Engineered arenavirus particles expressing mutant KRAS, mutated cancer driver genes, or tumor-associated antigens as cancer immunotherapy

Abstract

The present invention relates to genetically modified arenaviruses suitable for treating neoplastic diseases such as cancer. The arenaviruses described herein may be suitable for use in treating neoplastic diseases and / or immunotherapy. In particular, methods and compositions are provided herein for treating neoplastic diseases by administering genetically modified arenaviruses that are engineered to contain only nucleotide sequences encoding one or more antigenic fragments of mutant KRAS or further contain nucleotide sequences encoding one or more antigenic fragments of mutated cancer driver genes (e.g., mutant TP53) or tumor-associated antigens.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 404,008, filed September 6, 2022, U.S. Provisional Application No. 63 / 404,068, filed September 6, 2022, U.S. Provisional Application No. 63 / 277,049, filed November 8, 2021, and U.S. Provisional Application No. 63 / 277,052, filed November 8, 2021, the contents of each of which are incorporated by reference in their entirety herein and from which priority is claimed.

[0002] Sequence Listing This application contains a computer readable sequence listing submitted with this application in XML file format, the entire contents of which are incorporated herein by reference. The sequence listing XML file submitted with this application is named "13194-082-228_SequenceListing.xml", was created on November 4, 2022, and is 93,515 bytes in size.

[0003] 1. Introduction The present invention relates to a genetically modified arenavirus suitable for treating tumor diseases such as cancer. The arenaviruses described herein may be suitable for treating tumor diseases and / or for use in immunotherapy. In particular, methods and compositions are provided herein for treating tumor diseases by administering genetically modified arenaviruses that are engineered to contain only the nucleotide sequence encoding one or several antigenic fragments of mutant KRAS, or to further contain the nucleotide sequence encoding one or several different mutated cancer driver genes (e.g., mutant TP53) or one or several antigenic fragments of tumor-associated antigens. [Background technology]

[0004] 2.Background There is an unmet medical need for the treatment of tumor diseases such as cancer. The emerging field of immunotherapy shows promise for the treatment of these life-threatening diseases. In addition, combination therapy is also explored. However, as more and more therapies become available, possible combinations become more complex.

[0005] One strategy for immunotherapy involves arenavirus-based expression of mutant KRAS antigens, mutated cancer driver genes (e.g., mutant TP53), tumor-associated antigens. See, e.g., WO2009 / 083210, WO / 2016 / 075250, WO2017 / 198726, and WO2021 / 089853. Intratumoral administration of these immunotherapies has been described. See, e.g., WO2018 / 185307. Summary of the Invention

[0006] 3. Overview of the Invention The present application relates to genetically modified arenaviruses suitable for the treatment of neoplastic diseases such as cancer. In particular, the present application relates to arenavirus particles comprising: a. the arenavirus particle comprises an arenavirus genome comprising a nucleotide sequence encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising the mutation; b. At least one arenavirus open reading frame ("ORF") of the arenavirus genome is either (i) functionally inactivated or deleted, or (ii) located at a position other than the wild-type position of said at least one arenavirus ORF, or (iii) split into two or more functional fragments, wherein a fragment of at least one arenavirus ORF is located at a position other than the wild-type position of said at least one arenavirus ORF; Concerning arenavirus particles.

[0007] An arenavirus particle, wherein the KRAS mutation is present at amino acid position G12, G13, A18, A59, Q61, K117, A146, or D119 of KRAS.

[0008] The arenavirus particle, wherein the KRAS mutation is A18D, A59E, A59G, A59P, A59T, A59S, A59V, A146P, A146S, A146T, A146V, D119N, G12A, G12C, G12D, G12F, G12L, G12R, G12S, G12V, G13A, G13C, G13D, G13E, G13R, G13S, G13V, K117N, Q61E, Q61H, Q61K, Q61L, Q61P, Q61R, or a combination thereof.

[0009] The arenavirus particle, wherein the KRAS mutation is G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61R, A146T, or a combination thereof.

[0010] The arenavirus particle, wherein the mutation of KRAS is G13D, G12V, G12C, G12D, G12R, or a combination thereof. In a more specific embodiment, the arenavirus genome comprises a nucleotide sequence encoding a plurality of fragments of mutant KRAS, each of which comprises the mutations G13D, G12V, G12C, G12D, and G12R, from the N-terminus to the C-terminus. In another more specific embodiment, the arenavirus genome comprises a nucleotide sequence encoding a fragment of mutant KRAS comprising the mutations G13D, G12V, G12C, G12D, and G12R, in any possible order.

[0011] An arenavirus particle, wherein the arenavirus genome comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:20.

[0012] An arenavirus particle, wherein the arenavirus genome comprises a nucleotide sequence encoding an expression product whose amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:19.

[0013] The arenavirus particle, wherein the fragment of mutant KRAS is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In a more specific embodiment, the fragment of mutant KRAS is 18 amino acids in length.

[0014] An arenavirus particle, wherein the region adjacent to the N-terminal mutation of the antigenic fragment is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In a more specific embodiment, the region adjacent to the N-terminal mutation of the antigenic fragment is 8 or 9 amino acids in length.

[0015] An arenavirus particle, wherein the region adjacent to the C-terminal mutation of the antigenic fragment is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In a more specific embodiment, the region adjacent to the C-terminal mutation of the antigenic fragment is 8 or 9 amino acids in length.

[0016] An arenavirus particle, wherein the nucleotide sequence encodes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antigenic fragments of mutant KRAS, each of the antigenic fragments comprising one mutation of the mutant KRAS protein. In a more specific embodiment, the nucleotide sequence encodes 5 antigenic fragments of mutant KRAS, each of the antigenic fragments comprising one mutation of the mutant KRAS protein. In a more specific embodiment, the 5 antigenic fragments of mutant KRAS comprise the mutations G13D, G12V, G12C, G12D, and G12R.

[0017] Arenavirus particles, the antigenic fragments of which contain the same or different mutations of the mutant KRAS protein.

[0018] Arenavirus particles in which antigenic fragments are fused to each other via the same or different linkers.

[0019] An arenavirus particle in which the antigenic fragments are directly fused to each other with no intervening sequences.

[0020] An arenavirus particle, wherein the linker is an AAY linker (AAY), an AAA linker (AAA), a GS linker (GGSGGGGSGG) (SEQ ID NO: 42), or a variant of the AAY, AAA, and GS linker sequences optimized via in silico prediction.

[0021] An arenavirus particle in which the nucleotide sequence has been engineered to reduce or eliminate any CpG and TpA islands.

[0022] Removal of CpG and TpA islands is performed in three cycles: (i) removing CpG in the first cycle; (ii) removing TpA in the second cycle; (iii) removing CpGs in a third cycle to remove the newly introduced CpGs in the second cycle arenavirus particles comprising

[0023] The arenavirus genome (i) a first S segment comprising a nucleotide sequence encoding an antigenic fragment(s) under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus nucleoprotein ("NP") under the control of the arenavirus genome 3' UTR; (ii) a second S segment comprising a nucleotide sequence encoding an antigenic fragment(s) under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus glycoprotein ("GP") under the control of the arenavirus genome 3' UTR; (iii) L segment and arenavirus particles comprising

[0024] The arenavirus genome (i) a first S segment comprising the nucleotide sequence of SEQ ID NO:20 under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus nucleoprotein ("NP") under the control of the arenavirus genome 3' UTR; (ii) a second S segment comprising the nucleotide sequence of SEQ ID NO:20 under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus glycoprotein ("GP") under the control of the arenavirus genome 3' UTR; (iii) L segment and arenavirus particles comprising

[0025] The arenavirus genome (i) a first S segment comprising the nucleotide sequence of SEQ ID NO: 21; (ii) a second S segment comprising the nucleotide sequence of SEQ ID NO: 22; and (iii) L segment and arenavirus particles comprising

[0026] The arenavirus genome (i) a first S segment comprising the nucleotide sequence of SEQ ID NO: 23; (ii) a second S segment comprising the nucleotide sequence of SEQ ID NO: 24; and (iii) L segment and arenavirus particles comprising

[0027] The arenavirus genome (i) a first S segment comprising an ORF encoding the arenavirus GP1 and GP2 subunits fused to a heterologous signal peptide under the control of the arenavirus genome 5' UTR, and an ORF encoding a fusion of the arenavirus GP signal peptide and a nucleotide sequence encoding an antigenic fragment(s) under the control of the arenavirus genome 3' UTR; (ii) a second S segment comprising a nucleotide sequence encoding an antigenic fragment(s) under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus nucleoprotein ("NP") under the control of the arenavirus genome 3' UTR; (iii) L segment and arenavirus particles comprising

[0028] An arenavirus particle, wherein the nucleotide sequence encoding the antigenic fragment(s) on a first S segment differs from the nucleotide sequence encoding the antigenic fragment(s) on a second S segment.

[0029] An arenavirus particle, wherein the nucleotide sequence encoding the antigenic fragment(s) on the first S segment is the same as the nucleotide sequence encoding the antigenic fragment(s) on the second S segment.

[0030] An arenavirus particle, wherein the antigenic fragment(s) encoded on a first S segment differs from the antigenic fragment(s) encoded on a second S segment.

[0031] An arenavirus particle, wherein the antigenic fragment(s) encoded on the first S segment is the same as the antigenic fragment(s) encoded on the second S segment.

[0032] An arenavirus particle, wherein the antigenic fragments encoded on a first S segment are the same as the antigenic fragments encoded on a second S segment, but are fused to each other in an order different from the order in which the antigenic fragments encoded on the second S segment are fused to each other.

[0033] The arenavirus particle, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, mutant RET, mutant APC, mutant U2AF1, mutant EGFR, mutant FBXW7, mutant SMAD4, mutant GNAS, mutant ERBB2, mutant ERBB3, mutant CDKN2A, mutant TP53 and / or mutant CTNNB1, wherein the antigenic fragment comprises the respective mutation.

[0034] The arenavirus particle, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant PI3KCA, the antigenic fragment comprising an E545K, H1047R and / or E542K mutation.

[0035] The arenavirus particle, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant BRAF, the antigenic fragment comprising a V600E mutation.

[0036] An arenavirus particle, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant U2AF1, the antigenic fragment comprising an S34F mutation.

[0037] The arenavirus particle, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant TP53, the antigenic fragment comprising G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W and / or C277F.

[0038] The arenavirus particle, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of the BIRC family, CEACAM family, CTA family, EPH family, ERBB family, FOLR family, GAST family, GUCY2 family, IDO family, IL13RA family, KDR family, KLK family, MAGE family, MUC family, PEMT family, SDC family, SLAMF family, TERT family, TLR family, TPTE family, TYR family, WT family and / or XBP family.

[0039] Arenavirus particles derived from lymphocytic choriomeningitis virus (LCMV) or Pichinde virus.

[0040] A pharmaceutical composition comprising arenavirus particles.

[0041] A set of one or more nucleic acids that encode the genome of an arenavirus particle.

[0042] A host cell comprising a set of one or more nucleic acids encoding the genome of an arenavirus particle.

[0043] A method of producing arenavirus particles, the method comprising culturing a host cell and harvesting the arenavirus particles.

[0044] 1. A method for treating a neoplastic disease in a subject in need thereof, comprising administering to the subject arenavirus particles comprising: a. the arenavirus particle comprises an arenavirus genome comprising a nucleotide sequence encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising the mutation; b. At least one arenavirus open reading frame ("ORF") of the arenavirus genome is either (i) functionally inactivated or deleted, or (ii) located at a position other than the wild-type position of said at least one arenavirus ORF, or (iii) split into two or more functional fragments, wherein a fragment of at least one arenavirus ORF is located at a position other than the wild-type position of said at least one arenavirus ORF; A method comprising administering arenavirus particles.

[0045] A method for treating a neoplastic disease in a subject in need of treatment, wherein the KRAS mutation is present at amino acid position G12, G13, A18, A59, Q61, K117, A146, or D119 of KRAS.

[0046] 1. A method for treating a neoplastic disease in a subject in need thereof, wherein the KRAS mutation is A18D, A59E, A59G, A59P, A59T, A59S, A59V, A146P, A146S, A146T, A146V, D119N, G12A, G12C, G12D, G12F, G12L, G12R, G12S, G12V, G13A, G13C, G13D, G13E, G13R, G13S, G13V, K117N, Q61E, Q61H, Q61K, Q61L, Q61P, Q61R, or a combination thereof.

[0047] 1. A method for treating a neoplastic disease in a subject in need thereof, wherein the KRAS mutation is G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61R, A146T, or a combination thereof.

[0048] A method for treating a neoplastic disease in a subject in need of treatment, wherein the mutation of KRAS is G13D, G12V, G12C, G12D, G12R, or a combination thereof. In a more specific embodiment, the arenavirus genome comprises a nucleotide sequence encoding a plurality of fragments of mutant KRAS, each of which comprises the mutations G13D, G12V, G12C, G12D, and G12R, from the N-terminus to the C-terminus. In another more specific embodiment, the arenavirus genome comprises a nucleotide sequence encoding a fragment of mutant KRAS comprising the mutations G13D, G12V, G12C, G12D, and G12R, in any possible order.

[0049] A method for treating a neoplastic disease in a subject in need of treatment, wherein the arenavirus genome comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:20.

[0050] A method for treating a neoplastic disease in a subject in need of treatment, wherein the arenavirus genome comprises a nucleotide sequence encoding an expression product whose amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:19.

[0051] A method for treating a neoplastic disease in a subject in need of treatment, wherein the antigenic fragment of mutant KRAS is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In a more specific embodiment, the antigenic fragment of mutant KRAS is 18 amino acids in length.

[0052] A method for treating a neoplastic disease in a subject in need of treatment, wherein the region adjacent to the N-terminal mutation of the antigenic fragment is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In a more specific embodiment, the region adjacent to the N-terminal mutation of the antigenic fragment is 8 or 9 amino acids in length.

[0053] A method for treating a neoplastic disease in a subject in need of treatment, wherein the region adjacent to the C-terminal mutation of the antigenic fragment is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In a more specific embodiment, the region adjacent to the C-terminal mutation of the antigenic fragment is 8 or 9 amino acids in length.

[0054] A method for treating a neoplastic disease in a subject in need of treatment, wherein the nucleotide sequence encodes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antigenic fragments of mutant KRAS, each of the antigenic fragments comprising one mutation of the mutant KRAS protein.A method for treating a neoplastic disease in a subject in need of treatment, wherein the nucleotide sequence encodes 5 antigenic fragments of mutant KRAS, each of the antigenic fragments comprising one mutation of the mutant KRAS protein.A method for treating a neoplastic disease in a subject in need of treatment, wherein the 5 antigenic fragments of mutant KRAS comprise the mutations G13D, G12V, G12C, G12D, and G12R.

[0055] A method for treating a neoplastic disease in a subject in need of treatment, wherein the antigenic fragments comprise the same or different mutations of a mutant KRAS protein.

[0056] A method for treating a neoplastic disease in a subject in need of treatment, wherein antigenic fragments are fused to each other via the same or different linkers.

[0057] A method for treating a neoplastic disease in a subject in need of treatment, wherein antigenic fragments are directly fused to each other without any intervening sequences.

[0058] A method for treating a neoplastic disease in a subject in need of treatment, wherein the linker is an AAY linker (AAY), a AAA linker (AAA), a GS linker (GGSGGGGSGG) (SEQ ID NO: 42), or a variant of the AAY, AAA, and GS linker sequences optimized via in silico prediction.

[0059] A method for treating a neoplastic disease in a subject in need of treatment, wherein the nucleotide sequence has been engineered to reduce or eliminate any CpG and TpA islands.

[0060] A method for treating a neoplastic disease in a subject in need of treatment, comprising the steps of: (i) removing CpG in the first cycle; (ii) removing TpA in the second cycle; (iii) removing CpGs in a third cycle to remove the newly introduced CpGs in the second cycle A method comprising:

[0061] 1. A method for treating a neoplastic disease in a subject in need thereof, comprising: (i) a first S segment comprising a nucleotide sequence encoding an antigenic fragment(s) under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus nucleoprotein ("NP") under the control of the arenavirus genome 3' UTR; (ii) a second S segment comprising a nucleotide sequence encoding an antigenic fragment(s) under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus glycoprotein ("GP") under the control of the arenavirus genome 3' UTR; (iii) L segment and A method comprising:

[0062] 1. A method for treating a neoplastic disease in a subject in need thereof, comprising: (i) a first S segment comprising the nucleotide sequence of SEQ ID NO:20 under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus nucleoprotein ("NP") under the control of the arenavirus genome 3' UTR; (ii) a second S segment comprising the nucleotide sequence of SEQ ID NO:20 under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus glycoprotein ("GP") under the control of the arenavirus genome 3' UTR; (iii) L segment and A method comprising:

[0063] 1. A method for treating a neoplastic disease in a subject in need thereof, comprising: (i) a first S segment comprising the nucleotide sequence of SEQ ID NO: 21; (ii) a second S segment comprising the nucleotide sequence of SEQ ID NO: 22; and (iii) L segment and A method comprising:

[0064] 1. A method for treating a neoplastic disease in a subject in need thereof, comprising: (i) a first S segment comprising the nucleotide sequence of SEQ ID NO: 23; (ii) a second S segment comprising the nucleotide sequence of SEQ ID NO: 24; and (iii) L segment and A method comprising:

[0065] 1. A method for treating a neoplastic disease in a subject in need thereof, comprising: (i) a first S segment comprising an ORF encoding the arenavirus GP1 and GP2 subunits fused to a heterologous signal peptide under the control of the arenavirus genome 5' UTR, and an ORF encoding a fusion of the arenavirus GP signal peptide and a nucleotide sequence encoding an antigenic fragment(s) under the control of the arenavirus genome 3' UTR; (ii) a second S segment comprising a nucleotide sequence encoding an antigenic fragment(s) under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus nucleoprotein ("NP") under the control of the arenavirus genome 3' UTR; (iii) L segment and A method comprising:

[0066] A method for treating a neoplastic disease in a subject in need of treatment, wherein a nucleotide sequence encoding an antigenic fragment(s) on a first S segment is different from a nucleotide sequence encoding an antigenic fragment(s) on a second S segment.

[0067] A method for treating a neoplastic disease in a subject in need of treatment, wherein a nucleotide sequence encoding an antigenic fragment(s) on a first S segment is the same as a nucleotide sequence encoding an antigenic fragment(s) on a second S segment.

[0068] A method for treating a neoplastic disease in a subject in need of treatment, wherein the antigenic fragment(s) encoded on a first S segment is different from the antigenic fragment(s) encoded on a second S segment.

[0069] A method for treating a neoplastic disease in a subject in need of treatment, wherein the antigenic fragment(s) encoded on a first S segment is the same as the antigenic fragment(s) encoded on a second S segment.

[0070] A method for treating a neoplastic disease in a subject in need of treatment, wherein an antigenic fragment encoded on a first S segment is the same as an antigenic fragment encoded on a second S segment, but the antigenic fragments encoded on the second S segment are fused to each other in an order different from the order in which the antigenic fragments encoded on the second S segment are fused to each other.

[0071] A method for treating a neoplastic disease in a subject in need of treatment, wherein the neoplastic disease is pancreatic cancer, colorectal cancer, lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer (NSCLC).

[0072] 1. A method for treating a neoplastic disease in a subject in need of treatment, wherein the arenavirus genome comprises a nucleotide sequence encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprises a mutation, the KRAS mutation being G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A, and the neoplastic disease is pancreatic cancer.

[0073] 1. A method for treating a neoplastic disease in a subject in need thereof, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant TP53 and / or mutant U2AF1, wherein the antigenic fragment comprises mutations, the KRAS mutation being G12D, G12V, G12R, Q61H and / or Q61R, the TP53 mutation being R175H, R248W, G245S, R282W, R248Q and / or R273C, and the U2AF1 mutation being S34F, and the neoplastic disease is pancreatic cancer.

[0074] 1. A method for treating a neoplastic disease in a subject in need thereof, wherein the arenavirus genome comprises a nucleotide sequence encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising a mutation, the KRAS mutation being G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma.

[0075] A method for treating a neoplastic disease in a subject in need of treatment, wherein the arenavirus genome comprises a nucleotide sequence encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprises mutations, the KRAS mutations being G13D, G12V, G12C, G12D, and G12R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma.

[0076] 1. A method for treating a neoplastic disease in a subject in need thereof, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant TP53, the antigenic fragment comprising mutations, the KRAS mutations being G12D, G12R, G12V, Q61H and / or Q61R, the TP53 mutations being R175H, R248W, G245S, R282W, R248Q and / or R273C, and the neoplastic disease is pancreatic cancer.

[0077] 1. A method for treating a neoplastic disease in a subject in need thereof, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant BRAF, mutant TP53, mutant FBXW7, and / or mutant PIK3CA, wherein the antigenic fragment comprises mutations, the KRAS mutation being G12D, G12V, G13D, A146T, and / or G12C, the BRAF mutation being V600E, the TP53 mutation being R175H, R273H, and / or R248W, the FBXW7 mutation being R465H, and the PIK3CA mutation being E545K and / or H1047R, and the neoplastic disease is colorectal cancer or lung adenocarcinoma.

[0078] 1. A method for treating a neoplastic disease in a subject in need thereof, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant BRAF, mutant PIK3CA, mutant EGFR, mutant TP53, and / or mutant U2AF1, wherein the antigenic fragment comprises mutations, the KRAS mutation being G12C, G12D, G12R, G13D, and / or G12V, the BRAF mutation being V600E, the PIK3CA mutation being E545K, the EGFR mutation being L858R, the TP53 mutation being R175H, R273H, and / or R248W, and the U2AF1 mutation being S34F, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma.

[0079] 1. A method for treating a neoplastic disease in a subject in need thereof, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant TP53, mutant U2AF1, mutant PIK3CA, mutant EGFR and / or mutant BRAF, wherein the antigenic fragment comprises mutations, the KRAS mutation being G12C, G12V, G12D, and / or G12S, the TP53 mutation being R175H, the U2AF1 mutation being S34F, the EGFR mutation being L858R, L861Q and / or E746_A750del, the PIK3CA mutation being E545K and / or E542K, and the BRAF mutation being V600E, and the neoplastic disease is lung adenocarcinoma.

[0080] A method for treating a neoplastic disease in a subject in need of treatment, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA and / or mutant RET, wherein the antigenic fragment comprises a mutation, and wherein the neoplastic disease is non-small cell lung cancer (NSCLC).

[0081] 1. A method for treating a neoplastic disease in a subject in need thereof, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, mutant RET, mutant APC, mutant U2AF1, mutant EGFR, mutant FBXW7, mutant SMAD4, mutant GNAS, mutant ERBB2, mutant ERBB3, mutant CDKN2A, mutant TP53 and / or mutant CTNNB1, wherein the antigenic fragment comprises the respective mutation.

[0082] A method for treating a neoplastic disease in a subject in need of treatment, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant PI3KCA, the antigenic fragment comprising a mutation, the PI3KCA mutation being E545K, H1047R and / or E542K.

[0083] A method for treating a neoplastic disease in a subject in need of treatment, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant BRAF, the antigenic fragment comprising a mutation, and the BRAF mutation is V600E.

[0084] A method for treating a neoplastic disease in a subject in need of treatment, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutated U2AF1, the antigenic fragment comprising a mutation, and the U2AF1 mutation is S34F.

[0085] 1. A method for treating a neoplastic disease in a subject in need thereof, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant TP53, the antigenic fragment comprising a mutation, the TP53 mutation being G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W and / or C277F.

[0086] A method for treating a neoplastic disease in a subject in need of treatment, wherein the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of the BIRC family, CEACAM family, CTA family, EPH family, ERBB family, FOLR family, GAST family, GUCY2 family, IDO family, IL13RA family, KDR family, KLK family, MAGE family, MUC family, PEMT family, SDC family, SLAMF family, TERT family, TLR family, TPTE family, TYR family, WT family and / or XBP family.

[0087] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising a mutation, the KRAS mutation being G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A, and the neoplastic disease is pancreatic cancer.

[0088] 1. A method for treating a neoplastic disease in a subject in need thereof, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant KRAS, a mutant TP53, and / or a mutant U2AF1, the antigenic fragment comprising mutations, the KRAS mutation being G12D, G12V, G12R, Q61H and / or Q61R, the TP53 mutation being R175H, R248W, G245S, R282W, R248Q and / or R273C, and the U2AF1 mutation being S34F, and the neoplastic disease is pancreatic cancer.

[0089] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising a mutation, and the neoplastic disease is pancreatic cancer.

[0090] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising a mutation, and the neoplastic disease is colorectal cancer.

[0091] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising a mutation, the KRAS mutation being G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R, and the neoplastic disease being pancreatic cancer, colorectal cancer, or lung adenocarcinoma.

[0092] 1. A method for treating a neoplastic disease in a subject in need thereof, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of mutant KRAS and / or mutant TP53, the antigenic fragment comprising mutations, the KRAS mutations being G12D, G12R, G12V, Q61H and / or Q61R, the TP53 mutations being R175H, R248W, G245S, R282W, R248Q and / or R273C, and the neoplastic disease is pancreatic cancer.

[0093] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising mutations, the KRAS mutations being G13D, G12V, G12C, G12D, and G12R, and the neoplastic disease being pancreatic cancer, colorectal cancer, or lung adenocarcinoma.

[0094] 1. A method for treating a neoplastic disease in a subject in need thereof, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of mutant KRAS, mutant BRAF, mutant TP53, mutant FBXW7, and / or mutant PIK3CA, the antigenic fragment comprising mutations, the KRAS mutation being G12D, G12V, G13D, A146T, and / or G12C, the BRAF mutation being V600E, the TP53 mutation being R175H, R273H, and / or R248W, the FBXW7 mutation being R465H, and the PIK3CA mutation being E545K and / or H1047R, and the neoplastic disease is colorectal cancer or lung adenocarcinoma.

[0095] 1. A method for treating a neoplastic disease in a subject in need thereof, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of mutant KRAS, mutant BRAF, mutant PIK3CA, mutant EGFR, mutant TP53, and / or mutant U2AF1, the antigenic fragment comprising mutations, wherein the KRAS mutation is G12C, G12D, G12R, G13D, and / or G12V, the BRAF mutation is V600E, the PIK3CA mutation is E545K, the EGFR mutation is L858R, the TP53 mutation is R175H, R273H, and / or R248W, and the U2AF1 mutation is S34F, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma.

[0096] 1. A method for treating a neoplastic disease in a subject in need thereof, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of mutant KRAS, mutant TP53, mutant U2AF1, mutant PIK3CA, mutant EGFR and / or mutant BRAF, the antigenic fragment comprising mutations, the KRAS mutation being G12C, G12V, G12D, and / or G12S, the TP53 mutation being R175H, the U2AF1 mutation being S34F, the EGFR mutation being L858R, L861Q, and / or E746_A750del, the PIK3CA mutation being E545K and / or E542K, and the BRAF mutation being V600E, and the neoplastic disease is lung adenocarcinoma.

[0097] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant TP53, the antigenic fragment comprising the mutation.

[0098] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of mutant KRAS, mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA and / or mutant RET, the antigenic fragment comprising a mutation, and the neoplastic disease is non-small cell lung cancer (NSCLC).

