Shared antigens

Abstract

To provide compositions that include specific antigen-encoding nucleic acid sequences and / or specific antigen peptides, and also provide nucleotides, cells, and methods associated with the compositions, including their use as vaccines.SOLUTION: The present invention provides a composition comprising an antigen expression system, intended for the delivery of the antigen expression system, wherein the antigen expression system comprises one or more vectors, and the one or more vectors comprise a specific sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Nos. 62 / 675,649, filed May 23, 2018, and 62 / 675,559, filed May 23, 2018, each of which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy was created on May 22, 2019, is named GSO-019_SL.txt, and is 6,925,585 bytes in size. [Background technology]

[0003] background Therapeutic vaccines based on tumor-specific antigens hold great promise as the next generation of personalized cancer immunotherapy. 1~3 For example, cancers with high mutational burden, such as non-small cell lung cancer (NSCLC) and melanoma, are particularly promising targets for such therapies due to their relatively high likelihood of generating neoantigens. 4,5 Early evidence suggests that neoantigen-based vaccination induces T cell responses. 6 , Cell therapy targeting neoantigens can induce tumor regression in selected patients 7 It has been shown that:

[0004] One question regarding neoantigen vaccine design is which of the many coding mutations present in the target tumor can give rise to the "best" therapeutic neoantigen (e.g., an antigen capable of eliciting antitumor immunity and causing tumor regression).

[0005] Early methods have been proposed that incorporate mutation-based analysis using next-generation sequencing, RNA gene expression, and prediction of MHC binding affinity of neoantigen peptides. 8 However, these proposed methods involve many steps other than gene expression and MHC binding (e.g., TAP transport, proteasomal cleavage, and / or TCR recognition). 9 The entire epitope generation process cannot be modeled, and therefore existing methods tend to suffer from low positive predictive value (PPV).

[0006] Indeed, analyses of peptides presented by tumor cells conducted by several groups have shown that less than 5% of peptides predicted to be presented using gene expression and MHC binding affinity are found on tumor surface MHC. 10,11 This poor correlation between binding prediction and MHC presentation is further highlighted by the recent finding that mutation number alone does not improve prediction accuracy for binding-restricted neoantigens in response to checkpoint inhibitors. 12 .

[0007] Such low positive predictive value (PPV) of existing methods for predicting presentation presents a problem in the design of neoantigen-based vaccines. If a vaccine is designed using a low PPV prediction, it is unlikely that most patients will receive a therapeutic neoantigen, and even fewer will receive multiple neoantigens (even if all presented peptides are assumed to be immunogenic). Therefore, neoantigen vaccination using current methods is unlikely to be successful in a significant number of tumor-bearing subjects.

[0008] Furthermore, previous approaches have only used cis-acting mutations to generate candidate neoantigens, including splicing factor mutations that occur in multiple tumor types and lead to aberrant splicing of many genes. 13, and additional sources of nascent ORFs, including mutations that create or remove protease cleavage sites, were not considered in most cases.

[0009] Finally, standard approaches to tumor genome and transcriptome analysis may miss somatic mutations that give rise to candidate neoantigens due to suboptimal conditions in library construction, exome and transcriptome capture, sequencing, or data analysis. Similarly, standard tumor analysis approaches may erroneously promote sequence artifacts or germline polymorphisms as neoantigens, which can lead to inefficient utilization of vaccine doses or the risk of autoimmunity, respectively.

[0010] In addition to the challenges of current neoantigen prediction methods, existing vector systems that can be used to deliver neoantigens in humans, many of which are of human origin, also present certain challenges. For example, many humans have pre-existing immunity to human viruses as a result of previous natural exposure, and this immunity can pose a significant obstacle to the use of recombinant human viruses to deliver neoantigens for cancer therapy.

[0011] Furthermore, targeting antigens shared among cancer patients, including targeting both mutated neoantigens and non-mutated tumor antigens (e.g., inappropriately expressed tumor antigens), is a highly promising vaccine strategy. Challenges in shared antigen vaccine strategies include at least those mentioned above. Summary of the Invention

[0012] overview Disclosed herein is a composition for delivering an antigen expression system, comprising an antigen expression system, wherein the antigen expression system comprises one or more vectors, wherein the one or more vectors comprise: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; the vector backbone comprising (b) an antigen cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) at least one tumor-specific MHC class I antigen-encoding nucleic acid sequence, (A) an MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 57 to 29357; (B) optionally a 5' linker sequence, and (C) Optional 3' linker sequence the at least one tumor-specific MHC class I antigen-encoding nucleic acid sequence comprising: and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II antigen-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; The antigen cassette comprising:

[0013] Also disclosed herein is a composition for delivering an antigen expression system, comprising an antigen expression system, wherein the antigen expression system comprises one or more vectors, and the one or more vectors comprise: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; the vector backbone comprising (b) an antigen cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumor-specific MHC class I antigen-encoding nucleic acid sequences linearly linked to each other; (A) KRAS_G12A MHC class I epitope-encoding nucleic acid sequence encoding MHC class I comprising the sequence of SEQ ID NO: 19831; (B) KRAS_G12C MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope comprising the sequence of SEQ ID NO: 14954; (C) a KRAS_G12D MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19749 and 19865; and (D) a KRAS_G12V MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19976, 19979, 19779, 11495, and 19974; Including, each of the tumor-specific MHC class I antigen-encoding nucleic acid sequences comprises a class I epitope-encoding nucleic acid sequence, and optionally each MHC I epitope-encoding nucleic acid sequence encodes an MHC class I epitope selected from the group consisting of SEQ ID NOs: 57 to 29357; each of the tumor-specific MHC class I antigen-encoding nucleic acid sequences (A) optionally, a 5' linker sequence, and (B) Optionally, a 3' linker sequence. The tumor-specific MHC class I antigen-encoding nucleic acid sequence comprising and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II antigen-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; The antigen cassette comprising:

[0014] Also disclosed herein is a composition for delivering an antigen expression system, comprising an antigen expression system, wherein the antigen expression system comprises one or more vectors, wherein the one or more vectors comprise: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; the vector backbone comprising (b) an antigen cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) at least 20 tumor-specific MHC class I antigen-encoding nucleic acid sequences linearly linked to each other, (A) KRAS_G12A MHC class I epitope-encoding nucleic acid sequence encoding MHC class I comprising the sequence of SEQ ID NO: 19831; (B) KRAS_G12C MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope comprising the sequence of SEQ ID NO: 14954; (C) a KRAS_G12D MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19749 and 19865; and (D) a KRAS_G12V MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19976, 19979, 19779, 11495, and 19974; (E) KRAS_G13D MHC class I epitope-encoding nucleic acid sequence; (F) KRAS_Q61K MHC class I epitope-encoding nucleic acid sequence; (G) TP53_R249M MHC class I epitope-encoding nucleic acid sequence; (H) CTNNB1_S45P MHC class I epitope-encoding nucleic acid sequence; (I) CTNNB1_S45F MHC class I epitope-encoding nucleic acid sequence; (J)ERBB2_Y772_A775dup MHC class I epitope-encoding nucleic acid sequence, (K) KRAS_Q61R MHC class I epitope-encoding nucleic acid sequence; (L) CTNNB1_T41A MHC class I epitope-encoding nucleic acid sequence; (M) TP53_K132N MHC class I epitope-encoding nucleic acid sequence, (N) KRAS_Q61L MHC class I epitope-encoding nucleic acid sequence; (O) TP53_R213L MHC class I epitope-encoding nucleic acid sequence; (P)BRAF_G466V MHC class I epitope-encoding nucleic acid sequence, (Q)KRAS_Q61H MHC class I epitope-encoding nucleic acid sequence, (R)CTNNB1_S37F MHC class I epitope-encoding nucleic acid sequence, (S)TP53_S127Y MHC class I epitope-encoding nucleic acid sequence, (T)TP53_K132E MHC class I epitope-encoding nucleic acid sequence, (U)KRAS_G12C MHC class I epitope-encoding nucleic acid sequence Including, each of the tumor-specific MHC class I antigen-encoding nucleic acid sequences (A) optionally, a 5' linker sequence, and (B) Optionally, a 3' linker sequence. The tumor-specific MHC class I antigen-encoding nucleic acid sequence comprising and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II antigen-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; The antigen cassette comprising:

[0015] In some embodiments, the at least one antigen-encoding nucleic acid sequence does not comprise an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19749 and 19865.

[0016] Also disclosed herein is a composition for delivering an antigen expression system, comprising an antigen expression system, wherein the antigen expression system comprises one or more vectors, and the one or more vectors comprise: (a) a vector backbone comprising a chimpanzee adenovirus vector, optionally a ChAdV68 vector, or an alphavirus vector, optionally a Venezuelan equine encephalitis virus vector; (b) an antigen cassette integrated between a 26S promoter nucleotide sequence and a poly(A) sequence, (i) at least one antigen-encoding nucleic acid sequence, (I) at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumor-specific MHC class I antigen-encoding nucleic acid sequences linearly linked to each other, each of which comprises: (A) an MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope having a length of 7 to 15 amino acids, wherein at least one of the MHC class I epitopes is selected from the group consisting of SEQ ID NOs: 57 to 29357; (B) a 5' linker sequence that encodes the native N-terminal amino acid sequence of the MHC class I epitope and encodes a peptide that is at least 3 amino acids in length; (C) a 3' linker sequence encoding the native C-terminal amino acid sequence of the MHC class I epitope and encoding a peptide that is at least 3 amino acids in length. Including, the antigen cassette is operably linked to the 26S promoter nucleotide sequence, each of the MHC class I antigen-encoding nucleic acid sequences encodes a polypeptide having a length of 13 to 25 amino acids, and the 3' end of each MHC class I antigen-encoding nucleic acid sequence is linked to the 5' end of the subsequent MHC class I antigen-encoding nucleic acid sequence, except for the last MHC class I antigen-encoding nucleic acid sequence in the antigen cassette; the MHC class I antigen-encoding nucleic acid sequence and the at least one antigen-encoding nucleic acid sequence comprising: (ii) at least two MHC class II antigen-encoding nucleic acid sequences, (I) PADRE MHC class II sequence (SEQ ID NO: 48); (II) tetanus toxoid MHC class II sequence (SEQ ID NO: 46); (III) a first nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56) connecting the PADRE MHC class II sequence and the tetanus toxoid MHC class II sequence; (IV) a second nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56) that links the 5' ends of the at least two MHC class II antigen-encoding nucleic acid sequences to the tumor-specific MHC class I antigen-encoding nucleic acid sequence; (V) optionally, a third nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56) at the 3' ends of the at least two MHC class II antigen-encoding nucleic acid sequences; and the at least two MHC class II antigen-encoding nucleic acid sequences comprising: The antigen cassette comprising:

[0017] Also provided herein is a method of assessing a subject with cancer, comprising: a) 1) whether the subject has an HLA allele that is predicted or known to present the antigen included in the antigen-based vaccine; and below: 1) whether the subject's tumor expresses a gene associated with said antigen, and optionally whether said gene is aberrantly expressed compared to normal cells or tissues; 2) whether the subject's tumor has a mutation associated with the antigen one or both of a step of determining or having determined b) determining, or having already determined, from the results of (a) that the subject expresses the HLA allele and the subject's tumor expresses the gene and / or the subject's tumor has the mutation, that the subject is a candidate for treatment with the antigen-based vaccine; The antigen comprises at least one MHC class I epitope sequence selected from the group consisting of SEQ ID NOs: 57 to 29357. The process; c) optionally administering or having administered said antigen-based vaccine to said subject, said antigen-based vaccine comprising: 1) said at least one MHC class I epitope; or 2) an MHC class I epitope-encoding nucleic acid sequence encoding said at least one MHC class I epitope; The process comprising: Also disclosed is the method, comprising:

[0018] Also provided herein is a method of assessing a subject with cancer, comprising: a) the object is 1) expressing the A0301 HLA allele and the subject's tumor harboring a KRAS_G12A mutation; 2) expressing the A0201 HLA allele and the subject's tumor harboring the KRAS_G12C mutation; 3) expresses the C0802 HLA allele or the A1101 HLA allele, and the subject's tumor has a KRAS_G12D mutation; or 4) The subject expresses the A0301 HLA allele, the A1101 HLA allele, the A3101 HLA allele, the C0102 HLA allele, or the A0302 HLA allele, and the subject's tumor has the KRAS_G12V mutation. a step of determining or having determined b) From the results of (a), the subject is 1) if the subject expresses the A0301 allele and the subject's tumor has the KRAS_G12A mutation, 2) expressing the A0201 allele and the subject's tumor having the KRAS_G12C mutation; 3) expressing the C0802 HLA allele or the A1101 HLA allele and the subject's tumor harboring the KRAS_G12D mutation; or 4) The subject expresses the A0301 HLA allele, the A1101 HLA allele, the A3101 HLA allele, the C0102 HLA allele, or the A0302 HLA allele, and the subject's tumor has a KRAS_G12V mutation. determining, or having determined, that the subject is a candidate for treatment with the antigen-based vaccine; c) optionally administering or having administered said antigen-based vaccine to said subject, said antigen-based vaccine comprising: 1) at least one MHC class I epitope comprising the KRAS_G12A mutation, the KRAS_G12C mutation, the KRAS_G12D mutation, or the KRAS_G12V mutation, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding at least one MHC class I epitope comprising the KRAS_G12A mutation, the KRAS_G12C mutation, the KRAS_G12AD mutation, or the KRAS_G12V mutation, The process includes the steps of: Also disclosed is the method, comprising:

[0019] In some embodiments, steps (a) and / or (b) comprise obtaining a dataset from a third party that processed a sample from the subject. In some embodiments, step (a) comprises obtaining a sample from the subject and assaying the sample using a method selected from the group consisting of exome sequencing, targeted exome sequencing, transcriptome sequencing, Sanger sequencing, PCR-based genotyping assays, mass spectrometry-based methods, microarrays, nanostrings, ISH, and IHC. In some embodiments, the sample comprises a tumor sample, a normal tissue sample, or the tumor sample and the normal tissue sample. In some embodiments, the sample is selected from tissue, body fluid, blood, tumor biopsy, spinal fluid, and needle aspirate. In some embodiments, the gene is selected from the group consisting of any of the genes found in Table 34. In some embodiments, the gene is selected from the group consisting of any of the genes found in Table 32. In some embodiments, the cancer is selected from the group consisting of lung cancer, microsatellite-stable colon cancer, and pancreatic cancer. In some embodiments, the HLA allele has an HLA frequency of at least 5%. In some embodiments, the at least one MHC class I epitope is presented by an HLA allele on cells associated with the subject's tumor. In some embodiments, the antigen-based vaccine comprises an antigen expression system. In some embodiments, the antigen expression system comprises any one of the antigen expression systems disclosed herein. In some embodiments, the antigen-based vaccine comprises any one of the pharmaceutical compositions disclosed herein.

[0020] The present specification also discloses a method of treating a subject with cancer, comprising administering to the subject an antigen-based vaccine directed against the subject, wherein the antigen-based vaccine comprises 1) at least one MHC class I epitope, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding the at least one MHC class I epitope, wherein the at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs: 57-29357. In some embodiments, the at least one MHC class I antigen-encoding nucleic acid sequence is derived from a tumor of the subject with cancer. In some embodiments, the at least one MHC class I antigen-encoding nucleic acid sequence is not derived from a tumor of the subject with cancer.

[0021] Also disclosed herein are methods for inducing an immune response in a subject, comprising administering to the subject an antigen-based vaccine directed against the subject, wherein the antigen-based vaccine comprises 1) at least one MHC class I epitope, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding the at least one MHC class I epitope, wherein the at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs: 57-29357. In some embodiments, the subject expresses at least one HLA allele predicted or known to present the at least one MHC class I epitope sequence. In some embodiments, the subject expresses at least one HLA allele predicted or known to present the at least one MHC class I epitope sequence, wherein the at least one MHC class I epitope sequence comprises a mutation selected from the group consisting of the mutations set forth in Table 34. In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present said at least one MHC class I epitope sequence, and said at least one MHC class I epitope sequence comprises a mutation selected from the group consisting of the mutations set forth in Table 32.

[0022] The present specification also discloses a method for inducing an immune response in a subject, comprising administering to the subject an antigen-based vaccine directed against the subject, wherein the antigen-based vaccine comprises 1) at least one MHC class I epitope, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding the at least one MHC class I epitope, wherein the at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs: 57 to 29357, and the subject expresses at least one HLA allele that is predicted or known to present the at least one MHC class I epitope sequence.

[0023] The present specification also discloses a method for inducing an immune response in a subject, comprising administering to the subject an antigen-based vaccine directed against the subject, wherein the antigen-based vaccine comprises 1) at least one MHC class I epitope, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding the at least one MHC class I epitope, wherein the at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs: 57-29357, wherein the subject expresses at least one HLA allele predicted or known to present the at least one MHC class I epitope sequence, wherein the at least one MHC class I epitope sequence comprises a mutation selected from the group consisting of mutations set forth in Table 34, and wherein the subject expresses at least one HLA allele set forth in Table 34 associated with a corresponding mutation set forth in Table 34 (e.g., KRAS_G13D and C0802).

[0024] The present specification also provides a method for inducing an immune response in a subject, comprising administering to the subject an antigen-based vaccine, wherein the antigen-based vaccine comprises 1) at least one MHC class I epitope, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding the at least one MHC class I epitope, wherein the at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs: 57-29357, the subject expresses at least one HLA allele predicted or known to present the at least one MHC class I epitope sequence, and the at least one MHC class I epitope sequence comprises a mutation selected from the group consisting of mutations set forth in Table 32. In some embodiments, the antigen-based vaccine comprises an antigen expression system. In some embodiments, the antigen expression system comprises any one of the antigen expression systems described herein. In some embodiments, the antigen-based vaccine comprises any one of the pharmaceutical compositions described herein.

[0025] In some embodiments, the ordered sequence of each element of the neoantigen cassette is, from 5' to 3': Pa-(L5b-Nc-L3d)X-(G5e-Uf)Y-G3g It is described by a formula including During the ceremony, P comprises the second promoter nucleotide sequence, where a=0 or 1; N comprises one of the MHC class I epitope-encoding nucleic acid sequences, where c=1; L5 comprises the 5' linker sequence, where b=0 or 1; L3 comprises the 3' linker sequence, where d=0 or 1; G5 comprises one of said at least one nucleic acid sequence encoding a GPGPG amino acid linker (SEQ ID NO: 56), where e=0 or 1; G3 comprises one of said at least one nucleic acid sequence encoding a GPGPG amino acid linker (SEQ ID NO: 56), where g=0 or 1; U comprises one of the at least one MHC class II antigen-encoding nucleic acid sequences, where f=1; X=1 to 400, where for each X, the corresponding Nc is an epitope-encoding nucleic acid sequence; Y=0, 1, or 2, where for each Y, the corresponding Uf is an antigen-encoding nucleic acid sequence. In some embodiments, for each X, the corresponding Nc is a different MHC class I epitope-encoding nucleic acid sequence. In some embodiments, for each Y, the corresponding Uf is a different MHC class II antigen-encoding nucleic acid sequence.

[0026] In some embodiments, a=0, b=1, d=1, e=1, g=1, h=1, X=20, Y=2, the at least one promoter nucleotide sequence is a single 26S promoter nucleotide sequence provided by the backbone, the at least one polyadenylation poly(A) sequence is a poly(A) sequence of at least 100 consecutive A nucleotides provided by the backbone (SEQ ID NO: 29358), each N encodes an MHC class I epitope 7-15 amino acids in length, L5 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the MHC I epitope, the 5' linker sequence encodes a peptide that is at least 3 amino acids in length, and L3 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the MHC I epitope, the 5' linker sequence encodes a peptide that is at least 3 amino acids in length, and L4 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the MHC I epitope, the 5' linker sequence encodes a peptide that is at least 3 amino acids in length, and L5 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the MHC I epitope, the 5' linker sequence encodes a peptide that is at least 3 amino acids in length, and L6 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the MHC I epitope, the 5' linker sequence encodes a peptide that is at least 3 amino acids in length, and L7 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the MHC I epitope, the 5' linker sequence encodes a peptide that is at least 3 amino acids in length, and L8 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the MHC I epitope, the 5' linker sequence encodes a peptide that is at least 3 amino acids in length, and L9 is a native 5' linker and U is a natural 3' linker sequence encoding a natural terminal nucleic acid sequence of an MHC Class I epitope, wherein the 3' linker sequence encodes a peptide that is at least 3 amino acids in length; U is each of a PADRE class II sequence and a tetanus toxoid MHC Class II sequence; the vector backbone comprises a chimpanzee adenovirus vector, optionally a ChAdV68 vector, or an alphavirus vector, optionally a Venezuelan equine encephalitis virus vector; and each of the MHC Class I neoantigen-encoding nucleic acid sequences encodes a polypeptide that is 13 to 25 amino acids in length.

[0027] In some embodiments, the neoantigen cassette is incorporated between at least one promoter nucleotide sequence and at least one poly(A) sequence. In some embodiments, the at least one promoter nucleotide sequence is operably linked to the neoantigen-encoding nucleic acid sequence.

[0028] In some embodiments, the one or more vectors comprise one or more positive-strand RNA vectors. In some embodiments, the one or more positive-strand RNA vectors comprise a 5'7-methylguanosine (m7g) cap. In some embodiments, the one or more positive-strand RNA vectors are generated by in vitro transcription. In some embodiments, the one or more vectors are autonomously replicating in mammalian cells.

[0029] In some embodiments, the backbone comprises at least one nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. In some embodiments, the backbone comprises at least one nucleotide sequence of Venezuelan equine encephalitis virus. In some embodiments, the backbone comprises at least a sequence for nonstructural protein-mediated amplification, a 26S promoter sequence, a poly(A) sequence, a nonstructural protein 1 (nsP1) gene, a nsP2 gene, a nsP3 gene, and a nsP4 gene encoded by a nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. In some embodiments, the backbone comprises at least a sequence for nonstructural protein-mediated amplification, a 26S promoter sequence, and a poly(A) sequence encoded by a nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. In some embodiments, the sequence for nonstructural protein-mediated amplification is selected from the group consisting of an alphavirus 5' UTR, a 51 nt CSE, a 24 nt CSE, a 26S subgenomic promoter sequence, a 19 nt CSE, an alphavirus 3' UTR, or a combination thereof.

[0030] In some embodiments, the backbone does not encode the structural virion proteins capsid E2 and E1. In some embodiments, the neoantigen cassette is inserted in place of the structural virion proteins within the nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus.

[0031] In some embodiments, the Venezuelan equine encephalitis virus (VEE) comprises the TC-83 strain. In some embodiments, the Venezuelan equine encephalitis virus comprises the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, the Venezuelan equine encephalitis virus comprises the sequence of SEQ ID NO:3 or SEQ ID NO:5 further comprising a deletion between base pairs 7544 and 11175. In some embodiments, the backbone is the sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the neoantigen cassette is inserted to replace the deletion between base pairs 7544 and 11175 set forth in the sequence of SEQ ID NO:3 or SEQ ID NO:5.

[0032] In some embodiments, insertion of the neoantigen cassette results in transcription of a polycistronic RNA comprising the nsP1-4 genes and at least one antigen-encoding nucleic acid sequence, wherein the nsP1-4 genes and the at least one antigen-encoding nucleic acid sequence are in separate open reading frames.

[0033] In some embodiments, at least one promoter nucleotide sequence is a natural 26S promoter nucleotide sequence encoded by the backbone. In some embodiments, at least one promoter nucleotide sequence is an exogenous RNA promoter. In some embodiments, the second promoter nucleotide sequence is a 26S promoter nucleotide sequence. In some embodiments, the second promoter nucleotide sequence comprises multiple 26S promoter nucleotide sequences, each of which causes transcription of one or more of the separate open reading frames.

[0034] In some embodiments, the adenoviral vector is a chimpanzee adenovirus (ChAd) vector, optionally a C68 vector. In some embodiments, the adenoviral vector comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the adenoviral vector comprises the sequence set forth in SEQ ID NO: 1, except that at least one gene selected from the group consisting of chimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence set forth in SEQ ID NO: 1 is completely deleted or functionally deleted from the sequence, and optionally, the sequence is completely deleted or functionally deleted from (1) E1A and E1B, (2) E1A, E1B, and E3, or (3) E1A, E1B, E3, and E4 of the sequence set forth in SEQ ID NO: 1. In some aspects, the adenoviral vector comprises a gene or regulatory sequence derived from the sequence of SEQ ID NO: 1, and optionally the gene is selected from the group consisting of the inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of chimpanzee adenovirus of the sequence set forth in SEQ ID NO: 1.

[0035] In some embodiments, the neoantigen cassette is inserted into the adenoviral vector in the E1 region, the E3 region, and / or any deleted AdV region that allows for integration of the neoantigen cassette.

[0036] In some embodiments, at least one promoter sequence of the adenoviral vector is inducible. In some embodiments, at least one promoter sequence of the adenoviral vector is non-inducible. In some embodiments, at least one promoter sequence of the adenoviral vector is a CMV, SV40, EF-1, RSV, PGK or EBV promoter sequence.

[0037] In some embodiments, the adenoviral neoantigen cassette further comprises at least one polyA sequence operably linked to at least one of the multiple neoantigen-encoding nucleic acid sequences, optionally located 3' to at least one sequence within the multiple neoantigen-encoding nucleic acid sequences.

[0038] In some embodiments, the adenoviral vector is generated from one of a first generation, second generation, or helper-dependent adenoviral vector.

[0039] In some embodiments, the adenoviral vector comprises one or more deletions at base pairs 577 to 3407 of the sequence set forth in SEQ ID NO:1, and optionally, the adenoviral vector further comprises one or more deletions between base pairs 27141 to 32022 or at base pairs 27816 to 31332. In some embodiments, the adenoviral vector further comprises one or more deletions at base pairs 3957 to 10346, base pairs 21787 to 23370, and base pairs 33486 to 36193 of the sequence set forth in SEQ ID NO:1.

[0040] In some embodiments, the one or more neoantigen expression vectors are each at least 300 nt in size. In some embodiments, the one or more neoantigen expression vectors are each at least 1 kb in size. In some embodiments, the one or more neoantigen expression vectors are each 2 kb in size. In some embodiments, the one or more neoantigen expression vectors are each less than 5 kb in size.

[0041] In some embodiments, at least one of the at least one neoantigen-encoding nucleic acid sequence encodes a polypeptide sequence or a portion thereof that is presented by MHC class I on tumor cells. In some embodiments, each antigen-encoding nucleic acid sequence within each antigen-encoding nucleic acid sequence is directly linked to each other. In some embodiments, at least one of the at least one antigen-encoding nucleic acid sequence is linked to a different antigen-encoding nucleic acid sequence by a nucleic acid sequence encoding a linker. In some embodiments, the linker links two MHC class I sequences or one MHC class I sequence to one MHC class II sequence. In some embodiments, the linker is (1) at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive glycine residues in length (SEQ ID NO: 29359), (2) at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive alanine residues in length (SEQ ID NO: 29360), (3) two arginine residues (RR), (4) alanine, alanine, tyrosine (AAY), (5) a mammalian proteasome. and (6) one or more naturally occurring sequences flanking the antigen from the cognate protein of origin and having a length of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues. In some embodiments, the linker links two MHC class II sequences or one MHC class II sequence with one MHC class I sequence. In some embodiments, the linker comprises the sequence GPGPG (SEQ ID NO: 56).

[0042] In some embodiments, at least one of the at least one antigen-encoding nucleic acid sequences is operably or directly linked to a separate or contiguous sequence that enhances expression, stability, cellular trafficking, processing and presentation, and / or immunogenicity of the at least one antigen-encoding nucleic acid sequence. In some embodiments, the separate or contiguous sequence comprises at least one of a ubiquitin sequence, a ubiquitin sequence modified to enhance proteasome targeting (e.g., a ubiquitin sequence comprising a Gly to Ala substitution at position 76), an immunoglobulin signal sequence (e.g., IgK), a major histocompatibility class I sequence, a lysosomal-associated membrane protein (LAMP)-1, a human dendritic cell lysosomal-associated membrane protein, and a major histocompatibility class II sequence, optionally wherein the ubiquitin sequence modified to enhance proteasome targeting is A76.

[0043] In some embodiments, at least one of the at least one neoantigen-encoding nucleic acid sequence encodes a polypeptide sequence or portion thereof that has increased binding affinity for its corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence after translation. In some embodiments, at least one of the at least one neoantigen-encoding nucleic acid sequence in the plurality of neoantigen-encoding nucleic acid sequences encodes a polypeptide sequence or portion thereof that has increased binding stability for its corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence after translation. In some embodiments, at least one of the at least one neoantigen-encoding nucleic acid sequence in the plurality of neoantigen-encoding nucleic acid sequences encodes a polypeptide sequence or portion thereof that has increased likelihood of presentation on its corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence after translation.

