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

Abstract

To provide a method for treating cancer in an individual.SOLUTION: Methods of administering PD-1 axes binding antagonists (such as anti-PD-1 antibodies or anti-PD - L1 antibodies) and RNA vaccines (e.g., personalized cancer vaccines comprising one or more polynucleotides encoding one or more neo-epitopes resulting from cancer-specific somatic mutations present in a tumor specimen obtained from an individual) to an individual are provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 62 / 792,387, filed January 14, 2019, 62 / 795,476, filed January 22, 2019, and 62 / 887,410, filed August 15, 2019, the contents of each of which are incorporated herein by reference in their entirety. Submitting a sequence listing as an ASCII text file

[0002] The following submission in an ASCII text file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 146392046940SEQLIST.txt, Recorded: January 13, 2020, Size: 41 KB).

[0003] The present disclosure relates to methods, uses, and kits related to the treatment of cancer by administering a PD-1 axis-binding antagonist (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) in combination with an RNA vaccine. Further provided herein are RNA molecules (e.g., a personalized RNA cancer vaccine comprising one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen obtained from an individual), as well as DNA molecules and methods useful for making or using the RNA vaccine. [Background technology]

[0004] Melanoma is a potentially life-threatening form of skin cancer that originates in melanocytes. In 2012, there were approximately 232,000 new cases and 55,000 deaths from melanoma worldwide, with over 100,000 new cases and 22,000 deaths in Europe (Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, et al. Eur J Cancer 2013;49:1374-403). In the United States, an estimated 91,270 new cases of melanoma are expected to be diagnosed in 2018, and approximately 9,320 patients are expected to die from the disease (American Cancer Society 2018). Furthermore, estimates suggest that the incidence of melanoma doubles every 10 to 20 years (Garbe C, Leiter U. Clin Dermatol 2009;27:3-9).

[0005] The clinical outcome of melanoma patients depends largely on the stage of the disease at the time of presentation. Until recently, treatment options for metastatic melanoma were limited. Dacarbazine was considered the standard first-line treatment, but outcomes were poor, with response rates ranging from 5% to 12%, median progression-free survival (PFS) of less than 2 months, and median overall survival (OS) of 6.4 to 9.1 months (Middleton MR, Grob JJ, Aaronson N, et al. J Clin Oncol 2000;18:158-66; Bedikian AY, Millward M, Pehamberger H, et al. J Clin Oncol 2006;24:4738-45; Chapman PB, Hauschild A, Robert C, et al. N Engl J Med 2011;364:2507-16; Robert C, Thomas L, Bondarenko I, et al. N Engl J Med 2011;364:2517-26). Combination chemotherapy and chemotherapy combined with interferon-α (IFN)-α or interleukin-2 (IL-2) have shown improved response rates but have not resulted in improved OS (Chapman PB, Einhorn LH, Meyers ML, et al. J Clin Oncol 1999;17:2745-51; Ives NJ, Stowe RL, Lorigan P, et al. J Clin Oncol 2007;25:5426-34).

[0006] Immunotherapeutic agents that target co-inhibitory receptors, or "immune checkpoints," that suppress T-cell activation have improved outcomes for patients with advanced melanoma. Despite these advances, many patients do not respond to current therapies or remain refractory to their disease, highlighting an unmet medical need for more effective treatment options.

[0007] Currently available clinical and preclinical data on immunotherapy suggest that single-agent immunotherapy is unlikely to induce complete and durable antitumor responses in the majority of patients. Because host immunosuppression by malignant cells is mediated by multiple pathways, combination treatment regimens employing two or more targeted cancer immunotherapy (CIT) agents may be required to fully engage the antitumor potential of the host immune system.

[0008] Although promising, therapeutic vaccines have historically been disappointing, potentially due to the functional exhaustion of cancer-specific T cells during chronic exposure to cancer cells.

[0009] 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

[0010] Provided herein are methods, kits, and uses comprising a PD-1 axis binding antagonist (e.g., an anti-PD1 antibody or an anti-PD-L1 antibody) and an RNA vaccine for treating cancer.

[0011] In some aspects, provided herein are methods of treating cancer in an individual, the methods comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an 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 tumor specimen obtained from the individual.

[0012] 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 anti-PD-1 antibody is administered to the individual at a dose of about 200 mg.

[0013] 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 the following: (a) a heavy chain variable region (VH) comprising an HVR-H1 comprising the amino acid sequence of GFTFSDSWIH (SEQ ID NO: 1), an HVR-2 comprising the amino acid sequence of AWISPYGGSTYYADSVKG (SEQ ID NO: 2), and an HVR-3 comprising the amino acids RHWPGGFDY (SEQ ID NO: 3); and (b) a light chain variable region (VL) comprising an HVR-L1 comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 4), an HVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO: 5), and an HVR-L3 comprising the amino acid sequence of QQYLYHPAT (SEQ ID NO: 6). In some embodiments, 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 ) In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody is administered to the individual at a dose of about 1200 mg.

[0014] In some embodiments of any of the above embodiments, the PD-1 axis binding antagonist is administered to the individual at 21 day or 3 week intervals.

[0015] In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 10-20 neoepitopes arising from cancer-specific somatic mutations present in a tumor specimen. In some embodiments, the RNA vaccine is formulated into lipoplex nanoparticles or liposomes. In some embodiments, the RNA vaccine is administered to an individual at a dose of about 15 μg, about 25 μg, about 38 μg, about 50 μg, or about 100 μg. In some embodiments, the RNA vaccine is administered to an individual at 21-day or 3-week intervals.

[0016] In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are administered to the individual for eight 21-day cycles, with the RNA vaccine being administered to the individual on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3 through 7. In some embodiments, the PD-1 axis binding antagonist is administered to the individual on day 1 of cycles 1 through 8. In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are further administered to the individual after cycle 8. In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are further administered to the individual for 17 additional 21-day cycles, with the PD-1 axis binding antagonist being administered to the individual on day 1 of cycles 13 through 29, and the RNA vaccine being administered to the individual on days 1 of cycles 13, 21, and 29. In some embodiments, the PD-1 axis-binding antagonist and the RNA vaccine are administered to the individual in eight 21-day cycles, wherein the PD-1 axis-binding antagonist is pembrolizumab, and the PD-1 axis-binding antagonist is administered to the individual at a dose of about 200 mg on day 1 of cycles 1 through 8, and the RNA vaccine is administered to the individual at a dose of about 25 μg on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3 through 7. In some embodiments, the RNA vaccine is administered to the individual at a dose of about 25 μg on day 1 of cycle 2, about 25 μg on day 8 of cycle 2, about 25 μg on day 15 of cycle 2, and about 25 μg on day 1 of each of cycles 3 through 7. In some embodiments, the PD-1 axis-binding antagonist and the RNA vaccine are administered intravenously. In some embodiments, the individual is a human.

[0017] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, bladder cancer, colorectal cancer, triple-negative breast cancer, renal cancer, and head and neck cancer. In some embodiments, the cancer is melanoma. In some embodiments, the melanoma is cutaneous melanoma or mucosal melanoma. In some embodiments, the melanoma is not ocular or acral melanoma. In some embodiments, the melanoma is metastatic (e.g., stage IV (such as recurrent or de novo stage IV)) or unresectable locally advanced (e.g., stage IIIC or stage IIID) melanoma. In some embodiments, the melanoma is locally advanced melanoma. In some embodiments, the method results in improved progression-free survival (PFS). In some embodiments, the method results in an increased objective response rate (ORR).

[0018] In some aspects, provided herein is a kit or article of manufacture comprising a PD-1 axis binding antagonist for use in combination with an RNA vaccine for treating an individual with cancer according to the method of any one of the above embodiments.

[0019] In some embodiments, provided herein is a PD-1 axis-binding antagonist for use in a method of treating a human individual having cancer, the method comprising administering to the individual an effective amount of the PD-1 axis-binding antagonist in combination with an RNA vaccine, the RNA vaccine comprising one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen obtained from the individual. In some embodiments, provided herein is an RNA vaccine for use in a method of treating a human individual having cancer, the method comprising administering to the individual an effective amount of the RNA vaccine in combination with a PD-1 axis-binding antagonist, the RNA vaccine comprising one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen obtained from the individual.

[0020] In some aspects, provided herein are RNA molecules 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 at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule; (5) a 3' UTR, the 3' UTR comprising: (a) a 3' untranslated region of an amino-terminal enhancer of a 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.

[0021] In some embodiments, the 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 a secretory signal peptide (e.g., (3) above) and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule (e.g., (4) above). In some embodiments, the RNA molecule comprises polynucleotide sequences 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 molecule further comprises, in the 5' to 3' direction, a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope, wherein the polynucleotide sequences encoding the amino acid linker and the neoepitope form a first linker-neoepitope module, and the polynucleotide sequence forming the first linker-neoepitope module is located, in the 5' to 3' direction, between the polynucleotide sequence encoding a secretory signal peptide (e.g., (3) above) and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule (e.g., (4) above). 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).In some embodiments, the RNA molecule further comprises, in a 5' to 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, 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 a transmembrane domain and a cytoplasmic domain of an MHC molecule (e.g., (4) above), and the neoepitope of the first linker-epitope module is different from the neoepitope of the second linker-epitope module. In some embodiments, the 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, each linker-epitope module encoding a different neoepitope. In some embodiments, the 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 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 molecule further comprises a second polynucleotide sequence encoding an amino acid linker, wherein the second polynucleotide sequence encoding the amino acid linker is located between the polynucleotide sequence encoding the most distal neoepitope in the 3' direction and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule (e.g., (4) above).In some embodiments, the RNA molecule comprises a sequence as shown in Figure 4. In some embodiments, N in Figure 4 represents a polynucleotide sequence encoding one or more neoepitopes (e.g., 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, N in Figure 4 represents one or more (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, or 20 different) linker-neoepitope module(s), each module comprising, in the 5' to 3' direction, a polynucleotide sequence encoding one or more amino acid linkers and a polynucleotide sequence encoding a neoepitope.

[0022] In some embodiments, the 5' cap of an RNA molecule (e.g., (1) above) comprises a D1 diastereoisomer of the following structure: TIFF2026004341000002.tif53170 In some embodiments, the 5'UTR of the RNA molecule (e.g., (2) above) comprises the sequence UUCUUCUGGUCCCCACAGACUCAGAGAACCCGCCACC (SEQ ID NO: 23). In some embodiments, the 5'UTR of the RNA molecule (e.g., (2) above) comprises the sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 21). In some embodiments, the secretory signal peptide encoded by the RNA molecule (e.g., in (3) above) comprises the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 27). In some embodiments, the polynucleotide sequence encoding the secretory signal peptide of the RNA molecule (e.g., (3) above) comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 25). In some embodiments, at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule (e.g., (4) above) encoded by the RNA molecule comprises the amino acid sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 30). In some embodiments, a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule (e.g., (4) above) of the RNA molecule comprises the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC (SEQ ID NO: 28).In some embodiments, the 3' untranslated region of the AES mRNA of the RNA molecule (e.g., (5a) above) comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO: 33). In some embodiments, the non-coding RNA of the mitochondrially encoded 12S RNA of the RNA molecule (e.g., (5b) above) comprises the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO: 35). In some embodiments, the 3'UTR of the RNA molecule (e.g., (5) above) comprises the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 31). In some embodiments, the poly(A) sequence of the RNA molecule (e.g., (6) above) comprises 120 adenine nucleotides.

[0023] In some aspects, provided herein is, in 5' to 3' direction, the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 19), and the polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGU CACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGUACCCCGAGUCUCCCCCGACCUCGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAA UGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 20).

[0024] In some embodiments, provided herein is an RNA molecule comprising, in the 5' to 3' direction, the following polynucleotide sequence: GGGGCGAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCCGCC ACCAUGAGAG UGAUGGCCCC CAGAACCCUG AUCCUGCUGC UGUCUGGCGC CCUGGCCCUG ACAGAGACAU GGGCCGGAAG CNAUCGUGGGA AUUGUGGCAG GACUGGCAGU GCUGGCCGUG GUGGUGAUCG GAGCCGUGGU GGCUACCGUG AUGUGCAGAC GGAAGUCCAG CGGAGGCAAG GGCGGCAGCU ACAGCCAGGC CGCCAGCUCU GAUAGCGCCC AGGGCAGCGA CGUGUCACUG ACAGCCUAGU AACUCGAGCU GGUACUGCAU GCACGCAAUG CUAGCUGCCC CUUUCCCGUC CUGGGUACCC CGAGUCUCCC CCGACCUCGG GUCCCAGGUA UGCUCCCACC UCCACCUGCC CCACUCACCA CCUCUGCUAG UUCCAGACAC CUCCCAAGCA CGCAGCAAUG CAGCUCAAAA CGCUUAGCCU AGCCACACCC CCACGGGAAA CAGCAGUGAU UAACCUUUAG CAAUAAACGA AAGUUUAACU AAGCUAUACU AACCCCAGGG UUGGUCAAUU UCGUGCCAGC CACACCGAGA CCUGGUCCAG AGUCGCUAGC CGCGUCGCUA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA(SEQ ID NO: 42)

[0025] In some embodiments, the RNA molecule further comprises a polynucleotide sequence encoding at least one neoepitope, wherein the polynucleotide sequence encoding the at least one neoepitope is between the sequence of SEQ ID NO: 19 and the sequence of SEQ ID NO: 20, or at the position marked "N" in SEQ ID NO: 42. 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. In some embodiments, the RNA molecule further comprises, in the 5' to 3' direction (e.g., between the sequence of SEQ ID NO: 19 and the sequence of SEQ ID NO: 20, or at the position marked "N" in SEQ ID NO: 42): (a) at least a first linker-neoepitope module, the first linker-neoepitope module comprising a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope; and (b) a second polynucleotide sequence encoding the amino acid linker. In some embodiments, the 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, each linker-epitope module encoding a different neoepitope.In some embodiments, the 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 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 molecule further comprises a 5' cap, wherein the 5' cap is positioned 5' to the sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 19). In some embodiments, the 5' cap is positioned between two guanine nucleotides. In some embodiments, the RNA molecule further comprises a 5' cap, wherein the 5' cap is positioned between the first two G bases of SEQ ID NO: 42 (e.g., shown in Figure 4). In some embodiments, the 5' cap comprises a D1 diastereoisomer of the following structure: TIFF2026004341000003.tif55170

[0026] In some aspects, provided herein are liposomes comprising any one of the above-described embodiments of an RNA molecule (e.g., comprising any of the RNA molecules described herein or in the Sequence Listing or Figures) and one or more lipids, wherein the one or more lipids form a multilamellar structure encapsulating the RNA molecule. 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 liposome has a total positive to negative charge ratio of 1.3:2 (0.65). In some embodiments, at physiological pH, the liposome has a total positive to negative charge ratio of 1.0:2.0 or greater. In some embodiments, at physiological pH, the liposomes have a total charge ratio of positive to negative charges of 1.9:2.0 or less, hi some embodiments, at physiological pH, the liposomes have a total charge ratio of positive to negative charges of 1.0:2.0 or more and 1.9:2.0 or less.