[0099] A method for treating a neoplastic disease in a subject in need thereof, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, mutant RET, mutant APC, mutant U2AF1, mutant EGFR, mutant FBXW7, mutant SMAD4, mutant GNAS, mutant ERBB2, mutant ERBB3, mutant CDKN2A, mutant TP53 and / or mutant CTNNB1, and the antigenic fragment comprises the respective mutation.

[0100] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant PI3KCA, the antigenic fragment comprising a mutation, the PI3KCA mutation being E545K, H1047R and / or E542K.

[0101] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant BRAF, the antigenic fragment comprising a mutation, and the BRAF mutation is V600E.

[0102] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant U2AF1, the antigenic fragment comprising a mutation, and the U2AF1 mutation is S34F.

[0103] 1. A method for treating a neoplastic disease in a subject in need thereof, the method further comprising administering a second arenavirus particle, the second arenavirus particle encoding an antigenic fragment of a mutant TP53, the antigenic fragment comprising a mutation, the TP53 mutation being G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W and / or C277F.

[0104] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of the BIRC family, CEACAM family, CTA family, EPH family, ERBB family, FOLR family, GAST family, GUCY2 family, IDO family, IL13RA family, KDR family, KLK family, MAGE family, MUC family, PEMT family, SDC family, SLAMF family, TERT family, TLR family, TPTE family, TYR family, WT family and / or XBP family.

[0105] A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle comprising an arenavirus genome comprising the nucleotide sequence of SEQ ID NOs: 21 and 22. A method for treating a neoplastic disease in a subject in need of treatment, the method further comprising administering a second arenavirus particle, the second arenavirus particle comprising an arenavirus genome comprising the nucleotide sequence of SEQ ID NOs: 23 and 24.

[0106] A method for treating a neoplastic disease in a subject in need thereof, wherein the arenavirus particle is derived from lymphocytic choriomeningitis virus (LCMV) or Pichinde virus.

[0107] A method for treating a neoplastic disease in a subject in need of treatment, wherein the neoplastic disease is a solid tumor, and the method results in an increase in the concentration of T cells within the solid tumor.

[0108] 1. A method for treating a neoplastic disease in a subject in need thereof, the neoplastic disease being selected from the group consisting of acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphocytic leukemia; acute myelogenous leukemia; acute myeloid leukemia (adult / pediatric); adrenal cortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratoid / rhabdoid tumor; basal cell carcinoma; cholangiocarcinoma, extrahepatic (cholangiocarcinoma); bladder cancer; osteosarcoma of bone / malignant fibrous histiocytoma; brain cancer (adult / pediatric); brain tumor, cerebellar astrocytoma (adult / pediatric); brain tumor, cerebral astrocytoma / malignant glioma brain tumor; brain tumor, ependymoma; brain tumor, medulloblastoma; brain tumor, supratentorial primitive neuroectodermal tumor ;Brain tumors, optic tract and hypothalamic glioma;Brain stem glioma;Breast cancer;Bronchial adenoma / carcinoid;Bronchial tumor;Burkitt's lymphoma;Childhood cancer;Carcinoid gastrointestinal tumor;Carcinoid tumor;Adult carcinoma, unknown primary site;Carcinoma of unknown primary site;Central nervous system embryonal tumor;Central nervous system lymphoma, primary;Cervical cancer;Childhood adrenal cortical carcinoma;Childhood cancer;Childhood cerebral astrocytoma;Chordoma, childhood;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Chronic myeloproliferative disorder;Colon cancer;Colorectal cancer;Craniopharyngioma;Skin Cutaneous T-cell lymphoma;Desmoplastic small round cell tumor;Emphysema;Endometrial carcinoma;Ependymoblastoma;Ependymoma;Esophageal carcinoma;Ewing sarcoma in the Ewing family of tumors;Extracranial germ cell tumors;Extragonadal germ cell tumors;Extrahepatic bile duct carcinoma;Gallbladder carcinoma;Gastric (stomach) carcinoma;Gastric carcinoid;Gastrointestinal carcinoid tumors;Gastrointestinal stromal tumors;Germ cell tumors: extracranial, extragonadal, or ovarian;Gestational trophoblastic tumor, unknown primary site;Glioma;Glioma of the brain stem;Glioma, pediatric ophthalmology tract and hypothalamus;hairy cell leukemia;head and neck cancer;cardiac cancer;hepatocellular (liver) carcinoma;Hodgkin's lymphoma;hypopharyngeal cancer;hypothalamic and optic tract glioma;intraocular melanoma;islet cell carcinoma (pancreatic islet);Kaposi's sarcoma;renal carcinoma (renal cell carcinoma);Langerhans cell histiocytosis;laryngeal cancer;lip and oral cavity cancer;liposarcoma;hepatic cancer (primary);lung cancer, non-small cell;lung cancer, small cell;lymphoma, primary central nervous system;macroglobulinemia, Waldenstrom;male breast cancer;malignant fibrous histiocytoma / osteosarcoma of bone;medulloblastoma;medulloepithelioma;melanoma;melanoma, intraocular (eye);Merkel cell carcinoma;Merkel cell skin cancer;mesothelioma;mesothelioma, adult malignant;metastatic squamous neck cancer of unknown primary;oral cancer;multiple endocrine neoplasia syndrome;multiple myeloma / plasma cell neoplasm;mycosis fungoides, myelodysplastic syndrome;myelodysplastic / myeloproliferative disorders;myeloid leukemia, chronic;myeloid leukemia, adult acute;myeloid leukemia, childhood acute;myeloma, multiple (cancer of the bone marrow);myeloproliferative disorders, chronic;nasal and paranasal sinus cancer;nasopharyngeal carcinoma;neuroblastoma, non-small cell lung cancer;non-Hodgkin's lymphoma;oligodendrocyte glioma;oral cancer;oral cavity cancer cancer);oropharyngeal cancer;osteosarcoma / malignant fibrous histiocytoma of bone;ovarian cancer;ovarian epithelial carcinoma (superficial epithelial and stromal tumors);ovarian germ cell tumors;ovarian low malignant potential tumors;pancreatic cancer;pancreatic cancer, islet cell;papilloma;sino-nasal and nasal cancer;parathyroid carcinoma;penile cancer;pharyngeal cancer;pheochromocytoma;pineal astrocytoma;pineal germinoma;intermediately differentiated pineal parenchymal tumors;pineoblastoma and supratentorial primitive neuroectodermal tumors;pituitary tumors;pituitary adenomas;plasma cell neoplasms neoplasia) / multiple myeloma;pleuropulmonary blastoma;primary central nervous system lymphoma;prostate cancer;rectal cancer;renal cell carcinoma (kidney cancer);renal pelvis and ureter, transitional cell carcinoma;airway cancer involving the NUT gene on chromosome 15;retinoblastoma;rhabdomyosarcoma, childhood;salivary gland cancer;sarcoma, Ewing family tumor;Sezary syndrome;skin cancer (melanoma);skin cancer (non-melanoma);small cell lung cancer;small intestine cancer soft tissue sarcoma;soft tissue sarcoma;spinal tumor;squamous cell carcinoma;squamous cervical carcinoma of unknown primary, metastatic;gastric (stomach) carcinoma;supratentorial primitive neuroectodermal tumor;T-cell lymphoma, skin (mycosis fungoides and Sezary syndrome);testicular cancer;throat cancer;thymoma;thymoma and thymic carcinoma;thyroid cancer;pediatric thyroid cancer;transitional cell carcinoma of the renal pelvis and ureter;urethral cancer;uterine cancer, endometrium;uterine sarcoma;vaginal cancer;vulvar cancer;and Wilms' tumor.

[0109] A method for treating a neoplastic disease in a subject in need of treatment, wherein the neoplastic disease is a solid tumor and the route of administration of the arenavirus particles is intratumoral injection. [Table 1] [Brief description of the drawings]

[0110] 4. Brief description of the drawings [Figure 1] Schematic diagram of the artLCMV-4xKRASmut and artPICV-4xKRASmut vectors.

[0111] [Diagram 2] Schematic diagram of the artLCMV-4xKRASmut_E7 and artPICV-4xKRASmut_E7 vectors.

[0112] [Diagram 3] Schematic diagram of the artLCMV-4xKRASmut_EBV and artPICV-4xKRASmut_EBV vectors.

[0113] [Figure 4] Schematic diagram of the artLCMV-KRASmut and artPICV-KRASmut vectors.

[0114] [Figure 5A] Schematic diagram of the artLCMV-14xp53mut vector. [Figure 5B] Schematic diagram of the artPICV-14xp53mut vector.

[0115] [Figure 6A] Schematic diagram of the artLCMV-14xp53mut_E7 vector. [Figure 6B] Schematic diagram of the artPICV-14xp53mut_E7 vector.

[0116] [Figure 7A] Schematic diagram of the artLCMV-14xp53mut_EBV vector. [Figure 7B] Schematic diagram of the artPICV-14xp53mut_EBV vector.

[0117] [Figure 8] Schematic diagram of the artLCMV-p53mut and artPICV-p53mut vectors.

[0118] [Figure 9A] Schematic diagram of the artLCMV-p53mut / KRASmut and artPICV-p53mut / KRASmut vectors.

[0119] [Figure 9B] Schematic diagram of the artLCMV-KRASmut / p53mut and artPICV-KRASmut / p53mut vectors.

[0120] [Figure 9C] Schematic diagram of antigenic inserts in artLCMV and artPICV vectors encoding antigenic fragments of mutant KRAS and / or TP53.

[0121] [Figure 10] Schematic diagram of artLCMV-5xKRASmut-H2 and artPICV-5xKRASmut-H2 vectors.

[0122] [Figure 11] Induction of CD8+ T cell responses in HLA-A*11 transgenic mice (i.e., CB6F1-Tg(HLA-A*1101 / H2-Kb)A11.01 mice) following administration of arenavirus particles encoding different combinations of mutated KRAS epitopes. CD8+ T cell (i.e., IFN-γ+) responses to individual mutated KRAS epitopes were analyzed by ELISPOT in HLA-A*11 transgenic mice following prime-boost administration of the indicated vectors. Peptide stimulation was performed with wild-type and mutant-specific KRAS-based peptides. A mixture of NP-based peptides derived from LCMV and PICV was used as control. IFN-γ+ SFU (per 105 cells) are shown as arithmetic mean ± standard deviation for individual mice.

[0123] [Figure 12] Transgene stability of artLCMV-5xKRASmut-H2 (FIG. 12A) and artPICV-5xKRASmut-H2 (FIG. 12B). 5xKRASmut transgene stability was analyzed by PCR at the indicated passage levels (p1 to p6).

[0124] [Figure 13] Nucleotide sequence (SEQ ID NO:21) of artLCMV-5xKRASmut-H2-NP-S segment (S segment #1 in FIG. 10). The following elements are shown from 5' to 3' of the disclosed sequence: the 5' untranslated region (UTR) based on the LCMV cl13 S segment is shown in lower case, the KRASmut-H2 transgene is shown in bold, the intergenic region (IGR) based on the LCMV cl13 S segment is boxed, the nucleoprotein (NP) based on the LCMV cl13 is shown in underlined letters, and the 3' untranslated region (UTR) based on the LCMV cl13 S segment is shown in lower case. The genome segment is RNA, and the sequence of SEQ ID NO:21 is shown as DNA, however, the RNA sequence (SEQ ID NO:34) is obtained by replacing all thymidines ("T") in SEQ ID NO:21 with uridines ("U").

[0125] [Figure 14]Nucleotide sequence (SEQ ID NO:22) of artLCMV-5xKRASmut-H2-GP-S segment (S segment #2 in FIG. 10). The following elements are shown from 5' to 3' of the disclosed sequence: the 5' untranslated region (UTR) based on the LCMV cl13 S segment is shown in lower case, the KRASmut-H2 transgene is shown in bold, the intergenic region (IGR) based on the LCMV cl13 S segment is shown in a box, the glycoprotein (GP) based on the LCMV WE is underlined, and the 3' untranslated region (UTR) based on the LCMV cl13 S segment is shown in lower case. The genome segment is RNA and the sequence of SEQ ID NO:22 is shown in DNA, however, the RNA sequence (SEQ ID NO:35) is obtained by replacing all thymidines ("T") in SEQ ID NO:22 with uridines ("U").

[0126] [Figure 15] Nucleotide sequence (SEQ ID NO:23) of artPICV-5xKRASmut-H2-NP-S segment (S segment #1 in FIG. 10). The following elements are shown from 5' to 3' of the disclosed sequence: 5' untranslated region (UTR) based on PICV p18 S segment is shown in lower case, KRASmut-H2 transgene is shown in bold, intergenic region (IGR) based on PICV p18 S segment is shown in a box, nucleoprotein (NP) based on PICV p18 is underlined, and 3' untranslated region (UTR) based on PICV p18 S segment is shown in lower case. The genomic segment is RNA, and the sequence of SEQ ID NO:23 is shown in DNA, however, the RNA sequence (SEQ ID NO:36) is obtained by replacing all thymidines ("T") in SEQ ID NO:23 with uridines ("U").

[0127] [Figure 16]Nucleotide sequence (SEQ ID NO:24) of artPCIV-5xKRASmut-H2-GP-S segment (S segment #2 in FIG. 10). The following elements are shown from 5' to 3' of the disclosed sequence: 5' untranslated region (UTR) based on PICV p18 S segment is shown in lower case, KRASmut-H2 transgene is shown in bold, intergenic region (IGR) based on PICV p18 S segment is shown in a box, glycoprotein (GP) based on PICV p18 is underlined, and 3' untranslated region (UTR) based on PICV p18 S segment is shown in lower case. The genomic segment is RNA and the sequence of SEQ ID NO:24 is shown in DNA, however, the RNA sequence (SEQ ID NO:37) is obtained by replacing all thymidines ("T") in SEQ ID NO:24 with uridines ("U").

[0128] [Figure 17] Nucleotide sequence (SEQ ID NO:27) of artLCMV-5xKRASmut-H1-NP-S segment (S segment #1 in FIG. 10). The following elements are shown from 5' to 3' of the disclosed sequence: the 5' untranslated region (UTR) based on the LCMV cl13 S segment is shown in lower case, the KRASmut-H1 transgene is shown in bold, the intergenic region (IGR) based on the LCMV cl13 S segment is shown in a box, the nucleoprotein (NP) based on the LCMV cl13 is underlined, and the 3' untranslated region (UTR) based on the LCMV cl13 S segment is shown in lower case. The genome segment is RNA and the sequence of SEQ ID NO:27 is shown in DNA, however, the RNA sequence (SEQ ID NO:38) is obtained by replacing all thymidines ("T") in SEQ ID NO:27 with uridines ("U").

[0129] [Figure 18]Nucleotide sequence (SEQ ID NO:28) of artLCMV-5xKRASmut-H1-GP-S segment (S segment #2 in FIG. 10). The following elements are shown from 5' to 3' of the disclosed sequence: the 5' untranslated region (UTR) based on the LCMV cl13 S segment is shown in lower case, the KRASmut-H1 transgene is shown in bold, the intergenic region (IGR) based on the LCMV cl13 S segment is shown in a box, the glycoprotein (GP) based on the LCMV WE is underlined, and the 3' untranslated region (UTR) based on the LCMV cl13 S segment is shown in lower case. The genome segment is RNA and the sequence of SEQ ID NO:28 is shown in DNA, however, the RNA sequence (SEQ ID NO:39) is obtained by replacing all thymidines ("T") in SEQ ID NO:28 with uridines ("U").

[0130] [Figure 19] Nucleotide sequence (SEQ ID NO:29) of artPICV-5xKRASmut-H1-NP-S segment (S segment #1 in FIG. 10). The following elements are shown from 5' to 3' of the disclosed sequence: 5' untranslated region (UTR) based on PICV p18 S segment is shown in lower case, KRASmut-H1 transgene is shown in bold, intergenic region (IGR) based on PICV p18 S segment is shown in a box, nucleoprotein (NP) based on PICV p18 is underlined, and 3' untranslated region (UTR) based on PICV p18 S segment is shown in lower case. The genomic segment is RNA and the sequence of SEQ ID NO:29 is shown in DNA, however, the RNA sequence (SEQ ID NO:40) is obtained by replacing all thymidines ("T") of SEQ ID NO:29 with uridines ("U").

[0131] [Figure 20]Nucleotide sequence (SEQ ID NO:30) of artPCIV-5xKRASmut-H1-GP-S segment (S segment #2 in FIG. 10). The following elements are shown from 5' to 3' of the disclosed sequence: 5' untranslated region (UTR) based on PICV p18 S segment is shown in lower case, KRASmut-H1 transgene is shown in bold, intergenic region (IGR) based on PICV p18 S segment is shown in a box, glycoprotein (GP) based on PICV p18 is underlined, and 3' untranslated region (UTR) based on PICV p18 S segment is shown in lower case. The genomic segment is RNA and the sequence of SEQ ID NO:30 is shown in DNA, however, the RNA sequence (SEQ ID NO:41) is obtained by replacing all thymidines ("T") of SEQ ID NO:30 with uridines ("U").

[0132] [Figure 21] Induction of CD8 T cell responses in HLA-B*07 transgenic mice (i.e., CB6F1-Tg(HLA-B*0702 / H2-Kb)B7.xx mice) after administration of arenavirus particles encoding different combinations of mutated KRAS epitopes. CD8 T cell (i.e., IFN-γ+) responses against individual mutated KRAS epitopes were analyzed by ELISpot in HLA-B*07 transgenic mice after prime-boost administration of the indicated vectors. Peptide stimulation was performed with wild-type and mutant-specific KRAS-based peptides. A mixture of NP-based peptides derived from LCMV and PICV was used as control. IFN-γ+ SFU (per 105 cells) is shown as arithmetic mean ± standard deviation for individual mice.

[0133] [Figure 22] Example study design disclosed in Section 8.5 of this disclosure.

[0134] [Diagram 23] Example study design disclosed in Section 8.6 of this disclosure.

[0135] [Figure 24] Induction of CD8 T cell responses in HLA-A*11 transgenic mice (i.e., CB6F1-Tg(HLA-A*1101 / H2-Kb)A11.01 mice) after administration of arenavirus particles encoding combinations of mutated KRAS epitopes. CD8 T cell (i.e., IFN-γ+) responses against individual mutated KRAS epitopes were analyzed by ELISpot in HLA-A*11 transgenic mice after prime administration of the indicated vectors. Peptide stimulation was performed with wild-type and mutant-specific KRAS-based peptides. A mixture of LCMV-derived NP-based peptides was used as control. PICV-derived NP-based peptides were not detected due to technical errors. IFN-γ+ SFU (per 105 cells) are shown as arithmetic mean ± standard deviation for individual mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0136] 5. MODE FOR CARRYING OUT THEINVENTION Provided herein are methods for treating a neoplastic disease (see Section 5.1) in a subject in need thereof, comprising delivering to the subject an arenavirus particle (see Arenavirus Particles as defined in Sections 5.3-5.6) that has been engineered to contain an arenavirus genome that includes only a nucleotide sequence encoding an antigenic fragment of mutant KRAS (see Section 5.7) or that further includes a nucleotide sequence encoding an antigenic fragment of a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53) or a tumor-associated antigen (see Section 5.9). In certain embodiments, the method for treating a neoplastic disease comprises administering a first arenavirus particle and a second arenavirus particle, wherein the first and second arenavirus particles encode the same or different antigenic fragments (see Section 5.10).

[0137] In certain embodiments, the various antigenic fragments (derived from mutant KRAS, mutated cancer driver genes (e.g., mutant TP53), and / or tumor associated antigens) are present on the same transcript, at the same location in the arenavirus genome, on the same genomic segment, and / or in the same arenavirus genome. In certain embodiments, the various antigenic fragments (derived from mutant KRAS, mutated cancer driver genes (e.g., mutant TP53), and / or tumor associated antigens) are present on separate transcripts, at separate locations in the arenavirus genome, on separate genomic segments, and / or in separate arenavirus genomes.

[0138] 5.1 Neoplastic diseases In certain embodiments, the neoplastic diseases that can be treated with the methods and compositions described herein include the neoplastic diseases listed below. Thus, the mutant KRAS (see Section 5.7), mutated cancer driver genes (see Section 5.8, e.g., mutant TP53), or tumor-associated antigens (see Section 5.9) encoded by the genome of the arenavirus particles described herein may be associated with or specific to one of the listed neoplastic diseases. Neoplastic diseases include: acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphocytic leukemia; acute myeloid leukemia; acute myeloid leukemia (adult / child); adrenal cortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratoid / rhabdoid tumor; basal cell carcinoma; bile duct carcinoma, extrahepatic (cholangiocarcinoma); bladder cancer; osteosarcoma / malignant fibrous histiocytoma of bone; brain cancer (adult / child); brain tumor, cerebellar astrocytoma (adult / child); brain tumor, Cerebral astrocytoma / malignant glioma brain tumor;Brain tumor, ependymoma;Brain tumor, medulloblastoma;Brain tumor, supratentorial primitive neuroectodermal tumor;Brain tumor, optic and hypothalamic glioma;Brain stem glioma;Breast cancer;Bronchial adenoma / carcinoid;Bronchial tumor;Burkitt lymphoma;Cancer in children;Carcinoid gastrointestinal tumor;Carcinoid tumor;Adult carcinoma, unknown site of primary;Carcinoma of unknown primary;Central nervous system embryonal tumor;Central nervous system lymphoma, primary;Cervical cancer;Pediatric adrenocortical carcinoma;Pediatric cancer ;pediatric cerebral astrocytoma;chordomegalovirus, childhood;chronic lymphocytic leukemia;chronic myelogenous leukemia;chronic myeloproliferative disorder;colon cancer;colorectal cancer;craniopharyngioma;cutaneous T-cell lymphoma;desmoplastic small round cell tumor;emphysema;endometrial cancer;ependymoblastoma;ependymoma;esophageal cancer;Ewing's sarcoma in Ewing family of tumors;extracranial germ cell tumors;extragonadal germ cell tumors;extrahepatic bile duct cancer;gallbladder cancer;gastric (stomach) cancer;gastric carcinoid;gastrointestinal carcinoid tumors; Gastrointestinal stromal tumors;Germ cell tumors: extracranial, extragonadal, or ovarian;Gestational trophoblastic tumors, unknown primary site;Gliomas;Gliomas of the brain stem;Gliomas, childhood visual tract and hypothalamus;Hairy cell leukemia;Head and neck cancer;Carcinoma of the heart;Hepatocellular (liver) carcinoma;Hodgkin's lymphoma;Hypopharyngeal carcinoma;Hypothalamic and visual tract gliomas;Intraocular melanoma;Islet cell carcinoma (pancreatic islet);Kaposi's sarcoma;Kidney carcinoma (renal cell carcinoma);Langerhans cell histiocytosis;Laryngeal cancer;Cancers of the lip and oral cavity;Liposarcoma;Hepatocellular carcinoma (primary);Lung cancer, non-small cell;Lung cancer, small cell;Lymphoma, primary central nervous system;Macroglobulinemia, Waldenstrom;Male breast cancer;Malignant fibrous histiocytoma / osteosarcoma of bone;Medulloblastoma;Melanoma;Melanoma, intraocular (eye);Merkel cell carcinoma;Merkel cell skin cancer;Mesothelioma;Mesothelioma, adult malignant;Metastatic squamous cell neck cancer of unknown primary;Oral cancer;Multiple endocrine neoplasia syndromes;multiple myeloma / plasma cell neoplasms;mycosis fungoides, myelodysplastic syndromes;myelodysplastic / myeloproliferative disorders;myeloid leukemia, chronic;myeloid leukemia, adult acute;myeloid leukemia, childhood acute;myeloma, multiple (cancer of the bone marrow);myeloproliferative disorders, chronic;nasal and paranasal sinus cancer;nasopharyngeal cancer;neuroblastoma, non-small cell lung cancer;non-Hodgkin's lymphoma;oligodendrocyte glioma;oral cancer;oral cavity cancer;oropharyngeal Head cancer;Osteosarcoma / malignant fibrous histiocytoma of bone;Ovarian cancer;Ovarian epithelial carcinoma (superficial epithelial and stromal tumors);Ovarian germ cell tumors;Ovarian low malignant potential tumors;Pancreatic cancer;Pancreatic cancer, islet cell;Papilloma;Sino-nasal and nasal cancer;Parathyroid cancer;Penile cancer;Pharyngeal cancer;Pheochromocytoma;Pineal astrocytoma;Pineal germinoma;Intermediately differentiated pineal parenchymal tumors;Pineoblastoma and supratentorial primitive neuroectodermal tumors;Pituitary tumors;Pituitary gland Neoplasms;Plasma cell neoplasms / multiple myeloma;Pleuropulmonary blastoma;Primary central nervous system lymphoma;Prostate cancer;Rectal cancer;Renal cell carcinoma (kidney cancer);Renal pelvis and ureter, transitional cell carcinoma;Airway cancer involving the NUT gene on chromosome 15;Retinoblastoma;Rhabdomyosarcoma, childhood;Salivary gland cancer;Sarcoma, Ewing family tumors;Sezary syndrome;Skin cancer (melanoma);Skin cancer (non-melanoma);Small cell lung cancer;Small intestine cancer Soft tissue sarcoma;soft tissue sarcoma;spinal tumor;squamous cell carcinoma;squamous cell carcinoma of the neck, occult primary, metastatic;gastric (stomach) carcinoma;supratentorial primitive neuroectodermal tumor;T-cell lymphoma, skin (mycosis fungoides and Sézary syndrome);testicular cancer;throat cancer;thymoma;thymoma and thymic carcinoma;thyroid cancer;pediatric thyroid cancer;transitional cell carcinoma of the renal pelvis and ureter;urethral cancer;uterine cancer, endometrium;uterine sarcoma;vaginal cancer;vulvar cancer;and Wilms' tumor.

[0139] In certain embodiments, a method for treating a neoplastic disease in a subject in need thereof comprises administering to the subject arenavirus particles, wherein the arenavirus particles encode antigenic fragments of mutant KRAS (see Section 5.7) and / or mutant TP53 (see Section 5.8). In certain embodiments, the arenavirus particles encode antigenic fragments of mutant KRAS, wherein the mutation in KRAS may be associated with or specific to pancreatic cancer, colorectal cancer, lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer (NSCLC). In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject arenavirus particles, wherein the arenavirus particles encode an antigenic fragment of mutant KRAS, and the KRAS mutation may be associated with or specific to pancreatic cancer, colorectal cancer, lung adenocarcinoma, and the KRAS mutation is G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R. In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject arenavirus particles, wherein the arenavirus particles encode an antigenic fragment of mutant KRAS, and the KRAS mutation may be associated with or specific to pancreatic cancer, colorectal cancer, lung adenocarcinoma, and the KRAS mutation is G13D, G12V, G12C, G12D, and G12R. In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject an arenavirus particle, wherein the arenavirus particle encodes an antigenic fragment of a mutant KRAS, and the KRAS mutation may be associated with or specific to pancreatic cancer, and the KRAS mutation is G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A.In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject arenavirus particles, wherein the arenavirus particles encode antigenic fragments of mutant KRAS and / or mutant TP53, and wherein the KRAS and TP53 mutations may be associated with or specific to pancreatic cancer, and wherein the KRAS mutation is G12D, G12R, and / or G12V, and the TP53 mutation is R175H, R248W, and / or R273C. In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject arenavirus particles, wherein the arenavirus particles encode antigenic fragments of mutant KRAS and / or mutant TP53, and the antigenic fragments comprise mutations, and the KRAS and TP53 mutations may be associated with or specific to colorectal cancer or lung adenocarcinoma, and the KRAS mutations are G12D, G12V, G13D, A146T, and / or G12C, and the TP53 mutations are R175H, R273H, and / or R248W. In certain embodiments, the arenavirus particles encode antigenic fragments of mutant KRAS and / or mutant TP53, where the antigenic fragment comprises a mutation, and the KRAS and TP53 mutation may be associated with or specific for lung adenocarcinoma, where the KRAS mutation is G12C, G12V, G12D, and / or G12S, and the TP53 mutation is R175H. In certain embodiments, the arenavirus particles encode antigenic fragments of mutant KRAS and / or mutant TP53, where the antigenic fragment comprises a mutation, and the KRAS and TP53 mutation may be associated with or specific for pancreatic cancer, colorectal cancer, or lung adenocarcinoma, where the KRAS mutation is G12C, G12D, G12R, G13D, and / or G12V, and the TP53 mutation is R175H, R273H, and / or R248W.