[0044] In some embodiments, the at least one mutation comprises a point mutation, a frameshift mutation, a non-frameshift mutation, a deletion mutation, an insertion mutation, a splice variant, a genomic rearrangement, or a proteasome-generated splice antigen.

[0045] In some embodiments, the tumor is selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, bladder cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, adult acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0046] In some embodiments, the at least one neoantigen-encoding nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleic acid sequences. In some embodiments, the at least one neoantigen-encoding nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 nucleic acid sequences.

[0047] In some embodiments, the at least one neoantigen-encoding nucleic acid sequence comprises at least 2-400 nucleic acid sequences, and at least two of the neoantigen-encoding nucleic acid sequences encode a polypeptide sequence or a portion thereof that is presented by MHC class I on the surface of tumor cells. In some embodiments, at least two of the neoantigen-encoding nucleic acid sequences encode a polypeptide sequence or a portion thereof that is presented by MHC class I on the surface of tumor cells. In some embodiments, when administered to a subject and translated, at least one of the neoantigens encoded by the at least one neoantigen-encoding nucleic acid sequence is presented on antigen-presenting cells, resulting in an immune response that targets at least one of the neoantigens on the surface of tumor cells. In some embodiments, when administered to a subject and translated, at least one of the neoantigens encoded by the at least one neoantigen-encoding nucleic acid sequence is presented on antigen-presenting cells, resulting in an immune response that targets at least one of the neoantigens on the surface of tumor cells, and optionally, expression of each of the at least one neoantigen-encoding nucleic acid sequence is driven by the at least one promoter nucleotide sequence.

[0048] In some embodiments, each MHC class I neoantigen-encoding nucleic acid sequence encodes a polypeptide sequence that is 8 to 35 amino acids in length, optionally 9 to 17, 9 to 25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length.

[0049] In some embodiments, at least one MHC class II antigen-encoding nucleic acid sequence is present. In some embodiments, at least one MHC class II antigen-encoding nucleic acid sequence is present and comprises at least one MHC class II neoantigen-encoding nucleic acid sequence that includes at least one mutation that renders the at least one MHC class II neoantigen-encoding nucleic acid sequence different from a corresponding wild-type parent nucleic acid sequence. In some embodiments, the at least one MHC class II antigen-encoding nucleic acid sequence is 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length. In some embodiments, at least one MHC class II antigen-encoding nucleic acid sequence is present and comprises at least one universal MHC class II antigen-encoding nucleic acid sequence, optionally wherein the at least one universal sequence comprises at least one of tetanus toxoid and PADRE.

[0050] In some embodiments, at least one of the promoter nucleotide sequences or the second promoter nucleotide sequence is inducible. In some embodiments, at least one of the promoter nucleotide sequences or the second promoter nucleotide sequence is non-inducible.

[0051] In some embodiments, at least one poly(A) sequence comprises a poly(A) sequence naturally occurring in the backbone. In some embodiments, at least one poly(A) sequence comprises a poly(A) sequence exogenous to the backbone. In some embodiments, at least one poly(A) sequence is operably linked to at least one of the at least one antigen-encoding nucleic acid sequences. In some embodiments, at least one poly(A) sequence is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 consecutive A nucleotides (SEQ ID NO: 29361). In some embodiments, at least one poly(A) sequence is at least 100 consecutive A nucleotides (SEQ ID NO: 29358).

[0052] In some embodiments, the neoantigen cassette further comprises at least one of an intron sequence, a woodchuck hepatitis virus post-transcriptional regulator (WPRE) sequence, an internal ribosome entry sequence (IRES) sequence, a nucleotide sequence encoding a 2A self-cleaving peptide sequence, a nucleotide sequence encoding a furin cleavage site, or a sequence within the 5' or 3' non-coding region known to improve mRNA nuclear export, stability, or translation efficiency, operably linked to at least one of the at least one antigen-encoding nucleic acid sequence.

[0053] In some embodiments, the neoantigen cassette further comprises a reporter gene, including but not limited to, green fluorescent protein (GFP), a GFP variant, secreted alkaline phosphatase, luciferase, a luciferase variant, or a detectable peptide or epitope, hi some embodiments, the detectable peptide or epitope is selected from the group consisting of an HA tag, a Flag tag, a His tag, or a V5 tag.

[0054] In some embodiments, the one or more vectors further comprise one or more nucleic acid sequences encoding at least one immunomodulator. In some embodiments, the immunomodulator is an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a Fab fragment, a Fab' fragment, a single-chain Fv (scFv), a single-domain antibody (sdAb) (e.g., camelid antibody domains) in a monospecific or multispecific form linked together, or a full-length single-chain antibody (e.g., a full-length IgG in which the heavy and light chains are linked by a flexible linker). In some embodiments, the heavy and light chain sequences of the antibody are contiguous sequences separated by a self-cleaving sequence such as 2A or an IRES, or the heavy and light chain sequences of the antibody are linked by a flexible linker such as consecutive glycine residues.

[0055] In some embodiments, the immunomodulator is a cytokine, hi some embodiments, the cytokine is at least one of IL-2, IL-7, IL-12, IL-15, or IL-21, or each of their variants.

[0056] In some embodiments, the at least one MHC class I neoantigen-encoding nucleic acid sequence is (a) obtaining at least one of exome, transcriptome, or whole genome tumor nucleotide sequencing data from a tumor, wherein the tumor nucleotide sequencing data is used to obtain data representing the peptide sequences of each of a set of neoantigens; (b) inputting the peptide sequence of each neoantigen into a presentation model to generate a set of numerical likelihoods that each neoantigen is presented by one or more MHC alleles on the surface of tumor cells of the tumor, wherein the set of numerical likelihoods was determined based at least on the received mass spectrometry data; (c) selecting a subset of the set of neoantigens based on said set of numerical likelihoods to generate a set of selected neoantigens used to generate said at least one MHC class I neoantigen-encoding nucleic acid sequence; The selection is made by performing

[0057] In some embodiments, each of the at least one MHC class I neoantigen-encoding nucleic acid sequence comprises: (a) obtaining at least one of exome, transcriptome, or whole genome tumor nucleotide sequencing data from a tumor, wherein the tumor nucleotide sequencing data is used to obtain data representing the peptide sequences of each of a set of neoantigens; (b) inputting the peptide sequence of each neoantigen into a presentation model to generate a set of numerical likelihoods that each neoantigen is presented by one or more MHC alleles on the surface of tumor cells of the tumor, wherein the set of numerical likelihoods was determined based at least on the received mass spectrometry data; (c) selecting a subset of the set of neoantigens based on said set of numerical likelihoods to generate a set of selected neoantigens used to generate said at least one MHC class I neoantigen-encoding nucleic acid sequence; The selection is made by performing

[0058] In some embodiments, the number of sets of selected neoantigens is between 2 and 20.

[0059] In some embodiments, the presentation model describes the dependency between the presence of a pair of a particular one of the MHC alleles with a particular amino acid at a particular position in a peptide sequence and the likelihood of presentation of such a peptide sequence containing a particular amino acid at a particular position on the surface of a tumor cell by that particular one of the MHC alleles of the pair.

[0060] In some aspects, selecting the set of selected neoantigens comprises selecting neoantigens that have an increased likelihood of being presented on the surface of tumor cells compared to neoantigens not selected based on a presentation model. In some embodiments, the selected antigens are validated to be presented by one or more specific HLA alleles. In some aspects, selecting the set of selected neoantigens comprises selecting neoantigens that have an increased likelihood of inducing a tumor-specific immune response in a subject compared to neoantigens not selected based on a presentation model. In some aspects, selecting the set of selected neoantigens comprises selecting neoantigens that have an increased likelihood of being presented to naive T cells by professional antigen-presenting cells (APCs) compared to neoantigens not selected based on a presentation model, optionally the APCs are dendritic cells (DCs). In some aspects, selecting the set of selected neoantigens comprises selecting neoantigens that have a decreased likelihood of being inhibited by central or peripheral tolerance compared to neoantigens not selected based on a presentation model. In some embodiments, selecting a set of selected neoantigens comprises selecting neoantigens that have a reduced likelihood of inducing an autoimmune response against normal tissue in a subject compared to neoantigens that are not selected based on the presentation model. In some embodiments, the nucleotide sequencing data of exome or transcriptome is obtained by sequencing tumor tissue. In some embodiments, sequencing is next-generation sequencing (NGS) or any massively parallel sequencing approach.

[0061] In some embodiments, the neoantigen cassette comprises a junctional epitope sequence formed by adjacent sequences within the neoantigen cassette. In some embodiments, at least one or each junctional epitope sequence has an affinity for MHC greater than 500 nM. In some embodiments, each junctional epitope sequence is non-self. In some embodiments, each MHC class I epitope is predicted or verified to be capable of presentation by at least one HLA allele present in at least 5% of the population. In some embodiments, each MHC class I epitope is predicted or verified to be capable of presentation by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA frequency of at least 0.01% in the population. In some embodiments, each MHC class I epitope is predicted or verified to be capable of presentation by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA frequency of at least 0.1% in the population. In some embodiments, the neoantigen cassette does not encode a non-therapeutic MHC class I or class II epitope nucleic acid sequence comprising a wild-type nucleic acid sequence after translation, and the non-therapeutic epitope is predicted to be presented on the MHC allele of interest. In some embodiments, the predicted non-therapeutic MHC class I or class II epitope sequence is a junctional epitope sequence formed by adjacent sequences within the neoantigen cassette. In some embodiments, the prediction is based on a presentation likelihood generated by inputting the sequence of the non-therapeutic epitope into a presentation model. In some embodiments, the order of at least one antigen-encoding nucleic acid sequence in a neoantigen cassette is determined by a series of steps including: (a) generating a set of candidate neoantigen cassette sequences corresponding to various orders of the at least one antigen-encoding nucleic acid sequence; (b) determining a presentation score for each candidate neoantigen cassette sequence based on the presentation of non-therapeutic epitopes within the candidate neoantigen cassette sequence; and (c) selecting candidate cassette sequences associated with a presentation score below a predetermined threshold as neoantigen cassette sequences for a neoantigen vaccine.

[0062] In some embodiments, any of the above compositions further comprises a nanoparticulate delivery vehicle. In some embodiments, the nanoparticulate delivery vehicle can be a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable amino lipid. In some embodiments, the ionizable amino lipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule. In some embodiments, the nanoparticulate delivery vehicle encapsulates a neoantigen expression system.

[0063] In some embodiments, any of the above compositions further comprise a plurality of LNPs, the LNPs comprising a neoantigen expression system, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of the LNPs, wherein at least about 95% of the LNPs have either a non-lamellar morphology or are electron-dense.

[0064] In some embodiments, the non-cationic lipid is a mixture of (1) a phospholipid and (2) cholesterol or a cholesterol derivative.

[0065] In some embodiments, the conjugated lipid that inhibits aggregation of LNPs is a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate is selected from the group consisting of PEG-diacylglycerol (PEG-DAG) conjugates, PEG-dialkyloxypropyl (PEG-DAA) conjugates, PEG-phospholipid conjugates, PEG-ceramide (PEG-Cer) conjugates, and mixtures thereof. In some embodiments, the PEG-DAA conjugate is PEG-didecyloxypropyl (C 10 ) conjugate, PEG-dilauryloxypropyl (C 12 ) conjugate, PEG-dimyristyloxypropyl (C 14 ) conjugate, PEG-dipalmityloxypropyl (C 16 ) conjugate, PEG-distearyloxypropyl (C 18 ) complexes, and mixtures thereof.

[0066] In some embodiments, the neoantigen expression system is fully encapsulated in the LNP.

[0067] In some embodiments, the non-lamellar morphology of the LNPs is an inverted hexagonal (H II ) or cubic phase structure.

[0068] In some embodiments, the cationic lipids comprise about 10 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the cationic lipids comprise about 20 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the cationic lipids comprise about 20 mol% to about 40 mol% of the total lipids present in the LNP.

[0069] In some embodiments, non-cationic lipids comprise about 10 mol% to about 60 mol% of the total lipids present in the LNP. In some embodiments, cationic lipids comprise about 20 mol% to about 55 mol% of the total lipids present in the LNP. In some embodiments, cationic lipids comprise about 25 mol% to about 50 mol% of the total lipids present in the LNP.

[0070] In some embodiments, complex lipids comprise about 0.5 mol% to about 20 mol% of the total lipids present in the LNP. In some embodiments, complex lipids comprise about 2 mol% to about 20 mol% of the total lipids present in the LNP. In some embodiments, complex lipids comprise about 1.5 mol% to about 18 mol% of the total lipids present in the LNP.

[0071] In some embodiments, greater than 95% of the LNPs have a non-lamellar morphology. In some embodiments, greater than 95% of the LNPs are electron-dense.

[0072] In some embodiments, any of the above compositions further comprises a plurality of LNPs, the LNPs comprising a cationic lipid comprising 50 mol% to 65 mol% of the total lipids present in the LNP; a complex lipid that inhibits aggregation of the LNP comprising 0.5 mol% to 2 mol% of the total lipids present in the LNP; and a non-cationic lipid comprising either a mixture of phospholipids and cholesterol or a derivative thereof, wherein the phospholipids comprise 4 mol% to 10 mol% of the total lipids present in the LNP and the cholesterol or a derivative thereof comprises 30 mol% to 40 mol% of the total lipids present in the LNP; a mixture of phospholipids and cholesterol or a derivative thereof, wherein the phospholipids comprise 3 mol% to 15 mol% of the total lipids present in the LNP and the cholesterol or a derivative thereof comprises 30 mol% to 40 mol% of the total lipids present in the LNP; or a mixture of phospholipids and cholesterol or a derivative thereof that is 49.5 mol% or less of the total lipids present in the LNP, wherein the cholesterol or a derivative thereof comprises 30 mol% to 40 mol% of the total lipids present in the LNP.

[0073] In some embodiments, any of the above compositions further comprise a plurality of LNPs, the LNPs comprising cationic lipids comprising 50 mol% to 85 mol% of the total lipids present in the LNPs, complex lipids that inhibit aggregation of the LNPs comprising 0.5 mol% to 2 mol% of the total lipids present in the LNPs, and non-cationic lipids that comprise 13 mol% to 49.5 mol% of the total lipids present in the LNPs.

[0074] In some embodiments, the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.

[0075] In some embodiments, the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate comprises a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or a mixture thereof. In some embodiments, the PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate, a PEG-distearoyloxypropyl (PEG-DSA) conjugate, or a mixture thereof. In some embodiments, the PEG portion of the conjugate has an average molecular weight of about 2000 daltons.

[0076] In some embodiments, complex lipids comprise between 1 mol% and 2 mol% of the total lipids present in the LNP.

[0077] In some embodiments, the LNP has the structure of Formula I: TIFF2024178211000002.tif47128[where L 1 and L 2 are each independently -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) x -, -SS-, -C(=0)S-, -SC(=0)-, -R a C(=0)-, -C(=0)R a -, -R a C(=0)R a -, -OC(=0)R a -, -R a C(=0)0- or a direct bond, and G 1 is Ci to C2 alkylene, -(C=0)-, -0(C=0)-, -SC(=0)-, -R a C(=0)-, or a direct bond, -C(=0)-, -(C=0)0-, -C(=0)S-, -C(=0)R a - or a direct bond, G is a Ci-C6 alkylene, and R a is H or C1~C 12 alkyl, and R 1a and R 1b is, in each case independently: (a) H or C1-C 12alkyl, or (b) R 1a is H or C1~C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 2a and R 2b is, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 2a is H or C1~C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 3a and R 3b is, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 3a is H or C1~C 12 alkyl, and R 3b forms a carbon-carbon double bond with the carbon atom to which it is bonded, and with the adjacent R and the carbon atom to which it is bonded, and R 4a and R 4b is, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 4a is H or C1~C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 5 and R 6 are each independently H or methyl, and R 7 is C4~C 20 alkyl, and R 8 and R 9 are C1 to C 12 alkyl or R 8 and R 9together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle; a, b, c, and d are each independently an integer of 1 to 24; and x is 0, 1, or 2. or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0078] In some embodiments, the LNP has the structure of Formula II: TIFF2024178211000003.tif36128[where L 1 and L 2 are each independently -0(C=0)-, -(C=0)0-, or a carbon-carbon double bond; R 1a and R 1b is, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 1a is H or C1~C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 2a and R 2b is, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 2a is H or C1~C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 3a and R 3b is, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 3a is H or C1~C 12 alkyl, and R 3b together with the carbon atom to which it is attached, the adjacent R 3b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 4a and R 4bIn each case, (a) H or C1~C 12 alkyl, or (b) R 4a is H or C1~C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 5 and R 6 are each independently methyl or cycloalkyl, and R7, at each occurrence, is independently H or C1-C 12 alkyl, and R 8 and R 9 are each independently unsubstituted C1 to C 12 alkyl or R 8 and R 9 and together with the nitrogen atom to which they are attached, form a 5-, 6-, or 7-membered heterocycle containing one nitrogen atom, a and d each independently represent an integer of 0 to 24, b and c each independently represent an integer of 1 to 24, and e is 1 or 2, with the proviso that R 1a , R 2a , R 3a , or R 4a At least one of C1 to C 12 alkyl or L 1 or L 2 At least one of is -0(C=0)- or -(C=0)0-, and R 1a and R 1b is not isopropyl when a is 6 and is not n-butyl when a is 8] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0079] In some embodiments, any of the above compositions further comprise one or more excipients, including a neutral lipid, a steroid, and a polymer-bound lipid. In some embodiments, the neutral lipid comprises at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the neutral lipid is DSPC.

[0080] In some embodiments, the molar ratio of compound to neutral lipid ranges from about 2:1 to about 8:1.

[0081] In some embodiments, the steroid is cholesterol, hi some embodiments, the molar ratio of the compound to cholesterol ranges from about 2:1 to 1:1.

[0082] In some embodiments, the polymer-bound lipid is a PEGylated lipid. In some embodiments, the molar ratio of the compound to the PEGylated lipid ranges from about 100:1 to about 25:1. In some embodiments, the PEGylated lipid is PEG-DAG, PEG polyethylene (PEG-PE), PEG-succinoyl-diacylglycerol (PEG-S-DAG), PEG-cer, or PEG dialkyloxypropylcarbamate. In some embodiments, the PEGylated lipid has the following structure III: TIFF2024178211000004.tif25128[where, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain having 10 to 30 carbon atoms, said alkyl chain being optionally interrupted by one or more ester bonds, and z has an average value in the range of 30 to 60. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In some embodiments, R 10 and R 11 are each independently a linear saturated alkyl chain having 12 to 16 carbon atoms. In some embodiments, the average z is about 45. Start here

[0083] In some embodiments, the LNPs self-assemble into non-bilayer structures when mixed with polyanionic nucleic acids. In some embodiments, the non-bilayer structures have diameters of 60 nm to 120 nm. In some embodiments, the non-bilayer structures have diameters of about 70 nm, about 80 nm, about 90 nm, or about 100 nm. In some embodiments, the nanoparticulate delivery vehicles have diameters of about 100 nm.

[0084] Also disclosed herein are pharmaceutical compositions comprising any of the compositions disclosed herein (such as the alphavirus-based or ChAd-based vectors disclosed herein) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises an adjuvant. In some embodiments, the pharmaceutical composition further comprises an immunomodulator. In some embodiments, the immunomodulator is an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof.

[0085] Also disclosed herein is an isolated nucleotide sequence or set of isolated nucleotide sequences comprising a neoantigen cassette according to any of the preceding composition claims and one or more elements derived from the sequence of SEQ ID NO:3 or SEQ ID NO:5, wherein optionally, the one or more elements are selected from the group consisting of a sequence required for nonstructural protein-mediated amplification, a 26S promoter nucleotide sequence, a poly(A) sequence, and the nsP1-4 genes of the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, and optionally, the nucleotide sequences are cDNA. In some embodiments, the sequence or set of isolated nucleotide sequences comprises a neoantigen cassette disclosed herein inserted at position 7544 of the sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the isolated nucleotide sequence further comprises a nucleotide sequence for a T7 or SP6 RNA polymerase promoter located 5' to the one or more elements derived from the sequence of SEQ ID NO:3 or SEQ ID NO:5, and optionally, one or more restriction sites located 3' to the poly(A) sequence. In some embodiments, the neoantigen cassette disclosed herein is inserted at position 7563 of SEQ ID NO: 8 or SEQ ID NO: 9. In another embodiment, the sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9 further comprises an additional adenine nucleotide inserted at position 17.

[0086] Also disclosed herein is an isolated nucleotide sequence comprising a neoantigen cassette as disclosed herein and at least one promoter as disclosed herein. In some embodiments, the isolated nucleotide sequence further comprises a ChAd-based gene. In some embodiments, the ChAd-based gene is derived from the sequence of SEQ ID NO: 1, optionally, the gene is selected from the group consisting of the ITR, E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of a chimpanzee adenovirus of the sequence set forth in SEQ ID NO: 1, and optionally, the nucleotide sequence is cDNA.

[0087] Also provided herein is an isolated cell comprising an isolated nucleotide sequence disclosed herein, optionally wherein the cell is a BHK-21, CHO, HEK293 or a variant thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a cell.

[0088] Also disclosed herein are vectors that comprise the isolated nucleotide sequences disclosed herein.

[0089] Also disclosed herein are kits comprising a vector or composition disclosed herein and instructions for use.

[0090] Also disclosed herein are methods for treating a subject with cancer, comprising administering to the subject a vector disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the at least one MHC class I neoantigen-encoding nucleic acid sequence is derived from a tumor of the subject with cancer. In some embodiments, the at least one MHC class I neoantigen-encoding nucleic acid sequence is not derived from a tumor of the subject with cancer.

[0091] Also disclosed herein are methods for inducing an immune response in a subject, comprising administering to the subject any of the compositions, vectors, or pharmaceutical compositions described herein. In some embodiments, the subject expresses at least one HLA allele predicted or known to present an MHC class I epitope. In some embodiments, the subject expresses at least one HLA allele predicted or known to present an MHC class I epitope, wherein the MHC class I epitope comprises a mutation selected from the group consisting of the mutations set forth in Table 34. In some embodiments, the subject expresses at least one HLA allele predicted or known to present an MHC class I epitope, wherein the MHC class I epitope comprises a mutation selected from the group consisting of the mutations set forth in Table 32.

[0092] In some embodiments, the vector or composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV).

[0093] In some embodiments, the methods described herein further comprise administering one or more immunomodulatory agents, which may optionally be administered before, simultaneously with, or after administration of the composition or pharmaceutical composition. In some embodiments, the one or more immunomodulatory agents are selected from the group consisting of an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof. In some embodiments, the immunomodulatory agent is administered intravenously (IV), intramuscularly (IM), intradermally (ID), or subcutaneously (SC). In some embodiments, subcutaneous administration is near the site of administration of the composition or pharmaceutical composition or in proximity to the draining lymph nodes of the one or more vectors or compositions.

[0094] In some embodiments, the methods described herein further comprise administering a second vaccine composition to the subject. In some embodiments, the second vaccine composition is administered before administration of the composition or pharmaceutical composition described above. In some embodiments, the second vaccine composition is administered after administration of the composition or pharmaceutical composition described above. In some embodiments, the second vaccine composition is the same as the composition or pharmaceutical composition described above. In some embodiments, the second vaccine composition is different from the composition or pharmaceutical composition described above. In some embodiments, the second vaccine composition comprises a chimpanzee adenoviral vector encoding at least one antigen-encoding nucleic acid sequence. In some embodiments, the at least one antigen-encoding nucleic acid sequence encoded by the chimpanzee adenoviral vector is the same as at least one antigen-encoding nucleic acid sequence of any of the compositions or vectors described above.

[0095] Also disclosed herein are methods for producing one or more vectors of any of the above compositions, the method comprising: obtaining a linearized DNA sequence comprising the backbone and the neoantigen cassette; in vitro transcribing the linearized DNA sequence by adding the linearized DNA sequence to an in vitro transcription reaction containing all components necessary for transcribing the linearized DNA sequence into RNA, optionally further comprising adding the m7g cap to the resulting RNA in vitro; and isolating the one or more vectors from the in vitro transcription reaction. In some embodiments, the linearized DNA sequence is generated by linearizing a DNA plasmid sequence or by amplification using PCR. In some embodiments, the DNA plasmid sequence is generated using one of bacterial recombination, or total genome DNA synthesis, or total genome DNA synthesis with amplification of synthetic DNA in bacterial cells. In some embodiments, isolating one or more vectors from the in vitro transcription reaction comprises one or more of phenol-chloroform extraction, purification using a silica column, or similar RNA purification methods.

[0096] Also disclosed herein are methods of making any of the compositions disclosed herein, comprising providing components of a nanoparticulate delivery vehicle, providing a neoantigen expression system, and providing conditions sufficient for the nanoparticulate delivery vehicle and the neoantigen expression system to produce the composition for delivering the neoantigen expression system. In some aspects, such conditions are provided by microfluidic mixing.

[0097] Also disclosed herein is a method for producing the adenoviral vector disclosed herein, comprising obtaining a plasmid sequence comprising the at least one promoter sequence and the neoantigen cassette, transfecting the plasmid sequence into one or more host cells, and isolating the adenoviral vector from the one or more host cells.

[0098] In some embodiments, the isolating comprises lysing the host cells to obtain a cell lysate comprising the adenoviral vector, and purifying the adenoviral vector from the cell lysate.