[0027] In some aspects, provided herein are methods of treating or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of an RNA molecule of any one of the above embodiments (e.g., comprising any of the RNA molecules described herein or in the Sequence Listing or Figures) or a liposome of any one of the above embodiments. Also provided herein are RNA molecules of any one of the above embodiments or liposomes of any one of the above embodiments for use in a method of treating or delaying the progression of cancer in an individual, the method comprising administering to the individual an effective amount of the RNA molecule or liposome. Also provided herein are RNA molecules of any one of the above embodiments (e.g., comprising any of the RNA molecules described herein or in the Sequence Listing or Figures) or liposomes of any one of the above embodiments for use in the manufacture of a medicament for treating or delaying the progression of cancer in an individual. In some embodiments, the RNA molecule comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen obtained from the individual. In some embodiments, the method further comprises administering to the individual a PD-1 axis-binding antagonist (e.g., an anti-PDL1 antibody). In some embodiments, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, bladder cancer, colorectal cancer, triple-negative breast cancer, renal cancer, and head and neck cancer. In some embodiments, the RNA molecule or liposome is administered at a dose of about 15 μg, about 25 μg, about 38 μg, about 50 μg, or about 100 μg. In some embodiments, the RNA molecule or liposome is administered at a dose of about 15 μg, about 25 μg, about 38 μg, about 50 μg, or about 100 μg, and the PD-1 axis-binding antagonist (e.g., an anti-PDL1 antibody) is administered at a dose of about 200 or about 1200 mg.In some embodiments, the PD-1 axis-binding antagonist and the RNA molecule or liposome are administered to the individual in eight 21-day cycles, wherein the PD-1 axis-binding antagonist is pembrolizumab, the PD-1 axis-binding antagonist is administered to the individual at a dose of about 200 mg on day 1 of cycles 1-8, and the RNA vaccine is administered to the individual at a dose of about 25 μg on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3-7.

[0028] In some embodiments, provided herein are DNA molecules encoding any of the RNA molecules described herein. In some embodiments, provided herein are DNA molecules comprising, in a 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 3' UTR, the 3' UTR comprising (a) a 3' untranslated region of an amino-terminal enhancer of a split (AES) mRNA or a fragment thereof; and (b) a non-coding RNA of a mitochondrially encoded 12S RNA or a fragment thereof; and (5) a polynucleotide encoding a poly(A) sequence.

[0029] In some embodiments, the DNA 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 a secretory signal peptide (e.g., (2) above) and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule (e.g., (3) above). In some embodiments, the DNA molecule comprises polynucleotide sequences 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 further comprises, in the 5' to 3' direction, a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope, wherein the polynucleotide sequences encoding the amino acid linker and the neoepitope form a first linker-neoepitope module, and the polynucleotide sequence forming the first linker-neoepitope module is located, in the 5' to 3' direction, between the polynucleotide sequence encoding a secretory signal peptide (e.g., (2) above) and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule (e.g., (3) above). 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 GGCGGCTCTGGAGGAGGCGGCTCCGGAGGC (SEQ ID NO: 38).In some embodiments, the DNA molecule further comprises, in a 5' to 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, 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 an MHC molecule (e.g., (3) above), and the neoepitope of the first linker-epitope module is different from the neoepitope of the second linker-epitope module. 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, 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 further comprises a second polynucleotide sequence encoding an amino acid linker, wherein the second polynucleotide sequence encoding the amino acid linker is located between the polynucleotide sequence encoding the most distal neoepitope in the 3' direction and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule (e.g., (3) above).In some embodiments, a polynucleotide encoding the 5'UTR (e.g., (1) above) comprises the sequence TTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 24). In some embodiments, a polynucleotide encoding the 5'UTR (e.g., (1) above) comprises the sequence GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 22). In some embodiments, a secretory signal peptide (e.g., encoded by (2) above) comprises the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 27). In some embodiments, a polynucleotide sequence encoding a secretory signal peptide (e.g., (2) above) comprises the sequence ATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC (SEQ ID NO: 26). In some embodiments, at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule (e.g., encoded by (3) above) comprises the amino acid sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 30). In some embodiments, a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule (e.g., (3) above) comprises the sequence ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACAGCC (SEQ ID NO: 29).In some embodiments, a polynucleotide sequence encoding the 3' untranslated region of AES mRNA (e.g., (4a) above) comprises the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC (SEQ ID NO: 34). In some embodiments, a polynucleotide encoding a non-coding RNA of mitochondrially encoded 12S RNA (e.g., (4b) above) comprises the sequence CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG (SEQ ID NO: 36). In some embodiments, the polynucleotide encoding the 3'UTR (e.g., (4) above) comprises the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (SEQ ID NO: 32). In some embodiments, the poly(A) sequence (e.g., (5) above) comprises 120 adenine nucleotides.

[0030] In some aspects, provided herein are, in 5' to 3' direction, the polynucleotide sequence GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC (SEQ ID NO: 40), and the polynucleotide sequence ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGT CACTGACAGCCTAGTAACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAA TGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (SEQ ID NO: 41).

[0031] In some embodiments, the DNA molecule further comprises a polynucleotide sequence encoding at least one neoepitope, wherein the polynucleotide sequence encoding the at least one neoepitope is between the sequence of SEQ ID NO: 40 and the sequence of SEQ ID NO: 41. In some embodiments, the DNA 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. In some embodiments, the DNA molecule further comprises, in the 5' to 3' direction between the sequence of SEQ ID NO: 40 and the sequence of SEQ ID NO: 41, (a) at least a first linker-neoepitope module, the first linker-neoepitope module comprising a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope, and (b) a second polynucleotide sequence encoding the amino acid linker. 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, 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.

[0032] In some aspects, provided herein are methods of producing an RNA molecule, comprising transcribing a DNA molecule of any one of the above embodiments.

[0033] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows. [Brief explanation of the drawings]

[0034] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0035] [Figure 1] Figure 1 shows the study scheme for a phase II randomized, open-label trial designed to evaluate the efficacy and safety of an RNA-based personalized cancer vaccine (R07198457) plus an anti-PD1 antibody (pembrolizumab). During the randomization phase, patients were randomized (2:1) to either experimental treatment (Group B) or control treatment (Group A). ​​IMC = Internal Monitoring Committee; LDH = lactate dehydrogenase; Q3W = every 3 weeks; TBD = undetermined; ULN = upper limit of normal.

[0036] [Figure 2] Figure 2 shows the dosing scheme for Arm A (pembrolizumab) and the safety run-in phase and Arm B (R07198457 + pembrolizumab) of the Phase II study. C = cycle; D = day.

[0037] [Figure 3]Figure 3 shows the general structure of an exemplary RNA vaccine (i.e., polyneoepitope RNA). This figure is a schematic representation of the general structure of an RNA drug substance with patient-specific sequences encoding neoepitopes (neol-10) fused by a GS-rich linker, along with a constant 5' cap (β-S-ARCA(Dl)), 5' and 3' untranslated regions (hAg-Kozak and FI, respectively), N- and C-terminal fusion tags (sec2.0 and MITD, respectively), and a poly(A) tail (A120).

[0038] [Figure 4] Figure 4 shows the ribonucleotide sequence (5'→3') of the constant region of an exemplary RNA vaccine (SEQ ID NO: 42). The bond between the first two G residues is the unusual bond (5'→5')-ppsp- as shown in Table 5 and Figure 5 for the 5' cap structure. The insertion site for the patient's cancer-specific sequence is between residues C131 and A132 (marked in bold). "N" indicates the position of the polynucleotide sequence(s) encoding one or more (e.g., 1-20) neoepitopes (separated by optional linkers).

[0039] [Figure 5] Figure 5 shows the 5'-capping structure β-S-ARCA (D1) (m2 7 2' OGppspG) used at the 5' end of the RNA constant region. The stereogenic P center is in the Rp configuration in the "D1" isomer. Note: Differences between β-S-ARCA (D1) and the basic cap structure m7GpppG are shown in red; the component m7G ​​has an -OCH3 group at the C2' position, and the non-bridging oxygen of the β-phosphate is replaced with sulfur. Due to the presence of the stereogenic P center (labeled *), the phosphorothioate cap analog β-S-ARCA exists as two diastereomers. These were designated 01 and 02 based on their elution order in reversed-phase high-performance liquid chromatography. DETAILED DESCRIPTION OF THE INVENTION

[0040] 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.

[0041] 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" optionally includes a combination of two or more such molecules, and the like.

[0042] The term "about," as used herein, refers to a normal range of error for each value, which is 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.

[0043] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0044] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with any 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.

[0045] 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 particular 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, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, 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.

[0046] 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 particular aspects, PD-L1 binding antagonists inhibit 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.

[0047] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, or prevents signaling that occurs as a result of the interaction of PD-L2 with one or more of its binding partners, such as 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 particular 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 that results 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.

[0048] "Sustained response" refers to a sustained effect on tumor growth reduction after treatment is discontinued. For example, tumor size may remain the same or may be smaller compared to the size at the beginning of the administration period. In some embodiments, the sustained response has a duration at least equal to the treatment period, or at least 1.5, 2.0, 2.5, or 3.0 times the treatment period.

[0049] 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.

[0050] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cells being treated during the course of clinical pathology. Desirable effects of treatment include reducing the rate of disease progression, reversing or alleviating the disease state, and ameliorating or improving 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, reducing (or destroying) the proliferation of cancerous cells, alleviating symptoms caused by the disease, improving the quality of life of those suffering from the disease, reducing the dose of other medications required to treat the disease, and / or extending the individual's survival time.

[0051] As used herein, "delaying disease progression" means to postpone, prevent, slow, retard, stabilize, and / or prolong the onset of a disease (such as cancer). This delay can be of varying duration depending on the history of the disease and / or the individual being treated. As will be apparent to one skilled in the art, a sufficient or significant delay can effectively encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0052] An "effective amount" is at least the minimum amount necessary to achieve measurable improvement or prevention of a particular disorder. An 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 the individual. An effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of treatment. For prophylactic use, beneficial or desired results include results such as elimination or reduction of the risk of disease, reduction in the severity of disease, or delay in 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 alleviation of one or more symptoms attributable to the disease, improvement in the quality of life of a person suffering from the disease, reduction in the dose of other drugs required to treat the disease, enhancement of the effect of another drug (e.g., by targeting), delay in disease progression, and / or prolonged 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 to some extent or preferably stopping) cancer cell invasion into peripheral organs, inhibiting (i.e., slowing to some extent or preferably stopping) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating to some extent one or more symptoms associated with the disorder. An effective amount may be administered in a single dose or multiple doses. 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 clinically understood, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount when a desired result is obtained or achieved in combination with one or more other agents.

[0053] As used herein, "together with" or "in combination with" refers to the administration of one treatment modality in addition to another treatment modality. Thus, "together with" or "in combination with" refers to the administration of one treatment modality before, during, or after the administration of another treatment modality to an individual.

[0054] A "disorder" is any condition that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0055] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders involving 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.

[0056] As used herein, "tumor" refers to all neoblastic 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.

[0057] 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.

[0058] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0059] 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 experimental, diagnostic, or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, 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, 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.

[0060] "Native antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons, consisting 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 intrachain 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 constant domain of the light chain aligned with the first constant domain of the heavy chain and the variable domain of the light chain 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.

[0061] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portions of the immunoglobulin, the variable domains, which contain the antigen-binding sites. The constant domains contain the CH1, CH2, and CH3 domains (collectively CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0062] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. A heavy chain variable domain may be referred to as "VH." A light chain variable domain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0063] The term "variable" refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. The variability 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). Natural heavy and light-chain variable domains each contain four FR regions that primarily 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 participation of the antibody in antibody-dependent cellular toxicity.

[0064] 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.

[0065] The term IgG "isotype" or "subclass" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.

[0066] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. The five major classes of immunoglobulins are 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 generally 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.

[0067] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in a substantially intact form, rather than an antibody fragment as defined below. These terms specifically refer to an antibody having a heavy chain containing an Fc region.

[0068] A "naked antibody" for purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0069] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding region thereof. 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.

[0070] 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.

[0071] An "Fv" is the minimum antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- 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.

[0072] 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 by the addition of a few 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 herein for Fab' in which the cysteine ​​residue(s) of 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.

[0073] "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 review of scFvs, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0074] The term "diabody" refers to an antibody fragment having two antigen-binding sites; these fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on 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, creating 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).

[0075] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations that may be present in minor amounts, e.g., naturally occurring mutations. 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 an antibody comprising 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 the 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 can 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, or create a multispecific antibody, 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.

[0076] 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., 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., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 222:581-597 (1992)), and the like. al.,J.Mol.Biol.338(2):299-310(2004);Lee et 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 (see, 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)).

[0077] 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, for example, from an antibody produced by immunizing macaque monkeys with the antigen of interest.

[0078] "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 can 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. The humanized antibody optionally also comprises 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). See also, 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.

[0079] 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 produced using a variety of 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) are also available for preparing 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, e.g., immunized xenomouse, which 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). Also, for human antibodies produced by human B cell hybridoma technology, see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0080] A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than for a homolog of the antigen from a second mammalian species. Typically, a species-dependent antibody "specifically" binds to a human antigen (e.g., has a binding affinity (Kd) value of about 1×10 M or less, preferably about 1×10 M or less, and preferably about 1×10 M or less), but has a binding affinity 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 its 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.

[0081] The terms "hypervariable region," "HVR," or "HV," as used herein, refer to the region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Antibodies generally contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). In natural 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, e.g., 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).

[0082] Several HVR descriptions 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 H26-H32 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

[0083] 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.

[0084] 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.

[0085] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0086] 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. (see above). 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, etc.). The Kabat numbering of residues can be determined by alignment of the homologous regions of the sequence of an antibody with the "standard" Kabat-numbered sequence for a given antibody.

[0087] The Kabat numbering system is generally 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 generally used when referring to residues in immunoglobulin heavy chain constant regions (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.

[0088] 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.

[0089] 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.

[0090] The term "sample," as used herein, refers to a composition obtained or derived from a subject and / or individual of interest that contains a cellular and / or other molecular entity 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 known to contain the cellular and / or molecular entity 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 (e.g., a tumor sample) obtained from an individual's cancer, comprising tumor cells and optionally tumor-infiltrating immune cells. For example, the sample can 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 comprises 50 or more viable tumor cells (e.g., from a core needle biopsy, optionally embedded in a paraffin block; an excision biopsy, an incision biopsy, a punch biopsy, or a forceps biopsy; or from tumor tissue resection).

[0091] A "tissue sample" or "cell sample" refers to a collection of similar cells obtained from the tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue, 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 natural tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, or antibiotics.

[0092] 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 of the body (e.g., tissue or cells) 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 of the body (e.g., tissue or cells) 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 untreated tissues and / or cells from the body of an individual who is not the subject or individual.

[0093] A patient's "effective response" or patient "responsiveness" to treatment with a pharmaceutical 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.

[0094] A patient who "does not respond effectively" to treatment refers to a patient who does not have any of the following: prolonged 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.

[0095] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR); and the like. Such effector functions generally require association of the Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays, e.g., as disclosed in the definitions herein.

[0096] A cancer or biological sample "having human effector cells" is one that has human effector cells (e.g., infiltrating human effector cells) present in the sample in a diagnostic study.

