Common 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 of related applications This application asserts the interests of U.S. Provisional Patent Application No. 62 / 675,649 filed May 23, 2018 and U.S. Provisional Patent Application No. 62 / 675,559 filed May 23, 2018, which are incorporated herein by reference in their entirety for all purposes.

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

[0003] background Therapeutic vaccines based on tumor-specific antigens are extremely promising as the next generation of personalized cancer immunotherapy. 1~3 For example, cancers with high mutational loads, such as non-small cell lung cancer (NSCLC) and melanoma, are particularly promising targets for such therapies because they have a relatively high potential to generate neoantigens. 4,5 Early evidence suggests that vaccination based on newly synthesized antigens can trigger a T-cell response. 6 Cell therapy targeting newly generated antigens may induce tumor regression in selected patients. 7 This has been shown.

[0004] One question regarding the design of neoantigen vaccines is which of the numerous coding mutations present in the target tumor can generate the "best" therapeutic neoantigen (e.g., an antigen that can induce antitumor immunity and cause tumor regression).

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

[0006] Indeed, analysis of peptides presented by tumor cells, conducted by multiple groups, shows that less than 5% of peptides predicted to be presented using gene expression and MHC binding affinity are found on MHC on the tumor surface 10,11 This low correlation between binding prediction and MHC presentation is further emphasized by recent findings that no improvement in the prediction accuracy of binding-restricted neoantigens for checkpoint inhibitor response is observed relative to the number of mutations alone 12 .

[0007] Such low positive predictive value (PPV) of existing methods for predicting presentation presents problems in the design of neoantigen-based vaccines. When vaccines are designed using low PPV predictions, it is less likely that therapeutic neoantigens will be administered to the majority of patients, and it is expected that even fewer patients will receive multiple neoantigens, even assuming all presented peptides are immunogenic. Therefore, neoantigen vaccination according to current methods is unlikely to be effective in a substantial number of subjects with tumors.

[0008] Furthermore, previous approaches generate candidate neoantigens using only cis-acting mutations, which are mutations in splicing factors that occur in multiple tumor types and lead to abnormal splicing in many genes 13In most cases, further sources of nascent ORFs, including mutations that create or remove protease cleavage sites, were not considered.

[0009] Finally, standard approaches to tumor genome and transcriptome analysis may miss somatic mutations that give rise to candidate nascent antigens due to suboptimal conditions in library construction, exome and transcriptome capture, sequencing, or data analysis. Similarly, standard tumor analysis approaches may falsely promote sequence artifacts or germline polymorphisms as nascent antigens, potentially leading to inefficient vaccine capacity utilization or autoimmune risks, respectively.

[0010] In addition to the challenges of current methods for predicting neonatal antigens, existing vector systems that can be used for neonatal antigen delivery in humans, many of which are human-derived, also face specific challenges. For example, many humans have pre-existing immunity to human viruses as a result of past natural exposure, and this immunity can be a major obstacle to the use of recombinant human viruses to deliver neonatal antigens for cancer treatment.

[0011] Furthermore, targeting antigens shared among cancer patients, including both mutated neogenic antigens and non-mutated tumor antigens (e.g., inappropriately expressed tumor antigens), is an extremely promising vaccine strategy. The challenges in a shared antigen vaccine strategy include at least those mentioned above. [Overview of the project]

[0012] overview This specification discloses a composition for delivering an antigen expression system, wherein the antigen expression system comprises one or more vectors, and the one or more vectors comprise the following: (a) A vector skeleton, (i) at least one promoter nucleotide sequence, (ii) at least one polyadenylated (poly(A)) sequence and The vector skeleton, including, (b) Antigen cassette, (i) at least one antigen-coding nucleic acid sequence, (I) at least one tumor-specific MHC class I antigen-coding nucleic acid sequence, (A) MHC class I epitope coding nucleic acid sequences that encode an MHC class I epitope selected from the group consisting of sequence numbers 57 to 29357, (B) Optionally, a 5' linker sequence, and (C) Optional 3' linker array The at least one tumor-specific MHC class I antigen-coding nucleic acid sequence, including The at least one antigen-coding nucleic acid sequence, (ii) Optionally, a second promoter nucleotide sequence functionally linked to the antigen-coding nucleic acid sequence, (iii) Optionally, at least one MHC class II antigen-coding nucleic acid sequence, (iv) Optionally, at least one nucleic acid sequence encoding the GPGPG amino acid linker sequence (SEQ ID NO: 56), (v) Optionally, at least one second poly(A) sequence which is a native poly(A) sequence or an exogenous poly(A) sequence relative to the vector skeleton, The antigen cassette, including the antigen cassette.

[0013] This specification also discloses a composition for delivering an antigen expression system, wherein the antigen expression system comprises one or more vectors, and the one or more vectors comprise the following: (a) A vector skeleton, (i) at least one promoter nucleotide sequence, (ii) at least one polyadenylated (poly(A)) sequence and The vector skeleton, including, (b) Antigen cassette, (i) at least one antigen-coding nucleic acid sequence, (I) at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumor-specific MHC class I antigen-coding nucleic acid sequences linked linearly to each other, (A) KRAS_G12A MHC class I epitope coding nucleic acid sequence, which encodes MHC class I, including the sequence of sequence number 19831. (B) KRAS_G12C MHC class I epitope coding nucleic acid sequence that encodes an MHC class I epitope containing the sequence of sequence number 14954, (C) KRAS_G12D MHC class I epitope coding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs 19749 and 19865, and (D) KRAS_G12V MHC class I epitope coding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19976, 19979, 19779, 11495, and 19974. Includes, Each of the tumor-specific MHC class I antigen-coding nucleic acid sequences includes a class I epitope-coding nucleic acid sequence, and optionally, each MHC class I epitope-coding nucleic acid sequence codes for 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-coding nucleic acid sequences is, (A) Optionally, the 5' linker sequence, and (B) Optionally, 3' linker array The tumor-specific MHC class I antigen-coding nucleic acid sequence, including The at least one antigen-coding nucleic acid sequence, (ii) Optionally, a second promoter nucleotide sequence functionally linked to the antigen-coding nucleic acid sequence, (iii) Optionally, at least one MHC class II antigen-coding nucleic acid sequence, (iv) Optionally, at least one nucleic acid sequence encoding the GPGPG amino acid linker sequence (SEQ ID NO: 56), (v) Optionally, at least one second poly(A) sequence which is a native poly(A) sequence or an exogenous poly(A) sequence relative to the vector skeleton, The antigen cassette, including the antigen cassette.

[0014] This specification also discloses a composition for delivering an antigen expression system, wherein the antigen expression system comprises one or more vectors, and the one or more vectors comprise the following: (a) A vector skeleton, (i) at least one promoter nucleotide sequence, (ii) at least one polyadenylated (poly(A)) sequence and The vector skeleton, including, (b) Antigen cassette, (i) at least one antigen-coding nucleic acid sequence, (I) At least 20 tumor-specific MHC class I antigen-coding nucleic acid sequences linked linearly together, (A) KRAS_G12A MHC class I epitope coding nucleic acid sequence, which encodes MHC class I, including the sequence of sequence number 19831. (B) KRAS_G12C MHC class I epitope coding nucleic acid sequence that encodes an MHC class I epitope containing the sequence of sequence number 14954, (C) KRAS_G12D MHC class I epitope coding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs 19749 and 19865, and (D) KRAS_G12V MHC class I epitope coding 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 coding nucleic acid sequence, (F) KRAS_Q61K MHC class I epitope coding nucleic acid sequence, (G)TP53_R249M MHC class I epitope coding nucleic acid sequence, (H)CTNNB1_S45P MHC class I epitope coding nucleic acid sequence, (I)CTNNB1_S45F MHC class I epitope coding nucleic acid sequence, (J)ERBB2_Y772_A775dup MHC class I epitope coding nucleic acid sequence, (K)KRAS_Q61R MHC class I epitope coding nucleic acid sequence, (L)CTNNB1_T41A MHC class I epitope coding nucleic acid sequence, (M)TP53_K132N MHC class I epitope coding nucleic acid sequence, (N)KRAS_Q61L MHC class I epitope coding nucleic acid sequence, (O)TP53_R213L MHC class I epitope coding nucleic acid sequence, (P)BRAF_G466V MHC class I epitope coding nucleic acid sequence, (Q)KRAS_Q61H MHC class I epitope coding nucleic acid sequence, (R)CTNNB1_S37F MHC class I epitope coding nucleic acid sequence, (S)TP53_S127Y MHC class I epitope coding nucleic acid sequence, (T)TP53_K132E MHC class I epitope coding nucleic acid sequence, (U)KRAS_G12C MHC class I epitope coding nucleic acid sequence Includes, Each of the tumor-specific MHC class I antigen-coding nucleic acid sequences is, (A) Optionally, the 5' linker sequence, and (B) Optionally, 3' linker array The tumor-specific MHC class I antigen-coding nucleic acid sequence, including The at least one antigen-coding nucleic acid sequence, (ii) Optionally, a second promoter nucleotide sequence functionally linked to the antigen-coding nucleic acid sequence, (iii) Optionally, at least one MHC class II antigen-coding nucleic acid sequence, (iv) Optionally, at least one nucleic acid sequence encoding the GPGPG amino acid linker sequence (SEQ ID NO: 56), (v) Optionally, at least one second poly(A) sequence which is a native poly(A) sequence or an exogenous poly(A) sequence relative to the vector skeleton, The antigen cassette, including the antigen cassette.

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

[0016] This specification also discloses a composition for delivering an antigen expression system, wherein the antigen expression system comprises one or more vectors, and the one or more vectors comprise the following: (a) A vector skeleton comprising optionally a chimpanzee adenovirus vector which is a ChAdV68 vector, or optionally an alphavirus vector which is a Venezuelan encephalitis virus vector, (b) An antigen cassette incorporated between a 26S promoter nucleotide sequence and a poly(A) sequence, (i) at least one antigen-coding nucleic acid sequence, (I) At least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumor-specific MHC class I antigen-coding nucleic acid sequences linked linearly to each other, each of which is (A) An MHC class I epitope coding nucleic acid sequence encoding an MHC class I epitope of length 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 encoding a peptide that encodes the native N-terminal amino acid sequence of the MHC class I epitope and is at least three amino acids long, (C) A 3' linker sequence encoding a peptide that encodes the natural C-terminal acid sequence of the MHC class I epitope and is at least three amino acids long. Includes, The antigen cassette is functionally linked to the 26S promoter nucleotide sequence, each of the MHC class I antigen-coding nucleic acid sequences codes for a polypeptide of 13 to 25 amino acids, and the 3' end of each MHC class I antigen-coding nucleic acid sequence is ligated to the 5' end of the subsequent MHC class I antigen-coding nucleic acid sequence, except for the last MHC class I antigen-coding nucleic acid sequence in the antigen cassette. The aforementioned MHC class I antigen-coding nucleic acid sequence The at least one antigen-coding nucleic acid sequence, (ii) at least two MHC class II antigen-coding nucleic acid sequences, (I) PADRE MHC class II sequence (sequence number 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) that links 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-coding nucleic acid sequences with the tumor-specific MHC class I antigen-coding nucleic acid sequences, (V) Optionally, a third nucleic acid sequence encoding the GPGPG amino acid linker sequence (SEQ ID NO: 56) at the 3' end of at least two MHC class II antigen coding nucleic acid sequences, The at least two MHC class II antigen-coding nucleic acid sequences include The antigen cassette, including the antigen cassette.

[0017] This specification also provides a method for evaluating subjects having cancer, a)1) Whether the subject has an HLA allele that is predicted or known to present an antigen included in the antigen-based vaccine, and below: 1) Whether the target tumor expresses the gene related to the antigen, and optionally, whether the gene is expressed abnormally compared to normal cells or tissues. 2) Whether the target tumor has a mutation related to the antigen. one or both The process of determining, or already determining, b) A step in which, based on the results of (a) above, the subject expresses the HLA allele, the tumor of the subject expresses the gene, and / or the tumor of the subject has the mutation, the subject is determined to be a candidate for treatment with the antigen-based vaccine, or has already been determined to be so The antigen comprises at least one MHC class I epitope sequence selected from the group consisting of SEQ ID NOs. 57 to 29357. The above process, c) Optionally, administering the antigen-based vaccine to the subject, or having already administered the antigen-based vaccine, 1) The at least one MHC class I epitope, or 2) MHC class I epitope coding nucleic acid sequence encoding at least one MHC class I epitope The above process includes and The aforementioned method, including the above, is also disclosed.

[0018] This specification also provides a method for evaluating subjects having cancer, a) The above object is, 1) The tumor expresses the A0301 HLA allele and has the KRAS_G12A mutation, 2) The tumor expresses the A0201 HLA allele and has the KRAS_G12C mutation, 3) Expressing the C0802 HLA allele or the A1101 HLA allele, and the tumor in question has the KRAS_G12D mutation, or 4) The tumor expresses the A0301 HLA allele, or the A1101 HLA allele, or the A3101 HLA allele, or the C0102 HLA allele, or the A0302 HLA allele, and the tumor in question has the KRAS_G12V mutation. The process of determining, or already determining, b) From the results of (a) above, the subject is 1) If the A0301 allele is expressed and the target tumor has the KRAS_G12A mutation, 2) If the A0201 allele is expressed and the target tumor has the KRAS_G12C mutation, 3) If the tumor expresses the C0802 HLA allele or the A1101 HLA allele and the target tumor has the KRAS_G12D mutation, or 4) When the A0301 HLA allele, the A1101 HLA allele, the A3101 HLA allele, the C0102 HLA allele, or the A0302 HLA allele is expressed, and the target tumor has the KRAS_G12V mutation. The process includes determining, or having already determined, that the subject is a candidate for treatment with the antigen-based vaccine, c) Optionally, administering the antigen-based vaccine to the subject, or having already administered the antigen-based vaccine, 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, 2) MHC class I epitope coding nucleic acid sequences encoding at least one MHC class I epitope, each containing the KRAS_G12A mutation, the KRAS_G12C mutation, the KRAS_G12AD mutation, or the KRAS_G12V mutation. The above process includes The aforementioned method, including the above, is also disclosed.

[0019] In some embodiments, steps (a) and / or (b) include obtaining a dataset from a third party that has processed a sample from the subject. In some embodiments, step (a) includes 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 includes 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, cerebrospinal fluid, and needle aspiration. 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 tumor of interest. 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] This specification also discloses a method for treating a subject having cancer, 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-coding nucleic acid sequence encoding the at least one MHC class I epitope, and the at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs. 57 to 29357. In some embodiments, the at least one MHC class I antigen-coding nucleic acid sequence is derived from a tumor of the subject having cancer. In some embodiments, the at least one MHC class I antigen-coding nucleic acid sequence is not derived from a tumor of the subject having cancer.

[0021] This specification also discloses a method for inducing an immune response in a subject, comprising administering an antigen-based vaccine to the subject, wherein the antigen-based vaccine comprises 1) at least one MHC class I epitope, or 2) an MHC class I epitope-coding nucleic acid sequence encoding the at least one MHC class I epitope, and the at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs. 57 to 29357. In some embodiments, the subject expresses at least one HLA allele that is 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 that is 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 34. In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present at least one MHC class I epitope sequence, wherein the at least one MHC class I epitope sequence includes a mutation selected from the group of mutations shown in Table 32.

[0022] This specification also discloses 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-coding 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] This specification also discloses a method for inducing an immune response in a subject, comprising administering an antigen-based vaccine to the subject, wherein the antigen-based vaccine comprises 1) at least one MHC class I epitope, or 2) an MHC class I epitope-coding 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, wherein the subject expresses at least one HLA allele that is 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 shown in Table 34, and the subject expresses at least one HLA allele shown in Table 34 associated with the corresponding mutation shown in Table 34 (e.g., KRAS_G13D and C0802).

[0024] This specification also provides a method for inducing an immune response in a subject, comprising administering an antigen-based vaccine to the subject, wherein the antigen-based vaccine comprises 1) at least one MHC class I epitope, or 2) an MHC class I epitope-coding 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, the subject expresses at least one HLA allele that is 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. 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 nascent antigen cassette is from 5' to 3', Pa-(L5b-Nc-L3d)X-(G5e-Uf)Y-G3g It is explained by an expression that includes, During the ceremony, P comprises the second promoter nucleotide sequence, where a=0 or 1. N includes one of the MHC class I epitope coding nucleic acid sequences, where c=1. L5 includes the aforementioned 5' linker sequence, where b=0 or 1. L3 includes the 3' linker sequence, where d=0 or 1. G5 comprises one of the at least one nucleic acid sequences that encode the GPGPG amino acid linker (SEQ ID NO: 56), where e = 0 or 1. G3 comprises one of the at least one nucleic acid sequences that encode the 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-coding nucleic acid sequences, where f=1, X = 1 to 400, where for each X, the corresponding Nc is the epitope-coding nucleic acid sequence. Y = 0, 1, or 2, where for each Y, the corresponding Uf is the antigen-coding nucleic acid sequence. In some embodiments, for each X, the corresponding Nc is a different MHC class I epitope coding nucleic acid sequence. In some embodiments, for each Y, the corresponding Uf is a different MHC class II antigen coding 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 given by the backbone, the at least one polyadenylated poly(A) sequence is a poly(A) sequence of at least 100 consecutive A nucleotides given by the backbone (SEQ ID NO: 29358), each N encoding an MHC class I epitope of 7 to 15 amino acids, L5 being a native 5' linker sequence encoding the native N-terminal amino acid sequence of the MHC I epitope, the 5' linker sequence encoding a peptide of at least 3 amino acids, and L3 being the MHC The vector skeleton comprises a native 3' linker sequence encoding a native terminal nucleic acid sequence of an I epitope, wherein the 3' linker sequence encodes a peptide having a length of at least three amino acids, where U is a PADRE class II sequence and a tetanus toxoid MHC class II sequence, respectively, and the vector skeleton optionally comprises a chimpanzee adenovirus vector which is a ChAdV68 vector, or an alphavirus vector which is an alphavirus vector which is a Venezuelan encephalitis virus vector, and each of the MHC class I nascent antigen-coding nucleic acid sequences encodes a polypeptide having a length of 13 to 25 amino acids.

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

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

[0029] In some embodiments, the skeleton comprises at least one nucleotide sequence of Auravirus, Fort Morganvirus, Venezuelan Encephalitis Virus, Ross River Virus, Semryqui Forest Virus, Sindbisvirus, or Mayarovirus. In some embodiments, the skeleton comprises at least one nucleotide sequence of Venezuelan Encephalitis Virus. In some embodiments, the skeleton comprises at least a sequence for non-structural protein-mediated amplification, a 26S promoter sequence, a poly(A) sequence, a non-structural protein 1 (nsP1) gene, an nsP2 gene, an nsP3 gene, and an nsP4 gene, encoded by a nucleotide sequence of Auravirus, Fort Morganvirus, Venezuelan Encephalitis Virus, Ross River Virus, Semryqui Forest Virus, Sindbisvirus, or Mayarovirus. In some embodiments, the backbone includes at least a sequence for non-structural protein-mediated amplification, a 26S promoter sequence, and a poly(A) sequence encoded by a nucleotide sequence of Auravirus, Fort Morganvirus, Venezuelan Encephalitis Virus, Ross River Virus, Semlik Forest Virus, Sindbisvirus, or Mayarovirus. In some embodiments, the sequence for non-structural protein-mediated amplification is selected from the group consisting of alphavirus 5'UTR, 51nt CSE, 24nt CSE, 26S subgenomic promoter sequence, 19nt CSE, alphavirus 3'UTR, or a combination thereof.

[0030] In some embodiments, the backbone does not encode structural virion protein capsids E2 and E1. In some embodiments, the nascent antigen cassette is inserted in place of structural virion proteins within the nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semlik Forest virus, Sindbis virus, or Mayarovirus.

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

[0032] In some embodiments, insertion of a nascent antigen cassette results in the transcription of a polycistronic RNA containing the nsP1-4 genes and at least one antigen-coding nucleic acid sequence, where the nsP1-4 genes and at least one antigen-coding nucleic acid sequence reside in separate open reading frames.

[0033] In some embodiments, at least one promoter nucleotide sequence is a native 26S promoter nucleotide sequence encoded by a backbone. In some embodiments, at least one promoter nucleotide sequence is an exogenous RNA promoter. In some embodiments, a 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 26S promoter nucleotide sequence resulting in the transcription of one or more separate open reading frames.

[0034] In some embodiments, the adenovirus vector is a chimpanzee adenovirus (ChAd) vector, optionally a C68 vector. In some embodiments, the adenovirus vector includes the sequence described in Sequence ID No. 1. In some embodiments, the adenovirus vector includes the sequence described in Sequence 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 described in Sequence ID No. 1 is completely or functionally deleted from the sequence, and optionally, the sequence is one in which (1) E1A and E1B, (2) E1A, E1B, and E3, or (3) E1A, E1B, E3, and E4 of the sequence described in Sequence ID No. 1 are completely or functionally deleted. In some embodiments, the adenovirus vector comprises a gene or regulatory sequence derived from the sequence of Sequence ID No. 1, and optionally, the gene is selected from the group consisting of terminal inverted repeat sequences (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the chimpanzee adenovirus sequence described in Sequence ID No. 1.

[0035] In some embodiments, the nascent antigen cassette is inserted into the adenovirus vector in the E1 region, the E3 region, and / or any deleted AdV region into which the nascent antigen cassette can be incorporated.

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

[0037] In some embodiments, the adenovirus nascent antigen cassette further comprises at least one polyA sequence functionally linked to at least one of a plurality of nascent antigen-coding nucleic acid sequences, optionally, the polyA sequence located 3' to at least one sequence within the plurality of nascent antigen-coding nucleic acid sequences.

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

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

[0040] In some embodiments, one or more nascent antigen expression vectors are at least 300 nt in size. In some embodiments, one or more nascent antigen expression vectors are at least 1 kb in size. In some embodiments, one or more nascent antigen expression vectors are at least 2 kb in size. In some embodiments, one or more nascent antigen expression vectors are at least 5 kb in size.

[0041] In some embodiments, at least one of the at least one nascent antigen-coding nucleic acid sequences encodes a polypeptide sequence or a portion thereof presented by MHC class I on tumor cells. In some embodiments, each antigen-coding nucleic acid sequence within each antigen-coding nucleic acid sequence is directly linked to one another. In some embodiments, at least one of the at least one antigen-coding nucleic acid sequences is linked to a different antigen-coding 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) a sequence of glycine residues of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues (SEQ ID NO: 29359), (2) a sequence of alanine residues of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues (SEQ ID NO: 29360), (3) two arginine residues (RR), (4) alanine, alanine, tyrosine (AAY), (5) mammalian proteaso The linker is selected from the group consisting of a consensus sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, which is efficiently processed by the linker, and one or more native sequences of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2 to 20 amino acid residues, adjacent to an antigen derived from a congener protein of (6). In some embodiments, the linker aligns two MHC class II sequences or one MHC class II sequence with one MHC class I sequence. In some embodiments, the linker includes the sequence GPGPG (SEQ ID NO: 56).

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

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

[0044] In some embodiments, at least one mutation includes 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.

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

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

[0048] In some embodiments, each MHC class I nascent antigen-coding nucleic acid sequence encodes a polypeptide sequence of 8 to 35 amino acids in length, optionally 9 to 17 amino acids, 9 to 25 amino acids, 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.

[0049] In some embodiments, at least one MHC class II antigen-coding nucleic acid sequence is present. In some embodiments, at least one MHC class II antigen-coding nucleic acid sequence is present and includes at least one MHC class II nascent antigen-coding nucleic acid sequence that includes at least one mutation that causes the at least one MHC class II nascent antigen-coding nucleic acid sequence to be different from the corresponding wild-type parent nucleic acid sequence. In some embodiments, the at least one MHC class II antigen-coding nucleic acid sequence is 12–20 amino acids, 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-coding nucleic acid sequence is present and includes at least one universal MHC class II antigen-coding nucleic acid sequence, optionally, the at least one universal sequence includes at least one of tetanus toxoid and PADRE.

