatic cancer, PDAC, or resectable or resected PDAC.

146. 146. The method of claim 145, wherein the standard of care comprises gemcitabine combination therapy or mFOLFIRINOX chemotherapy.

147. The method of any one of claims 137, 139, 141 and 143-144, wherein the corresponding patient was treated with a control treatment comprising mFOLFIRINOX chemotherapy.

148. The mFOLFIRINOX chemotherapy is administered at a dose of about 85 mg / m ２ oxaliplatin at a dose of about 400 mg / m ２ Leucovorin at a dose of about 150 mg / m ２ and about 2400 mg / m ２ administered intravenously on day 1 of each 14 day cycle for up to a total of 12 cycles.

149. 149. The method of any one of claims 1-148, wherein two equal half doses of the RNA vaccine are administered to the patient.

150. 150. The method of claim 149, wherein the two equal half doses are administered sequentially, optionally with an observation period between the administered equal half doses.

151. 90. The method of claim 89, wherein the dose of about 25 μg is divided into two equal halves of about 12.5 μg, each administered over 1 minute, optionally with a 5 minute observation period between the administered equal halves.

152. 152. A personalized RNA vaccine for use in a method for treating a pancreatic cancer tumor in a human patient in need thereof, wherein said RNA vaccine is administered in combination with a PD-1 axis binding antagonist and chemotherapy treatment according to the method of any one of claims 1-151; A personalized RNA vaccine, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient.

153. 152. A PD-1 axis binding antagonist for use in a method for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the PD-1 axis binding antagonist is administered in combination with a personalized RNA vaccine and chemotherapy treatment according to the method of any one of claims 1 to 151; The PD-1 axis binding antagonist, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient.

154. 152. Use of a personalized RNA vaccine in the manufacture of a medicament for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the RNA vaccine is administered in combination with a PD-1 axis binding antagonist and a chemotherapy treatment according to the method of any one of claims 1 to 151; The use, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient.

155. 152. Use of a PD-1 axis binding antagonist in the manufacture of a medicament for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the PD-1 axis binding antagonist is administered in combination with a personalized RNA vaccine and chemotherapy treatment according to the method of any one of claims 1 to 151; The use, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient.

156. 152. A kit comprising a personalized RNA vaccine for use in a method for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the RNA vaccine is administered in combination with a PD-1 axis binding antagonist and chemotherapy treatment according to the method of any one of claims 1-151; A kit, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient.

157. 152. A kit comprising a PD-1 axis binding antagonist for use in a method for treating a pancreatic cancer tumor in a human patient in need thereof, wherein the PD-1 axis binding antagonist is administered in combination with a personalized RNA vaccine and chemotherapy treatment according to the method of any one of claims 1-151; A kit, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a pancreatic cancer tumor specimen obtained from the patient.

158. 1. A method for selecting a human patient having a cancer tumor who is likely to respond to a therapy comprising a personalized RNA vaccine, comprising: a) determining the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from said patient by T cell receptor sequencing; b) comparing the number and / or frequency of de novo SE TCR clones determined in a) with a reference number and / or reference frequency; c) selecting the patient as more likely to respond to the therapy comprising the personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency; the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient; wherein a number and / or frequency of de novo SE TCR clones greater than said reference number and / or reference frequency indicates that said patient is more likely to respond to said therapy comprising said personalized RNA vaccine.

159. 1. A method for selecting a human patient having a cancer tumor who is likely to respond to a therapy comprising a personalized RNA vaccine, comprising: a) comparing the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from said patient with a reference number and / or reference frequency; b) selecting the patient as more likely to respond to the therapy comprising the personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency; the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient; the number and / or frequency of de novo SE TCR clones is determined by T cell receptor sequencing; wherein a number and / or frequency of de novo SE TCR clones greater than said reference number and / or reference frequency indicates that said patient is more likely to respond to said therapy comprising said personalized RNA vaccine.

160. The method of claim 158 or claim 159, further comprising selecting the therapy comprising the personalized RNA vaccine or recommending the therapy comprising the personalized RNA vaccine.