[0099] In some embodiments, the plasmid sequence is generated using one of bacterial recombination, or total genomic DNA synthesis, or total genomic DNA synthesis with amplification of synthetic DNA in bacterial cells. In some embodiments, the one or more host cells are at least one of CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, and AE1-2a cells. In some embodiments, purifying the adenoviral vector from a cell lysate involves one or more of chromatographic separation, centrifugation, virus precipitation, and filtration. [The present invention 1001] A composition for delivering an antigen expression system, comprising the antigen expression system, the antigen expression system comprises one or more vectors; the composition wherein the one or more vectors comprise: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; the vector backbone comprising (b) an antigen cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) at least one tumor-specific MHC class I antigen-encoding nucleic acid sequence, (A) an MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 57 to 29357; (B) optionally a 5' linker sequence, and (C) Optional 3' linker sequence the at least one tumor-specific MHC class I antigen-encoding nucleic acid sequence comprising: and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II antigen-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; The antigen cassette comprising: [The present invention 1002] A composition for delivering an antigen expression system, comprising the antigen expression system, the antigen expression system comprises one or more vectors; the composition wherein the one or more vectors comprise: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; the vector backbone comprising (b) an antigen cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumor-specific MHC class I antigen-encoding nucleic acid sequences linearly linked to each other; (A) KRAS_G12A MHC class I epitope-encoding nucleic acid sequence encoding MHC class I comprising the sequence of SEQ ID NO: 19831; (B) KRAS_G12C MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope comprising the sequence of SEQ ID NO: 14954; (C) a KRAS_G12D MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19749 and 19865; and (D) A KRAS_G12V MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19976, 19979, 19779, 11495, and 19974. Including, each of the tumor-specific MHC class I antigen-encoding nucleic acid sequences comprises a class I epitope-encoding nucleic acid sequence, and optionally each MHC I epitope-encoding nucleic acid sequence encodes an MHC class I epitope selected from the group consisting of SEQ ID NOs: 57 to 29357; each of the tumor-specific MHC class I antigen-encoding nucleic acid sequences (A) optionally, a 5' linker sequence, and (B) Optionally, a 3' linker sequence. The tumor-specific MHC class I antigen-encoding nucleic acid sequence comprising and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II antigen-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; The antigen cassette comprising: [The present invention 1003] A composition for delivering an antigen expression system, comprising the antigen expression system, the antigen expression system comprises one or more vectors; the composition wherein the one or more vectors comprise: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; the vector backbone comprising (b) an antigen cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) at least 20 tumor-specific MHC class I antigen-encoding nucleic acid sequences linearly linked to each other, (A) KRAS_G12A MHC class I epitope-encoding nucleic acid sequence encoding MHC class I comprising the sequence of SEQ ID NO: 19831; (B) KRAS_G12C MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope comprising the sequence of SEQ ID NO: 14954; (C) a KRAS_G12D MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19749 and 19865; and (D) a KRAS_G12V MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19976, 19979, 19779, 11495, and 19974; (E) KRAS_G13D MHC class I epitope-encoding nucleic acid sequence; (F) KRAS_Q61K MHC class I epitope-encoding nucleic acid sequence; (G) TP53_R249M MHC class I epitope-encoding nucleic acid sequence; (H) CTNNB1_S45P MHC class I epitope-encoding nucleic acid sequence; (I) CTNNB1_S45F MHC class I epitope-encoding nucleic acid sequence; (J)ERBB2_Y772_A775dup MHC class I epitope-encoding nucleic acid sequence, (K) KRAS_Q61R MHC class I epitope-encoding nucleic acid sequence; (L) CTNNB1_T41A MHC class I epitope-encoding nucleic acid sequence; (M) TP53_K132N MHC class I epitope-encoding nucleic acid sequence, (N) KRAS_Q61L MHC class I epitope-encoding nucleic acid sequence; (O) TP53_R213L MHC class I epitope-encoding nucleic acid sequence; (P)BRAF_G466V MHC class I epitope-encoding nucleic acid sequence, (Q)KRAS_Q61H MHC class I epitope-encoding nucleic acid sequence, (R)CTNNB1_S37F MHC class I epitope-encoding nucleic acid sequence, (S)TP53_S127Y MHC class I epitope-encoding nucleic acid sequence, (T)TP53_K132E MHC class I epitope-encoding nucleic acid sequence, (U)KRAS_G12C MHC class I epitope-encoding nucleic acid sequence Including, each of the tumor-specific MHC class I antigen-encoding nucleic acid sequences (A) optionally, a 5' linker sequence, and (B) Optionally, a 3' linker sequence. The tumor-specific MHC class I antigen-encoding nucleic acid sequence comprising and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II antigen-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; The antigen cassette comprising: [The present invention 1004] A composition for delivering an antigen expression system, comprising the antigen expression system, the antigen expression system comprises one or more vectors; the composition wherein the one or more vectors comprise: (a) a vector backbone comprising a chimpanzee adenovirus vector, optionally a ChAdV68 vector, or an alphavirus vector, optionally a Venezuelan equine encephalitis virus vector; (b) an antigen cassette integrated between a 26S promoter nucleotide sequence and a poly(A) sequence, (i) at least one antigen-encoding nucleic acid sequence, (I) at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumor-specific MHC class I antigen-encoding nucleic acid sequences linearly linked to each other, each of which comprises: (A) an MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope having a length of 7 to 15 amino acids, wherein at least one of the MHC class I epitopes is selected from the group consisting of SEQ ID NOs: 57 to 29357; (B) a 5' linker sequence that encodes the native N-terminal amino acid sequence of the MHC class I epitope and encodes a peptide that is at least 3 amino acids in length; (C) a 3' linker sequence encoding the native C-terminal amino acid sequence of the MHC class I epitope and encoding a peptide that is at least 3 amino acids in length. Including, the antigen cassette is operably linked to the 26S promoter nucleotide sequence, each of the MHC class I antigen-encoding nucleic acid sequences encodes a polypeptide having a length of 13 to 25 amino acids, and the 3' end of each MHC class I antigen-encoding nucleic acid sequence is linked to the 5' end of the subsequent MHC class I antigen-encoding nucleic acid sequence, except for the last MHC class I antigen-encoding nucleic acid sequence in the antigen cassette; the MHC class I antigen-encoding nucleic acid sequence and the at least one antigen-encoding nucleic acid sequence comprising: (ii) at least two MHC class II antigen-encoding nucleic acid sequences, (I) PADRE MHC class II sequence (SEQ ID NO: 48); (II) tetanus toxoid MHC class II sequence (SEQ ID NO: 46); (III) a first nucleic acid sequence encoding a GPGPG amino acid linker sequence connecting the PADRE MHC class II sequence and the tetanus toxoid MHC class II sequence; (IV) a second nucleic acid sequence encoding a GPGPG amino acid linker sequence connecting the 5' ends of the at least two MHC class II antigen-encoding nucleic acid sequences to the tumor-specific MHC class I antigen-encoding nucleic acid sequence; (V) optionally, a third nucleic acid sequence encoding a GPGPG amino acid linker sequence at the 3' end of the at least two MHC class II antigen-encoding nucleic acid sequences; and and the at least two MHC class II antigen-encoding nucleic acid sequences comprising: The antigen cassette comprising: [The present invention 1005] The ordered sequence of each element of the antigen cassette, from 5' to 3', is: P a -(L5 b -N c -L3 d ) X -(G5 e -U f ) Y -G3 g It is described by a formula including During the ceremony, P comprises the second promoter nucleotide sequence, where a=0 or 1; N comprises one of the MHC class I epitope-encoding nucleic acid sequences, where c=1; L5 comprises the 5' linker sequence, where b=0 or 1; L3 comprises the 3' linker sequence, where d=0 or 1; G5 comprises one of said at least one nucleic acid sequence encoding a GPGPG amino acid linker, where e=0 or 1; G3 comprises one of said at least one nucleic acid sequence encoding a GPGPG amino acid linker, where g=0 or 1; U comprises one of the at least one MHC class II antigen-encoding nucleic acid sequences, where f=1; X = 1 to 400, where for each X, the corresponding N c is an epitope-encoding nucleic acid sequence, Y=0, 1, or 2, where for each Y, the corresponding U f is an antigen-encoding nucleic acid sequence, The composition of any one of 1001 to 1003 of the present invention. [The present invention 1006] For each X, the corresponding N c are different MHC class I epitope-encoding nucleic acid sequences. [The present invention 1007] For each Y, the corresponding U f are different MHC class II antigen-encoding nucleic acid sequences. [The present invention 1008] a=0, b=1, d=1, e=1, g=1, h=1, X=20, Y=2, the at least one promoter nucleotide sequence is a single 26S promoter nucleotide sequence provided by the backbone; the at least one polyadenylated poly(A) sequence is a poly(A) sequence of at least 100 consecutive A nucleotides provided by the backbone, each N encodes an MHC class I epitope 7 to 15 amino acids in length; L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of the MHC I epitope, the 5' linker sequence encoding a peptide that is at least 3 amino acids in length; L3 is a natural 3' linker sequence encoding the natural terminal nucleic acid sequence of the MHC I epitope, the 3' linker sequence encoding a peptide that is at least 3 amino acids in length; U is each of the PADRE class II sequence and the tetanus toxoid MHC class II sequence; the vector backbone comprises a chimpanzee adenovirus vector, optionally a ChAdV68 vector, or an alphavirus vector, optionally a Venezuelan equine encephalitis virus vector; Each of the MHC class I antigen-encoding nucleic acid sequences encodes a polypeptide having a length of 13 to 25 amino acids. The composition of any one of 1005 to 1007 of the present invention. [The present invention 1009] Any of the preceding compositions of the present invention further comprising a nanoparticulate delivery vehicle. [The present invention 1010] 1009. The composition of claim 10, wherein said nanoparticulate delivery vehicle is a lipid nanoparticle (LNP). [The present invention 1011] 10. The composition of claim 10, wherein the LNP comprises an ionizable amino lipid. [The present invention 1012] 1011. The composition of claim 10, wherein said ionizable amino lipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule. [The present invention 1013] 1013. The composition of any one of claims 1009 to 1012, wherein said nanoparticulate delivery vehicle encapsulates said antigen expression system. [The present invention 1014] The composition of any one of claims 1001 to 1003, 1005 to 1007, or 1009 to 1013, wherein the antigen cassette is incorporated between the at least one promoter nucleotide sequence and the at least one poly(A) sequence. [The present invention 1015] The composition of any of claims 1001 to 1003, 1005 to 1007, or 1009 to 1014, wherein said at least one promoter nucleotide sequence is operably linked to said antigen-encoding nucleic acid sequence. [The present invention 1016] The composition of any one of claims 1001 to 1003, 1005 to 1007, or 1009 to 1015, wherein the one or more vectors comprise one or more positive strand RNA vectors. [The present invention 1017] 1016. The composition of claim 1016, wherein said one or more positive strand RNA vectors comprise a 5'7-methylguanosine (m7g) cap. [The present invention 1018] The composition of any one of claims 1016 to 1017, wherein the one or more positive-strand RNA vectors are produced by in vitro transcription. [The present invention 1019] The composition of any one of claims 1001 to 1003, 1005 to 1007, or 1009 to 1018, wherein said one or more vectors are autonomously replicating in mammalian cells. [The present invention 1020] 1009-1019. Any of the compositions of claims 1001-1003, 1005-1007, or 1009-1019, wherein the backbone comprises at least one nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. [The present invention 1021] 1009-1019, wherein the backbone comprises at least one nucleotide sequence of a Venezuelan equine encephalitis virus. [The present invention 1022] The composition of claim 1020 or 1021, wherein the backbone comprises at least a sequence for nonstructural protein-mediated amplification, a 26S promoter sequence, a poly(A) sequence, a nonstructural protein 1 (nsP1) gene, a nsP2 gene, a nsP3 gene, and a nsP4 gene encoded by a nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. [The present invention 1023] The composition of claim 1020 or 1021, wherein the backbone comprises at least a sequence for nonstructural protein-mediated amplification encoded by a nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus, a 26S promoter sequence, and a poly(A) sequence. [The present invention 1024] The composition of invention 1022 or 1023, wherein the sequence for non-structural protein-mediated amplification is selected from the group consisting of an alphavirus 5'UTR, a 51 nt CSE, a 24 nt CSE, a 26S subgenomic promoter sequence, a 19 nt CSE, an alphavirus 3'UTR, or a combination thereof. [The present invention 1025] 1025. The composition of any of claims 1022 to 1024, wherein said backbone does not encode the structural virion proteins capsid E2 and E1. [The present invention 1026] 1025. The composition of the present invention, wherein the antigen cassette is inserted in place of a structural virion protein within the nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. [The present invention 1027] The composition of claim 1020 or 1021, wherein said Venezuelan equine encephalitis virus comprises the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5. [The present invention 1028] 1020 or 1021, wherein the Venezuelan equine encephalitis virus comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 5, further comprising a deletion between base pairs 7544 and 11175. [The present invention 1029] 1028. The composition of claim 1028, wherein said backbone comprises the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7. [The present invention 1030] 1028 or 1029, wherein said antigen cassette is inserted at position 7544 to replace said deletion between base pairs 7544 and 11175 set forth in the sequence of SEQ ID NO:3 or SEQ ID NO:5. [The present invention 1031] Any of the compositions of inventions 1026 to 1030, wherein insertion of the antigen cassette results in transcription of a polycistronic RNA comprising the nsP1-4 genes and the at least one antigen-encoding nucleic acid sequence, and the nsP1-4 genes and the at least one antigen-encoding nucleic acid sequence are in separate open reading frames. [The present invention 1032] 1009-1019, wherein the backbone comprises at least one nucleotide sequence of a chimpanzee adenoviral vector. [The present invention 1033] The composition of claim 1032, wherein the chimpanzee adenovirus vector is a ChAdV68 vector. [The present invention 1034] 10. The composition of any of claims 1001 to 1003, 1005 to 1007, or 1009 to 1033, wherein said at least one promoter nucleotide sequence is a native 26S promoter nucleotide sequence encoded by said backbone. [This invention 1035] The composition of any one of claims 1001 to 1003, 1005 to 1007, or 1009 to 1033, wherein said at least one promoter nucleotide sequence is an exogenous RNA promoter. [The present invention 1036] The composition of any one of inventions 1001 to 1003, 1005 to 1007, or 1009 to 1035, wherein the second promoter nucleotide sequence is a 26S promoter nucleotide sequence. [This invention 1037] Any of the compositions of claims 1001 to 1003, 1005 to 1007, or 1009 to 1035, wherein the second promoter nucleotide sequence comprises multiple 26S promoter nucleotide sequences, each 26S promoter nucleotide sequence resulting in transcription of one or more of the separate open reading frames. [The present invention 1038] Any of the aforementioned compositions of the invention, wherein the one or more vectors are each at least 300 nt in size. [This invention 1039] Any of the aforementioned compositions of the present invention, wherein the one or more vectors are each at least 1 kb in size. [The present invention 1040] Any of the aforementioned compositions of the present invention, wherein the one or more vectors are each 2 kb in size. [The present invention 1041] Any of the aforementioned compositions of the present invention, wherein the one or more vectors are each less than 5 kb in size. [The present invention 1042] Any of the compositions of the invention, wherein at least one of the at least one antigen-encoding nucleic acid sequences encodes a polypeptide sequence or a portion thereof that is presented by MHC class I on the tumor cell. [This invention 1043] The composition of any of claims 1001 to 1003, 1005 to 1007, or 1009 to 1042, wherein each antigen-encoding nucleic acid sequence is directly linked to each other. [This invention 1044] Any of the compositions of inventions 1001 to 1003, 1005 to 1007, or 1009 to 1043, wherein at least one of the at least one antigen-encoding nucleic acid sequence is linked to a different antigen-encoding nucleic acid sequence by a nucleic acid sequence encoding a linker. [This invention 1045] 104. The composition of claim 1044, wherein said linker links two MHC class I sequences or one MHC class I sequence to one MHC class II sequence. [The present invention 1046] The composition of the present invention 1045, wherein the linker is selected from the group consisting of: (1) a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive glycine residues in length; (2) a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive alanine residues in length; (3) two arginine residues (RR); (4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length that is efficiently processed by the mammalian proteasome; and (6) one or more naturally occurring sequences flanking the antigen from its cognate protein of origin and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length. [This invention 1047] 104. The composition of claim 1044, wherein said linker links two MHC class II sequences or one MHC class II sequence to one MHC class I sequence. [This invention 1048] 1047. The composition of claim 1047, wherein said linker comprises the sequence GPGPG. [This invention 1049] 9. The composition of any of claims 1001 to 1003, 1005 to 1007, or 1009 to 1048, wherein at least one of said at least one antigen-encoding nucleic acid sequences is operably or directly linked to a separate or contiguous sequence that enhances expression, stability, cellular trafficking, processing and presentation, and / or immunogenicity of said at least one antigen-encoding nucleic acid sequence. [The present invention 1050] The composition of claim 1049, wherein the separated or consecutive sequences comprise at least one of a ubiquitin sequence, a ubiquitin sequence modified to enhance proteasome targeting (e.g., a ubiquitin sequence comprising a Gly to Ala substitution at position 76), an immunoglobulin signal sequence (e.g., IgK), a major histocompatibility class I sequence, a lysosomal-associated membrane protein (LAMP)-1, a human dendritic cell lysosomal-associated membrane protein, and a major histocompatibility class II sequence, and optionally, the ubiquitin sequence modified to enhance proteasome targeting is A76. [This invention 1051] Any of the compositions of the invention, wherein at least one of the at least one antigen-encoding nucleic acid sequences encodes a polypeptide sequence or portion thereof that has increased binding affinity for its corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence after translation. [This invention 1052] Any of the compositions of the invention, wherein at least one of the at least one antigen-encoding nucleic acid sequences encodes a polypeptide sequence or portion thereof that has increased binding stability to its corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence after translation. [This invention 1053] Any of the compositions of the invention, wherein at least one of the at least one antigen-encoding nucleic acid sequences encodes a polypeptide sequence or portion thereof that has an increased likelihood of presentation on its corresponding MHC allele after translation compared to the corresponding wild-type nucleic acid sequence. [This invention 1054] Any of the compositions of the present invention, wherein the at least one alteration comprises a point mutation, a frameshift mutation, a non-frameshift mutation, a deletion mutation, an insertion mutation, a splice variant, a genomic rearrangement, or a splice antigen generated by the proteasome. [This invention 1055] Any of the compositions of the present invention, wherein the tumor is selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, bladder cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, adult acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer. [The present invention 1056] 1009-1055. Any of the compositions of claims 1001-1003, 1005-1007, or 1009-1055, wherein the at least one antigen-encoding nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleic acid sequences. [This invention 1057] Any of compositions 1001-1003, 1005-1007, or 1009-1055 of the present invention, wherein the at least one antigen-encoding nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 nucleic acid sequences. [This invention 1058] Any of the compositions of claims 1001 to 1003, 1005 to 1007, or 1009 to 1055, wherein the at least one antigen-encoding nucleic acid sequence comprises at least 2 to 400 nucleic acid sequences, and at least two of the antigen-encoding nucleic acid sequences encode a polypeptide sequence or a portion thereof that is presented by MHC class I on the surface of the tumor cell. [This invention 1059] The composition of any one of claims 1004 to 1008, wherein at least two of said antigen-encoding nucleic acid sequences encode a polypeptide sequence or a portion thereof that is presented by MHC class I on the surface of said tumor cells. [The present invention 1060] Any of the compositions of the present invention, which, when administered to the subject and translated, causes at least one of the antigens encoded by the at least one antigen-encoding nucleic acid sequence to be presented on antigen-presenting cells, resulting in an immune response targeting at least one of the antigens on the surface of the tumor cells. [This invention 1061] Any of the compositions of the present invention, wherein when the at least one antigen-encoding nucleic acid sequence is administered to the subject and translated, at least one of the MHC class I or class II antigens is presented on antigen-presenting cells, resulting in an immune response that targets at least one of the antigens on the surface of the tumor cells, and optionally, expression of each of the at least one antigen-encoding nucleic acid sequence is driven by the at least one promoter nucleotide sequence. [This invention 1062] Any of compositions 1001-1003, 1005-1007, or 1009-1061 of the invention, wherein each MHC class I antigen-encoding nucleic acid sequence encodes a polypeptide sequence that is 8-35 amino acids in length, optionally 9-17, 9-25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length. [The present invention 1063] The composition of any of claims 1001 to 1003, 1005 to 1007, or 1009 to 1062, wherein said at least one MHC class II antigen-encoding nucleic acid sequence is present. [The present invention 1064] Any of the compositions of claims 1001-1003, 1005-1007, or 1009-1062, wherein said at least one MHC class II antigen-encoding nucleic acid sequence is present and comprises at least one MHC class II antigen-encoding nucleic acid sequence that includes at least one alteration that causes the encoded peptide sequence to differ from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence. [This invention 1065] 1009-1064, wherein the at least one MHC class II antigen-encoding nucleic acid sequence is 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length. [The present invention 1066] The composition of any of claims 1001 to 1003, 1005 to 1007, or 1009 to 1065, wherein the at least one MHC class II antigen-encoding nucleic acid sequence is present and comprises at least one universal MHC class II antigen-encoding nucleic acid sequence, and optionally, the at least one universal sequence comprises at least one of tetanus toxoid and PADRE. [This invention 1067] The composition of any of claims 1001 to 1003, 1005 to 1007, or 1009 to 1066, wherein said at least one promoter nucleotide sequence or said second promoter nucleotide sequence is inducible. [The present invention 1068] The composition of any of claims 1001 to 1003, 1005 to 1007, or 1009 to 1066, wherein said at least one promoter nucleotide sequence or said second promoter nucleotide sequence is non-inducible. [The present invention 1069] 9. The composition of any one of claims 1001 to 1003, 1005 to 1007, or 1009 to 1068, wherein said at least one poly(A) sequence comprises a poly(A) sequence that is naturally occurring in said backbone. [The present invention 1070] 9. The composition of any one of claims 1001 to 1003, 1005 to 1007, or 1009 to 1068, wherein said at least one poly(A) sequence comprises a poly(A) sequence exogenous to said backbone. [This invention 1071] The composition of any of claims 1001 to 1003, 1005 to 1007, or 1009 to 1070, wherein the at least one poly(A) sequence is operably linked to at least one of the at least one antigen-encoding nucleic acid sequences. [This invention 1072] Any of compositions 1001-1003, 1005-1007, or 1009-1071, wherein the at least one poly(A) sequence is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 consecutive A nucleotides. [This invention 1073] The composition of any one of claims 1001 to 1003, 1005 to 1007, or 1009 to 1071, wherein said at least one poly(A) sequence is at least 100 consecutive A nucleotides. [This invention 1074] Any of the compositions of the present invention, wherein the antigen cassette further comprises at least one of an intron sequence, a woodchuck hepatitis virus post-transcriptional regulator (WPRE) sequence, an internal ribosome entry sequence (IRES) sequence, a nucleotide sequence encoding a 2A self-cleaving peptide sequence, a nucleotide sequence encoding a furin cleavage site, or a sequence within a 5' or 3' non-coding region known to improve nuclear export, stability, or translation efficiency of mRNA, operably linked to at least one of the at least one antigen-encoding nucleic acid sequence. [This invention 1075] Any of the compositions of the invention, wherein the antigen cassette further comprises a reporter gene, including, but not limited to, green fluorescent protein (GFP), a GFP variant, secreted alkaline phosphatase, luciferase, a luciferase variant, or a detectable peptide or epitope. [This invention 1076] 1075. The composition of claim 1075, wherein said detectable peptide or epitope is selected from the group consisting of an HA tag, a Flag tag, a His tag, or a V5 tag. [This invention 1077] Any of the aforementioned compositions of the invention, wherein the one or more vectors further comprise one or more nucleic acid sequences encoding at least one immunomodulator. [This invention 1078] 1077. The composition of the present invention, wherein the immunomodulator is an anti-CTLA4 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-4-1BB antibody or an antigen-binding fragment thereof, or an anti-OX-40 antibody or an antigen-binding fragment thereof. [This invention 1079] 1078. The composition of the invention, wherein the antibody or antigen-binding fragment thereof is a Fab fragment, a Fab' fragment, a single chain Fv (scFv), a single domain antibody (sdAb) (e.g., camelid antibody domains) either monospecific or multispecifically linked together, or a full-length single-chain antibody (e.g., full-length IgG in which the heavy and light chains are linked by a flexible linker). [The present invention 1080] The composition of claim 1078 or 1079, wherein the heavy chain sequence and the light chain sequence of the antibody are contiguous sequences separated by a self-cleaving sequence such as 2A or IRES, or the heavy chain sequence and the light chain sequence of the antibody are linked by a flexible linker such as consecutive glycine residues. [This invention 1081] The composition of claim 1077, wherein the immunomodulator is a cytokine. [This invention 1082] 1081. The composition of claim 1081, wherein said cytokine is at least one of IL-2, IL-7, IL-12, IL-15, or IL-21, or each of its variants. [This invention 1083] the at least one MHC class I antigen-encoding nucleic acid sequence is (a) obtaining at least one of exome, transcriptome, or whole genome tumor nucleotide sequencing data from a tumor, wherein the tumor nucleotide sequencing data is used to obtain data representing peptide sequences for each of a set of antigens; (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented by one or more of the MHC alleles on the surface of tumor cells of the tumor, the set of numerical likelihoods being determined based at least on the received mass spectrometry data; (c) selecting a subset of the set of antigens based on said set of numerical likelihoods to generate a set of selected antigens used to generate said at least one MHC class I antigen-encoding nucleic acid sequence; Any one of compositions 1001 to 1003, 1005 to 1007, or 1009 to 1082 of the present invention, selected by carrying out the above steps. [This invention 1084] each of said MHC class I epitope-encoding nucleic acid sequences comprising: (a) obtaining at least one of exome, transcriptome, or whole genome tumor nucleotide sequencing data from a tumor, wherein the tumor nucleotide sequencing data is used to obtain data representing peptide sequences for each of a set of antigens; (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented by one or more of the MHC alleles on the surface of tumor cells of the tumor, the set of numerical likelihoods being determined based at least on the received mass spectrometry data; (c) selecting a subset of the set of antigens based on said set of numerical likelihoods to generate a set of selected antigens used to generate said at least 20 MHC class I antigen-encoding nucleic acid sequences; The composition of the present invention 1004 or 1008, selected by carrying out the steps. [This invention 1085] The composition of the present invention 1083, wherein the number of sets of selected antigens is 2 to 20. [The present invention 1086] The presentation model: (a) the presence of a pair of a particular one of the MHC alleles and a particular amino acid at a particular position in the peptide sequence; (b) the likelihood of presentation on the surface of the tumor cell of such a peptide sequence containing the particular amino acid at the particular position by the particular one of the MHC alleles of the pair; and Any of compositions 1083 to 1085 of the present invention, which shows the dependency between. [This invention 1087] Any of the compositions of claims 1083 to 1086, wherein selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of being presented on the tumor cell surface compared to antigens not selected based on the presentation model, and optionally the selected antigens have been validated as being presented by one or more specific HLA alleles. [This invention 1088] Any of the compositions of claims 1083 to 1087, wherein selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of inducing a tumor-specific immune response in the subject compared to antigens that are not selected based on the presentation model. [This invention 1089] Any of the compositions of claims 1083 to 1088, wherein selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of being presented to naive T cells by professional antigen-presenting cells (APCs) compared to antigens not selected based on the presentation model, and optionally the APCs are dendritic cells (DCs). [The present invention 1090] 1089. The composition of any of claims 1083 to 1089, wherein selecting the set of selected antigens comprises selecting antigens that have a reduced likelihood of being inhibited by central or peripheral tolerance compared to antigens not selected based on the presentation model. [This invention 1091] Any of the compositions of claims 1083 to 1090, wherein selecting the set of selected antigens comprises selecting antigens that have a reduced likelihood of being able to induce an autoimmune response against normal tissue in the subject compared to antigens that are not selected based on the presentation model. [This invention 1092] Any of the compositions of claims 1083 to 1091, wherein the exome or transcriptome nucleotide sequencing data is obtained by sequencing tumor tissue. [This invention 1093] The composition of the present invention 1092, wherein said sequencing is next generation sequencing (NGS) or any massively parallel sequencing method. [This invention 1094] Any of the compositions of the present invention, wherein the antigen cassette comprises a junctional epitope sequence formed by adjacent sequences within the antigen cassette. [This invention 1095] 1094. The composition of the invention, wherein at least one or each junctional epitope sequence has an affinity for MHC greater than 500 nM. [This invention 1096] The composition of any one of claims 1094 and 1095, wherein each junctional epitope sequence is non-self. [This invention 1097] Any of the aforementioned compositions of the invention, wherein each of said MHC class I epitopes is predicted or verified to be capable of presentation by at least one HLA allele present in at least 5% of the population. [This invention 1098] Any of the compositions of the present invention, wherein each of the MHC class I epitopes is predicted or verified to be capable of being presented by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA occurrence rate in the population of at least 0.01%. [This invention 1099] Any of the compositions of the present invention, wherein each of the MHC class I epitopes is predicted or verified to be capable of being presented by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA occurrence rate of at least 0.1% in the population. [The present invention 1100] Any of the compositions of the invention, wherein the antigen cassette does not encode a non-therapeutic MHC class I or class II epitope nucleic acid sequence that comprises a translated wild-type nucleic acid sequence, and the non-therapeutic epitope is predicted to be presented on an MHC allele of the subject. [The present invention 1101] 110. The composition of claim 1100, wherein said non-therapeutic predicted MHC class I or class II epitope sequence is a junctional epitope sequence formed by adjacent sequences within said antigen cassette. [The present invention 1102] 1102. The composition of any of claims 1094 to 1101, wherein said prediction is based on a presentation likelihood generated by inputting the sequence of said non-therapeutic epitope into a presentation model. [The present invention 1103] The order of the at least one antigen-encoding nucleic acid sequence within the antigen cassette is (a) generating a set of candidate antigen cassette sequences corresponding to various permutations of the at least one antigen-encoding nucleic acid sequence; (b) determining for each candidate antigen cassette sequence a presentation score based on the presentation of non-therapeutic epitopes within the candidate antigen cassette sequence; (c) selecting candidate cassette sequences associated with presentation scores below a predetermined threshold as antigen cassette sequences for the antigen vaccine; Any of the compositions of inventions 1094 to 1102, which is determined by a series of steps including: [The present invention 1104] A pharmaceutical composition comprising any of the compositions of the present invention and a pharmaceutically acceptable carrier. [This invention 1105] The composition of the present invention 1104, further comprising an adjuvant. [The present invention 1106] The pharmaceutical composition of any one of claims 1104 to 1105, further comprising an immunomodulatory agent. [This invention 1107] 1106. The pharmaceutical composition of invention 1106, wherein the immunomodulator is an anti-CTLA4 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-4-1BB antibody or an antigen-binding fragment thereof, or an anti-OX-40 antibody or an antigen-binding fragment thereof. [This invention 1108] an antigen cassette of any of the preceding composition inventions; one or more elements obtained from the sequence of SEQ ID NO: 3 or SEQ ID NO: 5; 1. An isolated nucleotide sequence or set of isolated nucleotide sequences comprising: Optionally, the one or more elements are selected from the group consisting of sequences required for nonstructural protein-mediated amplification, a 26S promoter nucleotide sequence, a poly(A) sequence, and nsP1-4 genes of the sequences set forth in SEQ ID NO: 3 or SEQ ID NO: 5; Optionally, the nucleotide sequence is cDNA. The isolated nucleotide sequence or set of isolated nucleotide sequences. [This invention 1109] The isolated nucleotide sequence of the present invention 1108, wherein said sequence or set of isolated nucleotide sequences comprises an antigen cassette of any of the preceding composition inventions inserted at position 7544 of the sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. [The present invention 1110] the nucleotide sequence of a T7 or SP6 RNA polymerase promoter located 5' to the one or more elements derived from the sequence of SEQ ID NO:3 or SEQ ID NO:5; optionally, one or more restriction sites located 3' to said poly(A) sequence; The isolated nucleotide sequence of 1108 or 1109 of the invention further comprising: [The present invention 1111] 1108. The isolated nucleotide sequence of the present invention, wherein the antigen cassette of any of the preceding composition inventions is inserted at position 7563 of SEQ ID NO:8 or SEQ ID NO:9. [The present invention 1112] A vector or a set of vectors comprising any one of the nucleotide sequences of 1108 to 1111 of the present invention. [The present invention 1113] An isolated cell comprising the nucleotide sequence or set of isolated nucleotide sequences of any of 1108 to 1112 of the present invention, optionally wherein the cell is a BHK-21, CHO, HEK293 or a variant thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a cell. [This invention 1114] A kit comprising the composition of any of the preceding composition inventions and instructions for use. [This invention 1115] A method for treating a subject having cancer, comprising administering to the subject any of the compositions of the preceding composition inventions or any of the pharmaceutical compositions of inventions 1104 to 1107. [The present invention 1116] 1115. The method of claim 1115, wherein said at least one MHC class I antigen-encoding nucleic acid sequence is derived from a tumor in said subject with cancer. [This invention 1117] 1115. The method of claim 1115, wherein said at least one MHC class I antigen-encoding nucleic acid sequence is not derived from a tumor in said subject with cancer. [This invention 1118] A method for inducing an immune response in a subject, comprising administering to the subject a composition of any of the preceding composition inventions or a pharmaceutical composition of any of inventions 1104 to 1107. [This invention 1119] 1119. The method of any of claims 1115 to 1118, wherein said subject expresses at least one HLA allele predicted or known to present said MHC class I epitope. [The present invention 1120] Any of the methods of claims 1115 to 1118, wherein said subject expresses at least one HLA allele that is predicted or known to present said MHC class I epitope, and said MHC class I epitope comprises a mutation selected from the group consisting of the mutations set out in Table 34. [This invention 1121] Any of the methods of claims 1115 to 1118, wherein said subject expresses at least one HLA allele that is predicted or known to present said MHC class I epitope, and said MHC class I epitope comprises a mutation selected from the group consisting of the mutations set out in Table 32. [This invention 1122] The method of any of claims 1115 to 1121, wherein said composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). [This invention 1123] 1122. The method of any one of claims 1115 to 1121, wherein the composition is administered intramuscularly. [This invention 1124] The method of any of claims 1115 to 1123, further comprising administering one or more immunomodulatory substances, optionally administered before, simultaneously with, or after administration of said composition or pharmaceutical composition. [Invention 1125] 1124. The method of claim 1124, wherein said one or more immunomodulatory agents are selected from the group consisting of an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof. [The present invention 1126] The method of any one of claims 1124 to 1125, wherein said immunomodulatory agent is administered intravenously (IV), intramuscularly (IM), intradermally (ID), or subcutaneously (SC). [This invention 1127] 1127. The method of claim 1126, wherein said subcutaneous administration is near the site of administration of said composition or pharmaceutical composition or in proximity to a draining lymph node of one or more vectors or compositions. [This invention 1128] The method of any of claims 1115 to 1127, further comprising administering to said subject a second vaccine composition. [This invention 1129] The method of claim 1128, wherein said second vaccine composition is administered before the administration of the composition or pharmaceutical composition of any of claims 1115 to 1127. [The present invention 1130] The method of claim 1128, wherein said second vaccine composition is administered after administration of the composition or pharmaceutical composition of any of claims 1115 to 1127. [This invention 1131] The method of any one of claims 1129 or 1130, wherein said second vaccine composition is the same as the composition or pharmaceutical composition of any one of claims 1115 to 1127. [This invention 1132] The method of any one of claims 1129 or 1130, wherein said second vaccine composition is different from the composition or pharmaceutical composition of any one of claims 1115 to 1127. [This invention 1133] 1133. The method of claim 1132, wherein said second vaccine composition comprises a chimpanzee adenoviral vector encoding at least one antigen-encoding nucleic acid sequence. [This invention 1134] 1133. The method of claim 1133, wherein said at least one antigen-encoding nucleic acid sequence encoded by said chimpanzee adenoviral vector is the same as at least one antigen-encoding nucleic acid sequence of any of the preceding composition inventions. [This invention 1135] 1. A method of producing one or more vectors of any of the preceding composition inventions, comprising: (a) obtaining a linearized DNA sequence comprising the backbone and the antigen cassette; (b) in vitro transcribing the linearized DNA sequence by adding the linearized DNA sequence to an in vitro transcription reaction containing all components necessary to transcribe the linearized DNA sequence into RNA, optionally further comprising adding the m7g cap to the resulting RNA in vitro; (c) isolating the one or more vectors from the in vitro transcription reaction; and The method comprising: [This invention 1136] A method for producing 1135 of the present invention, wherein the linearized DNA sequence is generated by linearizing a DNA plasmid sequence or by amplification using PCR. [This invention 1137] 1136. The method of production of claim 1136, wherein said DNA plasmid sequence is generated using one of bacterial recombination, or total genome DNA synthesis, or total genome DNA synthesis with amplification of synthetic DNA within bacterial cells. [This invention 1138] The method of producing 1135 of the present invention, wherein isolating the one or more vectors from the in vitro transcription reaction comprises one or more of phenol-chloroform extraction, purification using a silica column, or similar RNA purification methods. [This invention 1139] 1. A method of making a composition of any of the preceding composition inventions for delivering said antigen expression system, comprising: (a) providing a nanoparticulate delivery vehicle component; (b) providing said antigen expression system; (c) providing conditions sufficient for said nanoparticulate delivery vehicle and said antigen expression system to produce said composition for delivering said antigen expression system; and The method comprising: [This invention 1140] The method for producing 1139 of the present invention, wherein the conditions are provided by microfluidic mixing. [This invention 1141] 1. A method of assessing a subject having cancer, comprising: a) 1) whether the subject has an HLA allele that is predicted or known to present the antigen included in the antigen-based vaccine; and below: 1) whether the subject's tumor expresses a gene associated with said antigen, and optionally whether said gene is aberrantly expressed compared to normal cells or tissues; 2) whether the subject's tumor has a mutation associated with the antigen one or both of a step of determining or having determined b) determining, or having already determined, from the results of (a) that the subject expresses the HLA allele and the subject's tumor expresses the gene and / or the subject's tumor has the mutation, that the subject is a candidate for treatment with the antigen-based vaccine; The antigen comprises at least one MHC class I epitope sequence selected from the group consisting of SEQ ID NOs: 57 to 29357. The process; c) optionally administering or having administered said antigen-based vaccine to said subject, said antigen-based vaccine comprising: 1) said at least one MHC class I epitope; or 2) an MHC class I epitope-encoding nucleic acid sequence encoding said at least one MHC class I epitope; The process comprising: The method comprising: [This invention 1142] 1. A method of assessing a subject having cancer, comprising: a) the object is 1) expressing the A0301 HLA allele and the subject's tumor harboring a KRAS_G12A mutation; 2) expressing the A0201 HLA allele and the subject's tumor harboring the KRAS_G12C mutation; 3) expresses the C0802 HLA allele or the A1101 HLA allele, and the subject's tumor has a KRAS_G12D mutation; or 4) The subject expresses the A0301 HLA allele, the A1101 HLA allele, the A3101 HLA allele, the C0102 HLA allele, or the A0302 HLA allele, and the subject's tumor has the KRAS_G12V mutation. a step of determining or having determined b) From the results of (a), the subject is 1) if the subject expresses the A0301 allele and the subject's tumor has the KRAS_G12A mutation, 2) expressing the A0201 allele and the subject's tumor having the KRAS_G12C mutation; 3) expressing the C0802 HLA allele or the A1101 HLA allele and the subject's tumor harboring the KRAS_G12D mutation; or 4) The subject expresses the A0301 HLA allele, the A1101 HLA allele, the A3101 HLA allele, the C0102 HLA allele, or the A0302 HLA allele, and the subject's tumor has a KRAS_G12V mutation. determining, or having determined, that the subject is a candidate for treatment with the antigen-based vaccine; c) optionally administering or having administered said antigen-based vaccine to said subject, said antigen-based vaccine comprising: 1) at least one MHC class I epitope comprising the KRAS_G12A mutation, the KRAS_G12C mutation, the KRAS_G12D mutation, or the KRAS_G12V mutation, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding at least one MHC class I epitope comprising the KRAS_G12A mutation, the KRAS_G12C mutation, the KRAS_G12D mutation, or the KRAS_G12V mutation, The process includes the steps of: The method comprising: [This invention 1143] The method of any one of claims 1141 to 1142, wherein steps (a) and / or (b) comprise obtaining a dataset from a third party that processed a sample from the subject. [This invention 1144] The method of any one of claims 1141 to 1142, wherein step (a) comprises obtaining a sample from the subject and assaying the sample using a method selected from the group consisting of exome sequencing, targeted exome sequencing, transcriptome sequencing, Sanger sequencing, PCR-based genotyping assays, mass spectrometry-based methods, microarrays, nanostrings, ISH, and IHC. [Invention 1145] 1145. The method of any one of claims 1143 to 1144, wherein said sample comprises a tumor sample, a normal tissue sample, or said tumor sample and said normal tissue sample. [Invention 1146] 1145. The method of claim 1145, wherein said sample is selected from tissue, body fluid, blood, tumor biopsy, spinal fluid, and needle aspirate. [This invention 1147] 1147. The method of any of claims 1141 or 1143 to 1146, wherein said gene is selected from the group consisting of any of the genes found in Table 34. [Invention 1148] 1147. The method of any of claims 1141 or 1143 to 1146, wherein said gene is selected from the group consisting of any of the genes found in Table 32. [This invention 1149] 1149. The method of any of claims 1141 to 1148, wherein said cancer is selected from the group consisting of lung cancer, microsatellite stable colon cancer, and pancreatic cancer. [This invention 1150] 1149. The method of any of claims 1141 to 1149, wherein said HLA allele has an HLA frequency of at least 5%. [This invention 1151] 1151. The method of any of claims 1141 to 1150, wherein said at least one MHC class I epitope is presented by said HLA allele on cells associated with a tumor of said subject. [This invention 1152] 1152. The method of any of claims 1141 to 1151, wherein said antigen-based vaccine comprises an antigen expression system. [This invention 1153] The method of claim 1152, wherein said antigen expression system comprises any one of the antigen expression systems of any of claims 1001 to 1103. [This invention 1154] The method of any one of claims 1141 to 1151, wherein said antigen-based vaccine comprises any one of the pharmaceutical compositions of any one of claims 1104 to 1107. [This invention 1155] 1. A method of treating a subject having cancer, comprising administering to said subject an antigen-based vaccine directed against said subject, said antigen-based vaccine comprising: 1) at least one MHC class I epitope, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding said at least one MHC class I epitope; Including, The at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs: 57 to 29357. The method. [Invention 1156] 1155. The method of claim 1155, wherein said at least one MHC class I antigen-encoding nucleic acid sequence is derived from a tumor in said subject with cancer. [This invention 1157] 1155. The method of claim 1155, wherein said at least one MHC class I antigen-encoding nucleic acid sequence is not derived from said tumor of said subject with cancer. [This invention 1158] 1. A method for inducing an immune response in a subject, comprising administering to said subject an antigen-based vaccine directed against said subject, said antigen-based vaccine comprising: 1) at least one MHC class I epitope, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding said at least one MHC class I epitope; Including, The at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs: 57 to 29357. The method. [This invention 1159] 1159. The method of any of claims 1155 to 1158, wherein said subject expresses at least one HLA allele predicted or known to present said at least one MHC class I epitope sequence. [The present invention 1160] 1159. The method of any of claims 1155 to 1158, wherein said subject expresses at least one HLA allele predicted or known to present said at least one MHC class I epitope sequence, and said at least one MHC class I epitope sequence comprises a mutation selected from the group consisting of the mutations set out in Table 34. [This invention 1161] 1159. The method of any of claims 1155 to 1158, wherein said subject expresses at least one HLA allele that is predicted or known to present said at least one MHC class I epitope sequence, and said at least one MHC class I epitope sequence comprises a mutation selected from the group consisting of the mutations set out in Table 32. [This invention 1162] 1. A method for inducing an immune response in a subject, comprising administering to said subject an antigen-based vaccine directed against said subject, said antigen-based vaccine comprising: 1) at least one MHC class I epitope, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding said at least one MHC class I epitope; Including, The at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs: 57 to 29357, and the subject expresses at least one HLA allele that is predicted or known to present the at least one MHC class I epitope sequence. The method. [This invention 1163] 1. A method for inducing an immune response in a subject, comprising administering to said subject an antigen-based vaccine directed against said subject, said antigen-based vaccine comprising: 1) at least one MHC class I epitope, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding said at least one MHC class I epitope; Including, the at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs: 57-29357, the subject expresses at least one HLA allele predicted or known to present the at least one MHC class I epitope sequence, the at least one MHC class I epitope sequence comprises a mutation selected from the group consisting of the mutations set forth in Table 34, and the subject expresses at least one HLA allele set forth in Table 34 associated with a corresponding mutation set forth in Table 34 (e.g., KRAS_G13D and C0802). The method. [This invention 1164] 1. A method for inducing an immune response in a subject, comprising administering to said subject an antigen-based vaccine directed against said subject, said antigen-based vaccine comprising: 1) at least one MHC class I epitope, or 2) an MHC class I epitope-encoding nucleic acid sequence encoding said at least one MHC class I epitope; Including, the at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs: 57-29357, the subject expresses at least one HLA allele predicted or known to present the at least one MHC class I epitope sequence, and the at least one MHC class I epitope sequence comprises a mutation selected from the group consisting of the mutations shown in Table 32. The method. [Invention 1165] 1165. The method of any one of claims 1155 to 1164, wherein said antigen-based vaccine comprises an antigen expression system. [Invention 1166] The method of claim 1165, wherein said antigen expression system comprises any one of the antigen expression systems of any of claims 1001 to 1103. [This invention 1167] The method of any one of claims 1155 to 1164, wherein said antigen-based vaccine comprises any one of the pharmaceutical compositions of any one of claims 1104 to 1107. [Brief explanation of the drawings]