[0140] In certain embodiments, the method for treating a neoplastic disease in a subject in need of treatment further comprises administering to the subject a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS (see Section 5.7) and / or mutant TP53 (see Section 5.8). In certain embodiments, the second arenavirus particle encodes an antigenic fragment of mutant KRAS, wherein the mutation in KRAS may be associated with or specific to pancreatic cancer. In certain embodiments, the second arenavirus particle encodes an antigenic fragment of mutant KRAS, wherein the mutation in KRAS may be associated with or specific to pancreatic cancer, wherein the mutation in KRAS is G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A. In certain embodiments, the second arenavirus particle encodes an antigenic fragment of mutant KRAS, where the KRAS mutation may be associated with or specific for pancreatic cancer, colorectal cancer, lung adenocarcinoma, and the KRAS mutation is G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R. In certain embodiments, the second arenavirus particle encodes an antigenic fragment of mutant KRAS and / or mutant TP53, where the KRAS and TP53 mutations may be associated with or specific for pancreatic cancer, where the KRAS mutation is G12D, G12R, and / or G12V, and the TP53 mutation is R175H, R248W, and / or R273C. In certain embodiments, the second arenavirus particle encodes an antigenic fragment of mutant KRAS and / or mutant TP53, where the KRAS and TP53 mutations may be associated with or specific to colorectal cancer or lung adenocarcinoma, and the KRAS mutation is G12D, G12V, G13D, A146T, and / or G12C, and the TP53 mutation is R175H, R273H, and / or R248W.In certain embodiments, the second arenavirus particle encodes an antigenic fragment of mutant KRAS and / or mutant TP53, where the KRAS and TP53 mutations may be associated with or specific to pancreatic cancer, colorectal cancer, or lung adenocarcinoma, and the KRAS mutation is G12C, G12D, G12R, G13D, and / or G12V, and the TP53 mutation is R175H, R273H, and / or R248W. In certain embodiments, the second arenavirus particle encodes an antigenic fragment of mutant KRAS and / or mutant TP53, where the KRAS and TP53 mutations may be associated with or specific to lung adenocarcinoma, and the KRAS mutation is G12C, G12V, G12D, and / or G12S, and the TP53 mutation is R175H.

[0141] In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject an arenavirus particle, wherein the arenavirus particle encodes an antigenic fragment of a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8), or a tumor-associated antigen (see Section 5.9). In certain embodiments, the arenavirus particle encodes an antigenic fragment of a mutant KRAS, wherein the mutation in KRAS may be associated with or specific to pancreatic cancer, colorectal cancer, lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer (NSCLC). In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject an arenavirus particle, wherein the arenavirus particle encodes an antigenic fragment of a mutant KRAS, and the KRAS mutation may be associated with or specific to pancreatic cancer, and the KRAS mutation is KRAS G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A. In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject arenavirus particles, wherein the arenavirus particles encode antigenic fragments of mutant KRAS, mutant TP53, and / or mutant U2AF1, and the KRAS, TP53, and / or U2AF1 mutations may be associated with or specific to pancreatic cancer, and the KRAS mutation is G12D, G12V, G12R, Q61H, and / or Q61R, the TP53 mutation is R175H, R248W, G245S, R282W, R248Q, and / or R273C, and the U2AF1 mutation is S34F. In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject an arenavirus particle, wherein the arenavirus particle encodes an antigenic fragment of a mutant KRAS, and the KRAS mutation may be associated with or specific to pancreatic cancer, colorectal cancer, or lung adenocarcinoma, and the KRAS mutation is KRAS G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R.In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject arenavirus particles, wherein the arenavirus particles encode antigenic fragments of mutant KRAS, mutant BRAF, mutant TP53, mutant FBXW7 and / or mutant PIK3CA, wherein the KRAS, BRAF, TP53, FBXW7 and / or PIK3CA mutations may be associated with or specific to colorectal cancer or lung adenocarcinoma, and wherein the KRAS mutation is G12D, G12V, G13D, A146T and / or G12C, the BRAF mutation is V600E, the TP53 mutation is R175H, R273H and / or R248W, the FBXW7 mutation is R465H, and the PIK3CA mutation is E545K and / or H1047R. In certain embodiments, a method for treating a neoplastic disease in a subject in need thereof comprises administering to the subject arenavirus particles, wherein the arenavirus particles encode antigenic fragments of mutant KRAS, mutant BRAF, mutant PIK3CA, mutant EGFR, mutant TP53 and / or mutant U2AF1, and wherein the mutations in KRAS, BRAF, PIK3CA, EGFR, TP53 and / or U2AF1 may be associated with or specific to pancreatic cancer, colorectal cancer or lung adenocarcinoma, and wherein the mutation in KRAS is G12C, G12D, G12R, G13D and / or G12V, the mutation in BRAF is V600E, the mutation in PIK3CA is E545K, the mutation in EGFR is L858R, the mutation in TP53 is R175H, R273H and / or R248W, and the mutation in U2AF1 is S34F.In certain embodiments, a method for treating a neoplastic disease in a subject in need thereof comprises administering to the subject arenavirus particles, wherein the arenavirus particles encode antigenic fragments of mutant KRAS, mutant TP53, mutant U2AF1, mutant PIK3CA, mutant EGFR and / or mutant BRAF, wherein the KRAS, TP53, U2AF1, PIK3CA, EGFR and / or BRAF mutations may be associated with or specific to lung adenocarcinoma, and wherein the KRAS mutation is G12C, G12V, G12D, and / or G12S, the TP53 mutation is R175H, the U2AF1 mutation is S34F, the EGFR mutation is L858R, L861Q and / or E746_A750del, the PIK3CA mutation is E545K and / or E542K, and the BRAF mutation is V600E. In certain embodiments, a method for treating a neoplastic disease in a subject in need of treatment comprises administering to the subject arenavirus particles, wherein the arenavirus particles encode antigenic fragments of mutant KRAS, mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA and / or mutant RET, and wherein mutations in KRAS, AKT1, BRAF, HER2, MEK1, MET, NRAS, PIK3CA and / or RET may be associated with or specific to NSCLC.

[0142] In certain embodiments, the method for treating a neoplastic disease in a subject in need of treatment further comprises administering to the subject a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of a mutant KRAS, a mutated cancer driver gene, or a tumor-associated antigen. In certain embodiments, the second arenavirus particle encodes an antigenic fragment of a mutant KRAS, wherein the mutation in KRAS may be associated with or specific to pancreatic cancer, and the mutation in KRAS is KRAS G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A. In certain embodiments, the method for treating a neoplastic disease in a subject in need of treatment further comprises administering to the subject a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS, mutant TP53, and / or mutant U2AF1, and the KRAS, TP53 and / or U2AF1 mutations may be associated with or specific to pancreatic cancer, the KRAS mutation is G12D, G12V, G12R, Q61H and / or Q61R, the TP53 mutation is R175H, R248W, G245S, R282W, R248Q and / or R273C, and the U2AF1 mutation is S34F. In certain embodiments, the method for treating a neoplastic disease in a subject in need of treatment further comprises administering to the subject a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of a mutant KRAS, and the KRAS mutation may be associated with or specific to pancreatic cancer, colorectal cancer or lung adenocarcinoma, and the KRAS mutation is KRAS G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R.In certain embodiments, the method for treating a neoplastic disease in a subject in need of treatment further comprises administering to the subject a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS, mutant BRAF, mutant TP53, mutant FBXW7 and / or mutant PIK3CA, wherein the KRAS, BRAF, TP53, FBXW7 and / or PIK3CA mutation may be associated with or specific to colorectal cancer or lung adenocarcinoma, wherein the KRAS mutation is G12D, G12V, G13D, A146T and / or G12C, the BRAF mutation is V600E, the TP53 mutation is R175H, R273H and / or R248W, the FBXW7 mutation is R465H, and the PIK3CA mutation is E545K and / or H1047R. In certain embodiments, the method for treating a neoplastic disease in a subject in need of treatment further comprises administering to the subject a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS, mutant BRAF, mutant PIK3CA, mutant EGFR, mutant TP53, and / or mutant U2AF1, and encodes an antigenic fragment of KRAS, BRAF, PIK3CA, EGFR, TP53, and / or U2AF1. The 2AF1 mutation may be associated with or specific to pancreatic cancer, colorectal cancer or lung adenocarcinoma, the KRAS mutation is G12C, G12D, G12R, G13D and / or G12V, the BRAF mutation is V600E, the PIK3CA mutation is E545K, the EGFR mutation is L858R, the TP53 mutation is R175H, R273H and / or R248W, and the U2AF1 mutation is S34F.In certain embodiments, the method for treating a neoplastic disease in a subject in need of treatment further comprises administering to the subject a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS, mutant TP53, mutant U2AF1, mutant PIK3CA, mutant EGFR, and / or mutant BRAF, and is capable of binding to KRAS, TP53, U2AF1, PIK3CA, EGFR, and / or BRAF. The AF mutation may be associated with or specific to lung adenocarcinoma, the KRAS mutation is G12C, G12V, G12D, and / or G12S, the TP53 mutation is R175H, the U2AF1 mutation is S34F, the EGFR mutation is L858R, L861Q, and / or E746_A750del, the PIK3CA mutation is E545K and / or E542K, and the BRAF mutation is V600E. In certain embodiments, the method for treating a neoplastic disease in a subject in need of treatment further comprises administering to the subject a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS, mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA and / or mutant RET, and the mutations in AKT1, BRAF, HER2, MEK1, MET, NRAS, PIK3CA and / or RET may be associated with or specific to NSCLC.

[0143] 5.2 Arenavirus constructs In certain embodiments, arenavirus particles that can be engineered for the methods and compositions herein include the constructs listed below. In certain embodiments, the arenavirus construct is a non-replicating arenavirus construct as described in International Patent Application Publication No. WO2009 / 083210 (incorporated herein in its entirety). See Section 5.3. In certain embodiments, the arenavirus construct is a replicating or non-replicating three-segment arenavirus construct as described in International Patent Application Publication No. WO2016 / 075250 and WO2021 / 089853 (both of which are incorporated herein in their entirety). See Sections 5.4 and 5.5.

[0144] The arenaviruses used in the methods and compositions provided herein can be, for example, Old World viruses, such as Lassa virus, Lymphocytic choriomeningitis virus (LCMV), Mobara virus, Mopeia virus, or Yippi virus, or New World viruses, such as Amapari virus, Flexar virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde virus, Piritar virus, Sabia virus, Tacaribe virus, Tamiami virus, Bear Canyon virus, Alpahuayo virus (ALLV), or Whitewater Arroyo virus. The arenaviruses used in the methods and compositions provided herein can be, for example, arenaviruses, mammarenaviruses, Old World mamma arenaviruses, New World mamma arenaviruses, clade A New World mamma arenaviruses, clade B New World mamma arenaviruses, clade C New World mamma arenaviruses, or clade D New World mamma arenaviruses. The arenaviruses used in the methods and compositions provided herein include, but are not limited to, Alpahuayovirus, Arshavirus, Juninvirus, Bear Canyonvirus, Sabiavirus, Pichindevirus, Chaparevirus, Lijiangvirus, Kupicivirus, Flexarvirus, Gairovirus, Guanaritovirus, Yippivirus, Lassavirus, Latinovirus, Loui Rivervirus, Lujovirus, Lunavirus, Lurivirus, Lunkvirus, and others. The virus may be a Marma arenavirus, including but not limited to, Lymphocytic choriomeningitis virus, Machupo virus, Mariental virus, Merinowalk virus, Mobara virus, Mopeia virus, Morogoro virus, Okahandja virus, Oliveros virus, Parana virus, Piritar virus, Apore virus, Ryukyu virus, Amapari virus, Solwezi virus, Souri virus, Tacaribe virus, Tamiami virus, Unshu virus, Whitewater Arroyo virus, Big Brushy Tank virus, Catalina virus, Skinner Tank virus, Tonto Creek virus, or Shapouri virus.In certain embodiments, the arenavirus used in the methods and compositions provided herein is a clade A arenavirus. In certain embodiments, the arenavirus used in the methods and compositions provided herein is a Pichinde virus.

[0145] 5.3 Replication-defective arenavirus particles Exemplary replication-defective arenavirus particles are described, for example, in International Patent Application Publication No. WO2009 / 083210, which is incorporated herein in its entirety. In certain embodiments, replication-defective (e.g., replication-deficient) arenavirus particles having a nucleotide sequence encoding a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein can be used in the methods and compositions provided herein. In specific embodiments, the replication-defective arenavirus particles described herein are used in the methods and compositions provided herein in combination with a replication-competent arenavirus particle described herein. In more specific embodiments, the replication-defective arenavirus particles described herein are used in the methods and compositions provided herein in combination with a replication-competent arenavirus particle described herein, wherein the replication-competent arenavirus particle is directly injected into a tumor of a subject.

[0146] In certain embodiments, arenavirus particles are provided herein in which the ORFs encoding GP, NP, Z, or L proteins are deleted or functionally inactivated, such that the resulting virus cannot produce further infectious progeny virus particles. Arenavirus particles containing genetically modified genomes in which one or more ORFs are deleted or functionally inactivated can be produced in complementing cells (i.e., cells expressing the deleted or functionally inactivated arenavirus ORFs). The genetic material of the resulting arenavirus particles can be transferred to a host cell upon infection of the host cell, where it can be expressed and amplified.

[0147] In certain embodiments, such heterologous nucleotide sequence may be polycistronic, so that multiple polypeptides are ultimately produced from a single heterologous nucleotide sequence / transcript.This can be achieved, for example, by using internal ribosome entry sites.In certain embodiments, one of such polypeptides may be mutant KRAS.In certain embodiments, such heterologous nucleotide sequence may code for mutant KRAS, mutated cancer driver gene (e.g., mutant TP53), or antigenic fragment of tumor-associated antigen.

[0148] In certain embodiments, the ORF of the arenavirus is deleted or functionally inactivated and replaced with a nucleotide sequence encoding a mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), or an antigenic fragment of a tumor-associated antigen described herein. In a specific embodiment, the ORF encoding the glycoprotein GP of the arenavirus is deleted or functionally inactivated. In certain embodiments, functional inactivation of a gene removes any translation product. In certain embodiments, functional inactivation refers to a genetic alteration that allows some translation, but the translation product is no longer functional and cannot replace the wild-type protein.

[0149] In certain embodiments, at least one of the four arenavirus ORFs encoding the GP, NP, Z, and L proteins is deleted and replaced with a nucleotide sequence encoding a mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), or an antigenic fragment of a tumor-associated antigen provided herein. In another embodiment, at least one ORF, at least two ORFs, at least three ORFs, or at least four ORFs encoding the GP, NP, Z, and L proteins may be deleted and replaced with a nucleotide sequence encoding a mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), or an antigenic fragment of a tumor-associated antigen provided herein. In a specific embodiment, only one of the four ORFs encoding the GP, NP, Z, and L proteins is deleted and replaced with a nucleotide sequence encoding a mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), or an antigenic fragment of a tumor-associated antigen provided herein. In a more specific embodiment, the ORF encoding the GP of the arenavirus genome segment is deleted. In another specific embodiment, the ORF encoding the NP of the arenavirus genome segment is deleted. In a more specific embodiment, the ORF encoding the Z protein of the arenavirus genome segment is deleted. In yet another specific embodiment, the ORF encoding the L protein of the arenavirus genome segment is deleted.

[0150] Thus, in certain embodiments, the arenavirus particles provided herein comprise a genome segment in which (i) an ORF encoding a GP, NP, Z protein, or L protein has been deleted, and (ii) the deleted ORF has been replaced with a nucleotide sequence encoding a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein.

[0151] In certain embodiments, growth and infectivity of arenavirus particles in complementing cells is not affected by nucleotide sequences encoding antigenic fragments of mutant KRAS (see Section 5.7), mutated cancer driver genes (see Section 5.8, e.g., mutant TP53), or tumor-associated antigens (see Section 5.9) provided herein.

[0152] In certain embodiments, the arenavirus particles or arenavirus genome segments provided herein that include a nucleotide sequence encoding an antigenic fragment of mutant KRAS (see Section 5.7) provided herein may further include at least one nucleotide sequence encoding at least one antigenic fragment of a mutated cancer driver gene or tumor associated antigen. In certain embodiments, the mutated cancer driver gene is mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, mutant RET, mutant APC, mutant TP53, mutant EGFR, mutant FBXW7, mutant SMAD4, mutant GNAS, mutant ERBB2, mutant ERBB3, mutant CDKN2A, mutant CTNNB1, and / or mutant U2AF1 (see Section 5.8). In certain embodiments, the tumor-associated antigen is from the BIRC family, CEACAM family, CTA family, EPH family, ERBB family, FOLR family, GAST family, GUCY2 family, IDO family, IL13RA family, KDR family, KLK family, MAGE family, MUC family, PEMT family, SDC family, SLAMF family, TERT family, TLR family, TPTE family, TYR family, WT family and / or XBP family (see Section 5.9).

[0153] In certain embodiments, the arenavirus particles described herein are suitable for use as vaccines, immunotherapies, or pharmaceutical compositions, and methods of using such arenavirus particles for the treatment of neoplastic diseases, such as cancer, are provided. A more detailed description of methods of using the arenavirus particles described herein is provided in Section (see Section 5.10).

[0154] 5.4 Three-segment arenavirus particles with ORF rearrangements Exemplary three-segmented arenavirus particles are described, for example, in International Patent Application Publication Nos. WO2016 / 075250 and WO2017 / 198726, which are incorporated by reference in their entireties.

[0155] In certain embodiments, three-segmented arenavirus particles with rearrangements of ORFs comprising nucleotide sequences encoding antigenic fragments of mutant KRAS (see Section 5.7), mutated cancer driver genes (see Section 5.8, e.g., mutant TP53), or tumor-associated antigens (see Section 5.9) provided herein may be used in the methods and compositions provided herein. In one aspect, three-segmented arenavirus particles comprising one L segment and two S segments, or two L segments and one S segment, are provided herein. See Section 5.6(b) for production methods. In certain embodiments, propagation of three-segmented arenavirus particles does not result in replicable two-segmented arenavirus particles. In other words, the three-segmented viruses provided herein are genetically stable. More specifically, in certain embodiments, two of the genome segments (e.g., two S segments or two L segments, respectively) cannot recombine to obtain a single viral segment that can replace the two parental segments. In certain embodiments, intersegmental recombination of two of the genome segments (e.g., two S segments or two L segments, respectively), integrating two arenavirus ORFs onto only one segment instead of two separate segments, abolishes viral promoter activity. In specific embodiments, the genome of a three-segmented arenavirus particle comprises an arenavirus ORF at a position other than its wild-type position and a nucleotide sequence encoding an antigenic fragment of a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein. In yet another specific embodiment, the genome of a three-segmented arenavirus particle comprises all four arenavirus ORFs. Thus, in certain embodiments, the three-segmented arenavirus particle is replicable and infectious.

[0156] In certain embodiments, the genome of such a replicative and infectious three-segmented arenavirus particle (see Section 5.6(b)) has two locations available for inclusion of a heterologous nucleotide sequence. These locations can be used to incorporate heterologous nucleotide sequences, for example, as described in Table 1 below. In certain embodiments, each such heterologous nucleotide sequence can be transcribed into a single transcript. In certain embodiments, each such heterologous nucleotide sequence encodes a polypeptide. In certain embodiments, such heterologous nucleotide sequences can be polycistronic, so that ultimately, multiple polypeptides are produced from a single heterologous nucleotide sequence / transcript. This can be achieved, for example, by using an internal ribosome entry site. In certain embodiments, one such polypeptide can be a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9). In certain embodiments, the heterologous nucleotide sequences at the two available locations together encode a mutant KRAS. In other embodiments, the heterologous nucleotide sequence at one of the two available positions encodes an antigenic fragment of mutant KRAS, and the heterologous nucleotide sequence at the other of the two available positions encodes an antigenic fragment of a mutated cancer driver gene (e.g., mutant TP53) or a tumor-associated antigen.

[0157] In certain embodiments, three-segmented arenavirus particles with rearrangements of ORFs that include nucleotide sequences that do not encode foreign antigens (see Section 5.6(b)) may be used in the methods and compositions provided herein. In specific embodiments, the three-segmented arenavirus particles include an ORF at a position other than the wild-type position of that ORF. In yet another specific embodiment, the three-segmented arenavirus particles include all four arenavirus ORFs. Thus, in certain embodiments, the three-segmented arenavirus particles are replication competent and infectious.

[0158] In certain embodiments, the ORFs encoding the GP, NP, Z, or L proteins of the three-segmented arenavirus particles described herein (see section 5.6(b)) may be under the control of the arenavirus genome 3'UTR or the arenavirus genome 5'UTR. In a more specific embodiment, the arenavirus genome 3'UTR is the 3'UTR of the arenavirus S segment. In another specific embodiment, the arenavirus genome 3'UTR is the 3'UTR of the arenavirus L segment. In a more specific embodiment, the arenavirus genome 5'UTR is the 5'UTR of the arenavirus S segment. In other specific embodiments, the arenavirus genome 5'UTR is the 5'UTR of the arenavirus L segment.

[0159] In other embodiments, the ORFs encoding the GP, NP, Z, or L proteins of the three-segmented arenavirus particles described herein (see Section 5.6(b) for methods of production) may be under the control of arenavirus conserved terminal sequence elements (regions 19-20 nt at the 5' and 3' ends) (see, e.g., Perez & de la Torre, 2003, J Virol. 77(2):1184-1194).

[0160] In certain embodiments, the ORFs encoding the GP, NP, Z or L proteins of the three-segmented arenavirus particle (see section 5.6(b) for production methods) may be under the control of the promoter element of the 5'UTR (see, e.g., Albarino et al., 2011, J Virol., 85(8):4020-4). In another embodiment, the ORFs encoding the GP, NP, Z or L proteins of the three-segmented arenavirus particle may be under the control of the promoter element of the 3'UTR (see, e.g., Albarino et al., 2011, J Virol., 85(8):4020-4). In a more specific embodiment, the promoter element of the 5'UTR is the 5'UTR promoter element of the S segment(s) or L segment(s). In another specific embodiment, the promoter element of the 3'UTR is the 3'UTR promoter element of the S segment(s) or L segment(s).

[0161] In certain embodiments, the ORFs encoding the GP, NP, Z or L proteins of the three-segmented arenavirus particle may be under the control of a truncated arenavirus 3'UTR or a truncated arenavirus 5'UTR (see, e.g., Perez & de la Torre, 2003, J Virol. 77(2):1184-1194; Albarino et al., 2011, J Virol., 85(8):4020-4). In more specific embodiments, the truncated 3'UTR is derived from the 3'UTR of an arenavirus S or L segment. In more specific embodiments, the truncated 5'UTR is derived from the 5'UTR of an arenavirus S or L segment(s).

[0162] Also provided herein is a cDNA of the genome of a three-segmented arenavirus particle comprising a nucleotide sequence encoding an antigenic fragment of a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein. In a more specific embodiment, provided herein is a DNA nucleic acid or set of DNA nucleic acids encoding the genome of a three-segmented arenavirus particle described in Table 1.

[0163] In certain embodiments, the nucleic acid encoding the genome of the three-segmented arenavirus genome (see section 5.6(b)) is part of or incorporated into one or more DNA expression vectors. In specific embodiments, the nucleic acid encoding the genome of the three-segmented arenavirus particle is part of or incorporated into one or more DNA expression vectors that facilitate the production of the three-segmented arenavirus particles described herein. In another embodiment, the cDNA described herein may be incorporated into a plasmid. Routine conventional techniques of molecular biology and DNA manipulation and production can be used, including techniques for the production of cDNA, as well as any cloning technique known to those of skill in the art. Such techniques are well known to those of skill in the art and are described in Sambrook and Russell, Molecular Cloning: A laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory NY (2001).

[0164] Provided herein are cell lines, cultures, and methods of culturing cells transfected with the nucleic acids, vectors, and compositions provided herein.

[0165] In a specific embodiment, the arenavirus particles described herein are attenuated.In a specific embodiment, the three-segment arenavirus particles are attenuated so that the virus remains at least partially replicable and can replicate in vivo, but can only produce low viral loads, resulting in non-pathogenic subclinical levels of infection.Such attenuated viruses can be used as immunogenic compositions.

[0166] In certain embodiments, a three segmented arenavirus particle has the same tropism as the two segmented arenavirus particle from which the three segmented virus was derived.

[0167] Also provided herein are pharmaceutical compositions comprising the three segmented arenavirus particles described herein.

[0168] (a) A three-segment arenavirus particle containing one L segment and two S segments. In one aspect, provided herein is a three-segmented arenavirus particle comprising one L segment and two S segments. In certain embodiments, propagation of a three-segmented arenavirus particle comprising one L segment and two S segments does not result in a replication-competent two-segmented virus particle. In a specific embodiment, propagation of a three-segmented arenavirus particle comprising one L segment and two S segments results in a replication-competent two-segmented virus particle lacking type I interferon receptor, type II interferon receptor, and recombination activating gene (RAG1). 4In mice infected with PFU of three segmented arenavirus particles (see section 5.12(m)), after persistent infection for at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, or at least 100 days, no replication-competent bi-segmented virus particles are produced. In other embodiments, propagation of three segmented arenavirus particles comprising one L segment and two S segments does not result in replication-competent bi-segmented virus particles after at least 10 passages, at least 20 passages, at least 30 passages, at least 40 passages, or at least 50 passages.

[0169] In particular, the genome of the three-segmented arenavirus particle comprises one L segment and two S segments, in which the nucleotide sequence encoding the mutant KRAS (see Section 5.7), mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or tumor-associated antigen (see Section 5.9) provided herein is inserted at one position on each S segment. More specifically, one S segment encodes the arenavirus GP and the mutant KRAS, the mutated cancer driver gene (e.g., mutant TP53), or their tumor-associated antigens, respectively. The other S segment encodes the arenavirus NP and the mutant KRAS, the mutated cancer driver gene (e.g., mutant TP53), or their tumor-associated antigens. The L segment encodes the arenavirus L protein and Z protein. All segments are flanked by their respective 5' and 3' UTRs.