[0097] 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 study. In some embodiments, the FcR is an FcγR. In some embodiments, the FcR is an activating FcγR. II. Overview

[0098] Provided herein are methods for treating or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) and an RNA vaccine. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in the cancer, e.g., present in a tumor specimen obtained from the individual. In some embodiments, the individual is human.

[0099] In some embodiments, provided herein are methods of treating or delaying the onset of cancer in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) and an RNA vaccine, where the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes identified based on somatic mutations present in a tumor sample obtained from the individual. In some embodiments, provided herein are methods of treating or delaying the onset of cancer in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) and an RNA vaccine, where the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes corresponding to somatic mutations present in a tumor sample obtained from the individual.

[0100] In some embodiments, the treatment extends the progression-free survival (PFS) and / or overall survival (OS) of the individual compared to a treatment comprising administration of a PD-1 axis-binding antagonist in the absence of an RNA vaccine. In some embodiments, the treatment improves the overall response rate (ORR) compared to a treatment comprising administration of a PD-1 axis-binding antagonist in the absence of an RNA vaccine. In some embodiments, ORR refers to the proportion of patients with a complete response (CR) or partial response (PR). In some embodiments, the treatment extends the duration of response (DOR) in the individual compared to a treatment comprising administration of a PD-1 axis-binding antagonist in the absence of an RNA vaccine. In some embodiments, the treatment improves the health-related quality of life (HRQoL) score in the individual compared to a treatment comprising administration of a PD-1 axis-binding antagonist in the absence of an RNA vaccine.

[0101] In some embodiments, the PD-1 axis-binding antagonist is administered to the individual at 21 day or 3 week intervals. In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-1 antibody (e.g., pembrolizumab) administered to the individual at 21 day or 3 week intervals, for example, at a dose of about 200 mg. In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-L1 antibody (e.g., atezolizumab) administered to the individual at 21 day or 3 week intervals, for example, at a dose of about 1200 mg.

[0102] In some embodiments, the RNA vaccine is administered to an individual at 21 day or 3 week intervals.

[0103] In some embodiments, the PD-1 axis-binding antagonist and the RNA vaccine are administered to the individual in eight 21-day cycles. In some embodiments, the RNA vaccine is administered to the individual on days 1, 8, and 15 of cycle 2 and on day 1 of cycles 3 through 7. In some embodiments, the PD-1 axis-binding antagonist is administered to the individual on day 1 of cycles 1 through 8. In some embodiments, the RNA vaccine is administered to the individual on days 1, 8, and 15 of cycle 2 and on day 1 of cycles 3 through 7, and the PD-1 axis-binding antagonist is administered to the individual on day 1 of cycles 1 through 8.

[0104] In some embodiments, the PD-1 axis-binding antagonist and the RNA vaccine are further administered to the individual after cycle 8. In some embodiments, the PD-1 axis-binding antagonist and the RNA vaccine are further administered to the individual for 17 additional 21-day cycles, the PD-1 axis-binding antagonist is administered to the individual on day 1 of cycles 13-29, and / or the RNA vaccine is administered to the individual on day 1 of cycles 13, 21, and 29.

[0105] In a specific embodiment, the PD-1 axis-binding antagonist and the RNA vaccine are administered to the individual in eight 21-day cycles, wherein the PD-1 axis-binding antagonist is pembrolizumab, and the PD-1 axis-binding antagonist is administered to the individual at a dose of about 200 mg on day 1 of cycles 1 through 8, and the RNA vaccine is administered to the individual at a dose of about 25 μg on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3 through 7. In a specific embodiment, the PD-L1 axis-binding antagonist and the RNA vaccine are administered to the individual in eight 21-day cycles, wherein the PD-L1 axis-binding antagonist is atezolizumab, and the PD-L1 axis-binding antagonist is administered to the individual at a dose of about 1200 mg on day 1 of cycles 1 through 8, and the RNA vaccine is administered to the individual at a dose of about 25 μg on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3 through 7. In some embodiments, the RNA vaccine is administered to an individual at doses of about 25 μg on day 1 of cycle 2, about 25 μg on day 8 of cycle 2, about 25 μg on day 15 of cycle 2, and about 25 μg on day 1 of each of cycles 3-7 (i.e., a total of about 75 μg of vaccine is administered to the individual over three doses during cycle 2). In some embodiments, a total of about 75 μg of vaccine is administered to the individual over three doses during cycle 1 in which the RNA vaccine is administered. In some embodiments, the PCV is administered intravenously at a dose of 15 μg, 25 μg, 38 μg, 50 μg, or 100 μg, for example, in a liposomal formulation. In some embodiments, 15 μg, 25 μg, 38 μg, 50 μg, or 100 μg of RNA is delivered per dose (i.e., the dose weight reflects the weight of the RNA administered, not the total weight of the formulation or lipoplex administered). III. RNA vaccines

[0106] Certain aspects of the present disclosure relate to personalized cancer vaccines (PCVs). In some embodiments, the PCV 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 of the features / sequences of the RNA vaccines described below. In some embodiments, the RNA polynucleotide is a single-stranded mRNA polynucleotide. In some other embodiments, the present disclosure provides a DNA polynucleotide encoding an RNA comprising one or more of the features / sequences of the RNA vaccines described below.

[0107] The personalized cancer vaccine comprises a personalized neoantigen (i.e., a tumor-associated antigen (TAA) specifically expressed in a patient's cancer) identified as having potential immunostimulatory activity. In the embodiments described herein, the PCV 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 the TAA-expressing cancer cell(s).

[0108] PCVs typically contain multiple neoantigenic epitopes ("neoepitopes"), for example, 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, 29, or 30 neoepitopes. The PCV may comprise a 5' mRNA cap analog, a 5' UTR, a signal sequence, a domain promoting antigen expression, a 3' UTR, and / or a poly(A) tail. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 10-20 neoepitopes arising from cancer-specific somatic mutations present in a tumor specimen. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding at least five neoepitopes arising from cancer-specific somatic mutations present in a tumor specimen. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 neoepitopes arising from cancer-specific somatic mutations present in a tumor specimen. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-10 neoepitopes arising from cancer-specific somatic mutations present in a tumor specimen.

[0109] 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.

[0110] 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 providing 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+ / CD ... + T cells and / or CD4 +Up to 20 MHC1 and MHCII neoepitopes predicted to induce 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 PCV and may help reduce the risk of immune escape that may occur if 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).

[0111] 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 patient-specific neoepitope sequences, between a patient-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 patient-specific neoepitope sequence. In some embodiments, the RNA vaccine encodes multiple linkers. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 neoepitopes arising 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 5-10 neoepitopes arising 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.

[0112] 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).

[0113] 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 sequence. 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 s In 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: TIFF2026004341000004.tif55170

[0114] In some embodiments, the 5'-cap is the D1 diastereoisomer of β-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). β-S-ARCA or the D1 diastereoisomer of β-S-ARCA (D1) is the diastereoisomer of β-S-ARCA that elutes first on an HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereoisomer of β-S-ARCA (beta-S-ARCA (D2)). The HPLC is preferably analytical HPLC. In one embodiment, a 5 μm, 4.6×250 mm format Supelcosil LC-18-T RP column is preferably used for the separation, 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, for example, 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.

[0115] 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 sequence 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).

[0116] 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 sequence(s) 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).

[0117] 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. The polypeptide chain has a length of slightly more 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. The domains and domains 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 α- and β-chain genes (i.e., DPα, DPβ, DQα, DQβ, DRα, and DRβ). 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 (a) or 26-29 kDa (beta). 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 peptide is 9 residues long in the alpha and beta chains, respectively. It 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 from 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 C-terminal to the one or more neoepitope sequence(s) 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 AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC (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).

[0118] In some embodiments, the RNA vaccine comprises both a polynucleotide sequence encoding a secretory signal peptide N-terminal to one or more neoepitope sequence(s) and a polynucleotide sequence encoding a transmembrane and / or cytoplasmic domain C-terminal to one or more neoepitope sequence(s). Combining such sequences has been shown to improve processing and presentation of MHC class I and II epitopes in human dendritic cells. See, e.g., Kreiter, S. et al. (2008) J. Immunol. 180:309-318.

[0119] In bone marrow DCs, the RNA is released into the cytosol and translated into polyneoepitopic peptides. The polypeptides contain additional sequences to enhance antigen presentation. In some embodiments, a signal sequence (sec) from the MHC1 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 MHC1 presentation efficiency. Without wishing to be bound by theory, it is believed that the transmembrane and cytoplasmic domains of the MHC1 heavy chain direct the polypeptide to endosomal / lysosomal compartments, which have been shown to improve MHC1 presentation.

[0120] In some embodiments, the RNA vaccine comprises a 3' UTR. Certain untranslated sequences found 3' to the protein-coding sequence 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. "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 as a hetero-oligomer with other family members to dominantly repress 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 in 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).

[0121] 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. Pat. No. 9,476,055.

[0122] 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) the 3' untranslated region of the 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 (6) a poly(A) sequence.

[0023] In some embodiments, an 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 AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGA CGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAG CAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (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.

[0123] 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. See, e.g., Figure 4. 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 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.

[0124] 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.

[0125] 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, 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.

[0126] 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 patient-specific neoepitopes (e.g., each encoding 27 amino acids) is greater than 1300 nucleotides in length. In some embodiments, an RNA vaccine comprising ten patient-specific neoepitopes (e.g., each encoding 27 amino acids) is greater than 1800 nucleotides in length.

[0127] In some embodiments, RNA vaccines are formulated into lipoplex nanoparticles or liposomes. In some embodiments, a lipoplex nanoparticle formulation for RNA (RNA-lipoplex) is used to enable IV delivery of the RNA vaccines 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.

[0128] In one embodiment, the nanoparticles comprise at least one lipid. In one embodiment, the nanoparticles comprise at least one cationic lipid. The cationic lipid may be monocationic or polycationic. Any cationic amphiphilic molecule, e.g., 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 nanoparticles comprise at least one helper lipid. The helper lipid may be a neutral or anionic lipid. The helper lipid may be a natural lipid, e.g., a phospholipid or an analog of a natural lipid, or a completely synthetic lipid, or a lipid-like molecule that bears no resemblance to a natural lipid. In one embodiment, the cationic lipid and / or the helper lipid is a bilayer-forming lipid.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] In one embodiment, the lipid is included in a vesicle that encapsulates the RNA. The vesicle may be a multilamellar vesicle, a unilamellar vesicle, or a mixture thereof. The vesicle may be a liposome.

[0133] The nanoparticles or liposomes described herein can be formed by mixing RNA and cationic lipids, adjusting the positive charge to negative charge ratio according to the (+ / -) charge ratio of the cationic lipid to the RNA. The + / - charge ratio of the cationic lipid to the RNA in the nanoparticles described herein can be calculated using the following formula: (+ / - charge ratio) = [(amount of cationic lipid (mol)) * (total number of positive charges in the cationic lipid)]: [(amount of RNA (mol)) * (total number of negative charges in the 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 nanoparticle preparation. For further description of exemplary nanoparticles, see, for example, U.S. Patent Application Publication No. 20150086612 to PG.

[0134] In one embodiment, the total charge ratio of positive to negative charges in the nanoparticles or liposomes (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, in particular 1:1.4 to 1:1.6, such as about 1:1.5. In some embodiments, at physiological pH, the total charge ratio of positive to negative charges in the nanoparticles is TIFF2026004341000005.tif10170. In some embodiments, at physiological pH, the total charge ratio of positive to negative charges of the nanoparticles or liposomes 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 total charge ratio of positive to negative charges of the nanoparticles or liposomes is 1.3:2 (0.65). In some embodiments, at physiological pH, the total charge ratio of positive to negative charges of the liposomes is 1.0:2.0 or greater. In some embodiments, at physiological pH, the total charge ratio of positive to negative charges of the liposomes is 1.9:2.0 or less. In some embodiments, at physiological pH, the total charge ratio of positive to negative charges of the liposomes is 1.0:2.0 or greater and 1.9:2.0 or less.

[0135] In one embodiment, the nanoparticles are lipoplexes comprising 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 nanoparticles are lipoplexes comprising 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 nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, where 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 nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 2:1 to 1:2, preferably 2:1 to 1:1, where the charge ratio of positive charges in DOTMA to negative charges in RNA is 1.4:1 or less. In one embodiment, the nanoparticles are lipoplexes comprising DOTMA and cholesterol in a molar ratio of 2:1 to 1:2, preferably 2:1 to 1:1, where the charge ratio of positive charges in DOTMA to negative charges in RNA is 1.4:1 or less. In one embodiment, the nanoparticles are lipoplexes comprising 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.

[0136] In one embodiment, the zeta potential of the nanoparticles or liposomes 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 nanoparticles or liposomes is -35 or more, -30 or more, or -25 or more. In one embodiment, the nanoparticles or liposomes have a zeta potential of 0 mV to -50 mV, preferably 0 mV to -40 mV or -10 mV to -30 mV.

[0137] In some embodiments, the polydispersity index of the nanoparticles or liposomes is 0.5 or less, 0.4 or less, or 0.3 or less, as measured by dynamic light scattering.

[0138] In some embodiments, the nanoparticles or liposomes 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.

[0139] In some embodiments, the PCV is administered intravenously, e.g., in a liposomal formulation, at a dose of 15 μg, 25 μg, 38 μg, 50 μg, or 100 μg. In some embodiments, 15 μg, 25 μg, 38 μg, 50 μg, or 100 μg of RNA is delivered per dose (i.e., the dose weight reflects the weight of the administered RNA, not the total weight of the administered formulation or lipoplex). Two or more PCVs may be administered to a subject; for example, a subject is administered one PCV having a combination of neoepitopes and a separate PCV having a different combination of neoepitopes. In some embodiments, a first PCV having 10 neoepitopes is administered in combination with a second PCV having 10 alternative epitopes.

[0140] In some embodiments, the PCV is administered to deliver the PCV to the spleen. For example, the PCV can be administered to deliver one or more antigen(s) (e.g., patient-specific neoantigens) to antigen-presenting cells (e.g., in the spleen).

[0141] Either the PCV or RNA vaccines of the present disclosure can 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 a personalized cancer vaccine (PCV), such as the RNA vaccines described above.

[0142] 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 comprise 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 3' UTR, the 3' UTR comprising (a) a 3' untranslated region of an amino-terminal enhancer of a split (AES) mRNA or a fragment thereof; and (b) a non-coding RNA of a mitochondrially encoded 12S RNA or a fragment thereof; and (5) a polynucleotide encoding a poly(A) sequence.In some embodiments, a DNA molecule of the disclosure comprises, from 5' to 3', the polynucleotide sequence GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC (SEQ ID NO: 40) and the polynucleotide sequence ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGT CACTGACAGCCTAGTAACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAA TGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (SEQ ID NO: 41).

[0143] 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, 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 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.

[0144] 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.

[0145] In some embodiments, the RNA or DNA molecules of the present disclosure contain a type IIS restriction cleavage site that allows the RNA to be transcribed under the control of a 5' RNA polymerase promoter and includes a polyadenylation cassette (poly(A) sequence), with the recognition sequence located 3' to the poly(A) sequence but the cleavage site located upstream, and thus 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. IV. PD-1 axis binding antagonists

[0146] In some embodiments, a PCV (e.g., an RNA vaccine) of the present disclosure is administered in combination with a PD-1 axis binding antagonist.

