Methods for homologous adenovirus vaccination

Abstract

Disclosed are nucleotides, cells, and methods relating to compositions including those for use as vaccines, including vectors and methods for allogeneic prime / boost vaccination strategies, such as adenoviral vectors for use in allogeneic prime / boost vaccination strategies.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 121,164, filed on 3 December 2020, which is incorporated in its entirety by reference for any purpose.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The above ASCII copy, created on 3 December 2020, is named GSO-096_Sequence_Listing.txt and has a size of 6,925,547 bytes. [Background technology]

[0003] background Therapeutic vaccines based on tumor-specific antigens show great promise 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 attractive targets for such therapies because they have a relatively high potential for neoantigen generation. 4、5 Early evidence suggests that neoantigen-based vaccination can induce a T-cell response. 6 , and that neoantigen-targeted cell therapy may induce tumor regression in certain patients under certain circumstances. 7 This is shown.

[0004] In both cancer and infectious disease situations, one of the challenges in antigen vaccine design is determining which of the many existing coding mutations will produce the "best" therapeutic antigen, such as an antigen that can induce immunity.

[0005] In addition to the challenges of current antigen prediction methods, there are also specific challenges regarding available vector systems, many of which are human-derived and can be used for antigen delivery in humans. 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 for antigen delivery in vaccination strategies, such as cancer treatment or vaccination against infectious diseases. While some progress has been made in vaccination strategies to address the above issues, improvements are still needed, particularly for clinical application, such as improvements in vaccine efficacy and effectiveness. [Overview of the project]

[0006] overview This specification discloses a method for delivering a composition comprising a chimpanzee adenovirus (ChAdV) vector to a target, the method comprising administering multiple doses of the composition to a target, wherein the multiple doses comprise at least an initial dose and a second dose, and the period between the initial dose and the second dose is at least 27 weeks.

[0007] Furthermore, this specification discloses a method for delivering a composition comprising a chimpanzee adenovirus (ChAdV) vector to a target, the method comprising administering multiple doses of the composition to a target, wherein the multiple doses include at least an initial dose and a second dose, and it is determined that the ChAdV-specific neutralizing antibody titer is below the neutralization threshold before the administration of the second dose.

[0008] Furthermore, this specification also discloses a method for delivering a composition containing a chimpanzee adenovirus (ChAdV) vector to a target, comprising: (a) administering an initial dose of the composition to the target; (b) determining or having determined a ChAdV-specific neutralizing antibody titer; and (c) if it is determined that the ChAdV-specific neutralizing antibody titer is below the neutralization threshold, administering a second dose of the composition to the target.

[0009] In some embodiments, the period between the first dose and the second dose is at least 27 weeks, at least 28 weeks, at least 29 weeks, at least 30 weeks, at least 31 weeks, or at least 32 weeks.

[0010] In some embodiments, the initial dose is a priming dose.

[0011] In some embodiments, the multiple doses include three or more doses. In some embodiments, one or more of the multiple doses are administered before the initial dose.

[0012] In some embodiments, no additional dose of the composition is administered between the first and second doses.

[0013] In some embodiments, the neutralizing antibody titer is an NT50 value calculated as the minimum dilution of serum from an immunized subject that neutralizes the ChAdV virus by 50%. In some embodiments, the neutralization threshold is an NT50 value of 900 or less. In some embodiments, determining the neutralizing antibody titer includes the steps of (1) contacting one or more dilutions of serum from an immunized subject with the ChAdV virus under conditions sufficient to neutralize the ChAdV virus, and (2) evaluating the neutralization of the ChAdV virus compared to an unneutralized virus. In some embodiments, the step of evaluating neutralization includes evaluating the expression of a reporter construct expressed by the ChAdV virus.

[0014] In some embodiments, the neutralization threshold is the minimum neutralizing antibody titer required for complete neutralization of the ChAdV virus at the second dose. In some embodiments, the neutralization threshold is the minimum neutralizing antibody titer at which the second dose induces an immune response in the subject.

[0015] In some embodiments, the ChAdV vector encodes at least one antigen. In some embodiments, the at least one antigen is a non-self peptide, which is not encoded by the wild-type gene of the subject. In some embodiments, the at least one antigen is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is a neoantigen.