[0170] More specifically, (i) a first S segment comprising a nucleotide sequence encoding an antigenic fragment(s) (e.g., a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor associated antigen (see Section 5.9)) under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus nucleoprotein ("NP") under the control of the arenavirus genome 3' UTR; (ii) a second S segment comprising a nucleotide sequence encoding an antigenic fragment(s) (e.g., a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9)) under the control of the arenavirus genome 5' UTR and an ORF encoding an arenavirus glycoprotein ("GP") under the control of the arenavirus genome 3' UTR; (iii) L segment and Provided herein arenaviruses comprising:

[0171] In certain embodiments, intersegmental recombination of the two S segments of the three-segmented arenavirus particles provided herein, which integrates two arenavirus ORFs onto one segment rather than two separate segments, results in a non-functional promoter (i.e., a genome segment of the following structure: 5'UTR--------5'UTR or 3'UTR--------3'UTR) in which each UTR forming one end of the genome becomes an inverted repeat of the other end of the same genome.

[0172] In certain embodiments, a three-segmented arenavirus particle comprising one L segment and two S segments is engineered to carry an arenavirus ORF at a position other than its wild-type position and a nucleotide sequence encoding a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein. In other embodiments, a three-segmented arenavirus particle comprising one L segment and two S segments is engineered to carry two arenavirus ORFs, or three arenavirus ORFs, or four arenavirus ORFs, or five arenavirus ORFs, or six arenavirus ORFs at a position other than their respective wild-type positions. In specific embodiments, a three-segmented arenavirus particle comprising one L segment and two S segments contains a full complement of all four arenavirus ORFs. Thus, in some embodiments, a three-segmented arenavirus particle is an infectious and replicable three-segmented arenavirus particle. In a specific embodiment, the two S segments of the three-segmented arenavirus particle are engineered to carry one of their ORFs at a position other than the wild-type position. In a more specific embodiment, the two S segments contain the complete complement of the S segment ORF. In certain specific embodiments, the L segment is engineered to carry an ORF at a position other than the wild-type position, or the L segment can be a wild-type genome segment.

[0173] In certain embodiments, one of the two S segments is (i) an arenavirus S segment in which the ORF encoding the Z protein is under the control of the arenavirus 5'UTR; (ii) an arenavirus S segment in which the ORF encoding the L protein is under the control of the arenavirus 5'UTR; (iii) an arenavirus S segment in which the ORF encoding NP is under the control of the arenavirus 5′UTR; (iv) an arenavirus S segment in which the ORF encoding GP is under the control of the arenavirus 3'UTR; (v) an arenavirus S segment in which the ORF encoding the L protein is under the control of the arenavirus 3'UTR; and (vi) an arenavirus S segment in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR It could be.

[0174] In certain embodiments, a three segmented arenavirus particle comprising an L segment and two S segments may comprise overlapping arenavirus ORFs (i.e., two wild-type ORFs encoding, for example, GP or NP). In specific embodiments, a three segmented arenavirus particle comprising an L segment and two S segments may comprise one overlapping ORF (e.g., (GP,GP)) or two overlapping ORFs (e.g., (GP,GP) and (NP,NP)).

[0175] Table 1 below illustrates an exemplary genomic organization of a three-segmented arenavirus particle comprising one L segment and two S segments, where intersegmental recombination of the two S segments in the three-segmented arenavirus genome does not result in a replicative two-segmented virus particle and abolishes arenavirus promoter activity (i.e., the resulting recombined S segment is composed of two 3'UTRs rather than a 3'UTR and a 5'UTR). [Table 2] TIFF2024540385000003.tif92165

[0176] In certain embodiments, the IGR between positions 1 and 2 can be an arenavirus S segment or L segment IGR, the IGR between positions 3 and 4 can be an arenavirus S segment or L segment IGR, and the IGR between positions 5 and 6 can be an arenavirus L segment IGR. In specific embodiments, the IGR between positions 1 and 2 can be an arenavirus S segment IGR, the IGR between positions 3 and 4 can be an arenavirus S segment IGR, and the IGR between positions 5 and 6 can be an arenavirus L segment IGR. In certain embodiments, other combinations are also possible. For example, a three-segmented arenavirus particle comprising one L segment and two S segments, where intersegmental recombination of the two S segments in the three-segmented arenavirus genome does not result in a replicative two-segmented virus particle and abolishes arenavirus promoter activity (i.e., the resulting recombined S segment is composed of two 5'UTRs rather than a 3'UTR and a 5'UTR).

[0177] In certain embodiments, intersegmental recombination between the S and L segments in a three-segmented arenavirus particle comprising one L and two S segments reconstitutes a functional segment with two viral genes on only one segment instead of two separate segments. In other embodiments, intersegmental recombination between the S and L segments in a three-segmented arenavirus particle comprising one L and two S segments does not result in a replicable two-segmented virus particle.

[0178] In certain embodiments, one of skill in the art could construct an arenavirus genome having the configuration illustrated in Table 1 and described herein, and then use the assays described in Section 5.12 to determine whether the three-segmented arenavirus particles are genetically stable, i.e., whether they do not give rise to the replication-competent two-segmented virus particles discussed herein.

[0179] 5.5 Division Arenavirus Vector Particles Arenaviruses can also be engineered by the means described in International Patent Application Publication No. WO2021 / 089853 and U.S. Provisional Application No. 63 / 188,317, filed May 13, 2021, which are incorporated herein in their entirety. This technology is also referred to as "split" vector technology. Similar to the three-segmented viruses described above, the technology described in WO2021 / 089853 can be used to generate three-segmented viruses with two open positions for heterologous nucleotide sequences. Such heterologous nucleotide sequences can encode polypeptides. In certain embodiments, one such polypeptide can be a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9).

[0180] Briefly, such "split" arenavirus particles are engineered such that the arenavirus ORFs are separated onto two or more mRNA transcripts. In certain embodiments, provided herein arenavirus genome or antigenome segments that have been engineered such that transcription results in one or more mRNA transcripts that contain nucleotide sequences encoding functional fragments of arenavirus GP, NP, L or Z proteins.

[0181] In certain embodiments, the ORF encoding the arenavirus GP is separated (or split) onto two mRNA transcripts, respectively at two locations in the arenavirus genome. For example, an arenavirus GP signal peptide or a functional fragment thereof can be expressed from a first mRNA transcript (e.g., a viral mRNA transcript), and arenavirus GP1 and GP2 subunits are expressed from a second mRNA transcript (e.g., a viral mRNA transcript). In certain embodiments, the first mRNA transcript is under the control of an arenavirus 3' genomic UTR. In certain embodiments, the second mRNA transcript further encodes a heterologous non-arenavirus signal peptide (e.g., a signal peptide of the vesicular stomatitis virus Indiana serotype glycoprotein). In certain embodiments, the first mRNA transcript further comprises a nucleotide sequence encoding a heterologous non-arenavirus polypeptide, i.e., a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., a mutant TP53), or a tumor-associated antigen (see Section 5.9).

[0182] In certain embodiments, the genome organization of such a "split" arenavirus vector is as follows: First S segment: Arenavirus GP1 and GP2 subunits fused to a heterologous signal peptide under the control of the Arenavirus genome 5'UTR; a fusion of the Arenavirus GP signal peptide and a nucleotide sequence encoding an antigenic fragment(s) under the control of the Arenavirus genome 3'UTR. Second S segment: A nucleotide sequence encoding the antigenic fragment(s) under the control of the arenavirus genome 5'UTR and an ORF encoding the nucleoprotein ("NP") under the control of the arenavirus genome 3'UTR. L segment: Arenavirus Z protein under the control of the Arenavirus genome 5'UTR; Arenavirus L protein under the control of the Arenavirus genome 3'UTR.

[0183] In certain embodiments, the nucleotide sequence encoding the antigenic fragment(s) on the first S segment is different from the nucleotide sequence encoding the antigenic fragment(s) on the second S segment, In certain embodiments, the nucleotide sequence encoding the antigenic fragment(s) on the first S segment is the same as the nucleotide sequence encoding the antigenic fragment(s) on the second S segment.

[0184] In certain embodiments, the antigenic fragment(s) encoded on the first S segment are different from the antigenic fragment(s) encoded on the second S segment. In certain embodiments, the antigenic fragment(s) encoded on the first S segment are the same as the antigenic fragment(s) encoded on the second S segment. In certain embodiments, the antigenic fragment(s) encoded on the first S segment are the same as the antigenic fragment(s) encoded on the second S segment, but are fused to each other in a different order than the order in which the antigenic fragments encoded on the second S segment are fused to each other.

[0185] 5.6 Generation of arenavirus particles In general, arenavirus particles for use in the methods and compositions provided herein can be recombinantly produced by standard reverse genetic techniques described for LCMV (see Flatz et al., 2006, Proc Natl Acad Sci USA 103:4663-4668; Sanchez et al., 2006, Virology 350:370; Ortiz-Riano et al., 2013, J Gen Virol. 94:1175-88, which are incorporated herein by reference). To generate arenavirus particles provided herein, these techniques can be applied as described below. The genome of the virus can be modified as described herein.

[0186] (a) Generation of replication-deficient arenavirus particles. Arenavirus particles engineered to contain a genome capable of amplifying and expressing genetic information in infected cells but unable to produce further infectious progeny particles in normal non-complementing cells, in which one arenavirus open reading frame has been removed and replaced with a nucleotide sequence encoding a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9), can be recombinantly produced by any reverse genetic technique known to those of skill in the art.

[0187] In certain embodiments, a method for generating infectious replication-defective arenavirus particles includes (i) transfecting a complementing host cell (expressing a deleted or functionally inactivated open reading frame of a genome segment) with a cDNA of a first arenavirus genome segment, (ii) transfecting the host cell with a cDNA of a second arenavirus genome segment, (iii) transfecting the host cell with a plasmid expressing arenavirus minimal trans-acting factors NP and L, (iv) maintaining the host cell under conditions suitable for virus formation, and (v) harvesting the arenavirus particles. In certain more specific embodiments, the cDNA is contained in a plasmid.

[0188] Once generated from cDNA, infectious replication-defective arenaviruses can be propagated in complementing cells, which are cells that provide the functionality removed from the replication-defective arenavirus by modification of the genome (e.g., if the ORF encoding a GP protein is deleted or functionally inactivated, the complementing cell provides the GP protein).

[0189] Due to the removal or functional inactivation of one or more of the ORFs in the arenavirus vector (here, the deletion of the glycoprotein, i.e., GP, is taken as an example), the arenavirus vector can be generated and propagated in cells that provide the deleted viral gene(s) in trans, e.g., GP in this example. Such complementing cell lines, hereafter referred to as C cells, are generated by transfecting cell lines, e.g., BHK-21, HEK 293, VERO, etc., with one or more plasmids (complementing plasmids, referred to as C plasmids) for the expression of the viral gene(s) of interest. The C plasmid(s) express the viral gene(s) deleted in the arenavirus vector, generated under the control of one or more expression cassettes suitable for expression in mammalian cells, e.g., a mammalian polymerase II promoter, such as the EF1 alpha promoter with a polyadenylation signal. In addition, the complementing plasmid comprises an expression cassette suitable for gene expression in mammalian cells, e.g., a mammalian selection marker under the control of the polymerase II expression cassette described above, e.g., puromycin resistance, or a viral gene transcript(s) followed by an internal ribosome entry site, e.g., an encephalomyocarditis virus internal ribosome entry site, followed by a mammalian resistance marker. For production in E. coli, the plasmid further comprises a bacterial selection marker, e.g., an ampicillin resistance cassette.

[0190] The cells that may be used, e.g., BHK-21, HEK 293, MC57G, etc., are kept in culture and transfected with the complementing plasmid(s) using any of the commonly used strategies, such as calcium phosphate, liposome-based protocols or electroporation. After a few days, a suitable selection agent, e.g., puromycin, is added in titrated concentrations. Surviving clones are isolated and subcloned according to standard procedures, and high-expressing C-cell clones are identified using Western blot or flow cytometry procedures with antibodies against the viral protein(s) of interest. Instead of using stably transfected C-cells, the missing viral gene(s) may be complemented in each of the following steps using C-cells by transient transfection of normal cells. In addition, helper viruses may be used to provide the missing functionality in trans.

[0191] In certain embodiments, the complementing host cells are continued in culture and transfected with one or more plasmids that encode arenavirus genome segment(s) for arenavirus particles to be generated under the control of a polymerase I promoter and terminator.

[0192] Plasmids that can be used for the production of arenavirus particles can include: i) a plasmid encoding an S genome segment, e.g., pol-I S; ii) a plasmid encoding an L genome segment, e.g., pol-I L. In certain embodiments, a plasmid encoding an arenavirus polymerase directing the intracellular synthesis of the viral L and S segments can be incorporated into the transfection mixture. For example, a plasmid encoding the L protein and / or a plasmid encoding NP (pC-L and pC-NP, respectively) can be present. The L protein and NP are the minimal trans-acting factors required for transcription and replication of viral RNA. Alternatively, the intracellular synthesis of the viral L and S segments together with the NP and L proteins can be performed using an expression cassette with pol-I and pol-II promoters that read from opposite sides of the L and S segment cDNAs, respectively, of two separate plasmids.

[0193] Typically, an RNA polymerase I-driven expression cassette, an RNA polymerase II-driven cassette or a T7 bacteriophage RNA polymerase-driven cassette can be used, the latter preferentially with a 3'-terminal ribozyme for processing of the primary transcript to obtain precise ends. In certain embodiments, the plasmids encoding the arenavirus genome segments can be the same, i.e., the genome sequence and trans-acting factors can be transcribed from one plasmid by the T7, polI, and polII promoters.

[0194] In other embodiments, transcription of the arenavirus genome segment is carried out using a bidirectional expression cassette (see, e.g., Ortiz-Riano et al., 2013, J Gen Virol., 94(Pt 6):1175-1188). In more specific embodiments, the bidirectional expression cassette contains both a polymerase I promoter and a polymerase II promoter that respectively read from opposite ends of the inserted arenavirus genome segment.

[0195] In other embodiments, transcription of the cDNA of the arenavirus genome segment described herein comprises a promoter, illustrative examples of which include an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, an SP6 promoter, or a T3 promoter.

[0196] For the recovery of arenavirus particles as described herein, the following procedure is envisaged: Day 1: Complementing cells are transfected with a mixture of plasmids as described above. For this purpose, any commonly used strategy can be utilized, such as calcium phosphate, liposome-based protocols or electroporation.

[0197] After 3-5 days: The cell suspension (i.e., cells and medium) is harvested. Arenavirus particles present in the medium are removed from the cells and debris by centrifugation, and the supernatant (i.e., arenavirus vector preparation) is aliquoted and stored at 4°C, -20°C, or -80°C. The infectious titer of the arenavirus vector preparation is assessed by immune focus assay. Alternatively, the transfected cells and supernatant may be passaged into larger vessels 3-5 days after transfection, and the vectors are harvested up to 5 days after passaging, as described above.

[0198] (b) Generation of three-segment, replication-competent arenavirus particles. Trisegmented arenavirus particles comprising a genomic segment engineered to carry a viral ORF in a position other than the wild-type position of the ORF and further comprising a nucleotide sequence encoding a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) can be recombinantly produced by reverse genetic techniques known in the art, for example as described by Emonet et al., 2008, PNAS, 106(9):3473-3478; Popkin et al., 2011, J. Virol., 85(15):7928-7932, which are incorporated herein by reference. Both vector systems described in Sections 5.4 and 5.5 can be generated using the methods described in this section.

[0199] In certain embodiments, a method for producing three-segmented arenavirus particles includes (i) transfecting a host cell with cDNAs of one arenavirus L segment and two arenavirus S segments, or two arenavirus L segments and one arenavirus S segment, (ii) transfecting the host cell with a plasmid expressing the arenavirus minimal trans-acting factor NP and L proteins, (iii) maintaining the host cell under conditions suitable for virus formation, and (iv) harvesting the arenavirus particles. In certain more specific embodiments, the cDNAs of the arenavirus S and L segments are contained in a plasmid.

[0200] Once generated from cDNA, the three-segmented arenavirus particles (i.e., infectious and replicable) can be propagated. In certain embodiments, the three-segmented arenavirus particles can be propagated in any host cell that allows the virus to grow to a titer that allows the use of the virus described herein. In one embodiment, the host cell allows the three-segmented arenavirus particles to grow to a titer comparable to the titer determined for the corresponding wild type.

[0201] In certain embodiments, the three-segmented arenavirus particles can grow in host cells.Specific examples of host cells that can be used include BHK-21, HEK 293, VERO, etc.In specific embodiments, the three-segmented arenavirus particles can grow in cell lines.

[0202] In certain embodiments, the host cells are continued to be cultured and transfected with one or more plasmids that encode arenavirus genome segment(s) for arenavirus particles to be generated under the control of a polymerase I promoter and terminator.

[0203] In a specific embodiment, the host cells are kept in culture and are transfected with one or more plasmids encoding the viral protein(s) to be produced under the control of one or more expression cassettes suitable for expression in mammalian cells, e.g., consisting of a polymerase II promoter and terminator.

[0204] Plasmids that can be used to generate a three segmented arenavirus containing one L segment and two S segments can include i) two plasmids each encoding a genomic S segment, e.g., pol-I S, ii) a plasmid encoding a genomic L segment, e.g., pol-I L. Plasmids required for a three segmented arenavirus containing two L segments and one S segment are i) two plasmids each encoding a genomic L segment, e.g., pol-L, ii) a plasmid encoding a genomic S segment, e.g., pol-I S.

[0205] In certain embodiments, a plasmid encoding an arenavirus polymerase directing intracellular synthesis of the viral L and S segments can be incorporated into the transfection mixture. For example, a plasmid encoding the L protein and a plasmid encoding NP (pC-L and pC-NP, respectively) can be used. The L protein and NP are the minimal trans-acting factors required for transcription and replication of viral RNA. Alternatively, intracellular synthesis of the viral L and S segments together with the NP and L proteins can be performed using an expression cassette with pol-I and pol-II promoters that read from opposite sides of the L and S segment cDNAs, respectively, of two separate plasmids.

[0206] In addition, the plasmid(s) comprise an expression cassette suitable for gene expression in mammalian cells, e.g., a mammalian selection marker under the control of the polymerase II expression cassette described above, e.g., puromycin resistance, or a viral gene transcript(s) followed by an internal ribosome entry site, e.g., an encephalomyocarditis virus internal ribosome entry site, followed by a mammalian resistance marker. For production in E. coli, the plasmid further comprises a bacterial selection marker, e.g., an ampicillin resistance cassette.

[0207] Transfection of the plasmid(s) into the host cells can be performed using any of the commonly used strategies such as calcium phosphate, liposome-based protocols or electroporation.

[0208] Typically, an RNA polymerase I-driven expression cassette, an RNA polymerase II-driven cassette or a T7 bacteriophage RNA polymerase-driven cassette can be used, the latter preferentially with a 3'-terminal ribozyme for processing of the primary transcript to obtain precise ends. In certain embodiments, the plasmids encoding the arenavirus genome segments can be the same, i.e., the genome sequence and trans-acting factors can be transcribed from one plasmid by the T7, polI, and polII promoters.

[0209] In other embodiments, transcription of the arenavirus genome segment is carried out using a bidirectional expression cassette (see, e.g., Ortiz-Riano et al., 2013, J Gen Virol., 94(Pt 6):1175-1188). In more specific embodiments, the bidirectional expression cassette contains both a polymerase I promoter and a polymerase II promoter that respectively read from opposite ends of the inserted arenavirus genome segment.

[0210] In other embodiments, transcription of the cDNA of the arenavirus genome segment described herein comprises a promoter, illustrative examples of which include an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, an SP6 promoter, or a T3 promoter.

[0211] For the recovery of 3-segmented arenavirus vectors, the following procedure is envisaged: Day 1: Cells are transfected with the mixture of plasmids as described above. For this, any commonly used strategy can be utilized, such as calcium phosphate, liposome-based protocols or electroporation.

[0212] After 3-5 days: The cell suspension (i.e., cells and medium) is harvested. Arenavirus particles present in the medium are removed from the cells and debris by centrifugation, and the supernatant (i.e., arenavirus vector preparation) is aliquoted and stored at 4°C, -20°C, or -80°C. The infectious titer of the arenavirus vector preparation is assessed by immune focus assay. Alternatively, the transfected cells and supernatant may be passaged into larger vessels 3-5 days after transfection, and the vectors are harvested up to 5 days after passaging, as described above.

[0213] In certain embodiments, expression of a nucleotide sequence encoding a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) is provided, where the plasmid encoding the genome segment is modified to incorporate the nucleotide sequence encoding the mutant KRAS, the mutated cancer driver gene (e.g., mutant TP53), or the tumor-associated antigen. The nucleotide sequence encoding the mutant KRAS, the mutated cancer driver gene (e.g., mutant TP53), or the tumor-associated antigen can be incorporated into the plasmid using a restriction enzyme.

[0214] 5.7 Mutant KRAS In the context of the present application, "mutated KRAS" means a polypeptide encoded by a mutated KRAS gene.

[0215] In certain embodiments, arenavirus particles having a nucleotide sequence encoding an antigenic fragment of mutant KRAS provided herein can be used in the methods and compositions provided herein. In certain embodiments, arenavirus particles having a nucleotide sequence encoding an antigenic fragment of mutant KRAS (i.e., the same arenavirus particle having a different nucleotide sequence) further comprising a nucleotide sequence encoding one or more antigenic fragments of mutated cancer driver gene(s) (e.g., mutant TP53) or tumor associated antigen(s) are provided herein and can be used in the methods and compositions provided herein. In certain embodiments, arenavirus particles having a nucleotide sequence encoding an antigenic fragment of mutant KRAS and one or more antigenic fragments of mutated cancer driver gene(s) (e.g., mutant TP53) or tumor associated antigen(s) (i.e., the same nucleotide sequence encoding different antigenic fragments) are provided herein and can be used in the methods and compositions provided herein. In certain embodiments, the KRAS mutation is present at amino acid position G12, G13, A18, A59, Q61, K117, A146, or D119 of KRAS. In certain embodiments, the KRAS mutation is A18D, A59E, A59G, A59P, A59T, A59S, A59V, A146P, A146S, A146T, A146V, D119N, G12A, G12C, G12D, G12F, G12L, G12R, G12S, G12V, G13A, G13C, G13D, G13E, G13R, G13S, G13V, K117N, Q61E, Q61H, Q61K, Q61L, Q61P, Q61R, or a combination thereof. In certain embodiments, the KRAS mutation is G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61R, A146T, or a combination thereof.In certain embodiments, the KRAS mutation is G13D, G12V, G12C, G12D, G12R, or a combination thereof.In more specific embodiments, the arenavirus genome comprises a nucleotide sequence encoding multiple fragments of mutant KRAS, each fragment comprising, from N-terminus to C-terminus, the mutations G13D, G12V, G12C, G12D, and G12R, In other more specific embodiments, the arenavirus genome comprises a nucleotide sequence encoding fragments of mutant KRAS comprising, in any possible order, the mutations G13D, G12V, G12C, G12D, and G12R.

[0216] In certain embodiments, the nucleotide sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:20.

[0217] In certain embodiments, the nucleotide sequence encodes an expression product whose amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:19.

[0218] In certain embodiments, the nucleotide sequence encodes a fragment of mutant KRAS, wherein the fragment is 8 amino acids long, 9 amino acids long, 10 amino acids long, 11 amino acids long, 12 amino acids long, 13 amino acids long, 14 amino acids long, 15 amino acids long, 16 amino acids long, 17 amino acids long, 18 amino acids long, 19 amino acids long, 20 amino acids long, 21 amino acids long, 22 amino acids long, 23 amino acids long, 24 amino acids long, 25 amino acids long, 26 amino acids long, 27 amino acids long, 28 amino acids long, 29 amino acids long, or 30 amino acids long, and the fragment comprises the mutation of mutant KRAS.In more specific embodiments, the nucleotide sequence encodes a fragment of mutant KRAS, wherein the fragment is 18 amino acids long.

[0219] In certain embodiments, the region adjacent to the N-terminal mutation of the antigenic fragment is 0 amino acids long, 1 amino acids long, 2 amino acids long, 3 amino acids long, 4 amino acids long, 5 amino acids long, 6 amino acids long, 7 amino acids long, 8 amino acids long, 9 amino acids long, 10 amino acids long, 11 amino acids long, 12 amino acids long, 13 amino acids long, 14 amino acids long, 15 amino acids long, 16 amino acids long, 17 amino acids long, 18 amino acids long, 19 amino acids long, or 20 amino acids long. In a more specific embodiment, the region adjacent to the N-terminal mutation of the antigenic fragment is 8 amino acids long. In another more specific embodiment, the region adjacent to the N-terminal mutation of the antigenic fragment is 9 amino acids long. In certain embodiments, the region adjacent to the mutation at the C-terminus of the antigenic fragment is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In a more specific embodiment, the region adjacent to the mutation at the C-terminus of the antigenic fragment is 8 amino acids long. In another more specific embodiment, the region adjacent to the mutation at the C-terminus of the antigenic fragment is 9 amino acids long.

[0220] In certain embodiments, the arenavirus particle comprises 2 antigenic fragments of mutant KRAS, 3 antigenic fragments of mutant KRAS, 4 antigenic fragments of mutant KRAS, 5 antigenic fragments of mutant KRAS, 6 antigenic fragments of mutant KRAS, 7 antigenic fragments of mutant KRAS, 8 antigenic fragments of mutant KRAS, 9 antigenic fragments of mutant KRAS, 10 antigenic fragments of mutant KRAS, 11 antigenic fragments of mutant KRAS, 12 antigenic fragments of mutant KRAS, The arenavirus genome comprises a nucleotide sequence encoding 13 antigenic fragments of mutant KRAS, 14 antigenic fragments of mutant KRAS, 15 antigenic fragments of mutant KRAS, 16 antigenic fragments of mutant KRAS, 17 antigenic fragments of mutant KRAS, 18 antigenic fragments of mutant KRAS, 19 antigenic fragments of mutant KRAS, or 20 antigenic fragments of mutant KRAS, wherein each of the antigenic fragments comprises one mutation of mutant KRAS protein. In certain embodiments, each of the antigenic fragments comprises the same mutation or different mutations of mutant KRAS protein. In a specific embodiment, each fragment comprises different mutations of KRAS. In a more specific embodiment, the arenavirus particle comprises an arenavirus genome comprising a nucleotide sequence encoding five antigenic fragments of mutant KRAS, where each of the antigenic fragments comprises a different mutation of mutant KRAS, the different mutations being G13D, G12V, G12C, G12D, and G12R, and each of the antigenic fragments is 18 amino acids in length.

[0221] In certain embodiments, the antigenic fragments of mutant KRAS are directly fused together. In certain embodiments, the antigenic fragments of mutant KRAS are fused together via the same or different peptide linkers. In a specific embodiment, the antigenic fragments of mutant KRAS are fused together via AAY linker (AAY), AAA linker (AAA), GS linker (GGSGGGGSGG) (SEQ ID NO: 42), or variants of AAY, AAA, and GS linker sequences optimized through in silico prediction.