[0100] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and accompanying drawings. [Figure 1] We describe the development of an in vitro T cell activating compound assay. This diagram shows the assay in which delivery of a vaccine cassette into antigen-presenting cells leads to the expression, processing, and MHC-restricted presentation of distinct peptide antigens. Reporter T cells engineered to carry T cell receptors matching specific peptide-MHC combinations are activated and express luciferase. [Figure 2] Figure 2A illustrates the evaluation of linker sequences within the short cassette, showing two universal class II MHC epitopes (MHC-II) joined to five class I MHC class-restricted epitopes (epitopes 1-5) linked at the same positions relative to each other. Various repeats were generated using different linkers. In some cases, the T cell epitopes are directly linked to each other. In other cases, the T cell epitopes are flanked on one or both sides by their native sequence. In other repeats, the T cell epitopes are linked by the non-native sequences AAY, RR, and DPP. Figure 2B illustrates the evaluation of linker sequences within the short cassette, showing sequence information for the T cell epitopes embedded within the short cassette. The figure discloses SEQ ID NOs: 29365-29366, 29369, 29368, 29367, and 29494-29495, respectively, in order of appearance. [Figure 3]We describe the evaluation of cell targeting sequences added to a model vaccine cassette. The targeting cassette consists of a short cassette design extended with ubiquitin (Ub), signal peptide (SP), and transmembrane (TM) domains, and contains five marker human T cell epitopes (epitopes 1-5) flanked by two murine T cell epitopes, SIINFEKL (SEQ ID NO: 29362) (SII) and SPSYAYHQF (SEQ ID NO: 29363) (A5), with either the non-natural linker AAY or natural linker sequences flanking the T cell epitopes (25-mer). [Figure 4] Illustrates the in vivo evaluation of linker sequences within short cassettes. A) Experimental design for the in vivo evaluation of vaccine cassettes using HLA-A2 transgenic mice. [Figure 5A] We describe an in vivo evaluation of the impact of epitope position within a long 21-marker set, showing that the design of the long cassette contains five marker class I epitopes (epitopes 1-5) contained within their 25-mer native sequences (linker = native flanking sequence) separated by additional well-known T cell class I epitopes (epitopes 6-21) contained within their 25-mer native sequences, and two universal class II epitopes (MHC-II0), differing only in the relative position of each class I epitope. [Figure 5B]

[0023] Figure 1 illustrates the in vivo evaluation of the influence of epitope position within the long 21-marker set and shows the sequence information of the T cell epitopes used. The figure discloses SEQ ID NOS: 29365-29366, 29369, 29368, 29367, 29496-29498, 29370, and 29499-29510, respectively, in order of appearance. [Figure 6A] The final cassette design for preclinical IND application experiments is described, showing that the final cassette design contains 20 MHC I epitopes within their 25-mer native sequence (linker = native flanking sequence), consisting of 6 non-human primate (NHP) epitopes, 5 human epitopes, 9 mouse epitopes, and 2 universal MHC class II epitopes. [Figure 6B] The final cassette design for preclinical IND application experiments is described, showing sequence information for the T cell epitopes used, presented on class I MHC from non-human primates, mice, and humans, as well as the sequences of two universal MHC class II epitopes, PADRE and tetanus toxoid. The figure discloses, in order of appearance, SEQ ID NOs: 29426-29431, 29362-29363, 29456, 29511, 29460-29462, 29458-29459, 29367-29369, 29365-29366, 29494, and 29512, respectively. [Figure 7] Figure 7A illustrates the generation of ChAdV68.4WTnt.GFP virus after transfection. HEK293A cells were transfected with ChAdV68.4WTnt.GFP DNA using the calcium phosphate protocol. Viral replication was observed 10 days after transfection, and ChAdV68.4WTnt.GFP viral plaques were visualized using a light microscope (40x magnification). Figure 7B illustrates the generation of ChAdV68.4WTnt.GFP virus after transfection. HEK293A cells were transfected with ChAdV68.4WTnt.GFP DNA using the calcium phosphate protocol. Viral replication was observed 10 days after transfection, and ChAdV68.4WTnt.GFP viral plaques were visualized using a fluorescent microscope (40x magnification). Figure 7C illustrates the generation of ChAdV68.4WTnt.GFP virus after transfection. HEK293A cells were transfected with ChAdV68.4WTnt.GFP DNA using a calcium phosphate protocol. Viral replication was observed 10 days after transfection, and ChAdV68.4WTnt.GFP viral plaques were visualized using a fluorescent microscope at 100x magnification. [Figure 8]Figure 8A illustrates the generation of ChAdV68.5WTnt.GFP virus after transfection. HEK293A cells were transfected with ChAdV68.5WTnt.GFP DNA using the Lipofectamine protocol. Viral replication (plaques) were observed 10 days after transfection. Lysates were prepared and used to reinfect 293A cells in T25 flasks. ChAdV68.5WTnt.GFP viral plaques were visualized and photographed 3 days later using a light microscope (40x magnification). Figure 8B illustrates the generation of ChAdV68.5WTnt.GFP virus after transfection. HEK293A cells were transfected with ChAdV68.5WTnt.GFP DNA using the Lipofectamine protocol. Viral replication (plaques) were observed 10 days after transfection. Lysates were prepared and used to reinfect 293A cells in T25 flasks. ChAdV68.5WTnt.GFP viral plaques were visualized and photographed 3 days later using a fluorescent microscope at 40x magnification. Figure 8C illustrates the generation of ChAdV68.5WTnt.GFP virus after transfection. HEK293A cells were transfected with ChAdV68.5WTnt.GFP DNA using the Lipofectamine protocol. Viral replication (plaques) was observed 10 days after transfection. Lysates were prepared and used to reinfect 293A cells in T25 flasks. ChAdV68.5WTnt.GFP viral plaques were visualized and photographed 3 days later using a fluorescent microscope at 100x magnification. [Figure 9] A scheme for generating virus particles is described. [Figure 10] Alphavirus-derived VEE self-replicating RNA (srRNA) vectors are described. [Figure 11] In vivo reporter expression after inoculation of C57BL / 6J mice with VEE-luciferase srRNA is demonstrated. Representative images of luciferase signals after immunization of C57BL / 6J mice with VEE-luciferase srRNA (bilateral intramuscular injection of 10 μg / mouse in MC3) at different time points are shown. [Figure 12A] This study describes T cell responses measured 14 days after immunization with VEE srRNA formulated in MC3 LNPs in B16-OVA tumor-bearing mice. B16-OVA tumor-bearing C57BL / 6J mice were injected with 10 μg of VEE-luciferase srRNA (control), VEE-UbAAY srRNA (Vax), VEE-luciferase srRNA and anti-CTLA-4 (aCTLA-4), or VEE-UbAAY srRNA and anti-CTLA-4 (Vax+aCTLA-4). Additionally, all mice were treated with anti-PD-1 mAb starting on day 7. Each group consisted of eight mice. 14 days after immunization, mice were sacrificed, and spleens and lymph nodes were harvested. SIINFEKL (SEQ ID NO: 29362)-specific T cell responses were assessed by IFN-γ ELISPOT and are reported as spot-forming cells (SFC) per 10 6 splenocytes. Each line represents the median value. [Figure 12B] This section describes T cell responses measured 14 days after immunization with VEE srRNA formulated in MC3 LNPs in B16-OVA tumor-bearing mice. B16-OVA tumor-bearing C57BL / 6J mice were injected with 10 μg of VEE-luciferase srRNA (control), VEE-UbAAY srRNA (Vax), VEE-luciferase srRNA and anti-CTLA-4 (aCTLA-4), or VEE-UbAAY srRNA and anti-CTLA-4 (Vax + aCTLA-4). Additionally, all mice were treated with anti-PD-1 mAb starting on day 7. Each group consisted of eight mice. 14 days after immunization, mice were sacrificed, and spleens and lymph nodes were harvested. SIINFEKL (SEQ ID NO: 29362)-specific T cell responses were assessed by MHCI pentamer staining and are reported as pentamer-positive cells and the percentage of CD8-positive cells. Each line indicates the median value. [Figure 13A]Antigen-specific T cell responses after heterologous prime / boost in B16-OVA tumor-bearing mice are described. B16-OVA tumor-bearing C57BL / 6J mice were injected with adenovirus expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA formulated in MC3 LNPs (control) or with Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both the control and Vax groups were also treated with an IgG control mAb. A third group was treated with Ad5-GFP prime / VEE-luciferase srRNA boost in combination with anti-CTLA-4 (aCTLA-4), and a fourth group was treated with Ad5-UbAAY prime / VEE-UbAAY boost in combination with anti-CTLA-4 (Vax+aCTLA-4). Additionally, all mice were treated with anti-PD-1 mAb starting on day 21. T cell responses were measured by IFN-γ ELISPOT. Mice were sacrificed 14 days after adenovirus immunization, and spleens and lymph nodes were harvested. [Figure 13B] Antigen-specific T cell responses after heterologous prime / boost in B16-OVA tumor-bearing mice are described. B16-OVA tumor-bearing C57BL / 6J mice were injected with adenovirus expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA formulated in MC3 LNPs (control) or with Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both the control and Vax groups were also treated with an IgG control mAb. A third group was treated with Ad5-GFP prime / VEE-luciferase srRNA boost in combination with anti-CTLA-4 (aCTLA-4), and a fourth group was treated with Ad5-UbAAY prime / VEE-UbAAY boost in combination with anti-CTLA-4 (Vax+aCTLA-4). Additionally, all mice were treated with anti-PD-1 mAb starting on day 21. T cell responses were measured by IFN-γ ELISPOT. Mice were sacrificed 14 days after adenovirus immunization and 14 days after srRNA boost (28 days after prime), and spleens and lymph nodes were harvested. [Figure 13C] Antigen-specific T cell responses after heterologous prime / boost in B16-OVA tumor-bearing mice are described. B16-OVA tumor-bearing C57BL / 6J mice were injected with adenovirus expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA formulated in MC3 LNPs (control) or with Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both the control and Vax groups were also treated with an IgG control mAb. A third group was treated with Ad5-GFP prime / VEE-luciferase srRNA boost in combination with anti-CTLA-4 (aCTLA-4), and a fourth group was treated with Ad5-UbAAY prime / VEE-UbAAY boost in combination with anti-CTLA-4 (Vax+aCTLA-4). Additionally, all mice were treated with anti-PD-1 mAb starting on day 21. T cell responses were measured by MHC class I pentamer staining. 14 days after adenovirus immunization, mice were sacrificed and the spleens and lymph nodes were harvested. [Figure 13D]Antigen-specific T cell responses after heterologous prime / boost in B16-OVA tumor-bearing mice are described. B16-OVA tumor-bearing C57BL / 6J mice were injected with adenovirus expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA formulated in MC3 LNPs (control) or with Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both the control and Vax groups were also treated with an IgG control mAb. A third group was treated with Ad5-GFP prime / VEE-luciferase srRNA boost in combination with anti-CTLA-4 (aCTLA-4), and a fourth group was treated with Ad5-UbAAY prime / VEE-UbAAY boost in combination with anti-CTLA-4 (Vax+aCTLA-4). Additionally, all mice were treated with anti-PD-1 mAb starting on day 21. T cell responses were measured by MHC class I pentamer staining. Mice were sacrificed 14 days after adenovirus immunization and 14 days after the srRNA boost (28 days after prime), and spleens and lymph nodes were harvested. [Figure 14A] Antigen-specific T cell responses after heterologous prime / boost in CT26 (Balb / c) tumor-bearing mice are described. Mice were immunized with Ad5-GFP and boosted 15 days after adenoviral prime with VEE-luciferase srRNA formulated in MC3 LNP (control) or primed with Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both the control and Vax groups were also treated with an IgG control mAb. Another group received a combination of Ad5-GFP / VEE-luciferase srRNA prime / boost and anti-PD-1 (aPD1), and a fourth group received a combination of Ad5-UbAAY / VEE-UbAAY srRNA prime / boost and anti-PD-1 mAb (Vax+aPD1). T cell responses to the AH1 peptide were measured using IFN-γ ELISPOT. Twelve days after adenovirus immunization, the mice were sacrificed and the spleens and lymph nodes were collected. [Figure 14B]Antigen-specific T cell responses after heterologous prime / boost in CT26 (Balb / c) tumor-bearing mice are described. Mice were immunized with Ad5-GFP and boosted 15 days after adenoviral prime with VEE-luciferase srRNA formulated in MC3 LNP (control) or primed with Ad5-UbAAY and boosted with VEE-UbAAY srRNA (Vax). Both the control and Vax groups were also treated with an IgG control mAb. Another group received a combination of Ad5-GFP / VEE-luciferase srRNA prime / boost and anti-PD-1 (aPD1), and a fourth group received a combination of Ad5-UbAAY / VEE-UbAAY srRNA prime / boost and anti-PD-1 mAb (Vax+aPD1). T cell responses to the AH1 peptide were measured using IFN-γ ELISPOT. Twelve days after adenovirus immunization and six days after srRNA boost (21 days after prime), mice were sacrificed and spleens and lymph nodes were harvested. [Figure 15]