[0147] 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.

[0148] 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 a specific aspect, the ligand binding partner of PD-1 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(s). In a specific aspect, the binding partner(s) of PDL1 are 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(s). In a specific aspect, the binding partner of PDL2 is PD-1. The antagonist may be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide.

[0149] 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).

[0150] 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), and (b), wherein the light chain comprises the amino acid sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

[0151] 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 from SEQ ID NO:11 and three light chain HVRs from 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.

[0152] 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), and (b), wherein the light chain comprises the amino acid sequence: Contains EIVLTQSPAT LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14).

[0153] 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 from SEQ ID NO: 13 and three light chain HVRs from 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.

[0154] In some embodiments, the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.

[0155] 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.

[0156] In some embodiments, the anti-PD-1 antibody is REGN2810 (Regeneron), a human anti-PD1 antibody also known as LIBTAYO® and cemiplimab-rwlc.

[0157] In some embodiments, the anti-PD-1 antibody is BGB-108 (BeiGene). In some embodiments, the anti-PD-1 antibody is BGB-A317 (BeiGene).

[0158] In some embodiments, the anti-PD-1 antibody is JS-001 (Shanghai Junshi). JS-001 is a humanized anti-PD1 antibody.

[0159] In some embodiments, the anti-PD-1 antibody is STI-A1110 (Sorrento). STI-A1110 is a human anti-PD-1 antibody.

[0160] In some embodiments, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR-1210 is a human IgG4 anti-PD1 antibody.

[0161] In some embodiments, the anti-PD-1 antibody is PF-06801591 (Pfizer).

[0162] In some embodiments, the anti-PD-1 antibody is TSR-042 (also known as ANB011; Tesaro / AnaptysBio).

[0163] In some embodiments, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).

[0164] In some embodiments, the anti-PD-1 antibody is ENUM 244C8 (Enumeral Biomedical Holdings). ENUM 244C8 is an anti-PD-1 antibody that inhibits the function of PD-1 without inhibiting the binding of PDL1 to PD-1.

[0165] 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.

[0166] In some embodiments, the PD-1 antibody comprises six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and / or a combination of HVR sequences as described in 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 Medicine), WO 2014 / 206107 (applicant: Shanghai Junshi Pharmaceuticals ... and WO 2014 / 179664 (AnaptysBio), WO 2016 / 106160 (Enumeral), and WO 2014 / 194302 (Sorrento).

[0167] 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.

[0168] In some embodiments, the PD-1 binding antagonist is a peptide or 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.

[0169] 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.

[0170] 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 embodiments herein, the isolated anti-PDL1 antibody can bind to human PDL1, e.g., human PDL1 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 No. 2010 / 077634A1 and U.S. Patent No. 8,217,149, which are incorporated herein by reference.

[0171] 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;

[0172] 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 the 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 is the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7), and (b), wherein the light chain variable region sequence comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 8).

[0173] 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: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 9), and (b), wherein the light chain comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

[0174] 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 heavy and light chain sequences: (a) the heavy chain has the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 15), and (b) the light chain comprises the amino acid sequence: QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 16).

[0175] 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.

[0176] In some embodiments, the anti-PDL1 antibody is durvalumab (CAS Registry Number: 1428935-60-7), also known as MEDI4736, 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: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 17), and (b) the light chain comprises the amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18).

[0177] 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.

[0178] 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.

[0179] In some embodiments, the anti-PDL1 antibody is LY3300054 (Eli Lilly).

[0180] In some embodiments, the anti-PDL1 antibody is STI-A1014 (Sorrento). STI-A1014 is a human anti-PDL1 antibody.

[0181] In some embodiments, the anti-PDL1 antibody is KN035 (Suzhou Alphamab), a single domain antibody (dAB) generated from a camel phage display library.

[0182] 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).

[0183] In some embodiments, the PDL1 antibody comprises six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and / or heavy and light chain variable domains from the PDL1 antibodies described in U.S. Patent Application Publication No. 20160108123 (assigned to Novartis), WO 2016 / 000619 (applicant: Beigene), WO 2012 / 145493 (applicant: Amplimmune), U.S. Patent No. 9205148 (assigned to Medimmune), WO 2013 / 181634 (applicant: Sorrento), and WO 2016 / 061142 (applicant: Novartis).

[0184] In a still further specific aspect, the antibody further comprises a human or mouse constant region. In a still further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In a still further specific aspect, the human constant region is IgG1. In a still further aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In a still further aspect, the mouse constant region is IgG2A.

[0185] In a still further specific aspect, the antibody has reduced or minimal effector function. In a more specific aspect, the minimal effector function results from an "effector-less Fc mutation" or glycosylation mutation. In a still further embodiment, 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 within a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of the sugars N-acetylgalactosamine, galactose, or xylose to one of the hydroxyamino acids, 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 the asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

[0186] In yet a further embodiment, the present disclosure provides a composition comprising any of the above-mentioned anti-PDL1 antibodies in combination with at least one pharmaceutically acceptable carrier.

[0187] 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. V. Antibody preparation

[0188] The antibodies described herein are prepared using techniques available in the art for generating antibodies, exemplary methods of which are described in more detail in the following sections.

[0189] The antibody is directed against an antigen of interest (e.g., PD-1 or PD-L1, such as human PD-1 or PD-L1). Preferably, the antigen is a biologically important polypeptide, such that administration of the antibody to a mammal suffering from a disorder can provide a therapeutic benefit to the mammal.

[0190] In certain embodiments, the antibodies provided herein have a cytotoxicity of 1 μM or less, 150 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0191] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed using the Fab version of the antibody of interest and its antigen, as illustrated by the following assay: The solution binding affinity of the Fab for the antigen is determined by the lowest concentration ( 125I) Fab is equilibrated with labeled antigen, followed by measurement by capturing the bound antigen on a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol., 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / mL of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc No. 269620), 100 pM or 26 pM [ 125 [I] The antigen is mixed with serial dilutions of the Fab of interest. The Fab of interest is then incubated overnight, although incubation may be continued for a longer period (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that results in 20% or less of maximum binding is selected for use in the competitive binding assay.

[0192] According to another embodiment, Kd is measured using a surface plasmon resonance assay with a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip at approximately 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) are injected at a flow rate of approximately 25 μl / min at 25° C. Association rates (k) and dissociation rates (k) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software Version 3.2). The equilibrium dissociation constant (K) is calculated as the ratio k / k. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).If the association rate by the surface plasmon resonance assay described above exceeds 10 M s, the association rate can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing antigen concentrations, as measured in a spectrometer such as a spectrophotometer equipped with stopped flow (Aviv Instruments) or an 8000 Series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette. Chimeric antibodies, humanized antibodies, human antibodies

[0193] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0194] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Optionally, a humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0195] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (description of SDR (a-CDR) grafting), Padlan, Mol. Immunol. 28:489-498 (1991) (description of "resurfacing"), Dall'Acqua et al., Methods 36:43-60 (2005) (description of "FR shuffling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (description of a "guided selection" approach to FR shuffling).

[0196] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997), and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0197] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0198] Human antibodies can be prepared by administering immunogens to transgenic animals that have been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci, or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0199] Human antibodies can also be produced using hybridoma techniques. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006), which describes human-human hybridomas. Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0200] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below. antibody fragment

[0201] Antibody fragments can be produced by conventional means such as enzymatic digestion or by recombinant techniques. Under certain circumstances, there are advantages to using antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and may result in improved access to solid tumors. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

[0202] Various techniques have been developed to produce antibody fragments. Traditionally, these fragments were obtained via proteolysis of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli, allowing for the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having increased in vivo half-lives are described in U.S. Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single-chain Fv fragment (scFv). See WO 93 / 16185, U.S. Pat. Nos. 5,571,894, and 5,587,458. Fvs and scFvs are the only species with intact binding sites lacking constant regions and, therefore, may be suitable for reducing nonspecific binding during in vivo use. scFv fusion proteins can be constructed, resulting in fusion of an effector protein at either the amino or carboxy terminus of the scFv. See Antibody Engineering, ed. Borrebaeck, supra. An antibody fragment may also be a "linear antibody," for example, as described in U.S. Patent No. 5,641,870. Such linear antibodies may be monospecific or bispecific. Single Domain Antibodies

[0203] In some embodiments, the antibodies of the present disclosure are single-domain antibodies. A single-domain antibody is a single polypeptide chain that comprises all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In one embodiment, a single-domain antibody consists of all or a portion of the heavy chain variable domain of an antibody. Antibody variants

[0204] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid alterations can be introduced into the amino acid sequence of the subject antibodies when that sequence is generated. Substitutional, insertional, and deletion mutants

[0205] In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 3. More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions," and as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. TIFF2026004341000006.tif133170

[0206] Amino acids can be grouped according to common side chain properties. a. Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. Acidic: Asp, Glu; d. Basic: His, Lys, Arg; e. Affected residues Chain orientation: Gly, Pro; f. Aromatics: Trp, Tyr, Phe.

[0207] Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0208] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have a modification (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which may be conveniently generated using, for example, phage-display-based affinity maturation techniques described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0209] Modifications (e.g., substitutions) can be made to HVRs to, for example, improve antibody affinity. Such modifications may be made in HVR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted.

[0210] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such modifications may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains no more than one, two, or three amino acid substitutions.

[0211] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody's interaction with the antigen is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex may be used to identify contact points between the antibody and antigen. Such contact and neighboring residues may be targeted as candidates for substitution or removed. Mutants may be screened to determine whether they possess desired properties.

[0212] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody (e.g., for ADEPT). Glycosylation variants

[0213] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0214] If the antibody comprises an Fc region, the carbohydrate attached to the antibody may be varied. Natural antibodies produced by mammalian cells typically contain biantennary oligosaccharides, generally attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNac), galactose, and sialic acid, as well as fucose attached to the GlcNac in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present disclosure may be made to generate antibody variants with improved certain properties.

[0215] In one embodiment, antibody variants comprising an Fc region are provided, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function. Specifically, antibodies having reduced fucose compared to the amount of fucose in the same antibody produced in wild-type CHO cells are contemplated herein. That is, they are characterized by having a lower amount of fucose than they would otherwise have if produced by native CHO cells (e.g., CHO cells producing a native glycosylation pattern, such as CHO cells containing a native FUT8 gene). In certain embodiments, the antibody is one in which less than about 50%, 40%, 30%, 20%, 10%, or 5% of its N-linked glycans contain fucose. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In certain embodiments, the antibody does not contain fucose in any of its N-linked glycans, i.e., the antibody has no fucose, no fucose, or is afucosylated. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297 relative to the sum of all glycan structures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures) as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues) within the Fc region; however, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; WO 2002 / 0164328; WO 2004 / 0093621; WO 2004 / 01 32140; 2004 / 0110704; 2004 / 0110282; 2004 / 0109865; WO 2003 / 085119; 2003 / 084570; 2005 / 035586; 2005 / 035778; 2005 / 053742; 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108A1, Presta, L; and WO 2004 / 056312A1, Adams et al., especially in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng, 94(4):680-688 (2006); and WO 2003 / 085107).

[0216] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody, bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.); and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0217] In certain embodiments, antibody variants comprising an Fc region described herein are capable of binding to FcγRIII. In certain embodiments, antibody variants comprising an Fc region described herein have ADCC activity in the presence of human effector cells, or have increased ADCC activity in the presence of human effector cells compared to the same antibody but comprising a human wild-type IgG1 Fc region. Fc region variants

[0218] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0219] In certain embodiments, the present disclosure contemplates antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications in which antibody half-life in vivo is important, but where certain effector functions (e.g., complement and ADCC, etc.) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express Fc(RIII) only, whereas monocytes express Fc(RI), Fc(RII), and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (Bruggemann, M. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985)). al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo in an animal model, e.g., as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0220] Antibodies with reduced effector function include antibodies with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0221] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0222] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at Fc region positions 298, 333, and / or 334 (EU numbering of residues). In one exemplary embodiment, the antibody comprises the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.

[0223] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0224] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve binding to FcRn. Such Fc variants include variants with substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826)). See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Pat. No. 5,648,260, U.S. Pat. No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants. VI. Pharmaceutical Compositions and Formulations

[0225] Also provided herein are pharmaceutical compositions and formulations, for example, for the treatment of cancer. In some embodiments, the pharmaceutical compositions and formulations further comprise a pharmaceutically acceptable carrier.

[0226] After the antibody of interest is prepared (e.g., techniques for producing antibodies that can be formulated as disclosed herein are extensively described herein and known in the art), a pharmaceutical formulation comprising it is prepared. In certain embodiments, the antibody to be formulated 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, in which case issues that do not occur with full-length antibodies (e.g., clipping of the antibody to Fab) may need to be addressed. The therapeutically effective amount of the antibody present in the formulation is determined, for example, by considering the desired dose volume and mode(s) 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 antibodies described herein (e.g., atezolizumab) are 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).

[0227] In some embodiments, the RNA vaccines described herein are administered at a dose of about 15 μg, about 25 μg, about 38 μg, about 50 μg, or about 100 μg.

[0228] 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 (e.g., 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 employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10 residues) polypeptides. Proteins such as serum 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 dextrin; 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 intercalating drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as 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 (e.g., chondroitinases).

[0229] 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 comprising a histidine acetate buffer.

[0230] The compositions and formulations herein may contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0231] The active ingredient may also be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in 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).

[0232] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films or microcapsules. Preparations to be used for in vivo administration are generally sterilized. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0233] 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 commercial products. VII. Treatment Methods

[0234] Provided herein are methods for treating or delaying the progression of cancer in an individual, comprising administering to the individual effective amounts of a PD-1 axis binding antagonist and an RNA vaccine. In some embodiments, the individual is a human.

[0235] Both the PD-1 axis binding antagonists and RNA vaccines of the present disclosure may find use in the treatment methods described herein. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 10-20 neoepitopes arising from cancer-specific somatic mutations present in a tumor specimen. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 neoepitopes arising from cancer-specific somatic mutations present in a tumor specimen. In some embodiments, the RNA vaccine is formulated in lipoplex nanoparticles or liposomes. In some embodiments, lipoplex nanoparticle formulations for RNA (RNA-lipoplexes) are used to enable IV delivery of the RNA vaccines of the present disclosure. In some embodiments, the PCV is administered intravenously at a dose of 15 μg, 25 μg, 38 μg, 50 μg, or 100 μg, for example, in a liposomal formulation. In some embodiments, 15 μg, 25 μg, 38 μg, 50 μg, or 100 μg of RNA is delivered per dose (i.e., the dose weight reflects the weight of the RNA administered, not the total weight of the formulation or lipoplex administered). More than one PCV may be administered to a subject; for example, a subject is administered one PCV having a combination of neoepitopes and a separate PCV having a different combination of neoepitopes. In some embodiments, a first PCV having 10 neoepitopes is administered in combination with a second PCV having 10 alternative epitopes. In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-1 antibody, including but not limited to, pembrolizumab. In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-L1 antibody, including but not limited to, atezolizumab.