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

[0051] In some embodiments, at least one poly(A) sequence includes a poly(A) sequence naturally occurring in the backbone. In some embodiments, at least one poly(A) sequence includes an exogenous poly(A) sequence relative to the backbone. In some embodiments, at least one poly(A) sequence is functionally linked to at least one of at least one antigen-coding 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 nascent antigen cassette further comprises at least one of the following: an intron sequence, a woodchuck hepatitis virus posttranscriptional regulator (WPRE) sequence, an internal ribosome entry sequence (IRES) sequence, a nucleotide sequence encoding a 2A self-cleavage peptide sequence, a nucleotide sequence encoding a furin cleavage site, or a sequence in a 5' or 3' terminal non-coding region known to improve mRNA nuclear export, stability, or translation efficiency, functionally ligated to at least one of the at least one antigen-coding nucleic acid sequences.

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

[0054] In some embodiments, 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 its antigen-binding fragment, an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, an anti-4-1BB antibody or its antigen-binding fragment, or an anti-OX-40 antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment is a Fab fragment, a Fab' fragment, a single-chain Fv (scFv), a single-domain antibody (sdAb) as monospecific or linked multispecific (e.g., an antibody domain from a camelid), or a full-length single-chain antibody (e.g., a full-length IgG with heavy and light chains linked by a flexible linker). In some embodiments, the heavy chain sequence and 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 light chain sequence of the antibody are linked by a flexible linker such as a contiguous glycine residue.

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

[0056] In some embodiments, at least one MHC class I nascent antigen-coding nucleic acid sequence is (a) A step of 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 each peptide sequence of a set of nascent antigens, (b) A step of inputting the peptide sequence of each nascent antigen into a presentation model in order to generate a set of numerical likelihoods that each nascent antigen is presented by one or more MHC alleles on the surface of tumor cells of the tumor, wherein the set of numerical likelihoods is identified at least based on received mass spectrometry data, (c) A step of selecting a subset of the set of nascent antigens based on the set of numerical likelihoods in order to generate a selected set of nascent antigens used to generate the at least one MHC class I nascent antigen coding nucleic acid sequence. It is selected by doing so.

[0057] In some embodiments, each of at least one MHC class I nascent antigen-coding nucleic acid sequence is (a) A step of 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 each peptide sequence of a set of nascent antigens, (b) A step of inputting the peptide sequence of each nascent antigen into a presentation model in order to generate a set of numerical likelihoods that each nascent antigen is presented by one or more MHC alleles on the surface of tumor cells of the tumor, wherein the set of numerical likelihoods is identified at least based on received mass spectrometry data, (c) A step of selecting a subset of the set of nascent antigens based on the set of numerical likelihoods in order to generate a selected set of nascent antigens used to generate the at least one MHC class I nascent antigen coding nucleic acid sequence. It is selected by doing so.

[0058] In some embodiments, the number of selected sets of nascent antigens is 2 to 20.

[0059] In some embodiments, the presentation model represents a dependence between the presence of a pair of a specific MHC allele and a specific amino acid at a specific position in a peptide sequence, and the likelihood of presentation of such a peptide sequence containing the specific amino acid at the specific position on the tumor cell surface by the specific MHC allele of the pair.

[0060] In some embodiments, selecting a set of selected nascent antigens involves selecting nascent antigens that have a higher likelihood of being presented on the surface of tumor cells compared to nascent antigens that have not been selected based on a presentation model. In some embodiments, the selected antigens are verified to be presented by one or more specific HLA alleles. In some embodiments, selecting a set of selected nascent antigens involves selecting nascent antigens that have a higher likelihood of inducing a tumor-specific immune response in a target compared to nascent antigens that have not been selected based on a presentation model. In some embodiments, selecting a set of selected nascent antigens involves selecting nascent antigens that have a higher likelihood of being presented to naive T cells by professional antigen-presenting cells (APCs), where APCs are optionally dendritic cells (DCs), compared to nascent antigens that have not been selected based on a presentation model. In some embodiments, selecting a set of selected nascent antigens involves selecting nascent antigens that have a lower likelihood of being inhibited by central or peripheral tolerance compared to nascent antigens that have not been selected based on a presentation model. In some embodiments, selecting a set of chosen nascent antigens involves selecting nascent antigens that have a reduced likelihood of inducing an autoimmune response against normal tissue in the subject compared to nascent antigens that have not been selected based on the presentation model. In some embodiments, exome or transcriptome nucleotide sequencing data are obtained by sequencing with respect to tumor tissue. In some embodiments, sequencing is next-generation sequencing (NGS) or any large-scale parallel processing sequencing approach.

[0061] In some embodiments, the nascent antigen cassette includes a junctional epitope sequence formed by adjacent sequences within the nascent antigen cassette. In some embodiments, at least one, or each junctional epitope sequence, has an affinity greater than 500 nM for MHC. In some embodiments, each junctional epitope sequence is non-self. In some embodiments, each MHC class I epitope is predicted or verified to be presentable 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 presentable by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA occurrence rate of at least 0.01% in the population. In some embodiments, each MHC class I epitope is predicted or verified to be presentable 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. In some embodiments, the nascent antigen cassette does not encode a non-therapeutic MHC class I or class II epitope nucleic acid sequence, including a post-translational wild-type nucleic acid sequence, and it is predicted that the non-therapeutic epitope will be presented on the target MHC allele. 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 nascent antigen cassette. In some embodiments, the prediction is based on the 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-coding nucleic acid sequence in a nascent antigen cassette is determined by a series of steps including: (a) generating a set of candidate nascent antigen cassette sequences corresponding to various orders of the at least one antigen-coding nucleic acid sequence; (b) determining a presentation score for each candidate nascent antigen cassette sequence based on the presentation of non-therapeutic epitopes within the candidate nascent antigen cassette sequence; and (c) selecting candidate cassette sequences associated with presentation scores below a predetermined threshold as nascent antigen cassette sequences for a nascent antigen vaccine.

[0062] In some embodiments, any of the above compositions further comprises a nanoparticle-like delivery vehicle. In some embodiments, the nanoparticle-like delivery vehicle may be lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise ionizable aminolipids. In some embodiments, the ionizable aminolipids comprise MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecules. In some embodiments, the nanoparticle-like delivery vehicle encapsulates a nascent antigen expression system.

[0063] In some embodiments, any of the above compositions further comprises a plurality of LNPs, the LNPs comprising a nascent antigen expression system, a cationic lipid, a non-cationic lipid, and a complex lipid that inhibits aggregation of the LNPs, wherein at least about 95% of the plurality of LNPs are either non-layered or highly electron-densitic.

[0064] In some embodiments, the noncationic lipid is a mixture of (1) phospholipids and (2) cholesterol or cholesterol derivatives.

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

[0066] In some embodiments, the nascent antigen expression system is completely encapsulated within the LNP.

[0067] In some embodiments, the non-layered morphology of LNP is inverse hexagonal (H II ) or containing a cubic crystal structure.

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

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

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

[0071] In some embodiments, more than 95% of LNPs have a non-layered morphology. In some embodiments, more than 95% of LNPs have high electron density.

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

[0073] In some embodiments, any of the above compositions further comprises a plurality of LNPs, the LNPs comprising a cationic lipid constituting 50 mol% to 85 mol% of the total lipids present in the LNPs, a complex lipid that inhibits aggregation of the LNPs constituting 0.5 mol% to 2 mol% of the total lipids present in the LNPs, and a non-cationic lipid constituting 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 complex lipid comprises a polyethylene glycol (PEG)-lipid complex. In some embodiments, the PEG-lipid complex comprises a PEG-diacylglycerol (PEG-DAG) complex, a PEG-dialkyloxypropyl (PEG-DAA) complex, or a mixture thereof. In some embodiments, the PEG-DAA complex comprises a PEG-dimyristyloxypropyl (PEG-DMA) complex, a PEG-distearoyloxypropyl (PEG-DSA) complex, or a mixture thereof. In some embodiments, the PEG portion of the complex has an average molecular weight of about 2000 daltons.

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

[0077] In some embodiments, LNP has the structure of formula I: TIFF0007923078000001.tif47128[where L 1 and L 2 These are independently -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, and -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, G 1 These are Ci~C2 alkylene, -(C=0)-, -0(C=0)-, -SC(=0)-, -R a C(=0)-, or direct bond, -C(=0)-, -(C=0)0-, -C(=0)S-, -C(=0)R a -, or a direct bond, where G is a Ci~C6 alkylene, and R a H or C1~C 12 It is alkyl, R 1a and R 1b In each case, independently, (a) H or C1~C 12(b) Alkyl or R 1a H or C1~C 12 It is alkyl, R 1b Along with the carbon atom it bonds to, the adjacent R 1b and together with the carbon atom to which it is bonded, it forms a carbon-carbon double bond, R 2a and R 2b In each case, independently, (a) H or C1~C 12 (b) Alkyl or R 2a H or C1~C 12 It is alkyl, R 2b Along with the carbon atom it bonds to, the adjacent R 2b and together with the carbon atom to which it is bonded, it forms a carbon-carbon double bond, R 3a and R 3b In each case, independently, (a) H or C1~C 12 (b) Alkyl or R 3a H or C1~C 12 It is alkyl, R 3b It forms a carbon-carbon double bond with the carbon atom it bonds to, and with the adjacent R and the carbon atom it bonds to, 4a and R 4b In each case, independently, (a) H or C1~C 12 (b) Alkyl or R 4a H or C1~C 12 It is alkyl, R 4b Along with the carbon atom it bonds to, the adjacent R 4b and together with the carbon atom to which it is bonded, it forms a carbon-carbon double bond, R 5 and R 6 Each is independently either H or methyl, and R 7 C4~C 20 It is alkyl, R 8 and R 9 These are C1~C, each independent of the others. 12 Alkyl or R 8 and R 9, together with the nitrogen atom to which they are bound, form a 5-, 6-, or 7-membered heterocyclic ring; a, b, c, and d are each independently an integer of 1 to 24, and x is 0, 1, or 2] a compound having, or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0078] In some aspects, the LNP has the structure of Formula II: TIFF0007923078000002.tif36128 [wherein L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a carbon-carbon double bond, and R 1a and R 1b in each occurrence are independently (a) H or C1 to C 12 alkyl, or (b) R 1a is H or C1 to C 12 alkyl, and R 1b together with the carbon atom to which it is bonded and the adjacent R 1b together with the carbon atom to which it is bonded form a carbon-carbon double bond, R 2a and R 2b in each occurrence are independently (a) H or C1 to C 12 alkyl, or (b) R 2a is H or C1 to C 12 alkyl, and R 2b together with the carbon atom to which it is bonded and the adjacent R 2b together with the carbon atom to which it is bonded form a carbon-carbon double bond, R 3a and R 3b in each occurrence are independently (a) H or C1 to C 12 alkyl, or (b) R 3a is H or C1 to C 12 alkyl, and R 3b together with the carbon atom to which it is bonded and the adjacent R 3b together with the carbon atom to which it is bonded form a carbon-carbon double bond, R 4a and R 4bin each case, is (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 bonded, together with adjacent R 4b and the carbon atom to which it is bonded form a carbon-carbon double bond, R 5 and R 6 are each independently methyl or cycloalkyl; R7, in each case, is independently H or C1~C 12 alkyl, R 8 and R 9 are each independently unsubstituted C1~C 12 alkyl, or R 8 and R 9 together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered heterocyclic ring containing one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; e is 1 or 2; provided that at least one of R 1a , R 2a , R 3a , or R 4a is C1~C 12 alkyl, or at least one of L 1 and L 2 is -O(C=O)- or -(C=O)O-; R 1a and R 1b are not isopropyl when a is 6, and are not n-butyl when a is 8] which comprises a compound having the above definition, or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0079] In some embodiments, any of the above compositions further comprises one or more excipients comprising 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 the compound to the neutral lipid is in the range of approximately 2:1 to approximately 8:1.

[0081] In some embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to cholesterol is in the range of approximately 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 is in the range of 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 dialkyloxypropyl carbamate. In some embodiments, the PEGylated lipid has the structure shown in Structure III below: TIFF0007923078000003.tif25128[where, R 10 and R 11 Each of these is an alkyl chain, either linear or branched, saturated or unsaturated, having 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds, and z has an average value in the range of 30 to 60. It has, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In some embodiments, R 10 and R 11 Each of these is an independent, linear saturated alkyl chain having 12 to 16 carbon atoms. In some embodiments, the average value of z is approximately 45. Start from here

[0083] In some embodiments, LNPs self-assemble into a non-bilayer structure when mixed with polyanionic nucleic acids. In some embodiments, the non-bilayer structure has a diameter of 60 nm to 120 nm. In some embodiments, the non-bilayer structure has a diameter of approximately 70 nm, approximately 80 nm, approximately 90 nm, or approximately 100 nm. In some embodiments, the nanoparticle-like delivery vehicle has a diameter of approximately 100 nm.

[0084] This specification also discloses pharmaceutical compositions comprising any of the compositions disclosed herein (such as a vector based on an alphavirus or ChAd 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 its antigen-binding fragment, an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, an anti-4-1BB antibody or its antigen-binding fragment, or an anti-OX-40 antibody or its antigen-binding fragment.

[0085] Also disclosed herein are isolated nucleotide sequences or sets of isolated nucleotide sequences comprising a nascent antigen cassette described in any of the claims of a prior composition and one or more elements obtained 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 sequences required for unstructured protein-mediated amplification, 26S promoter nucleotide sequences, poly(A) sequences, and nsP1-4 genes of the sequence described 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 the nascent antigen cassette disclosed herein, inserted at position 7544 of the sequence described in SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the isolated nucleotide sequence further comprises a nucleotide sequence of a T7 or SP6 RNA polymerase promoter located at the 5' end of the one or more elements obtained from the sequence of SEQ ID NO: 3 or SEQ ID NO: 5, and optionally, one or more restriction sites located at the 3' end of the poly(A) sequence. In some embodiments, the nascent antigen cassette disclosed herein is inserted at position 7563 of SEQ ID NO: 8 or SEQ ID NO: 9. In other embodiments, the sequence described in SEQ ID NO: 8 or SEQ ID NO: 9 further comprises an additional adenine nucleotide inserted at position 17.

[0086] This specification also discloses an isolated nucleotide sequence comprising a nascent antigen cassette disclosed herein and at least one promoter disclosed herein. In some embodiments, the isolated nucleotide sequence further comprises a ChAd-based gene. In some embodiments, the ChAd-based gene is obtained from the sequence of Sequence ID No. 1, optionally selected from the group consisting of the ITR, E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of chimpanzee adenovirus of the sequence described in Sequence ID No. 1, and optionally the nucleotide sequence is cDNA.

[0087] This specification also provides isolated cells comprising an isolated nucleotide sequence disclosed herein, wherein the cells are optionally BHK-21, CHO, HEK293 or a variant thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a cells.

[0088] This specification also discloses vectors containing isolated nucleotide sequences disclosed herein.

[0089] This specification also discloses a kit comprising the vector or composition disclosed herein and instructions for use.

[0090] This specification also discloses a method for treating a subject having 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 nascent antigen-coding nucleic acid sequence is derived from a tumor of the subject having cancer. In some embodiments, the at least one MHC class I nascent antigen-coding nucleic acid sequence is not derived from a tumor of the subject having cancer.

[0091] This specification also discloses 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 that is predicted or known to present an MHC class I epitope. In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present an MHC class I epitope, and the MHC class I epitope includes a mutation selected from the group of mutations shown in Table 34. In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present an MHC class I epitope, and the MHC class I epitope includes a mutation selected from the group of mutations shown 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 include administering one or more immunomodulators, which may optionally be administered before, concurrently with, or after administration of the composition or pharmaceutical composition. In some embodiments, one or more immunomodulators are selected from the group consisting of anti-CTLA4 antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, anti-PD-L1 antibody or its antigen-binding fragment, anti-4-1BB antibody or its antigen-binding fragment, or anti-OX-40 antibody or its antigen-binding fragment. In some embodiments, the immunomodulators are administered intravenously (IV), intramuscularly (IM), intradermally (ID), or subcutaneously (SC). In some embodiments, subcutaneous administration is performed near the administration site of the composition or pharmaceutical composition, or in close proximity to the lymph nodes in the inflow area of ​​one or more vectors or compositions.

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

[0095] This specification also discloses a method for producing one or more vectors of any of the above compositions, comprising: obtaining a linearized DNA sequence comprising the backbone and the nascent antigen cassette; in vitro transcription of the linearized DNA sequence by adding the linearized DNA sequence to an in vitro transcription reaction comprising all components necessary for transcribing the linearized DNA sequence to RNA, further optionally comprising in vitro addition of the m7g cap to the resulting RNA; and isolating the one or more vectors from the in vitro transcription reaction. In some embodiments, the linearized DNA sequence is produced by linearizing a DNA plasmid sequence or by amplification using PCR. In some embodiments, the DNA plasmid sequence is produced using one of bacterial recombination or whole-genome DNA synthesis or whole-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 includes one or more of phenol-chloroform extraction, purification using a silica column, or similar RNA purification methods.

[0096] This specification also discloses a method for producing any of the compositions disclosed herein, comprising providing components of a nanoparticle delivery vehicle, providing a nascent antigen expression system, and providing conditions sufficient for the nanoparticle delivery vehicle and the nascent antigen expression system to produce the composition for delivering the nascent antigen expression system. In some embodiments, such conditions are provided by microfluidic mixing.

[0097] This specification also discloses a method for producing an adenovirus vector disclosed herein, comprising: obtaining a plasmid sequence comprising the at least one promoter sequence and the nascent antigen cassette; transfecting the plasmid sequence into one or more host cells; and isolating the adenovirus vector from the one or more host cells.

[0098] In some embodiments, isolation includes lysing host cells to obtain a cell lysate containing the adenovirus vector, and purifying the adenovirus vector from the cell lysate.