[0033] Figure 1 illustrates ChAdV68-induced T cell responses to murine tumor antigens in mice. Mice were immunized with ChAdV68.5WTnt.MAG2 pentamer, and T cell responses to the MHC class I epitope SIINFEKL (SEQ ID NO: 29362) (OVA) were measured in C57BL / 6J female mice, and the MHC class I epitope AH1-A5 was measured in Balb / c mice. The mean spot-forming cells (SFC) per 10 splenocytes measured by ELISpot assay are shown. Error bars indicate standard deviation. [Figure 16]Cellular immune responses in the CT26 tumor model after a single immunization with either ChAdV6, ChAdV + anti-PD-1, srRNA, srRNA + anti-PD-1, or anti-PD-1 alone are described. Antigen-specific IFN-γ production was measured in splenocytes from six mice from each group using ELISpot. Results are shown as spot-forming cells (SFC) per 10 splenocytes. The median value for each group is indicated by a horizontal line. P values ​​were determined using Dunnett's multiple comparison test; i.e., ***P<0.0001, **P<0.001, *P<0.05. ChAdV = ChAdV68.5WTnt.MAG25mer; srRNA = VEE-MAG25mer-srRNA. [Figure 17] This figure illustrates the CD8 T cell immune response in a CT26 tumor model after a single immunization with ChAdV6, ChAdV + anti-PD-1, srRNA, srRNA + anti-PD-1, or anti-PD-1 alone. Antigen-specific IFN-γ production in CD8 T cells was measured using ICS, and results were expressed as antigen-specific CD8 T cells as a percentage of total CD8 T cells. The median value for each group is indicated by a horizontal line. P values ​​were determined using Dunnett's multiple comparison test; i.e., ***P<0.0001, **P<0.001, *P<0.05. ChAdV = ChAdV68.5WTnt.MAG25mer, srRNA = VEE-MAG25mer-srRNA. [Figure 18] Tumor growth in the CT26 tumor model after immunization with ChAdV / srRNA heterologous prime / boost, srRNA / ChAdV heterologous prime / boost, or srRNA / srRNA homologous prime / boost is shown. Prime / boost immunizations with or without anti-PD-1 administration in the prime and boost are also shown for comparison. Tumor volumes were measured twice weekly, and the mean tumor volume is shown for the first 21 days of the experiment. Each group consisted of 22–28 mice at the start of the experiment. Error bars indicate the standard error of the mean (SEM). P values ​​were determined using Dunnett's test; i.e., ***P<0.0001, **P<0.001, *P<0.05. ChAdV = ChAdV68.5WTnt.MAG25mer, srRNA = VEE-MAG25mer-srRNA. [Figure 19] Survival rates in the CT26 tumor model after immunization with ChAdV / srRNA heterologous prime / boost, srRNA / ChAdV heterologous prime / boost, or srRNA / srRNA homologous prime / boost are described. Prime / boost immunization with or without anti-PD-1 administration in the prime and boost are also shown for comparison. P values ​​were determined using the log-rank test; i.e., ***P<0.0001, **P<0.001, *P<0.01. ChAdV = ChAdV68.5WTnt.MAG25mer, srRNA = VEE-MAG25mer-srRNA. [Figure 20A] Figure 20 shows antigen-specific cellular immune responses measured by ELISpot. Antigen-specific IFN-γ production against six different mamuA01-restricted epitopes was measured in PBMCs (six macaques per group) 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeks after the initial boost immunization using ELISpot for the homologous prime / boost groups with VEE-MAG25mer srRNA-LNP1 (30 μg) (Figure 20A), VEE-MAG25mer srRNA-LNP1 (100 μg) (Figure 20B), or VEE-MAG25mer srRNA-LNP2 (100 μg) (Figure 20C), or the heterologous prime / boost group with ChAdV68.5WTnt.MAG25mer / VEE-MAG25mer srRNA (Figure 20D). Results are shown in stacked bar graph format as the mean spot-forming cells (SFC) per 10 PBMC for each epitope. Values ​​for each animal were normalized to prebleed (week 0) levels. [Figure 20B] See legend to Figure 20A. [Figure 20C] See legend to Figure 20A. [Figure 20D] See legend to Figure 20A. [Figure 21]Antigen-specific cellular immune responses measured using ELISpot are shown. Antigen-specific IFN-γ production against six different mamuA01-restricted epitopes was measured in PBMCs after immunization with a heterologous prime / boost regimen of ChAdV68.5WTnt.MAG25mer / VEE-MAG25mer srRNA using ELISpot before immunization and 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks after the initial immunization. Results are shown in stacked bar graph format as the mean spot-forming cells (SFC) per 10 PBMCs for each epitope (six rhesus macaques per group). [Figure 22] Figure 1 shows antigen-specific cellular immune responses measured using ELISpot. Antigen-specific IFN-γ production against six different mamuA01-restricted epitopes was measured in PBMCs after immunization with the allogeneic prime / boost regimen of VEE-MAG25mer srRNA LNP2 by ELISpot before immunization and at 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, or 15 weeks after the initial immunization. Results are shown in stacked bar graph format as the mean spot-forming cells (SFC) per 10 PBMCs for each epitope (six rhesus macaques per group). [Figure 23] Figure 1 shows antigen-specific cellular immune responses measured using ELISpot. Antigen-specific IFN-γ production against six different mamuA01-restricted epitopes was measured in PBMCs after immunization with the allogeneic prime / boost regimen of VEE-MAG25mer srRNA LNP1 using ELISpot before immunization and 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, or 15 weeks after the initial immunization. Results are shown in stacked bar graph format as the mean spot-forming cells (SFC) per 10 PBMCs for each epitope (six rhesus macaques per group). [Figure 24A] 1 shows an exemplary peptide spectrum generated from a dynamic range standard from Promega. The figure discloses SEQ ID NO: 29364. [Figure 24B] An exemplary peptide spectrum generated from a Promega dynamic range standard is shown. [Figure 25] 1 shows the correlation between EDGE score and probability of detection of candidate shared neoantigenic peptides by targeted MS. [Figure 26] Expanded TILs from a patient stained with mutant peptide HLA-A*11:01 tetramer are shown. Flow cytometry gating strategy for CD8+ cells (left panel) and staining of CD8+ cells with KRAS-G12V / HLA-A*11:01 tetramer (right panel) are shown. [Figure 27] A general TCR sequencing methodology and workflow is shown. [Figure 28] A representative example of a TCR sequencing method using the KRAS_G12V / HLA-A*11:01 tetramer is shown. [Figure 29] The general organization of model epitopes from different tumors in large antigen cassettes with 30 (L), 40 (XL) or 50 (XXL) epitopes is shown. [Figure 30] The ChAd vector expresses a long cassette, as shown by the Western blot analysis using an anti-class II (PADRE) antibody that recognizes a sequence common to all cassettes. HEK293 cells were infected with chAd68 vectors expressing different sized large cassettes (chAd68-50XXL, chAd68-40XL, and chAd68-30L). Infection was performed at an MOI of 0.2. 24 h postinfection, the proteasome inhibitor MG132 was added to one set of infected wells (indicated by a + sign). Another set of virus-treated wells was not treated with MG132 (indicated by a - sign). Uninfected HEK293 cells (293F) served as a negative control. 48 h postinfection, cell pellets were collected, analyzed by SDS / PAGE electrophoresis, and immunoblotted using a rabbit anti-class II PADRE antibody. HRP anti-rabbit antibody and ECL chemiluminescent substrate were used for detection. [Figure 31]Figure 1 shows CD8+ immune responses in mice immunized with the large cassette of chAd68 detected by ICS against AH1 (top panel) and SIINFEKL (SEQ ID NO: 29362) (bottom panel). Data are presented as IFNg+ cells against model epitopes and as a % of total CD8 cells. [Figure 32] Figure 1 shows CD8+ responses to LD-AH1+ (top panel) and Kb-SIINFEKL (SEQ ID NO: 29362)+ (bottom panel) tetramers after vaccination with the chAd68 large cassette. Data are presented as % of total CD8 cells with reactivity to the model tetramer-peptide complex. *p<0.05, **p<0.01 by ANOVA with Tukey's test. All p values ​​were compared to the MAG20 antigen cassette. [Figure 33] Figure 1 shows CD8+ immune responses in mice treated with alphavirus large cassettes detected by ICS against AH1 (top panel) and SIINFEKL (SEQ ID NO: 29362) (bottom panel). Data are presented as IFNg+ cells against model epitopes and as a % of total CD8 cells. *p<0.05, **P<0.01, **P<0.01 by ANOVA with Tukey's test. All p-values ​​were compared to the MAG20 antigen cassette. [Figure 34] Figure 1 shows the vaccination strategy for assessing the immunogenicity of antigen cassette-containing vectors in rhesus macaques. Triangles indicate vaccination with chAd68 (1e12 vp / animal) at weeks 0 and 32. Circles represent alphavirus vaccination at weeks 0, 4, 12, 12, 20, 28, and 32. Squares represent administration of anti-CTLA-4 antibody. [Figure 35] 1 shows the time course of CD8+ anti-epitope responses in rhesus monkeys administered with chAd-MAG alone (Group 4). The mean SFC / 1e6 splenocytes is shown. [Figure 36] 1 shows the time course of CD8+ anti-epitope responses in rhesus macaques (Group 5) administered chAd-MAG and an anti-CTLA4 antibody (ipilimumab) by IV administration. The mean SFC / 1e6 splenocytes is shown. [Figure 37]1 shows the time course of CD8+ anti-epitope responses in rhesus macaques (Group 6) administered with chAd-MAG and an anti-CTLA4 antibody (ipilimumab) by SC administration. The mean SFC / 1e6 splenocytes is shown. [Figure 38] Figure 1 shows antigen-specific memory responses generated by the chAdV68 / samRNA vaccine protocol as measured by ELISpot. Results are shown as individual dot plots, with each dot representing one animal. Pre-immunization baseline (left panel) and memory responses 18 months post-prime (right panel) are shown. [Figure 39] FIG. 1 shows memory cell phenotyping of antigen-specific CD8+ T cells by flow cytometry using combinatorial tetramer staining and CD45RA / CCR7 co-staining. [Figure 40] The distribution of memory cell types among the four total Mamu-A*01 tetramer CD8 T cell populations at month 18 of the study is shown. Memory cells were characterized as follows: CD45RA CCR7 = naive, CD45RA CCR7 = effector (Teff), CD45RA CCR7 = central memory (Tcm), and CD45RA CCR7 = effector memory (Tem). [Figure 41] The frequency of CD8+ T cells recognizing the CT26 tumor antigen AH1 in CT26 tumor-bearing mice is shown. P values ​​were determined using one-way ANOVA with Tukey's multiple comparison test (**P<0.001, *P<0.05). ChAdV = ChAdV68.5WTnt.MAG25mer; aCTLA4 = anti-CTLA4 antibody, clone 9D9. DETAILED DESCRIPTION OF THE INVENTION

[0101] Detailed Description I. Definition In general, terms used in the claims and the specification shall be interpreted as having their ordinary meaning as understood by one of ordinary skill in the art. Certain terms are defined below to provide further clarity. If there is a conflict between the ordinary meaning and a given definition, the given definition shall control.

[0102] As used herein, the term "antigen" refers to a substance that induces an immune response. An antigen may be a neoantigen. An antigen may be a "shared antigen," which is an antigen found in a particular population, for example, a particular population of cancer patients.

[0103] As used herein, the term "neoantigen" refers to an antigen that has at least one change that makes it different from the corresponding wild-type antigen, for example, due to a tumor cell mutation or a tumor cell-specific post-translational modification. Neoantigens may include polypeptide or nucleotide sequences. Mutations can include frameshift or non-frameshift insertion / deletion (indel), missense or nonsense substitution, splice site alteration, genomic rearrangement or gene fusion, or any genomic or expression change that results in a new ORF. Mutations can also include splice variants. Tumor cell-specific post-translational modifications can include aberrant phosphorylation. Tumor cell-specific post-translational modifications can also include splice antigens generated by the proteasome. See Liepe et al., "A large fraction of HLA class I ligands are proteasome-generated spliced ​​peptides"; Science. 2016 Oct 21;354(6310):354-358. Exemplary shared neoantigens are shown in Table A and in the AACR GENIE results (SEQ ID NOS: 10755-29357), with the corresponding HLA allele(s) for each antigen. Such shared neoantigens are useful for inducing an immune response in subjects upon administration. Subjects can be identified for administration by various diagnostic methods, such as the patient selection methods described further below.

[0104] As used herein, the term "tumor antigen" is an antigen that is present in tumor cells or tissues of a subject but not in the corresponding normal cells or tissues of the subject, or an antigen derived from a polypeptide that is known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues.

[0105] As used herein, the term "antigen-based vaccine" refers to a vaccine composition based on one or more antigens, e.g., multiple antigens. The vaccine may be nucleotide-based (e.g., virus-based, RNA-based, or DNA-based), protein-based (e.g., peptide-based), or a combination thereof.

[0106] As used herein, the term "candidate antigen" refers to a mutation or other abnormality that results in a sequence that may represent an antigen.

[0107] As used herein, the term "coding region" refers to the portion of a gene that codes for a protein.

[0108] As used herein, the term "coding mutation" refers to a mutation that occurs in a coding region.

[0109] As used herein, the term "ORF" means open reading frame.

[0110] As used herein, the term "neo-ORF" refers to a tumor-specific ORF that arises due to mutation or other abnormalities such as splicing.

[0111] As used herein, the term "missense mutation" is a mutation that results in the substitution of one amino acid for another.

[0112] As used herein, the term "nonsense mutation" is a mutation that results in the substitution of an amino acid with a stop codon or the removal of the canonical start codon.

[0113] As used herein, the term "frameshift mutation" is a mutation that causes an alteration in the frame of a protein.

[0114] As used herein, the term "indel" refers to the insertion or deletion of one or more nucleic acids.

[0115] As used herein, the term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences in which a certain percentage of nucleotides or amino acid residues are the same when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to those of skill in the art), or by visual inspection. Depending on the application, the "percent identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or over the full length of the two sequences being compared.

[0116] In sequence comparison, generally, one sequence serves as a reference sequence to which test sequences are compared.When using a sequence comparison algorithm, test sequences and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity (%) of the test sequence to the reference sequence based on the designated program parameters.Alternatively, sequence similarity or difference can also be established by the combination of the presence or absence of a specific nucleotide at a selected sequence position (e.g., sequence motif) or an amino acid in a translated sequence.

[0117] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0118] One example of an algorithm that is suitable for determining percent sequence identity and percent sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0119] As used herein, the term "non-stop or read-through" refers to a mutation that results in the removal of the natural stop codon.

[0120] As used herein, the term "epitope" refers to a specific portion of an antigen that is typically bound by an antibody or T-cell receptor.

[0121] As used herein, the term "immunogenic" refers to the ability to elicit an immune response, for example, via T cells, B cells, or both.

[0122] As used herein, the terms "HLA binding affinity" and "MHC binding affinity" refer to the affinity of binding between a specific antigen and a specific MHC allele.

[0123] As used herein, the term "bait" refers to a nucleic acid probe used to enrich a specific sequence of DNA or RNA from a sample.

[0124] As used herein, the term "variant" refers to a difference between a nucleic acid of interest and a reference human genome used as a control.

[0125] As used herein, the term "variant calling" is the algorithmic determination, typically from sequencing, of the presence of a variant.

[0126] As used herein, the term "polymorphism" refers to a germline variant, i.e., a variant found in all DNA-bearing cells of an individual.

[0127] As used herein, the term "somatic variant" is a variant that occurs in the non-germline cells of an individual.

[0128] As used herein, the term "allele" refers to one version of a gene or one version of a gene sequence or one version of a protein.

[0129] As used herein, the term "HLA type" refers to the complement of HLA gene alleles.

[0130] As used herein, the term "nonsense-mediated decay" or "NMD" refers to the degradation of mRNA by the cell due to a premature stop codon.

[0131] As used herein, the term "truncal mutation" is a mutation that occurs early in the development of a tumor and is present in the majority of the cells of the tumor.

[0132] As used herein, the term "subclonal mutation" is a mutation that occurs late in the development of a tumor and is present in only a portion of the cells of the tumor.

[0133] As used herein, the term "exome" refers to the subset of the genome that encodes proteins. The exome can be the collection of exons of the genome.

[0134] As used herein, the term "logistic regression" is a regression model for binary data from statistics in which the logit of the probability that the dependent variable is equal to 1 is modeled as a linear function of the dependent variable.

[0135] As used herein, the term "neural network" refers to a machine learning model for classification or regression that consists of multiple layers of linear transformations followed by element-wise nonlinear transformations typically trained by stochastic gradient descent and backpropagation.

[0136] As used herein, the term "proteome" refers to the set of all proteins expressed and / or translated by a cell, a group of cells, or an individual.

[0137] As used herein, the term "peptidome" refers to the set of all peptides presented by MHC-I or MHC-II on the cell surface. Peptidome can also refer to the properties of a cell or a collection of cells (e.g., a tumor peptidome refers to the union of the peptidomes of all cells that comprise a tumor).

[0138] As used herein, the term "ELISPOT" refers to enzyme-linked immunosorbent spot assay, a common method for monitoring immune responses in humans and animals.

[0139] As used herein, the term "dextramer" refers to a dextran-based peptide-MHC multimer used for antigen-specific T cell staining in flow cytometry.

[0140] As used herein, the term "tolerance or immune tolerance" refers to a state of immune unresponsiveness to one or more antigens, eg, self-antigens.

[0141] As used herein, the term "central tolerance" is tolerance conferred in the thymus by either deleting autoreactive T cell clones or promoting their differentiation into immunosuppressive regulatory T cells (Tregs).

[0142] As used herein, the term "peripheral tolerance" refers to tolerance conferred in the peripheral system by downregulating or anergizing autoreactive T cells that survive central tolerance or by promoting the differentiation of these T cells into Tregs.

[0143] The term "sample" can include a single cell, or multiple cells, or fragments of cells, or an aliquot of bodily fluid obtained from a subject by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspiration, lavage sample, scraping, surgical incision, or intervention, or other means known in the art.

[0144] The term "subject" includes cells, tissues, or organisms, human or non-human, whether male or female, in vivo, ex vivo, or in vitro. The term subject includes mammals, including humans.

[0145] The term "mammal" encompasses both humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0146] The term "clinical factor" refers to a measurement of a subject's condition, e.g., disease activity or severity. "Clinical factor" encompasses all markers of a subject's health status, including non-sample markers, and / or other characteristics of the subject, such as, but not limited to, age and sex. A clinical factor can be a score, value, or set of values ​​that can be obtained from assessing a subject or a sample (or a population of samples) from a subject under a given condition. A clinical factor can also be predicted by other parameters, such as markers and / or gene expression surrogates. Clinical factors can include tumor type, tumor subtype, and smoking history.

[0147] "Tumor-derived antigen-encoding nucleic acid sequence" refers to nucleic acid sequence extracted directly from the tumor, e.g., by RT-PCR, or sequence data obtained by sequencing the tumor and then using the sequencing data to synthesize nucleic acid sequence, e.g., by various synthetic or PCR-based methods well known in the art.

[0148] The term "alphavirus" refers to members of the Togaviridae family, which are single-stranded, positive-sense RNA viruses. Alphaviruses are generally classified as Old World types, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World types, such as Eastern equine encephalitis virus, Aura, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83. Alphaviruses are generally self-replicating RNA viruses.

[0149] The term "alphavirus backbone" refers to the minimal sequence(s) of an alphavirus that allows for autonomous replication of the viral genome. The minimal sequences can include conserved sequences for nonstructural protein-mediated amplification, the nonstructural protein 1 (nsP1), nsP2, nsP3, nsP4 genes, and polyA sequences, as well as sequences for expression of subgenomic viral RNA, including the 26S promoter element.

[0150] The term "conserved sequence for nonstructural protein-mediated amplification" includes alphavirus conserved sequence elements (CSEs) well known in the art, including, but not limited to, the alphavirus 5' UTR, a 51-nt CSE, a 24-nt CSE, or other 26S subgenomic promoter sequence, a 19-nt CSE, and the alphavirus 3' UTR.

[0151] The term "RNA polymerase" includes polymerases that catalyze the production of an RNA polynucleotide from a DNA template, including, but not limited to, bacteriophage-derived polymerases, including T3, T7, and SP6.

[0152] The term "lipid" includes hydrophobic and / or amphipathic molecules. Lipids may be cationic, anionic, or neutral. Lipids may be synthetic or naturally derived, and in certain instances may be biodegradable. Lipids may include cholesterol, phospholipids, lipid conjugates, including but not limited to polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, fats, and fat-soluble vitamins. Lipids may also include dilinoleylmethyl-4-dimethylaminobutyrate (MC3) and MC3-like molecules.

[0153] The term "lipid nanoparticle" or "LNP" includes vesicle-like structures, also known as liposomes, formed with a lipid-containing membrane surrounding an aqueous interior. Lipid nanoparticles include lipid-based compositions with a solid lipid core stabilized by surfactants. The core lipid can be fatty acids, acylglycerols, waxes, and mixtures of these surfactants. Biological membrane lipids, such as phospholipids, sphingomyelin, bile acids (taurocholate), and sterols (cholesterol), can be used as stabilizers. Lipid nanoparticles can be formed using defined ratios of different lipid molecules, including, but not limited to, defined ratios of one or more cationic, anionic, or neutral lipids. Lipid nanoparticles can encapsulate molecules within their outer membrane shell and then be contacted with target cells to deliver the encapsulated molecules to the host cell cytosol. Lipid nanoparticles can be modified or functionalized with non-lipid molecules, such as their surface. Lipid nanoparticles can be monolamellar (unilamellar) or multilamellar (multilamellar). Lipid nanoparticles can be complexed with nucleic acids. Unilamellar lipid nanoparticles can be complexed with nucleic acids, where the nucleic acid is in the aqueous interior. Multilamellar lipid nanoparticles can be complexed with nucleic acids, where the nucleic acid is in the aqueous interior, or forming or sandwiched between them.

[0154] Abbreviations: MHC: major histocompatibility complex; HLA: human leukocyte antigen, or human MHC locus; NGS: next-generation sequencing; PPV: positive predictive value; TSNA: tumor-specific neoantigen; FFPE: formalin-fixed, paraffin-embedded; NMD: nonsense-mediated decay; NSCLC: non-small cell lung cancer; DC: dendritic cell.

[0155] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0156] Unless otherwise specified or clear from the context, the term "about" used herein is understood to be within the range of normal tolerance in the art, for example, within two standard deviations from the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​set forth herein are modified by the word "about."

[0157] Terms not directly defined herein should be understood to have the meanings generally associated with them as understood within the technical field of the present invention. Certain terms are discussed herein to provide further guidance to the practitioner in describing the compositions, devices, methods, etc. of embodiments of the present invention, as well as how to make or use them. It will be recognized that multiple ways of saying the same thing may be used. Accordingly, alternative terms and synonyms may be used for any one or more of the terms discussed herein. No weight should be placed on whether a term is detailed or discussed herein. Several synonyms or alternative methods, materials, etc. are provided. The recitation of one or more synonyms or equivalents does not exclude the use of other synonyms or equivalents, unless expressly stated. The use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the inventive embodiments herein.

[0158] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0159] II. Methods for Identifying Antigens Methods for identifying shared antigens (e.g., neoantigens) include identifying antigens from a subject's tumor that are likely to be presented on the cell surface of tumor or immune cells, including professional antigen-presenting cells such as dendritic cells, and / or that are likely to be immunogenic. By way of example, one such method includes: obtaining at least one of exome, transcriptome, or whole genome tumor nucleotide sequencing and / or expression data from tumor cells of the subject, wherein the tumor nucleotide sequencing and / or expression data is used to obtain data representing the peptide sequences of each of a set of antigens (e.g., in the case of neoantigens, the peptide sequence of each neoantigen contains at least one alteration that makes the peptide sequence different from the corresponding wild-type parent peptide sequence, or in the case of unmutated shared antigens, the peptides are derived from any polypeptide known or found to be altered in expression in tumor cells or cancerous tissues compared to normal cells or tissues); inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical likelihoods that each of the antigens will be presented by one or more MHC alleles on the tumor cell surface of tumor cells of the subject or by cells present within the tumor, wherein the set of numerical likelihoods has been determined based at least on the received mass spectrometry data; selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens; may include:

[0160] The proposed model can include a statistical regression or machine learning (e.g., deep learning) model trained with a set of reference data (also referred to as a training dataset) including a corresponding set of labels, the set of reference data optionally being obtained from each of a plurality of separate subjects, some of whom may have tumors, and the set of reference data includes at least one of data representing exome nucleotide sequences from tumor tissue, data representing exome nucleotide sequences from normal tissue, data representing transcriptome nucleotide sequences from tumor tissue, data representing proteome sequences from tumor tissue, and data representing MHC peptidome sequences from tumor tissue, and data representing MHC peptidome sequences from normal tissue. The reference data can further include mass spectrometry data, sequencing data, RNA sequencing data, expression profiling data, and proteomics data of synthetic proteins, normal and tumor human cell lines, and single-allelic cell lines engineered to express a predetermined MHC allele that are subsequently exposed to fresh and frozen primary samples, as well as T cell assays (e.g., ELISPOT). In certain embodiments, the set of reference data includes each form of reference data.

[0161] The proposed model can include a set of features derived at least in part from a set of reference data, the set of features including at least one of an allele-dependent feature and an allele-independent feature. In certain embodiments, each feature is included.

[0162] The method for identifying shared antigens also includes generating output for constructing a personalized cancer vaccine by identifying one or more antigens from one or more tumor cells of a subject that are likely to be presented on the surface of the tumor cells. By way of example, one such method includes obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing and / or expression data from tumor cells and normal cells of a subject, wherein the nucleotide sequencing and / or expression data includes a set of identified antigens (e.g., in the case of neoantigens, the peptide sequence of each neoantigen includes at least one alteration that makes the peptide sequence different from the corresponding wild-type parent peptide sequence, or in the case of shared antigens without mutations, a peptide sequence that contains at least one alteration that makes the peptide sequence different from the corresponding wild-type parent peptide sequence) by comparing the nucleotide sequencing and / or expression data from the tumor cells with the nucleotide sequencing and / or expression data from normal cells and peptide sequences identified from the normal cells of the subject. a step of obtaining data representing a peptide sequence of each of the antigens (e.g., a polypeptide derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues); a step of encoding the peptide sequence of each of the antigens into a corresponding numerical vector, each numerical vector including information about a set of amino acids constituting the peptide sequence and the positions of the amino acids within the peptide sequence; a step of inputting the numerical vectors into a deep learning presentation model using a computer processor to generate a set of presentation likelihoods for the set of antigens, wherein each presentation likelihood in the set represents the likelihood that a corresponding antigen will be presented on the surface of the tumor cells of the subject by one or more class II MHC alleles, and the deep learning presentation model selects a subset of the set of antigens based on the set of presentation likelihoods to generate a set of selected antigens; and a step of generating the output for constructing the personalized cancer vaccine based on the set of selected antigens.

[0163] Specific methods for identifying antigens, including neoantigens, are well known to those of skill in the art and are described in more detail, for example, in International Patent Application Publication Nos. WO / 2017 / 106638, WO / 2018 / 195357, and WO / 2018 / 208856, each of which is incorporated by reference in its entirety for all purposes.

[0164] Disclosed herein is a method for treating a subject with a tumor, comprising performing any of the steps of the antigen identification methods described herein, and further comprising obtaining a tumor vaccine comprising a selected set of antigens, and administering the tumor vaccine to the subject.

[0165] The methods disclosed herein can also further include identifying one or more T cells that are antigen-specific for at least one of the antigens in the subset. In some embodiments, the identifying comprises co-culturing one or more T cells with one or more of the antigens in the subset under conditions that expand the one or more antigen-specific T cells. In further embodiments, the identifying comprises contacting one or more T cells with a tetramer comprising one or more of the antigens in the subset under conditions that allow binding of the T cells to the tetramer. In even further embodiments, the methods disclosed herein can also further comprise identifying one or more T cell receptors (TCRs) of the one or more identified T cells. In certain embodiments, identifying one or more T cell receptors comprises sequencing the T cell receptor sequences of the one or more identified T cells. The methods disclosed herein can further comprise genetically engineering a plurality of T cells to express at least one of the one or more identified T cell receptors, culturing the plurality of T cells under conditions that expand the plurality of T cells, and infusing the expanded T cells into a subject. In some embodiments, engineering a plurality of T cells to express at least one of the one or more identified T cell receptors comprises cloning the T cell receptor sequences of the one or more identified T cells into an expression vector and transfecting each of the plurality of T cells with the expression vector. In certain embodiments, the methods disclosed herein further comprise culturing the one or more identified T cells under conditions that expand the one or more T cells and injecting the expanded T cells into a subject.

[0166] Also disclosed herein are isolated T cells that are antigen-specific for at least one selected antigen within said subset.

[0167] Also provided herein is a method for producing a tumor vaccine, comprising: obtaining at least one of exome, transcriptome, or whole genome tumor nucleotide sequencing and / or expression data from tumor cells of the subject, wherein the tumor nucleotide sequencing and / or expression data is used to obtain data representing the peptide sequences of each of a set of antigens (e.g., in the case of neoantigens, the peptide sequence of each neoantigen includes at least one alteration that makes the peptide sequence different from the corresponding wild-type parent peptide sequence, or in the case of unmutated shared antigens, the peptides are derived from any polypeptide known or found to be altered in expression in tumor cells or cancerous tissues compared to normal cells or tissues); inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical likelihoods that each of the antigens will be presented by one or more MHC alleles on the tumor cell surface of the tumor cells of the subject, wherein the set of numerical likelihoods was determined based at least on the received mass spectrometry data; selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens; producing or having produced a tumor vaccine comprising said selected set of antigens; Also disclosed is a method comprising:

[0168] Also herein, obtaining at least one of exome, transcriptome, or whole genome tumor nucleotide sequencing and / or expression data from tumor cells of the subject, wherein the tumor nucleotide sequencing and / or expression data is used to obtain data representing the peptide sequences of each of a set of antigens, wherein the peptide sequence for each antigen (e.g., in the case of neoantigens, the peptide sequence for each neoantigen includes at least one alteration that makes the peptide sequence different from the corresponding wild-type parent peptide sequence, or in the case of unmutated shared antigens, the peptide is derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissue compared to normal cells or tissue); inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical likelihoods that each of the antigens will be presented by one or more MHC alleles on the tumor cell surface of the tumor cells of the subject, wherein the set of numerical likelihoods was determined based at least on the received mass spectrometry data; selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens; producing or having produced a tumor vaccine comprising said selected set of antigens; Also disclosed is a tumor vaccine comprising a set of selected antigens selected by performing a method comprising:

[0169] The tumor vaccine may comprise one or more of a nucleotide sequence, a polypeptide sequence, RNA, DNA, a cell, a plasmid, or a vector.

[0170] A tumor vaccine may comprise one or more antigens displayed on the surface of tumor cells.

[0171] A tumor vaccine may comprise one or more antigens that are immunogenic in a subject.