[0236] In some embodiments, the PD-1 axis-binding antagonist is administered to an individual at 21-day or 3-week intervals. In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-1 antibody (e.g., pembrolizumab) administered to an individual at 21-day or 3-week intervals, for example, at a dose of about 200 mg. In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-1 antibody (e.g., cemiplimab-rwlc) administered to an individual at 21-day or 3-week intervals, for example, at a dose of about 350 mg. In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-L1 antibody (e.g., atezolizumab) administered to an individual at 21-day or 3-week intervals, for example, at a dose of about 1200 mg.

[0237] In some embodiments, the PD-1 axis-binding antagonist is administered to the individual at 14-day or 28-day intervals. In some embodiments, the PD-1 axis-binding antagonist is administered to the individual at 2-week or 4-week intervals. In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-1 antibody (e.g., nivolumab) administered to the individual at 14-day, 2-week, 28-day, or 4-week intervals, e.g., at a dose of about 240 mg at 14-day or 2-week intervals, or at a dose of about 480 mg at 28-day or 4-week intervals. In some embodiments, the PD-1 axis binding antagonist is an anti-PD-1 antibody (e.g., nivolumab) that is administered to an individual at 21 day or 3 week intervals, e.g., at a dose of about 1 mg / kg in 1, 2, 3, or 4 doses, optionally in combination with an anti-CTLA-4 antibody (e.g., ipilimumab), optionally followed by administration of the anti-PD-1 antibody (e.g., nivolumab) alone at 14 day, 2 week, 28 day, or 4 week intervals, e.g., at a dose of about 240 mg at 14 day or 2 week intervals, or at a dose of about 480 mg at 28 day or 4 week intervals.

[0238] In some embodiments, the PD-1 axis-binding antagonist is administered to the individual at intervals of 14 days or 2 weeks. In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-L1 antibody (e.g., durvalumab) administered to the individual at intervals of 14 days or 2 weeks, for example, at a dose of about 10 mg / kg (optionally by intravenous infusion over 60 minutes). In some embodiments, the PD-1 axis-binding antagonist is an anti-PD-L1 antibody (e.g., avelumab) administered to the individual at intervals of 14 days or 2 weeks, for example, at a dose of about 10 mg / kg (optionally by intravenous infusion over 60 minutes).

[0239] In some embodiments, the RNA vaccine is administered to an individual at 21 day or 3 week intervals.

[0240] In some embodiments, the PD-1 axis-binding antagonist and the RNA vaccine are administered to the individual in eight 21-day cycles. In some embodiments, the RNA vaccine is administered to the individual on days 1, 8, and 15 of cycle 2 and on day 1 of cycles 3 through 7. In some embodiments, the PD-1 axis-binding antagonist is administered to the individual on day 1 of cycles 1 through 8. In some embodiments, the RNA vaccine is administered to the individual on days 1, 8, and 15 of cycle 2 and on day 1 of cycles 3 through 7, and the PD-1 axis-binding antagonist is administered to the individual on day 1 of cycles 1 through 8.

[0241] In some embodiments, the PD-1 axis-binding antagonist and the RNA vaccine are further administered to the individual after cycle 8. In some embodiments, the PD-1 axis-binding antagonist and the RNA vaccine are further administered to the individual for 17 additional 21-day cycles, the PD-1 axis-binding antagonist is administered to the individual on day 1 of cycles 13-29, and / or the RNA vaccine is administered to the individual on day 1 of cycles 13, 21, and 29.

[0242] In a specific embodiment, the PD-1 axis-binding antagonist and the RNA vaccine are administered to the individual in eight 21-day cycles, wherein the PD-1 axis-binding antagonist is pembrolizumab, and the PD-1 axis-binding antagonist is administered to the individual at a dose of about 200 mg on day 1 of cycles 1 through 8, and the RNA vaccine is administered to the individual at a dose of about 25 μg on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3 through 7. In a specific embodiment, the PD-L1 axis-binding antagonist and the RNA vaccine are administered to the individual in eight 21-day cycles, wherein the PD-L1 axis-binding antagonist is atezolizumab, and the PD-L1 axis-binding antagonist is administered to the individual at a dose of about 1200 mg on day 1 of cycles 1 through 8, and the RNA vaccine is administered to the individual at a dose of about 25 μg on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3 through 7. In some embodiments, the RNA vaccine is administered to an individual at doses of about 25 μg on day 1 of cycle 2, about 25 μg on day 8 of cycle 2, about 25 μg on day 15 of cycle 2, and about 25 μg on day 1 of each of cycles 3 through 7 (i.e., a total of about 75 μg of vaccine is administered to an individual over three doses during cycle 2). In some embodiments, a total of about 75 μg of vaccine is administered to an individual over three doses during cycle 1 in which the RNA vaccine is administered.

[0243] In a specific embodiment, the PD-1 axis-binding antagonist and the RNA vaccine are administered to the individual over eight 21-day cycles, wherein the PD-1 axis-binding antagonist is pembrolizumab, and the PD-1 axis-binding antagonist is administered to the individual at a dose of 200 mg on day 1 of cycles 1 through 8, and the RNA vaccine is administered to the individual at a dose of 25 μg on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3 through 7. In a specific embodiment, the PD-L1 axis-binding antagonist and the RNA vaccine are administered to the individual over eight 21-day cycles, wherein the PD-L1 axis-binding antagonist is atezolizumab, and the PD-L1 axis-binding antagonist is administered to the individual at a dose of 1200 mg on day 1 of cycles 1 through 8, and the RNA vaccine is administered to the individual at a dose of 25 μg on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3 through 7. In some embodiments, the RNA vaccine is administered to an individual at doses of 25 μg on day 1 of cycle 2, 25 μg on day 8 of cycle 2, 25 μg on day 15 of cycle 2, and 25 μg on day 1 of each of cycles 3 through 7 (i.e., a total of 75 μg of vaccine is administered to an individual over three doses during cycle 2). In some embodiments, a total of 75 μg of vaccine is administered to an individual over three doses during cycle 1, in which the RNA vaccine is administered.

[0244] The PD-1 axis binding antagonist and the RNA vaccine 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 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.

[0245] In some embodiments, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, bladder cancer, colorectal cancer, triple-negative breast cancer, renal cancer, and head and neck cancer. In some embodiments, the cancer is locally advanced or metastatic melanoma, non-small cell lung cancer, bladder cancer, colorectal cancer, triple-negative breast cancer, renal cancer, or head and neck cancer. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, bladder cancer, colorectal cancer, triple-negative breast cancer, renal cancer, and head and neck cancer. In some embodiments, the cancer is locally advanced or metastatic non-small cell lung cancer, bladder cancer, colorectal cancer, triple-negative breast cancer, renal cancer, or head and neck cancer.

[0246] In some embodiments, the cancer is melanoma. In some embodiments, the melanoma is cutaneous melanoma or mucosal melanoma. In some embodiments, the melanoma is cutaneous, mucosal, or acral melanoma. In some embodiments, the melanoma is not ocular or acral melanoma. In some embodiments, the melanoma is metastatic or unresectable locally advanced melanoma. In some embodiments, the melanoma is stage IV melanoma. In some embodiments, the melanoma is stage IIIC or stage IIID melanoma. In some embodiments, the melanoma is unresectable or metastatic melanoma. In some embodiments, the method provides adjuvant treatment of melanoma.

[0247] In some embodiments, the cancer (e.g., melanoma) has not been previously treated. In some embodiments, the cancer is locally advanced melanoma.

[0248] In some embodiments, prior to treatment with a PD-1 axis binding antagonist and an RNA vaccine according to any of the methods described herein, the individual has progressed after treatment with a PD-1 axis binding antagonist-based monotherapy, e.g., treatment with pembrolizumab in the absence of the RNA vaccine, or has not responded adequately to treatment.

[0249] The PD-1 axis binding antagonist and the RNA vaccine 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 lipoplex particles or liposomes). In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are administered via intravenous infusion. Effective amounts of the PD-1 axis binding antagonist and the RNA vaccine can be administered for the prevention or treatment of disease.

[0250] In some embodiments, these methods may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery (e.g., tumor resection and mastectomy), 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. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. VIII. Products or Kits

[0251] Further provided herein are articles of manufacture or kits comprising a PD-1 axis binding antagonist (e.g., atezolizumab or pembrolizumab). In some embodiments, the articles of manufacture or kits further comprise a package insert containing instructions for using the PD-1 axis binding antagonist in conjunction with an RNA vaccine to treat or delay the progression of cancer in an individual, or to enhance immune function in an individual with cancer. Also provided herein are articles of manufacture or kits comprising a PD-1 axis binding antagonist (e.g., atezolizumab or pembrolizumab) and an RNA vaccine.

[0252] 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 perspective, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture 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.

[0253] 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]

[0254] The present disclosure will be more fully understood by reference to the following examples. However, these examples should not be construed as limiting the scope of the present invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art in light of these, which should be included within the spirit and scope of this application and the scope of the appended claims. Example 1: A Phase II, Open-Label, Multicenter, Randomized Study of the Efficacy and Safety of an RNA Vaccine in Combination with Pembrolizumab in Patients with Previously Untreated Advanced Melanoma Rationale

[0255] As noted above, checkpoint inhibitors are currently the standard of care for metastatic melanoma. However, the durable clinical benefits observed with agents targeting PD-L1 / PD-1 across a variety of malignancies, including melanoma, appear to be limited to a subset of patients. Despite advances in OS with the development of currently widely administered immunotherapies, such as PD-1 therapy (nivolumab, pembrolizumab) or the combination of anti-PD1 and anti-CTLA-4 therapy (nivolumab and ipilimumab), a significant proportion of patients fail to respond to checkpoint inhibitor treatment or experience only transient disease stabilization (Robert C, Long GV, Brady B, et al. N Engl J Med 2015a;372:320-30; Rosenberg JE, Hoffman-Censits J, Powles T, et al. Lancet 2016;387:1909-20), demonstrating a continuing unmet need for treatment for patients with metastatic solid tumors. Although objective responses in approximately 10%–30% of patients who respond to treatment with PD-1 inhibitors tend to be durable, these patients nonetheless remain at risk of progression. In a recent study of melanoma patients treated with PD-1 blockade, 53 of 205 patients (26%) who had an objective response to pembrolizumab had disease progression at a median follow-up of 21 months (Ribas A, Hamid O, Daud A, et al. JAMA 2016;315:1600-9).

[0256] Anti-PD1 and anti-PD1 plus anti-CTLA-4 combinations have significantly improved long-term outcomes in melanoma patients, but melanoma has come at the cost of increased treatment-related toxicity. Despite these improvements, a significant proportion of patients remain at risk for disease progression and succumb to disease. Combination therapies that address mechanisms of resistance checkpoint blockade associated with increased toxicity are needed.

[0257] Resistance can occur at the level of effector T cells, whose activity can be limited by insufficient T cell stimulation. In preclinical models, induction of antigen-specific immunity in combination with simultaneous blockade of the PD-L1 / PD-1 pathway demonstrated superior efficacy to single-agent inhibitors of each of these pathways, even in models in which single-agent vaccines have limited activity. In these studies, tumor-infiltrating T cells showed increased IFN-γ expression (a hallmark of T cell activation and antitumor activity) only when PD-L1 was blocked, but not when single-agent vaccines were used (Duraiswamy J, Kaluza KM, Freeman GJ, et al. Cancer Res 2013;73:3591-603; Fu J, Malm IJ, Kadayakkara DK, et al. Cancer Res 2014;74:4042-52). Based on these studies, it is hypothesized that the combination of RO7198457 and anti-PD-L1 / PD-1 may result in the activation of anti-tumor immune responses, leading to enhanced tumor cell killing and improved clinical responses in cancer patients. the purpose

[0258] This study will evaluate the efficacy, safety, pharmacokinetics, and patient-reported outcomes (PROs) of a personalized RNA neoepitope vaccine (PCV), RO7198457 plus pembrolizumab, compared with pembrolizumab alone in patients with previously untreated advanced melanoma. The specific objectives and corresponding endpoints of the study are outlined below.

[0259] The primary efficacy endpoint of this study is to evaluate the efficacy of RO7198457 plus pembrolizumab compared with pembrolizumab alone based on the following endpoints: Progression-free survival (PFS) after randomization (defined as the time from randomization to first disease progression or death from any cause, whichever occurs first, as determined by the investigator according to Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1)). Objective response rate (ORR) (defined as the proportion of patients with a complete response (CR) or partial response (PR) on two consecutive occasions ≥ 4 weeks apart, as determined by the investigator according to RECIST v1.1)

[0260] The secondary efficacy endpoints of this study are to evaluate the efficacy of RNA neoepitope vaccine plus pembrolizumab compared with pembrolizumab alone based on the following endpoints: Overall survival (OS) after randomization (defined as the time from randomization to death from any cause) Duration of response (DOR) (defined as the time from the first occurrence of a demonstrated objective response to disease progression or death from any cause as determined by the investigator according to RECIST v1.1) Mean change from baseline in health-related quality of life (HRQoL) scores, assessed by using the two-item global health status (GHS) / HRQoL subscale (questions 29 and 30) of the European Organization for Research and Treatment of Cancer Quality of Life-Core 30 (EORTC QLQ-C30), at specified time points

[0261] Another secondary efficacy endpoint of this study is to evaluate the proportion of participants with an objective response of CR or PR after crossover from pembrolizumab monotherapy to combination therapy (e.g., RNA neoepitope vaccine + pembrolizumab).

[0262] Another secondary endpoint is to evaluate the efficacy of RNA neoepitope vaccine plus pembrolizumab in patients who have progressed after pembrolizumab monotherapy, based on the following endpoints: ORR: At the time of crossover, defined as the proportion of patients with CR or PR on two consecutive occasions ≥ 4 weeks apart, as determined by the investigator according to RECIST v1.1

[0263] Another objective of this study is to assess the incidence and severity of adverse events (AEs). Research Plan

[0264] This is a Phase II, randomized, open-label, multicenter study designed to evaluate the efficacy and safety of RO7198457 (PCV) plus pembrolizumab compared with pembrolizumab alone in patients with previously untreated advanced melanoma. The patient population includes patients with unresectable locally advanced (stages IIIC and IIID) and metastatic (recurrent or de novo stage IV) melanoma. The study will be conducted comprehensively.

[0265] The study consisted of two phases: an initial safety run-in phase and a randomization phase (Figure 1). Each phase had a two-part screening period, a treatment period, and a post-treatment follow-up period.

[0266] The safety run-in phase will consist of a single arm enrolling approximately 6-12 patients to receive one cycle (21 days) of 200 mg pembrolizumab administered by IV infusion, followed by 25 μg RO7198457 + 200 mg pembrolizumab IV (Q3W) every 3 weeks for subsequent cycles. Accrual in the randomized phase will not begin until the Internal Monitoring Committee (IMC) has reviewed the safety data from the first 6 patients treated in the safety run-in phase.