[0099] In some embodiments, the plasmid sequence is generated using one of the following methods: bacterial recombination, whole-genome DNA synthesis, or whole-genome DNA synthesis with amplification of synthetic DNA within a bacterial cell. In some embodiments, the one or more host cells are at least one of the following: CHO, HEK293 or its variants, 911, HeLa, A549, LP-293, PER.C6, and AE1-2a cells. In some embodiments, the purification of the adenovirus vector from the cell lysate is performed by one or more of the following methods: chromatographic separation, centrifugation, viral precipitation, and filtration. [Invention 1001] A composition for delivering an antigen expression system, which includes an antigen expression system, The antigen expression system comprises one or more vectors, The composition comprises one or more vectors: (a) A vector skeleton, (i) at least one promoter nucleotide sequence, (ii) at least one polyadenylated (poly(A)) sequence and The vector skeleton, including, (b) Antigen cassette, (i) at least one antigen-coding nucleic acid sequence, (I) at least one tumor-specific MHC class I antigen-coding nucleic acid sequence, (A) MHC class I epitope coding nucleic acid sequences that encode an MHC class I epitope selected from the group consisting of sequence numbers 57 to 29357, (B) Optionally, a 5' linker sequence, and (C) Optional 3' linker array The at least one tumor-specific MHC class I antigen-coding nucleic acid sequence, including The at least one antigen-coding nucleic acid sequence, (ii) Optionally, a second promoter nucleotide sequence functionally linked to the antigen-coding nucleic acid sequence, (iii) Optionally, at least one MHC class II antigen-coding nucleic acid sequence, (iv) Optionally, at least one nucleic acid sequence encoding the GPGPG amino acid linker sequence (SEQ ID NO: 56), (v) Optionally, at least one second poly(A) sequence which is a native poly(A) sequence or an exogenous poly(A) sequence relative to the vector skeleton, The antigen cassette, including the antigen cassette. [Invention 1002] A composition for delivering an antigen expression system, which includes an antigen expression system, The antigen expression system comprises one or more vectors, The composition comprises one or more vectors: (a) A vector skeleton, (i) at least one promoter nucleotide sequence, (ii) at least one polyadenylated (poly(A)) sequence and The vector skeleton, including, (b) Antigen cassette, (i) at least one antigen-coding nucleic acid sequence, (I) at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumor-specific MHC class I antigen-coding nucleic acid sequences linked linearly to each other, (A) KRAS_G12A MHC class I epitope coding nucleic acid sequence, which encodes MHC class I, including the sequence of sequence number 19831. (B) KRAS_G12C MHC class I epitope coding nucleic acid sequence that encodes an MHC class I epitope containing the sequence of sequence number 14954, (C) KRAS_G12D MHC class I epitope coding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs 19749 and 19865, and (D) KRAS_G12V MHC class I epitope coding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19976, 19979, 19779, 11495, and 19974 Includes, Each of the tumor-specific MHC class I antigen-coding nucleic acid sequences includes a class I epitope-coding nucleic acid sequence, and optionally, each MHC class I epitope-coding nucleic acid sequence codes for 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-coding nucleic acid sequences is, (A) Optionally, the 5' linker sequence, and (B) Optionally, 3' linker array The tumor-specific MHC class I antigen-coding nucleic acid sequence, including The at least one antigen-coding nucleic acid sequence, (ii) Optionally, a second promoter nucleotide sequence functionally linked to the antigen-coding nucleic acid sequence, (iii) Optionally, at least one MHC class II antigen-coding nucleic acid sequence, (iv) Optionally, at least one nucleic acid sequence encoding the GPGPG amino acid linker sequence (SEQ ID NO: 56), (v) Optionally, at least one second poly(A) sequence which is a native poly(A) sequence or an exogenous poly(A) sequence relative to the vector skeleton, The antigen cassette, including the antigen cassette. [Invention 1003] A composition for delivering an antigen expression system, which includes an antigen expression system, The antigen expression system comprises one or more vectors, The composition comprises one or more vectors: (a) A vector skeleton, (i) at least one promoter nucleotide sequence, (ii) at least one polyadenylated (poly(A)) sequence and The vector skeleton, including, (b) Antigen cassette, (i) at least one antigen-coding nucleic acid sequence, (I) At least 20 tumor-specific MHC class I antigen-coding nucleic acid sequences linked linearly together, (A) KRAS_G12A MHC class I epitope coding nucleic acid sequence, which encodes MHC class I, including the sequence of sequence number 19831. (B) KRAS_G12C MHC class I epitope coding nucleic acid sequence that encodes an MHC class I epitope containing the sequence of sequence number 14954, (C) KRAS_G12D MHC class I epitope coding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs 19749 and 19865, and (D) KRAS_G12V MHC class I epitope coding 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 coding nucleic acid sequence, (F) KRAS_Q61K MHC class I epitope coding nucleic acid sequence, (G)TP53_R249M MHC class I epitope coding nucleic acid sequence, (H)CTNNB1_S45P MHC class I epitope coding nucleic acid sequence, (I)CTNNB1_S45F MHC class I epitope coding nucleic acid sequence, (J)ERBB2_Y772_A775dup MHC class I epitope coding nucleic acid sequence, (K)KRAS_Q61R MHC class I epitope coding nucleic acid sequence, (L)CTNNB1_T41A MHC class I epitope coding nucleic acid sequence, (M)TP53_K132N MHC class I epitope coding nucleic acid sequence, (N)KRAS_Q61L MHC class I epitope coding nucleic acid sequence, (O)TP53_R213L MHC class I epitope coding nucleic acid sequence, (P)BRAF_G466V MHC class I epitope coding nucleic acid sequence, (Q)KRAS_Q61H MHC class I epitope coding nucleic acid sequence, (R)CTNNB1_S37F MHC class I epitope coding nucleic acid sequence, (S)TP53_S127Y MHC class I epitope coding nucleic acid sequence, (T)TP53_K132E MHC class I epitope coding nucleic acid sequence, (U)KRAS_G12C MHC class I epitope coding nucleic acid sequence Includes, Each of the tumor-specific MHC class I antigen-coding nucleic acid sequences is, (A) Optionally, the 5' linker sequence, and (B) Optionally, 3' linker array The tumor-specific MHC class I antigen-coding nucleic acid sequence, including The at least one antigen-coding nucleic acid sequence, (ii) Optionally, a second promoter nucleotide sequence functionally linked to the antigen-coding nucleic acid sequence, (iii) Optionally, at least one MHC class II antigen-coding nucleic acid sequence, (iv) Optionally, at least one nucleic acid sequence encoding the GPGPG amino acid linker sequence (SEQ ID NO: 56), (v) Optionally, at least one second poly(A) sequence which is a native poly(A) sequence or an exogenous poly(A) sequence relative to the vector skeleton, The antigen cassette, including the antigen cassette. [Invention 1004] A composition for delivering an antigen expression system, which includes an antigen expression system, The antigen expression system comprises one or more vectors, The composition comprises one or more vectors: (a) A vector skeleton comprising optionally a chimpanzee adenovirus vector which is a ChAdV68 vector, or optionally an alphavirus vector which is a Venezuelan encephalitis virus vector, (b) An antigen cassette incorporated between a 26S promoter nucleotide sequence and a poly(A) sequence, (i) at least one antigen-coding nucleic acid sequence, (I) At least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumor-specific MHC class I antigen-coding nucleic acid sequences linked linearly to each other, each of which is (A) An MHC class I epitope coding nucleic acid sequence encoding an MHC class I epitope of length 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 encoding a peptide that encodes the native N-terminal amino acid sequence of the MHC class I epitope and is at least three amino acids long, (C) A 3' linker sequence encoding a peptide that encodes the natural C-terminal acid sequence of the MHC class I epitope and is at least three amino acids long. Includes, The antigen cassette is functionally linked to the 26S promoter nucleotide sequence, each of the MHC class I antigen-coding nucleic acid sequences codes for a polypeptide of 13 to 25 amino acids, and the 3' end of each MHC class I antigen-coding nucleic acid sequence is ligated to the 5' end of the subsequent MHC class I antigen-coding nucleic acid sequence, except for the last MHC class I antigen-coding nucleic acid sequence in the antigen cassette. The aforementioned MHC class I antigen-coding nucleic acid sequence The at least one antigen-coding nucleic acid sequence, (ii) at least two MHC class II antigen-coding nucleic acid sequences, (I) PADRE MHC class II sequence (sequence number 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 that links 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 that links the 5' ends of the at least two MHC class II antigen-coding nucleic acid sequences with the tumor-specific MHC class I antigen-coding nucleic acid sequences, (V) Optionally, a third nucleic acid sequence encoding the GPGPG amino acid linker sequence at the 3' end of at least two MHC class II antigen-coding nucleic acid sequences and The at least two MHC class II antigen-coding nucleic acid sequences include The antigen cassette, including the antigen cassette. [Invention 1005] 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 explained by an expression that includes, During the ceremony, P comprises the second promoter nucleotide sequence, where a=0 or 1. N includes one of the MHC class I epitope coding nucleic acid sequences, where c=1. L5 includes the aforementioned 5' linker sequence, where b=0 or 1. L3 includes the 3' linker sequence, where d=0 or 1. G5 comprises one of the at least one nucleic acid sequences encoding the GPGPG amino acid linker, where e=0 or 1. G3 comprises one of the at least one nucleic acid sequences encoding the GPGPG amino acid linker, where g=0 or 1. U comprises one of the at least one MHC class II antigen-coding nucleic acid sequences, where f=1, X = 1 to 400, and here for each X, the corresponding N c This is an epitope-coding nucleic acid sequence, Y = 0, 1, or 2, where for each Y, the corresponding U f This is an antigen-coding nucleic acid sequence. A composition according to any of the present invention 1001 to 1003. [Invention 1006] For each X, the corresponding N c However, the composition of the present invention 1005 is a different MHC class I epitope coding nucleic acid sequence. [Invention 1007] For each Y, the corresponding U f However, the composition of the present invention 1005 or 1006 is a different MHC class II antigen-coding nucleic acid sequence. [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 given by the skeleton, The at least one polyadenylated poly(A) sequence is a poly(A) sequence of at least 100 consecutive A nucleotides given by the backbone, Each N encodes an MHC class I epitope with a length of 7 to 15 amino acids. L5 is a natural 5' linker sequence that encodes the natural N-terminal amino acid sequence of the MHC I epitope, and the 5' linker sequence encodes a peptide having a length of at least three amino acids. L3 is a natural 3' linker sequence that encodes the natural terminal nucleic acid sequence of the MHC I epitope, and the 3' linker sequence encodes a peptide having a length of at least three amino acids. U is a PADRE class II sequence and a tetanus toxoid MHC class II sequence, respectively. The vector skeleton optionally comprises a chimpanzee adenovirus vector which is a ChAdV68 vector, or an alphavirus vector which is a Venezuelan encephalitis virus vector. Each of the aforementioned MHC class I antigen-coding nucleic acid sequences encodes a polypeptide with a length of 13 to 25 amino acids. A composition according to any of the present invention 1005 to 1007. [Invention 1009] Any of the compositions of the present invention, further comprising a nanoparticle-shaped delivery vehicle. [Invention 1010] The composition of the present invention 1009, wherein the nanoparticle-like delivery vehicle is lipid nanoparticles (LNPs). [Invention 1011] The composition of the present invention 1010, wherein the LNP contains an ionizable aminolipid. [Invention 1012] The composition of the present invention 1011, comprising the ionizable aminolipid molecule MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate). [Invention 1013] A composition according to any one of the present invention 1009 to 1012, wherein the nanoparticle-like delivery vehicle encapsulates the antigen expression system. [Invention 1014] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1013, wherein the antigen cassette is incorporated between the at least one promoter nucleotide sequence and the at least one poly(A) sequence. [Invention 1015] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1014, wherein the at least one promoter nucleotide sequence is functionally linked to the antigen-coding nucleic acid sequence. [Invention 1016] The composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1015, wherein the one or more vectors comprises one or more +-chain RNA vectors. [Invention 1017] The composition of the present invention 1016, wherein one or more positive-chain RNA vectors contain a 5'7-methylguanosine (m7g) cap. [Invention 1018] The composition of the present invention 1016 or 1017, wherein one or more positive-chain RNA vectors are produced by in vitro transcription. [Invention 1019] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1018, wherein one or more vectors self-replicate within mammalian cells. [Invention 1020] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1019, wherein the aforementioned skeleton comprises at least one nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semliki forest virus, Sindbis virus, or Mayarovirus. [Invention 1021] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1019, wherein the aforementioned skeleton comprises at least one nucleotide sequence of Venezuelan encephalitis virus. [Invention 1022] The composition of the present invention 1020 or 1021, wherein the aforementioned skeleton comprises a sequence for non-structural protein-mediated amplification, a 26S promoter sequence, a poly(A) sequence, a non-structural protein 1 (nsP1) gene, an nsP2 gene, an nsP3 gene, and an nsP4 gene, which are encoded by at least the nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semliki forest virus, Sindbis virus, or Mayarovirus. [Invention 1023] The composition of the present invention 1020 or 1021, wherein the aforementioned skeleton comprises a sequence for non-structural protein-mediated amplification, a 26S promoter sequence, and a poly(A) sequence, which are encoded by at least the nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semliki forest virus, Sindbis virus, or Mayarovirus. [Invention 1024] The composition of the present invention 1022 or 1023, wherein the sequence for non-structural protein-mediated amplification is selected from the group consisting of alphavirus 5'UTR, 51nt CSE, 24nt CSE, 26S subgenomic promoter sequence, 19nt CSE, alphavirus 3'UTR, or a combination thereof. [Invention 1025] A composition according to any one of the present invention 1022 to 1024, wherein the aforementioned skeleton does not encode structural virion protein capsids E2 and E1. [Invention 1026] The composition of the present invention 1025, wherein the antigen cassette is inserted in place of a structural virion protein in the nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semliki forest virus, Sindbis virus, or Mayarovirus. [Invention 1027] The composition of the present invention 1020 or 1021, wherein the Venezuelan encephalitis virus comprises the sequence described in SEQ ID NO: 3 or SEQ ID NO: 5. [Invention 1028] A composition of the present invention 1020 or 1021, wherein the Venezuelan 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. [Invention 1029] The composition of the present invention 1028, wherein the skeleton comprises the sequence described in Sequence ID No. 6 or Sequence ID No. 7. [Invention 1030] A composition of the present invention 1028 or 1029, wherein the antigen cassette is inserted at position 7544 to replace the deletion between base pair 7544 and 11175 as described in the sequence of SEQ ID NO: 3 or SEQ ID NO: 5. [Invention 1031] A composition according to any one of the present invention 1026 to 1030, wherein the insertion of the antigen cassette results in the transcription of a polycistronic RNA containing the nsP1-4 genes and the at least one antigen-coding nucleic acid sequence, and the nsP1-4 genes and the at least one antigen-coding nucleic acid sequence are located in separate open reading frames. [Invention 1032] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1019, wherein the aforementioned skeleton comprises at least one nucleotide sequence of a chimpanzee adenovirus vector. [Invention 1033] The composition of the present invention 1032, wherein the chimpanzee adenovirus vector is the ChAdV68 vector. [Invention 1034] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1033, wherein the at least one promoter nucleotide sequence is a native 26S promoter nucleotide sequence encoded by the skeleton. [Invention 1035] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1033, wherein the at least one promoter nucleotide sequence is an exogenous RNA promoter. [Invention 1036] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1035, wherein the second promoter nucleotide sequence is a 26S promoter nucleotide sequence. [Invention 1037] The composition according to any one of Inventions 1001 to 1003, 1005 to 1007, or 1009 to 1035, wherein the second promoter nucleotide sequence comprises a plurality of 26S promoter nucleotide sequences, and each 26S promoter nucleotide sequence drives transcription of one or more of the separate open reading frames. [Invention 1038] The composition according to any one of the present invention described above, wherein each of the one or more vectors has a size of at least 300 nt. [Invention 1039] The composition according to any one of the present invention described above, wherein each of the one or more vectors has a size of at least 1 kb. [Invention 1040] The composition according to any one of the present invention described above, wherein each of the one or more vectors has a size of 2 kb. [Invention 1041] The composition according to any one of the present invention described above, wherein each of the one or more vectors has a size of less than 5 kb. [Invention 1042] The composition according to any one of the present invention described above, wherein at least one of the at least one antigen-encoding nucleic acid sequence encodes a polypeptide sequence or a portion thereof presented by MHC class I on tumor cells. [Invention 1043] The composition according to any one of Inventions 1001 to 1003, 1005 to 1007, or 1009 to 1042, wherein each antigen-encoding nucleic acid sequence is directly linked to each other. [Invention 1044] The composition according to any one 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 via a nucleic acid sequence encoding a linker. [Invention 1045] The composition according to Invention 1044, wherein the linker links two MHC class I sequences or one MHC class I sequence to one MHC class II sequence. [Invention 1046] The composition of the present invention 1045, wherein the linker is selected from the group consisting of (1) consecutive glycine residues of a length of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues, (2) consecutive alanine residues of a length of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues, (3) two arginine residues (RR), (4) alanine, alanine, tyrosine (AAY), (5) a consensus sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues that is efficiently processed by the mammalian proteasome, and (6) one or more native sequences of 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 to 20 amino acid residues that are adjacent to an antigen derived from a congener protein of origin. [Invention 1047] The composition of the present invention 1044, wherein the linker links two MHC class II sequences or one MHC class II sequence to one MHC class I sequence. [Invention 1048] The composition of the present invention 1047, wherein the linker comprises the sequence GPGPG. [Invention 1049] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1048, wherein at least one of the at least one antigen-coding nucleic acid sequences is functionally or directly linked to a separated or continuous sequence that enhances the expression, stability, cell trafficking, processing and presentation, and / or immunogenicity of the at least one antigen-coding nucleic acid sequence. [Invention 1050] The composition of Invention 1049, wherein the separated or continuous sequence comprises at least one of the following: a ubiquitin sequence, a ubiquitin sequence modified to enhance proteasome targeting (e.g., a ubiquitin sequence including a Gly-to-Ala substitution at position 76), an immunoglobulin signaling sequence (e.g., IgK), a major histocompatibility class I sequence, lysosome-associated membrane protein (LAMP)-1, a human dendritic cell lysosome-associated membrane protein, and a major histocompatibility class II sequence, wherein the ubiquitin sequence modified to enhance proteasome targeting is optionally A76. [Invention 1051] Any of the compositions of the present invention, wherein at least one of the at least one antigen-coding nucleic acid sequences encodes a polypeptide sequence or a portion thereof that has increased binding affinity to the corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence after translation. [Invention 1052] Any of the compositions of the present invention, wherein at least one of the at least one antigen-coding nucleic acid sequences encodes a polypeptide sequence or a portion thereof that has increased binding stability to the corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence after translation. [Invention 1053] Any of the compositions of the present invention, wherein at least one of the at least one antigen-coding nucleic acid sequences encodes a polypeptide sequence or a portion thereof that has an increased presentation likelihood on the corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence after translation. [Invention 1054] Any of the compositions of the present invention wherein the at least one change 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 a proteasome. [Invention 1055] Any composition 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, 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. [Invention 1056] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1055, wherein the at least one antigen-coding nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleic acid sequences. [Invention 1057] A composition according to any of the present inventions 1001-1003, 1005-1007, or 1009-1055, wherein the at least one antigen-coding 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. [Invention 1058] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1055, wherein the at least one antigen-coding nucleic acid sequence comprises at least 2 to 400 nucleic acid sequences, and at least two of the antigen-coding 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. [Invention 1059] A composition of the present invention 1004 or 1008, wherein at least two of the antigen-coding 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. [Invention 1060] Any composition of the present invention, wherein, when administered to the subject and translated, at least one of the antigens encoded by the at least one antigen-coding nucleic acid sequence is presented on an antigen-presenting cell, resulting in an immune response targeting at least one of the antigens on the surface of the tumor cell. [Invention 1061] Any of the compositions of the present invention, wherein, when the at least one antigen-coding 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 targeting at least one of the antigens on the surface of the tumor cells, and optionally, the expression of each of the at least one antigen-coding nucleic acid sequences is driven by the at least one promoter nucleotide sequence. [Invention 1062] A composition according to any of the Invention 1001-1003, 1005-1007, or 1009-1061, wherein each MHC class I antigen-coding nucleic acid sequence encodes a polypeptide sequence of a length of 8-35 amino acids, optionally 9-17 amino acids, 9-25 amino acids, 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. [Invention 1063] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1062, wherein at least one MHC class II antigen-coding nucleic acid sequence is present. [Invention 1064] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1062, comprising at least one MHC class II antigen-coding nucleic acid sequence having the aforementioned at least one MHC class II antigen-coding nucleic acid sequence and comprising at least one modification that causes the encoded peptide sequence to be different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence. [Invention 1065] A composition according to any of the present invention 1001-1003, 1005-1007, or 1009-1064, wherein the at least one MHC class II antigen-coding nucleic acid sequence has a length of 12-20 amino acids, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids. [Invention 1066] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1065, wherein the present invention contains at least one MHC class II antigen-coding nucleic acid sequence and at least one universal MHC class II antigen-coding nucleic acid sequence, wherein the at least one universal sequence contains at least one of tetanus toxoid and PADRE. [Invention 1067] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1066, wherein at least one promoter nucleotide sequence or the second promoter nucleotide sequence is inducible. [Invention 1068] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1066, wherein the at least one promoter nucleotide sequence or the second promoter nucleotide sequence is non-inducible. [Invention 1069] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1068, wherein the at least one poly(A) sequence comprises a poly(A) sequence that is naturally present in the skeleton. [Invention 1070] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1068, wherein the at least one poly(A) sequence comprises an exogenous poly(A) sequence relative to the skeleton. [Invention 1071] A composition according to any one of the present invention 1001-1003, 1005-1007, or 1009-1070, wherein the at least one poly(A) sequence is functionally linked to at least one of the at least one antigen-coding nucleic acid sequences. [Invention 1072] The composition according to any one of Inventions 1001 to 1003, 1005 to 1007, or 1009 to 1071, wherein said 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. [Invention 1073] The composition according to any one of Inventions 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. [Invention 1074] The composition according to any one of the preceding Inventions, wherein said antigen cassette further comprises at least one selected from the group consisting of: an intron sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence, an internal ribosome entry sequence (IRES), a nucleotide sequence encoding a 2A self-cleaving peptide, a nucleotide sequence encoding a furin cleavage site, or a sequence within the 5' or 3' non-coding region operably linked to at least one of said at least one antigen-encoding nucleic acid sequence, which is known to improve nuclear export, stability, or translation efficiency of mRNA. [Invention 1075] The composition according to any one of the preceding Inventions, wherein said 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. [Invention 1076] The composition according to Invention 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. [Invention 1077] The composition according to any one of the preceding Inventions, wherein said one or more vectors further comprise one or more nucleic acid sequences encoding at least one immunomodulator. [Invention 1078] The composition of the present invention 1077, wherein the immunomodulatory substance is an anti-CTLA4 antibody or its antigen-binding fragment, an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, an anti-4-1BB antibody or its antigen-binding fragment, or an anti-OX-40 antibody or its antigen-binding fragment. [Invention 1079] The composition of the present invention 1078, wherein the antibody or its antigen-binding fragment is a Fab fragment, a Fab' fragment, a single-chain Fv (scFv), a single-domain antibody (sdAb) as monospecific or linked multiplespecific (e.g., an antibody domain from a camelid), 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). [Invention 1080] A composition according to the present invention 1078 or 1079, wherein the heavy chain sequence and the light chain sequence of the antibody are continuous 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 a continuous glycine residue. [Invention 1081] The composition of the present invention 1077, wherein the immunomodulatory substance is a cytokine. [Invention 1082] The composition of the present invention 1081, wherein the cytokine is at least one of IL-2, IL-7, IL-12, IL-15, or IL-21, or each of their variants. [Invention 1083] The aforementioned at least one MHC class I antigen-coding nucleic acid sequence is (a) A step of 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 each peptide sequence of a set of antigens, (b) A step of inputting the peptide sequence of each antigen into a presentation model in order to generate a set of numerical likelihoods that each antigen is presented by one or more MHC alleles on the surface of tumor cells of the tumor, wherein the set of numerical likelihoods is identified at least based on received mass spectrometry data, (c) A step of selecting a subset of the set of antigens based on the set of numerical likelihoods in order to generate the set of selected antigens used to generate the set of at least one MHC class I antigen coding nucleic acid sequence. A composition selected by performing the following: any of the compositions 1001-1003, 1005-1007, or 1009-1082 of the present invention. [Invention 1084] Each of the aforementioned MHC class I epitope coding nucleic acid sequences is (a) A step of 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 each peptide sequence of a set of antigens, (b) A step of inputting the peptide sequence of each antigen into a presentation model in order to generate a set of numerical likelihoods that each antigen is presented by one or more MHC alleles on the surface of tumor cells of the tumor, wherein the set of numerical likelihoods is identified at least based on received mass spectrometry data, (c) A step of selecting a subset of the set of antigens based on the set of numerical likelihoods in order to generate the set of selected antigens used to generate the set of at least 20 MHC class I antigen coding nucleic acid sequences. A composition of the present invention 1004 or 1008, selected by performing the following. [Invention 1085] The composition of the present invention 1083, wherein the number of sets of selected antigens is 2 to 20. [Invention 1086] The aforementioned model is, (a) The presence of a pair of a specific MHC allele and a specific amino acid at a specific position in the peptide sequence, (b) The likelihood of presentation on the tumor cell surface of such peptide sequence, which includes the specific amino acid at the specific position, by the specific one of the MHC alleles of the pair, A composition of any of the present invention 1083 to 1085 that exhibits a dependency between [the specified values]. [Invention 1087] A composition of any of the present invention 1083 to 1086, wherein the selection of the set of selected antigens includes selecting antigens that have a higher likelihood of being presented on the surface of the tumor cells compared to antigens not selected based on the presentation model, and optionally, the selected antigens are verified to be presented by one or more specific HLA alleles. [Invention 1088] A composition according to any one of the present invention 1083 to 1087, wherein the selection of the set of selected antigens includes selecting antigens that have a higher likelihood of inducing a tumor-specific immune response in the target compared to antigens not selected based on the presented model. [Invention 1089] The composition of any of the present invention 1083 to 1088, wherein the selection of the set of selected antigens comprises selecting antigens that have a higher 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). [Invention 1090] A composition according to any one of the present invention 1083 to 1089, wherein the selection of the set of selected antigens includes 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. [Invention 1091] A composition according to any one of the present invention 1083 to 1090, wherein the selection of the selected set of antigens includes selecting antigens that have a reduced likelihood of inducing an autoimmune response against normal tissue in the subject compared to antigens not selected based on the presented model. [Invention 1092] A composition according to any one of the present invention 1083 to 1091, wherein exome or transcriptome nucleotide sequencing data is obtained by sequencing with respect to tumor tissue. [Invention 1093] The composition of the present invention 1092, wherein the sequencing is next-generation sequencing (NGS) or any large-scale parallel processing sequencing method. [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. [Invention 1095] The composition of the present invention 1094, wherein at least one, or each, junctional epitope sequence has an affinity for MHC that is higher than 500 nM. [Invention 1096] A composition of the present invention 1094 or 1095, wherein each junctional epitope sequence is non-self. [Invention 1097] Any of the compositions of the present invention, wherein each of the MHC class I epitopes is predicted or verified to be presentable by at least one HLA allele present in at least 5% of the population. [Invention 1098] Any of the compositions of the present invention, wherein each of the MHC class I epitopes is predicted or verified to be presentable by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA occurrence rate of at least 0.01% in the population. [Invention 1099] Any of the compositions of the present invention, wherein each of the MHC class I epitopes is predicted or verified to be presentable 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. [Invention 1100] Any of the compositions of the present invention, wherein the antigen cassette does not encode a non-therapeutic MHC class I or class II epitope nucleic acid sequence containing a post-translational wild-type nucleic acid sequence, and the non-therapeutic epitope is expected to be presented on the target MHC allele. [Invention 1101] The composition of the present invention 1100, wherein the non-therapeutic predicted MHC class I or class II epitope sequence is a junctional epitope sequence formed by adjacent sequences within the antigen cassette. [Invention 1102] A composition according to any one of the present invention 1094 to 1101, wherein the prediction is based on the presentation likelihood generated by inputting the sequence of the non-therapeutic epitope into a presentation model. [Invention 1103] The order of the at least one antigen-coding nucleic acid sequence within the antigen cassette is (a) A step of generating a set of candidate antigen cassette sequences corresponding to various orders of the at least one antigen-coding nucleic acid sequence, (b) A step of determining a presentation score for each candidate antigen cassette sequence based on the presentation of non-therapeutic epitopes within the candidate antigen cassette sequence, (c) A step of selecting candidate cassette sequences associated with presentation scores below a predetermined threshold as antigen cassette sequences for an antigen vaccine. A composition of any of the present inventions 1094 to 1102, determined by a series of steps including the above. [Invention 1104] A pharmaceutical composition comprising any of the compositions of the present invention described above and a pharmaceutically acceptable carrier. [Invention 1105] A composition according to the present invention 1104, further comprising an adjuvant. [Invention 1106] A pharmaceutical composition according to invention 1104 or 1105, further comprising an immunomodulator. [Invention 1107] The pharmaceutical composition of the present invention 1106, wherein the immunomodulatory substance is an anti-CTLA4 antibody or its antigen-binding fragment, an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, an anti-4-1BB antibody or its antigen-binding fragment, or an anti-OX-40 antibody or its antigen-binding fragment. [Invention 1108] An antigen cassette of any of the prior compositions, One or more elements obtained from the array of sequence number 3 or sequence number 5 and An isolated nucleotide sequence or set of isolated nucleotide sequences comprising, Optionally, one or more of the above elements are selected from the group consisting of sequences required for non-structural protein-mediated amplification, 26S promoter nucleotide sequences, poly(A) sequences, and nsP1-4 genes of sequences described in SEQ ID NO: 3 or SEQ ID NO: 5. Optionally, the nucleotide sequence is cDNA. The aforementioned isolated nucleotide sequence or set of isolated nucleotide sequences. [Invention 1109] The isolated nucleotide sequence of the present invention 1108, comprising the aforementioned sequence or set of isolated nucleotide sequences, which includes an antigen cassette of any of the prior composition inventions, inserted at position 7544 of the sequence described in SEQ ID NO: 6 or SEQ ID NO: 7. [Invention 1110] A nucleotide sequence of a T7 or SP6 RNA polymerase promoter located at the 5' end of one or more elements obtained from the sequence of SEQ ID NO: 3 or SEQ ID NO: 5, Optionally, one or more restriction sites located on the 3' side of the poly(A) sequence and The isolated nucleotide sequence of the present invention 1108 or 1109, further comprising: [Invention 1111] The isolated nucleotide sequence of the present invention 1108, wherein an antigen cassette of any of the prior compositions is inserted at position 7563 of SEQ ID NO: 8 or SEQ ID NO: 9. [Invention 1112] A vector or set of vectors containing any of the nucleotide sequences 1108 to 1111 of the present invention. [Invention 1113] An isolated cell comprising any nucleotide sequence or set of isolated nucleotide sequences according to invention 1108 to 1112, wherein the isolated cell is optionally BHK-21, CHO, HEK293 or a variant thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a cell. [Invention 1114] A kit comprising any of the prior compositions and instructions for use. [Invention 1115] A method for treating a subject having cancer, comprising administering to the subject any of the compositions of the prior art or any of the pharmaceutical compositions 1104 to 1107 of the present invention. [Invention 1116] The method of the present invention 1115, wherein the at least one MHC class I antigen-coding nucleic acid sequence is derived from the target tumor having cancer. [Invention 1117] The method of the present invention 1115, wherein the at least one MHC class I antigen-coding nucleic acid sequence does not originate from the target tumor having cancer. [Invention 1118] A method for inducing an immune response in a subject, comprising administering to the subject any of the compositions of the prior art or any of the pharmaceutical compositions 1104 to 1107 of the present invention. [Invention 1119] The method according to any one of the present invention 1115 to 1118, wherein the subject expresses at least one HLA allele that is predicted or known to present the MHC class I epitope. [Invention 1120] The method according to any one of the present invention 1115 to 1118, wherein the subject expresses at least one HLA allele that is predicted or known to present the MHC class I epitope, and the MHC class I epitope includes a mutation selected from the group of mutations shown in Table 34. [Invention 1121] The method according to any one of the present invention 1115 to 1118, wherein the subject expresses at least one HLA allele that is predicted or known to present the MHC class I epitope, and the MHC class I epitope includes a mutation selected from the group of mutations shown in Table 32. [Invention 1122] The method according to any one of items 1115 to 1121 of the present invention, wherein the composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). [Invention 1123] A method according to any of the present invention 1115 to 1121, wherein the composition is administered intramuscularly. [Invention 1124] A method according to any one of the present invention 1115 to 1123, further comprising administering one or more immunomodulators, wherein the immunomodulators are optionally administered before, simultaneously with, or after the administration of the composition or pharmaceutical composition. [Invention 1125] The method of the present invention 1124, wherein the one or more immunomodulators are selected from the group consisting of an anti-CTLA4 antibody or its antigen-binding fragment, an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, an anti-4-1BB antibody or its antigen-binding fragment, or an anti-OX-40 antibody or its antigen-binding fragment. [Invention 1126] The method of the present invention 1124 or 1125, wherein the immunomodulator is administered intravenously (IV), intramuscularly (IM), intradermally (ID), or subcutaneously (SC). [Invention 1127] The method of the present invention 1126, wherein the subcutaneous administration is performed near the administration site of the composition or pharmaceutical composition, or in close proximity to the lymph nodes in the inflow area of ​​one or more vectors or compositions. [Invention 1128] Any method according to 1115 to 1127 of the present invention, further comprising administering a second vaccine composition to the subject. [Invention 1129] The method of the present invention 1128, wherein the second vaccine composition is administered before the administration of any of the compositions or pharmaceutical compositions of the present invention 1115 to 1127. [Invention 1130] The method of the present invention 1128, wherein the second vaccine composition is administered after the administration of any of the compositions or pharmaceutical compositions of the present invention 1115 to 1127. [Invention 1131] The method of the present invention 1129 or 1130, wherein the second vaccine composition is the same as any of the compositions or pharmaceutical compositions of the present invention 1115 to 1127. [Invention 1132] The method of the present invention 1129 or 1130, wherein the second vaccine composition is different from any of the compositions or pharmaceutical compositions of the present invention 1115 to 1127. [Invention 1133] The method of the present invention 1132, wherein the second vaccine composition comprises a chimpanzee adenovirus vector encoding at least one antigen-coding nucleic acid sequence. [Invention 1134] The method of the present invention 1133, wherein the at least one antigen-coding nucleic acid sequence encoded by the chimpanzee adenovirus vector is the same as the at least one antigen-coding nucleic acid sequence of any of the prior compositions of the invention. [Invention 1135] A method for producing one or more vectors of any of the prior composition inventions, (a) To obtain a linearized DNA sequence including the skeleton and the antigen cassette, (b) In vitro transcription of the linearized DNA sequence by adding the linearized DNA sequence to an in vitro transcription reaction containing all the components necessary for transcribing the linearized DNA sequence into RNA, further comprising optionally adding the m7g cap to the resulting RNA in vitro, (c) Isolating one or more vectors from the in vitro transcription reaction. The method, including the method described above. [Invention 1136] The method for producing the present invention 1135, wherein the linearized DNA sequence is produced by linearizing a DNA plasmid sequence or by amplification using PCR. [Invention 1137] The method for producing the DNA plasmid sequence of the present invention 1136, wherein the DNA plasmid sequence is produced using one of the following: bacterial recombination, whole genome DNA synthesis, or whole genome DNA synthesis accompanied by amplification of synthetic DNA within a bacterial cell. [Invention 1138] The method for producing the present invention 1135, wherein isolating one or more vectors from the in vitro transcription reaction comprises one or more of the following: phenol-chloroform extraction, purification using a silica column, or a similar RNA purification method. [Invention 1139] A method for producing any of the compositions of the prior compositions for delivering the antigen expression system, (a) To provide components of a nanoparticle-like delivery vehicle, (b) To provide the antigen expression system, (c) The nanoparticle-like delivery vehicle and the antigen expression system provide sufficient conditions for generating the composition for delivering the antigen expression system. The method, including the method described above. [Invention 1140] A method for producing the present invention 1139, wherein the conditions are provided by microfluidic mixing. [Invention 1141] A method for evaluating subjects with cancer, a)1) Whether the subject has an HLA allele that is predicted or known to present an antigen included in the antigen-based vaccine, and below: 1) Whether the target tumor expresses the gene related to the antigen, and optionally, whether the gene is expressed abnormally compared to normal cells or tissues. 2) Whether the target tumor has a mutation related to the antigen. one or both The process of determining, or already determining, b) A step in which, based on the results of (a) above, the subject expresses the HLA allele, the tumor of the subject expresses the gene, and / or the tumor of the subject has the mutation, the subject is determined to be a candidate for treatment with the antigen-based vaccine, or has already been determined to be so The antigen comprises at least one MHC class I epitope sequence selected from the group consisting of SEQ ID NOs. 57 to 29357. The above process, c) Optionally, administering the antigen-based vaccine to the subject, or having already administered the antigen-based vaccine, 1) The at least one MHC class I epitope, or 2) MHC class I epitope coding nucleic acid sequence encoding at least one MHC class I epitope The above process includes and The method, including the method described above. [Invention 1142] A method for evaluating subjects with cancer, a) The above object is, 1) The tumor expresses the A0301 HLA allele and has the KRAS_G12A mutation, 2) The tumor expresses the A0201 HLA allele and has the KRAS_G12C mutation, 3) Expressing the C0802 HLA allele or the A1101 HLA allele, and the tumor in question has the KRAS_G12D mutation, or 4) The tumor expresses the A0301 HLA allele, or the A1101 HLA allele, or the A3101 HLA allele, or the C0102 HLA allele, or the A0302 HLA allele, and the tumor in question has the KRAS_G12V mutation. The process of determining, or already determining, b) From the results of (a) above, the subject is 1) If the A0301 allele is expressed and the target tumor has the KRAS_G12A mutation, 2) If the A0201 allele is expressed and the target tumor has the KRAS_G12C mutation, 3) If the tumor expresses the C0802 HLA allele or the A1101 HLA allele and the target tumor has the KRAS_G12D mutation, or 4) When the A0301 HLA allele, the A1101 HLA allele, the A3101 HLA allele, the C0102 HLA allele, or the A0302 HLA allele is expressed, and the target tumor has the KRAS_G12V mutation. The process includes determining, or having already determined, that the subject is a candidate for treatment with the antigen-based vaccine, c) Optionally, administering the antigen-based vaccine to the subject, or having already administered the antigen-based vaccine, 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, 2) MHC class I epitope coding nucleic acid sequences encoding at least one MHC class I epitope, each containing the KRAS_G12A mutation, the KRAS_G12C mutation, the KRAS_G12D mutation, or the KRAS_G12V mutation. The above process includes The method, including the method described above. [Invention 1143] The method of the present invention 1141 or 1142, wherein step (a) and / or (b) includes obtaining a dataset from a third party that has processed a sample from the subject. [Invention 1144] The method of the present invention 1141 or 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 assay, mass spectrometry-based method, microarray, nanostring, ISH, and IHC. [Invention 1145] The method of the present invention 1143 or 1144, wherein the sample comprises a tumor sample, a normal tissue sample, or the tumor sample and the normal tissue sample. [Invention 1146] The method of the present invention 1145, wherein the sample is selected from tissue, body fluid, blood, tumor biopsy, cerebrospinal fluid, and needle aspiration. [Invention 1147] The method according to invention 1141 or any of 1143-1146, wherein the gene is selected from the group consisting of any of the genes shown in Table 34. [Invention 1148] The method according to invention 1141 or any of 1143-1146, wherein the gene is selected from the group consisting of any of the genes shown in Table 32. [Invention 1149] The method according to any one of the present invention 1141 to 1148, wherein the cancer is selected from the group consisting of lung cancer, microsatellite-stable colon cancer, and pancreatic cancer. [Invention 1150] The method according to any one of the present invention 1141 to 1149, wherein the HLA allele has an HLA frequency of at least 5%. [Invention 1151] Any method of the present invention 1141 to 1150, wherein the at least one MHC class I epitope is presented by the HLA allele on cells associated with the tumor of interest. [Invention 1152] The antigen-based vaccine comprises an antigen expression system, according to any method 1141 to 1151 of the present invention. [Invention 1153] The method of the present invention 1152, wherein the antigen expression system comprises one of the antigen expression systems of the present invention 1001 to 1103. [Invention 1154] A method according to any of the inventions 1141 to 1151, wherein the antigen-based vaccine comprises one of the pharmaceutical compositions of any of the inventions 1104 to 1107. [Invention 1155] A method for treating a subject having cancer, comprising administering an antigen-based vaccine to the subject, wherein the antigen-based vaccine is 1) At least one MHC class I epitope, or 2) MHC class I epitope coding nucleic acid sequence encoding at least one MHC class I epitope Includes, The at least one MHC class I epitope sequence is selected from the group consisting of sequence numbers 57 to 29357. The aforementioned method. [Invention 1156] The method of the present invention 1155, wherein the at least one MHC class I antigen-coding nucleic acid sequence is derived from the target tumor having cancer. [Invention 1157] The method of the present invention 1155, wherein the at least one MHC class I antigen-coding nucleic acid sequence does not originate from the tumor of the subject having cancer. [Invention 1158] A method for inducing an immune response in a subject, comprising administering an antigen-based vaccine to the subject, wherein the antigen-based vaccine is 1) At least one MHC class I epitope, or 2) MHC class I epitope coding nucleic acid sequence encoding at least one MHC class I epitope Includes, The at least one MHC class I epitope sequence is selected from the group consisting of sequence numbers 57 to 29357. The aforementioned method. [Invention 1159] The method according to any one of the present invention 1155 to 1158, wherein the subject expresses at least one HLA allele that is predicted or known to present the at least one MHC class I epitope sequence. [Invention 1160] The method according to any one of the present invention 1155 to 1158, wherein the subject expresses at least one HLA allele that is predicted or known to present the at least one MHC class I epitope sequence, and the at least one MHC class I epitope sequence includes a mutation selected from the group consisting of the mutations shown in Table 34. [Invention 1161] The method according to any one of the present invention 1155 to 1158, wherein the subject expresses at least one HLA allele that is predicted or known to present the at least one MHC class I epitope sequence, and the at least one MHC class I epitope sequence includes a mutation selected from the group consisting of the mutations shown in Table 32. [Invention 1162] A method for inducing an immune response in a subject, comprising administering an antigen-based vaccine to the subject, wherein the antigen-based vaccine is 1) At least one MHC class I epitope, or 2) MHC class I epitope coding nucleic acid sequence encoding at least one MHC class I epitope Includes, 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 aforementioned method. [Invention 1163] A method for inducing an immune response in a subject, comprising administering an antigen-based vaccine to the subject, wherein the antigen-based vaccine is 1) At least one MHC class I epitope, or 2) MHC class I epitope coding nucleic acid sequence encoding at least one MHC class I epitope Includes, The at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs. 57 to 29357, 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 at least one MHC class I epitope sequence includes a mutation selected from the group consisting of the mutations shown in Table 34, and the subject expresses at least one HLA allele shown in Table 34 that is associated with the corresponding mutation shown in Table 34 (e.g., KRAS_G13D and C0802), The aforementioned method. [Invention 1164] A method for inducing an immune response in a subject, comprising administering an antigen-based vaccine to the subject, wherein the antigen-based vaccine is 1) At least one MHC class I epitope, or 2) MHC class I epitope coding nucleic acid sequence encoding at least one MHC class I epitope Includes, The at least one MHC class I epitope sequence is selected from the group consisting of SEQ ID NOs. 57 to 29357, the subject expresses at least one HLA allele that is predicted or known to present the at least one MHC class I epitope sequence, and the at least one MHC class I epitope sequence contains a mutation selected from the group consisting of the mutations shown in Table 32. The aforementioned method. [Invention 1165] The antigen-based vaccine comprises an antigen expression system, according to any method 1155 to 1164 of the present invention. [Invention 1166] The method of the present invention 1165, wherein the antigen expression system comprises one of the antigen expression systems of the present invention 1001 to 1103. [Invention 1167] A method of any of the present inventions 1155 to 1164, wherein the antigen-based vaccine comprises any one of the pharmaceutical compositions of any of the present inventions 1104 to 1107. [Brief explanation of the drawing]