[0172] A tumor vaccine may not include one or more antigens that induce an autoimmune response in a subject against normal tissue.

[0173] The tumor vaccine may include an adjuvant.

[0174] The tumor vaccine may include an excipient.

[0175] The methods disclosed herein may also include selecting antigens that have an increased likelihood of being presented on the tumor cell surface compared to antigens that are not selected based on the presentation model.

[0176] The methods disclosed herein may also include selecting an antigen that has an increased likelihood of being able to induce a tumor-specific immune response in a subject compared to an antigen that is not selected based on the presentation model.

[0177] The methods disclosed herein may also include selecting an antigen that has an increased likelihood of being presented to naive T cells by a professional antigen-presenting cell (APC) compared to an antigen that is not selected based on the presentation model, and optionally the APC is a dendritic cell (DC).

[0178] The methods disclosed herein may also include selecting antigens that have a reduced likelihood of being inhibited by central or peripheral tolerance compared to antigens that are not selected based on the presentation model.

[0179] The methods disclosed herein may also include selecting an antigen that has a reduced likelihood of being able to induce an autoimmune response against normal tissue in a subject compared to an antigen that is not selected based on the presentation model.

[0180] Nucleotide sequencing and / or expression data of the exome or transcriptome can be obtained by sequencing tumor tissue.

[0181] Sequencing may be next generation sequencing (NGS) or any massively parallel sequencing approach.

[0182] The set of numerical likelihoods can be further specified by at least MHC allele interaction properties, including at least one of the following: predicted binding affinity between the MHC allele and the antigen-encoding peptide; predicted stability of the antigen-encoding peptide-MHC complex; sequence and length of the antigen-encoding peptide; probability of presentation of an antigen-encoding peptide with a similar sequence in cells from other individuals expressing the particular MHC allele, as assessed by mass spectrometry proteomics or other means; expression level of the particular MHC allele in the subject of interest (e.g., as measured by RNA-seq or mass spectrometry); probability of presentation by a particular MHC allele in other distinct individuals expressing the particular MHC allele, independent of the overall neoantigen-encoding peptide sequence; probability of presentation by MHC alleles of the same molecular family (e.g., HLA-A, HLA-B, HLA-C, HLA-DQ, HLA-DR, HLA-DP) in other distinct subjects, independent of the overall neoantigen-encoding peptide sequence.

[0183] The set of numerical likelihoods is further specified by at least MHC allele non-interacting properties, including at least one of the following: C-terminal and N-terminal sequences flanking the neoantigen-encoded peptide within its source protein sequence; the presence of protease cleavage motifs within the neoantigen-encoded peptide, optionally weighted according to the expression of the corresponding protease in tumor cells (as measured by RNA-seq or mass spectrometry); the turnover rate of the source protein measured in the appropriate cell type; the tumor specificity measured by RNA-seq or proteomic mass spectrometry or predicted from the annotation of germline or somatic splicing variants detected in DNA or RNA sequence data. the length of the source protein, optionally taking into account the particular splice variants ("isoforms") that are most highly expressed in tumor cells; the level of expression of the proteasome, immunoproteasome, thymoproteasome, or other proteases in tumor cells (which can be measured by RNA-seq, proteome mass spectrometry, or immunohistochemistry); the expression of the source gene of the neoantigen-encoding peptide (e.g., measured by RNA-seq or mass spectrometry); the typical tissue-specific expression of the source gene of the neoantigen-encoding peptide in different stages of the cell cycle; e.g., as determined by uniProt or PDB a comprehensive catalog of the properties of the source protein and / or its domains, such as can be found at http: / / www.rcsb.org / pdb / home / home.do; properties describing the nature of the source protein domain containing the peptide, e.g., secondary or tertiary structure (e.g., alpha helix versus beta sheet); alternative splicing; the probability of presentation of the peptide derived from the source protein of the neoantigen-encoded peptide of interest in other distinct subjects; the probability that the peptide will be undetected or over-represented by mass spectrometry due to technical bias; the expression of various gene modules / pathways (not necessarily including the source protein of the peptide) as measured by RNASeq, which gives information about the status of tumor cells, stroma, or tumor-infiltrating lymphocytes (TILs); the copy number of the source gene of the neoantigen-encoded peptide in tumor cells;The probability that the peptide will bind to TAP, or the measured or predicted binding affinity of the peptide to TAP; the expression level of TAP in tumor cells (which can be measured by RNA-seq, proteome mass spectrometry, immunohistochemistry); the presence or absence of tumor mutations, including, but not limited to, driver mutations in known cancer driver genes such as EGFR, KRAS, ALK, RET, ROS1, TP53, CDKN2A, CDKN2B, NTRK1, NTRK2, NTRK3, and genes encoding proteins involved in antigen presentation machinery (e.g., B2M, HLA- A, HLA-B, HLA-C, TAP-1, TAP-2, TAPBP, CALR, CNX, ERP57, HLA-DM, HLA-DMA, HLA-DMB, HLA-DO, HLA-DOA, HLA-DOB, HLA-DP, HLA-DPA1, HLA-DPB1, HLA-DQ, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DR, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, or any of the genes encoding components of the proteasome or immunoproteasome. Peptides whose presentation depends on components of the antigen presentation machinery that carry loss-of-function mutations in the tumor have a low probability of presentation; including, but not limited to, the presence or absence of functional germline polymorphisms in genes encoding proteins involved in antigen presentation machinery (e.g., B2M, HLA-A, HLA-B, HLA-C, TAP-1, TAP-2, TAPBP, CALR, CNX, ERP57, HLA-DM, HLA-DMA, HLA-DMB, HLA-DO, HLA-DOA, HLA-DOB, HLA-D ... polymorphisms in HLA-DP, HLA-DPA1, HLA-DPB1, HLA-DQ, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DR, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, or any of the genes encoding components of the proteasome or immunoproteasome; tumor type (e.g., NSCLC, melanoma); clinical tumor subtype (e.g., squamous cell lung cancer vs. non-squamous); smoking history;Typical expression of the peptide's source gene in relevant tumor types or clinical subtypes, optionally stratified by driver mutations;

[0184] The at least one mutation may be a frameshift or non-frameshift indel, a missense or nonsense substitution, a splice site alteration, a genomic rearrangement or gene fusion, or any genomic or expression alteration that results in a de novo ORF.

[0185] The tumor cells can be selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and T-lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0186] The methods disclosed herein may also include obtaining a tumor vaccine comprising the selected set of neoantigens or a subset thereof, and may optionally further include administering the tumor vaccine to a subject.

[0187] At least one of the neoantigens in the set of selected neoantigens, when in polypeptide form, may comprise at least one of the following: a binding affinity to MHC with an IC50 value of less than 1000 nM; a length of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids for MHC class I polypeptides; or a length of 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids for MHC class II polypeptides. , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length, the presence of a sequence motif within or near the polypeptide in the parent protein sequence that promotes proteasomal cleavage, and the presence of a sequence motif that promotes TAP transport, and for MHC class II, the presence of a sequence motif within or near the peptide-promoted cleavage site by extracellular or lysosomal proteases (e.g., cathepsins) or the HLA-binding site catalyzed by HLA-DM.

[0188] Disclosed herein is a method for identifying one or more neoantigens likely to be presented on the tumor cell surface of tumor cells, the method comprising the steps of: receiving mass spectrometry data including data relating to a plurality of isolated peptides eluted from major histocompatibility complex (MHC) antigens from a plurality of fresh or frozen samples; obtaining a training dataset by identifying at least a set of training peptide sequences present in the tumor samples and presented on one or more MHC alleles associated with each training peptide sequence; obtaining a set of training protein sequences based on the training peptide sequences; and training a set of numerical parameters of a presentation model using the training protein sequences and the training peptide sequences, wherein the presentation model provides a plurality of numerical likelihoods that a peptide sequence from the tumor cell will be presented by one or more MHC alleles on the tumor cell surface.

[0189] The presentation model can represent the dependency between the presence of a pair of a particular one of the MHC alleles with a particular amino acid at a particular position in a peptide sequence, and the likelihood of presentation of such a peptide sequence comprising said particular amino acid at said particular position on the surface of a tumor cell by said particular one of the MHC alleles of said pair.

[0190] The methods disclosed herein may also include selecting a subset of neoantigens, each of which has an increased likelihood of being presented on the cell surface of a tumor relative to one or more distinct tumor neoantigens.

[0191] The methods disclosed herein may also include selecting a subset of neoantigens, each of which has an increased likelihood of being able to induce a tumor-specific immune response in a subject against one or more distinct tumor neoantigens.

[0192] The methods disclosed herein may also include selecting a subset of neoantigens, each selected for having an increased likelihood relative to one or more distinct tumor neoantigens that they can be presented to naive T cells by professional antigen-presenting cells (APCs), optionally the APCs being dendritic cells (DCs).

[0193] The methods disclosed herein may also include selecting a subset of neoantigens, each of which has a reduced likelihood of being inhibited by central or peripheral tolerance to one or more distinct tumor neoantigens.

[0194] The methods disclosed herein may also include selecting a subset of neoantigens, each of which has a reduced likelihood of inducing an autoimmune response against normal tissue in a subject relative to one or more distinct tumor neoantigens.

[0195] The methods disclosed herein may also include selecting a subset of neoantigens, each selected because it has a reduced likelihood of being differentially post-translationally modified in tumor cells relative to APCs, and optionally, the APCs are dendritic cells (DCs).

[0196] The practice of the methods herein will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are fully explained in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry, 3rd Ed. (Plenum Press), Vols. A and B (1992).

[0197] III. Identification of tumor-specific mutations in neoantigens Also disclosed herein are methods for identifying certain mutations (e.g., variants or alleles present in cancer cells). In particular, these mutations may be present in the genome, transcriptome, proteome, or exome of cancer cells of a subject with cancer, but may not be present in normal tissues derived from the subject. Specific methods for identifying tumor-specific antigens, including shared neoantigens, are well known to those skilled in the art, and are described in more detail in, for example, International Patent Application Publication Nos. WO / 2017 / 106638, WO / 2018 / 195357, and WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.

[0198] Genetic mutations in tumors can be considered useful for immunological targeting of tumors if they result in changes in the amino acid sequence of proteins exclusively in tumors. Useful mutations include: (1) non-synonymous mutations that result in different amino acids in proteins; (2) read-through mutations in which the stop codon is modified or deleted, resulting in the translation of a longer protein with a new tumor-specific sequence at the C-terminus; (3) splice site mutations that result in the inclusion of an intron in mature mRNA, thus resulting in a unique tumor-specific protein sequence; (4) chromosomal rearrangements (i.e., gene fusions) that result in chimeric proteins with tumor-specific sequences at the junction of two proteins; (5) frameshift mutations or deletions that result in new open reading frames with new tumor-specific protein sequences. Mutations can also include one or more of non-frameshift insertions / deletions, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic or expression changes that result in new ORFs.

[0199] For example, mutated peptides or mutated polypeptides resulting from splice site, frameshift, readthrough, or gene fusion mutations in tumor cells can be identified by sequencing DNA, RNA, or protein in tumor versus normal cells.

[0200] Mutations can also include previously identified tumor-specific mutations. Known tumor mutations can be found in the Catalogue of Somatic Mutations in Cancer (COSMIC) database.

[0201] Various methods are available for detecting the presence of specific mutations or alleles in an individual's DNA or RNA. Advances in this field have provided accurate, easy, and inexpensive large-scale SNP genotyping. Several techniques have been described, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and various DNA "chip" technologies such as the Affymetrix SNP chip. These methods utilize amplification of target gene regions, typically by PCR. Still other methods rely on the generation of small signal molecules by invasive cleavage followed by mass spectrometry, or on immobilized padlock probes and rolling circle amplification. Some of the methods known in the art for detecting specific mutations are summarized below.

[0202] PCR-based detection means can involve the multiplex amplification of multiple markers simultaneously.For example, it is well known in the art to select PCR primers so as to generate PCR products that do not overlap in size and can be analyzed simultaneously.Alternatively, it is possible to amplify different markers with primers that are differentially labeled and therefore can be differentially detected.Of course, hybridization-based detection means allows the differential detection of multiple PCR products in a sample.Other techniques that allow multiplex analysis of multiple markers are known in the art.

[0203] Several methods have been developed to facilitate the analysis of single nucleotide polymorphisms in genomic DNA or cellular RNA.For example, single nucleotide polymorphisms can be detected by using special exonuclease-resistant nucleotides, as disclosed in Mundy, CR (US Patent No. 4,656,127).According to this method, a primer complementary to the allele sequence immediately 3' of the polymorphic site is hybridized to a target molecule obtained from a specific animal or human.If the polymorphic site on the target molecule contains a nucleotide that is complementary to the specific exonuclease-resistant nucleotide derivative present, this derivative will be incorporated onto the end of the hybridized primer.This incorporation makes the primer resistant to exonucleases, thereby enabling its detection.Since the identity of the exonuclease-resistant derivative of the sample is known, the knowledge that the primer has become resistant to exonucleases reveals that the nucleotide present at the polymorphic site of the target molecule is complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of exogenous sequence data.

[0204] Solution-based methods can be used to determine the identity of the nucleotide at a polymorphic site (Cohen, D. et al. (French Patent No. 2,650,840; PCT Application No. WO 91 / 02087)). As in the method of Mundy, U.S. Pat. No. 4,656,127, a primer is used that is complementary to the allelic sequence immediately 3' to the polymorphic site. This method uses a labeled dideoxynucleotide derivative that becomes incorporated onto the end of the primer if it is complementary to the nucleotide at the polymorphic site to determine the identity of the nucleotide at that site.

[0205] An alternative method known as Genetic Bit Analysis or GBA has been described by Goelet, P. et al. (PCT Application No. 92 / 15712). The method of Goelet, P. et al. uses a mixture of labeled terminators and primers that are complementary to the sequence 3' of the polymorphic site. The incorporated labeled terminators are determined by and complementary to the nucleotides present at the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent No. 2,650,840; PCT Application No. WO91 / 02087), the method of Goelet, P. et al. can be a heterogeneous phase assay in which the primers or target molecules are immobilized on a solid phase.

[0206] Several primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Komher, J. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990); Kuppuswamy, M. et al., Proc. Natl. Acad. Sci. (USA) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al. al., Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA in that they utilize the incorporation of labeled deoxynucleotides to discriminate between bases at the polymorphic site. In such formats, signal is proportional to the number of incorporated deoxynucleotides, so that polymorphisms occurring in runs of the same nucleotide can result in a signal proportional to the length of the run (Syvanen, A.-C., et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0207] Numerous initiatives obtain sequence information directly from millions of individual molecules of DNA or RNA in parallel. Real-time single-molecule sequencing by synthesis techniques rely on the detection of fluorescent nucleotides as they are incorporated into nascent strands of DNA complementary to the template being sequenced. In one method, oligonucleotides 30–50 bases in length are covalently anchored at their 5′ ends to glass coverslips. These anchored strands serve two functions. First, they act as capture sites for the target template strands when the template is constructed with a capture tail complementary to the surface-bound oligonucleotide. They also act as primers for template-directed primer extension, which forms the basis for sequence reading. The capture primers serve as fixed-location sites for sequencing using multiple cycles of synthesis, detection, and chemical cleavage of the dye-linker to remove the dye. Each cycle consists of the addition of a polymerase / labeled nucleotide mixture, rinsing, imaging, and dye cleavage. In an alternative method, the polymerase is modified with a fluorescent donor molecule and immobilized on a glass slide, while each nucleotide is color-coded with an acceptor fluorescent moiety attached to the γ-phosphate. As the nucleotide becomes incorporated into the new strand, the system detects the interaction between the fluorescently tagged polymerase and the fluorescently modified nucleotide. Other sequencing-by-synthesis techniques also exist.

[0208] Any suitable sequencing-by-synthesis platform can be used to identify mutations. As mentioned above, four major sequencing-by-synthesis platforms are currently available: the Genome Sequencer sold by Roche / 454 Life Sciences, the 1G Analyzer sold by Illumina / Solexa, the SOLiD system sold by Applied BioSystems, and the Heliscope system sold by Helicos Biosciences. Sequencing-by-synthesis platforms have also been described by Pacific BioSciences and VisiGen Biotechnologies. In some embodiments, multiple nucleic acid molecules to be sequenced are attached to a support (e.g., a solid support). To immobilize the nucleic acid on the support, a capture sequence / universal priming site can be added to the 3' and / or 5' end of the template. The nucleic acid can be bound to the support by hybridizing the capture sequence to a complementary sequence covalently attached to the support. The capture sequence (also called a universal capture sequence) is a nucleic acid sequence complementary to the sequence attached to the support, which can double as a universal primer.

[0209] As an alternative to capture sequences, a member of a coupling pair (e.g., antibody / antigen, receptor / ligand, or avidin-biotin pair, e.g., as described in U.S. Patent Application Publication No. 2006 / 0252077) can be linked to each fragment and captured on a surface coated with the respective second member of the coupling pair.

[0210] Following capture, the sequence can be analyzed by single-molecule detection / sequencing, including, for example, template-dependent sequencing by synthesis, as described, for example, in the Examples and in U.S. Patent No. 7,283,337. In sequencing by synthesis, surface-bound molecules are exposed to a multitude of labeled nucleotide triphosphates in the presence of a polymerase. The sequence of the template is determined by the order of labeled nucleotides incorporated into the 3' end of the growing strand. This can be done in real time, in a step-and-repeat mode. For real-time analysis, a different optical label can be incorporated for each nucleotide, and multiple lasers can be utilized for stimulation of the incorporated nucleotides.

[0211] Sequencing can also include other massively parallel sequencing or next-generation sequencing (NGS) techniques and platforms. Additional examples of massively parallel sequencing techniques and platforms are Illumina HiSeq or MiSeq, ThermoPGM or Proton, Pac Bio RS II or Sequel, Qiagen's Gene Reader, and Oxford Nanopore MinION. Additional similar current massively parallel sequencing technologies, and future generations of these technologies, can be used.

[0212] Any cell type or tissue can be used to obtain nucleic acid samples for use in the methods described herein.For example, DNA or RNA samples can be obtained from tumor or body fluids, for example, blood obtained by known techniques (for example, venipuncture) or saliva.Alternatively, nucleic acid testing can be performed on dry samples (for example, hair or skin).In addition, a sample can be obtained from tumor for sequencing, and another sample can be obtained from normal tissue for sequencing, if the normal tissue is of the same tissue type as tumor.A sample can be obtained from tumor for sequencing, and another sample can be obtained from normal tissue for sequencing, if the normal sample is of a different tissue type from tumor.

[0213] The tumor may include one or more of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and T-lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0214] Alternatively, protein mass spectrometry can be used to identify or demonstrate the presence of mutated peptides bound to MHC proteins on tumor cells. Peptides can be acid-eluted from tumor cells or from HLA molecules immunoprecipitated from tumors, and then identified using mass spectrometry.

[0215] IV. Antigen An antigen can comprise a nucleotide or a polynucleotide. For example, an antigen can be an RNA sequence that encodes a polypeptide sequence. Antigens useful in vaccines can therefore comprise a nucleotide sequence or a polypeptide sequence. Shared neoantigens are shown in Table A (see SEQ ID NOS: 10755-21015) and the AACR GENIE results (see SEQ ID NOS: 21016-29357). Shared antigens are shown in Table 1.2 (see SEQ ID NOS: 57-10754).

[0216] Disclosed herein are isolated peptides comprising tumor-specific mutations identified by the methods disclosed herein, peptides comprising known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. Neoantigen peptides can be described in the context of their coding sequences when the neoantigen comprises nucleotide sequences (e.g., DNA or RNA) that encode the associated polypeptide sequence.

[0217] The present specification also discloses peptides derived from any polypeptide known or found to be altered in expression in tumor cells or cancerous tissues compared to normal cells or tissues, for example, any polypeptide known or found to be abnormally expressed in tumor cells or cancerous tissues compared to normal cells or tissues. Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database. COSMIC is a comprehensive curation of somatic mutation information in human cancers. Peptides include tumor-specific mutations.

[0218] The one or more polypeptides encoded by the antigen nucleotide sequence can comprise at least one of the following: a binding affinity to MHC with an IC50 value of less than 1000 nM; a length of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids for MHC class I peptides; the presence of a sequence motif within or near the peptide that promotes proteasomal cleavage and the presence of a sequence motif that promotes TAP transport; a length of 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids for MHC class II polypeptides; the presence of a sequence motif within or near the peptide-promoted cleavage site by an extracellular or lysosomal protease (e.g., cathepsin) or the HLA binding site catalyzed by HLA-DM.

[0219] The one or more antigens can be present on the surface of the tumor.

[0220] The one or more antigens can be immunogenic in a tumor-bearing subject, for example, capable of eliciting a T cell or B cell response in the subject.

[0221] One or more antigens that induce an autoimmune response in a subject can be eliminated from consideration in the context of generating a vaccine for a tumor-bearing subject.

[0222] The size of the at least one antigenic peptide molecule can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, The antigenic peptide molecule can include, but is not limited to, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, or more amino acid residues, and any range derivable therein. In a specific embodiment, the antigenic peptide molecule is 50 amino acids or less.

[0223] Antigenic peptides and polypeptides can be 15 residues or less in length, typically between about 8 and about 11 residues, particularly 9 or 10 residues, for MHC class I; and 6 to 30 residues for MHC class II.

[0224] If desired, longer peptides can be designed in several ways. In one example, if the likelihood of peptide presentation on HLA alleles is predicted or known, the longer peptides can consist of either (1) individual presented peptides with extensions of 2-5 amino acids toward the N- and C-termini of each corresponding gene product; or (2) a concatenation of some or all of the presented peptides, each with its extended sequence. In another example, if sequencing reveals long (more than 10 residues) neo-epitope sequences present in the tumor (e.g., due to frameshifts, readthrough, or intron inclusion resulting in novel peptide sequences), the longer peptides would (3) consist of the entire novel tumor-specific stretch of amino acids, thus avoiding the need for computational or in vitro test-based selection of shorter peptides that are presented to the strongest HLA alleles. In either example, the use of longer peptides may allow for endogenous processing by patient cells, resulting in more effective antigen presentation and induction of T cell responses.

[0225] Antigenic peptides and polypeptides can be presented on HLA proteins. In some embodiments, the antigenic peptides and polypeptides are presented on HLA proteins with stronger affinity than wild-type peptides. In some embodiments, the neoantigenic peptide or polypeptide can have an IC50 of at least 5000 nM, at least 1000 nM, at least 500 nM, at least 250 nM, at least 200 nM, at least 150 nM, at least 100 nM, at least 50 nM, or less.

[0226] In some embodiments, the antigenic peptides and polypeptides do not induce an autoimmune response and / or do not cause immune tolerance when administered to a subject.

[0227] Also provided are compositions comprising at least two or more antigenic peptides. In some embodiments, the composition contains at least two different peptides. The at least two different peptides can be derived from the same polypeptide. Different polypeptides mean that the peptides differ in length, amino acid sequence, or both. The peptides can be derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues, e.g., any polypeptide known or found to contain tumor-specific mutations or peptides derived from any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissues compared to normal cells or tissues. Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database or the AACR GENIE (Genomics Evidence Neoplasia Information Exchange) database. COSMIC curates comprehensive information on somatic mutations in human cancers. AACR GENIE aggregates and links clinical-grade cancer genomic data with clinical outcomes from tens of thousands of cancer patients. The peptides contain tumor-specific mutations. In some embodiments, the tumor-specific mutation is a driver mutation for a particular cancer type.

[0228] Antigenic peptides and polypeptides with desired activities or properties can be modified to confer certain desirable attributes, e.g., improved pharmacological characteristics, while enhancing or at least retaining substantially all of the biological activity of the unmodified peptide, which binds to desired MHC molecules and activates appropriate T cells. For example, antigenic peptides and polypeptides can be further subjected to various modifications, such as conservative or non-conservative substitutions, which may provide certain advantages in their use, such as improved MHC binding, stability, or presentation. Conservative substitutions refer to the replacement of an amino acid residue with another that is biologically and / or chemically similar, e.g., one hydrophobic residue with another hydrophobic residue, or one polar residue with another polar residue. Substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effects of single amino acid substitutions can also be explored using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2nd Ed. (1984).

[0229] Modification of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful for increasing peptide and polypeptide stability in vivo. Stability can be assayed in a number of ways. For example, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Peptide half-life can be conveniently determined using a 25% human serum (v / v) assay. The protocol generally follows: Pooled human serum (type AB, non-heat-inactivated) is defatted by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, small aliquots of the reaction solution are removed and added to either 6% aqueous trichloroacetic acid or ethanol. The cloudy reaction sample is cooled (4°C) for 15 minutes and then spun to precipitate the precipitated serum proteins. The presence of the peptide is then determined by reverse-phase HPLC using stability-specific chromatography conditions.

[0230] Peptides and polypeptides can be modified to provide desirable attributes other than improved serum half-life. For example, the ability of a peptide to induce CTL activity can be enhanced by linking it to a sequence containing at least one epitope capable of inducing a T helper cell response. The immunogenic peptide / T helper conjugate can be linked by a spacer molecule. The spacer is typically composed of relatively small, neutral molecules, such as amino acids or amino acid mimetics, that are substantially uncharged under physiological conditions. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that the optional spacer need not be composed of the same residues and can therefore be a hetero- or homo-oligomer. If present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the peptide can be linked to the T helper peptide without a spacer.

[0231] The antigenic peptide can be linked to the T helper peptide at either the amino or carboxy terminus of the peptide, either directly or via a spacer. The amino terminus of either the antigenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid at 830-843, influenza at 307-319, and malaria sporozoites at around 382-398 and 378-389.

[0232] Proteins or peptides can be produced by any technique known to those of skill in the art, including expressing proteins, polypeptides, or peptides through standard molecular biology techniques, isolating proteins or peptides from natural sources, or chemically synthesizing proteins or peptides. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed and can be found in computerized databases known to those of skill in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, located on the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as known to those of skill in the art. Alternatively, various commercial preparations of proteins, polypeptides, and peptides are known to those of skill in the art.

[0233] In a further embodiment, the antigen comprises a nucleic acid (e.g., a polynucleotide) encoding an antigenic peptide or a portion thereof. The polynucleotide can be, for example, a single-stranded and / or double-stranded polynucleotide, such as DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), or a polynucleotide having a phosphorothioate backbone, either in a natural or stabilized form, or a combination thereof, and may or may not contain introns. Yet a further embodiment provides an expression vector capable of expressing the polypeptide or a portion thereof. Expression vectors for various cell types are well known in the art and can be selected without undue experimentation. Generally, the DNA is inserted into an expression vector, such as a plasmid, in the proper orientation and correct reading frame for expression. If necessary, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host; such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0234] V. Vaccine Compositions Also disclosed herein are immunogenic compositions, e.g., vaccine compositions, that can generate specific immune responses, e.g., tumor-specific immune responses. Vaccine compositions typically contain one or multiple antigens selected using, for example, the methods described herein or in Table A, Table 1.2, or the AACR GENIE results. Vaccine compositions can also be referred to as vaccines.

[0235] The vaccine can contain 1 to 30 different peptides, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different peptides, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different peptides, or 12, 13, or 14 different peptides. The peptides can include post-translational modifications. The vaccine may contain 1 to 100 or more nucleotide sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, It may contain 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different nucleotide sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different nucleotide sequences, or 12, 13, or 14 different nucleotide sequences.The vaccine contains 1 to 30 antigen sequences: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different antigen sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different antigen sequences, or 12, 13, or 14 different antigen sequences.

[0236] In one embodiment, the different peptides and / or polypeptides, or the nucleotide sequences encoding them, are selected such that the peptides and / or polypeptides are capable of binding to different MHC molecules, such as different MHC class I molecules and / or different MHC class II molecules. In some embodiments, a vaccine composition comprises coding sequences for peptides and / or polypeptides capable of binding to the most frequently occurring MHC class I molecules and / or different MHC class II molecules. Thus, the vaccine composition can comprise different fragments capable of binding to at least two preferred, at least three preferred, or at least four preferred MHC class I molecules and / or different MHC class II molecules.

[0237] The vaccine composition may generate a specific cytotoxic T cell response and / or a specific helper T cell response.

[0238] The vaccine composition can further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are provided herein below. The composition can be combined with a carrier, such as a protein, or an antigen-presenting cell, such as a dendritic cell (DC), which can present peptides to T cells.

[0239] An adjuvant is any substance whose incorporation into a vaccine composition enhances or otherwise modifies the immune response to an antigen. The carrier can be a scaffold structure, such as a polypeptide or polysaccharide, to which the antigen can be bound. Optionally, the adjuvant is covalently or non-covalently conjugated.

[0240] The ability of adjuvant to increase the immune response to antigen is typically manifested by a significant or substantial increase in immune-mediated reaction or a reduction in disease symptoms.For example, the increase in humoral immunity is typically manifested by a significant increase in the titer of antibody produced against antigen, and the increase in T cell activity is typically manifested in an increase in cell proliferation, or cellular cytotoxicity, or cytokine secretion.Adjuvant can also change immune response, for example, by changing mainly humoral or Th response to mainly cellular or Th response.