161. 1. A method of treating a human patient having a cancer tumor, comprising: a) determining the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from said patient by T cell receptor sequencing; b) comparing the number and / or frequency of de novo SE TCR clones determined in a) with a reference number and / or reference frequency; c) if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency, selecting the patient as more likely to respond to the therapy comprising a personalized RNA vaccine, thereby treating the cancer tumor; the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient; wherein a number and / or frequency of de novo SE TCR clones greater than said reference number and / or reference frequency indicates that said patient is more likely to respond to said therapy comprising said personalized RNA vaccine.

162. 1. A method of treating a human patient having a cancer tumor, comprising: a) comparing the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from said patient with a reference number and / or reference frequency; b) selecting the patient as more likely to respond to the therapy comprising a personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency, thereby treating the cancer tumor; the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient; the number and / or frequency of de novo SE TCR clones is determined by T cell receptor sequencing; wherein a number and / or frequency of de novo SE TCR clones greater than said reference number and / or reference frequency indicates that said patient is more likely to respond to said therapy comprising said personalized RNA vaccine.

163. The method of claim 161 or claim 162, further comprising administering the therapy comprising the personalized RNA vaccine to the patient if the number and / or frequency of de novo SE TCR clones in the sample from the patient exceeds the reference number and / or reference frequency, thereby treating the cancer tumor.

164. The method of claim 163, further comprising selecting the therapy comprising the personalized RNA vaccine, thereby treating the cancer tumor, if the number and / or frequency of de novo SE TCR clones in the sample from the patient exceeds the reference number and / or reference frequency.

165. 165. The method of any one of claims 158-164, wherein said number and / or frequency is measured after six administrations of said personalized cancer vaccine.

166. 166. The method of any one of claims 158 to 165, wherein the reference number is six de novo SE TCR clones.

167. The reference frequency is 10 －４ The method of any one of claims 158 to 165, wherein the TCR clone is a de novo SE TCR clone.

168. The method of any one of claims 158 to 167, wherein the cancer tumor is a pancreatic cancer tumor.

169. 169. The method of any one of claims 158 to 168, wherein the cancer tumor is a pancreatic ductal adenocarcinoma (PDAC) tumor.

170. The method of any one of claims 158-169, wherein the therapy comprising the personalized RNA vaccine further comprises a PD-1 axis binding antagonist.

171. the therapy further comprises chemotherapy treatment, and the RNA vaccine, the PD-1 axis binding antagonist, and the chemotherapy treatment are administered to the patient during a priming phase, a chemotherapy phase after the priming phase, and a boost phase after the chemotherapy phase; (i) the priming phase comprises at least one administration of the RNA vaccine and at least one administration of the PD-1 axis binding antagonist to the patient; (ii) the chemotherapy phase comprises administering the chemotherapy treatment to the patient; and (iii) the boost phase comprises at least one administration of the RNA vaccine and at least one administration of the PD-1 axis binding antagonist to the patient. The method of claim 170.

172. 172. The method of claim 170 or 171, wherein the PD-1 axis binding antagonist is atezolizumab.

173. 173. The method of claim 171 or 172, wherein the chemotherapy treatment is a FOLFIRINOX treatment or mFOLFIRINOX treatment.

174. Prior to the administering step, the patient is a) determining the number and / or frequency of significantly de novo expanded (SE) TCR clones in a sample from said patient by T cell receptor sequencing; b) comparing the number and / or frequency of de novo SE TCR clones determined in a) with a reference number and / or reference frequency; c) selecting the patient as more likely to respond to the therapy comprising the personalized RNA vaccine if the number and / or frequency of de novo SE TCR clones in the sample from the patient is greater than the reference number and / or reference frequency; Selecting by a method including the personalized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a cancer tumor specimen obtained from the patient; The method of any one of claims 1 to 151, wherein a number and / or frequency of de novo SE TCR clones above the reference number and / or reference frequency indicates that the patient is more likely to respond to the therapy comprising the personalized RNA vaccine.

175. In vitro use of the number and / or frequency of significantly de novo expanded (SE) TCR clones to select patients with cancer tumors who are more likely to respond to a therapy comprising a personalized RNA vaccine, wherein the number and / or frequency of de novo SE TCR clones in a sample from the patient that exceeds a reference number and / or reference frequency selects the patient as more likely to respond to the therapy comprising the personalized RNA vaccine.

176. Use of the number and / or frequency of significantly de novo expanded (SE) TCR clones for the manufacture of a diagnostic agent for assessing the likelihood of a patient with a cancer tumor responding to a therapy including a personalized RNA vaccine.

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