[0267] The randomization phase will enroll approximately 120 patients randomized in a 2:1 ratio to either the experimental or control group: Group A (control): 200 mg pembrolizumab administered by IV infusion Q3W Arm B (experimental): 200 mg pembrolizumab administered by IV infusion for one cycle, followed by 25 μg RO7198457 + 200 mg pembrolizumab IV Q3W in subsequent cycles

[0268] Upon confirmed disease progression (as assessed by the investigator according to RECIST v1.1), patients randomized to Arm A will have the option to receive crossover and combination treatment with RO7198457 and pembrolizumab, provided they meet the eligibility criteria.

[0269] During the first part of the screening period (Part A), consenting patients will be evaluated for preliminary eligibility (e.g., Eastern Cooperative Oncology Group [ECOG] Performance Status, blood chemistries, HIV serology, hepatitis B virus [HBV], and hepatitis C virus [HCV]), tumor tissue and blood samples will be collected to define tumor-specific somatic mutations, and human leukocyte antigen (HLA) typing will be performed to enable manufacturing of RO7198457. The currently planned manufacturing turnaround time is approximately 4-6 weeks after receipt of sufficient quantity and quality of blood and tumor samples. The second part of the screening period (Part B) is the 28 days prior to Day 1 to confirm patient eligibility.

[0270] Eligible patients include male and female patients ≥18 years of age with histologically confirmed measurable stage IIIC or IIID (unresectable) or metastatic (recurrent or de novo stage IV) invasive cutaneous or mucosal melanoma and an ECOG performance status of 0 or 1 who have not received prior treatment for progressive disease. Patients with ocular or acral melanoma or untreated CNS metastases are not eligible. Prior adjuvant therapy with ipilimumab, BRAF inhibitors, and / or MEK inhibitors is permitted. Prior adjuvant therapy with anti-PD-1 / PD-L1 agents is permitted, provided the last dose was administered at least 6 months prior to Day 1 of Cycle 1. Patients must be able to provide tumor specimens for vaccine manufacturing and PD-L1 testing.

[0271] As shown in Figure 2, patients in Group A (pembrolizumab) received 200 mg of pembrolizumab by IV infusion Q3W starting in Cycle 1. Patients in Group B (25 μg RO7198457 + 200 mg pembrolizumab) in the safety run-in and randomization phases receive pembrolizumab administered by IV infusion Q3W starting in Cycle 1. Cycle 1 is a pembrolizumab monotherapy run-in to allow time for vaccine production. RO7198457 + pembrolizumab begins in Cycle 2, with RO7198457 administered by IV infusion 30 minutes after completion of the pembrolizumab infusion. For the safety run-in phase and Group B, RO7198457 will be administered starting on Day 1 of Cycle 2, then on Days 8 and 15 of Cycle 2, Day 1 of Cycles 3-7, and then every 8 cycles (Cycles 13, 21, and 29) starting with Cycle 13 as a maintenance treatment. Patients who experience a delay in initiation of combination treatment with RO7198457 (e.g., RO7198457 is not available until Day 1 of Cycle 2) or an interruption during induction of RO7198457 may be permitted to start combination treatment after Day 1 of Cycle 2 and / or receive a top-up dose of RO7198457 after the initial treatment period to achieve a total of 8 induction doses, with medical monitor approval (e.g., a patient who misses Day 1 of Cycle 2 starts RO7198457 on Day 8 of Cycle 2 and receives a top-up dose on Day 8 of Cycle 3 as an unscheduled visit; a patient who starts RO7198457 on Day 15 of Cycle 2 receives top-up doses on both Days 8 and 15 of Cycle 3 as unscheduled visits, etc.).

[0272] The treatment duration for this study will be a maximum of 24 months for all patients, as long as they experience clinical benefit as assessed by the investigator in the absence of unacceptable toxicity or symptomatic worsening due to disease progression after an integrated assessment of radiographic data and clinical status. Patients may be permitted to continue treatment after RECIST v1.1 criteria for progressive disease are met. Patients in Arm A may have the option to switch to combination treatment with RO7198457 plus pembrolizumab after confirmed disease progression if crossover eligibility criteria are met. Furthermore, if patients in Arm A complete 24 months of pembrolizumab and experience confirmed disease progression <6 months after discontinuing pembrolizumab, they may have the option to undergo crossover treatment with RO7198457 plus pembrolizumab.

[0273] Patients will undergo tumor assessments at baseline (Cycle 1, Day 1), Week 12, and every 6 weeks thereafter (every 2 cycles) for the first 48 weeks after Cycle 1, Day 1. If indicated, digital photography of skin lesions will be performed at screening and the first visit after each tumor assessment. From Cycle 1, Day 1 through Week 48, patients will undergo tumor assessments every 12 (±1) weeks (approximately every 4 cycles). Tumor assessments will continue until discontinuation of study treatment, withdrawal of consent, sponsor-initiated study discontinuation, or death, whichever occurs first. After experiencing disease progression resulting in treatment interruption, patients will also be asked to return to the clinic approximately 6 (±2) weeks later for a confirmatory tumor assessment, if feasible. Patients who discontinue treatment for reasons other than disease progression (e.g., toxicity) should continue scheduled tumor assessments until disease progression, withdrawal of consent, sponsor-initiated study discontinuation, or death, whichever occurs first. Primary imaging data used for tumor assessment will be collected by the sponsor to allow centralized and independent review of response endpoints, if necessary.

[0274] In addition, patients will also be asked to complete PRO assessments at the start of each cycle until disease progression or treatment discontinuation, whichever occurs later. Inclusion and Exclusion Criteria

[0275] Patients must meet the following study entry criteria: -Age 18 or older at the time of signing the informed consent document Histologically confirmed metastatic (recurrent or de novo stage IV) or unresectable locally advanced (stage IIIC or IIID) cutaneous or mucosal melanoma as defined by AJCC v8.0 (Amin MB, Edge SB, Greene FL, et al., editors. AJCC cancer staging manual. 8th rev ed. New York: Springer; 2017) Enrollment of patients with mucosal melanoma will be limited to approximately 10 patients. ECOG performance status 0 or 1 Life expectancy ≥ 12 weeks Adequate hematologic and end-organ function as defined by the following laboratory test results obtained within 28 days prior to the first study treatment (Cycle 1, Day 1): ANC ≥ 1,500 cells / μL (no granulocyte colony-stimulating factor [GCSF] support within 2 weeks prior to Day 1 of Cycle 1) 〇White blood cell count ≧2,500 / μL Platelet count ≥ 100,000 / μL (no transfusion within 14 days prior to Day 1 of Cycle 1) Hemoglobin ≥ 9 g / dL (patients may be transfused or undergo erythropoietic treatment according to local standard of care) Total bilirubin ≤ 1.5 × ULN, with the following exceptions: Patients with known Gilbert's disease: serum bilirubin concentration ≤ 3 × ULN. AST and ALT ≤ 3 × ULN o ALP≦2.5×ULN, with the following exception: patients with documented liver or bone metastases may have an ALP≦5×ULN. Serum albumin ≥ 2.5g / dL Measured or calculated creatinine CL ≥ 50 mL / min based on Cockcroft-Gault glomerular filtration rate estimates: (140 - age) x (weight in kilograms) x (0.85 for women) 72×(serum creatinine mg / dL) Measured disease per RECIST v1.1. Previously irradiated lesions should not be counted as target lesions unless progression within the lesion is demonstrated and other target lesions are available. Lesions intended for biopsy should not be counted as target lesions. Skin lesions and other superficial lesions detectable only by physical examination should not be counted as target lesions but may be included as non-target lesions. Naive to previous systemic anti-cancer therapy for advanced melanoma (e.g., chemotherapy, hormonal therapy, targeted therapy, immunotherapy, or other biologic therapy), except for the following adjuvant therapies: Adjuvant treatment with anti-PD1 / PD-L1 or anti-CTLA-4, if discontinued at least 6 months prior to Cycle 1, Day 1, and does not meet any of the following criteria: Any history of immune-related grade 4 adverse events attributable to previous CIT (other than endocrine deficiencies managed with replacement therapy or asymptomatic elevations of serum amylase or lipase) Any history of immune-related grade 3 adverse events attributable to prior CIT that required permanent discontinuation of the prior immunotherapy agent according to local prescribing information, European Society for Medical Oncology (ESMO) guidelines (Haanen JBAG, Carbonnel F, Robert C, et al. Ann Oncol 2017;28:iv119-iv142), or American Society of Clinical Oncology (ASCO) guidelines (Brahmer JR, Lacchetti C, Schneider BJ, et al. J Clin Oncol 2018;36:1714-68). Adverse events from prior anticancer therapy that have not resolved to Grade ≤1, except for alopecia, vitiligo, or endocrinopathy managed with replacement therapy. Patients with asymptomatic elevations of lipase / amylase may be eligible after discussion with the medical monitor. Immune-mediated adverse events related to previous CIT (other than endocrinopathy or stable vitiligo controlled with replacement therapy) that have not resolved to baseline. Patients treated with corticosteroids for immune-mediated adverse events must demonstrate freedom from related symptoms or signs for ≥ 4 weeks after discontinuation of corticosteroids. Adjuvant treatment with targeted therapy (e.g., BRAFi / MEKi) if discontinued at least 2 months before initiation of study treatment Adjuvant treatment with herbal therapy if discontinued at least 7 days before the start of study treatment The availability of representative tumor specimens in formalin-fixed, paraffin-embedded blocks (preferred) or sectioned tissue (as described in the laboratory manual) was confirmed along with the relevant pathology report. Acceptable specimens may also include core needle biopsies (minimum 5 cores) for deep tumor tissue, and excisional, incisional, punch, or forceps biopsies for skin, subcutaneous, or mucosal lesions. Patients with fewer than 5 cores may be considered eligible with approval from the medical monitor. Fine needle aspirates, brushings, cell pellets from effusions or ascites, and lavage fluid samples are not acceptable. Tumor tissue from bone metastases is difficult to assess for PD-L1 expression and should be avoided. However, if the bone metastasis site is the only viable tissue source, it may be an acceptable tumor specimen with approval from the medical monitor. Decalcified bone tissue may be acceptable before decalcification, as many reagents contain strong acids that damage the antigen used for PD-L1 IHC and nucleic acids for sequencing. If suitable tissue is available from different time points (e.g., at initial diagnosis and disease recurrence) and / or from multiple metastatic tumors, preference should be given to the most recently collected tissue (ideally after the most recent systemic adjuvant therapy). While multiple samples may be collected from a given patient based on availability, the requirement for block or sectioned tissue should be met by a single biopsy or resection specimen. Patients with insufficient or unavailable archival tissue are ineligible due to the need for evaluable tumor tissue to create a PCV unless the patient consents to and is willing to undergo a pretreatment biopsy sample of the tumor (see above for acceptable samples). Enrollment is limited to patients with at least five identified tumor neoantigens and sufficient tumor material (both quality and quantity) to allow for donor-defined vaccine production. Archival tumor tissue is acceptable for CIT-naive patients and must be submitted and evaluated for mutation assessment before enrollment. A baseline tumor biopsy is required for CIT-experienced patients (i.e., patients treated with immune checkpoint inhibitors in the adjuvant setting) and must be submitted and evaluated for mutation assessment before enrollment. CIT-experienced patients who have undergone a tumor biopsy after CIT but before enrollment may use that tissue for screening if sufficient material exists. If available, patients should also submit archival tumor tissue for evaluation. Archival tissue may also be used for CIT-experienced patients if the baseline fresh tumor biopsy is insufficient for production. Patients with unevaluable tumor tissue or an insufficient number of mutations for vaccine production are ineligible. For women of childbearing potential: agreement to abstain (refrain from heterosexual intercourse) or use contraception, and agreement to refrain from egg donation. For men: Agreement to abstain (refrain from sexual intercourse with the opposite sex) or use condoms, and agreement to refrain from donating sperm.