[0100] These, as well as other features, embodiments, and advantages of the present invention will be better understood by referring to the following description and accompanying drawings. [Figure 1] This paper describes the development of an in vitro T cell activating compound assay. It outlines an assay in which delivery of a vaccine cassette to antigen-presenting cells leads to the expression, processing, and MHC restriction presentation of different peptide antigens. Reporter T cells, engineered to possess T cell receptors matching specific peptide-MHC combinations, are activated, and luciferase is expressed. [Figure 2] Figure 2A illustrates the evaluation of linker sequences within a short cassette, showing two universal class II MHC epitopes (MHC-II) linked to five class I MHC class restriction epitopes (epitopes 1-5) linked at the same position relative to each other. Various repeats were generated using different linkers. In some cases, T cell epitopes are directly linked to each other. In other cases, their native sequences are adjacent to one or both sides of the T cell epitope. In other repeats, T cell epitopes are linked by non-native sequences AAY, RR, and DPP. Figure 2B illustrates the evaluation of linker sequences within a short cassette, showing sequence information of T cell epitopes embedded within the short cassette. The figure discloses sequence numbers 29365-29366, 29369, 29368, 29367, and 29494-29495 in order of appearance. [Figure 3]This paper describes the evaluation of cell targeting sequences attached to a model vaccine cassette. The targeting cassette extends a short cassette design with ubiquitin (Ub), signal peptide (SP), and transmembrane (TM) domains, and further contains two mouse T cell epitopes SIINFEKL (SEQ ID NO: 29362) (SII) and SPSYAYHQF (SEQ ID NO: 29363) (A5) adjacent to five marker human T cell epitopes (epitopes 1-5), and uses non-natural linker AAY- or natural linker sequences adjacent to both sides of the T cell epitopes (25 markers). [Figure 4] This section describes the in vivo evaluation of linker sequences within short cassettes. A) Experimental design for in vivo evaluation of vaccine cassettes using HLA-A2 transgenic mice. [Figure 5A] This paper describes an in vivo evaluation of the effect of epitope location within a long 21-marker cassette, showing that the long cassette design includes five marker class I epitopes (epitopes 1-5) contained within their 25-marker natural sequences (linker = natural flanking sequences), separated by further known T cell class I epitopes (epitopes 6-21) contained within those 25-marker natural sequences, and two universal class II epitopes (MHC-II0), with only the relative position of each class I epitope differing. [Figure 5B] This document describes the in vivo evaluation of the effect of epitope location within a long 21-marker set and shows the sequence information of the T cell epitopes used. The figure discloses sequence numbers 29365-29366, 29369, 29368, 29367, 29496-29498, 29370, and 29499-29510 in order of appearance. [Figure 6A] This paper describes the final cassette design for the preclinical IND application experiment and shows that the final cassette design includes 20 MHC I epitopes contained within their 25-mar natural sequences (linkers = natural flanking sequences), and consists 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 the preclinical IND application experiment is described, and the sequence information of the T cell epitopes used, presented on non-human primate, mouse, and human-derived class I MHC, as well as the sequences of two universal MHC class II epitopes, PADRE and tetanus toxoid, is shown. The figure discloses sequence numbers 29426-29431, 29362-29363, 29456, 29511, 29460-29462, 29458-29459, 29367-29369, 29365-29366, 29494, and 29512, respectively, in order of appearance. [Figure 7] Figure 7A 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 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 a calcium phosphate protocol. Viral replication was observed 10 days after transfection, and ChAdV68.4WTnt.GFP viral plaques were visualized using a fluorescence 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 at 100x magnification using a fluorescence microscope. [Figure 8]Figure 8A illustrates the generation of ChAdV68.5WTnt.GFP virus after transfection. HEK293A cells were transfected with ChAdV68.5WTnt.GFP DNA using a 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 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 a 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 at 3 days using a fluorescence 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 a lipofectamine protocol. Viral replication (plaques) was observed 10 days after transfection. Lysates were prepared and used to reinfect 293A cells in a T25 flask. ChAdV68.5WTnt.GFP viral plaques were visualized and photographed at 3 days using a fluorescence microscope at 100x magnification. [Figure 9] This explains the scheme for generating virus particles. [Figure 10] This explains the alphavirus-derived VEE self-replicating RNA (srRNA) vector. [Figure 11] This paper describes in vivo reporter expression in C57BL / 6J mice after inoculation with VEE-luciferase srRNA. Representative images of luciferase signaling are shown after immunization of C57BL / 6J mice with VEE-luciferase srRNA at different time points (10 ug / mouse intramuscular injection, encapsulated in MC3). [Figure 12A] This report describes the T cell response measured 14 days after immunization with MC3 LNP-formulated VEE srRNA in B16-OVA tumor-carrying mice. B16-OVA tumor-carrying C57BL / 6J mice were injected with 10ug 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). Furthermore, all mice were treated with an anti-PD-1 mAb starting on day 7. Each group consisted of 8 mice. Mice were sacrificed 14 days after immunization, and their spleens and lymph nodes were collected. SIINFEKL (SEQ ID NO: 29362)-specific T cell responses were evaluated using IFN-γ ELISPOT and reported as spot-forming cells (SFCs) per 106 splenocytes. Each line represents the median. [Figure 12B] This report describes the T cell response measured 14 days after immunization with MC3 LNP-formulated VEE srRNA in B16-OVA tumor-carrying mice. B16-OVA tumor-carrying C57BL / 6J mice were injected with 10ug 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). Furthermore, all mice were treated with anti-PD-1 mAb starting on day 7. Each group consisted of 8 mice. Mice were sacrificed 14 days after immunization, and spleens and lymph nodes were collected. SIINFEKL (SEQ ID NO: 29362)-specific T cell response was evaluated by MHCI pentamer staining, and the percentage of pentamer-positive cells relative to CD8-positive cells (%) is reported. Each line represents the median. [Figure 13A]This study describes the antigen-specific T cell response after xenogeneic priming / boosting in B16-OVA tumor-carrying mice. B16-OVA tumor-carrying C57BL / 6J mice were either injected with adenovirus-expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA formulated with MC3 LNP (control), or injected 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 a combination of Ad5-GFP prime / VEE-luciferase srRNA boost and anti-CTLA-4 (aCTLA-4), and a fourth group was treated with a combination of Ad5-UbAAY prime / VEE-UbAAY boost and anti-CTLA-4 (Vax + aCTLA-4). Furthermore, all mice were treated with an anti-PD-1 mAb starting on day 21. T cell responses were measured using IFN-γ ELISPOT. Mice were sacrificed 14 days after adenovirus immunization, and their spleens and lymph nodes were collected. [Figure 13B] This study describes the antigen-specific T cell response after xenogeneic priming / boosting in B16-OVA tumor-carrying mice. B16-OVA tumor-carrying C57BL / 6J mice were either injected with adenovirus-expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA formulated with MC3 LNP (control), or injected 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 a combination of Ad5-GFP prime / VEE-luciferase srRNA boost and anti-CTLA-4 (aCTLA-4), and a fourth group was treated with a combination of Ad5-UbAAY prime / VEE-UbAAY boost and anti-CTLA-4 (Vax + aCTLA-4). Furthermore, all mice were treated with an anti-PD-1 mAb starting on day 21. T cell responses were measured using IFN-γ ELISPOT. Mice were sacrificed 14 days after adenovirus immunization and 14 days after srRNA boosting (28 days after prime), and their spleens and lymph nodes were collected. [Figure 13C] This study describes the antigen-specific T cell response after xenogeneic priming / boosting in B16-OVA tumor-carrying mice. B16-OVA tumor-carrying C57BL / 6J mice were either injected with adenovirus-expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA formulated with MC3 LNP (control), or injected 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 a combination of Ad5-GFP prime / VEE-luciferase srRNA boost and anti-CTLA-4 (aCTLA-4), and a fourth group was treated with a combination of Ad5-UbAAY prime / VEE-UbAAY boost and anti-CTLA-4 (Vax + aCTLA-4). Furthermore, all mice were treated with an 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 their spleens and lymph nodes were collected. [Figure 13D]This study describes the antigen-specific T cell response after xenogeneic priming / boosting in B16-OVA tumor-carrying mice. B16-OVA tumor-carrying C57BL / 6J mice were either injected with adenovirus-expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA formulated with MC3 LNP (control), or injected 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 a combination of Ad5-GFP prime / VEE-luciferase srRNA boost and anti-CTLA-4 (aCTLA-4), and a fourth group was treated with a combination of Ad5-UbAAY prime / VEE-UbAAY boost and anti-CTLA-4 (Vax + aCTLA-4). Furthermore, all mice were treated with an 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 srRNA boosting (28 days after priming), and the spleen and lymph nodes were collected. [Figure 14A] This study describes antigen-specific T cell responses after xenovirus priming / boost in mice carrying CT26 (Balb / c strain) tumors. Mice were immunized with Ad5-GFP and boosted 15 days after adenovirus priming with VEE-luciferase srRNA formulated with 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 priming / boost and anti-PD-1 (aPD1), and a fourth group received a combination of Ad5-UbAAY / VEE-UbAAY srRNA priming / boost and anti-PD-1 mAb (Vax + aPD1). T cell responses to the AH1 peptide were measured using IFN-γ ELISPOT. The mice were sacrificed 12 days after immunization with adenovirus, and their spleens and lymph nodes were collected. [Figure 14B]This study describes antigen-specific T cell responses after xenovirus priming / boost in mice carrying CT26 (Balb / c strain) tumors. Mice were immunized with Ad5-GFP and boosted 15 days after adenovirus priming with VEE-luciferase srRNA formulated with 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 priming / boost and anti-PD-1 (aPD1), and a fourth group received a combination of Ad5-UbAAY / VEE-UbAAY srRNA priming / boost and anti-PD-1 mAb (Vax + aPD1). T cell responses to the AH1 peptide were measured using IFN-γ ELISPOT. Mice were sacrificed 12 days after adenovirus immunization and 6 days after srRNA boosting (21 days after prime), and their spleens and lymph nodes were collected. [Figure 15] This study describes the ChAdV68-induced T cell response to mouse tumor antigens in mice. Mice were immunized with ChAdV68.5WTnt.MAG25, and the T cell response to the MHC class I epitope SIINFEKL (SEQ ID NO: 29362) (OVA) was measured in female C57BL / 6J mice, while the response to the MHC class I epitope AH1-A5 was measured in Balb / c mice. The mean spot-forming cells (SFCs) per 106 splenocytes measured by the ELISpot assay are shown. Error bars indicate the standard deviation. [Figure 16]This study describes the cellular immune response in a CT26 tumor model after a single immunization with either ChAdV6, ChAdV + anti-PD-1, srRNA, srRNA + anti-PD-1, or anti-PD-1 alone. Antigen-specific IFN-γ production was measured using ELISpot in splenocytes from six mice in each group. Results are shown as spot-forming cells (SFCs) per 106 splenocytes. The median for each group is shown as a horizontal line. P-values ​​were determined using Dunnett's multiple comparison test: ***P<0.0001, **P<0.001, *P<0.05. ChAdV = ChAdV6 8.5WTnt.MAG25, srRNA = VEE-MAG25 srRNA. [Figure 17] This study describes the CD8 T cell immune response in a CT26 tumor model after a single immunization with either 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 the results are presented as the percentage of total CD8 T cells, representing antigen-specific CD8 T cells. The median values ​​for each group are shown with horizontal lines. P-values ​​were determined using Dunnett's multiple comparison test: ***P<0.0001, **P<0.001, *P<0.05. ChAdV = ChAdV6 8.5WTnt.MAG25, srRNA = VEE-MAG25 srRNA. [Figure 18] This paper describes tumor growth in CT26 tumor models after immunization with ChAdV / srRNA heterogeneous prime / boost, srRNA / ChAdV heterogeneous prime / boost, or srRNA / srRNA allogeneic primer / boost. Prime / boost immunization with and without anti-PD-1 administration is also presented for comparison. Tumor volume was 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 (SEM) of the mean. P-values ​​were calculated using Dunnett's test: ***P<0.0001, **P<0.001, *P<0.05. ChAdV = ChAdV68.5WTnt.MAG25mer, srRNA = VEE-MAG25mer srRNA. [Figure 19] This paper describes the survival rates in CT26 tumor models after immunization with ChAdV / srRNA heterogeneous prime / boost, srRNA / ChAdV heterogeneous prime / boost, or srRNA / srRNA allogeneic primer / boost. Prime / boost immunization with and without anti-PD-1 administration is also presented for comparison. P-values ​​were determined using the log-rank test: ***P<0.0001, **P<0.001, *P<0.01. ChAdV = ChAdV68.5WTnt.MAG25-mer, srRNA = VEE-MAG25-mer srRNA. [Figure 20A] Figure 20 shows antigen-specific cellular immune responses measured using ELISpot. Antigen-specific IFN-γ production against six different mamuA01 restriction epitopes was measured in PBMCs (6 rhesus macaques per group) 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeks after the initial boost immunization using ELISpot in allogeneic prime / boost groups with VEE-MAG25-mer srRNA-LNP1 (30 μg) (Figure 20A), VEE-MAG25-mer srRNA-LNP1 (100 μg) (Figure 20B), or VEE-MAG25-mer srRNA-LNP2 (100 μg) (Figure 20C), or heterogeneic prime / boost groups with ChAdV68.5WTnt.MAG25-mer / VEE-MAG25-mer srRNA (Figure 20D). The results are presented in a stacked bar graph format, showing the average spot-forming cells (SFCs) per 106 PBMCs for each epitope. The values ​​for each animal were normalized to the pre-bred (week 0) level. [Figure 20B] See the explanation in Figure 20A. [Figure 20C] See the explanation in Figure 20A. [Figure 20D] See the explanation in Figure 20A. [Figure 21]This report shows antigen-specific cellular immune responses measured using ELISpot. Antigen-specific IFN-γ production against six different mamuA01 restriction epitopes was measured in PBMCs using ELISpot before immunization and at 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks after initial immunization, using heterologous prime / boost regimens with ChAdV68.5WTnt.MAG25mer / VEE-MAG25mer srRNA. Results are presented in a stacked bar graph format, showing the average spot-forming cells (SFCs) per 106 PBMCs for each epitope (6 rhesus monkeys per group). [Figure 22] This report shows antigen-specific cellular immune responses measured using ELISpot. Antigen-specific IFN-γ production against six different mamuA01 restriction epitopes was measured in PBMCs using ELISpot before immunization and at 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, or 15 weeks after initial immunization, following allogeneic prime / boost regimen with VEE-MAG25-mer srRNA LNP2. Results are presented in a stacked bar graph format, showing the average spot-forming cells (SFCs) per 106 PBMCs for each epitope (6 rhesus monkeys per group). [Figure 23] This report shows antigen-specific cellular immune responses measured using ELISpot. Antigen-specific IFN-γ production against six different mamuA01 restriction epitopes was measured in PBMCs using ELISpot before immunization and at 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, or 15 weeks after initial immunization, following allogeneic prime / boost regimen with VEE-MAG25-mer srRNA LNP1. Results are presented in a stacked bar graph format, showing the average spot-forming cells (SFCs) per 106 PBMCs for each epitope (6 rhesus monkeys per group). [Figure 24A] An exemplary peptide spectrum, generated from Promega's dynamic range standard, is shown. The figure discloses Sequence ID No. 29364. [Figure 24B] An exemplary peptide spectrum, generated from Promega's dynamic range standard, is shown. [Figure 25] This shows the correlation between the EDGE score obtained by targeted MS and the detection probability of candidate shared nascent antigen peptides. [Figure 26] The image shows tissue lithotripsy (TIL) augmented from patients and stained with the mutant peptide HLA-A*11:01 tetramer. Flow cytometry (left panel) and staining of CD8+ cells with KRAS-G12V / HLA-A*11:01 tetramer (right panel) are also shown. [Figure 27] This document outlines typical TCR sequencing methods and workflows. [Figure 28] This shows a typical TCR sequencing method using the KRAS_G12V / HLA-A*11:01 tetramer. [Figure 29] This shows a typical organization of different tumor-derived model epitopes in large antigen cassettes with 30 (L), 40 (XL), or 50 (XXL) epitopes. [Figure 30] This study demonstrates that the ChAd vector expresses long cassettes, as shown by the Western blot above using an anti-class II (PADRE) antibody that recognizes a sequence common to all cassettes. HEK293 cells were infected with chAd68 vectors expressing large cassettes of different sizes (chAd68-50XXL, chAd68-40XL, and chAd68-30L). Infection was set to an MOI of 0.2. 24 hours after infection, the proteasome inhibitor MG132 was added to a 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) were used as a negative control. 48 hours after infection, the cell pellet was harvested, analyzed by SDS / PAGE electrophoresis, and immunoblotting was performed using rabbit anti-class II PADRE antibody. HRP anti-rabbit antibody and ECL chemiluminescent substrate were used for detection. [Figure 31]This shows the CD8+ immune response in mice immunized with a large chAd68 cassette, detected by ICS against AH1 (upper figure) and SIINFEKL (SEQ ID NO: 29362) (lower figure). Data are shown as IFNg+ cells relative to the model epitope, and as a percentage of total CD8 cells. [Figure 32] This figure shows the CD8+ response to LD-AH1+ (upper figure) and Kb-SIINFEKL (SEQ ID NO: 29362)+ (lower figure) tetramers after vaccination with a large chAd68 cassette. Data are shown as % of total CD8 cells that react to the model tetramer peptide complex. *p<0.05, **p<0.01 by ANOVA using Tukey's test. All p-values ​​were compared to those for the MAG20 antigen cassette. [Figure 33] This shows the CD8+ immune response in mice treated with large alphavirus cassettes detected by ICS against AH1 (upper figure) and SIINFEKL (SEQ ID NO: 29362) (lower figure). Data are shown as IFNg+ cells relative to the model epitope, as a percentage of total CD8 cells. *p<0.05, **p<0.01, **p<0.01 by ANOVA using Tukey's test. All p-values ​​were compared to those for the MAG20 antigen cassette. [Figure 34] This diagram shows a vaccination method for evaluating the immunogenicity of antigen cassette-containing vectors in rhesus monkeys. Triangles represent vaccination with chAd68 at weeks 0 and 32 (1e12vp / animal). Circles represent alphavirus vaccination at weeks 0, 4, 12, 12, 20, 28, and 32. Squares represent administration of anti-CTLA-4 antibody. [Figure 35] This shows the temporal progression of the CD8+ anti-epitope response in rhesus monkeys alone (group 4) administered chAd-MAG. The mean SFC / 1e6 splenocyte ratio is shown. [Figure 36] This shows the temporal progression of the CD8+ anti-epitope response in rhesus monkeys (Group 5) administered the anti-CTLA4 antibody (ipilimumab) via chAd-MAG and IV. The mean SFC / 1e6 splenocyte ratio is shown. [Figure 37]This shows the temporal progression of the CD8+ anti-epitope response in rhesus monkeys (group 6) administered with the anti-CTLA4 antibody (ipilimumab) via chAd-MAG and SC. The mean SFC / 1e6 splenocyte ratio is shown. [Figure 38] This shows antigen-specific memory responses induced by the chAdV68 / samRNA vaccine protocol as measured by ELISpot. Results are presented as individual dot plots, each dot representing a single animal. Baseline before immunization (left panel) and memory responses 18 months after priming (right panel) are shown. [Figure 39] This shows the determination of the memory cell phenotype of antigen-specific CD8+ T cells by flow cytometry using combinatorial tetramer staining and CD45RA / CCR7 co-staining. [Figure 40] This shows the distribution of memory cell types within the sum of four types of Mamu-A*01 tetramer + CD8+ T cell populations at 18 months of the experiment. Memory cells were characterized as follows: CD45RA+CCR7+ = naive, CD45RA+CCR7- = effector (Teff), CD45RA-CCR7+ = central memory (Tcm), CD45RA-CCR7- = effector memory (Tem). [Figure 41] This study shows the frequency of CD8+ T cells that recognize the CT26 tumor antigen AH1 in mice carrying CT26 tumors. P values ​​were determined using Tukey's multiple comparison test with one-way ANOVA (**P<0.001, *P<0.05). ChAdV = ChAdV68.5WTnt.MAG25; aCTLA4 = anti-CTLA4 antibody, clone 9D9. [Modes for carrying out the invention]