[0016] In some embodiments, at least one antigen is an exogenous antigen. In some embodiments, the exogenous antigen is from a pathogen, virus, bacterium, fungus, or parasite.

[0017] In some embodiments, each of the multiple doses contains the same antigen.

[0018] In some embodiments, the composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). In some embodiments, the composition is administered (IM). In some embodiments, IM administration is performed at separate injection sites. In some embodiments, the separate injection sites are injection sites in opposing deltoid muscles. In some embodiments, the separate injection sites are injection sites in bilateral gluteal muscles or rectus femoris muscles.

[0019] In some embodiments, the method does not involve the administration of an immunomodulator, or the method is carried out in the absence of an immunomodulator, and optionally the immunomodulator is a checkpoint inhibitor.

[0020] In some embodiments, the method further comprises administering 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.

[0021] In some embodiments, the method further includes determining or predetermining the HLA haplotype of the subject.

[0022] In some embodiments, the ChAdV vector comprises (a) a ChAdV skeleton comprising (i) at least one promoter nucleotide sequence and (ii) at least one polyadenylated (poly(A)) sequence, and (b) a cassette comprising (i) at least one antigen-coding nucleic acid sequence comprising a. an epitope-coding nucleic acid sequence which optionally comprises at least one change that makes the encoded epitope sequence different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence, b. optionally a 5' linker sequence, and c. optionally a 3' linker sequence, wherein the cassette is functionally linked to at least one promoter nucleotide sequence and at least one poly(A) sequence.

[0023] In some embodiments, the ChAdV vector comprises (a) a ChAdV skeleton, (i) a modified ChAdV68 sequence comprising at least nucleotides 2-36,518 of the sequence described in SEQ ID NO: 1, wherein nucleotides 2-36,518 lack (1) nucleotides 577-3403 of the sequence shown in SEQ ID NO: 1, corresponding to an E1 deletion, (2) nucleotides 27,125-31,825 of the sequence shown in SEQ ID NO: 1, corresponding to an E3 deletion, and (3) nucleotides 34,916-35,642 of the sequence shown in SEQ ID NO: 1, corresponding to a partial E4 deletion, (ii) a CMV promoter nucleotide sequence, and (iii) SV40 polyadenylation (Poly(A (b) a ChAdV skeleton comprising a sequence, and (b) a cassette comprising (i) at least one antigen-coding nucleic acid sequence comprising a. an epitope-coding nucleic acid sequence which optionally comprises at least one change that makes the encoded epitope sequence different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence, b. optionally a 5' linker sequence, and c. optionally a 3' linker sequence, wherein the cassette is inserted into the E1 deletion and the cassette is functionally linked to the CMV promoter nucleotide sequence and the SV40 poly(A) sequence.

[0024] In some embodiments, the epitope-encoding nucleic acid sequence encodes an epitope known or suspected to be presented by MHC class I and / or MHC class II on the surface of a cell, and optionally, the surface of the cell is the surface of a tumor cell or an infected cell, and optionally, the cell is a cell of a subject. In some embodiments, at least one antigen-encoding nucleic acid sequence encodes a polypeptide sequence capable of undergoing antigen processing to an epitope, and optionally, the epitope is known or suspected to be presented by MHC class I on the surface of a cell, and optionally, the surface of the cell is the surface of a tumor cell or an infected cell. In some embodiments, the cell is a tumor cell 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 tumor, B cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer, or the cell is an infected cell selected from the group consisting of pathogen-infected cells, virus-infected cells, bacteria-infected cells, fungus-infected cells, and parasite-infected cells.

[0025] In some embodiments, the virus-infected cell is an HIV-infected cell. In some embodiments, the epitope-encoding nucleic acid sequence encodes an HIV GAG protein or an epitope.