[0222] In certain embodiments, the nucleotide sequence of the arenavirus particle is engineered to reduce or eliminate any CpG and TpA islands. In a specific embodiment, the elimination of CpG and TpA islands comprises three cycles: (i) removing CpG in the first cycle, (ii) removing TpA in the second cycle, and (iii) removing CpG in the third cycle to remove the newly introduced CpG in the second cycle.

[0223] 5.8 Mutated cancer driver genes In the context of this application, "mutated cancer driver gene" means a polypeptide encoded by a mutated cancer driver gene.

[0224] In certain embodiments, the mutated cancer driver genes used in the methods and compositions disclosed herein include mutated AKT1, mutated BRAF, mutated HER2, mutated MEK1, mutated MET, mutated NRAS, mutated PIK3CA, mutated RET, mutated APC, mutated U2AF1, mutated EGFR, mutated FBXW7, mutated SMAD4, mutated GNAS, mutated ERBB2, mutated ERBB3, mutated CDKN2A, mutated TP53, and mutated CTNNB1.

[0225] In certain embodiments, arenavirus particles having nucleotide sequences encoding an antigenic fragment of mutant KRAS (i.e., the same arenavirus particle having a different nucleotide sequence) that further comprises a nucleotide sequence encoding one or more antigenic fragments of a mutated cancer driver gene(s) (e.g., mutant TP53(s)) are provided herein and can be used in the methods and compositions provided herein. In certain embodiments, arenavirus particles having nucleotide sequences encoding an antigenic fragment of mutant KRAS and one or more antigenic fragments of a mutated cancer driver gene(s) (e.g., mutant TP53(s)) (i.e., the same nucleotide sequence encoding different antigenic fragments) are provided herein and can be used in the methods and compositions provided herein. In certain embodiments, the arenavirus particle having a nucleotide sequence encoding an antigenic fragment of mutant KRAS may further encode an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, mutant RET, mutant APC, mutant U2AF1, mutant EGFR, mutant FBXW7, mutant SMAD4, mutant GNAS, mutant ERBB2, mutant ERBB3, mutant CDKN2A, mutant TP53, and / or mutant CTNNB1, where the antigenic fragment comprises the respective mutation. In certain embodiments, the arenavirus particle having a nucleotide sequence encoding an antigenic fragment of mutant KRAS may further encode an antigenic fragment of mutant PI3KCA. In particular, the mutation of PI3KCA is E545K, H1047R, and / or E542K. In certain embodiments, the arenavirus particle having the nucleotide sequence encoding the antigenic fragment of mutant KRAS can further encode the antigenic fragment of mutant BRAF.In particular, the mutation of BRAF is V600E.In certain embodiments, the arenavirus particle having the nucleotide sequence encoding the antigenic fragment of mutant KRAS can further encode the antigenic fragment of mutant U2AF1.In particular, the mutation of U2AF1 is S34F.In certain embodiments, the arenavirus particle having a nucleotide sequence encoding an antigenic fragment of mutant KRAS may further encode an antigenic fragment of mutant TP53. In particular, the mutation of TP53 is G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W, and / or C277F mutation. In certain embodiments, the arenavirus particle having a nucleotide sequence encoding an antigenic fragment of mutant KRAS may further encode an antigenic fragment of mutant PI3KCA having E545K mutation, H1047R mutation, or E542K mutation, mutant BRAF having V600E mutation, or mutant TP53 having R175H mutation.

[0226] In certain embodiments, the method for treating a neoplastic disease further comprises administering a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of a mutated cancer driver gene (e.g., mutant TP53). In certain embodiments, the antigenic fragment of mutant KRAS, mutant BRAF with a V600E mutation, or mutant PIK3CA encoded by the genome of the arenavirus particle may be associated with or specific to colorectal cancer. In certain embodiments, the antigenic fragment of mutant KRAS, mutant BRAF, or mutant PIK3CA encoded by the genome of the arenavirus particle may be associated with or specific to lung adenocarcinoma. In certain embodiments, the antigenic fragment of mutant KRAS and mutant PIK3CA encoded by the genome of the arenavirus particle may be associated with or specific to lung squamous cell carcinoma. In certain embodiments, the antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, or mutant RET encoded by the genome of the arenavirus particle may be associated with or specific to non-small cell lung cancer (NSCLC).

[0227] In certain embodiments, the nucleotide sequence encodes a fragment of a mutated cancer driver gene (e.g., mutant TP53), where the fragment is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length, and where the fragment comprises a mutation in the mutated cancer driver gene (e.g., mutant TP53).

[0228] In certain embodiments, the nucleotide sequence encodes a fragment of a mutated cancer driver gene (e.g., mutant TP53), where the fragment is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length, and where the fragment comprises a mutation in the mutated cancer driver gene (e.g., mutant TP53).

[0229] In certain embodiments, the region adjacent to the mutation at the N-terminus of the antigenic fragment is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In certain embodiments, the region adjacent to the mutation at the C-terminus of the antigenic fragment is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0230] In certain embodiments, two antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), three antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), four antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), five antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), six antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), seven antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), eight antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), nine antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), ten antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), eleven antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), arenavirus particles having a nucleotide sequence encoding 12 antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), 13 antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), 14 antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), 15 antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), 16 antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), 17 antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), 18 antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), 19 antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), or 20 antigenic fragments of a mutated cancer driver gene (e.g., mutant TP53), wherein each of the antigenic fragments comprises one mutation of a mutated cancer driver gene (e.g., mutant TP53) protein. In certain embodiments, each of the antigenic fragments comprises the same or different mutations of a mutated cancer driver gene (e.g., mutant TP53) protein. In a specific embodiment, each fragment comprises a different mutation of a cancer driver gene.

[0231] In certain embodiments, antigenic fragments of mutated cancer driver genes (e.g., mutant TP53) are directly fused together. In certain embodiments, antigenic fragments of mutated cancer driver genes (e.g., mutant TP53) are fused together via the same or different peptide linkers. In specific embodiments, antigenic fragments of mutated cancer driver genes (e.g., mutant TP53) are fused together via AAY linker (AAY), AAA linker (AAA), GS linker (GGSGGGGSGG) (SEQ ID NO: 42), or variants of AAY, AAA, and GS linker sequences optimized via in silico prediction.

[0232] In the context of this application, "mutated TP53" refers to a polypeptide encoded by a mutated TP53 gene. In certain embodiments, arenavirus particles having a nucleotide sequence encoding an antigenic fragment of mutant KRAS (i.e., the same arenavirus particle having a different nucleotide sequence) further comprising a nucleotide sequence encoding one or more antigenic fragments of one or more mutant TP53 are provided herein and can be used in the methods and compositions provided herein. In certain embodiments, arenavirus particles having a nucleotide sequence encoding an antigenic fragment of mutant KRAS and one or more antigenic fragments of one or more mutant TP53 (i.e., the same nucleotide sequence encoding different antigenic fragments) are provided herein and can be used in the methods and compositions provided herein. In certain embodiments, arenavirus particles having a nucleotide sequence encoding an antigenic fragment of mutant KRAS may further encode an antigenic fragment of mutant TP53, where the antigenic fragment comprises the respective mutation. In certain embodiments, arenavirus particles having a nucleotide sequence encoding an antigenic fragment of mutant KRAS may further encode an antigenic fragment of mutant TP53. In particular, the TP53 mutation is a G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W, or C277F mutation.

[0233] In certain embodiments, a method for treating a neoplastic disease comprises administering arenavirus particles, wherein the arenavirus particles encode an antigenic fragment of mutant KRAS and mutant TP53, wherein the antigenic fragment comprises mutations, wherein the KRAS mutation is G12D, G12R, G12V, Q61H, and / or Q61R, and the TP53 mutation is R175H, R248W, G245S, R282W, R248Q, and / or R273C, and the neoplastic disease is pancreatic cancer. In certain embodiments, a method for treating a neoplastic disease comprises administering arenavirus particles, wherein the arenavirus particles encode antigenic fragments of mutant KRAS and mutant TP53, wherein the antigenic fragments comprise mutations, wherein the KRAS mutations are G12D, G12V, G13D, A146T, and / or G12C, and the TP53 mutations are R175H, R273H, and / or R248W, and the neoplastic disease is colorectal cancer or lung adenocarcinoma. In certain embodiments, the method for treating a neoplastic disease comprises administering arenavirus particles, wherein the arenavirus particles encode an antigenic fragment of mutant KRAS and mutant TP53, the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12C, G12D, G12R, G13D, and / or G12V, and the TP53 mutation is R175H, R273H, and / or R248W, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. In certain embodiments, the method for treating a neoplastic disease comprises administering arenavirus particles, wherein the arenavirus particles encode an antigenic fragment of mutant KRAS and mutant TP53, the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12C, G12V, G12D, and / or G12S, and the TP53 mutation is R175H, and the neoplastic disease is lung adenocarcinoma.In certain embodiments, the method for treating a neoplastic disease further comprises administering a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS and mutant TP53, wherein the antigenic fragment comprises mutations, the KRAS mutations are G12D, G12R, G12V, Q61H and / or Q61R, the TP53 mutations are R175H, R248W, G245S, R282W, R248Q and / or R273C, and the neoplastic disease is pancreatic cancer. In certain embodiments, the method for treating a neoplastic disease further comprises administering a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS and mutant TP53, wherein the antigenic fragment comprises mutations, the KRAS mutations are G12D, G12R, G12V, Q61H and / or Q61R, the TP53 mutations are R175H, R248W, G245S, R282W, R248Q and / or R273C, and the neoplastic disease is pancreatic cancer. In certain embodiments, the method for treating a neoplastic disease further comprises administering a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS and mutant TP53, wherein the antigenic fragment comprises mutations, the KRAS mutation is G12D, G12V, G13D, A146T, and / or G12C, the TP53 mutation is R175H, R273H, and / or R248W, and the neoplastic disease is colorectal cancer or lung adenocarcinoma. In certain embodiments, the method for treating a neoplastic disease further comprises administering a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS and mutant TP53, wherein the antigenic fragment comprises mutations, the KRAS mutation is G12C, G12D, G12R, G13D and / or G12V, the TP53 mutation is R175H, R273H and / or R248W, and the neoplastic disease is pancreatic cancer, colorectal cancer or lung adenocarcinoma.In certain embodiments, the method for treating a neoplastic disease further comprises administering a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of mutant KRAS and mutant TP53, wherein the antigenic fragment comprises mutations, the KRAS mutation is G12C, G12V, G12D, and / or G12S, the TP53 mutation is R175H, and the neoplastic disease is lung adenocarcinoma.

[0234] 5.9 Tumor-associated antigens In certain embodiments, tumor-associated antigens used in the methods and compositions disclosed herein include antigens from the BIRC family, CEACAM family, CTA family, EPH family, ERBB family, FOLR family, GAST family, GUCY2 family, IDO family, IL13RA family, KDR family, KLK family, MAGE family, MUC family, PEMT family, SDC family, SLAMF family, TERT family, TLR family, TPTE family, TYR family, WT family, and XBP family.

[0235] In certain embodiments, arenavirus particles having nucleotide sequences encoding an antigenic fragment of mutant KRAS (i.e., the same arenavirus particles having different nucleotide sequences) that further comprise a nucleotide sequence encoding one or more antigenic fragments of the tumor-associated antigen(s) are provided herein and can be used in the methods and compositions provided herein. In certain embodiments, arenavirus particles having nucleotide sequences encoding an antigenic fragment of mutant KRAS and one or more antigenic fragments of the tumor-associated antigen(s) (i.e., the same nucleotide sequence encoding different antigenic fragments) are provided herein and can be used in the methods and compositions provided herein. In certain embodiments, the tumor-associated antigens used in the methods and compositions described herein are immunogenic proteins expressed in or on the surface of neoplastic cells or tumors, such as cancer cells or malignant tumors. In certain embodiments, the tumor-associated antigens used in the methods and compositions described herein are non-specific, mutated, overexpressed, or aberrantly expressed proteins that may be present on the surface of both neoplastic cells or tumors and normal cells or tissues. In certain embodiments, the tumor-associated antigen used in the methods and compositions described herein is a tumor-specific antigen that is restricted to tumor cells.In certain embodiments, the tumor-associated antigen used in the methods and compositions described herein is a cancer-specific antigen that is restricted to cancer cells.In certain embodiments, the method for treating neoplastic disease further comprises administering a second arenavirus particle, wherein the second arenavirus particle encodes an antigenic fragment of the tumor-associated antigen.

[0236] In certain embodiments, tumor-associated antigens may exhibit one, two, or more than three, e.g., all, of the following characteristics: overexpressed / accumulated (i.e., expressed by both normal and neoplastic tissues, but highly expressed in neoplasms), oncofetal (i.e., typically expressed only in fetal tissues and cancerous somatic cells), oncoviral or oncoviral (i.e., encoded by an oncogenic transforming virus), onco-testicular (i.e., expressed only by cancer cells and adult reproductive tissues, e.g., testes), lineage-restricted (i.e., expressed primarily by a single cancer tissue type), mutated (i.e., expressed only in neoplastic tissues as a result of genetic mutations or transcriptional changes), post-translationally altered (e.g., tumor-associated changes in glycosylation), or idiotypic (i.e., arising from malignant clonal expansion of B or T lymphocytes).

[0237] In certain embodiments, tumor associated antigens used in the methods and compositions described herein include acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphocytic leukemia; acute myeloid leukemia; acute myeloid leukemia (adult / childhood); adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratoid / rhabdoid tumor; basal cell carcinoma; cholangiocarcinoma, extrahepatic (cholangiocarcinoma); bladder cancer; osteosarcoma of bone / malignant fibrous histiocytoma; brain cancer (adult / childhood); brain tumor, cerebellar astrocytoma (adult / childhood); brain tumor, cerebral astrocytoma / malignant glioma encephaloma tumors;brain tumors, ependymoma;brain tumors, medulloblastoma;brain tumors, supratentorial primitive neuroectodermal tumor;brain tumors, optic and hypothalamic gliomas;brain stem gliomas;breast cancer;bronchial adenoma / carcinoid;bronchial tumors;Burkitt's lymphoma;cancer of childhood;carcinoid gastrointestinal tumors;carcinoid tumors;adult carcinoma, unknown primary site;carcinoma of unknown primary site;central nervous system embryonal tumors;central nervous system lymphoma, primary;cervical cancer;pediatric adrenocortical carcinoma;pediatric cancer;pediatric cerebral astrocytoma;chordoma, childhood;chronic lymphocytic leukemia;chronic myeloid leukemia;chronic myeloproliferative disorder;colon cancer;colorectal cancer;craniopharyngioma; Cutaneous T-cell lymphoma;Desmoplastic small round cell tumor;Emphysema;Endometrial carcinoma;Ependymoblastoma;Ependymoma;Esophageal carcinoma;Ewing sarcoma in Ewing family of tumors;Extracranial germ cell tumors;Extragonadal germ cell tumors;Extrahepatic bile duct carcinoma;Gallbladder carcinoma;Gastric (stomach) carcinoma;Gastric carcinoid;Gastrointestinal carcinoid tumors;Gastrointestinal stromal tumors;Germ cell tumors: extracranial, extragonadal, or ovarian gestational trophoblastic tumors;Genomic trophoblastic tumors, unknown primary site;Glioma;Glioma of the brain stem;Glioma, childhood visual pathway and hypothalamus;Hairy cell leukemia;Head and neck cancer;Carcinoma of the heart;Hepatocellular (liver) carcinoma;Hodgkin's lymphoma;Hypopharyngeal carcinoma;Hypothalamus and optic tract glioma;intraocular melanoma;islet cell carcinoma (pancreatic islet);Kaposi's sarcoma;renal carcinoma (renal cell carcinoma);Langerhans cell histiocytosis;laryngeal carcinoma;cancer of the lip and oral cavity;liposarcoma;hepatic carcinoma (primary);lung cancer, non-small cell;lung cancer, small cell;lymphoma, primary central nervous system;macroglobulinemia, Waldenstrom;male breast cancer;malignant fibrous histiocytoma / osteosarcoma of bone;medulloblastoma;medulloepithelioma;melanoma;melanoma, intraocular (eye);Merkel cell carcinoma;Merkel cell skin cancer;mesothelioma;mesothelioma, adult malignant;metastatic squamous neck cancer of unknown primary;mouth cancer;multiple endocrine neoplasia syndrome;multiple myeloma / plasma cell neoplasm;Mycosis fungoides, myelodysplastic syndromes;Myelodysplastic / myeloproliferative disorders;Myeloid leukemia, chronic;Myeloid leukemia, adult acute;Myeloid leukemia, childhood acute;Myeloma, multiple (cancer of the bone marrow);Myeloproliferative disorders, chronic;Nasal and paranasal sinus cancer;Nasopharyngeal carcinoma;Neuroblastoma, non-small cell lung cancer;Non-Hodgkin's lymphoma;Oligodendroglioma;Oral cavity cancer;Oral cancer;Oropharyngeal cancer;Osteosarcoma / malignant fibrous histiocytoma of bone;Ovarian cancer;Ovarian epithelial carcinoma (superficial epithelial and stromal tumor);Ovarian germ cell tumor;Ovarian low malignant potential tumor;Pancreatic cancer;Pancreatic cancer, islet cell;Papilloma;Sinonasal and nasal cancer;Parathyroid cancer;Penis Cancer;Pharyngeal carcinoma;Pheochromocytoma;Pineal astrocytoma;Pineal germinoma;Intermediately differentiated pineal parenchymal tumor;Pineoblastoma and supratentorial primitive neuroectodermal tumor;Pituitary tumor;Pituitary adenoma;Plasma cell neoplasm / multiple myeloma;Pleuropulmonary blastoma;Primary central nervous system lymphoma;Prostate cancer;Rectal cancer;Renal cell carcinoma (kidney cancer);Renal pelvis and ureter, transitional cell carcinoma;Airway cancer involving the NUT gene on chromosome 15;Retinoblastoma;Rhabdomyosarcoma, childhood;Salivary gland carcinoma;Sarcoma, Ewing family tumor;Sezary syndrome;Skin cancer (melanoma);Skin cancer (non-melanoma);Small cell lung cancer;Small intestine cancer These include antigens from neoplastic diseases including soft tissue sarcoma; soft tissue sarcoma; spinal tumor; squamous cell carcinoma; squamous cell cervical carcinoma of unknown primary, metastatic; gastric (stomach) carcinoma; supratentorial primitive neuroectodermal tumor; T-cell lymphoma, skin (mycosis fungoides and Sézary syndrome); testicular cancer; throat cancer; thymoma; thymoma and thymic carcinoma; thyroid cancer; thyroid cancer, childhood; transitional cell carcinoma of the renal pelvis and ureter; urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; vulvar cancer; and Wilms' tumor.

[0238] In certain embodiments, arenavirus particles having a nucleotide sequence encoding an antigenic fragment of mutant KRAS may further encode antigenic fragments of the BIRC family, CEACAM family, CTA family, EPH family, ERBB family, FOLR family, GAST family, GUCY2 family, IDO family, IL13RA family, KDR family, KLK family, MAGE family, MUC family, PEMT family, SDC family, SLAMF family, TERT family, TLR family, TPTE family, TYR family, WT family, and XBP family.

[0239] In certain embodiments, a fragment of a tumor-associated antigen is antigenic if it is capable of (i) eliciting an antibody immune response in a host (e.g., a mouse, rabbit, goat, donkey or human) in which the resulting antibodies specifically bind to an immunogenic protein expressed within or on the surface of a neoplastic cell (e.g., a cancer cell), and / or (ii) eliciting a specific T cell immune response.

[0240] In certain embodiments, the nucleotide sequence encoding an antigenic fragment of a tumor associated antigen provided herein is 8 to 100 nucleotides in length, 15 to 100 nucleotides in length, 25 to 100 nucleotides in length, 50 to 200 nucleotides in length, 50 to 400 nucleotides in length, 200 to 500 nucleotides in length, or 400 to 600 nucleotides in length, 500 to 800 nucleotides in length. In other embodiments, the nucleotide sequence encoding the antigenic fragment provided herein is 750-900 nucleotides in length, 800-100 nucleotides in length, 850-1000 nucleotides in length, 900-1200 nucleotides in length, 1000-1200 nucleotides in length, 1000-1500 nucleotides in length, or 10-1500 nucleotides in length, 1500-2000 nucleotides in length, 1700-2000 nucleotides in length, 2000-2300 nucleotides in length, 2200-2500 nucleotides in length, 2500-3000 nucleotides in length, 3000-3200 nucleotides in length, 3000-3500 nucleotides in length, or 4000-4200 nucleotides in length. The length of the nucleotide may be 3200-3600 nucleotides, 3300-3800 nucleotides, 4000-4400 nucleotides, 4200-4700 nucleotides, 4800-5000 nucleotides, 5000-5200 nucleotides, 5200-5500 nucleotides, 5500-5800 nucleotides, 5800-6000 nucleotides, 6000-6400 nucleotides, 6200-6800 nucleotides, 6600-7000 nucleotides, 7000-7200 nucleotides, 7200-7500 nucleotides, or 7500 nucleotides. In some embodiments, the nucleotide sequence encodes a peptide or polypeptide that is 5-10 amino acids long, 10-25 amino acids long, 25-50 amino acids long, 50-100 amino acids long, 100-150 amino acids long, 150-200 amino acids long, 200-250 amino acids long, 250-300 amino acids long, 300-400 amino acids long, 400-500 amino acids long, 500-750 amino acids long, 750-1000 amino acids long, 1000-1250 amino acids long, 1250-1500 amino acids long, 1500-1750 amino acids long, 1750-2000 amino acids long, 2000-2500 amino acids long, or greater than or equal to 2500 amino acids long.In some embodiments, the nucleotide sequence encodes a polypeptide that does not exceed 2500 amino acids in length. In specific embodiments, the nucleotide sequence does not contain a stop codon. In certain embodiments, the nucleotide sequence is codon-optimized. In certain embodiments, the nucleotide composition, the nucleotide pair composition, or both can be optimized. Such techniques for optimization are known in the art and can be applied to optimize the nucleotide sequence that codes for tumor-associated antigen or its antigenic fragment.

[0241] In certain embodiments, the arenavirus genome segment, arenavirus particle, or three-segmented arenavirus particle may comprise one or more nucleotide sequences encoding a tumor-associated antigen or an antigenic fragment thereof. In other embodiments, the arenavirus genome segment, arenavirus particle, or three-segmented arenavirus particle may comprise at least one nucleotide sequence encoding a tumor-associated antigen or an antigenic fragment thereof, at least two nucleotide sequences encoding a tumor-associated antigen or an antigenic fragment thereof, at least three nucleotide sequences encoding a tumor-associated antigen or an antigenic fragment thereof, or four or more nucleotide sequences encoding a tumor-associated antigen or an antigenic fragment thereof.

[0242] In certain embodiments, arenavirus particles provided herein a) have a removal or functional inactivation of an ORF present in the wild-type form of the genome segment, and b) (i) comprise a genome segment that encodes (in either sense or antisense) one or more mutant KRAS, mutated cancer driver genes, or tumor-associated antigens provided herein. In other embodiments, arenavirus particles provided herein a) comprise an arenavirus ORF at a position other than the wild-type position of that ORF, and b) (i) comprise a genome segment that encodes (in either sense or antisense) one or more mutant KRAS, mutated cancer driver genes, or tumor-associated antigens provided herein.

[0243] In certain embodiments, the arenavirus particles generated to encode one or more mutant KRAS, mutated cancer driver genes, or tumor-associated antigens include one or more nucleotide sequences encoding the mutant KRAS, mutated cancer driver genes, or tumor-associated antigens provided herein. In specific embodiments, the mutant KRAS, mutated cancer driver genes, or tumor-associated antigens provided herein are separated by one or more of the various linkers, spacers, or cleavage sites described herein.

[0244] 5.10 How to use Provided herein are methods for preventing and / or treating a neoplastic disease in a subject, comprising administering to the subject arenavirus particles, wherein the arenavirus particles encode a mutant KRAS provided herein (see Section 5.7). In certain embodiments, the arenavirus particles encode a mutant KRAS provided herein, a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9). In certain embodiments, these methods result in suppression of tumor growth. In certain embodiments, these methods result in a lower recurrence rate.

[0245] In certain embodiments, provided herein are methods for treating a neoplastic disease in a subject, the methods comprising: (a) administering to the subject a first arenavirus particle, the first arenavirus particle encoding a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), and / or a tumor-associated antigen (see Section 5.9); and (b) administering to the subject a second arenavirus particle expressing a mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), and / or a tumor-associated antigen. In certain embodiments, (a) and (b) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the first and second arenavirus particles are administered simultaneously. In other embodiments, the interval between (a) and (b) is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In other embodiments, the interval between (a) and (b) is 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 23 weeks, or 24 weeks. In other embodiments, the interval between (a) and (b) is 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, or 36 weeks. Furthermore, during the repetition of (a) and (b), the interval may be the same as the original cycle of (a) and (b) or may be different from the original cycle of (a) and (b). Thus, the interval between (a) and (b) when repeated may be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks. In certain embodiments, the first arenavirus particle and the second arenavirus particle encode the same antigen(s), e.g., the two arenavirus particles encode the same mutant KRAS(s), mutated cancer driver gene(s) (e.g., mutant TP53(s)), and / or tumor-associated antigen(s).In certain embodiments, the first arenavirus particle and the second arenavirus particle encode different antigens, e.g., the first arenavirus particle encodes only mutant KRAS, and the second arenavirus particle encodes a mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), and / or a tumor-associated antigen provided herein. In certain embodiments, the first arenavirus particle encodes only mutant KRAS, and the second arenavirus particle encodes a mutated cancer driver gene (e.g., mutant TP53), and / or a tumor-associated antigen. In certain embodiments, the first arenavirus particle encodes a mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), and / or a tumor-associated antigen, and the second arenavirus particle encodes only mutant KRAS. In certain embodiments, the first arenavirus particle encodes a mutated KRAS, a mutated cancer driver gene (e.g., mutated TP53) and / or a tumor-associated antigen, and the second arenavirus particle encodes a mutated cancer driver gene (e.g., mutated TP53) and / or a tumor-associated antigen.

[0246] In certain embodiments, the first arenavirus particle and the second arenavirus particle are the same. In certain embodiments, the first arenavirus particle and the second arenavirus particle are derived from the same (i.e., have the same backbone) arenavirus but express different mutant KRAS (see Section 5.7), mutated cancer driver genes (see Section 5.8, e.g., mutant TP53), or tumor-associated antigens (see Section 5.9). In certain embodiments, the first arenavirus particle and the second arenavirus particle are derived from different (i.e., have different backbone) arenaviruses but express the same mutant KRAS(es), mutant TP53(s), mutated cancer driver genes(s), or tumor-associated antigen(s). In certain embodiments, the first arenavirus particle and the second arenavirus particle are derived from different arenaviruses (i.e., have different backbones) and express different mutant KRAS, mutant TP53(s), mutated cancer driver genes, or tumor-associated antigens.