[0101] Detailed explanation I. Definition In general, terms used in the claims and specification shall be interpreted as having the ordinary meaning understood by those skilled in the art. Certain terms are defined below for further clarity. In the event of any conflict between the ordinary meaning and the given definition, the given definition shall prevail.

[0102] As used herein, the term “antigen” refers to a substance that induces an immune response. Antigens may be neonatal antigens. Antigens may be “covalent antigens,” which are antigens found within a specific population, for example, a specific population of cancer patients.

[0103] As used herein, the term “nascent antigen” refers to an antigen that has at least one change that makes it different from the corresponding wild-type antigen, for example, due to tumor cell mutations or tumor cell-specific post-translational modifications. Nascent antigens may include polypeptide sequences or nucleotide sequences. Mutations may include frameshift or non-frameshift insertions or deletions (indels), missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic or expression changes that result in nascent ORFs. Mutations may also include splice variants. Tumor cell-specific post-translational modifications may include abnormal phosphorylation. Tumor cell-specific post-translational modifications may also include splice antigens produced 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 covalent neogeneic antigens are shown in Table A and the results of the AACR GENIE (SEQ ID NOs: 10755-29357), with the corresponding HLA allele(s) also indicated for each antigen. Such covalent neogeneic antigens 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 further described below.

[0104] As used herein, the term "tumor antigen" refers to an antigen present in the tumor cells or tissue of interest but not in the corresponding normal cells or tissue of interest, or an antigen derived from a polypeptide whose expression is known or has been found to be altered in tumor cells or cancerous tissue compared to normal cells or tissue.

[0105] As used herein, the term “antigen-based vaccine” refers to a vaccine composition based on one or more antigens, for example, 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 the coding region.

[0109] As used herein, the term "ORF" means Open Reading Frame.

[0110] As used herein, the term “new-onset ORF” refers to tumor-specific ORFs resulting from mutations or other abnormalities such as splicing.

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

[0112] As used herein, the term "nonsense mutation" refers to a mutation that results in the substitution of an amino acid for a stop codon or the removal of a reference start codon.

[0113] As used herein, the term "frameshift mutation" refers to a mutation that causes a change in the frame of a protein.

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

[0115] As used herein, the term “identity” (%) in relation to the sequences of two or more nucleic acids or polypeptides means two or more sequences or subsequences that, when compared and aligned for the greatest match, have the same specific ratio (%) of nucleotides or amino acid residues, either using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the “identity” (%) may be present across regions of the sequences being compared, for example, across functional domains, or across the full lengths of the two sequences being compared.

[0116] In sequence comparison, typically, one sequence functions as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, subsequence coordinates are specified if necessary, and parameters for the sequence algorithm program are specified. The sequence comparison algorithm then calculates the sequence identity percentage (%) of the test sequence to the reference sequence based on the specified program parameters. Alternatively, sequence similarity or difference can also be established by the presence or absence of specific nucleotides at selected sequence locations (e.g., sequence motifs), or, in the post-translated sequence, by the presence or absence of amino acids.

[0117] The 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), the homology alignment algorithm of Needleman & Wunsch, J.Mol.Biol.48:443 (1970), the similarity search method of Pearson & Lipman, Proc.Nat'l.Acad.Sci.USA 85:2444 (1988), by computer execution 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 (generally, see Ausubel et al. below).

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

[0119] As used herein, the terms “nonstop” or “readthrough” refer to mutations that result in the removal of a natural stop codon.

[0120] As used herein, the term “epitope” refers to a specific portion of an antigen to which an antibody or T cell receptor commonly binds.

[0121] As used herein, the term “immunogenicity” means, for example, the ability to induce an immune response via T cells, B cells, or both.

[0122] As used herein, the terms "HLA binding affinity" and "MHC binding affinity" refer to the affinity for 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 concentrate a specific sequence of DNA or RNA from a sample.

[0124] As used herein, the term “variant” refers to the difference between the nucleic acid in question and a reference human genome used as a control.

[0125] As used herein, the term “variant call” typically refers to the algorithmic determination of the existence of a variant, from sequencing.

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

[0127] As used herein, the term “somatic cell variant” refers to 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, 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 an HLA gene allele.

[0130] As used herein, the term “nonsense mutation-dependent degradation mechanism” or “NMD” refers to the degradation of mRNA by cells due to immature stop codons.

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

[0132] As used herein, the term “subclonal mutation” refers to a mutation that occurs later in tumor development and is present in only a subset of tumor cells.

[0133] As used herein, the term “exome” refers to a subset of the genome that codes for proteins. An exome can be a collection of exons within a genome.

[0134] As used herein, the term “logistic regression” refers to a regression model for binary data from statistics, in which the logit, with a probability of the dependent variable being 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 performing multi-layer 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 organism.

[0137] As used herein, the term “peptideome” refers to the set of all peptides presented by MHC-I or MHC-II on the surface of a cell. The peptideome may also refer to the properties of a cell or a collection of cells (for example, the tumor peptideome means the union of the peptideomes of all cells, including a tumor).

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

[0139] As used herein, the term "dexatormer" 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 non-response to one or more antigens, such as autoantigens.

[0141] As used herein, the term “central tolerance” refers to tolerance induced in the thymus by either deleting autoreactive T cell clones or promoting the differentiation of autoreactive T cell clones into immunosuppressive regulatory T cells (Tregs).

[0142] As used herein, the term “peripheral tolerance” refers to tolerance handed down in the peripheral system by downregulating or anergizing autoreactive T cells that have survived central tolerance, or by promoting the differentiation of these T cells into Tregs.

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

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

[0145] The term “mammal” encompasses both humans and non-humans, including but not limited to humans, non-human primates, dogs, cats, mice, cattle, horses, and pigs.

[0146] The term “clinical factors” refers to a measure of the subject’s condition, such as the activity or severity of a disease. “Clinical factors” encompass all markers of the subject’s health status, including non-sample markers, and / or, non-limitingly, other characteristics of the subject, such as age and sex. Clinical factors can be scores, values, or sets of values ​​that can be obtained from assessments of a subject or a sample (or population of samples) derived from a subject under given conditions. Clinical factors can also be predicted by other parameters, such as markers and / or gene expression substitutes. Clinical factors may include tumor type, tumor subtype, and smoking history.

[0147] "Tumor-derived antigen-coding nucleic acid sequences" refer to nucleic acid sequences directly extracted from a tumor by, for example, RT-PCR, or sequence data obtained by sequencing a tumor and then synthesizing the nucleic acid sequence using sequencing data, for example, by various synthesis methods or PCR-based methods well known in the relevant field.

[0148] The term "alphavirus" refers to members of the Togaviridae family, which are single-stranded positive-sense RNA viruses. Alphaviruses are generally classified into Old World types, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semlik Forest virus, 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 skeleton" refers to the smallest sequence(s) of an alphavirus that enables the self-replication of the viral genome. Examples of these smallest sequences include conserved sequences for non-structural protein-mediated amplification, the non-structural protein 1 (nsP1) gene, the nsP2 gene, the nsP3 gene, the nsP4 gene, the poly(A) sequence, and sequences for the expression of subgenomic viral RNA, including the 26S promoter factor.

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

[0151] The term "RNA polymerase" includes polymerases that catalyze the synthesis of RNA polynucleotides from a DNA template. RNA polymerases are not limited to these, but include bacteriophage-derived polymerases such as T3, T7, and SP6.

[0152] The term "lipid" includes hydrophobic and / or amphiphilic molecules. Lipids may be cationic, anionic, or neutral. Lipids may be synthetic or naturally occurring and, in certain cases, biodegradable. Lipids may include (but not limited to) cholesterol, phospholipids, polyethylene glycol (PEG) complexes (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 nanoparticles" or "LNPs" includes vesicle-like structures formed using a lipid-containing membrane surrounding an aqueous interior, also known as liposomes. Lipid nanoparticles include lipid-based compositions having a solid lipid core stabilized by a surfactant. The core lipid may be a mixture of fatty acids, acylglycerols, waxes, and these surfactants. Biomembrane lipids such as phospholipids, sphingomyelin, bile acids (taurocholic acid), and sterols (cholesterol) can be used as stabilizers. Lipid nanoparticles can be formed using lipid molecules in specified ratios of different types, including (but not limited to) one or more cationic, anionic, or neutral lipids. Lipid nanoparticles can encapsulate molecules within an outer membrane shell, and then deliver the encapsulated molecules to the host cell's cytosol by contact with a target cell. Lipid nanoparticles can be modified or functionalized with non-lipid molecules on their surface, etc. Lipid nanoparticles can be monolayered or multilayered. Lipid nanoparticles can be complexed with nucleic acids. Monolayer lipid nanoparticles can be complexed with nucleic acids, in which case the nucleic acids are located inside the aqueous layer. Multilayer lipid nanoparticles can also be complexed with nucleic acids, in which case the nucleic acids are located inside the aqueous layer, form a layer between them, or are 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 Neo-Antigenic Antigen; FFPE: Formalin-Fixed Paraffin-Embedded; NMD: Nonsense Mutation-Dependent Degradation Mechanism; NSCLC: Non-Small Cell Lung Cancer; DC: Dendritic Cell.

[0155] When used in this specification and the appended claims, the singular forms "a," "an," and "the" refer to plural nouns unless the context explicitly indicates otherwise.

[0156] Unless otherwise specified or as is evident from the context, the term “approximately” as used herein is understood to mean within the nominal tolerance of the art, for example, within two standard deviations from the mean. “Approximately” 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 evident from the context, all numerical values ​​presented herein are modified by the term “approximately.”

[0157] Terms not directly defined herein should be understood to have the general meanings associated with them as understood within the art of the present invention. Certain terms are considered herein for the purpose of providing further guidance to practitioners in describing compositions, apparatus, methods, etc., of embodiments of the present invention, as well as their manufacture or use. It will be recognized that there may be multiple ways of saying the same thing. Accordingly, alternative words and synonyms may be used for any one or more of the terms considered herein. Emphasis should not be placed on whether a term is detailed or considered herein. Several synonyms or alternative methods, materials, etc., are provided. The listing of one or more synonyms or equivalents does not exclude the use of other synonyms or equivalents unless explicitly stated. The use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of embodiments of the invention herein.

[0158] All references, published patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0159] II. Methods for identifying antigens A method for identifying co-antigens (e.g., nascent antigens) involves identifying antigens from a tumor of interest 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 are likely to be immunogenic. For example, one such method is: A step of obtaining at least one of exome, transcriptome, or whole-genome tumor nucleotide sequencing and / or expression data from target tumor cells, wherein the tumor nucleotide sequencing and / or expression data is used to obtain data representing the peptide sequence of each antigen (for example, in the case of a nascent antigen, the peptide sequence of each nascent antigen includes at least one change that makes the peptide sequence different from the corresponding wild-type parent peptide sequence, or in the case of a non-mutated covalent antigen, the peptide is derived from any polypeptide that is known or found to have altered expression in tumor cells or cancerous tissue compared to normal cells or tissues), A step of inputting the peptide sequence of each antigen into one or more presentation models in order to generate a set of numerical likelihoods for each antigen being presented by one or more MHC alleles on the surface of target tumor cells or by cells present within the tumor, wherein the set of numerical likelihoods is identified at least based on received mass spectrometry data, To generate a set of selected antigens, a step is taken to select a subset of the set of antigens based on the set of numerical likelihoods. It can include...

[0160] The presented model may include a statistical regression or machine learning (e.g., deep learning) model trained on a set of reference data (also called a training dataset) containing a set of corresponding labels, wherein the set of reference data is optionally obtained from each of several distinct subjects, some of which may have tumors, and the set of reference data includes at least one of the following: 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, data representing MHC peptidomethic sequences from tumor tissue, and data representing MHC peptidomethic sequences from normal tissue. The reference data may 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 presented model may include a set of characteristics derived at least in part from a set of reference data, the set of characteristics including at least one of allele-dependent characteristics and allele-independent characteristics. In certain embodiments, each characteristic is included.