[0276] Patients who meet any of the following criteria will be excluded from study enrollment: Ocular or acral melanoma Pregnant or nursing, or intending to become pregnant during the study or within 1 month after the last dose of RO7198457 or within 4 months after the last dose of pembrolizumab, whichever occurs later. Women of childbearing potential (including those who have had a tubal ligation) must have a negative serum pregnancy test result within 14 days prior to starting study drug (i.e., Cycle 1, Day 1). ·Significant cardiovascular disease, e.g., New York Heart Association heart disease (class II or higher), myocardial infarction within the past 3 months, unstable arrhythmia, and / or unstable angina. Known clinically significant liver disease, including active viral, alcoholic or other hepatitis, cirrhosis, and inherited liver disease or current alcohol abuse Major surgical procedure within 28 days prior to Day 1 of Cycle 1 or anticipated need for major surgical procedure during the course of the study Any other disease, metabolic dysfunction, physical examination finding, or clinical laboratory finding that raises a reasonable suspicion of a disease or condition that contraindicates the use of the investigational drug, that may affect the interpretation of the results, and / or that may place the patient at high risk for treatment complications. Corticosteroids in doses greater than 7.5 mg (unless for physiological replacement) Prior splenectomy Known primary immunodeficiency, either a cellular immunodeficiency (e.g., DiGeorge syndrome, T-negative severe combined immunodeficiency [SCID]) or a combined T-cell and B-cell immunodeficiency (e.g., T- and B-negative SCID, Wiskott-Aldrich syndrome, ataxia-telangiectasia, common variable immunodeficiency) Symptomatic, untreated, or actively progressing CNS metastases. Patients with a history of CNS disease are eligible if all of the following criteria are met: Measurable disease per RECIST v1.1 must be located outside the CNS Only supratentorial and cerebellar metastases were allowed (i.e., no metastases to the midbrain, pons, medulla oblongata, or spinal cord) History of metastasis to the optic nerve (optic nerve and optic chiasm) within 10 mm No ongoing need for corticosteroids to treat CNS disease No stereotactic radiation therapy within 7 days No prior whole brain irradiation No clinical evidence of interim progression between completion of CNS-directed therapy and screening radiography Patients with newly detected asymptomatic CNS metastases on screening must have undergone radiation therapy and / or surgery for CNS metastases. After treatment, these patients may be eligible without requiring an additional brain scan before Day 1 of Cycle 1 if all other criteria are met. Treatment with stable doses of anticonvulsants is possible No history of intracranial hemorrhage from CNS lesions History of leptomeningeal metastatic disease Uncontrolled tumor-related pain. Patients requiring narcotic analgesics must be on a stable regimen at the time of study enrollment. Symptomatic lesions amenable to palliative radiation therapy (e.g., bone metastases or metastases causing nerve impingement) must be treated before enrollment. Patients must recover from the effects of radiation. There is no minimum required recovery period. Asymptomatic metastatic lesions likely to cause functional impairment or intractable pain with further growth (e.g., epidural metastases not currently associated with spinal cord compression) should be considered for locoregional therapy, if appropriate, before enrollment. Uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage multiple times every 28 days. An indwelling drainage catheter (e.g., PleurX®) is possible. Any anti-cancer therapy in the metastatic setting, whether investigational or approved, including chemotherapy, hormonal therapy, and / or radiation therapy, prior to the initiation of study treatment, with the following exceptions: Herbal Therapy > Cycle 1, 1 week before Day 1 Palliative radiotherapy for painful metastases or metastases in potentially sensitive locations (e.g., epidural space) >2 weeks prior to Cycle 1, Day 1 Prior cancer vaccines (e.g., T-vec) are not permitted. Malignancies other than those under investigation within 5 years prior to Day 1 of Cycle 1, except those with a negligible risk of metastasis or death (e.g., adequately treated non-invasive cervical cancer, basal cell or squamous cell skin cancer, localized prostate cancer, or ductal carcinoma in situ of the breast). Uncontrolled hypercalcemia (>1.5 mmol / L ionized calcium or Ca +2 Patients with symptomatic hypercalcemia requiring continued use of bisphosphonate therapy (>12 mg / dL or corrected serum calcium ≥ ULN) or bisphosphonate therapy, particularly those receiving bisphosphonate therapy or denosumab to prevent skeletal events, and with no history of clinically significant hypercalcemia, are eligible. Spinal cord compression has not been definitively treated with surgery and / or radiation, or previously diagnosed and treated spinal cord compression has no evidence that the disease has been clinically stable for ≥ 2 weeks prior to screening. · A history of autoimmune disease, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, vascular thrombosis associated with antiphospholipid syndrome, Wegener's granulomatosis, Sjogren's syndrome, Bell's palsy, Guillain-Barré syndrome, multiple sclerosis, vasculitis, or glomerulonephritis; Patients with a history of autoimmune hypothyroidism who are receiving a stable dose of thyroid replacement hormone may be eligible. Patients with controlled type 1 diabetes receiving stable insulin therapy may be eligible. Patients with eczema, psoriasis, lichen simplex chronicus, or vitiligo with only skin manifestations (e.g., without psoriatic arthritis) may be eligible if they meet the following conditions: The rash must cover less than 10% of the body surface area Disease is well controlled at baseline and requires only low-potency topical steroids No acute exacerbation of underlying disease within the past 12 months (e.g., no need for psoralen + ultraviolet A radiation, methotrexate, retinoids, biologic agents, oral calcineurin inhibitors, or high-potency or oral steroids) Treatment with a monoamine oxidase inhibitor (MAOI) within 3 weeks prior to Cycle 1, Day 1 Treatment with systemic immunosuppressants (including, but not limited to, prednisone ≥ 7.5 mg / day, cyclophosphamide, azathioprine, methotrexate, thalidomide, and TNFα antagonists) within 2 weeks prior to Cycle 1, Day 1 Patients receiving acute low-dose systemic immunosuppressants (e.g., a single dose of dexamethasone for nausea) may be enrolled in the study after consultation with and approval from the medical monitor: Use of inhaled corticosteroids (e.g., fluticasone for chronic obstructive pulmonary disease) is permitted. The use of oral mineralocorticoids (e.g., fludrocortisone for patients with orthostatic hypotension) is acceptable. Physiological doses of corticosteroids for adrenal insufficiency are tolerated. History of idiopathic pulmonary fibrosis, pneumonitis (including drug-induced), organizing pneumonia (i.e., bronchiolitis obliterans, latent organizing pneumonia, etc.), or active pneumonitis on screening chest computed tomography (CT) scan. A history of radiation pneumonitis (fibrosis) in the radiology field is acceptable. - Positive test for HIV infection Active hepatitis B (defined as having a positive hepatitis B surface antigen [HBsAg] test at screening). Patients with previous or resolved hepatitis B infection (defined as having a negative HBsAg test and positive IgG antibody to hepatitis B core antigen [anti-HBc]) are eligible. HBV DNA must be obtained in these patients before Day 1 of Cycle 1 and must not show active infection. Active hepatitis C. Patients who are positive for HCV antibodies are eligible only if the polymerase chain reaction (PCR) is negative for HCV RNA. Known active or latent tuberculosis infection. If the investigator considers that the potential patient is at high risk of infection with Mycobacterium tuberculosis, diagnostic procedures for latent tuberculosis should be followed during the screening period in accordance with local standard of practice. Severe infection within 4 weeks prior to Day 1 of Cycle 1, including but not limited to infection, bacteremia, or severe pneumonia complications Recent infections that do not meet the criteria for severe infection, including: Signs or symptoms of infection within 2 weeks prior to Cycle 1, Day 1 〇 Received oral or IV antibiotics within 2 weeks prior to Cycle 1, Day 1 Patients receiving prophylactic antibiotics (e.g., for urinary tract infection or chronic obstructive pulmonary disease prophylaxis) are eligible. Prior allogeneic bone marrow transplant or prior solid organ transplant Administration of a live attenuated vaccine within 4 weeks prior to Cycle 1, Day 1, or anticipation that such a live attenuated vaccine will be required during the study. Influenza vaccination should only be administered during influenza season. Patients must not receive a live attenuated influenza vaccine (e.g., FluMist®) within 4 weeks prior to Cycle 1, Day 1, or at any time during the study, and for 5 months after their last study treatment. Known hypersensitivity to any of the active substances or excipients in the vaccine · History of severe allergic, anaphylactic, or other hypersensitivity reactions to chimeric or humanized antibodies or fusion proteins Known hypersensitivity to Chinese hamster ovary cell products Allergy or hypersensitivity to any component of the pembrolizumab formulation Example 2:

[0277] This example describes an exemplary RNA vaccine for use in the methods described herein. Overall explanation

[0278] RNA vaccines are single-stranded messenger ribonucleic acid (mRNA) molecules that encode constant sequences and patient-specific tumor neoantigen sequences. Specifically, they are 5'-capped single-stranded messenger RNAs (mRNAs). Each mRNA encodes up to 20 neoepitopes defined by identified and selected patient tumor-specific mutations. The sequence containing the patient tumor-specific mutations typically consists of 81 nucleotides. A schematic diagram of the mRNA (in this example, mRNAs encoding 10 patient-specific neoepitopes) is shown in Figure 3.

[0279] The constant sequence elements include: a 5' cap (β-S-ARCA), 5'- and 3'-untranslated regions [UTRs], a secretory signal peptide [sec 2.0

[00100] , the MHC [major histocompatibility complex] class I transmembrane and cytoplasmic domains [MITD], and the poly(A) tail. These constant sequences are optimized for mRNA translation efficiency and stability and are identical for each batch and therefore for every patient. The role of all constant sequence elements is summarized in Table 4, and they flank the patient-specific neoepitope region and the glycine / serine (GS)-rich linker. TIFF2026004341000007.tif135170Abbreviations: AES = amino-terminal enhancer of split; MHC = major histocompatibility complex; MITD = MHC class I transmembrane and cytoplasmic domain; UTR = untranslated region. Constant Array Description

[0280] RNA[1,2-[m2 7·2’·O G-(5'→5')-pp s pG (Rp-isomer)] (constant 5'UTR + sec linked to constant MITD) 2.0 + 3'UTR and poly(A) tail) Sequence length: 739 nucleotides (A: 255, C: 204, G: 168, U: 112)

[0281] The RNA sequences of the constant regions of exemplary RNA vaccines are shown in Figure 4. The insertion sites of patient-specific sequences (C131-A132) are shown in bold. See Table 5 for modified bases and unusual linkages in the RNA sequences. TIFF2026004341000008.tif34170

[0282] Overall, the length of each RNA ranges from approximately 1,000 to 2,000 nucleotides, depending on the size of each neoepitope and the number of neoepitopes encoded by each RNA. The constant region of the RNA comprises 739 ribonucleotides, regardless of the patient-specific sequence.

[0283] References Holtkamp S,Kreiter S,Selmi A,et al.Modification of antigen-encoding RNA increases stability,translational efficacy,and T-cell stimulatory capacity of dendritic cells.Blood 2006;108:4009-17 Kozak M.At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells.J Mol Biol 1987;196:947-50. Kreiter S, Selmi A, Diken M, et al.Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals.J lmmunol 2008;180:309-18. Kuhn AN,Diken M,Kreiter S,et al.Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo.Gene Ther 2010;17:961-71. Trinh R, Gurbaxani B, Morrison SL, et al.Optimization of codon pair use within the(GGGGS)3 linker sequence results in enhanced protein expression.Mol lmmunol 2004;40:717-22. array All polynucleotide sequences are shown in 5' to 3' orientation. All polypeptide sequences are shown in N- to C-terminal orientation. Anti-PDL1 antibody HVR-H1 sequence (SEQ ID NO: 1) GFTFSDSWIH Anti-PDL1 antibody HVR-H2 sequence (SEQ ID NO: 2) AWISPYGGSTYYADSVKG Anti-PDL1 antibody HVR-H3 sequence (SEQ ID NO: 3) RHWPGGFDY Anti-PDL1 antibody HVR-L1 sequence (SEQ ID NO: 4) RASQDVSTAVA Anti-PDL1 antibody HVR-L2 sequence (SEQ ID NO: 5) SASFLYS Anti-PDL1 antibody HVR-L3 sequence (SEQ ID NO: 6) QQYLYHPAT Anti-PDL1 antibody VH sequence (SEQ ID NO: 7) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS Anti-PDL1 antibody VL sequence (SEQ ID NO: 8) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR Anti-PDL1 antibody heavy chain sequence (SEQ ID NO: 9) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHNHYTQKSLSLSPG Anti-PDL1 antibody light chain sequence (SEQ ID NO: 10) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Nivolumab heavy chain sequence (SEQ ID NO: 11) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWY DGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Nivolumab light chain sequence (SEQ ID NO: 12) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Pembrolizumab heavy chain sequence (SEQ ID NO: 13) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGG INPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Pembrolizumab light chain sequence (SEQ ID NO: 14) EIVLTQSPAT LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Avelumab heavy chain sequence (SEQ ID NO: 15) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Avelumab light chain sequence (SEQ ID NO: 16) QSALTQPASVGSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVT VLGQPKANTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Durvalumab heavy chain sequence (SEQ ID NO: 17) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Durvalumab light chain sequence (SEQ ID NO: 18) EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Complete PCV RNA 5' constant sequence (SEQ ID NO: 19) GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC Complete PCV RNA 3' constant sequence (SEQ ID NO: 20) AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU Full-length PCV Kozak RNA (SEQ ID NO: 21) GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC Full-length PCV Kozak DNA (SEQ ID NO: 22) GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC Short Kozak RNA (SEQ ID NO: 23) UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC Short Kozak DNA (SEQ ID NO: 24) TTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC sec RNA (SEQ ID NO: 25) AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC sec DNA (SEQ ID NO: 26) ATGAGAGTGATGGCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC sec protein (SEQ ID NO: 27) MRVMAPRTLILLLSGALALTETWAGS MITD RNA (SEQ ID NO: 28) AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC MITD DNA (SEQ ID NO: 29) ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACAGCC MITD protein (SEQ ID NO: 30) IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA Complete PCV FI RNA (SEQ ID NO: 31) CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU Full-length PCV FI DNA (SEQ ID NO: 32) CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT F element RNA (SEQ ID NO: 33) CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC F element DNA (SEQ ID NO: 34) CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC I element RNA (SEQ ID NO: 35) CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG I element DNA (SEQ ID NO: 36) CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG Linker RNA (SEQ ID NO: 37) GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC Linker DNA (SEQ ID NO: 38) GGCGGCTCTGGAGGAGGCGGCTCCGGAGGC Linker protein (SEQ ID NO: 39) GGSGGGGSGG Complete PCV DNA 5' constant sequence (SEQ ID NO: 40) GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC Complete PCV DNA 3' constant sequence (SEQ ID NO: 41) ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACAGCCTAGTAACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT Full-length PCV RNA (SEQ ID NO: 42) with 5’GG from the cap GGGGCGAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCCGCC ACCAUGAGAG UGAUGGCCCC CAGAACCCUG AUCCUGCUGC UGUCUGGCGC CCUGGCCCUG ACAGAGACAU GGGCCGGAAG CNAUCGUGGGA AUUGUGGCAG GACUGGCAGU GCUGGCCGUG GUGGUGAUCG GAGCCGUGGU GGCUACCGUG AUGUGCAGAC GGAAGUCCAG CGGAGGCAAG GGCGGCAGCU ACAGCCAGGC CGCCAGCUCU GAUAGCGCCC AGGGCAGCGA CGUGUCACUG ACAGCCUAGU AACUCGAGCU GGUACUGCAU GCACGCAAUG CUAGCUGCCC CUUUCCCGUC CUGGGUACCC CGAGUCUCCC CCGACCUCGG GUCCCAGGUA UGCUCCCACC UCCACCUGCC CCACUCACCA CCUCUGCUAG UUCCAGACAC CUCCCAAGCA CGCAGCAAUG CAGCUCAAAA CGCUUAGCCU AGCCACACCC CCACGGGAAA CAGCAGUGAU UAACCUUUAG CAAUAAACGA AAGUUUAACU AAGCUAUACU AACCCCAGGG UUGGUCAAUU UCGUGCCAGC CACACCGAGA CCUGGUCCAG AGUCGCUAGC CGCGUCGCUA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA

Claims

1. 1. A method of treating or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an 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 tumor specimen obtained from the individual.

2. 2. The method of claim 1, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

3. The method of claim 2, wherein the PD-1 binding antagonist is an anti-PD-1 antibody.

4. The method of claim 3, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.

5. 5. The method of claim 3 or claim 4, wherein the anti-PD-1 antibody is administered to the individual at a dose of about 200 mg.

6. 2. The method of claim 1, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

7. The method of claim 6, wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.

8. The method of claim 7, wherein the anti-PD-L1 antibody is avelumab or durvalumab.

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

10. 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 8. The method of claim 7, comprising:

11. The method of claim 7, wherein the anti-PD-L1 antibody is atezolizumab.

12. The method of any one of claims 7 to 11, wherein the anti-PD-L1 antibody is administered to the individual at a dose of about 1200 mg.

13. 13. The method of any one of claims 1 to 12, wherein the PD-1 axis binding antagonist is administered to the individual at intervals of 21 days or 3 weeks.

14. 14. The method of any one of claims 1 to 13, wherein the RNA vaccine comprises one or more polynucleotides encoding 10 to 20 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen.

15. The method of any one of claims 1 to 14, wherein the RNA vaccine is formulated in a lipoplex nanoparticle or a liposome.

16. The method of any one of claims 1 to 15, wherein the RNA vaccine is administered to the individual at a dose of about 15 μg, about 25 μg, about 38 μg, about 50 μg, or about 100 μg.

17. The method of any one of claims 1 to 16, wherein the RNA vaccine is administered to the individual at intervals of 21 days or 3 weeks.

18. 17. The method of any one of claims 1-16, wherein the PD-1 axis binding antagonist and the RNA vaccine are administered to the individual in eight 21-day cycles, and the RNA vaccine is administered to the individual on days 1, 8, and 15 of cycle 2 and days 1 of cycles 3-7.

19. 19. The method of claim 18, wherein the PD-1 axis binding antagonist is administered to the individual on day 1 of cycles 1-8.

20. The method of claim 18 or claim 19, wherein the PD-1 axis binding antagonist and the RNA vaccine are further administered to the individual after cycle 8.

21. 21. The method of claim 20, wherein the PD-1 axis binding antagonist and RNA vaccine are further administered to the individual for 17 additional 21-day cycles, wherein the PD-1 axis binding antagonist is administered to the individual on day 1 of cycles 13-29, and the RNA vaccine is administered to the individual on day 1 of cycles 13, 21, and 29.