[0241] Suitable adjuvants include 1018 ISS, alum, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA206, Montanide ISA 50V, and Montanide. Adjuvants include, but are not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are also useful. Several immunological adjuvants specific for dendritic cells (e.g., MF59) and their preparations have been previously described (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison AC; Dev Biol Stand. 1998;92:3-11). Cytokines can also be used. Several cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J ImmunotherEmphasis Tumor Immunol. 1996(6):414-418).

[0242] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine settings. Other TLR-binding molecules, such as RNA that binds to TLR 7, TLR 8, and / or TLR 9, may also be used.

[0243] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), Poly(I:C) (e.g., polyi:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives can be readily determined by one of ordinary skill in the art without undue experimentation. Additional adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0244] A vaccine composition can include more than one different adjuvant. Additionally, a therapeutic composition can include any adjuvant material, including any of the above or combinations thereof. It is also contemplated that the vaccine and adjuvant can be administered together or separately in any suitable sequence.

[0245] The carrier (or excipient) can exist independently of the adjuvant. The function of the carrier can be, for example, to increase activity or immunogenicity, to provide stability, to increase biological activity, or to increase serum half-life, particularly to increase the molecular weight of the variant. Furthermore, the carrier can help present the peptide to T cells. The carrier can be any suitable carrier known to those skilled in the art, such as a protein or an antigen-presenting cell. The carrier protein can be, but is not limited to, a serum protein such as keyhole limpet hemocyanin, transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, an immunoglobulin, or a hormone such as insulin, or palmitic acid. For human immunization, the carrier is generally a physiologically acceptable carrier that is tolerated and safe for humans. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier can be a dextran, such as Sepharose.

[0246] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules rather than the intact foreign antigen itself. MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, CTL activation is possible when a trimeric complex of peptide antigen, MHC molecules, and APCs is present. Correspondingly, not only when peptides are used to activate CTLs, but also when APCs bearing the respective MHC molecules are added, it can enhance the immune response. Therefore, in some embodiments, the vaccine composition additionally contains at least one antigen-presenting cell.

[0247] Antigens can also be derived from vaccinia, fowlpox, self-replicating alphaviruses, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including, but not limited to, second, third, or hybrid second / third generation lentiviruses, and any generation of recombinant lentiviruses designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human The ubiquitin C promoter, Nucl. Acids Res. (2015) 43(1):682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72(12):9873-9880), etc. Depending on the packaging capacity of the viral vector-based vaccine platform described above, this approach can deliver one or more nucleotide sequences encoding one or more neoantigen peptides.The sequence may be flanked by non-mutated sequences, separated by linkers, or preceded by one or more sequences that target intracellular compartments (see, e.g., Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22(4):433-8; Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science. (2016) 352(6291):1337-41; Lu et al., Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20( 13):3401-10). Upon introduction into the host, infected cells express the antigen, thereby eliciting a host immune (e.g., CTL) response against the peptide. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette-Guerin). BCG vectors are described by Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful for therapeutic administration of antigens or immunization, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the description herein.

[0248] VA antigen cassette The methods used to select one or more antigens, cloning and construction of a "cassette," and its insertion into a viral vector are within the skill of the art in light of the teachings provided herein. An "antigen cassette" refers to a combination of a selected antigen or antigens with other regulatory elements required to transcribe the antigen(s) and express the transcript. The antigen or antigens can be operably linked to regulatory elements in a manner that allows transcription. Such elements include conventional regulatory elements capable of driving expression of the antigen(s) in cells transfected with the viral vector. Thus, the antigen cassette can also include a selected promoter linked to the antigen(s) and positioned, along with other optional regulatory elements, within the selected viral sequence of the recombinant vector. The cassette can include one or more neoantigens listed in Table A and / or the AACR GENIE results, and / or one or more antigens listed in Table 1.2.

[0249] Useful promoters may be constitutive promoters or regulated (inducible) promoters that allow for control of the amount of antigen(s) expressed. For example, a desirable promoter is the cytomegalovirus immediate early promoter / enhancer [see, e.g., Boshart et al., Cell, 41:521-530 (1985)]. Another desirable promoter is the Rous sarcoma virus LTR promoter / enhancer. Yet another promoter / enhancer sequence is the chicken β-actin promoter [TAKost et al., Nucl. Acids Res., 11(23):8287 (1983)]. Those skilled in the art can also select other suitable or desirable promoters.

[0250] The antigen cassette may also contain nucleic acid sequences heterologous to the viral vector sequence, including sequences providing signals for efficient polyadenylation of the transcript (poly(A), polyA, or pA) and introns containing functional splice donor and acceptor sites. A common polyA sequence used in the exemplary vectors herein is derived from the papovavirus SV-40. The polyA sequence can be inserted into the cassette after the antigen-based sequence and before the viral vector sequence. A common intron sequence may also be derived from SV-40 and is referred to as the SV-40T intron sequence. The antigen cassette may also contain an intron located between the promoter / enhancer sequence and the antigen(s). The selection of these and other common vector elements is conventional (see, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual," 2d ed., Cold Spring Harbor Laboratory, New York (1989) and references cited therein), and many such sequences are available from commercial and industrial sources, as well as Genbank.

[0251] An antigen cassette can have one or more antigens. For example, a particular cassette can include 1-10, 1-20, 1-30, 10-20, 15-25, 15-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens. The antigens may be directly linked to each other. The antigens may be linked to each other by a linker. The antigens can be in any orientation relative to each other, including N-C or C-N.

[0252] As noted above, the antigen cassette can be placed in any selected deletion site in the viral vector, such as a deletion of the E1 gene region, or a deletion of the E3 gene region, among other choices.

[0253] The antigen cassette has the following formula describing the ordered sequence of each element from 5' to 3': (P a -(L5 b -N c -L3 d ) X ) Z -(P2 h -(G5 e -U f ) Y ) W -G3 g It can be explained using During the ceremony, P and P2 comprise a promoter nucleotide sequence, N comprises an MHC class I epitope-encoding nucleic acid sequence; L5 comprises a 5' linker sequence, L3 comprises a 3' linker sequence, G5 comprises a nucleic acid sequence encoding an amino acid linker, G3 comprises one of at least one nucleic acid sequence encoding an amino acid linker; U comprises an MHC class II antigen-encoding nucleic acid sequence, where for each X, the corresponding Nc is an epitope-encoding nucleic acid sequence; For each Y, the corresponding Uf is an antigen-encoding nucleic acid sequence. The compositions and ordered sequences can be further defined by selecting the number of elements present, for example, a=0 or 1, b=0 or 1, c=1, d=0 or 1, e=0 or 1, f=1, g=0 or 1, h=0 or 1, X=1-400, Y=0, 1, 2, 3, 4 or 5, Z=1-400, and W=0, 1, 2, 3, 4 or 5.

[0254] In one example, the elements present are a=0, b=1, d=1, e=1, g=1, h=0, X=10, Y=2, Z=1, and W=1, and when the case is described where there is no additional promoter (i.e., only the promoter nucleotide sequence provided by the RNA alphavirus backbone is present), there are 20 MHC class I epitopes, there is a 5' linker for each N, there is a 3' linker for each N, there are two MHC class II epitopes, there is a linker connecting the two MHC class II epitopes, there is a linker connecting the 5' ends of the two MHC class II epitopes to the 3' linker of the last MHC class I epitope, and there is a linker connecting the 3' ends of the two MHC class II epitopes to the RNA alphavirus backbone. Examples of linkage of the 3' end of the antigen cassette to the RNA alphavirus backbone include linking it directly to a 3' UTR element provided by the RNA alphavirus backbone, such as the 3' 19 nt CSE. Examples of linkages of the 5' end of the antigen cassette to the RNA alphavirus backbone include direct linkage to the 26S promoter sequence, the alphavirus 5'UTR, the 51 nt CSE, or the 24 nt CSE.

[0255] Other examples include when a=1, describing the presence of a promoter other than the promoter nucleotide sequence provided by the RNA alphavirus backbone; when a=1 and Z is greater than 1, describing the presence of multiple promoters other than the promoter nucleotide sequence provided by the RNA alphavirus backbone, each of which results in the expression of one or more different MHC Class I epitope-encoding nucleic acid sequences; when h=1, describing the presence of another promoter that results in the expression of an MHC Class II antigen-encoding nucleic acid sequence; and when g=0, describing the presence of an MHC Class II antigen-encoding nucleic acid sequence (if present) directly linked to the RNA alphavirus backbone.

[0256] Other examples include cases where each MHC class I epitope present has a 5' linker, a 3' linker, neither, or both. In examples where multiple MHC class I epitopes are present in the same antigen cassette, some MHC class I epitopes may have both a 5' linker and a 3' linker, while other MHC class I epitopes may have a 5' linker, a 3' linker, or neither. In other examples where multiple MHC class I epitopes are present in the same antigen cassette, some MHC class I epitopes may have either a 5' linker or a 3' linker, while other MHC class I epitopes may have a 5' linker, a 3' linker, or neither.

[0257] In instances where multiple MHC Class II epitopes are present within the same antigen cassette, some MHC Class II epitopes may have both a 5' linker and a 3' linker, while other MHC Class II epitopes may have a 5' linker, a 3' linker, or neither. In other instances where multiple MHC Class II epitopes are present within the same antigen cassette, some MHC Class II epitopes may have either a 5' linker or a 3' linker, while other MHC Class II epitopes may have a 5' linker, a 3' linker, or neither.

[0258] The promoter nucleotide sequences P and / or P2 may be the same as the promoter nucleotide sequences provided by the RNA alphavirus backbone. For example, the promoter sequences Pn and P2 provided by the RNA alphavirus backbone may each comprise a 26S subgenomic promoter. The promoter nucleotide sequences P and / or P2 may be different from the promoter nucleotide sequences provided by the RNA alphavirus backbone and may also be different from each other.

[0259] The 5' linker L5 can be a natural or non-natural sequence. Non-natural sequences include, but are not limited to, AAY, RR, and DPP. The 3' linker L3 can also be a natural or non-natural sequence. Furthermore, L5 and L3 can both be natural sequences, both can be non-natural sequences, or one can be natural and the other can be non-natural. For each X, the amino acid linker may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, The lengths may be 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more. For each X, the amino acid linker may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0260] For each Y, the amino acid linker G5 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more lengths. For each Y, the amino acid linker may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0261] The amino acid linker G3 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more lengths. G3 may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0262] For each X, each N can encode an MHC class I epitope 7 to 15 amino acids in length. For each X, each N may encode an MHC class I epitope 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. For each X, each N may also encode an MHC class I epitope at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0263] VB immune checkpoint A vector described herein, such as a C68 vector described herein, or an alphavirus vector described herein, can contain a nucleic acid encoding at least one antigen, and the same or a separate vector can contain a nucleic acid encoding at least one immune modulator (e.g., an antibody such as an scFv) that binds to and blocks the activity of an immune checkpoint molecule. The vector can contain an antigen cassette and one or more nucleic acid molecules encoding a checkpoint inhibitor.

[0264] Exemplary immune checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (a member of the CD2 family of molecules expressed on all NK (gamma delta) and memory CD8+ (alpha beta) T cells), CD160 (also known as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, or antigen-binding fragments thereof, or other binding proteins that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (a CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), ipilimumab, MK-3475 (a PD-1 blocker), nivolumab (an anti-PD1 antibody), CT-011 (an anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (an anti-PDL1 antibody), BMS-936559 (an anti-PDL1 antibody), MPLDL3280A (an anti-PDL1 antibody), MSB0010718C (an anti-PDL1 antibody), and yervoy / ipilimumab (an anti-CTLA-4 checkpoint inhibitor). The antibody-encoding sequence can be engineered into a vector, e.g., C68, using ordinary skill in the art. One exemplary method is described in Fang et al., Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol. 2005 May;23(5):584-90. Epub 2005 Apr 17; which is incorporated herein by reference for all purposes.

[0265] Further considerations for VC vaccine design and manufacturing VC1. Determining a set of peptides covering all tumor subclones Truncal peptides, meaning those presented by all or most tumor subclones, are prioritized for inclusion in the vaccine. 53 Optionally, if there are no truncal peptides that are predicted to be highly likely to be presented and immunogenic, or if the number of truncal peptides that are predicted to be highly likely to be presented and immunogenic is small enough that additional non-truncal peptides can be included in the vaccine, additional peptides can be prioritized by estimating the number and identity of tumor subclones and selecting peptides to maximize the number of tumor subclones covered by the vaccine. 54 .

[0266] VC2. Antigen prioritization After applying all of the above antigen filters, there may still be more candidate antigens available for vaccine inclusion than vaccine technology can accommodate. Additionally, there may remain uncertainties about various aspects of antigen analysis, and trade-offs may exist between various properties of candidate vaccine antigens. Therefore, instead of predetermined filters at each stage of the selection process, one can consider an integral multidimensional model, in which candidate antigens are placed in a space with at least the following axes, and an integral approach is used to optimize selection: 1. Risk of autoimmunity or tolerance (germline risk) (lower autoimmune risk is typically preferred) 2. Probability of sequencing artifacts (lower artifact probabilities are typically preferred) 3. Probability of immunogenicity (higher probability of immunogenicity is typically preferred) 4. Probability of presentation (higher probability of presentation is typically preferable) 5. Gene Expression (higher expression is typically preferred) 6. HLA gene coverage (a greater number of HLA molecules involved in presenting a set of antigens may decrease the probability that a tumor will evade immune attack through downregulation or mutation of HLA molecules) 7. HLA class coverage (covering both HLA-I and HLA-II may increase the probability of therapeutic response and decrease the probability of tumor immune evasion)

[0267] Furthermore, in some cases, neoantigens can be deprioritized (e.g., excluded) from vaccination if they are predicted to be presented by HLA alleles that are lost or inactivated in all or part of the patient's tumor. Loss of HLA alleles can occur through somatic mutation, loss of heterozygosity, or homozygous deletion of the locus. Methods for detecting somatic mutations of HLA alleles are well known in the art (e.g., Shukla et al., 2015). Methods for detecting somatic loss of heterozygosity and homozygous deletion (including HLA loci) have also been described (Carter et al., 2012; McGranahan et al., 2017; Van Loo et al., 2010). Antigens may be deprioritized if mass spectrometry data indicates that the predicted antigen is not presented by the predicted HLA allele.

[0268] VD alphavirus VD1. Alphavirus Biology Alphaviruses are members of the Togaviridae family and are single-stranded, positive-sense RNA viruses. Members are generally classified as either Old World types, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World types, such as Eastern equine encephalitis virus, Aura, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83 (Strauss Microbrial Review 1994). Natural alphavirus genomes are typically approximately 12 kb in length, with the first two-thirds containing genes encoding nonstructural proteins (nsPs) that form the RNA replication complex for autonomous replication of the viral genome, and the last one-third containing subgenomic expression cassettes encoding structural proteins for virion production (Frolov RNA 2001).

[0269] The model life cycle of alphaviruses involves multiple distinct steps (Strauss Microbrial Review 1994, Jose Future Microbiol 2009). After viral adsorption to the host cell, the virion fuses with membranes within the intracellular compartment, ultimately releasing the genomic RNA into the cytosol. The genomic RNA, which has a positive-strand orientation, a 5'-methylguanylate cap, and a 3'-poly(A) tail, is translated to generate nonstructural proteins nsP1-4, which form a replication complex. Early in infection, the positive-strand RNA is replicated onto a negative-strand template by this complex. In the current model, the replication complex undergoes further processing as infection progresses, and the resulting processed complex switches to transcribing the negative-strand full-length positive-strand genomic RNA and the 26S subgenomic positive-strand RNA containing the structural genes. Several conserved sequence elements (CSEs) in alphaviruses have been identified as potentially playing roles in various steps of RNA synthesis, including the complement of the 5' UTR in replication of plus-strand RNA from a minus-strand template, a 51-nt CSE in replication of minus-strand synthesis from a genomic template, a 24-nt CSE within the junction region between nsP and 26S RNA in transcription of subgenomic RNA from the minus strand, and a 19-nt CSE at the 3' end in minus-strand synthesis from a plus-strand template.

[0270] In the natural life cycle of viruses, viral particles typically assemble after replication of different RNA species. The 26S RNA is translated, and the resulting proteins are further processed to generate structural proteins, including capsid proteins, glycoproteins E1 and E2, and two small polypeptides, E3 and 6K (Strauss 1994). After encapsidation of the viral RNA occurs and capsid proteins, typically specific only for the genomic RNA, are packaged, virions assemble and bud onto the membrane surface.

[0271] VD2. Alphaviruses as Delivery Vectors Alphaviruses (alphavirus sequences, characteristics, and other elements) can be used to generate alphavirus-based delivery vectors (also referred to as alphavirus vectors, alphavirus viral vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying RNA (samRNA) vectors). Alphaviruses have traditionally been genetically engineered for use as expression vector systems (Pushko 1997, Rheme 2004). Alphaviruses offer several advantages in vaccine settings, where expression of heterologous antigens may be desirable. Because of their ability to replicate autonomously in the host cytosol, alphaviruses typically achieve high intracellular copy numbers of the expression cassette, thereby enabling high levels of heterologous antigen production. Furthermore, vectors are generally transient, resulting in high biosafety and low induction of immune tolerance to the vector. The general public generally lacks pre-existing immunity to alphaviruses compared to other standard viral vectors, such as human adenoviruses. Alphavirus-based vectors also typically produce cytotoxic responses to infected cells. Cytotoxicity may be of some importance in vaccine settings to adequately induce an immune response to the expressed heterologous antigen. However, the desired degree of cytotoxicity is a matter of balance, and for this reason, several attenuated alphaviruses have been developed, including the TC-83 strain of VEE. Thus, one example of an antigen expression vector described herein utilizes an alphavirus backbone that allows for high levels of antigen expression, induces a strong immune response to the antigen, does not induce an immune response to the vector itself, and is safe to use. Furthermore, antigen expression cassettes can be designed to induce different levels of immune responses through optimization of which alphavirus sequences the vector uses, including, but not limited to, sequences derived from VEE or its attenuated derivative, TC-83.

[0272] Several strategies for designing expression vectors using alphavirus sequences have been developed (Pushko 1997). In one strategy, alphavirus vector design involves inserting a second copy of the 26S promoter sequence element downstream of the structural protein gene followed by the insertion of a heterologous gene (Frolov 1993). This results in the production of subgenomic RNA that expresses additional heterologous proteins in addition to the native nonstructural and structural proteins. In this system, all the factors for producing infectious virions are present, and therefore repeated rounds of infection of the expression vector in uninfected cells can be carried out.

[0273] Another expression vector design utilizes a helper virus system (Pushko 1997). In this strategy, structural proteins are replaced by heterologous genes. Thus, after autonomous replication of viral RNA mediated by the still-intact nonstructural genes, the 26S subgenomic RNA drives expression of the heterologous protein. Traditionally, an additional vector expressing the structural proteins is provided in trans, for example, by cotransfection of a cell line, to generate infectious virus. One system is described in U.S. Patent No. 8,093,021, the entire contents of which are incorporated herein by reference for all purposes. Helper vector systems offer the advantage of limiting the potential for infectious particle formation, thus improving biosafety. Furthermore, helper vector systems may shorten the overall vector length and improve replication and expression efficiency. Thus, one example of an antigen expression vector described herein can use an alphavirus backbone in which structural proteins are replaced with an antigen cassette, and the resulting vector promotes efficient expression by reducing biosafety concerns while simultaneously reducing the overall expression vector size.

[0274] VD3. In vitro generation of alphavirus Alphavirus delivery vectors are generally positive-stranded RNA polynucleotides. A conventional method for RNA production, known in the art, is in vitro translation (IVT). In this method, a DNA template of the desired vector is first generated by standard molecular biology methods well known in the art, including cloning, restriction digestion, ligation, gene synthesis, and polymerase chain reaction (PCR). This DNA template carries an RNA polymerase promoter at the 5' end of the sequence desired to be transcribed into RNA. Examples of promoters include, but are not limited to, promoters for bacteriophage polymerases such as T3, T7, or SP6. The DNA template is then incubated with an appropriate RNA polymerase enzyme, buffering agents, and nucleotides (NTPs). The resulting RNA polynucleotide can optionally be further modified by methods including, but not limited to, the addition of a 5' cap structure, such as 7-methylguanosine or a related structure, and optionally modifying the 3' end to include a polyadenylated (polyA) tail. The RNA can then be purified using methods well known in the art, such as phenol chloroform extraction.

[0275] VD4. Delivery via lipid nanoparticles One important aspect to consider in vaccine vector design is immunity to the vector itself (Riley 2017). This can be in the form of pre-existing immunity to the vector itself, for example, certain human adenovirus systems, or immunity to the vector that develops after administration of the vaccine. The latter is an important consideration when multiple doses of the same vaccine are administered, for example, separate priming and booster doses, or when the same vaccine vector system is used to deliver different antigen cassettes.

[0276] In the case of alphavirus vectors, the standard delivery method is the helper virus system described above, which generates infectious viral particles by delivering the capsid, E1, and E2 proteins in trans. However, it is important to note that E1 and E2 proteins are often the primary targets of neutralizing antibodies (Strauss 1994). Therefore, the effectiveness of using alphavirus vectors to deliver antigens of interest to target cells may be reduced if the infectious particles are targeted by neutralizing antibodies.

[0277] An alternative to viral particle-mediated gene delivery is the delivery of expression vectors using nanoparticles (Riley 2017). Importantly, nanomaterial carriers can be formed from non-immunogenic materials, generally avoiding the induction of immunity against the delivery vector itself. These materials can include, but are not limited to, lipids, inorganic nanomaterials, and other polymeric materials. Lipids can be cationic, anionic, or neutral. Such materials can be synthetic or naturally derived and, in certain instances, biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins.

[0278] Lipid nanoparticles (LNPs) are attractive delivery systems due to the amphiphilic nature of lipids, which allows them to form membrane- and vesicular structures (Riley 2017). These vesicles typically deliver expression vectors by absorbing into the membrane of target cells and releasing the nucleic acid into the cytosol. Furthermore, LNPs can be further modified or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity. Lipid compositions typically contain defined mixtures of cationic, neutral, anionic, and amphiphilic lipids. In some instances, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide chemical functional groups that facilitate the attachment of additional moieties. The lipid composition can affect the size and stability of the overall LNP. In one example, the lipid composition includes dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. Compositions of MC3 and MC3-like lipids can be formulated to include one or more other lipids, such as, for example, PEG or PEG-conjugated lipids, sterols, or neutral lipids.

[0279] Nucleic acid vectors, such as expression vectors, directly exposed to serum can have several undesirable effects, including degradation of the nucleic acid by nucleases in serum or stimulation of off-targets of the immune system by free nucleic acids. Therefore, encapsulation of alphavirus vectors can be used to prevent degradation while also preventing potential off-target effects. In certain examples, the alphavirus vector is fully encapsulated within the delivery vehicle, such as the aqueous interior of an LNP. Encapsulation of the alphavirus vector within the LNP can be achieved by methods well known in the art, such as microfluidic mixing and droplet generation performed in a microfluidic droplet generator. Such devices include, but are not limited to, standard T-junction or flow-focusing devices. In one example, a desired lipid formulation, such as an MC3- or MC3-like molecule-containing composition, is fed into the droplet generator in parallel with the alphavirus delivery vector and other desired substances, thereby fully encapsulating the delivery vector and desired substances within the MC3- or MC3-like molecule-based LNP. In one example, the droplet generator can control the particle size range and size distribution of the generated LNPs. For example, LNPs can have particle sizes ranging from 1 to 1000 nm in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nm. After droplet generation, the delivery vector encapsulating the expression vector can be further processed or modified in preparation for administration.

[0280] VE chimpanzee adenovirus (ChAd) VE1. Viral delivery by chimpanzee adenovirus Vaccine compositions for delivering one or more antigens (e.g., including one or more neoantigens listed in Table A and the AACR GENIE results, and / or one or more antigens listed in Table 1.2, via an antigen cassette) can be generated by providing chimpanzee-derived adenovirus nucleotide sequences, various novel vectors, and cell lines expressing chimpanzee adenovirus genes. The nucleotide sequence of chimpanzee C68 adenovirus (also referred to herein as ChAdV68) can be used in vaccine compositions for delivering antigens (see SEQ ID NO: 1). The use of C68 adenovirus-derived vectors is described in further detail in U.S. Patent No. 6,083,716, the entire contents of which are incorporated herein by reference for all purposes.

[0281] In a further aspect, provided herein is a recombinant adenovirus comprising a DNA sequence of a chimpanzee adenovirus, such as C68, and an antigen cassette operably linked to a regulatory sequence that directs expression. The recombinant virus is capable of infecting mammalian, preferably human, cells and expressing the product of the neoantigen cassette in the cells. The vector can be deleted for the native chimpanzee E1 gene, E3 gene, and / or E4 gene. An antigen cassette can be inserted into any of these gene deletion sites. The antigen cassette can include an antigen against which a primed immune response is desired.

[0282] In another aspect, provided herein are mammalian cells infected with a chimpanzee adenovirus, such as C68.

[0283] In yet another embodiment, novel mammalian cell lines are provided that express chimpanzee adenovirus genes (eg, from C68) or functional fragments thereof.

[0284] In an even further aspect, provided herein is a method for delivering an antigen cassette into a mammalian cell, the method comprising introducing into the cell an effective amount of a chimpanzee adenovirus, such as C68, engineered to express the antigen cassette.

[0285] Yet another embodiment provides a method for eliciting an immune response in a mammalian host to treat cancer, the method comprising administering to the host an effective amount of a recombinant chimpanzee adenovirus, such as C68, that includes an antigen cassette encoding one or more antigens derived from the tumor to which the immune response is targeted.

[0286] Also disclosed are non-simian mammalian cells expressing chimpanzee adenovirus genes obtained from the sequence of SEQ ID NO: 1. The genes can be selected from the group consisting of adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 of SEQ ID NO: 1.

[0287] Also disclosed are nucleic acid molecules comprising a DNA sequence of a chimpanzee adenovirus comprising a gene obtained from the sequence of SEQ ID NO: 1. The gene can be selected from the group consisting of the chimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 1. In some embodiments, the nucleic acid molecule comprises the sequence of SEQ ID NO: 1, deleted for at least one gene selected from the group consisting of the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of SEQ ID NO: 1.

[0288] Also disclosed are vectors comprising a chimpanzee adenoviral DNA sequence derived from SEQ ID NO: 1 and an antigen cassette operably linked to one or more regulatory sequences that direct expression of the cassette in a heterologous host cell, optionally wherein the chimpanzee adenoviral DNA sequence comprises at least cis elements required for replication and encapsidation, the cis elements flanking the antigen cassette and regulatory sequences. In some embodiments, the chimpanzee adenoviral DNA sequence comprises genes selected from the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 gene sequences of SEQ ID NO: 1. In some embodiments, the vector may be deleted for the E1A and / or E1B genes.

[0289] Also disclosed are host cells transfected with the vectors disclosed herein, such as the C68 vector engineered to express an antigen cassette. Also disclosed are human cells that express a selected gene introduced into the cell by introducing into the cell a vector disclosed herein.

[0290] Also provided is a method for delivering an antigen cassette to a mammalian cell, comprising introducing into said cell an effective amount of a vector disclosed herein, such as a C68 vector engineered to express the antigen cassette.

[0291] Also disclosed is a method for producing an antigen, comprising introducing a vector disclosed herein into a mammalian cell, culturing the cell under appropriate conditions, and producing the antigen.

[0292] Complementing cell lines expressing VE2.E1 To generate recombinant chimpanzee adenoviruses (Ad) having deletions in any of the genes described herein, the function of the deleted gene region (if essential for viral replication and infectivity) can be supplied to the recombinant virus by a helper virus or cell line (i.e., a complementation or packaging cell line). For example, to generate replication-deficient chimpanzee adenovirus vectors, cell lines expressing the E1 gene product of human or chimpanzee adenovirus can be used, and such cell lines can include HEK293 or variants thereof. Cell lines expressing any selected chimpanzee adenovirus gene can be generated according to the protocol for generating cell lines expressing the chimpanzee E1 gene (Examples 3 and 4 of U.S. Pat. No. 6,083,716).

[0293] AAV enrichment assay can be used to identify chimpanzee adenovirus E1-expressing cell lines. This assay is useful for identifying E1 function in cell lines created using E1 genes from other uncharacterized adenoviruses (e.g., from other species). This assay is described in Example 4B of U.S. Patent No. 6,083,716.