[0162] Methods for identifying co-antigens also include generating output for constructing personalized cancer vaccines by identifying one or more antigens derived from one or more target tumor cells that are likely to be presented on the surface of tumor cells. For example, one such method is a step of obtaining at least one of exome, transcriptome, or whole-genome nucleotide sequencing and / or expression data from target tumor cells and normal cells, wherein the nucleotide sequencing and / or expression data is a set of antigens identified by comparing the nucleotide sequencing and / or expression data from tumor cells with the nucleotide sequencing and / or expression data from normal cells, and peptide sequences identified from target normal cells (for example, in the case of neonatal antigens, the peptide sequence of each neonatal antigen includes at least one change that makes the peptide sequence different from the corresponding wild-type parent peptide sequence, or in the case of unmutated co-antigens, the peptide sequence is The method may include: a step of obtaining data representing the peptide sequence of each antigen (derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissue compared to normal cells or tissues); a step of encoding each of the peptide sequences of the antigen into a corresponding numerical vector, each numerical vector including information about a set of amino acids constituting the peptide sequence and a set of positions of the amino acids within the peptide sequence; a step of using a computer processor to input the numerical vectors into a deep learning presentation model to generate a set of presentation likelihoods for the set of antigens, each presentation likelihood in the set representing the likelihood that the corresponding antigen is presented on the surface of the target tumor cells by one or more class II MHC alleles, and the deep learning presentation model generating a selected set of antigens by selecting a subset of the set of antigens based on the set of presentation likelihoods; and a step of generating the output for constructing the personalized cancer vaccine based on the selected set of antigens.

[0163] Specific methods for identifying antigens, including nascent antigens, are well known to those skilled in the art, and such methods are described in detail, for example, in International Patent Application Publications WO / 2017 / 106638, WO / 2018 / 195357, and WO / 2018 / 208856, which are incorporated herein by reference in their entirety for all purposes.

[0164] This specification discloses a method for treating a subject having a tumor, further comprising the steps of: performing one of the antigen identification methods described herein to obtain a tumor vaccine comprising the selected set of antigens; and administering the tumor vaccine to the subject.

[0165] The methods disclosed herein may further include identifying one or more antigen-specific T cells for at least one of the antigens in a subset. In some embodiments, the identification includes co-culturing one or more T cells with one or more antigens in a subset under conditions that allow the growth of one or more antigen-specific T cells. In further embodiments, the identification includes contacting one or more T cells with a tetramer containing one or more antigens in a subset under conditions that allow binding between the T cells and the tetramer. In even further embodiments, the methods disclosed herein may further include 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 includes sequencing the T cell receptor sequences of the one or more identified T cells. The methods disclosed herein may further include 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 allow the growth of the plurality of T cells, and injecting the grown T cells into a target. In some embodiments, genetically engineering multiple T cells to express at least one of one or more identified T cell receptors includes cloning the T cell receptor sequence of the one or more identified T cells into an expression vector and transfecting each of the multiple T cells with the expression vector. In certain embodiments, the methods disclosed herein further include culturing the one or more identified T cells under conditions that cause the one or more T cells to grow and injecting the grown T cells into a target.

[0166] This specification also discloses isolated T cells that are antigen-specific to at least one selected antigen from the subset.

[0167] This specification also describes a method for producing a tumor vaccine, A step of obtaining at least one of exome, transcriptome, or whole-genome tumor nucleotide sequencing and / or expression data from target tumor cells, wherein the tumor nucleotide sequencing and / or expression data is used to obtain data representing the peptide sequence of each antigen (for example, in the case of a nascent antigen, the peptide sequence of each nascent antigen includes at least one change that makes the peptide sequence different from the corresponding wild-type parent peptide sequence, or in the case of a non-mutated covalent antigen, the peptide is derived from any polypeptide that is known or found to have altered expression in tumor cells or cancerous tissue compared to normal cells or tissues), A step of inputting the peptide sequence of each antigen into one or more presentation models in order to generate a set of numerical likelihoods that each of the antigens is presented by one or more MHC alleles on the surface of the target tumor cells, wherein the set of numerical likelihoods is identified at least based on received mass spectrometry data, To generate a set of selected antigens, a step is taken to select a subset of the set of antigens based on the set of numerical likelihoods, To produce a tumor vaccine containing the aforementioned selected set of antigens, or to combine with a process that has been used to produce such vaccines. Methods including this are also disclosed.

[0168] This specification also states, A step of obtaining at least one of exome, transcriptome, or whole-genome tumor nucleotide sequencing and / or expression data from target tumor cells, wherein the tumor nucleotide sequencing and / or expression data is used to obtain data representing each peptide sequence of a set of antigens, the peptide sequence of each antigen (for example, in the case of a nascent antigen, the peptide sequence of each nascent antigen includes at least one change that makes the peptide sequence different from the corresponding wild-type parent peptide sequence, or in the case of a non-mutated covalent antigen, the peptide is derived from any polypeptide that is known or found to have altered expression in tumor cells or cancerous tissue compared to normal cells or tissues), the step of obtaining the above, A step of inputting the peptide sequence of each antigen into one or more presentation models in order to generate a set of numerical likelihoods that each of the antigens is presented by one or more MHC alleles on the surface of the target tumor cells, wherein the set of numerical likelihoods is identified at least based on received mass spectrometry data, To generate a set of selected antigens, a step is taken to select a subset of the set of antigens based on the set of numerical likelihoods, To produce a tumor vaccine containing the aforementioned selected set of antigens, or to combine with a process that has been used to produce such vaccines. A tumor vaccine comprising a selected set of antigens, selected by performing a method including the following, is also disclosed.

[0169] Tumor vaccines may contain one or more of the following: nucleotide sequences, polypeptide sequences, RNA, DNA, cells, plasmids, or vectors.

[0170] Tumor vaccines may contain one or more antigens presented on the surface of tumor cells.

[0171] Tumor vaccines may contain one or more antigens that exhibit immunogenicity in the target organism.

[0172] Tumor vaccines do not necessarily need to contain one or more antigens that induce an autoimmune response against normal tissue in the target organism.

[0173] Tumor vaccines may contain adjuvants.

[0174] Tumor vaccines may contain excipients.

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

[0176] The methods disclosed herein may also include selecting antigens that have a higher likelihood of inducing a tumor-specific immune response in a target compared to antigens not selected based on the presented model.

[0177] The methods disclosed herein may also include selecting antigens that have a higher likelihood of being presented to naive T cells by professional antigen-presenting cells (APCs) compared to antigens not selected based on a presentation model, and optionally, the APCs are dendritic cells (DCs).

[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 not selected based on the presented model.

[0179] The methods disclosed herein may also include selecting antigens that have a reduced likelihood of inducing an autoimmune response against normal tissue in a subject compared to antigens not selected based on the presented model.

[0180] Exome or transcriptome nucleotide sequencing and / or expression data can be obtained by sequencing tumor tissue.

[0181] Sequencing may be next-generation sequencing (NGS) or any large-scale parallel processing sequencing approach.

[0182] The set of numerical likelihoods can be further specified by at least one of the following MHC allele interaction properties: namely, the predicted affinity for binding between the MHC allele and the antigen-coding peptide; the predicted stability of the antigen-coding peptide-MHC complex; the sequence and length of the antigen-coding peptide; the probability of presentation of an antigen-coding peptide with a similar sequence in cells from other individuals expressing a particular MHC allele, as assessed by mass spectrometry proteomics or other means; the expression level of the particular MHC allele in the subject of interest (e.g., measured by RNA-seq or mass spectrometry); the probability of presentation by the particular MHC allele in other distinct individuals expressing the particular MHC allele, independent of the overall nascent antigen-coding peptide sequence; and the 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 nascent antigen-coding peptide sequence.

[0183] The set of numerical likelihoods is further identified by at least one of the following MHC allele non-interaction properties: namely, the C-terminal and N-terminal sequences adjacent to the nascent antigen-coding peptide within its source protein sequence; optionally, the presence of protease cleavage motifs within the nascent antigen-coding peptide, weighted according to the expression of the corresponding protease in tumor cells (measured by RNA-seq or mass spectrometry); the turnover rate of the source protein as measured in appropriate cell types; and the tumor, predicted from annotations of germline or somatic splicing mutations measured by RNA-seq or proteome-mass spectrometry, or detected in DNA or RNA sequence data. Length of the source protein, optionally considering the specific splice variant ("isoform") most highly expressed in tumor cells; level of expression of proteasomes, immunoproteasomes, thymic proteasomes, or other proteases in tumor cells (which can be measured by RNA-seq, proteome-mass spectrometry, or immunohistochemistry); expression of the source gene of the nascent antigen-coding peptide (e.g., measured by RNA-seq or mass spectrometry); typical tissue-specific expression of the source gene of the nascent antigen-coding peptide at different stages of the cell cycle; e.g., uniProt or PDB A comprehensive catalog of the properties of source proteins and / or their domains, as can be found at http: / / www.rcsb.org / pdb / home / home.do; properties describing the nature of the domains of source proteins, including peptides, e.g., secondary or tertiary structure (e.g., α-helix relative to β-sheet); alternative splicing; the probability of presentation of peptides derived from the source protein of the target nascent antigen-coding peptide in other distinct subjects; the probability that peptides are not detected or are overexpressed by mass spectrometry due to technical bias; the expression of various gene modules / pathways, as measured by RNASeq, providing information about the state of tumor cells, stroma, or tumor-infiltrating lymphocytes (TILs) (not necessarily including the source protein of the peptide); the copy number of the source gene of the nascent antigen-coding peptide in tumor cells;The probability of a peptide binding to TAP, or the measurement 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, or 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- Mutations in any of the following genes: 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 gene encoding components of the proteasome or immunoproteasome. Peptides whose presentation depends on components of the antigen-presenting machinery that produce loss-of-function mutations in tumors have a low probability of presentation; presence or absence of functional germline polymorphisms, including but not limited to: genes encoding proteins involved in the antigen-presenting 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, H Polymorphisms in any of the genes encoding components of the proteasome or immunoproteasome (LA-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 cell lung cancer); smoking history;Optionally, typical expression of peptide source genes in associated tumor types or clinical subtypes, stratified by driver mutations.

[0184] At least one mutation may be a frameshift or non-frameshift insertion or deletion, a missense or nonsense substitution, a splice site change, a genome rearrangement or gene fusion, or any genomic or expression change that results in a nascent ORF.

[0185] Tumor cells can be 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, 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 a selected set or subset thereof of neoplastic antigens, and optionally further including administering the tumor vaccine to a target.

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

[0188] This specification discloses a method for identifying one or more nascent antigens likely to be presented on the surface of tumor cells, comprising: receiving mass spectrometry data including data relating to a plurality of isolated peptides eluted from major histocompatibility complexes (MHCs) derived 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 sample 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 one or more MHC alleles on the surface of tumor cells present peptide sequences derived from tumor cells.

[0189] The presentation model can represent the dependence between the presence of a pair of a specific MHC allele and a specific amino acid at a specific position in a peptide sequence, and the likelihood of presentation of such a peptide sequence containing the specific amino acid at the specific position on the tumor cell surface by the specific MHC allele of the pair.

[0190] The methods disclosed herein may also include selecting a subset of neoplastic antigens, each of which is selected because it has an increased likelihood of being presented on the surface of tumor cells for one or more distinct neoplastic antigens.

[0191] The methods disclosed herein may also include selecting a subset of neoplastic antigens, each of which is selected because it has a high likelihood of inducing a tumor-specific immune response in a target against one or more distinct neoplastic antigens.

[0192] The methods disclosed herein may also include selecting a subset of neonatal antigens, each of which is selected because of the increased likelihood that it can be presented to naive T cells by professional antigen-presenting cells (APCs) for one or more distinct neoplastic antigens, and optionally the APCs are dendritic cells (DCs).

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

[0194] The methods disclosed herein may also include selecting a subset of neoplastic antigens, each of which is selected because it has a reduced likelihood of inducing an autoimmune response against normal tissue in a subject compared to one or more distinct neoplastic antigens.

[0195] The methods disclosed herein may also include selecting a subset of nascent antigens, each of which is selected because of its reduced likelihood of differential post-translational modification of APCs in tumor cells, and optionally, APCs are dendritic cells (DCs).

[0196] In carrying out the methods described herein, unless otherwise specified, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the scope of the skills in the art shall be used. Such techniques are adequately described in the literature. For example, see TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); ALLehninger, 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 neogeneic antigens Methods for identifying certain mutations (e.g., variants or alleles present in cancer cells) are also disclosed herein. In particular, these mutations may be present in the genome, transcriptome, proteome, or exome of cancer cells of a subject having cancer, but not in normal tissue from which the subject originates. Specific methods for identifying tumor-specific antigens, including covalently generated antigens, are well known to those skilled in the art, and such methods are described in detail, for example, in International Patent Application Publications WO / 2017 / 106638, WO / 2018 / 195357, and WO / 2018 / 208856, which are incorporated herein by reference in their entirety for all purposes.

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

[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 proteins in tumor versus normal cells.

[0200] Furthermore, mutations may 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 to detect 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. For example, 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 typically utilize amplification of the target gene region by PCR. Further methods are based on the generation of small signal molecules by invasive cleavage and subsequent 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 methods can simultaneously involve multiple amplification of numerous markers. For example, it is well known in the art to select PCR primers to produce PCR products of non-overlapping sizes that can be analyzed simultaneously. Alternatively, it is possible to amplify different markers with primers that are differentially labeled and therefore can be differentially detected. Naturally, hybridization-based detection methods enable differential detection of multiple PCR products in a sample. Other techniques that enable multiple 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 specialized exonuclease-resistant nucleotides, such as those disclosed in Mundy, CR (U.S. 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 complementary to a specific exonuclease-resistant nucleotide derivative, that derivative is incorporated into the end of the hybridized primer. Such incorporation makes the primer resistant to the exonuclease, thereby enabling its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, the finding that the primer has become resistant to the exonuclease reveals that the nucleotide present in the polymorphic site of the target molecule is complementary to that of the nucleotide derivative used in the reaction. This method has the advantage of not requiring the determination of a large amount of exogenous sequence data.

[0204] To determine the identity of nucleotides at polymorphic sites, a solution-based method can be used (Cohen, D. et al. (French Patent No. 2,650,840; PCT Application No. WO91 / 02087)). This method uses a primer complementary to the allele sequence immediately 3' of the polymorphic site, as in Mundy's method, U.S. Patent No. 4,656,127. This method determines the identity of the nucleotide at that site using a labeled dideoxynucleotide derivative, which, if complementary to the nucleotide at the polymorphic site, is incorporated onto the end of the primer.

[0205] An alternative method, known as Genetic Bit Analysis or GBA, is described by Goelet, P. et al. (PCT application 92 / 15712). Goelet, P. et al.'s method uses a mixture of a labeled terminator and a primer complementary to the 3' sequence of the polymorphic site. The incorporated labeled terminator is determined by and complementary to the nucleotide 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 WO91 / 02087), Goelet, P. et al.'s method can be a heterogeneous phase assay in which the primer or target molecule is immobilized on a solid phase.

[0206] Several primer guide nucleotide insertion procedures for assaying polymorphism sites in DNA are described (Komher, J. Set al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, BP, Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990); Kuppuswamy, M. Net al., Proc. Natl. Acad. Sci. (USA) 88:1143-1147 (1991); Prezant, TR et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al. (Syvanen, A.-C., et al., Amer. J. Hum. Genet. 52:46-59 (1993)). These methods differ from GBA in that they utilize the incorporation of labeled deoxynucleotides to distinguish between bases at polymorphic sites. In such a form, the signal is proportional to the number of incorporation of deoxynucleotides, so polymorphisms occurring in a run 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 and in parallel from millions of individual molecules of DNA or RNA. Real-time single-molecule synthesis sequencing techniques rely on the detection of fluorescent nucleotides as they are incorporated into the nascent strand of DNA that is complementary to the template being sequenced. In one method, oligonucleotides 30–50 bases long are covalently immobilized at their 5' ends to a glass coverslip. These immobilized strands serve two functions. First, they act as capture sites on the target template strand when the template is composed of a capture tail complementary to the surface-bound oligonucleotide. They also act as primers for template-directed primer extension, forming the basis for sequence reading. The capture primers function as fixed positional sites for sequencing, using multiple cycles of synthesis, detection, and chemical cleavage of a dye-linker to remove the dye. Each cycle consists of adding a polymerase / labeled nucleotide mixture, rinsing, imaging, and dye cleavage. In an alternative method, polymerase is modified with a fluorescent donor molecule and immobilized on a glass slide, while each nucleotide is color-coded by an acceptor fluorescent moiety attached to γ-phosphate. As the nucleotides are incorporated into a new chain, the system detects the interaction between the fluorescently tagged polymerase and the fluorescently modified nucleotides. Other synthetic sequencing techniques also exist.

[0208] Any suitable synthetic sequencing platform can be used to identify mutations. As described above, four major synthetic sequencing platforms are currently available: the Genome Sequencer from Roche / 454 Life Sciences, the 1G Analyzer from Illumina / Solexa, the SOLiD system from Applied BioSystems, and the Heliscope system from Helicos Biosciences. Synthetic sequencing platforms have also been described by Pacific BioSciences and VisiGen Biotechnologies. In some embodiments, a number of nucleic acid molecules to be sequenced are bound 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' ends of the template. The nucleic acid can be immobilized 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 function dually as a universal primer.

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

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

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

[0212] Nucleic acid samples for use in the methods described herein can be obtained using any cell type or tissue. For example, DNA or RNA samples can be obtained from tumors or body fluids, such as blood or saliva obtained by known techniques (e.g., venipuncture). Alternatively, nucleic acid testing can be performed on dry samples (e.g., hair or skin). In addition, samples can be obtained from tumors for sequencing, and other samples can be obtained from normal tissue for sequencing if the normal tissue is of the same tissue type as the tumor. Samples can be obtained from tumors for sequencing, and other samples can be obtained from normal tissue for sequencing if the normal sample is of a different tissue type than the tumor.

[0213] Tumors may include one or more of the following: lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric 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. These peptides can be acid-eluted from tumor cells or from HLA molecules immunoprecipitated from tumors, and then identified using mass spectrometry.

[0215] IV. Antigen Antigens can include nucleotides or polynucleotides. For example, an antigen can be an RNA sequence encoding a polypeptide sequence. Antigens useful in vaccines can therefore include nucleotide sequences or polypeptide sequences. Covalent nascent antigens are shown in Table A (see SEQ ID NOs. 10755-21015) and the results of AACR GENIE (see SEQ ID NOs. 21016-29357). Covalent antigens are shown in Table 1.2 (see SEQ ID NOs. 57-10754).

[0216] Disclosed herein are isolated peptides containing tumor-specific mutations identified by the methods disclosed herein, peptides containing known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. Nascent antigen peptides may be described in the context of their coding sequences if the nascent antigen includes a nucleotide sequence (e.g., DNA or RNA) encoding the polypeptide sequence to which it relates.

[0217] This specification also discloses peptides derived from any polypeptide known or found to exhibit altered 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 may be derived can be found, for example, in the COSMIC database, which is a curated collection of comprehensive information on somatic mutations in human cancers. The peptides include tumor-specific mutations.

[0218] One or more polypeptides encoded by an antigen nucleotide sequence may include at least one of the following: binding affinity to MHC with an IC50 value of less than 1000 nM; for MHC class I peptides, a length of 8 to 15 amino acids, 8, 9, 10, 11, 12, 13, 14, or 15; presence of a sequence motif within or near the peptide that promotes proteasome cleavage; and presence of a sequence motif that promotes TAP transport; for MHC class II polypeptides, a length of 6 to 30 amino acids, 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; and presence of a sequence motif within or near an extracellular or lysosomal protease (e.g., cathepsin) peptide-promoting cleavage site or an HLA-DM-catalyzed HLA-binding site.

[0219] One or more antigens can be present on the surface of a tumor.

[0220] One or more antigens may be immunogenic in a tumor-bearing subject and, for example, may elicit a T-cell response or a B-cell response in the subject.

[0221] One or more antigens that induce an autoimmune response in a subject can be excluded from consideration in the context of vaccine production for subjects with tumors.

[0222] The size of at least one antigenic peptide molecule is approximately 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, The amino acid molecule may include, but is not limited to, approximately 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, or more amino acid residues, and any range derived from these ranges. In a specific embodiment, the antigenic peptide molecule has 50 amino acids or less.

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

[0224] Where desirable, longer peptides can be designed in several ways. In one example, if the likelihood of peptide presentation on an HLA allele is predicted or known, the longer peptide may consist of (1) individual presented peptides having an extension of 2-5 amino acids toward the N-terminus and C-terminus of their respective corresponding gene products; or (2) a chain of some or all of the presented peptides, each having an extended sequence. In another example, if sequencing reveals long (longer than 10 residues) nascent epitope sequences present in the tumor (e.g., by frameshift, read-through, or intron inclusion resulting in a novel peptide sequence), the longer peptide may consist of (3) the entire stretch of novel tumor-specific amino acids, thus avoiding the need for computational or in vitro test-based selection of shorter peptides to present to the strongest HLA. In either example, the use of longer peptides may enable endogenous processing by patient cells, potentially leading to more effective antigen presentation and induction of T cell responses.

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

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

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

[0228] Antigenic peptides and polypeptides possessing desirable activity or properties can be modified to enhance the biological activity of the unmodified peptide in binding to the desired MHC molecule and activating appropriate T cells, or to give it certain desirable attributes, such as improved pharmacological features, while retaining at least substantially all of the biological activity of the unmodified peptide. 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. A conservative substitution means replacing an amino acid residue with another that is biologically and / or chemically similar, for example, 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 may also be explored using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, for example, as described 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), 2d Ed. (1984).

[0229] Modification of peptides and polypeptides with various amino acid mimes or non-natural amino acids can be particularly useful in increasing the in vivo stability of peptides and polypeptides. Stability can be assayed in many ways. For example, peptidases, as well as various biological media such as human plasma and serum, have been used to test stability. See, for example, Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). The half-life of peptides can be conveniently determined using a 25% human serum (v / v) assay. The protocol is generally as follows: Pooled human serum (type AB, non-thermally inactivated) is degreased by centrifugation before use. The serum is then diluted to 25% in RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, small amounts of the reaction solution are taken and added to either 6% aqueous trichloroacetic acid or ethanol. The turbid reaction sample is cooled for 15 minutes (4°C), and then spun to precipitate the serum protein. 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 ligation to a sequence containing at least one epitope capable of inducing a T helper cell response. Immunogenic peptide / T helper conjugates can be ligated by spacer molecules. Spacers typically consist of relatively small neutral molecules, such as amino acids or amino acid mimes, which are substantially uncharged under physiological conditions. Spacers are typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that optionally present spacers do not need to consist of the same residues and can therefore be heterooligomers or homooligomers. If present, spacers will usually consist of at least one or two residues, more typically three to six residues. Alternatively, peptides can be ligated to T helper peptides without spacers.

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

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

[0233] In a further embodiment, the antigen comprises a nucleic acid (e.g., polynucleotide) encoding an antigenic peptide or a portion thereof. The polynucleotide can be single-stranded and / or double-stranded, in its native or stabilized form, or a combination thereof, such as DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), or polynucleotides having a phosphorothioate backbone, and may or may not contain introns. Further embodiments provide an expression vector capable of expressing a polypeptide or a portion thereof. Expression vectors for various cell types are well known in the art and can be selected without excessive experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, in the correct orientation and with a precise reading frame for expression. If necessary, the DNA can be ligated to a suitable transcriptional and translational regulatory nucleotide sequence recognized by the desired host, although such regulation is generally available in the expression vector. The vector is then introduced into the host through standard techniques. A guide 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 composition Also disclosed herein are immunogenic compositions, such as vaccine compositions, that can induce a specific immune response, for example, a tumor-specific immune response. Vaccine compositions typically comprise one or more antigens selected using, for example, the methods described herein or in Table A, Table 1.2, or the AACR GENIE results. Vaccine compositions may also be referred to as vaccines.