22. 2. The method of claim 1, wherein the PD-1 axis binding antagonist and the RNA vaccine are administered to the individual in eight 21-day cycles, wherein the PD-1 axis binding antagonist is pembrolizumab and is administered to the individual at a dose of about 200 mg on day 1 of cycles 1-8, and the RNA vaccine is administered to the individual at a dose of about 25 μg on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3-7.

23. 23. The method of claim 22, wherein the RNA vaccine is administered to the individual at a dose of about 25 μg on day 1 of cycle 2, about 25 μg on day 8 of cycle 2, about 25 μg on day 15 of cycle 2, and about 25 μg on day 1 of each of cycles 3-7.

24. 24. The method of any one of claims 1 to 23, wherein the PD-1 axis binding antagonist and the RNA vaccine are administered intravenously.

25. The method of any one of claims 1 to 24, wherein the individual is a human.

26. 26. The method of any one of claims 1 to 25, wherein the cancer is selected from the group consisting of non-small cell lung cancer, bladder cancer, colorectal cancer, triple-negative breast cancer, renal cancer, and head and neck cancer.

27. The method of any one of claims 1 to 25, wherein the cancer is melanoma.

28. 28. The method of claim 27, wherein the melanoma is cutaneous or mucosal melanoma.

29. 28. The method of claim 27, wherein the melanoma is not ocular or acral melanoma.

30. The method of any one of claims 27 to 29, wherein the melanoma is metastatic melanoma or unresectable locally advanced melanoma.

31. 31. The method of claim 30, wherein the melanoma is stage IV melanoma.

32. 31. The method of claim 30, wherein the melanoma is stage IIIC melanoma or stage IIID melanoma.

33. 28. The method of claim 27, wherein the melanoma is an aggressive melanoma that has not been previously treated.

34. 34. The method of any one of claims 1 to 33, wherein said method results in improved progression-free survival (PFS).

35. 35. The method of any one of claims 1 to 34, wherein said method results in an increased objective response rate (ORR).

36. A kit comprising a PD-1 axis binding antagonist for use in combination with an RNA vaccine for treating an individual with cancer according to the method of any one of claims 1 to 35.

37. 1. A PD-1 axis binding antagonist for use in a method of treating a human individual having cancer, the method comprising administering to the individual an effective amount of the PD-1 axis binding antagonist in combination with an RNA vaccine, the RNA vaccine comprising one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen obtained from the individual.

38. 1. An RNA vaccine for use in a method of treating a human individual having cancer, the method comprising administering to the individual an effective amount of the RNA vaccine in combination with a PD-1 axis binding antagonist, the RNA vaccine comprising one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen obtained from the individual.

39. In the 5'→3' direction, (1) 5′ cap; (2) 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, (a) the 3' untranslated region of the 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 (6) a poly(A) sequence; An RNA molecule comprising:

40. 40. The RNA molecule of claim 39, further comprising 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.

41. further comprising, in the 5' to 3' direction, a polynucleotide sequence encoding an amino acid linker, and a polynucleotide sequence encoding a neoepitope; the polynucleotide sequence encoding the amino acid linker and the neoepitope forms a first linker-neoepitope module; 40. The RNA molecule of claim 39, wherein 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.

42. 42. The RNA molecule of claim 41, wherein the amino acid linker comprises the sequence GGSGGGGGSGG (SEQ ID NO: 39).

43. 42. The RNA molecule of claim 41, wherein the polynucleotide sequence encoding the amino acid linker comprises the sequence GGCGGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO: 37).

44. and further comprising, in the 5' to 3' direction, at least a second linker-epitope module, said at least second linker-epitope 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 is 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 the at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule; 44. The RNA molecule of any one of claims 41 to 43, wherein the neoepitope of the first linker-epitope module is different from the neoepitope of the second linker-epitope module.

45. 45. The RNA molecule of claim 44, wherein the RNA molecule comprises five linker-epitope modules, each of the five linker-epitope modules encoding a different neoepitope.

46. 45. The RNA molecule of claim 44, wherein the RNA molecule comprises 10 linker-epitope modules, each of the 10 linker-epitope modules encoding a different neoepitope.

47. 45. The RNA molecule of claim 44, wherein the RNA molecule comprises 20 linker-epitope modules, each of the 20 linker-epitope modules encoding a different neoepitope.

48. 48. The RNA molecule of any one of claims 40 to 47, further comprising a second polynucleotide sequence encoding an amino acid linker, wherein the second polynucleotide sequence encoding the amino acid linker is located in the 3' direction between the polynucleotide sequence encoding the distal-most neoepitope and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

49. The 5' cap has the structure: The RNA molecule of any one of claims 39 to 48, comprising the D1 diastereoisomer of

50. 50. The RNA molecule of any one of claims 39 to 49, wherein the 5'UTR comprises the sequence UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 23).

51. 50. The RNA molecule of any one of claims 39 to 49, wherein the 5'UTR comprises the sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 21).

52. 52. The RNA molecule of any one of claims 39 to 51, wherein the secretory signal peptide comprises the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 27).

53. 52. The RNA molecule of any one of claims 39 to 51, wherein the polynucleotide sequence encoding the secretory signal peptide comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 25).

54. 54. The RNA molecule of any one of claims 39 to 53, 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).

55. 54. The RNA molecule of any one of claims 39 to 53, 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).

56. 56. The RNA molecule of any one of claims 39 to 55, wherein the 3' untranslated region of the AES mRNA comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCCUGGGUACCCCGAGUCUCCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO: 33).

57. 57. The RNA molecule of any one of claims 39 to 56, wherein the non-coding RNA of the mitochondrially encoded 12S RNA comprises the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO: 35).

58. The 3'UTR has the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGGUACCCCGAGUCUCCCCCGACCUCGGGU CCCAGGUAUGCUCCACCUCCACCUGCCCCACUCUCACCACCUGCUAGUUCCAGACACCUCCCCAAGCACGCAGCAAUGCAGCUCAAAACGC 58. The RNA molecule of any one of claims 39 to 57, comprising UUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 31).

59. 59. The RNA molecule of any one of claims 39 to 58, wherein the poly(A) sequence comprises 120 adenine nucleotides.

60. From the 5' to 3' direction, the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 19); and the polynucleotide sequence Column AUCGUGGGAAUUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAA GUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCCUAG UAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGGUACCCCGAGUCUCCCCCGACCUCGGGUC CCAGGUAUGCUCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCCAAGCACGCAGCAAUGCA AACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 20).

61. 61. The RNA molecule of claim 60, further comprising a polynucleotide sequence encoding at least one neoepitope between the sequences of SEQ ID NO: 19 and SEQ ID NO:

20.

62. In the 5'→3' direction between the sequences of SEQ ID NO: 19 and SEQ ID NO: 20 (a) at least a first linker-neoepitope module, the at least a first linker-neoepitope module comprising a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope; 61. The RNA molecule of claim 60, further comprising: (b) a second polynucleotide sequence encoding an amino acid linker.

63. 63. The RNA molecule of claim 62, comprising five linker-epitope modules, each of the five linker-epitope modules encoding a different neoepitope.

64. 63. The RNA molecule of claim 62, comprising 10 linker-epitope modules, each of the 10 linker-epitope modules encoding a different neoepitope.

65. 63. The RNA molecule of claim 62, comprising 20 linker-epitope modules, each of the 20 linker-epitope modules encoding a different neoepitope.

66. 66. The RNA molecule of any one of claims 60 to 65, further comprising a 5' cap, wherein the 5' cap is located 5' to the sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 19).

67. The 5' cap has the structure:

67. The RNA molecule of claim 66, comprising the D1 diastereoisomer of

68. A liposome comprising the RNA molecule of any one of claims 39 to 67 and one or more lipids, wherein the one or more lipids form a multilayer structure that encapsulates the RNA molecule.

69. 69. The liposome of claim 68, wherein the one or more lipids comprise at least one cationic lipid and at least one helper lipid.

70. 69. The liposome of claim 68, 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).

71. 71. The liposome of claim 70, wherein the liposome has an overall charge ratio of positive to negative charges of 1.3:2 (0.65) at physiological pH.

72. 72. A method of treating or delaying the progression of cancer in an individual, the method comprising administering to said individual an effective amount of an RNA molecule according to any one of claims 39 to 67 or a liposome according to any one of claims 68 to 71.

73. 73. The method of Claim 72, wherein said RNA molecule comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen obtained from said individual.

74. 74. The method of claim 72 or claim 73, further comprising administering to the individual a PD-1 axis binding antagonist.

75. 75. The method of any one of claims 72 to 74, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, bladder cancer, colorectal cancer, triple-negative breast cancer, renal cancer, and head and neck cancer.

76. 72. An RNA molecule according to any one of claims 39 to 67 or a liposome according to any one of claims 68 to 71 for use in a method for treating or delaying the progression of cancer in an individual.

77. In the 5'→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 3'UTR, (a) the 3' untranslated region of the 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 (5) a polynucleotide sequence encoding a poly(A) sequence; A DNA molecule comprising:

78. 78. The DNA molecule of claim 77, further comprising 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.

79. further comprising, in the 5' to 3' direction, a polynucleotide sequence encoding an amino acid linker, and a polynucleotide sequence encoding a neoepitope; the polynucleotide sequence encoding the amino acid linker and the neoepitope forms a first linker-neoepitope module; 78. The DNA molecule of Claim 77, wherein 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.

80. 80. The DNA molecule of claim 79, wherein the amino acid linker comprises the sequence GGSGGGGGSGG (SEQ ID NO: 39).

81. 80. The DNA molecule of claim 79, wherein the polynucleotide sequence encoding the amino acid linker comprises the sequence GGCGGCTCTGGAGGAGGGCGGCTCCGGAGGC (SEQ ID NO: 38).

82. and further comprising, in the 5' to 3' direction, at least a second linker-epitope module, said at least second linker-epitope 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 is 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 the at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule; 82. The DNA molecule of any one of claims 79 to 81, wherein the neoepitope of the first linker-epitope module is different from the neoepitope of the second linker-epitope module.

83. 83. The DNA molecule of claim 82, wherein the DNA molecule comprises five linker-epitope modules, each of the five linker-epitope modules encoding a different neoepitope.

84. 83. The DNA molecule of claim 82, wherein the DNA molecule comprises 10 linker-epitope modules, each of the 10 linker-epitope modules encoding a different neoepitope.

85. 83. The DNA molecule of claim 82, wherein the DNA molecule comprises 20 linker-epitope modules, each of the 20 linker-epitope modules encoding a different neoepitope.

86. 86. The DNA molecule of any one of claims 78-85, further comprising a second polynucleotide sequence encoding an amino acid linker, wherein the second polynucleotide sequence encoding the amino acid linker is located in the 3' direction between the polynucleotide sequence encoding the distal-most neoepitope and the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

87. 85. The DNA molecule of any one of claims 77 to 84, wherein the polynucleotide encoding the 5'UTR comprises the sequence TTCTTCTGGTCCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 24).

88. 85. The DNA molecule of any one of claims 77 to 84, wherein the polynucleotide encoding the 5'UTR comprises the sequence GGCGAACTAGTATTCTTCTGGTCCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 22).

89. 89. The DNA molecule of any one of claims 77 to 88, wherein the secretory signal peptide comprises the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 27).

90. 89. The DNA molecule of any one of claims 77 to 88, wherein the polynucleotide sequence encoding the secretory signal peptide comprises the sequence ATGAGAGTGATGGCCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC (SEQ ID NO: 26).

91. 91. The DNA molecule of any one of claims 77 to 90, 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).

92. 91. The DNA molecule of any one of claims 77 to 90, wherein the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule comprises the sequence ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGGCAGCGACGTGTCACTGACAGCC (SEQ ID NO: 29).

93. 93. The DNA molecule of any one of claims 77 to 92, wherein the polynucleotide sequence encoding the 3' untranslated region of the AES mRNA comprises the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC (SEQ ID NO: 34).

94. 94. The DNA molecule of any one of claims 77 to 93, wherein the polynucleotide encoding the non-coding RNA of the mitochondrially encoded 12S RNA comprises the sequence CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG (SEQ ID NO: 36).

95. the polynucleotide encoding the 3'UTR has the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCCAAGCAACGCAGCAATGCAGCTCAA 95. The DNA molecule of any one of claims 77 to 94, comprising: AACGCTTAGCCTAGCCACACCCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (SEQ ID NO: 32).

96. 96. The DNA molecule of any one of claims 77 to 95, wherein the poly(A) sequence comprises 120 adenine nucleotides.

97. From the 5' to 3' direction, the polynucleotide sequence GGCGAACTAGTATTCTTCTGGTCCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGATGGCCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC (SEQ ID NO: 40) and the polynucleotide sequence ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAG TCCAGCGGAGGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACAGCCTAGT AACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCC CAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAA A DNA molecule comprising: AACGCTTAGCCTAGCCACACCCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (SEQ ID NO: 41).

98. 98. The DNA molecule of claim 97, further comprising a polynucleotide sequence encoding at least one neoepitope in the 5'→3' direction between the sequences of SEQ ID NO: 40 and SEQ ID NO:

41.

99. In the 5'→3' direction between the sequences of SEQ ID NO: 40 and SEQ ID NO: 41 (a) at least a first linker-neoepitope module, the at least a first linker-neoepitope module comprising a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope; (b) a second polynucleotide sequence encoding an amino acid linker; and 98. The DNA molecule of claim 97, further comprising:

100. 100. The DNA molecule of claim 99, comprising five linker-epitope modules, each of said five linker-epitope modules encoding a different neoepitope.

101. 100. The DNA molecule of claim 99, comprising 10 linker-epitope modules, each of said 10 linker-epitope modules encoding a different neoepitope.

102. 100. The DNA molecule of claim 99, comprising 20 linker-epitope modules, each of said 20 linker-epitope modules encoding a different neoepitope.

103. A method for producing an RNA molecule, the method comprising transcribing a DNA molecule according to any one of claims 77 to 102.

104. 72. A method of treating or delaying the progression of cancer in an individual, comprising administering to said individual an RNA molecule of any one of claims 39 to 67 or a liposome of any one of claims 68 to 71 according to a method of any one of claims 1 to 35.

105. 72. A method of treating or delaying the progression of cancer in an individual, comprising administering to said individual an RNA molecule of any one of claims 39-67 or a liposome of any one of claims 68-71 in combination with a PD-1 axis binding antagonist.

106. 106. The method of claim 105, wherein the RNA molecule or liposome is administered to the individual at a dose of about 15 μg, about 25 μg, about 38 μg, about 50 μg, or about 100 μg, and the PD-1 axis binding antagonist is administered to the individual at a dose of about 200 mg or about 1200 mg.

107. 107. The method of claim 105 or claim 106, wherein the RNA molecule or liposome and the PD-1 axis binding antagonist are administered to the individual in eight 21-day cycles.

108. 108. The method of claim 107, wherein the PD-1 axis binding antagonist is pembrolizumab and is administered to the individual at a dose of about 200 mg on day 1 of cycles 1-8, and the RNA molecule or liposome is administered to the individual at a dose of about 25 μg on days 1, 8, and 15 of cycle 2 and day 1 of cycles 3-7.

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