[0247] In certain embodiments, provided herein is a method for treating a neoplastic disease in a subject, the method comprising: (a) administering to the subject a first arenavirus particle, the first arenavirus particle being replication-competent and expressing a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein; and (b) administering to the subject a second arenavirus particle, the second arenavirus particle expressing a mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), or a tumor-associated antigen. In certain embodiments, the first arenavirus particle is three-segmented (see Section 5.6(b)). In specific embodiments, the first arenavirus particle is replication-competent. In specific embodiments, the second arenavirus particle is replication-defective. In specific embodiments, the second arenavirus particle is replication-competent. In certain embodiments, the second arenavirus particle is three-segmented. In a specific embodiment, the second arenavirus particle is three-segmented and replication-competent.

[0248] In certain embodiments, provided herein is a method of treating a neoplastic disease in a subject, comprising administering an arenavirus particle comprising a nucleotide sequence encoding an antigenic fragment of mutant KRAS (see Section 5.7), wherein the neoplastic disease is pancreatic cancer, colorectal cancer, lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer (NSCLC). In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS (see Section 5.8), wherein the antigenic fragment comprises a mutation, wherein the mutation in KRAS is G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A, and the neoplastic disease is pancreatic cancer. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding antigenic fragments of mutant KRAS, mutant TP53 and / or mutant U2AF1 (see Section 5.8), where the KRAS mutation is G12D, G12V, G12R, Q61H and / or Q61R, the TP53 mutation is R175H, R248W, G245S, R282W, R248Q and / or R273C, the U2AF1 mutation is S34F, and the neoplastic disease is pancreatic cancer. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, where the KRAS mutations are G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, where the KRAS mutations are G13D, G12V, G12C, G12D, and G12R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma.In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, mutant BRAF, mutant TP53, mutant FBXW7, and / or mutant PIK3CA, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12D, G12V, G13D, A146T, and / or G12C, the BRAF mutation is V600E, the TP53 mutation is R175H, R273H, and / or R248W, the FBXW7 mutation is R465H, the PIK3CA mutation is E545K and / or H1047R, and the neoplastic disease is colorectal cancer or lung adenocarcinoma. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, mutant BRAF, mutant PIK3CA, mutant EGFR, mutant TP53, and / or mutant U2AF1, wherein the antigenic fragment comprises mutations, the KRAS mutation is G12C, G12D, G12R, G13D, and / or G12V, the BRAF mutation is V600E, the PIK3CA mutation is E545K, the EGFR mutation is L858R, the TP53 mutation is R175H, R273H, and / or R248W, the U2AF1 mutation is S34F, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, mutant TP53, mutant U2AF1, mutant PIK3CA, mutant EGFR, and / or mutant BRAF, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12C, G12V, G12D, and / or G12S, the TP53 mutation is R175H, the U2AF1 mutation is S34F, the EGFR mutation is L858R, L861Q, and / or E746_A750del, the PIK3CA mutation is E545K and / or E542K, the BRAF mutation is V600E, and the neoplastic disease is lung adenocarcinoma.In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, and / or mutant RET, where the antigenic fragment comprises a mutation, and the neoplastic disease is non-small cell lung cancer (NSCLC). In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, mutant RET, mutant APC, mutant U2AF1, mutant EGFR, mutant FBXW7, mutant SMAD4, mutant GNAS, mutant ERBB2, mutant ERBB3, mutant CDKN2A, mutant TP53, and / or mutant CTNNB1, where the antigenic fragment comprises the respective mutation. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant U2AF1, wherein the antigenic fragment comprises a mutation, and the U2AF1 mutation is S34F. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant TP53, wherein the antigenic fragment comprises a mutation, and the TP53 mutation is G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W, or C277F. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of the BIRC family, CEACAM family, CTA family, EPH family, ERBB family, FOLR family, GAST family, GUCY2 family, IDO family, IL13RA family, KDR family, KLK family, MAGE family, MUC family, PEMT family, SDC family, SLAMF family, TERT family, TLR family, TPTE family, TYR family, WT family, and / or XBP family (see Section 5.9), where the antigenic fragment comprises a mutation.

[0249] In certain embodiments, provided herein is a method of treating a neoplastic disease in a subject, further comprising administering a second arenavirus particle, wherein the second arenavirus particle comprises a nucleotide sequence encoding an antigenic fragment of a mutant KRAS, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A, and wherein the neoplastic disease is pancreatic cancer. In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, mutant TP53, and / or mutant U2AF1, wherein the antigenic fragment comprises a mutation, the KRAS mutation is G12D, G12V, G12R, Q61H, and / or Q61R, the TP53 mutation is R175H, R248W, G245S, R282W, R248Q, and / or R273C, the U2AF1 mutation is S34F, and the neoplastic disease is pancreatic cancer. In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G13D, G12V, G12C, G12D, and G12R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma.In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, mutant BRAF, mutant TP53, mutant FBXW7, and / or mutant PIK3CA, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12D, G12V, G13D, A146T, and / or G12C, the BRAF mutation is V600E, the TP53 mutation is R175H, R273H, and / or R248W, the FBXW7 mutation is R465H, the PIK3CA mutation is E545K and / or H1047R, and the neoplastic disease is colorectal cancer or lung adenocarcinoma. In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, mutant BRAF, mutant PIK3CA, mutant EGFR, mutant TP53, and / or mutant U2AF1, wherein the antigenic fragment comprises mutations, the KRAS mutation is G12C, G12D, G12R, G13D, and / or G12V, the BRAF mutation is V600E, the PIK3CA mutation is E545K, the EGFR mutation is L858R, the TP53 mutation is R175H, R273H, and / or R248W, the U2AF1 mutation is S34F, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, mutant TP53, mutant U2AF1, mutant PIK3CA, mutant EGFR, and / or mutant BRAF, wherein the antigenic fragment comprises mutations, the KRAS mutation is G12C, G12V, G12D, and / or G12S, the TP53 mutation is R175H, the U2AF1 mutation is S34F, the EGFR mutation is L858R, L861Q, and / or E746_A750del, the PIK3CA mutation is E545K and / or E542K, the BRAF mutation is V600E, and the neoplastic disease is lung adenocarcinoma.In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA and / or mutant RET, where the antigenic fragment comprises a mutation and the neoplastic disease is non-small cell lung cancer (NSCLC). In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, mutant RET, mutant APC, mutant U2AF1, mutant EGFR, mutant FBXW7, mutant SMAD4, mutant GNAS, mutant ERBB2, mutant ERBB3, mutant CDKN2A, mutant FBXW7, mutant TP53, and / or mutant CTNNB1 (see Section 5.8), where the antigenic fragment comprises a mutation. In certain embodiments, the second arenavirus particle comprises a nucleotide sequence encoding an antigenic fragment of mutant U2AF1, where the antigenic fragment comprises a mutation, and where the mutation in U2AF1 is S34F. In certain embodiments, the second arenavirus particle comprises a nucleotide sequence encoding an antigenic fragment of a mutant TP53, wherein the antigenic fragment comprises a mutation, the TP53 mutation being G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W and / or C277F (see Section 5.8). In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of the BIRC family, CEACAM family, CTA family, EPH family, ERBB family, FOLR family, GAST family, GUCY2 family, IDO family, IL13RA family, KDR family, KLK family, MAGE family, MUC family, PEMT family, SDC family, SLAMF family, TERT family, TLR family, TPTE family, TYR family, WT family, and / or XBP family (see Section 5.9).

[0250] In certain embodiments, provided herein is a method of treating a neoplastic disease in a subject, comprising administering an arenavirus particle comprising a nucleotide sequence encoding an antigenic fragment of mutant KRAS (see Section 5.7), wherein the neoplastic disease is pancreatic cancer, colorectal cancer, lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer (NSCLC). In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, wherein the mutations of KRAS are G13D, G12V, G12C, G12D, and G12R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, wherein the antigenic fragment comprises a mutation, wherein the mutations of KRAS are G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A, and the neoplastic disease is pancreatic cancer. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, wherein the antigenic fragment comprises a mutation, wherein the mutations of KRAS are G12D, G12R. G12V, Q61H and / or Q61R, the TP53 mutation is R175H, R248W, G245S, R282W, R248Q and / or R273C, and the neoplastic disease is pancreatic cancer. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, wherein the antigenic fragment comprises a mutation, the KRAS mutation is G12D, G12V, G13D, A146T and / or G12C, the TP53 mutation is R175H, R273H and / or R248W, and the neoplastic disease is colorectal cancer or lung adenocarcinoma.In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12C, G12D, G12R, G13D, and / or G12V, wherein the TP53 mutation is R175H, R273H, and / or R248W, and wherein the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12C, G12V, G12D, and / or G12S, wherein the TP53 mutation is R175H, and wherein the neoplastic disease is lung adenocarcinoma.

[0251] In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, wherein the antigenic fragment comprises the respective mutation. In certain embodiments, the arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, wherein the antigenic fragment comprises the respective mutation, wherein the TP53 mutation is G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W, or C277F.

[0252] In certain embodiments, provided herein is a method of treating a neoplastic disease in a subject, further comprising administering a second arenavirus particle, wherein the second arenavirus particle comprises a nucleotide sequence encoding an antigenic fragment of mutant KRAS (see Section 5.7), wherein the antigenic fragment comprises a mutation, and wherein the neoplastic disease is pancreatic cancer. In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R, and wherein the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma.

[0253] In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of a mutant KRAS, wherein the antigenic fragment comprises a mutation, the KRAS mutation is G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A, and the neoplastic disease is pancreatic cancer.

[0254] In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, wherein the antigenic fragment comprises a mutation, the KRAS mutation is G12D, G12R, G12V, Q61H and / or Q61R, the TP53 mutation is R175H, R248W, G245S, R282W, R248Q and / or R273C, and the neoplastic disease is pancreatic cancer. In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, wherein the antigenic fragment comprises a mutation, the KRAS mutation is G12D, G12V, G13D, A146T, and / or G12C, the TP53 mutation is R175H, R273H, and / or R248W, and the neoplastic disease is colorectal cancer or lung adenocarcinoma. In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12C, G12D, G12R, G13D, and / or G12V, wherein the TP53 mutation is R175H, R273H, and / or R248W, and wherein the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, wherein the antigenic fragment comprises a mutation, wherein the KRAS mutation is G12C, G12V, G12D, and / or G12S, wherein the TP53 mutation is R175H, and wherein the neoplastic disease is lung adenocarcinoma. In certain embodiments, the second arenavirus particle comprises one or more nucleotide sequences encoding an antigenic fragment of mutant KRAS and / or mutant TP53, where the antigenic fragment comprises the mutation.In certain embodiments, the second arenavirus particle comprises a nucleotide sequence encoding an antigenic fragment of mutant KRAS and / or mutant TP53, where the antigenic fragment comprises a mutation, and the TP53 mutation is G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W and / or C277F (see Section 5.8).

[0255] Successful treatment of a cancer patient can be evaluated as an increase in expected survival, induction of an anti-tumor immune response, or improvement of a particular characteristic of the cancer. Examples of cancer characteristics that may be improved include tumor size (e.g., T0, T1, or T1-4), metastatic status (e.g., M0, M1), number of observable tumors, lymph node metastasis (e.g., N0, N1-4, Nx), grade (i.e., grades 1, 2, 3, or 4), stage (e.g., 0, I, II, III, or IV), the presence or concentration of certain markers on cells or in bodily fluids (e.g., AFP, B2M, beta-HCG, B TA, CA15-3, CA27.29, CA125, CA72.4, CA19-9, calcitonin, CEA, chromogranin A, EGFR, hormone receptors, HER2, HCG, immunoglobulins, NSE, NMP22, PSA, PAP, PSMA, S-100, TA-90, and thyroglobulin), and / or associated pathology (e.g., ascites or edema) or symptoms (e.g., cachexia, fever, anorexia, or pain). When measurable by percentage, the improvement can be at least a 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or 90% improvement (e.g., survival time, or tumor volume or linear dimension).

[0256] In another embodiment, arenavirus particles expressing mutant KRAS (see Section 5.7), mutated cancer driver genes (see Section 5.8, e.g., mutant TP53), or tumor associated antigens (see Section 5.9) provided herein are administered to a subject by intratumoral injection. In another embodiment, arenavirus particles expressing mutant KRAS, mutated cancer driver genes (e.g., mutant TP53), or tumor associated antigens provided herein are administered to a subject by intravenous injection.

[0257] In another embodiment, an arenavirus particle or composition thereof expressing mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein is administered to a subject with risk factors. Exemplary risk factors include aging, tobacco, sun exposure, radiation exposure, chemical exposure, family history, alcohol, an unhealthy diet, lack of physical activity, or being overweight.

[0258] In another embodiment, arenavirus particles or compositions thereof expressing mutant KRAS (see Section 5.7), mutated cancer driver genes (see Section 5.8, e.g., mutant TP53), or tumor-associated antigens (see Section 5.9) provided herein are administered to a subject suffering from one or more types of cancer. In other embodiments, any type of neoplastic disease, such as cancer, susceptible to treatment with the compositions described herein may be targeted.

[0259] In another embodiment, administering to a subject an arenavirus particle or composition thereof expressing a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein confers cell-mediated immunity (CMI) against a neoplastic cell or tumor, e.g., a cancer cell or tumor. Without being bound by theory, in another embodiment, an arenavirus particle or composition thereof expressing a mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), or a tumor-associated antigen provided herein infects a host antigen-presenting cell (APC) (e.g., macrophage) for direct presentation of the antigen on major histocompatibility complex (MHC) class I and II, and expresses the antigen of interest therein. In another embodiment, administering to a subject an arenavirus particle or composition thereof expressing mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), or a tumor-associated antigen provided herein induces a large-scale, multifunctional IFN-γ and TNF-α co-producing cancer-specific CD4+ and CD8+ T cell response (IFN-γ is produced by CD4+ and CD8+ T cells, and TNF-α is produced by CD4+ T cells) to treat a neoplastic disease.

[0260] In another embodiment, administering an arenavirus particle or composition thereof expressing mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein improves or improves one or more clinical outcomes associated with cancer treatment. Non-limiting examples of such outcomes are overall survival, progression-free survival, time to progression, time to treatment failure, event-free survival, time to initiation of next treatment, overall response rate, and duration of response. The improvement or improvement in one or more of the clinical outcomes can be an improvement or improvement of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a patient or group of patients having the same neoplastic disease in the absence of such treatment.

[0261] The change in cell-mediated immune (CMI) response function against neoplastic cells or tumors, including cancer cells or tumors, induced by administering to a subject an arenavirus particle or composition thereof expressing a mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein can be measured using any assay known to those of skill in the art, including, but not limited to, flow cytometry (see, e.g., Perfetto SP et al., Nat Rev Immun. 2004;4(8):648-55), lymphocyte proliferation assays (see, e.g., Bonilla FA et al., Ann Allergy Asthma Immunol. 2008;101:101-4, and Hicks MJ et al., Am J Clin Pathol. 1983;80:159-63), assays measuring lymphocyte activation including determining changes in surface marker expression following measurement of T lymphocyte cytokines (see, e.g., Caruso A. et al., Cytometry. 1997;27:71-6), ELISPOT assays (see, e.g., Czerkinsky CC et al., J Immunol Methods. 1983;65:109-121, and Hutchings PR, et al., J Immunol Methods. 1989;120:1-8), or natural killer cell cytotoxicity assays (see, e.g., Bonilla FA et al., Ann Allergy Asthma Immunol. 2005 May;94(5 Suppl 1):S1-63).

[0262] In certain embodiments, the treatment provided herein can be further combined with a chemotherapeutic agent. The chemotherapeutic agent can include an alkylating agent (e.g., cyclophosphamide), a platinum-based therapeutic agent, an antimetabolite, a topoisomerase inhibitor, a cytotoxic antibiotic, an intercalating agent, a mitotic inhibitor, a taxane, or a combination of two or more thereof. In certain embodiments, the alkylating agent is a nitrogen mustard, a nitrosourea, an alkylsulfonate, a non-classical alkylating agent, or a triazene. In certain embodiments, the chemotherapeutic agent is cyclophosphamide, thiotepa, mechlorethamine (chlormethine / mustine), uramustine, melphalan, chlorambucil, ifosfamide, chlornaphazine, chlorophosphamide, estramustine, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, bendamustine, busulfan, improper Sulfan, piposulfan, carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine, streptozocin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, dacarbazine, mitozolomide, temozolomide, paclitaxel, docetaxel, vinblastine, vincristine, vinorelbine , cabazitaxel, dactinomycin (actinomycin D), calicheamicin, dynemicin, amsacrine, doxorubicin, daunorubicin, epirubicin, mitoxantrone, idarubicin, pirarubicin, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, triethylenediamine, triethylenediaminetetraacetic acid ... Limethylolmelamine, Bullatacin, Bullatacinone, Camptothecin, Topotecan, Bryostatin, Kallistatin, CC-1065, Adzelesin, Carzelesin, Bizelesin, Cryptophycin, Dolastatin, Duocarmycin, KW-2189, CB1-TM1, Eleutherobin, Pancratistatin, Sarcodictin, Spongestatin, Clodronate, Esperamicin, Neocarzinostatin Chromophore,Aclacinomycin, Anthramycin, Azaserine, Bleomycin, Cactinomycin, Carabicin, Carminomycin, Carzinophilin, Chromomycin, Detorubicin, 6-Diazo-5-oxo-L-norleucine, Esorubicin, Idarubicin, Marcellomycin, Mitomycin, Mycophenolic acid, Nogalamycin, Olivomycin, Peplomycin, Potofilomycin, Puromycin, Querramycin, Rodorubicin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex, Zinostatin, Zorubicin, Methotrexate, xate, 5-fluorouracil (5-FU), denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, mitotane, trilostane, furoic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, enyl Uracil, Bestravcil, Bisantrene, Edatraxate, Defofamine, Demecolcine, Diaziquone, Eflornithine, Elliptinium Acetate, Etoglucide, Gallium Nitrate, Hydroxyurea, Lentinan, Lonidamine, Maytansine, Ansamitocin, Mitoguazone, Mopidamol, Nitraerin, Pentostatin, Phenamet, Pirarubicin, Rosoxantrone, Podophyllic Acid, 2-Ethylhydrazide, PSK Polysaccharide Complex, Razoxane, Rhizoxin, Sizofiran, Spirogermanium, Tenuazonic Acid, Triaziquone, 2,2',2"-Tet trichlorotriethylamine; T-2 toxin, veraculin A, roridin A and anguidine, urethan, vindesine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), etoposide (VP-16), vinorelbine, novantrone, teniposide, edatrexate, aminopterin, xeloda, ibandronate, irinotecan (e.g., CPT-11), topoisomerase inhibitors RFS2000, difluoromethylornithine (DMFO), retinoic acid, capecitabine,plicomycin, gemcitabine, navelbine, transplatinum, and one or more of the pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0263] In certain embodiments, one or more arenavirus particles or compositions thereof expressing mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein are administered in two or more separate injections over a 1 hour period, a 2 hour period, a 3 hour period, a 6 hour period, a 12 hour period, a 24 hour period, or a 48 hour period.

[0264] In certain embodiments, one or more arenavirus particles or compositions thereof expressing mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein are administered in two or more separate injections over a 3 day period, a 5 day period, a 1 week period, a 2 week period, a 3 week period, a 4 week period, or a 12 week period.

[0265] In certain embodiments, one or more arenavirus particles or compositions thereof expressing mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), or a tumor-associated antigen (see Section 5.9) provided herein are administered in two or more separate injections over a 6 month period, a 12 month period, a 24 month period, or a 48 month period.

[0266] In certain embodiments, two arenavirus particles provided herein are administered in a treatment regimen, and the administration can be in a molar ratio ranging from about 1:1 to 1:1000, in particular, for example, a 1:1 ratio, a 1:2 ratio, a 1:5 ratio, a 1:10 ratio, a 1:20 ratio, a 1:50 ratio, a 1:100 ratio, a 1:200 ratio, a 1:300 ratio, a 1:400 ratio, a 1:500 ratio, a 1:600 ​​ratio, a 1:700 ratio, a 1:800 ratio, a 1:900 ratio, a 1:1000 ratio.

[0267] In certain embodiments, provided herein are methods of treating neoplastic disease, in which a first arenavirus particle is initially administered as a "prime" and a second arenavirus particle is administered as a "boost". The first and second arenavirus particles can express the same or different mutant KRAS, mutated cancer driver genes (e.g., mutant TP53), or tumor-associated antigens. Alternatively or additionally, in some certain embodiments, the "prime" and "boost" administrations are performed with arenavirus particles derived from different arenavirus species. In certain specific embodiments, the "prime" administration is performed with arenavirus particles derived from LCMV, and the "boost" is performed with arenavirus particles derived from Pichindevirus. In certain specific embodiments, the "prime" administration is performed with arenavirus particles derived from Pichindevirus, and the "boost" is performed with arenavirus particles derived from LCMV.

[0268] In certain embodiments, administering a first arenavirus particle expressing mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), and / or a tumor-associated antigen (see Section 5.9), followed by administering a second arenavirus particle expressing mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), and / or a tumor-associated antigen, results in a greater antigen-specific CD8+ T cell response than administering a single arenavirus particle expressing mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), and / or a tumor-associated antigen. In certain embodiments, the number of antigen-specific CD8+ T cells increases by 50%, 100%, 150%, or 200% after the second administration compared to the first administration.

[0269] In certain embodiments, administering a first arenavirus particle expressing mutant KRAS (see Section 5.7), a mutated cancer driver gene (see Section 5.8, e.g., mutant TP53), and / or a tumor associated antigen (see Section 5.9) and a second heterologous arenavirus particle expressing mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), and / or a tumor associated antigen elicits a greater CD8+ T cell response than administering a first arenavirus particle expressing mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), and / or a tumor associated antigen and a second homologous arenavirus particle expressing mutant KRAS, a mutated cancer driver gene (e.g., mutant TP53), and / or a tumor associated antigen.

[0270] 5.11 Compositions, Administration, and Dosages Also provided herein are vaccines, immunogenic compositions (e.g., vaccine formulations), and pharmaceutical compositions comprising the arenavirus particles provided herein. Such vaccines, immunogenic compositions, and pharmaceutical compositions can be formulated according to standard procedures in the art.

[0271] In certain embodiments, provided herein are immunogenic compositions comprising the arenavirus particles described herein (or a combination of different arenavirus particles). In certain embodiments, such immunogenic compositions further comprise a pharma- ceutically acceptable excipient. In certain embodiments, such immunogenic compositions further comprise an adjuvant. An adjuvant administered in combination with a composition described herein can be administered before, simultaneously with, or after administration of said composition. In some embodiments, the term "adjuvant" refers to a compound that amplifies, enhances, and / or boosts an immune response to infectious replication-deficient arenavirus particles when administered with or as part of a composition described herein, but does not generate an immune response to infectious replication-deficient arenavirus particles when administered alone. In some embodiments, an adjuvant generates an immune response to infectious replication-deficient arenavirus particles and does not cause an allergic or other adverse reaction. An adjuvant can enhance an immune response by several mechanisms, including, for example, lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. When the vaccine or immunogenic composition of the invention comprises an adjuvant or is administered together with one or more adjuvants, adjuvants that can be used include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particle adjuvants, particulate adjuvants, mucosal adjuvants, and immune stimulating adjuvants.Examples of adjuvants include, but are not limited to, aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (see GB2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate 80 (Tween 80, ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT / US2007 / 064857, published as WO2007 / 109812), imidazoquinoxaline compounds (see International Application No. PCT / US2007 / 064858, published as WO2007 / 109813), and saponins, such as QS21 (Kensil et al., J. D. Med. Soc., 2006). al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); see U.S. Patent No. 5,057,540. In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (e.g., squalene or peanut oil), optionally combined with an immunostimulant such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)).

[0272] In certain embodiments, the compositions described herein further comprise a preservative, such as the mercury derivative thimerosal. In specific embodiments, the pharmaceutical compositions described herein comprise 0.001% to 0.01% thimerosal. In other embodiments, the pharmaceutical compositions described herein do not comprise a preservative.

[0273] The pharmaceutical composition is about 10 3 ~about 10 11 The genetically engineered arenavirus particles contain focus forming units. The unit dosage form for parenteral administration may be, for example, an ampoule or a vial, for example, about 10 3 ~1010 Focus formation unit or 10 5 ~10 15 A vial containing a genetically engineered arenavirus particle of physical particles.

[0274] In another embodiment, the vaccine or immunogenic composition provided herein is administered to a subject by intratumoral injection. In another embodiment, the vaccine or immunogenic composition provided herein is administered to a subject by intravenous injection.

[0275] The dosage of the active ingredient depends on the type of vaccination and on the subject, and on its age, weight, individual condition, individual pharmacokinetic data, and the mode of administration.

[0276] In certain embodiments, the composition is administered to the patient as a single dose, followed by a second dose 3-6 weeks later. In these embodiments, a booster vaccination may be administered to the subject at intervals of 6-12 months after the second vaccination. In certain embodiments, the booster vaccination may utilize a different arenavirus particle or composition thereof. In some embodiments, administration of the same composition described herein may be repeated and spaced at least 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months apart.

[0277] In certain embodiments, a suitable dosage of arenavirus particles or three segmented arenavirus particles is 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 1×10 9 , 5×10 9, 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 or 10 12 pfu and can be administered to a subject once, twice, or three or more times at intervals, as needed.

[0278] In certain embodiments, the methods and compositions provided herein are used in combination with personalized medicine. Personalized medicine aims to benefit patients by using information from a patient's unique genetic and / or epigenetic profile to predict a patient's response to various therapies and identify which therapies are more likely to be effective. Techniques that can be used in combination with the methods and compositions provided herein to obtain a patient's unique genetic and / or epigenetic profile include, but are not limited to, genome sequencing, RNA sequencing, gene expression analysis, and identification of tumor antigens (e.g., neoantigens), tumor-associated antigens, or antigenic fragments thereof. In certain embodiments, the selection of Arenavirus tumor antigens or tumor-associated antigens for use in the methods and compositions provided herein is performed based on the patient's genetic profile. In certain embodiments, the selection of Arenavirus tumor antigens or tumor-associated antigens for use in the methods and compositions provided herein is performed based on the genetic profile of the tumor or tumor cells.

[0279] Also provided herein is a kit that can be used to carry out the methods described herein. In certain embodiments, the kit provided herein can include one or more containers. These containers can hold the compositions provided herein (e.g., pharmaceutical compositions, immunogenic compositions, or vaccine compositions) for storage. The kit also includes instructions for use. These instructions fully describe the treatment protocol for using the compositions contained therein. For example, the instructions can include dosage and administration instructions for the methods of treating neoplastic disease provided herein.

[0280] 5.12 Assays to Demonstrate Activity (a) Arenavirus detection assay A person skilled in the art can use techniques known in the art to detect arenavirus genome segments or arenavirus particles as described herein.For example, RT-PCR can be used with primers specific for arenavirus to detect and quantify arenavirus genome segments or three-segment arenavirus particles.Western blot, ELISA, radioimmunoassay, immunoprecipitation, immunocytochemistry, or immunocytochemistry combined with FACS can be used to quantify gene products of arenavirus genome segments or arenavirus particles.