[0026] In some embodiments, the sequences of the elements of the cassette in the ChAdV vector arranged in order are, in the 5' to 3' direction, as follows: P a -(L5 b -N c -L3 d ) X -(G5 e -U f ) Y -G3 g represented by the formula comprising In the formula, P comprises at least one promoter sequence functionally linked to at least one of at least one antigen-coding nucleic acid sequences, a=1; N comprises one of the epitope-coding nucleic acid sequences, c=1; L5 comprises a 5' linker sequence, b=0 or 1; L3 comprises a 3' linker sequence, d=0 or 1; G5 comprises one of at least one nucleic acid sequences encoding a GPGPG amino acid linker, e=0 or 1; G3 comprises one of at least one nucleic acid sequences encoding a GPGPG amino acid linker, g=0 or 1; U comprises one of at least one MHC class II epitope-coding nucleic acid sequences, f=1; X=1 to 400, and for each X, the corresponding N c is an epitope-coding nucleic acid sequence, where Y = 0, 1, or 2, and for each Y, the corresponding U f This is an MHC class II epitope-coding nucleic acid sequence.

[0027] In some embodiments, for each X, the corresponding N c is a separate epitope-coding nucleic acid sequence. In some embodiments, for each Y, the corresponding U f These are separate MHC class II epitope-coding nucleic acid sequences.

[0028] In some embodiments, b=1, d=1, e=1, g=1, h=1, X=10, Y=2, P is the CMV promoter sequence, each N codes for an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, with a length of 7-15 amino acids, L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of the epitope, where the 5' linker sequence codes for a peptide of at least 3 amino acids, L3 is a natural 3' linker sequence encoding the natural C-terminal amino acid sequence of the epitope, where the 3' linker sequence codes for a peptide of at least 3 amino acids, and U is the PADRE class II sequence Each of the tetanus toxoid MHC class II sequences is a modified ChAdV vector comprising a modified ChAdV68 sequence containing at least nucleotides 2-36,518 of the sequence described in SEQ ID NO: 1, where nucleotides 2-36,518 lack (1) nucleotides 577-3403 of the sequence shown in SEQ ID NO: 1, corresponding to the E1 deletion; (2) nucleotides 27,125-31,825 of the sequence shown in SEQ ID NO: 1, corresponding to the E3 deletion; and (3) nucleotides 34,916-35,642 of the sequence shown in SEQ ID NO: 1, corresponding to the partial E4 deletion. The neoantigen cassette is inserted within the E1 deletion, and each of the I antigen-coding nucleic acid sequences encodes a polypeptide with a length of 25 amino acids.

[0029] In some embodiments, the 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 ligated to the cassette.

[0030] In some embodiments, the ChAdV skeleton includes the ChAdV68 vector skeleton. In some embodiments, the ChAdV68 vector skeleton includes the sequence described in Sequence ID No. 1. In some embodiments, the ChAdV68 vector backbone includes a functional deletion in at least one gene selected from the group consisting of adenovirus E1A, E1B, E2A, E2B, E3, L1, L2, L3, L4, and L5 genes with respect to the ChAdV68 genome or the sequence shown in SEQ ID NO: 1, and optionally, the adenovirus backbone or modified ChAdV68 sequence is completely deleted or functionally deleted in (1) E1A and E1B, or (2) E1A, E1B, and E3 with respect to the adenovirus genome or the sequence shown in SEQ ID NO: 1, and optionally, the E1 gene is functionally deleted via an E1 deletion of at least nucleotides 577-3403 with respect to the sequence shown in SEQ ID NO: 1, and optionally, the E3 gene is functionally deleted via an E3 deletion of at least nucleotides 27,125-31,825 with respect to the sequence shown in SEQ ID NO: 1. In some embodiments, the ChAdV68 vector backbone comprises one or more genes or regulatory sequences with respect to the ChAdV68 genome or the sequence shown in Sequence ID No. 1, and optionally, one or more genes or regulatory sequences are selected from the group consisting of chimpanzee adenovirus terminal inverted repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes.