[0235] The vaccine may 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 may include post-translational modifications. Vaccines contain 1 to 100 or more nucleotide sequences, such as 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, 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 types of 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 It may contain 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, different peptides and / or polypeptides, or encoding nucleotide sequences, are selected so that the peptides and / or polypeptides can bind to different MHC molecules, such as different MHC class I molecules and / or different MHC class II molecules. In some embodiments, a single vaccine composition contains an encoding sequence of a peptide and / or polypeptide that can bind to the most frequently present MHC class I molecules and / or different MHC class II molecules. Thus, a vaccine composition may contain different fragments that can bind 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 can induce a specific cytotoxic T cell response and / or a specific helper T cell response.

[0238] The vaccine composition may further comprise an adjuvant and / or carrier. Examples of useful adjuvants and carriers are given below in this specification. The composition may be conjugated to a carrier, such as a protein, or to an antigen-presenting cell, such as a dendritic cell (DC) capable of presenting peptides to T cells.

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

[0240] The ability of adjuvants to enhance the immune response to an antigen is typically demonstrated by a significant or substantial increase in the immune-mediated response or a reduction in disease symptoms. For example, an increase in humoral immunity is typically demonstrated by a significant increase in the titer of antibodies produced against the antigen, and an increase in T cell activity is typically demonstrated by increased cell proliferation, cellular cytotoxicity, or cytokine secretion. Adjuvants can also alter the immune response, for example, by changing a primarily humoral or Th response to a primarily cellular or Th response.

[0241] Suitable adjuvants include 1018 ISS, alum, aluminum salt, 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 This includes, but is not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector systems, PLG microparticles, reciquimod, SRL172, virosoms and other virus-like particles, YF-17D, VEGF traps, 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 dendritic cell-specific immunological adjuvants (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. Some cytokines are directly linked to their effects on dendritic cell migration to lymphoid tissue (e.g., TNF-α), to accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (specifically, U.S. Patent No. 5,849,589, which is incorporated herein by reference in its entirety), and to their action as immunoadjuvants (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 adjuvant effect in vaccine settings. Other TLR-binding molecules, such as RNAs that bind 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, as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as adjuvants. The amount and concentration of adjuvants and additives can be readily determined by those skilled in the art without excessive experimentation. Additional adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, salglamostim).

[0244] The vaccine composition may contain more than one different adjuvant. Furthermore, the therapeutic composition may contain any adjuvant substance, including any or a combination thereof. It is also intended that the vaccine and adjuvant may 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 may be, for example, to increase activity or immunogenicity, to provide stability, to increase biological activity, or to increase serum half-life, particularly by increasing the molecular weight of the variant. Furthermore, the carrier can assist in the presentation of peptides to T cells. The carrier can be any suitable carrier known to those skilled in the art, e.g., a protein or antigen-presenting cell. Carrier proteins may be, but are not limited to, serum proteins such as keyhole limpet hemocyanin, transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones such as insulin, or palmitic acid. For human immunization, the carrier is generally a physiologically acceptable carrier that is tolerable and safe for humans. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier may be dextran, e.g., cepharose.

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

[0247] Antigens also include, but are not limited to, vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or second, third, or hybrid second / third generation lentiviruses, and recombinant lentiviruses of any generation 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 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 It can also be incorporated into viral vector-based vaccine platforms, such as ubiquitin C promoter, Nucl. AcidsRes. (2015) 43(1):682-690, and Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72(12):9873-9880). Depending on the packaging capabilities of the aforementioned viral vector-based vaccine platforms, this approach can deliver one or more nucleotide sequences encoding one or more nascent antigen peptides.The sequence may be adjacent to a non-mutant sequence, separated by a linker, or preceded by one or more sequences targeting an intracellular compartment (see, for example, 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, and 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 response (e.g., CTLs) to the peptide. Useful vaccinia vectors and methods in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus calmette-Guérin). The BCG vector is described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful for the therapeutic administration or immunization of antigens, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the description herein.

[0248] VA antigen cassette Methods used for selecting one or more antigens, cloning and construction of “cassettes,” and their insertion into viral vectors are within the scope of the art, given the teachings provided herein. “Antigen cassette” means a combination of a selected antigen or group of antigens and other regulatory elements required to transcribe k-antigens and express the transcript. The antigen or group of antigens can be functionally linked to regulatory elements in a manner that enables transcription. Such elements include conventional regulatory elements that can promote the expression of antigens in cells transfected with a viral vector. Thus, an antigen cassette may also include a selected promoter linked to the antigens and positioned within the selected viral sequence of a recombinant vector together with other optional regulatory elements. A cassette may include one or more nascent antigens shown in Table A and / or the AACR GENIE results, and / or one or more antigens shown in Table 1.2.

[0249] A useful promoter may be a constitutive promoter or a regulated (inducible) promoter capable of controlling the amount of antigen(s) expressed. For example, a preferred promoter is the cytomegalovirus early promoter / enhancer [see, e.g., Boshart et al, Cell, 41:521-530 (1985)]. Another preferred promoter is the Roussarcoma 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 may select other suitable or preferred promoters.

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

[0251] An antigen cassette can have one or more antigens. For example, a particular cassette can contain 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. Antigens may be linked to each other directly. Antigens may be linked to each other by linkers. Antigens can be oriented in any direction relative to each other, including N-C or C-N.

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

[0253] The antigen cassette is described by the following formula, which describes 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 include promoter nucleotide sequences. N contains an MHC class I epitope coding nucleic acid sequence. L5 contains the 5' linker sequence, L3 contains the 3' linker sequence, G5 contains a nucleic acid sequence that encodes an amino acid linker. G3 contains one of at least one nucleic acid sequences that encode an amino acid linker. U contains an MHC class II antigen-coding nucleic acid sequence, where for each X, the corresponding Nc is an epitope-coding nucleic acid sequence. For each Y, the corresponding Uf is the antigen-coding nucleic acid sequence. The composition and ordered arrangement 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 to 400, Y=0, 1, 2, 3, 4 or 5, Z=1 to 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 the case where no further promoters are present is described (i.e., only the promoter nucleotide sequence provided by the RNA alphavirus backbone is present). In this case, there are 20 MHC class I epitopes, each with a 5' linker, each with a 3' linker, 2 MHC class II epitopes, a linker linking the 2 MHC class II epitopes, a linker linking the 5' ends of the 2 MHC class II epitopes to the 3' linker of the last MHC class I epitope, and a linker linking the 3' ends of the 2 MHC class II epitopes to the RNA alphavirus backbone. An example of linking the 3' end of the antigen cassette to the RNA alphavirus backbone is direct linking to the 3'UTR element provided by the RNA alphavirus backbone, such as a 19nt CSE at 3'. Examples of ligation of the 5' end of the antigen cassette to the RNA alphavirus backbone include direct ligation to the 26S promoter sequence, the alphavirus 5'UTR, the 51nt CSE, or the 24nt CSE.

[0255] Other examples include cases where a=1 and a promoter other than the promoter nucleotide sequence provided by the RNA alphavirus backbone is present; cases where a=1 and Z is greater than 1 and there are multiple promoters other than the promoter nucleotide sequence provided by the RNA alphavirus backbone, each resulting in the expression of one or more different MHC class I epitope coding nucleic acid sequences; cases where h=1 and another promoter is present resulting in the expression of an MHC class II antigen coding nucleic acid sequence; and cases where g=0 and the MHC class II antigen coding nucleic acid sequence (if present) is 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 cases 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 either a 5' linker or a 3' linker, or neither. In other cases 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 either a 5' linker or a 3' linker, or neither.

[0257] In cases where multiple MHC class II epitopes are present in 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 either a 5' linker or a 3' linker, or neither. In other cases where multiple MHC class II epitopes are present in 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 either a 5' linker or 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 skeleton. For example, the promoter sequences Pn and P2 provided by the RNA alphavirus skeleton may each contain 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 skeleton, and may also be different from each other.

[0259] The 5' linker L5 may be a natural or non-natural sequence. Non-natural sequences include, but are not limited to, AAY, RR, and DPP. The 3' linker L3 may also be a natural or non-natural sequence. Furthermore, L5 and L3 may both be natural sequences, both be non-natural sequences, or one may be natural and the other non-natural. For each X, the amino acid linker consists of 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 amino acids. The length 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 of a length of 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.

[0260] For each Y, the amino acid linker G5 consists of 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, and 51 amino acids. The length 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 Y, the amino acid linker may also be of a length of 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.

[0261] Amino acid linker G3 contains 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, and 52 amino acids. The length may be 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. G3 may also be a length of 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.

[0262] For each X, each N can encode an MHC class I epitope of a length of 7 to 15 amino acids. For each X, each N may encode an MHC class I epitope of a length of 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. For each X, each N may also encode an MHC class I epitope of length of 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.

[0263] VB Immunity Checkpoint The vectors described herein, for example, the C68 vector or the alphavirus vector described herein, may contain a nucleic acid encoding at least one antigen, and the same or a different vector may also contain a nucleic acid encoding at least one immunomodulator (e.g., an antibody such as scFv) that binds to an immune checkpoint molecule and blocks its activity. The vector may contain an antigen cassette and one or more nucleic acid molecules encoding checkpoint inhibitors.

[0264] Examples of immune checkpoint molecules that may be targeted for blockade 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 molecule belonging to the CD2 family and expressed in all NK(γδ) and memory CD8+(αβ) T cells), CD160 (also known as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, antigen-binding fragments thereof, or other binding proteins that bind to one or more of the following and block or inhibit their activity: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN-15049. Examples of immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), ipilimumab, MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody), and Yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor). The sequence encoding the antibody can be incorporated into a vector, such as C68, by manipulation using the usual skills 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. Determination of the set of peptides covering all tumor subclones. Truncal peptides, which refer to those presented by all or most tumor subclones, are preferred for inclusion in vaccines. 53 Optionally, if there are no truncal peptides that are likely to be presented and are predicted to be immunogenic, or if the number of truncal peptides that are likely to be presented and are predicted to be immunogenic is small enough that additional non-truncal peptides can be included in the vaccine, further 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. Prioritization of antigens Even after applying all of the above antigen filters, there may still be more candidate antigens available for vaccine inclusion than the vaccine technology can accommodate. Additionally, uncertainties may remain regarding various aspects of antigen analysis, and trade-offs may exist between the various properties of candidate vaccine antigens. Therefore, instead of predetermined filters at each stage of the selection process, we can consider an integral multidimensional model that places candidate antigens in a space with at least the following axes and optimizes selection using an integral approach. 1. Risk of autoimmunity or tolerance (germline risk) (lower autoimmunity risk is typically preferable) 2. Probability of sequencing artifacts (a lower artifact probability is generally preferable) 3. Probability of immunogenicity (a higher probability of immunogenicity is generally preferable) 4. Probability of presentation (a higher probability of presentation is generally preferable) 5. Gene expression (higher expression is generally preferable) 6. HLA gene coverage (a greater number of HLA molecules involved in the presentation of a set of antigens may reduce the likelihood that tumors 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 treatment response and decrease the probability of tumor immune evasion).

[0267] Furthermore, in some cases, if an antigen is expected to be presented by an HLA allele that is lost or inactivated in all or part of the patient's tumor, these neonatal antigens may be given lower priority (e.g., excluded) in vaccination. Loss of an HLA allele can result from somatic mutation, loss of heterozygosity, or homozygous deletion of a 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 LOH and homozygous deletions (including HLA loci) are similarly described (Carter et al., 2012; McGranahan et al., 2017; Van Loo et al., 2010). Antigens may be de-prioritized if mass spectrometry data indicate that the predicted antigen is not presented by the predicted HLA allele.

[0268] VD Alpha Virus VD1. Biology of Alphaviruses Alphaviruses are members of the Togaviridae family and are single-stranded positive-sense RNA viruses. Members are generally classified into Old World types, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest virus, or New World types, such as Eastern Equine Encephalitis Virus, Aura, Fort Morgan, or Venezuelan Equine Encephalitis Virus and its derivative TC-83 (Strauss Microbrial Review 1994). The natural alphavirus genome is typically about 12 kb long, with the first two-thirds containing genes encoding non-structural proteins (nsPs) that form RNA replication complexes for self-replication of the viral genome, and the last third containing subgenome expression cassettes encoding structural proteins for virion production (Frolov RNA 2001).

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

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

[0271] VD2. Alphavirus as a delivery vector Alphavirus-based delivery vectors (also called alphavirus vectors, alphavirus viral vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying RNA (samRNA) vectors) can be constructed using alphaviruses (alphavirus sequences, characteristics, and other elements). Alphaviruses have traditionally been genetically engineered for use as expression vector systems (Pushko 1997, Rheme 2004). Alphaviruses have several advantages in vaccine settings where the expression of heterologous antigens is desirable. Due to their ability to self-replicate in the host cytosol, alphaviruses can generally achieve high copy numbers of expression cassettes within cells, thus enabling the production of high levels of heterologous antigens. Furthermore, because the vectors are generally transient, they have high biosafety and induce little immune tolerance to the vectors. Also, the general public generally does not have pre-existing immunity to alphaviruses compared to other standard viral vectors such as human adenoviruses. Alphavirus-based vectors also generally produce cytotoxic reactions to infected cells. Cytotoxicity can be of some importance in vaccine design to appropriately 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, including the TC-83 strain of VEE, have been developed. Accordingly, an example of an antigen expression vector described herein can utilize an alphavirus scaffold that enables high levels of antigen expression, induces a strong immune response to the antigen, does not induce an immune response to the vector itself, and can be used safely. Furthermore, the antigen expression cassette can be designed to induce different levels of immune response by optimizing which alphavirus sequences the vector uses, including (but not limited to) sequences derived from VEE or its attenuated derivative strain TC-83.

[0272] Several expression vector design strategies using alphaviral sequences have been developed (Pushko 1997). One strategy involves designing an alphaviral vector by inserting a second copy of a 26S promoter sequence factor downstream of a structural protein gene, followed by the insertion of a heterologous gene (Frolov 1993). This produces a subgenomic RNA that expresses additional heterologous proteins in addition to native non-structural and structural proteins. In this system, all the factors necessary to produce an infectious virion are present, and therefore, rounds of repeated infection with the expression vector can occur in uninfected cells.

[0273] Another expression vector design utilizes a helper virus system (Pushko 1997). In this strategy, the structural protein is replaced by a heterologous gene. Thus, after self-replication of viral RNA mediated by still intact non-structural genes, the 26S subgenomic RNA results in the expression of the heterologous protein. Traditionally, an infectious virus is generated by providing a further vector expressing the structural protein in-trans, for example, by simultaneous transfection of a cell line. One such system is described in U.S. Patent No. 8,093,021, the entirety of which is incorporated herein by reference for all purposes. Helper vector systems offer the advantage of limiting the possibility of forming infectious particles and thus improving biosafety. Furthermore, helper vector systems can shorten the overall vector length and improve replication and expression efficiency. For example, in one antigen expression vector described herein, an alphaviral scaffold in which the structural protein is replaced by an antigen cassette can be used, and the resulting vector promotes efficient expression by reducing the overall size of the expression vector while simultaneously reducing biosafety issues.

[0274] VD3. Alphavirus generation in vitro Alphavirus delivery vectors are generally positive-strand RNA polynucleotides. A conventional method well known in the art for RNA generation is in vitro translation (IVT). In this method, a DNA template for the desired vector is first generated by methods well known in the art, including standard molecular biological methods such as cloning, restriction digestion, ligation, gene synthesis, and polymerase chain reaction (PCR). This DNA template has an RNA polymerase promoter at the 5' end of the sequence to be transcribed into RNA. Promoters include, but are not limited to, bacteriophage polymerase promoters such as T3, T7, or SP6. The DNA template is then incubated with a suitable RNA polymerase enzyme, buffer, and nucleotide (NTP). The resulting RNA polynucleotide can be optionally 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 modification of the 3' end to optionally have a polyadenylated (poly-A) tail. Next, RNA can be purified using methods well known in this field, such as phenol-chloroform extraction.

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

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

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

[0278] Lipid nanoparticles (LNPs) are an attractive delivery system due to the amphiphilic nature of lipids, which allows for the formation of membrane and vesicular structures (Riley 2017). Typically, these vesicles are absorbed into the membrane of target cells and deliver expression vectors by releasing nucleic acids 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 generally include a defined mixture of cationic, neutral, anionic, and amphiphilic lipids. In some examples, specific lipids are included to provide chemical functional groups that prevent LNP aggregation, prevent lipid oxidation, or promote the attachment of further parts. The lipid composition can affect the overall size and stability of the 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 PEG or PEG-compounded 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 serum nucleases or off-target stimulation of the immune system by free nucleic acids. Therefore, encapsulation of alphaviral vectors can be used to prevent degradation while simultaneously preventing potential off-target effects. In certain cases, the alphaviral vector is completely encapsulated within a delivery carrier, such as inside an aqueous LNP. Encapsulation of the alphaviral vector within an LNP can be carried out 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, the delivery vector and desired substance are completely encapsulated inside an MC3 or MC3-like molecule-based LNP by supplying a desired lipid formulation, such as an MC3 or MC3-like molecule-containing composition, to the droplet generator in parallel with the alphaviral delivery vector and other desired substances. In one example, the droplet generator can control the particle size range and particle size distribution of the generated LNP. For example, LNPs can have particle sizes in the range of 1 to 1000 nm in diameter, such as 1, 10, 50, 100, 500, or 1000 nm. After droplet generation, the delivery vector containing the expression vector can be further processed or modified in preparation for administration.

[0280] VE Chimpanzee Adenovirus (ChAd) VE1. Virus delivery by chimpanzee adenovirus Vaccine compositions for delivering one or more antigens (e.g., one or more nascent antigens shown in Table A and the AACR GENIE results, and / or one or more antigens shown in Table 1.2, delivered via an antigen cassette) can be produced 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 herein as ChAdV68) can be used in vaccine compositions for delivering antigens (see Sequence ID No. 1). The use of C68 adenovirus-derived vectors is described in further detail in U.S. Patent No. 6,083,716, the entirety of which is incorporated herein by reference for all purposes.

[0281] In a further embodiment, this specification provides a recombinant adenovirus comprising a chimpanzee adenovirus DNA sequence, such as C68, and an antigen cassette functionally linked to a regulatory sequence that induces expression. This recombinant virus can infect mammalian, preferably human, cells and express the product of the nascent antigen cassette within the cells. The vector can be used to delete the native chimpanzee E1 gene and / or E3 gene and / or E4 gene. The antigen cassette can be inserted into any of these gene deletion sites. The antigen cassette may contain an antigen against which a primed immune response is desired.

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

[0283] In yet another embodiment, a novel mammalian cell line expressing a chimpanzee adenovirus gene (e.g., derived from C68) or a functional fragment thereof is provided.

[0284] In a further embodiment, the Specified Reference Provision provides a method for delivering an antigen cassette into a mammalian cell, comprising the step of introducing into the cell an effective amount of a chimpanzee adenovirus, such as C68, which has been engineered to express the antigen cassette.

[0285] A further embodiment provides a method for treating cancer by inducing an immune response in a mammalian host. This method may include administering to a host an effective amount of recombinant chimpanzee adenovirus, such as C68, which contains an antigen cassette encoding one or more antigens derived from the tumor to be targeted by the immune response.

[0286] Furthermore, non-monkey mammalian cells expressing a chimpanzee adenovirus gene obtained from the sequence of Sequence ID No. 1 are also disclosed. This gene can be selected from the group consisting of adenoviruses E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 of Sequence ID No. 1.

[0287] Furthermore, a nucleic acid molecule is disclosed that contains a DNA sequence of a chimpanzee adenovirus containing a gene obtained from the sequence of Sequence ID No. 1. This gene can be selected from the group consisting of the chimpanzee adenovirus genes E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 of Sequence ID No. 1. In some embodiments, the nucleic acid molecule contains Sequence ID No. 1. In some embodiments, the nucleic acid molecule contains a sequence of Sequence ID No. 1 in which at least one gene selected from the group consisting of the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 of Sequence ID No. 1 is deleted.

[0288] Furthermore, a vector is disclosed comprising a chimpanzee adenovirus DNA sequence obtained from Sequence ID No. 1 and an antigen cassette functionally linked to one or more regulatory sequences that induce cassette expression in a heterologous host cell, wherein optionally, the chimpanzee adenovirus DNA sequence includes at least a cis element required for replication and capsid formation, and the cis element is adjacent to the antigen cassette and regulatory sequences. In some embodiments, the chimpanzee adenovirus DNA sequence includes genes selected from the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 gene sequences of Sequence ID No. 1. In some embodiments, the vector may be deleting the E1A and / or E1B genes.

[0289] Furthermore, host cells transfected with vectors disclosed herein, such as a C68 vector engineered to express an antigen cassette, are also disclosed. In addition, human cells expressing selected genes introduced intracellularly by introducing the vectors disclosed herein into the cells are also disclosed.

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

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

[0292] Complementary cell lines expressing VE2.E1 To produce recombinant chimpanzee adenovirus (Ad) having a deletion 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., complementary or packaging cell line). For example, to produce a replication-deficient chimpanzee adenovirus vector, a cell line expressing the E1 gene product of a human or chimpanzee adenovirus can be used, and such cell lines may include HEK293 or its variants. A cell line expressing any selected chimpanzee adenovirus gene can be produced according to the protocol for producing a cell line expressing the chimpanzee E1 gene (Examples 3 and 4 of U.S. Patent No. 6,083,716).

[0293] AAV-enhanced assays can be used to identify chimpanzee adenovirus E1-expressing cell lines. This assay is useful for identifying the function of E1 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 parental cell line. Inducible or constitutive promoters can be used for this purpose. Inducible promoters include the zinc-induced sheep metallothione promoter, or the glucocorticoid-induced mouse mammalian oncovirus (MMTV), particularly dexamethasone. Other inducible promoters, such as those specified in international application WO95 / 13392, incorporated herein by reference, can also be used to construct packaging cell lines. Constitutive promoters that control the expression of the chimpanzee adenovirus gene can also be used.

[0295] Parental cells can be selected to create novel cell lines expressing any desired C68 gene. Such parental cell lines may include, but are not limited to, HeLa [ATCC deposit number CCL2], A549 [ATCC deposit number CCL185], KB [CCL17], Detroit [e.g., Detroit 510, CCL72], and WI-38 [CCL75] cells. Other suitable parental cell lines may be obtained from other sources. Examples of parental cell lines include CHO, HEK293 or its variants, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a.

[0296] E1-expressing cell lines can be useful in the production of recombinant chimpanzee adenovirus E1 deletion vectors. Cell lines constructed using essentially the same procedure to express one or more other chimpanzee adenovirus gene products are useful in the production of recombinant chimpanzee adenovirus vectors with deletions in the genes encoding these products. Furthermore, cell lines expressing other human AdE1 gene products are also useful in the production of recombinant chimpanzee AdE1.

[0297] VE3. Recombinant viral particles as vectors The compositions disclosed herein may include a viral vector for delivering at least one antigen to a cell. Such a vector comprises a chimpanzee adenovirus DNA sequence, such as C68, and an antigen cassette functionally linked to a regulatory sequence for direct expression of the cassette. The C68 vector is capable of expressing the cassette in infected mammalian cells. The C68 vector may have functional deletions in one or more viral genes. The antigen cassette contains at least one antigen under the control of one or more regulatory sequences, such as a promoter. The chimpanzee virus vector can be supplied with any desired product of the deleted adenovirus gene by an optional helper virus and / or packaging cell line.

[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, so that the gene region is unable to produce one or more functional products of gene expression. Mutations or modifications that may lead to functional deletion include, but are not limited to, nonsense mutations such as the introduction of immature stop codons and the removal of standard and non-standard start codons, mutations that alter mRNA splicing or other transcriptional processing, or combinations thereof. If necessary, the entire gene region may be removed.