[0281] (b) Infectivity assay To measure the infectivity of arenavirus vector preparations, any assay known to those skilled in the art can be used. For example, the determination of virus / vector titer can be done by "focus forming unit assay" (FFU assay). Briefly, complementation cells, e.g., HEK293-TVL cells, are plated and inoculated with various dilutions of virus / vector samples. After an incubation period, the cells form a monolayer, which is covered with methylcellulose to allow the virus to attach to the cells. If the plate is further incubated, the original infected cells release virus progeny. Due to the methylcellulose overlay, the spread of new viruses is restricted to nearby cells. As a result, each infectious particle generates a circular zone of infected cells called a focus. Such foci can be visualized and therefore countable using antibodies against LCMV-NP or arenavirus particles or another protein expressed by three-segmented arenavirus particles and an HRP-based color reaction. The virus / vector titer can be calculated as focus forming units / milliliter (FFU / mL). Similarly, the percentage of three-segmented replicative virus particles can be determined. Instead of complementing cells, a non-complementing cell line, e.g., HEK293, is used. This allows only the three-segmented viral particles to infect nearby cells. The titer of replication-competent virus / vector (RCV) can be calculated as focus-forming units / milliliter (RCV FFU / mL).

[0282] (c) Growth of arenavirus particles The growth of the arenavirus particles described herein can be assessed by any method (e.g., cell culture) known in the art or described herein. Virus growth can be determined by inoculating a cell culture (e.g., Vero cells or BHK-21 cells) with a defined amount / concentration of the arenavirus particles described herein. After incubating the virus for a specified time, virus-containing supernatants can be harvested using standard methods and infectivity can be measured using the assays described herein.

[0283] (d) Serum ELISA Determination of humoral immune response upon vaccination of animals (e.g. mice, guinea pigs) can be performed by antigen-specific serum ELISA (enzyme-linked immunosorbent assay). Briefly, plates are coated with antigen (e.g. recombinant protein), blocked to avoid non-specific binding of antibodies, and incubated with serial dilutions of serum. After incubation, bound serum-antibodies can be detected, for example, using enzyme-linked anti-species (e.g. mouse, guinea pig) specific antibodies (detecting total IgG or IgG subclasses) and subsequent color reaction. Antibody titers can be determined, for example, as endpoint geometric mean titers.

[0284] (e) Assay to measure the neutralizing activity of induced antibodies Determination of neutralizing antibodies in serum is performed using the following cellular assay using ATCC ARPE-19 cells and GFP-tagged virus, plus supplemental guinea pig serum as an exogenous complement source. The assay is performed by plating 6.5×10 IgG antibodies into 384-well plates 1 or 2 days before use for neutralization. 3Start with seeding cells / well (50 μl / well). Neutralization is performed in 96-well sterile tissue culture plates without cells for 1 h at 37° C. After the neutralization incubation step, the mixture is added to the cells and incubated for another 4 days for GFP detection on a plate reader. To ensure the reliability of all results, a positive neutralizing human serum is used as an assay positive control in each plate. The titer (EC50) is determined using a 4-parameter logistic curve fitting. As an additional test, the wells are checked using a fluorescent microscope.

[0285] (f) Plaque reduction assay Briefly, plaque reduction (neutralization) assays for LCMV can be performed by using replication-competent or replication-deficient LCMV encoding a reporter gene (e.g., green fluorescent protein), 5% rabbit serum may be used as an exogenous complement source, and plaques can be counted by fluorescence microscopy. Neutralization titers can be defined as the highest dilution of serum that results in a 50%, 75%, 90% or 95% reduction in plaques compared to control (pre-immune) serum samples. qPCR LCMV RNA genomes are isolated using the QIAamp Viral RNA mini Kit (QIAGEN) according to the protocol provided by the manufacturer. LCMV RNA genome equivalents are detected by quantitative PCR carried out in a StepOnePlus Real Time PCR System (Applied Biosystems) equipped with a SuperScript® III Platinum® One-Step qRT-PCR Kit (Invitrogen) and primers and probes (FAM reporter and NFQ-MGB quencher) specific for a part of the LCMV NP coding region or another genomic stretch of arenavirus particles or three-segmented arenavirus particles. The temperature profile of the reaction can be 30 min at 60° C., 2 min at 95° C., followed by 45 cycles of 15 s at 95° C. and 30 s at 56° C. The RNA can be quantified by comparing the sample results with a standard curve made from a log10 dilution series of spectrophotometrically quantified in vitro transcribed RNA fragments corresponding to fragments of the LCMV NP coding sequence or another genomic stretch of arenavirus particles or three-segmented arenavirus particles containing primer and probe binding sites.

[0286] (g) Western blotting Infected cells grown in tissue culture flasks or in suspension were lysed using RIPA buffer (Thermo Scientific) at the indicated time points post-infection or used directly without cell lysis. Samples were heated to 99 °C for 10 min with reducing agent and NuPage LDS Sample buffer (NOVEX) and cooled to room temperature before loading onto 4-12% SDS gels for electrophoresis. Proteins were blotted onto membranes using Invitrogen's iBlot Gel transfer Device and visualized by Ponceau staining. Finally, preparations were probed with primary antibodies against the protein of interest and alkaline phosphatase-conjugated secondary antibodies, followed by staining with 1-Step NBT / BCIP solution (INVITROGEN).

[0287] (h) MHC-peptide multimer staining assay for detection of antigen-specific CD8+ T cells Any assay known to those skilled in the art can be used to test antigen-specific CD8+ T cell responses. For example, MHC-peptide tetramer staining assay can be used (see, for example, Altman JDet al., Science. 1996; 274: 94-96, and Murali-Krishna K. et al., Immunity. 1998; 8: 177-187). Briefly, the assay includes the following steps, using tetramer assay to detect the presence of antigen-specific T cells. To detect antigen-specific T cells, they need to bind both peptides and tetramers of MHC molecules (typically fluorescently labeled) that are customized for the defined antigen specificity and MHC haplotype of the T cells. The tetramers are then detected by flow cytometry via fluorescent labeling.

[0288] (i) ELISPOT assay for detection of antigen-specific T cells Any assay known to those skilled in the art can be used to test antigen-specific T cell response. For example, ELISPOT assay can be used (see, for example, Czerkinsky CC et al., J Immunol Methods. 1983; 65: 109-121, and Hutchings PR et al., J Immunol Methods. 1989; 120: 1-8). For example, cytokines such as, but not limited to, IFN-γ can be measured by ELISPOT assay. Briefly, the assay includes the following steps: coat an immunospot plate with anti-cytokine antibody. Incubate cells with peptides derived from the antigen of interest in the immunospot plate. Antigen-specific cells secrete cytokines that bind to the coated antibody. Then, wash off the cells, add a second biotinylated anti-cytokine antibody to the plate, and use avidin-HRP system or other suitable methods to visualize.

[0289] (j) Intracellular cytokine assays for detecting CD8+ and CD4+ T cell functionality Any assay known to those skilled in the art can be used to test the functionality of CD8+ and CD4+ T cell responses. For example, an intracellular cytokine assay combined with flow cytometry can be used (see, for example, Suni MA et al., J Immunol Methods. 1998; 212: 89-98, Nomura LE et al., Cytometry. 2000; 40: 60-68, and Ghanekar SA et al., Clinical and Diagnostic Laboratory Immunology. 2001; 8: 628-63). Briefly, the assay includes the following steps: Immediately after activation of cells with a specific peptide or protein, a protein transport inhibitor (e.g., brefeldin A) is added to retain the cytokines inside the cells. After a defined incubation period, typically 5 hours, followed by a washing step, antibodies against other cellular markers can be added to the cells. The cells are then fixed and permeabilized. Fluorochrome-conjugated anti-cytokine antibodies can be added and the cells can be analyzed by flow cytometry.

[0290] (k) Assays to confirm replication deficiency of viral vectors Any assay known to those skilled in the art that determines the concentration of infectious and replicative viral particles can also be used to measure replication-deficient viral particles in a sample. For example, an FFU assay using non-complementing cells can be used for this purpose.

[0291] Furthermore, plaque-based assays are the standard method used to determine virus concentration in terms of plaque-forming units (PFU) in a virus sample. Specifically, confluent monolayers of non-complementing host cells are infected with various dilutions of the virus, and the cells are covered with a semi-solid medium, such as agar, to prevent the indiscriminate spread of the virus infection. Viral plaques form when the virus successfully infects and replicates in cells within the fixed cell monolayer, and spread to surrounding cells (see, for example, Kaufmann, SH; Kabelitz, D. (2002). Methods in Microbiology Vol. 32: Immunology of Infection. Academic Press. ISBN 0-12-521532-0). Plaque formation can take 2-14 days depending on the virus being analyzed. Plaques are generally counted manually, and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque-forming units per unit volume of sample (PFU / mL). Results in PFU / mL represent the number of infectious, replication-competent particles in the sample. When using C cells, the same assay can be used to titer replication-deficient or three-segment arenavirus particles.

[0292] (l) Assay for expression of viral antigens Any assay known to those skilled in the art can be used to measure the expression of viral antigens.For example, FFU assay can be carried out.For detection, use monoclonal or polyclonal antibody preparation(s) against each viral antigen (transgene-specific FFU).

[0293] (m) Animal Model Animal models can be used to study the in vivo recombination and infectivity of the arenavirus particles described herein. In certain embodiments, animal models that can be used to study the recombination and infectivity of three-segment arenavirus particles include mice, guinea pigs, rabbits, and monkeys. In a preferred embodiment, animal models that can be used to study the recombination and infectivity of arenaviruses include mice. In a more specific embodiment, mice that can be used to study the recombination and infectivity of arenavirus particles are triple-deficient mice of type I interferon receptor, type II interferon receptor, and recombination activating gene 1 (RAG1).

[0294] In certain embodiments, animal models can be used to determine arenavirus infectivity and transgene stability. In some embodiments, viral RNA can be isolated from the serum of the animal model. Techniques are readily known by those skilled in the art. Viral RNA can be reverse transcribed, and cDNA carrying arenavirus ORFs can be PCR amplified using gene-specific primers. Flow cytometry can also be used to examine arenavirus infectivity and transgene stability.

[0295] 6. Equivalents All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. From the foregoing description, it will be apparent that variations and modifications of the invention described herein can be made to adapt it to various uses and conditions. Such embodiments also fall within the scope of the following claims. [Table 3] TIFF2024540385000005.tif77165TIFF2024540385000006.tif210165TIFF2024540385000007.tif205 165TIFF2024540385000008.tif220165TIFF2024540385000009.tif220165TIFF2024540385000010.ti f236165TIFF2024540385000011.tif231165TIFF2024540385000012.tif221165TIFF202454038500001 3.tif237165TIFF2024540385000014.tif236165TIFF2024540385000015.tif200165TIFF20245403850 00016.tif214165TIFF2024540385000017.tif220165TIFF2024540385000018.tif220165TIFF2024540 385000019.tif230165TIFF2024540385000020.tif205165TIFF2024540385000021.tif230165TIFF202 4540385000022.tif236165TIFF2024540385000023.tif215165TIFF2024540385000024.tif236165TIF F2024540385000025.tif215165TIFF2024540385000026.tif220165TIFF2024540385000027.tif212165 EXAMPLES

[0296] 8. Working Example 8.1 Arenavirus vector design (a)artLCMV-4xKRASmut artLCMV-4xKRASmut is an attenuated, replicable, three-segment vector based on LCMV clone 13 (LCMV cl13) that expresses the GP of LCMV strain WE in place of its endogenous glycoprotein (LCMV cl13 / WE) (Figure 1). The NP-S segment contains a nucleotide sequence (SEQ ID NO: 1) encoding four copies of KRAS amino acids 1-29, each with G12D, G12V, G12C, and G13D mutations, and the GP-S segment contains a nucleotide sequence (SEQ ID NO: 1) encoding four copies of KRAS amino acids 1-29, each with G12D, G12V, G12C, and G13D mutations. The nucleotide sequence of the KRAS epitope has been modified to lack the CpG dinucleotide motif. Vectors are generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0297] (b) artLCMV-4xKRASmut_E7 artLCMV-4xKRASmut_E7 is an attenuated, replicable, three-segment vector based on LCMV clone 13 (LCMV cl13) that expresses the GP of LCMV strain WE instead of its endogenous glycoprotein (LCMV cl13 / WE) (FIG. 2). The NP-S segment contains a nucleotide sequence (SEQ ID NO:2) encoding four copies of KRAS amino acids 1-29, each with G12D, G12V, G12C, and G13D mutations, and an HPV E7 epitope, and the GPS-S segment contains a nucleotide sequence (SEQ ID NO:2) encoding four copies of KRAS amino acids 1-29, each with G12D, G12V, G12C, and G13D mutations, and an HPV E7 epitope. The nucleotide sequences of the KRAS epitope and the HPV E7 epitope have been modified to lack CpG dinucleotide motifs. Vectors are generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0298] (c)artLCMV-4xKRASmut_EBV artLCMV-4xKRASmut_EBV is an attenuated, replicable, three-segment vector based on LCMV clone 13 (LCMV cl13) that expresses the GP of LCMV strain WE instead of its endogenous glycoprotein (LCMV cl13 / WE) (Figure 3). The NP-S segment contains a nucleotide sequence (SEQ ID NO: 3) encoding four copies of KRAS amino acids 1-29 with G12D, G12V, G12C, and G13D mutations, respectively, and an EBV epitope, and the GPS-S segment contains a nucleotide sequence (SEQ ID NO: 3) encoding four copies of KRAS amino acids 1-29 with G12D, G12V, G12C, and G13D mutations, respectively, and an EBV epitope. The nucleotide sequences of the KRAS epitope and the HPV E7 epitope have been modified to lack CpG dinucleotide motifs. Vectors are generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0299] (d)artPICV-4xKRASmut artPICV-4xKRASmut is an attenuated, replicable, three-segment vector based on the virulent strain of Pichinde virus, passage 18 (designated PIC, or alternatively PICV p18) (Figure 1). The NP-S segment contains a nucleotide sequence (SEQ ID NO: 1) encoding four copies of KRAS amino acids 1-29, each with G12D, G12V, G12C, and G13D mutations, and the GPS-S segment contains a nucleotide sequence (SEQ ID NO: 1) encoding four copies of KRAS amino acids 1-29, each with G12D, G12V, G12C, and G13D mutations. The nucleotide sequence of the KRAS epitope has been modified to lack CpG dinucleotide motifs. The vector is generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0300] (e)artPICV-4xKRASmut_E7 artPICV-4xKRASmut_E7 is an attenuated, replicable, three-segment vector based on the virulent strain of Pichinde virus, passage 18 (designated PIC, or PICV p18) (FIG. 2). The NP-S segment contains four copies of KRAS amino acids 1-29, each with G12D, G12V, G12C, and G13D mutations, and a nucleotide sequence encoding an HPV E7 epitope (SEQ ID NO: 2). The GPS-S segment contains four copies of KRAS amino acids 1-29, each with G12D, G12V, G12C, and G13D mutations, and a nucleotide sequence encoding an HPV E7 epitope (SEQ ID NO: 2). The nucleotide sequences of the KRAS epitope and the HPV E7 epitope are modified to lack CpG dinucleotide motifs. Vectors are generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0301] (f)artPICV-4xKRASmut_EBV artPICV-4xKRASmut_EBV is an attenuated, replicable, three-segment vector based on the virulent strain of Pichinde virus, passage 18 (designated PIC, or PICV p18) (Figure 3). The NP-S segment contains four copies of KRAS amino acids 1-29, each with G12D, G12V, G12C, and G13D mutations, and a nucleotide sequence encoding an EBV epitope (SEQ ID NO: 3). The GPS-S segment contains four copies of KRAS amino acids 1-29, each with G12D, G12V, G12C, and G13D mutations, and a nucleotide sequence encoding an EBV epitope (SEQ ID NO: 3). The nucleotide sequences of the KRAS epitope and the HPV E7 epitope are modified to lack CpG dinucleotide motifs. Vectors are generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0302] (g)artLCMV-14xp53mut artLCMV-14xp53mut is an attenuated, replicable, three-segment vector based on LCMV clone 13 (LCMV cl13) that expresses the GP of LCMV strain WE instead of its endogenous glycoprotein (LCMV cl13 / WE) (Figure 5A). The NP-S segment contains nucleotide sequences (SEQ ID NO: 7) encoding 14 p53 neoepitopes of 29 amino acids each, and the GP-S segment contains nucleotide sequences (SEQ ID NO: 7) encoding 14 p53 neoepitopes of 29 amino acids each. The nucleotide sequences encoding the p53 neoepitopes have been modified to lack CpG dinucleotide motifs. The vector is generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0303] (h)artLCMV-14xp53mut_E7 artLCMV-14xp53mut_E7 is an attenuated, replicable, three-segment vector based on LCMV clone 13 (LCMV cl13) that expresses the GP of LCMV strain WE instead of its endogenous glycoprotein (LCMV cl13 / WE) (Figure 6A). The NP-S segment contains nucleotide sequences (SEQ ID NO: 8) encoding 14 p53 neoepitopes and HPV E7 epitopes, each consisting of 29 amino acids, and the GP-S segment contains nucleotide sequences (SEQ ID NO: 8) encoding 14 p53 neoepitopes and HPV E7 epitopes, each consisting of 29 amino acids. The nucleotide sequences encoding the p53 neoepitopes and HPV E7 epitopes have been modified to lack CpG dinucleotide motifs. The vector is generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0304] (i) artLCMV-14xp53mut_EBV artLCMV-14xp53mut_EBV is an attenuated, replicable, three-segment vector based on LCMV clone 13 (LCMV cl13) that expresses the GP of LCMV strain WE instead of its endogenous glycoprotein (LCMV cl13 / WE) (Figure 7A). The NP-S segment contains nucleotide sequences (SEQ ID NO: 9) encoding 14 p53 neoepitopes and an EBV epitope, each consisting of 29 amino acids, and the GP-S segment contains nucleotide sequences (SEQ ID NO: 9) encoding 14 p53 neoepitopes and an EBV epitope, each consisting of 29 amino acids. The nucleotide sequences encoding the p53 neoepitopes have been modified to lack CpG dinucleotide motifs. The vector is generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0305] (j)artPICV-14xp53mut artPICV-14xp53mut is an attenuated, replicable, three-segment vector based on the virulent strain of Pichinde virus, passage 18 (designated PIC, or alternatively PICV p18) (Figure 5B). The NP-S segment contains nucleotide sequences (SEQ ID NO: 10) encoding 14 p53 neoepitopes, each consisting of 29 amino acids, and the GPS-S segment contains nucleotide sequences (SEQ ID NO: 10) encoding 14 p53 neoepitopes, each consisting of 29 amino acids. The nucleotide sequences encoding the p53 neoepitopes have been modified to lack CpG dinucleotide motifs. The vector is generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0306] (k)artPICV-14xp53mut_E7 artPICV-14xp53mut_E7 is an attenuated, replicable three-segment vector based on the virulent strain of Pichinde virus, passage 18 (designated PIC, or alternatively PICV p18) (Figure 6B). The NP-S segment contains nucleotide sequences (SEQ ID NO: 11) encoding 14 p53 neoepitopes and HPV E7 epitopes, each consisting of 29 amino acids, and the GP-S segment contains nucleotide sequences (SEQ ID NO: 11) encoding 14 p53 neoepitopes and HPV E7 epitopes, each consisting of 29 amino acids. The nucleotide sequences encoding the p53 neoepitopes and HPV E7 epitopes have been modified to lack CpG dinucleotide motifs. The vector is generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0307] (l)artPICV-14xp53mut_EBV artPICV-14xp53mut_EBV is an attenuated, replicable, three-segment vector based on the virulent strain of Pichinde virus, passage 18 (designated PIC, or alternatively PICV p18) (Figure 7B). The NP-S segment contains nucleotide sequences (SEQ ID NO: 12) encoding 14 p53 neoepitopes and EBV epitopes, each consisting of 29 amino acids, and the GPS-S segment contains nucleotide sequences (SEQ ID NO: 12) encoding 14 p53 neoepitopes and EBV epitopes, each consisting of 29 amino acids. The nucleotide sequences encoding the p53 neoepitopes have been modified to lack CpG dinucleotide motifs. The vector is generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327.

[0308] (m)artLCMV-5xKRASmut-H2 artLCMV-5xKRASmut-H2 is an attenuated, replicative three-segment vector based on LCMV clone 13 (LCMV cl13) that expresses the GP of LCMV strain WE instead of its endogenous glycoprotein (LCMV cl13 / WE). The NP-S and GP-S segments each encode an antigenic insert (SEQ ID NO: 19) consisting of five mutant epitopes of KRAS, each consisting of 18 amino acids. The nucleotide sequence of the antigenic insert was modified to lack CpG dinucleotide motifs. The vector was generated de novo by electroporation of producer cells using a five-plasmid co-transfection system as previously described by Kallert et al. Nat Commun 2017;8:15327. Figure 10 shows a schematic diagram of artLCMV-5xKRASmut-H2.

[0309] (n)artPICV-5xKRASmut-H2 artPICV-5xKRASmut-H2 is an attenuated, replicable three-segment vector based on the virulent strain of Pichinde virus, passage 18 (designated PIC, or alternatively PICV p18). The NP-S and GPS-S segments each encode an antigenic insert (SEQ ID NO: 19) consisting of five mutant epitopes of KRAS, each consisting of 18 amino acids. The nucleotide sequence of the antigenic insert was modified to lack CpG dinucleotide motifs. The vector was generated de novo by electroporation of producer cells using a five-plasmid co-transfection system, as previously described by Kallert et al. Nat Commun 2017;8:15327. Figure 10 shows a schematic diagram of artPICV-5xKRASmut-H2.

[0310] 8.2 In vitro T cell activation and function (a) Human PBMC activation assay Human PBMCs are isolated from healthy HLA-A2+ donors and pulsed with HLA-A2-restricted KRASmut and control peptides. T cell activation is measured by ELISPOT, proliferation (FACS CFSE) and cytokine expression profiles (supernatants, intracellular FACS staining).

[0311] (b) In vitro T cell activation system Monocyte-derived dendritic cells (moDCs) or artificial antigen presenting cells (aAPCs) are infected with arenavirus particles encoding the antigenic fragment(s) of mutant KRAS (KRASmut vector) and a control vector (i.e., arenavirus particles not encoding the antigenic fragment of mutant KRAS) and incubated with isolated human T cells. T cell activation is measured by ELISPOT, proliferation (FACS CFSE) and cytokine expression profiles (supernatant, intracellular FACS staining).

[0312] (c) Cytotoxicity In vitro killing of KRASmut human cancer cell lines (i.e., human cancer cell lines expressing mutant KRAS) by KRASmut vector-primed human T cells was measured using LDH release, 51 Assess by Cr release, flow cytometry or live cell imaging assays.

[0313] (d) KRAS-reporter cell assay Activation of KRAS-mut-specific T cell receptor (TCR) is measured using reporter cell lines engineered to express KRAS-mut-specific TCR and produce a bioluminescent or fluorescent signal induced by TCR signaling. These cells are incubated with HLA-matched KRASmut vector-transfected cell lines.

[0314] 8.3 In vivo immunogenicity and efficacy (a) Immunogenicity Human HLA transgenic mice are immunized with KRASmut and control vectors. PBMCs and / or splenocytes from immunized animals are isolated and KRASmut-specific T cell responses are measured by ELISPOT, intracellular cytokine staining, MHC multimer staining, and by multiplex cytokine analysis.

[0315] Humanized mice are immunized with KRASmut and control vectors. PBMCs and / or splenocytes from immunized animals are isolated and KRASmut-specific T cell responses are measured by ELISPOT, intracellular cytokine staining, MHC multimer staining, and by multiplex cytokine analysis.

[0316] To analyze the ability of vector constructs encoding various combinations of mutated KRAS epitopes (i.e., mutant KRAS fragments) to induce antigen-specific immune responses in HLA-A*11 transgenic mice (i.e., CB6F1-Tg(HLA-A*1101 / H2-Kb)A11.01 mice), intravenous (iv) immunization was performed using 1 × 10 5 RCV FFU / dose was performed using the indicated vector constructs.

[0317] Table 2 below is illustrative of experiments testing the in vivo immunogenicity of the vectors described in this application. [Table 4]

[0318] On day 0, HLA-A*11 transgenic mice (i.e., CB6F1-Tg(HLA-A*1101 / H2-Kb)A11.01 mice) were inoculated at 1 × 10 5Mice were immunized intravenously with RCV FFU / dose of artPICV-5xKRASmut-H2 (Group 2), artPICV-5xKRASmut-H1 (Group 3), artPICV-4xKRASmut(18-mer) (Group 4), or an artPICV-based vector encoding the corresponding 18-mer wild-type epitope of KRAS (i.e., artPICV-KRASwt) (Group 5). Control mice were treated with formulation buffer only (Group 1). On day 21, mice were immunized intravenously with 1×10 5 Mice were immunized intravenously with RCV FFU / dose of artLCMV-5xKRASmut-H2 (group 2), artLCMV-5xKRASmut-H1 (group 3), artLCMV-4xKRASmut(18-mer) (group 4), artLCMV-KRASwt (group 5) or treated with formulation buffer (group 1). On day 26, KRAS epitope-specific CD8+ T cell responses were analyzed by ELISPOT analysis using wild-type and mutant-specific KRAS-based peptides as stimuli. A mixture of NP-based peptides derived from LCMV and PICV was used as a control.

[0319] As shown in Figure 11, CD8+ T cell responses against two of the encoded mutated KRAS epitopes (i.e., KRAS G12D and KRAS G12V) could be detected in group 2 animals (treated with a combination of artPICV and artLCMV-based vectors encoding a 5xKRASmut-H2 epitope cassette) and group 4 mice (treated with a combination of artPICV and artLCMV-based vectors encoding a 4xKRASmut (18-mer) epitope cassette). Surprisingly, in contrast, in group 3 animals (treated with a combination of artPICV and artLCMV-based vectors encoding a 5xKRASmut-H1 epitope cassette), CD8+ T cell responses could only be observed against one of the encoded mutated KRAS epitopes (i.e., KRAS G12D), and this vector combination did not induce a detectable immune response against the KRAS G12V epitope. Importantly, none of the tested vector constructs encoding mutated epitopes of KRAS induced detectable CD8+ T cell responses against the wild-type KRAS protein.

[0320] (b) Validity (i) CT26 model Balb / c mice bearing subcutaneously implanted CT26 (KRAS-mut) tumors are injected with KRASmut vector, control vector, or buffer. Tumor control is assessed by monitoring tumor growth after vector treatment (vernier caliper measurement). KRASmut-specific T cell responses are analyzed by MHC multimer staining and flow cytometry.

[0321] (ii) KPC PDAC model KPC mice with mutations in KRAS (e.g., G12D) and Tp53 (e.g., R172H) spontaneously develop tumors and metastases in the lungs and liver. KPC mice or mice implanted with KPC tumors are injected with KRASmut vector, control vector, or buffer. Primary tumor and metastatic control are assessed by histological analysis of pancreas, liver, and lymph nodes after vector treatment. KRASmut-specific T cell responses are analyzed by MHC multimer staining and flow cytometry.

[0322] (iii) Humanized PDX and KRAS-mut tumor cell lines Humanized mice are implanted with patient-derived HLA-matched human KRASmut xenografts or human KRASmut tumor cell lines and injected with KRASmut vector, control vector, or buffer. Tumor control is assessed by monitoring tumor growth after vector treatment (caliper measurement). KRASmut-specific T cell responses are analyzed by MHC multimer staining and flow cytometry.