[0294] The selected chimpanzee adenovirus gene (e.g., E1) may be under the transcriptional control of a promoter for expression in the selected parent cell line. Inducible or constitutive promoters can be used for this purpose. Inducible promoters include the sheep metallothionine promoter, which is induced by zinc, or the mouse mammary tumor virus (MMTV) promoter, which is induced by glucocorticoids, particularly dexamethasone. Other inducible promoters, such as those identified in International Application No. WO 95 / 13392, incorporated herein by reference, can also be used to generate packaging cell lines. Constitutive promoters controlling the expression of chimpanzee adenovirus genes can also be used.

[0295] Parent cells can be selected to generate novel cell lines expressing any desired C68 gene. Such parent cell lines can be, but are not limited to, HeLa [ATCC Deposit No. CCL2], A549 [ATCC Deposit No. CCL185], KB [CCL17], Detroit [e.g., Detroit 510, CCL72], and WI-38 [CCL75] cells. Other suitable parent cell lines can be obtained from other sources. Parent cell lines can include CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a.

[0296] E1-expressing cell lines may be useful in producing recombinant chimpanzee adenovirus E1-deleted vectors. Cell lines constructed using essentially the same procedures that express one or more other chimpanzee adenovirus gene products are useful in producing recombinant chimpanzee adenovirus vectors with deletions in the genes encoding these products. In addition, cell lines expressing other human Ad E1 gene products are also useful in producing chimpanzee recombinant Ad.

[0297] VE3. Recombinant viral particles as vectors The compositions disclosed herein can include a viral vector that delivers at least one antigen to cells. Such a vector includes a chimpanzee adenovirus DNA sequence, such as C68, and an antigen cassette operably linked to a regulatory sequence for directing expression of the cassette. The C68 vector is capable of expressing the cassette in infected mammalian cells. The C68 vector can have functional deletions in one or more viral genes. The antigen cassette includes at least one antigen under the control of one or more regulatory sequences, such as a promoter. Optional helper viruses and / or packaging cell lines can supply the chimpanzee viral vector with any necessary products of the deleted adenovirus genes.

[0298] The term "functionally deleted" means that a sufficient amount of the gene region has been removed or otherwise altered, for example, by mutation or modification, such that the gene region is no longer able to produce one or more functional products of gene expression. Mutations or modifications that can lead to functional deletions include, but are not limited to, nonsense mutations such as the introduction of premature stop codons and the removal of canonical and non-canonical start codons, mutations that alter mRNA splicing or other transcription processing, or combinations thereof. If necessary, the entire gene region can be removed.

[0299] Modifications of the nucleic acid sequences forming the vectors disclosed herein, including sequence deletions, insertions, and other mutations, can be produced using standard molecular biology techniques and are within the scope of the present invention.

[0300] VE4. Viral Plasmid Vector Construction Chimpanzee adenovirus C68 vectors useful in the present invention include recombinant defective adenoviruses, i.e., chimpanzee adenovirus sequences having functional deletions in the E1a or E1b genes and, optionally, other mutations, such as temperature-sensitive mutations or deletions in other genes. These chimpanzee sequences are also expected to be useful in forming hybrid vectors from other adenovirus and / or adeno-associated virus sequences. Homologous adenovirus vectors prepared from human adenoviruses have been described in the published literature [see, e.g., Kozarsky I and II, cited above, and references cited therein, U.S. Patent No. 5,240,846].

[0301] A wide range of adenoviral nucleic acid sequences can be used in constructing chimpanzee adenovirus C68 vectors useful for delivering antigen cassettes into human (or other mammalian) cells. Vectors containing minimal chimpanzee C68 adenovirus sequences can be used in conjunction with a helper virus to generate infectious recombinant viral particles. The helper virus provides the essential gene products required for viral infectivity and propagation of the minimal chimpanzee adenovirus vector. When only one or more selected deletions of chimpanzee adenovirus genes are introduced into an otherwise functional viral vector, the deleted gene products can be supplied during the viral vector production process by propagating the virus that provides the deleted gene function in trans in a selected packaging cell line.

[0302] VE5. Recombinant Minimal Adenovirus The minimal chimpanzee Ad C68 virus is a viral particle that contains only the adenoviral cis elements necessary for replication and virion encapsidation. That is, the vector contains the adenoviral cis-acting 5' and 3' inverted terminal repeats (ITRs) (which function as origins of replication) and the native 5' packaging / enhancer domain (which contains sequences necessary for packaging the linear Ad genome and the enhancer element of the E1 promoter). See, for example, the methods described for preparing a "minimal" human Ad vector in International Application No. WO 96 / 13597, incorporated herein by reference.

[0303] VE6. Other defective adenoviruses Recombinant replication-deficient adenoviruses may contain more than the minimal chimpanzee adenovirus sequences. These other Ad vectors can be characterized by deletion of different portions of the viral gene region and the infectious viral particles formed by the use of helper viruses and / or packaging cell lines as needed.

[0304] As one example, suitable vectors can be generated by deleting all or a sufficient portion of the immediate-early gene E1a and the delayed-early gene E1b of C68 adenovirus, thereby eliminating their normal biological function. Replication-deficient E1-deleted viruses are capable of replicating and generating infectious virus when grown in chimpanzee adenovirus-transformed complementation cell lines containing functional adenovirus E1a and E1b genes that provide the corresponding gene products in trans. Based on homology to known adenovirus sequences, the resulting recombinant chimpanzee adenovirus is capable of infecting many cell types and expressing antigen(s), as are known human recombinant E1-deleted adenoviruses, but is expected to be unable to replicate in many cells lacking chimpanzee E1 region DNA unless the cells are infected at an extremely high multiplicity of infection.

[0305] As another example, all or part of the C68 adenovirus immediate early gene E3 can be removed from the chimpanzee adenovirus sequences that form part of the recombinant virus.

[0306] Chimpanzee adenovirus C68 vectors can also be constructed with a deletion of the E4 gene. Yet another vector can have a deletion in the delayed-early gene E2a.

[0307] Deletions can be introduced into any of the late genes L1 through L5 of the chimpanzee C68 adenovirus genome. Similarly, deletions within intermediate genes IX and IVa2 may be useful for certain purposes. Other deletions can also be introduced into other structural or nonstructural adenovirus genes.

[0308] The deletions described above can be used individually. That is, the adenoviral sequence can have a deletion of only E1. Also, any combination of whole or partial genes effective to disrupt or reduce their biological activity can be used. For example, in one exemplary vector, the adenoviral C68 sequence can have a deletion of the E1 and E4 genes, or the E1, E2a, and E3 genes, or the E1 and E3 genes, or the E1, E2a, and E4 genes, with or without a deletion of E3. As noted above, such deletions can be used in combination with other mutations, such as temperature-sensitive mutations, to achieve the desired results.

[0309] The cassette containing the antigen(s) can optionally be inserted into any deleted region of the chimpanzee C68Ad virus, or, if desired, can be inserted within an existing gene region to disrupt the function of that region.

[0310] VE7.Helper virus Depending on the chimpanzee adenovirus gene content of the viral vector used to deliver the antigen cassette, helper adenovirus or non-replicating viral fragments can be used to provide sufficient chimpanzee adenovirus gene sequences to generate infectious recombinant viral particles containing the cassette.

[0311] Useful helper viruses contain selected adenoviral gene sequences that are not present in the adenoviral vector construct and / or are not expressed by the packaging cell line transfected with the vector. The helper virus may be replication-deficient and may contain various adenoviral genes other than those described above. Helper viruses can be used in combination with the E1-expressing cell lines described herein.

[0312] For C68, the "helper" virus can be a fragment formed by truncating the C-terminus of the C68 genome with SspI, which removes approximately 1300 bp from the left end of the virus. This truncated virus is then co-transfected with plasmid DNA into an E1-expressing cell line to form recombinant virus by homologous recombination with the C68 sequences in the plasmid.

[0313] The helper virus can also be formulated as a polycation complex, as described in Wu et al., J. Biol. Chem., 264:16985-16987 (1989); KJ Fisher and JM Wilson, Biochem. J., 299:49 (Apr. 1, 1994). The helper virus can optionally contain a reporter gene. Many such reporter genes are known in the art. The presence of a reporter gene on the helper virus that is different from the antigen cassette on the adenovirus vector allows for independent monitoring of the Ad vector and the helper virus. This second reporter gene allows for the separation of the resulting recombinant virus from the helper virus during purification.

[0314] VE8. Assembly of Viral Particles and Infection of Cell Lines Assembly of selected DNA sequences of adenovirus, antigen cassettes, and other vector elements into various intermediate plasmids and shuttle vectors, as well as use of the plasmids and shuttle vectors to generate recombinant viral particles, can all be accomplished using conventional techniques, including conventional cDNA cloning, in vitro recombination (e.g., Gibson assembly), use of overlapping oligonucleotide sequences of the adenovirus genome, polymerase chain reaction, and any suitable method that provides the desired nucleotide sequence. Standard transfection and cotransfection techniques, such as CaPO precipitation or liposome-mediated transfection methods such as lipofectamine, can be used. Other conventional methods that can be used include homologous recombination of the viral genome, viral plaque formation in agar overlays, and signal generation assays.

[0315] For example, after construction and assembly of a viral vector containing a desired antigen, the vector can be transfected in vitro into a packaging cell line in the presence of a helper virus, and homologous recombination occurs between the helper and vector sequences, allowing the adenovirus-antigen sequences within the vector to be replicated and packaged into virion capsids, resulting in recombinant viral vector particles.

[0316] The resulting recombinant chimpanzee C68 adenovirus is useful for transferring antigen cassettes into selected cells. In vivo experiments using recombinant viruses propagated in packaging cell lines demonstrate the utility of the E1-deleted recombinant chimpanzee adenovirus for transferring cassettes into non-chimpanzee cells, preferably human cells.

[0317] VE9. Use of Recombinant Viral Vectors Thus, the resulting recombinant chimpanzee C68 adenovirus containing the antigen cassette (produced by the adenoviral vector in combination with a helper virus or the adenoviral vector in combination with a packaging cell line, as described above) provides an efficient gene transfer vehicle capable of delivering antigen(s) to a subject in vivo or ex vivo.

[0318] The recombinant vectors described above are administered to humans according to published methods for gene therapy. The chimpanzee virus vector carrying the antigen cassette can be administered to patients preferably suspended in a biocompatible solution or pharmaceutically acceptable delivery vehicle. Suitable vehicles include sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known as pharmaceutically acceptable carriers and familiar to those skilled in the art can also be used for this purpose.

[0319] The chimpanzee adenoviral vector is administered in an amount sufficient to transform human cells and result in the introduction and expression of antigens at levels sufficient to provide a therapeutic effect without adverse effects or with a medically acceptable physiological effect, as can be determined by those skilled in the medical field. Conventional pharmaceutically acceptable routes of administration include, but are not limited to, intrahepatic, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parenteral routes of administration. Routes of administration can be combined as needed.

[0320] The dosage of the viral vector depends primarily on factors such as the condition being treated, the patient's age, weight, and health status, and may therefore vary between patients. The dosage is adjusted to balance the therapeutic effect against any side effects, and may vary depending on the therapeutic application for which the recombinant vector is used. The frequency of administration can be determined by observing the expression level of the antigen(s).

[0321] The recombinant replication-deficient adenovirus can be administered in a "pharmacologically effective amount," i.e., an amount of recombinant adenovirus effective by a given route of administration to transfect the desired cells and provide sufficient expression levels of the selected gene to produce a vaccine effect, i.e., a measurable level of protective immunity. The C68 vector containing the antigen can be co-administered with an adjuvant. The adjuvant can be separate from the vector (e.g., alum) or encoded within the vector, particularly if the adjuvant is a protein. Adjuvants are well known in the art.

[0322] Conventional pharmaceutically acceptable routes of administration include, but are not limited to, intranasal, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral, and other parenteral routes of administration. Routes of administration can be combined as needed or adjusted depending on the immunogen or disease. For example, for the prevention of rabies, subcutaneous, intratracheal, and intranasal routes are preferred. The route of administration is primarily determined by the nature of the disease being treated.

[0323] By observing the expression level of the antigen(s), the need for boosters, if any, can be determined. For example, after evaluation of antibody titers in the serum, booster immunizations may be desirable, if necessary.

[0324] VI. Methods of Treatment and Manufacturing Also provided are methods for inducing a tumor-specific immune response in a subject, vaccinating against the tumor, and treating and / or alleviating symptoms of cancer in a subject by administering to the subject one or more antigens, such as a plurality of antigens identified using the methods disclosed herein.

[0325] In some embodiments, the subject has been diagnosed with cancer or is at risk of developing cancer. The subject can be a human, dog, cat, horse, or any animal in which a tumor-specific immune response is desired. The tumor can be any solid tumor, such as breast, ovarian, prostate, lung, kidney, stomach, colon, testicular, head and neck, pancreas, brain, melanoma, and other tissue organ tumors, as well as hematological tumors, such as lymphomas and leukemias, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-lymphocytic leukemia, and B-cell lymphoma.

[0326] The antigen can be administered in an amount sufficient to induce a CTL response.

[0327] The antigen can be administered alone or in combination with other therapeutic agents, such as chemotherapeutic agents, radiation, or immunotherapy. Any suitable therapeutic treatment for the particular cancer can be administered.

[0328] In addition, the subject can be further administered with an anti-immunosuppressive / immunostimulatory substance, such as a checkpoint inhibitor.For example, the subject can be further administered with an anti-CTLA antibody, or anti-PD-1 or anti-PD-L1.Blocking CTLA-4 or PD-L1 with an antibody can enhance the immune response against cancerous cells in patients.In particular, blocking CTLA-4 has been shown to be effective when used in vaccination protocols.

[0329] The optimal amount of each antigen to be included in the vaccine composition and the optimal dosing regimen can be determined. For example, the antigen or its variant can be prepared for intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. Methods of injection include sc, id, ip, im, and iv. Methods of DNA or RNA injection include id, im, sc, ip, and iv. Other methods of administering vaccine compositions are known to those skilled in the art.

[0330] Vaccines can be edited so that the selection, number, and / or amount of antigens present in the composition are tissue-, cancer-, and / or patient-specific. For example, the exact peptide selection can be guided by the expression pattern of the parent protein in a given tissue or by the patient's mutational status. Selection can depend on the specific type of cancer, the state of the disease, earlier treatment regimens, the patient's immune status, and, of course, the patient's HLA haplotype. Furthermore, vaccines can contain components that are personalized according to the individual needs of a particular patient. Examples include altering the antigen selection according to the expression of the antigen in a particular patient, or adjusting for secondary treatments after a first round or regimen of treatment.

[0331] Patients for administration of antigen vaccines can be identified using various diagnostic methods, such as the patient selection methods described further below. Patient selection can include identifying mutations or expression patterns of one or more genes. Optionally, patient selection can include identifying a patient's haplotype. Various patient selection methods can be performed in parallel, for example, sequencing diagnostics can identify both a patient's mutation and haplotype. Various patient selection methods can also be performed sequentially, for example, one diagnostic test can identify a mutation and another diagnostic test can identify a patient's haplotype, where each test can be the same (e.g., both high-throughput sequencing) or different (e.g., one high-throughput sequencing and the other Sanger sequencing) diagnostic method.

[0332] For compositions to be used as vaccines for cancer, antigens with similar normal self-peptides that are abundantly expressed in normal tissues can be avoided or present in low amounts in the compositions described herein. On the other hand, if a patient's tumor is known to express a high amount of a particular antigen, the respective pharmaceutical composition for treating that cancer can be present in high amount and / or can include more than one antigen specific for that particular antigen or pathway of that antigen.

[0333] Compositions containing antigens can be administered to individuals already suffering from cancer. In therapeutic applications, the compositions are administered to patients in an amount sufficient to elicit an effective CTL response against tumor antigens and cure or at least partially halt symptoms and / or complications. An amount adequate to achieve this is defined as a "therapeutically effective dose." The amount effective for this use will depend, for example, on the composition, the mode of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician. It should be kept in mind that compositions can generally be used in severe disease states, i.e., life-threatening or potentially life-threatening situations, especially when cancer has metastasized. In such instances, it is possible, and the treating physician may find it desirable, to administer substantial excesses of these compositions, taking into account the minimization of foreign substances and the relatively non-toxic nature of the antigen.

[0334] For therapeutic use, administration can begin at the time of detection or surgical removal of a tumor, followed by boosting doses until at least symptoms are substantially abated, and for a period thereafter.

[0335] Pharmaceutical compositions for therapeutic treatment (e.g., vaccine compositions) are intended for parenteral, topical, nasal, oral, or local administration. Pharmaceutical compositions can be administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. The compositions can be administered at the site of surgical resection to induce a local immune response against tumors. Disclosed herein are compositions for parenteral administration that contain a solution of an antigen, where the vaccine composition is dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. Various aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional, well-known sterilization techniques or sterile filtered. The resulting aqueous solutions can be packaged for use as is or lyophilized, with the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0336] Antigens can also be administered via liposomes, which target them to specific cellular tissues, such as lymphoid tissues. Liposomes are also useful for increasing half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these preparations, the antigen to be delivered is incorporated as part of the liposome, either alone or in combination with a molecule that binds to a receptor dominant among lymphoid cells, such as a monoclonal antibody that binds to the CD45 antigen, or with other therapeutic or immunogenic compositions. Liposomes filled with the desired antigen can thus be directed to the site of lymphoid cells, where they then deliver the selected therapeutic / immunogenic composition. Liposomes can generally be formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid is generally guided by considerations, for example, of liposome size, acid lability, and stability of the liposomes in the bloodstream. Various methods are available for preparing liposomes, as described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

[0337] For targeting to immune cells, the ligand to be incorporated into the liposome can include, for example, an antibody or fragment thereof specific for a cell surface determinant of the desired immune system cell. The liposome suspension can be administered intravenously, topically, locally, etc., at doses that vary according to, inter alia, the mode of administration, the peptide being delivered, and the stage of the disease being treated.

[0338] The peptides described herein, and optionally nucleic acids encoding one or more of the peptides, can also be administered to a patient for therapeutic or immunization purposes. Numerous methods are conveniently used to deliver nucleic acids to a patient. For example, nucleic acids can be delivered directly as "naked DNA." This approach is described, for example, in Wolff et al., Science 247:1465-1468 (1990), and U.S. Pat. Nos. 5,580,859 and 5,589,466. Nucleic acids can also be administered using ballistic delivery, as described, for example, in U.S. Pat. No. 5,204,253. Particles consisting solely of DNA can be administered. Alternatively, DNA can be attached to particles, such as gold particles. Approaches for delivering nucleic acid sequences include viral vectors, mRNA vectors, and DNA vectors, with or without electroporation.

[0339] Nucleic acids can also be delivered by complexing them with cationic compounds, such as cationic lipids. Lipid-mediated gene delivery meth...

Claims

1. A composition for delivering an antigen expression system comprising an antigen expression system, the antigen expression system comprises one or more vectors; The composition, wherein the one or more vectors comprise: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; the vector backbone comprising (b) an antigen cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) at least one tumor-specific antigen-encoding nucleic acid sequence, (A) an MHC class I epitope-encoding nucleic acid sequence encoding a KRAS MHC class I epitope selected from the group consisting of SEQ ID NOs: 14954, 19749, 19865, 19976, 19779, and 11495; The antigen cassette comprising:

2. 2. The composition of claim 1, wherein the MHC class I epitope-encoding nucleic acid sequence encodes at least two KRAS MHC class I epitopes selected from the group consisting of SEQ ID NOs: 14954, 19749, 19865, 19976, 19779, and 11495.

3. The composition of claim 1, wherein the MHC class I epitope-encoding nucleic acid sequence further encodes a KRAS MHC class I epitope of SEQ ID NO: 19850, 14409 or 11855.

4. The composition of claim 1 , wherein the MHC class I epitope-encoding nucleic acid sequence encodes each of the KRAS MHC class I epitopes having SEQ ID NOs: 14954, 19749, 19865, 19976, 19779, and 11495.

5. The ordered sequence of each element of the antigen cassette is, from 5' to 3': P ａ -(L5 ｂ -N ｃ -L3 ｄ ) Ｘ -(G5 ｅ -U ｆ ) Ｙ -G3 ｇ It is described by a formula including During the ceremony, P comprises the promoter nucleotide sequence, where a=0 or 1; N comprises one of said MHC class I epitope-encoding nucleic acid sequences, where c=1; L5 comprises a 5' linker sequence, where b=0 or 1; L3 comprises a 3' linker sequence, where d=0 or 1; G5 comprises a nucleic acid sequence encoding a GPGPG amino acid linker (SEQ ID NO:56), where e=0 or 1; G3 comprises a nucleic acid sequence encoding a GPGPG amino acid linker (SEQ ID NO:56), where g=0 or 1; U comprises at least one MHC class II antigen-encoding nucleic acid sequence, where f=1; X=1 to 400, where for each X, the corresponding N ｃ is an epitope-encoding nucleic acid sequence, Y=0, 1, or 2, where for each Y, the corresponding U ｆ is an antigen-encoding nucleic acid sequence, The composition according to any one of claims 1 to 4.

6. (a) For each X, the corresponding N ｃ are different MHC class I epitope-encoding nucleic acid sequences; and / or (b) For each Y, the corresponding U ｆ are different MHC class II antigen-encoding nucleic acid sequences; The composition of claim 5.

7. a=0, b=1, d=1, e=1, g=1, h=1, X=20, Y=2; the at least one promoter nucleotide sequence is a single 26S promoter nucleotide sequence provided by the vector backbone; said at least one polyadenylation (poly(A)) sequence is a poly(A) sequence of at least 100 consecutive A nucleotides provided by said vector backbone (SEQ ID NO: 29358); each N encodes an MHC class I epitope 7-15 amino acids in length; L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of said MHC class I epitope, said 5' linker sequence encoding a peptide that is at least 2 amino acids in length; L3 is a natural 3' linker sequence encoding a natural terminal nucleic acid sequence of said MHC class I epitope, said 3' linker sequence encoding a peptide that is at least 2 amino acids in length; U is each of the PADRE class II sequence and the tetanus toxoid MHC class II sequence; the vector backbone comprises a chimpanzee adenovirus vector or an alphavirus vector; Each of the tumor-specific antigen-encoding nucleic acid sequences encodes a polypeptide having a length of 13 to 25 amino acids. The composition according to claim 5 or 6.

8. The composition of any one of claims 1 to 7, further comprising a nanoparticulate delivery vehicle, The nanoparticulate delivery vehicle is a lipid nanoparticle (LNP); composition.

9. the one or more vectors comprise one or more positive strand RNA vectors; and / or the one or more vectors are autonomously replicating in mammalian cells; and / or the vector backbone comprises at least one nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus; (a) the vector backbone comprises at least sequences for nonstructural protein-mediated amplification encoded by nucleotide sequences of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus, a 26S promoter sequence, and a poly(A) sequence; the sequence for nonstructural protein mediated amplification is selected from the group consisting of an alphavirus 5′UTR, a 51 nt CSE, a 24 nt CSE, a 26S subgenomic promoter sequence, a 19 nt CSE, an alphavirus 3′UTR, or a combination thereof; or (b) the Venezuelan equine encephalitis virus is comprising the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, further comprising a deletion between base pairs 7544 and 11175, The antigen cassette is inserted at position 7544 to replace the deletion between base pairs 7544 and 11175 as set forth in the sequence of SEQ ID NO: 3 or SEQ ID NO: 5; The composition according to any one of claims 1 to 6 or 8.

10. 10. The composition of any one of claims 1 to 6, 8, or 9, wherein the vector backbone comprises at least one nucleotide sequence of a chimpanzee adenoviral vector, and the chimpanzee adenoviral vector is a ChAdV68 vector.

11. the one or more vectors are each at least 300 nt in size, each at least 1 kb in size, each 2 kb in size, or each less than 5 kb in size; and / or At least one of the at least one antigen-encoding nucleic acid sequences encodes a polypeptide sequence that is presented by MHC class I on tumor cells. The composition according to any one of claims 1 to 10.

12. The at least one antigen-encoding nucleic acid sequence is and / or comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 nucleic acid sequences; and / or each tumor-specific antigen-encoding nucleic acid sequence encodes a polypeptide sequence that is 8-35 amino acids in length, 9-17, 9-25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length; The composition according to any one of claims 1 to 6 or 8 to 11.

13. (i) the antigen cassette comprises a junctional epitope sequence formed by adjacent sequences within the antigen cassette, and at least one, or each, of the junctional epitope sequences has an affinity for MHC greater than 500 nM; and / or each junctional epitope sequence is non-self; and / or (ii) the antigen cassette does not encode a non-therapeutic MHC class I or class II epitope nucleic acid sequence that comprises a wild-type nucleic acid sequence after translation, and the non-therapeutic MHC class I or class II epitope is predicted to be presented on an MHC allele of the subject; The composition according to any one of claims 1 to 12.

14. A pharmaceutical composition comprising the composition of any one of claims 1 to 13 and a pharma- ceutically acceptable carrier.

15. An isolated nucleotide or set of isolated nucleotides comprising the antigen cassette of the composition of any one of claims 1 to 13 and one or more elements taken from the sequence of SEQ ID NO: 3 or SEQ ID NO: 5, the one or more elements are selected from the group consisting of sequences required for nonstructural protein-mediated amplification, 26S promoter nucleotide sequences, poly(A) sequences, and nsP1-4 genes of the sequences set forth in SEQ ID NO:3 or SEQ ID NO:5; the isolated nucleotide or set of isolated nucleotides comprises an antigen cassette; The isolated nucleotide or set of isolated nucleotides.

16. A vector or a set of vectors comprising the nucleotide of claim 15.

17. 17. An isolated cell comprising a nucleotide or set of isolated nucleotides according to claim 15 or a vector or set of vectors according to claim 16.

18. A kit comprising the composition according to any one of claims 1 to 13 and instructions for use.

19. A pharmaceutical composition comprising the composition according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 14, for treating a subject having cancer.

20. The pharmaceutical composition of claim 19, wherein the subject expresses at least one HLA allele predicted or known to present a KRAS MHC class I epitope selected from the group consisting of SEQ ID NOs: 14954, 19749, 19865, 19976, 19779, and 11495.

21. 21. The pharmaceutical composition of claim 19 or 20 for use in combination with a second vaccine composition.

22. 22. The pharmaceutical composition of claim 21, wherein the second vaccine composition is the same as the pharmaceutical composition.

23. 17. A method for producing one or more vectors according to claim 16, comprising the steps of: (a) obtaining a linearized DNA sequence comprising the vector backbone and the antigen cassette; (b) in vitro transcribing the linearized DNA sequence by adding the linearized DNA sequence to an in vitro transcription reaction that contains all components necessary to transcribe the linearized DNA sequence into RNA; (c) isolating the one or more vectors from the in vitro transcription reaction; and Including, The method.

24. 1. A method for assessing a subject having cancer, comprising: a) 1) whether the subject has an HLA allele that is predicted or known to present the antigen included in the antigen-based vaccine; and below: 1) whether the subject's tumor expresses a gene associated with the antigen; 2) whether the subject's tumor has a mutation associated with the antigen Either or both determining or having determined; b) determining, or having already determined, from the result of (a) that the subject expresses the HLA allele and the subject's tumor expresses the gene and / or has the mutation, that the subject is a candidate for treatment with the antigen-based vaccine; The antigen comprises at least one KRAS MHC class I epitope sequence selected from the group consisting of SEQ ID NOs: 14954, 19749, 19865, 19976, 19779, and 11495; The process The method comprising:

25. a) the subject is At least one KRAS MHC class I epitope selected from the group consisting of SEQ ID NOs: 14954, 19749, 19865, 19976, 19779, and 11495; and HLA alleles predicted or known to present said at least one MHC class I epitope determining whether the gene is expressed or has been expressed; b) From the result of (a), the subject is 1) expressing at least one KRAS MHC class I epitope; and 2) expressing said HLA allele predicted or known to present said at least one KRAS MHC class I epitope. determining, or having determined, that the subject is a candidate for treatment with the antigen-based vaccine if 25. The method of claim 24, comprising:

26. The composition of any one of claims 1 to 13, wherein the at least one tumor-specific antigen-encoding nucleic acid sequence comprises (i) a 5' linker sequence, (ii) a 3' linker sequence, or (iii) a 5' linker sequence and a 3' linker sequence, and the at least one tumor-specific antigen-encoding nucleic acid sequence is linked to a different MHC class I epitope-encoding nucleic acid sequence by the 5' linker sequence and / or the 3' linker sequence.

27. 27. The composition of claim 26, wherein the 5' linker sequence and / or the 3' linker sequence comprises one or more naturally occurring sequences adjacent to the antigen from the cognate protein of origin and is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length.

28. the at least one promoter nucleotide sequence is a native 26S promoter nucleotide sequence encoded by an RNA alphavirus backbone or is an exogenous RNA promoter; and / or the antigen cassette comprises a second promoter nucleotide sequence; the second promoter nucleotide sequence comprises a 26S promoter nucleotide sequence or a plurality of 26S promoter nucleotide sequences; The composition of any one of claims 1 to 13, wherein each 26S promoter nucleotide sequence directs transcription of one or more of the separate open reading frames.

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