[0299] Modifications of nucleic acid sequences that form the vectors disclosed herein, including deletions, insertions, and other mutations, can be generated using standard molecular biological techniques and are within the scope of this specification.

[0300] VE4. Construction of a viral plasmid vector The chimpanzee adenovirus C68 vectors useful in the present invention include recombinant knockout adenoviruses, i.e., chimpanzee adenovirus sequences having a functional deletion in the E1a or E1b gene, and optionally having 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 adenoviruses and / or adeno-associated virus sequences. Allogeneic adenovirus vectors prepared from human adenoviruses are described in publications [see, for example, Kozarsky I and II cited above, and the references cited therein, and U.S. Patent No. 5,240,846].

[0301] In constructing useful chimpanzee adenovirus C68 vectors for delivering antigen cassettes to human (or other mammalian) cells, a wide range of adenovirus nucleic acid sequences can be used in the vector. A vector containing the smallest chimpanzee C68 adenovirus sequence can be used with a helper virus to produce infectious recombinant viral particles. The helper virus provides the basic gene products necessary for the viral infectivity and replication of the smallest chimpanzee adenovirus vector. If only one or more selected deletions of the chimpanzee adenovirus gene are introduced into a viral vector that would be functional without the deletion, the deleted gene product can be supplied in a viral vector production process by growing a virus that gives the deleted gene function in trans within a selected packaging cell line.

[0302] VE5. Recombinant Minimal Adenovirus The minimal chimpanzee Ad C68 virus is a viral particle containing only the cis elements of the adenovirus necessary for replication and virion capsid formation. Specifically, this vector comprises the cis-active 5' and 3' terminal inverted repeats (ITRs) of the adenovirus (which function as the origin of replication) and the native 5' packaging / enhancer domain (which contains the sequences necessary to package the linear Ad genome and the enhancer elements of the E1 promoter). See, for example, International Application WO96 / 13597, which describes the preparation of the “minimal” human Ad vector and the method incorporated herein by reference.

[0303] VE6. Other deficient adenoviruses Recombinant non-replicating adenoviruses may contain sequences greater than or equal to the smallest chimpanzee adenovirus sequence. These other Ad vectors can be characterized by deletions of different parts of the viral gene region, as well as by the use of helper viruses and / or packaging cell lines as needed to form infectious viral particles.

[0304] As one example, a suitable vector can be formed by deleting all or a significant portion of the C68 adenovirus's earliest gene E1a and later early gene E1b, thereby causing them to lose their normal biological function. The non-replicating E1 deletion virus can replicate and produce an infectious virus when grown in chimpanzee adenovirus-transfected complementary cell lines containing functional adenovirus E1a and E1b genes that donate the corresponding gene products in trans. Based on homology to known adenovirus sequences, the resulting recombinant chimpanzee adenovirus is expected to be able to infect many cell types and express antigens, as is the case with human recombinant E1 deletion adenoviruses in the art, but will not be able to replicate in many cells that do not possess chimpanzee E1 region DNA unless the cells are infected with a very high degree of infection multiplicity.

[0305] As another example, all or part of the post-initial gene E3 of the C68 adenovirus can be removed from the chimpanzee adenovirus sequence that forms part of the recombinant virus.

[0306] The chimpanzee adenovirus C68 vector can also be constructed to have a deletion in the E4 gene. Yet another vector can have a deletion in the post-early gene E2a.

[0307] The deletion can be introduced into any of the late-stage genes L1–L5 of the chimpanzee C68 adenovirus genome. Similarly, deletions within the mid-stage genes IX and IVa2 may be useful for specific purposes. Other deletions can also be introduced into other structural or non-structural adenovirus genes.

[0308] The deletions described above can also be used individually. That is, an adenovirus sequence may have only an E1 deletion. Alternatively, any combination of whole genes or parts thereof that are effective in disrupting or reducing their biological activity can be used. For example, in one exemplary vector, the adenovirus C68 sequence may have deletions of the E1 and E4 genes, or E1, E2a, and E3 genes, or E1 and E3 genes, or E1, E2a, and E4 genes with or without an E3 deletion. As described above, such deletions can be used in combination with other mutations, such as temperature-sensitive mutations, to obtain the desired result.

[0309] A cassette containing antigen(s) can be optionally inserted into any deletion region of the chimpanzee C68Ad virus. Furthermore, if necessary, the cassette can be inserted into an existing gene region to disrupt its function.

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

[0311] Useful helper viruses include selected adenovirus gene sequences that are not present in the adenovirus vector construct and / or not expressed by the packaging cell line transfected with the vector. Helper viruses may be non-replicating and may contain a variety of adenovirus genes other than those described above. Helper viruses can be used in combination with the E1-expressing cell lines described herein.

[0312] In C68, the "helper" virus can be formed as a fragment by shortening the C-terminus of the C68 genome using SspI, which removes approximately 1300 bp from the left end of the virus. Next, by co-transfecting this shortened virus with plasmid DNA into an E1-expressing cell line, recombinant viruses are formed through homologous recombination with the C68 sequence in the plasmid.

[0313] Helper viruses can also be formed as polycation complexes, as described in Wu et al, J. Biol. Chem., 264:16985-16987 (1989); K.J. Fisher and J.M. Wilson, Biochem. J., 299:49 (Apr. 1, 1994). Helper viruses may optionally contain a reporter gene. Many such reporter genes are known in the art. The presence of a reporter gene on the helper virus, distinct from the antigen cassette on the adenovirus vector, makes it possible to observe the Ad vector and the helper virus independently. Using this second reporter gene, it is possible to separate the recombinant virus obtained during purification from the helper virus.

[0314] VE8. Assembly of viral particles and infection of cell lines The assembly of selected DNA sequences of adenoviruses, antigen cassettes, and other vector factors into various intermediate plasmids and shuttle vectors, as well as the use of plasmids and shuttle vectors to produce recombinant viral particles, can all be achieved using conventional methods. Such methods include conventional cDNA cloning, in vitro recombination (e.g., Gibson assembly), the use of redundant oligonucleotide sequences of the adenovirus genome, polymerase chain reaction, and any suitable method for obtaining the desired nucleotide sequence. Standard transfection and co-transfection methods, such as CaPO4 precipitation or liposome-mediated transfection methods like lipofectamine, are employed. Other conventional methods used include homologous recombination of viral genomes, viral plaque formation in agar overlays, and signal generation assays.

[0315] For example, after constructing and assembling a viral vector containing the desired antigen, the vector can be transfected in vitro into a packaging cell line in the presence of a helper virus. Homologous recombination occurs between the helper and the vector sequence, thereby replicating the adenovirus-antigen sequence within the vector and packaging it within a virion capsid, resulting in recombinant viral vector particles.

[0316] The resulting recombinant chimpanzee C68 adenovirus is useful for introducing antigen cassettes into selected cells. In in vivo experiments using recombinant viruses grown in packaging cell lines, the E1 deletion recombinant chimpanzee adenovirus has been demonstrated to be practical for introducing cassettes into non-chimpanzee cells, preferably human cells.

[0317] VE9. Use of Recombinant Viral Vectors Therefore, the resulting recombinant chimpanzee C68 adenovirus containing an antigen cassette (produced, as described above, by the cooperation of an adenovirus vector and a helper virus, or by the cooperation of an adenovirus vector and a packaging cell line) provides an efficient gene delivery carrier that can deliver antigen(s) in vivo or ex vivo to a target.

[0318] The recombinant vectors described above are administered to humans in accordance with publicly available methods for gene therapy. Chimpanzee virus vectors with antigen cassettes can preferably be administered to patients suspended in a biocompatible solution or a pharmaceutically acceptable delivery solvent. Suitable solvents include sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions, as well as aqueous and non-aqueous sterile suspensions, known as pharmaceutically acceptable carriers and well known to those skilled in the art, can also be used for this purpose.

[0319] Chimpanzee adenovirus vectors are administered in amounts sufficient to transform human cells and produce a therapeutic effect by introducing and expressing an antigen at a level sufficient to deliver a therapeutic effect, without adverse effects that can be determined by those skilled in the medical field, or with medically acceptable physiological effects. Conventional pharmaceutically acceptable routes of administration include, but are not limited to, hepatic, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parenteral routes of administration. Routes of administration may be combined as needed.

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

[0321] Recombinant non-replicating adenoviruses can be administered in a "pharmaceutically effective dose," that is, a quantity of recombinant adenovirus effective via a given route of administration to transfect the desired cells and give sufficient expression levels of selected genes that result in a vaccine effect, i.e., a certain measurable level of protective immunity. The C68 vector containing the antigen can be administered co-administered with an adjuvant. The adjuvant may be separate from the vector (e.g., alum) or may be encoded within the vector, especially if the adjuvant is a protein. Adjuvants are well known in the art.

[0322] Conventional pharmacovigilant routes of administration, though not limited to these, include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral, and other parenteral routes. The routes of administration can be combined as needed or modified according to the immunogen or disease. For example, in 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 levels of the antigen(s), the need for a booster (if any) can be determined. For example, after evaluating the antibody titer in serum, a booster immunization may be desirable if necessary.

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

[0325] In some embodiments, subjects are diagnosed with cancer or are at risk of developing cancer. Subjects may be humans, dogs, cats, horses, or any animal for which a tumor-specific immune response is desirable. Tumors may be any solid tumors, such as tumors of the mammary gland, ovaries, prostate, lungs, kidneys, stomach, colon, testes, head and neck, pancreas, brain, melanoma, and other tissue organs, as well as hematological malignancies, 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 a sufficient amount to induce a CTL response.

[0327] Antigens can be administered alone or in combination with other therapeutic substances. These therapeutic substances may include, for example, chemotherapy agents, radiation, or immunotherapy. Any suitable therapeutic treatment for a particular cancer may be administered.

[0328] In addition, the subjects may be further administered immunosuppressive / immunostimulatory substances such as checkpoint inhibitors. For example, the subjects may be further administered anti-CTLA antibodies or anti-PD-1 or anti-PD-L1 antibodies. Blocking CTLA-4 or PD-L1 with antibodies can enhance the immune response against cancer cells in patients. In particular, CTLA-4 blockade has been shown to be effective when a vaccination protocol is adopted.

[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, and 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 the vaccine composition 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 specific to the tissue, cancer, and / or patient. For example, the precise selection of peptides may be guided by the expression pattern of the parent protein in a given tissue or by the patient's mutational status. The selection may depend on the specific type of cancer, the disease state, earlier treatment regimens, the patient's immune status, and, of course, the patient's HLA halotype. Furthermore, vaccines may contain individualized components according to the personal needs of a particular patient. Examples include altering the selection of antigens according to the expression of antigens in a particular patient, or making adjustments for secondary treatments after the first round or scheme of treatment.

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

[0332] With respect to compositions to be used as vaccines for cancer, antigens having similar normal self-peptides that are expressed in large quantities in normal tissues may be avoided or present in small quantities in the compositions described herein. On the other hand, if it is known that a patient's tumor expresses a particular antigen in large quantities, each pharmaceutical composition for the treatment of this cancer may be present in large quantities and / or may contain more than one antigen specific to this particular antigen or the pathway of this antigen.

[0333] Compositions containing antigens can be administered to individuals already suffering from cancer. In therapeutic applications, the composition is administered to the patient in an amount sufficient to induce an effective CTL response to the tumor antigen and to cure or at least partially cessate the symptoms and / or complications. The amount appropriate to achieve this is defined as the "therapeutic effective dose." The effective dose 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 overall health condition, and the judgment of the prescribing physician. It should be kept in mind that the composition is generally not for use in serious disease conditions, i.e., life-threatening or potentially life-threatening situations, especially when the cancer has metastasized. In such cases, given the minimization of exogenous substances and the relatively non-toxic nature of the antigen, it may be possible and desirable for the treating physician to administer substantially excessive amounts of these compositions.

[0334] For therapeutic use, administration can be initiated at the time of tumor detection or surgical removal. This is followed by a boost dose until the symptoms have substantially subsided, and then 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 may be administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. Compositions may be administered to the site of surgical excision to induce a local immune response against a tumor. Compositions for parenteral administration comprising a solution of an antigen are disclosed herein, and the vaccine composition is dissolved or suspended in an acceptable carrier, such as an aqueous carrier. Various aqueous carriers can be used, such as water, buffer water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized by conventional, well-known sterilization techniques or by sterile filtration. The resulting aqueous solutions can be packaged for use as is or lyophilized, and the lyophilized preparations are combined with a sterile solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters, buffers, isotonic agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0336] Antigens can also be administered via liposomes, targeting them to specific cell tissues such as lymphoid tissue. Liposomes are also useful for increasing half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. In these preparations, the antigen to be delivered is incorporated either alone or as part of a liposome, together with a molecule that binds to a receptor dominant among lymphoid cells, such as a monoclonal antibody that binds to the CD45 antigen, or other therapeutic or immunogenic compositions. Thus, liposomes filled with the desired antigen can be directed to a site on lymphoid cells, where the liposome then delivers 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. Lipid selection is generally guided by considerations such as liposome size, acid instability, and liposome stability in the bloodstream. For example, various methods are available for preparing liposomes, as described in Szoka et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Patents No. 4,235,871, No. 4,501,728, No. 4,501,728, No. 4,837,028, and No. 5,019,369.

[0337] For targeting immune cells,...

Claims

1. A composition for delivering an antigen expression system, which includes an antigen expression system, The antigen expression system comprises one or more vectors, The composition comprises one or more vectors, including the following: (a) A vector skeleton, (i) at least one promoter nucleotide sequence, (ii) at least one polyadenylated (poly(A)) sequence and The vector skeleton, and (b) Antigen cassette, (i) at least one antigen-coding nucleic acid sequence, (I) at least one tumor-specific antigen-coding nucleic acid sequence, (A) MHC class I epitope coding nucleic acid sequences encoding an EGFR_L858R MHC class I epitope selected from the group consisting of SEQ ID NOs: 14915, 23759, 14913, 23758, 13816, 14183, 14729, 14730, 14731, 14732, 14733, 23760, 23753, 23755, 23756, 23757, and 23754. The antigen cassette, including the antigen cassette.

2. The composition according to claim 1, wherein the MHC class I epitope coding nucleic acid sequence codes for at least two EGFR_L858R MHC class I epitopes selected from the group consisting of SEQ ID NOs: 14915, 23759, 14913, 23758, 13816, 14183, 14729, 14730, 14731, 14732, 14733, 23760, 23753, 23755, 23756, 23757, and 23754.

3. The composition according to claim 1, wherein the MHC class I epitope coding nucleic acid sequence codes for an EGFR_L858R MHC class I epitope selected from the group consisting of SEQ ID NOs: 14915, 14913, 13816, 14183, 14729, 14730, 14731, 14732, and 14733.

4. The composition according to claim 1, wherein the MHC class I epitope coding nucleic acid sequence codes for an EGFR_L858R MHC class I epitope selected from the group consisting of SEQ ID NOs: 23753, 23754, 23755, 23756, 23757, 23758, 23759, and 23760.

5. The composition according to claim 1, wherein the MHC class I epitope coding nucleic acid sequence codes for the EGFR_L858R MHC class I epitope of sequence number 14915 or sequence number 14913.

6. 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 explained by an expression that includes, During the ceremony, P comprises the promoter nucleotide sequence, where a = 0 or 1. N includes one of the MHC class I epitope coding nucleic acid sequences, where c = 1. L5 contains the 5' linker sequence, where b = 0 or 1. L3 contains the 3' linker sequence, where d = 0 or 1. G5 contains a nucleic acid sequence encoding the GPPGG amino acid linker (SEQ ID NO: 56), where e = 0 or 1. G3 contains a nucleic acid sequence encoding the GPPGG amino acid linker (SEQ ID NO: 56), where g = 0 or 1. U contains at least one MHC class II antigen-coding nucleic acid sequence, where f = 1. X = 1 to 400, and here, for each X, the corresponding N ｃ This is an epitope-coding nucleic acid sequence, Y = 0, 1, or 2, where for each Y, the corresponding U ｆ This is an antigen-coding nucleic acid sequence. The composition according to any one of claims 1 to 5.

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

8. 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 skeleton, The at least one polyadenylated (poly(A)) sequence is a poly(A) sequence of at least 100 consecutive A nucleotides provided by the vector skeleton (SEQ ID NO: 29358), Each N encodes an MHC class I epitope with a length of 7 to 15 amino acids. L5 is a natural 5' linker sequence that encodes the natural N-terminal amino acid sequence of the MHC class I epitope, and the 5' linker sequence encodes a peptide that is at least two amino acids long. L3 is a natural 3' linker sequence that codes for the natural terminal nucleic acid sequence of the MHC class I epitope, and the 3' linker sequence codes for a peptide that is at least two amino acids long. U is the PADRE class II sequence and the tetanus toxoid MHC class II sequence, respectively. The vector skeleton comprises a chimpanzee adenovirus vector or an alphavirus vector. Each of the tumor-specific antigen-coding nucleic acid sequences encodes a polypeptide with a length of 13 to 25 amino acids. The composition according to claim 6 or 7.

9. A composition according to any one of claims 1 to 8, further comprising a nanoparticle-shaped delivery vehicle, The aforementioned nanoparticle-like delivery vehicle is lipid nanoparticles (LNPs). composition.

10. The one or more vectors include one or more +-strand RNA vectors; and / or The one or more vectors self-replicate within mammalian cells; and / or The vector skeleton comprises at least one nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semliki forest virus, Sindbis virus, or Mayarovirus. (a) The vector skeleton comprises at least a sequence for non-structural protein-mediated amplification, a 26S promoter sequence, and a poly(A) sequence encoded by the nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semryqui forest virus, Sindbis virus, or Mayarovirus, The sequence for non-structural protein-mediated amplification is selected from the group consisting of alphavirus 5'UTR, 51nt CSE, 24nt CSE, 26S subgenomic promoter sequence, 19nt CSE, alphavirus 3'UTR, or a combination thereof; or (b) The Venezuelan encephalitis virus, The sequence described in Sequence ID No. 3 or Sequence ID No. 5, or the sequence described in Sequence ID No. 3 or Sequence 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 pair 7544 and 11175 as described in the sequence of sequence number 3 or sequence number 5. The composition according to any one of claims 1 to 7 or 9.

11. The composition according to any one of claims 1 to 7, 9, or 10, wherein the vector skeleton comprises at least one nucleotide sequence of a chimpanzee adenovirus vector, and the chimpanzee adenovirus vector is a ChAdV68 vector.

12. 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 aforementioned at least one antigen-coding nucleic acid sequences encodes a polypeptide sequence presented by MHC class I on tumor cells. The composition according to any one of claims 1 to 11.

13. The at least one antigen-coding nucleic acid sequence is Containing at least 2 to 10, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 to 20, 15 to 20, 11 to 100, 11 to 200, 11 to 300, 11 to 400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 nucleic acid sequences; and / or Each tumor-specific antigen-coding nucleic acid sequence encodes a polypeptide sequence of length 8 to 35 amino acids, 9 to 17 amino acids, 9 to 25 amino acids, 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. The composition according to any one of claims 1 to 7 or 9 to 12.

14. (i) The antigen cassette includes a junctional epitope sequence formed by adjacent sequences within the antigen cassette, and at least one, or each junctional epitope sequence, has an affinity greater than 500 nM for MHC; 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 containing a post-translational wild-type nucleic acid sequence, and the non-therapeutic MHC class I or class II epitope is expected to be presented on the target MHC allele, The composition according to any one of claims 1 to 13.

15. A pharmaceutical composition comprising the composition according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. An isolated nucleotide or set of isolated nucleotides comprising an antigen cassette of the composition according to any one of claims 1 to 14 and one or more elements obtained 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 necessary for non-structural protein-mediated amplification, 26S promoter nucleotide sequences, poly(A) sequences, and nsP1-4 genes of sequences described in SEQ ID NO: 3 or SEQ ID NO:

5. The isolated nucleotide or set of isolated nucleotides includes an antigen cassette. The aforementioned isolated nucleotide or set of isolated nucleotides.

17. A vector or set of vectors comprising the nucleotides described in claim 16.

18. Isolated cells comprising an isolated nucleotide or a set of isolated nucleotides as described in claim 16, or a vector or a set of vectors as described in claim 17.

19. A kit comprising the composition according to any one of claims 1 to 14 and instructions for use.

20. A pharmaceutical composition comprising the composition according to any one of claims 1 to 14, or the pharmaceutical composition according to claim 15, for treating a subject having cancer.

21. The pharmaceutical composition according to claim 20, wherein the subject expresses at least one HLA allele that is predicted or known to present an EGFR_L858R MHC class I epitope selected from the group consisting of SEQ ID NOs: 14915, 23759, 14913, 23758, 13816, 14183, 14729, 14730, 14731, 14732, 14733, 23760, 23753, 23755, 23756, 23757, and 23754.

22. The pharmaceutical composition according to claim 20 or 21, to be used in combination with a second vaccine composition.

23. The pharmaceutical composition according to claim 22, wherein the second vaccine composition is the same as the pharmaceutical composition.

24. A method for producing one or more vectors according to claim 17, (a) To obtain a linearized DNA sequence including the vector skeleton and the antigen cassette, (b) Transcribing the linearized DNA sequence in vitro by adding it to an in vitro transcription reaction containing all the components necessary to transcribe the linearized DNA sequence into RNA, (c) Isolating one or more vectors from the in vitro transcription reaction. including, The aforementioned method.

25. A method for evaluating subjects with cancer, a) 1) Whether the subject has an HLA allele that is predicted or known to present an antigen included in the antigen-based vaccine, and below: 1) Whether the target tumor expresses the gene related to the antigen, 2) Whether the target tumor has a mutation related to the antigen. one or both The process of determining, or already determining, b) A step in which, based on the results of (a) above, the subject expresses the HLA allele, the tumor of the subject expresses the gene, and / or the tumor of the subject has the mutation, the subject is determined to be a candidate for treatment with the antigen-based vaccine, or has already been determined to be so The antigen comprises at least one EGFR_L858R MHC class I epitope selected from the group consisting of SEQ ID NOs: 14915, 23759, 14913, 23758, 13816, 14183, 14729, 14730, 14731, 14732, 14733, 23760, 23753, 23755, 23756, 23757, and 23754. The aforementioned process and The method, including the method described above.

26. a) The subject is, At least one EGFR_L858R MHC class I epitope selected from the group consisting of SEQ ID NOs: 14915, 23759, 14913, 23758, 13816, 14183, 14729, 14730, 14731, 14732, 14733, 23760, 23753, 23755, 23756, 23757, and 23754, and HLA alleles that are predicted or known to present at least one MHC class I epitope A process of determining whether or not the expression occurs, b) From the results of (a) above, the subject is 1) Expressing at least one EGFR_L858R MHC class I epitope, 2) Expressing the HLA allele that is predicted or known to present at least one EGFR_L858R MHC class I epitope In this case, the process involves determining, or having already determined, that the subject is a candidate for treatment with the antigen-based vaccine. The method according to claim 25, including the method described in claim 25.

27. The composition according to any one of claims 1 to 14, wherein the at least one tumor-specific antigen-coding 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-coding nucleic acid sequence is linked to different MHC class I epitope-coding nucleic acid sequences by the 5' linker sequence and / or the 3' linker sequence.

28. The composition according to claim 27, wherein the 5' linker sequence and / or 3' linker sequence comprises one or more natural sequences adjacent to an antigen derived from a 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 to 20 amino acid residues.

29. The at least one promoter nucleotide sequence is a native 26S promoter nucleotide sequence encoded by an RNA alphavirus skeleton, or is an exogenous RNA promoter; and / or The antigen cassette comprises a second promoter nucleotide sequence, The second promoter nucleotide sequence comprises one 26S promoter nucleotide sequence or multiple 26S promoter nucleotide sequences. The composition according to any one of claims 1 to 14, wherein each 26S promoter nucleotide sequence results in the transcription of one or more separate open reading frames.

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