[0323] 8.4 Transgene stability (i) artLCMV-5xKRASmut-H2 The genetic stability of the encoded transgene after generation of the artLCMV-5xKRASmut-H2 vector was analyzed by PCR at increasing passage levels (FIG. 12A). The 5xKRASmut transgene was stable over all passage levels tested.

[0324] (ii) artPICV-5xKRASmut-H2 The genetic stability of the encoded transgene after generation of the artPICV-5xKRASmut-H2 vector was analyzed by PCR at increasing passage levels (FIG. 12B). The 5xKRASmut transgene was stable over all passage levels tested.

[0325] 8.5 Vector immunogenicity To analyze the ability of vector constructs encoding various combinations of mutated KRAS epitopes to induce antigen-specific immune responses, intravenous immunization was performed in mice with 1 × 10 5 RCV FFU / dose was performed with the indicated vector constructs (see FIG. 22 for study design).

[0326] On day 0, HLA-B*07 transgenic mice (i.e., CB6F1-Tg(HLA-B*0702 / H2-Kb)B7.xx mice) were inoculated at 1 × 10 5 Mice were immunized intravenously with RCV FFU / dose of artPICV-5xKRASmut-H2 (Group 2), artPICV-5xKRASmut-H1 (Group 3), artPICV-4xKRASmut (Group 4), or an artPICV-based vector encoding the corresponding 18-mer wild-type epitope of KRAS (i.e., artPICV-KRASwt) (Group 5). Control mice were treated with formulation buffer only (Group 1). After 21 days, mice were immunized intravenously with 1×10 5 Mice were immunized intravenously with RCV FFU / dose of artLCMV-5xKRASmut-H2 (group 2), artLCMV-5xKRASmut-H1 (group 3), artLCMV-4xKRASmut (group 4), artLCMV-KRASwt (group 5) or treated with formulation buffer (group 1). On day 26, KRAS epitope-specific CD8 T cell responses were analyzed by ELISpot analysis using wild-type and mutant-specific KRAS-based peptides as stimuli. A mixture of NP-based peptides derived from LCMV and PICV was used as a control.

[0327] As shown in FIG. 21, CD8 T cell responses against two of the encoded mutated KRAS epitopes (i.e., KRAS G12C and KRAS G12R) could be detected in group 2 (2 out of 5) animals (treated with a combination of artPICV and artLCMV-based vectors encoding a 5xKRASmut-H2 epitope cassette) and group 3 (2 out of 5) mice (treated with a combination of artPICV and artLCMV-based vectors encoding a 5xKRASmut-H1 epitope cassette). In contrast, no CD8 T cell responses could be observed in group 4 animals (treated with a combination of artPICV and artLCMV-based vectors encoding a 4xKRASmut epitope cassette). Furthermore, none of the tested vector constructs encoding mutated epitopes of KRAS induced detectable CD8 T cell responses against wild-type KRAS protein.

[0328] 8.6 Vector immunogenicity To analyze the ability of vector constructs encoding mutated KRAS epitopes to induce antigen-specific immune responses, intravenous immunization was performed in mice with 1 × 10 5 RCV FFU / dose was performed with the indicated vector constructs (see FIG. 23 for study design).

[0329] On day 0, HLA-A*11 transgenic mice (i.e., CB6F1-Tg(HLA-A*1101 / H2-Kb)A11.01 mice) were inoculated at 1 × 10 5Mice were immunized intravenously with RCV FFU / dose of artLCMV-5xKRASmut-H2 (group 2), artPICV-5xKRASmut-H2 (group 4), or artLCMV- and artPICV-based vectors encoding the corresponding 18-mer wild-type epitope of KRAS (i.e., artLCMV-KRASwt and artPICV-KRASwt) (groups 3 and 5). Control mice were treated with formulation buffer only (group 1). Seven days after immunization, KRAS epitope-specific CD8 T cell responses were analyzed by ELISpot analysis using wild-type and mutant-specific KRAS-based peptides as stimuli. A mixture of LCMV-derived NP-based peptides was used as a control. PICV-derived NP-based peptides were not detected due to technical errors.

[0330] As shown in Figure 24, CD8 T cell responses against KRAS G12V were detectable in animals in group 2 (treated with artLCMV-5xKRASmut-H2) and mice in group 4 (treated with artLCMV-5xKRASmut-H2). Furthermore, none of the tested vector constructs encoding mutated epitopes of KRAS induced detectable CD8 T cell responses against the wild-type KRAS protein.

Claims

1. 1. An arenavirus particle, comprising: a. the arenavirus particle comprises an arenavirus genome comprising a nucleotide sequence encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising a mutation in KRAS; and b. At least one arenavirus open reading frame ("ORF") of the arenavirus genome is either (i) functionally inactivated or deleted, or (ii) located at a position other than the wild-type position of the at least one arenavirus ORF, or (iii) divided into two or more functional fragments, and the fragments of the at least one arenavirus ORF are located at a position other than the wild-type position of the at least one arenavirus ORF; The arenavirus particle.

2. The arenavirus particle of claim 1, (i) the KRAS mutation is at amino acid position G12, G13, A18, A59, Q61, K117, A146, or D119 of KRAS; (ii) the KRAS mutation is A18D, A59E, A59G, A59P, A59T, A59S, A59V, A146P, A146S, A146T, A146V, D119N, G12A, G12C, G12D, G12F, G12L, G12R, G12S, G12V, G13A, G13C, G13D, G13E, G13R, G13S, G13V, K117N, Q61E, Q61H, Q61K, Q61L, Q61P, Q61R, or a combination thereof; (iii) the KRAS mutation is G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61R, A146T, or a combination thereof; (iv) the KRAS mutation is one, more, or all of G13D, G12V, G12C, G12D, and G12R, and optionally the KRAS mutation is all of G13D, G12V, G12C, G12D, and G12R in any possible order; and / or (v) the nucleotide sequence encodes, from N-terminus to C-terminus, multiple fragments of mutant KRAS, each fragment containing the mutations G13D, G12V, G12C, G12D, and G12R; The arenavirus particle.

3. 2. The arenavirus particle of claim 1, wherein the arenavirus genome comprises: (i) a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 20; or (ii) a nucleotide sequence encoding an expression product whose amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19; The arenavirus particle comprising:

4. 2. The arenavirus particle of claim 1, wherein the fragment of mutant KRAS is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length, and / or the fragment of mutant KRAS is 18 amino acids in length.

5. The arenavirus particle of claim 1, (i) the region adjacent to the N-terminal mutation of the antigenic fragment is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length; (ii) the region adjacent to the N-terminal mutation of the antigenic fragment is 8 or 9 amino acids in length; (iii) the region adjacent to the mutation at the C-terminus of the antigenic fragment is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length; and / or (iv) the region adjacent to the C-terminal mutation of the antigenic fragment is 8 or 9 amino acids in length; The arenavirus particle.

6. The arenavirus particle of claim 1, (i) the nucleotide sequence encodes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antigenic fragments of mutant KRAS, each of the antigenic fragments comprising one of the mutations in the mutant KRAS protein; or (ii) the nucleotide sequence encodes five antigenic fragments of mutant KRAS, each of the antigenic fragments comprising one of the mutations of the mutant KRAS protein, and optionally, the five antigenic fragments of mutant KRAS comprise the mutations G13D, G12V, G12C, G12D, and G12R; The arenavirus particle.

7. 7. The arenavirus particle of claim 6, (i) the antigenic fragments contain the same or different mutations of the mutant KRAS protein; (ii) the antigenic fragments are fused to each other via the same or different linkers, optionally the linkers being an AAY linker (AAY), an AAA linker (AAA), a GS linker (GGSGGGGSGG) (SEQ ID NO: 42), or variants of the AAY, AAA, and GS linker sequences optimized via in silico prediction; and / or (iii) the antigenic fragments are directly fused to each other without any intervening sequences; The arenavirus particle.

8. The arenavirus particle of claim 1, The nucleotide sequence has been engineered to reduce or eliminate any CpG and TpA islands, and optionally The removal of the CpG and TpA islands is carried out in three cycles: (i) a first cycle to remove CpGs; (ii) a second cycle to remove TpA, and (iii) a third cycle of CpG removal to remove newly introduced CpGs in the second cycle; The arenavirus particle comprising:

9. 2. The arenavirus particle of claim 1, wherein the arenavirus genome comprises: (a) (i) a first S segment comprising the nucleotide sequence encoding the antigenic fragment(s) under the control of an arenavirus genome 5′ UTR, and an ORF encoding an arenavirus nucleoprotein (“NP”) under the control of an arenavirus genome 3′ UTR; and (ii) a second S segment comprising the nucleotide sequence encoding the antigenic fragment(s) under the control of the arenavirus genome 5′ UTR, and an ORF encoding an arenavirus glycoprotein (“GP”) under the control of the arenavirus genome 3′ UTR; and (iii) an L segment, or (b) (i) a first S segment comprising the nucleotide sequence of SEQ ID NO:20 under the control of the arenavirus genome 5'UTR and an ORF encoding an arenavirus nucleoprotein ("NP") under the control of the arenavirus genome 3'UTR; and (ii) a second S segment comprising the nucleotide sequence of SEQ ID NO:20 under the control of the arenavirus genome 5'UTR and an ORF encoding an arenavirus glycoprotein ("GP") under the control of the arenavirus genome 3'UTR; and (iii) an L segment, or (c) (i) a first S segment comprising the nucleotide sequence of SEQ ID NO: 21; and (ii) a second S segment comprising the nucleotide sequence of SEQ ID NO: 22; and (iii) an L segment, or (d) (i) a first S segment comprising the nucleotide sequence of SEQ ID NO: 23; and (ii) a second S segment comprising the nucleotide sequence of SEQ ID NO: 24; and (iii) an L segment, or (e) (i) a first S segment comprising an ORF encoding the arenavirus GP1 and GP2 subunits fused to a heterologous signal peptide under the control of the arenavirus genome 5' UTR, and an ORF encoding a fusion of the arenavirus GP signal peptide and a nucleotide sequence encoding the antigenic fragment(s) under the control of the arenavirus genome 3' UTR; and (ii) a second S segment comprising the nucleotide sequence encoding the antigenic fragment(s) under the control of the arenavirus genome 5′ UTR, and an ORF encoding an arenavirus nucleoprotein (“NP”) under the control of the arenavirus genome 3′ UTR; and (iii) L segment; The arenavirus particle comprising:

10. 10. The arenavirus particle of claim 9, (i) the nucleotide sequence encoding the antigenic fragment(s) on the first S segment is different from the nucleotide sequence encoding the antigenic fragment(s) on the second S segment; (ii) the nucleotide sequence encoding the antigenic fragment(s) on the first S segment is the same as the nucleotide sequence encoding the antigenic fragment(s) on the second S segment; (iii) the antigenic fragment(s) encoded on the first S segment are different from the antigenic fragment(s) encoded on the second S segment; or (iv) the antigenic fragment(s) encoded on the first S segment are the same as the antigenic fragment(s) encoded on the second S segment, optionally wherein the antigenic fragments encoded on the first S segment are the same as the antigenic fragments encoded on the second S segment but are fused to each other in a different order than the order in which the antigenic fragments encoded on the second S segment are fused to each other; The arenavirus particle.

11. The arenavirus particle of claim 1, (i) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, mutant RET, mutant APC, mutant U2AF1, mutant EGFR, mutant FBXW7, mutant SMAD4, mutant GNAS, mutant ERBB2, mutant ERBB3, mutant CDKN2A, mutant TP53, and / or mutant CTNNB1, wherein the antigenic fragment comprises the respective mutation; and / or (ii) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant PI3KCA, wherein the antigenic fragment comprises an E545K, H1047R, and / or E542K mutation; and / or (iii) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant BRAF, wherein the antigenic fragment comprises a V600E mutation; and / or (iv) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant U2AF1, wherein the antigenic fragment comprises the S34F mutation; and / or (v) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant TP53, wherein the antigenic fragment comprises G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W, and / or C277F mutations; and / or (vi) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of the BIRC family, CEACAM family, CTA family, EPH family, ERBB family, FOLR family, GAST family, GUCY2 family, IDO family, IL13RA family, KDR family, KLK family, MAGE family, MUC family, PEMT family, SDC family, SLAMF family, TERT family, TLR family, TPTE family, TYR family, WT family, and / or XBP family; The arenavirus particle.

12. The arenavirus particle of claim 1 , which is derived from lymphocytic choriomeningitis virus (LCMV) or Pichinde virus.

13. A pharmaceutical composition comprising the arenavirus particle according to any one of claims 1 to 12.

14. A set of one or more nucleic acids encoding the genome of an arenavirus particle according to any one of claims 1 to 12.

15. A host cell comprising the set of one or more nucleic acids of claim 14.

16. 16. A method for producing arenavirus particles according to any one of claims 1 to 12, comprising culturing a host cell according to claim 15, and harvesting the arenavirus particles.

17. Use of an arenavirus particle according to any of claims 1 to 12 in the manufacture of a medicament for treating a neoplastic disease in a subject in need thereof.

18. 18. The use according to claim 17, wherein the neoplastic disease is pancreatic cancer, colorectal cancer, lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer (NSCLC).

19. 18. The use according to claim 17, (i) the arenavirus genome comprises a nucleotide sequence encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising a mutation, the KRAS mutation being G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A, and the neoplastic disease is pancreatic cancer; (ii) the arenavirus genome comprises a nucleotide sequence encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising mutations, the KRAS mutations being G13D, G12V, G12C, G12D, and G12R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma; (iii) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of a mutant TP53, wherein the antigenic fragment comprises mutations, the KRAS mutations are G12D, G12R, G12V, Q61H, and / or Q61R, the TP53 mutations are R175H, R248W, G245S, R282W, R248Q, and / or R273C, and the neoplastic disease is pancreatic cancer. (iv) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant TP53 and / or mutant U2AF1, wherein the antigenic fragment comprises mutations, the KRAS mutations are G12D, G12V, G12R, Q61H, and / or Q61R, the TP53 mutations are R175H, R248W, G245S, R282W, R248Q, and / or R273C, the U2AF1 mutation is S34F, and the neoplastic disease is pancreatic cancer. (v) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant BRAF, mutant TP53, mutant FBXW7, and / or mutant PIK3CA, wherein the antigenic fragment comprises mutations, the KRAS mutation being G12D, G12V, G13D, A146T, and / or G12C, the BRAF mutation being V600E, the TP53 mutation being R175H, R273H, and / or R248W, the FBXW7 mutation being R465H, and the PIK3CA mutation being E545K and / or H1047R, and the neoplastic disease is colorectal cancer. (vi) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant BRAF, mutant TP53, mutant FBXW7, and / or mutant PIK3CA, wherein the antigenic fragment comprises mutations, the KRAS mutation being G12D, G12V, G13D, A146T, and / or G12C, the BRAF mutation being V600E, the TP53 mutation being R175H, R273H, and / or R248W, the FBXW7 mutation being R465H, and the PIK3CA mutation being E545K and / or H1047R, and the neoplastic disease is lung adenocarcinoma. (vii) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant BRAF, mutant PIK3CA, mutant EGFR, mutant TP53, and / or mutant U2AF1, wherein the antigenic fragment comprises mutations, wherein the KRAS mutation is G12C, G12D, G12R, G13D, and / or G12V, the BRAF mutation is V600E, the PIK3CA mutation is E545K, the EGFR mutation is L858R, the TP53 mutation is R175H, R273H, and / or R248W, and the U2AF1 mutation is S34F; and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. (viii) the arenavirus genome comprises a nucleotide sequence encoding an antigenic fragment of a mutant KRAS, the antigenic fragment comprising a mutation, the KRAS mutation being G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. (ix) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant TP53, mutant U2AF1, mutant PIK3CA, mutant EGFR, and / or mutant BRAF, wherein the antigenic fragment comprises mutations, wherein the KRAS mutation is G12C, G12V, G12D, and / or G12S, the TP53 mutation is R175H, the U2AF1 mutation is S34F, the EGFR mutation is L858R, L861Q, and / or E746_A750del, the PIK3CA mutation is E545K and / or E542K, and the BRAF mutation is V600E; and the neoplastic disease is lung adenocarcinoma; or (x) the arenavirus genome further comprises a nucleotide sequence encoding an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, and / or mutant RET, wherein the antigenic fragment comprises a mutation, and the neoplastic disease is non-small cell lung cancer (NSCLC); The above use.

20. 18. The use according to claim 17, wherein the medicament is for administration together with a second arenavirus particle, (i) the second arenavirus particle encodes an antigenic fragment of a mutant KRAS, the antigenic fragment comprises a mutation, and the neoplastic disease is pancreatic cancer or colorectal cancer; (ii) the second arenavirus particle encodes an antigenic fragment of a mutant KRAS, the antigenic fragment comprises a mutation, the KRAS mutation is G12D, G12V, G12R, Q61H, Q61R, G12C, G12S, and / or G12A, and the neoplastic disease is pancreatic cancer; (iii) the second arenavirus particle encodes an antigenic fragment of mutant KRAS and / or mutant TP53, the antigenic fragment comprising a mutation, the KRAS mutation being G12D, G12R, G12V, Q61H, and / or Q61R, the TP53 mutation being R175H, R248W, G245S, R282W, R248Q, and / or R273C, and the neoplastic disease is pancreatic cancer; (iv) the second arenavirus particle encodes an antigenic fragment of mutant KRAS, mutant TP53, and / or mutant U2AF1, and the antigenic fragment comprises mutations, the KRAS mutations are G12D, G12V, G12R, Q61H, and / or Q61R, the TP53 mutations are R175H, R248W, G245S, R282W, R248Q, and / or R273C, the U2AF1 mutation is S34F, and the neoplastic disease is pancreatic cancer. (v) the second arenavirus particle encodes an antigenic fragment of mutant KRAS, mutant BRAF, mutant TP53, mutant FBXW7, and / or mutant PIK3CA, wherein the antigenic fragment comprises mutations, wherein the KRAS mutation is G12D, G12V, G13D, A146T, and / or G12C, the BRAF mutation is V600E, the TP53 mutation is R175H, R273H, and / or R248W, the FBXW7 mutation is R465H and / or R465C, and the PIK3CA mutation is E545K and / or H1047R, and the neoplastic disease is colorectal cancer. (vi) the second arenavirus particle encodes an antigenic fragment of mutant KRAS, mutant BRAF, mutant TP53, mutant FBXW7, and / or mutant PIK3CA, wherein the antigenic fragment comprises mutations, the KRAS mutation being G12D, G12V, G13D, A146T, and / or G12C, the BRAF mutation being V600E, the TP53 mutation being R175H, R273H, and / or R248W, the FBXW7 mutation being R465H, and the PIK3CA mutation being E545K and / or H1047R, and the neoplastic disease is lung adenocarcinoma. (vii) the second arenavirus particle encodes an antigenic fragment of mutant KRAS, mutant BRAF, mutant PIK3CA, mutant EGFR, mutant TP53, and / or mutant U2AF1, wherein the antigenic fragment comprises mutations, wherein the KRAS mutation is G12C, G12D, G12R, G13D, and / or G12V, the BRAF mutation is V600E, the PIK3CA mutation is E545K, the EGFR mutation is L858R, the TP53 mutation is R175H, R273H, and / or R248W, and the U2AF1 mutation is S34F; and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. (viii) the second arenavirus particle encodes an antigenic fragment of a mutant KRAS, the antigenic fragment comprising a mutation, the KRAS mutation being G12D, G12V, G12C, G12R, G13D, A146T, G12S, Q61H, G12A, and / or Q61R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma. (ix) the second arenavirus particle encodes an antigenic fragment of a mutant KRAS, the antigenic fragment comprises mutations, the KRAS mutations are G13D, G12V, G12C, G12D, and G12R, and the neoplastic disease is pancreatic cancer, colorectal cancer, or lung adenocarcinoma; (x) the second arenavirus particle encodes an antigenic fragment of mutant KRAS, mutant TP53, mutant U2AF1, mutant PIK3CA, mutant EGFR, and / or mutant BRAF, wherein the antigenic fragment comprises mutations, wherein the KRAS mutation is G12C, G12V, G12D, and / or G12S, the TP53 mutation is R175H, the U2AF1 mutation is S34F, the EGFR mutation is L858R, L861Q, and / or E746_A750del, the PIK3CA mutation is E545K and / or E542K, and the BRAF mutation is V600E; and the neoplastic disease is lung adenocarcinoma. (xi) the second arenavirus particle encodes an antigenic fragment of a mutant TP53, and the antigenic fragment comprises a mutation; (xii) the second arenavirus particle encodes an antigenic fragment of mutant AKT1, mutant BRAF, mutant HER2, mutant MEK1, mutant MET, mutant NRAS, mutant PIK3CA, mutant RET, mutant APC, mutant U2AF1, mutant EGFR, mutant FBXW7, mutant SMAD4, mutant GNAS, mutant ERBB2, mutant ERBB3, mutant CDKN2A, mutant TP53, and / or mutant CTNNB1, and each of the antigenic fragments contains a mutation; (xiii) the second arenavirus particle encodes an antigenic fragment of a mutant PI3KCA, the antigenic fragment comprises a mutation, and the PI3KCA mutation is E545K, H1047R, and / or E542K; (xiv) the second arenavirus particle encodes an antigenic fragment of a mutant BRAF, the antigenic fragment comprises a mutation, and the BRAF mutation is V600E. (xv) the second arenavirus particle encodes an antigenic fragment of a mutant U2AF1, the antigenic fragment comprises a mutation, and the U2AF1 mutation is S34F; (xvi) the second arenavirus particle encodes an antigenic fragment of a mutant TP53, the antigenic fragment comprises a mutation, and the TP53 mutation is G245S, Y220C, R248Q, R282W, H179R, V157F, R273C, R213L, R273H, R273L, R175H, R158L, R196P, R248W, and / or C277F; (xvii) the second arenavirus particle encodes an antigenic fragment of the BIRC family, CEACAM family, CTA family, EPH family, ERBB family, FOLR family, GAST family, GUCY2 family, IDO family, IL13RA family, KDR family, KLK family, MAGE family, MUC family, PEMT family, SDC family, SLAMF family, TERT family, TLR family, TPTE family, TYR family, WT family, and / or XBP family; (xviii) the second arenavirus particle comprises an arenavirus genome comprising the nucleotide sequence of SEQ ID NOs: 21 and 22; or (xix) the second arenavirus particle comprises an arenavirus genome comprising the nucleotide sequence of SEQ ID NOs: 23 and 24; The above use.

21. 18. The use according to claim 17, (i) the neoplastic disease is a solid tumor, and administration of the medicament results in an increase in the concentration of T cells within the solid tumor; and / or (ii) the neoplastic disease is a solid tumor, and the arenavirus particles are administered via intratumoral injection; The above use.

22. 18. The use according to claim 17, wherein the neoplastic disease is acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphocytic leukemia; acute myeloid leukemia; acute myeloid leukemia (adult / child); adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratoid / rhabdoid tumor; basal cell carcinoma; bile duct cancer, extrahepatic (cholangiocarcinoma); bladder cancer; bone tumor, osteosarcoma / malignant fibrous histiocytoma; brain cancer (adult / child); brain tumor, cerebellar astrocytoma (adult Human / Child); Brain tumor, cerebral astrocytoma / malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumor; Brain tumor, optic tract and hypothalamic glioma; Brain stem glioma; Breast cancer; Bronchial adenoma / carcinoid; Bronchial tumor; Burkitt lymphoma; Cancer of childhood; Carcinoid gastrointestinal tumor; Carcinoid tumor; Adult carcinoma, unknown primary site; Carcinoma of unknown primary site; Central nervous system embryonal tumor; Central nervous system lymphoma, primary; Cervical cancer; Pediatric adrenocortical carcinoma; Pediatric cancer; Pediatric cerebral astrocytoma; Chordoma, childhood; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Chronic myeloproliferative disorder; Colon cancer; Colorectal cancer; Craniopharyngioma; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Emphysema; Endometrial cancer; Ependymoblastoma; Ependymoma; Esophageal cancer; Ewing's sarcoma in Ewing's family of tumors; Extracranial germ cell tumor; Extragonadal germ cell tumor; Extrahepatic bile duct cancer; Gallbladder cancer; Gastric (stomach) cancer; Gastric carcinoid; Gastrointestinal carcinoid tumor; Digestive ductal stromal tumors; germ cell tumors: extracranial, extragonadal, or ovarian gestational trophoblastic tumors; gestational trophoblastic tumors, site of unknown origin; gliomas; gliomas of the brain stem; gliomas, childhood visual pathways, and hypothalamus; hairy cell leukemia; head and neck cancer; cardiac cancer; hepatocellular (liver) carcinoma; Hodgkin's lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway gliomas; intraocular melanoma; islet cell carcinoma (pancreatic islet); Kaposi's sarcoma; kidney cancer (renal cell carcinoma); Langerhans cell histiocytosis; laryngeal cancer; lip and oral cavity cancer; liposarcoma; Liver cancer (primary); lung cancer, non-small cell; lung cancer, small cell; lymphoma, primary central nervous system; macroglobulinemia, Waldenstrom; male breast cancer; malignant fibrous histiocytoma / osteosarcoma of bone; medulloblastoma; medulloepithelioma; melanoma; melanoma, intraocular (eye); Merkel cell carcinoma; Merkel cell skin cancer; mesothelioma; Mesothelioma, adult malignancy; metastatic squamous cell carcinoma of the neck with unknown primary; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell neoplasm; Mycosis fungoides, myelodysplastic syndrome; myelodysplastic / myeloproliferative disorders; myeloid leukemia, chronic; myeloid leukemia, adult acute; myeloid leukemia, childhood acute; myeloma, multiple (cancer of the bone marrow); myeloproliferative disorders, chronic; nasal cavity and paranasal sinus cancer; nasopharyngeal carcinoma; neuroblastoma, non-small cell lung cancer; non-Hodgkin's lymphoma; oligodendroglioma; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytosis of bone tumor; ovarian cancer; ovarian epithelial carcinoma (superficial epithelial and stromal tumors); ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; pancreatic carcinoma, islet cell; papilloma; paranasal sinus and nasal cancer; parathyroid carcinoma; penile cancer; pharyngeal cancer; pheochromocytoma; pineal astrocytoma; pineal germ cell tumor; intermediately differentiated pineal parenchymal tumor; pineoblastoma and supratentorial primitive neuroectodermal tumor; pituitary tumor; pituitary adenoma; plasma cell neoplasm / multiple myeloma; breast Membrane pulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell carcinoma; Airway cancer involving the NUT gene on chromosome 15; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family tumor; Sezary syndrome; Skin cancer (melanoma); Skin cancer (non-melanoma); Small cell lung cancer; Small intestine cancer, soft tissue sarcoma; Soft tissue sarcoma; Spinal cord tumor tumor; squamous cell carcinoma; squamous cell cervical carcinoma of occult primary, metastatic; gastric (stomach) cancer; supratentorial primitive neuroectodermal tumor; T-cell lymphoma, skin (mycosis fungoides and Sézary syndrome); testicular cancer; throat cancer; thymoma; thymoma and thymic carcinoma; thyroid cancer; pediatric thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; vulvar cancer; or Wilms' tumor.

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