[0031] In some embodiments, the ChAdV68 vector backbone includes a partially deleted E4 gene. In some embodiments, the partially deleted E4 gene is: A. an E4 gene sequence shown in SEQ ID NO: 1 and lacking at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1; B. an E4 gene sequence shown in SEQ ID NO: 1 and lacking at least nucleotides 34,916 to 34,942, nucleotides 34,952 to 35,305, and nucleotides 35,302 to 35,642 of the sequence shown in SEQ ID NO: 1, wherein the vector includes at least nucleotides 2 to 36,518 of the sequence shown in SEQ ID NO: 1; C. an E4 gene sequence shown in SEQ ID NO: 1 and lacking at least nucleotides 34,980 to 36,516 of the sequence shown in SEQ ID NO: 1, wherein the vector includes at least nucleotides 2 to 36 The vector includes an E4 gene sequence containing ,518, D. an E4 gene sequence shown in SEQ ID NO: 1 and lacking at least nucleotides 34,979 to 35,642 of the sequence shown in SEQ ID NO: 1, and the vector includes an E4 gene sequence containing at least nucleotides 2 to 36,518 of the sequence shown in SEQ ID NO: 1, E. an E4 deletion which is at least a partial deletion of E4Orf2, a complete deletion of E4Orf3, and at least a partial deletion of E4Orf4, F. an E4 deletion which is at least a partial deletion of E4Orf2, at least a partial deletion of E4Orf3, and at least a partial deletion of E4Orf4, G. an E4 deletion which is at least a partial deletion of E4Orf1, a complete deletion of E4Orf2, and at least a partial deletion of E4Orf3, or H. an E4 deletion which is at least a partial deletion of E4Orf2 and at least a partial deletion of E4Orf3.

[0032] In some embodiments, the ChAdV68 vector skeleton comprises at least nucleotides 2-36,518 of the sequence described in SEQ ID NO: 1, wherein nucleotides 2-36,518 lack (1) nucleotides 577-3403 of the sequence shown in SEQ ID NO: 1, corresponding to the E1 deletion; (2) nucleotides 27,125-31,825 of the sequence shown in SEQ ID NO: 1, corresponding to the E3 deletion; and (3) nucleotides 34,916-35,642 of the sequence shown in SEQ ID NO: 1, corresponding to the partial E4 deletion, and optionally an antigen cassette is inserted into the E1 deletion. In some embodiments, the ChAdV68 vector skeleton comprises the sequence described in SEQ ID NO: 29369, and optionally an antigen cassette is inserted into the E1 deletion. In some embodiments, the ChAdV68 vector skeleton includes at least nucleotides 2-36,518 of the sequence described in SEQ ID NO: 1, wherein nucleotides 2-36,518 lack A. nucleotides 577-3403 of the sequence shown in SEQ ID NO: 1, corresponding to the E1 deletion; B. nucleotides 27,125-31,825 of the sequence shown in SEQ ID NO: 1, corresponding to the E3 deletion; C. nucleotides 34,916-35,642 of the sequence shown in SEQ ID NO: 1, corresponding to the partial E4 deletion; D. nucleotides 456-3014 of the sequence shown in SEQ ID NO: 1; E. nucleotides 27,816-31,333 of the sequence shown in SEQ ID NO: 1; F. nucleotides 3957-10346 of the sequence shown in SEQ ID NO: 1; G. nucleotides 21787-23370 of the sequence shown in SEQ ID NO: 1; H. nucleotides 33486-36193 of the sequence shown in SEQ ID NO: 1; or a combination thereof.

[0033] In some embodiments, the ChAdV68 vector skeleton comprises at least nucleotides 2-36,518 of the sequence described in SEQ ID NO: 1, wherein nucleotides 2-36,518 lack (1) nucleotides 577-3403 of the sequence shown in SEQ ID NO: 1, corresponding to the E1 deletion, and (2) nucleotides 27,125-31,825 of the sequence shown in SEQ ID NO: 1, corresponding to the E3 deletion.

[0034] In some embodiments, the cassette is inserted into the ChAdV skeleton in the E1 region, in the E3 region, and / or in any missing AdV region that enables the incorporation of the cassette.

[0035] In some embodiments, the ChAdV backbone is constructed from one of a first-generation, second-generation, or helper-dependent adenovirus vector. In some embodiments, at least one promoter nucleotide sequence is selected from the group consisting of CMV promoter sequences, SV40 promoter sequences, EF-1 promoter sequences, RSV promoter sequences, PGK promoter sequences, HSA promoter sequences, MCK promoter sequences, and EBV promoter sequences. In some embodiments, at least one promoter nucleotide sequence is a CMV promoter sequence.

[0036] In some embodiments, at least one of the epitope-coding nucleic acid sequences encodes an epitope that, when expressed and translated, can be presented on the target cell by MHC class I. In some embodiments, at least one of the epitope-coding nucleic acid sequences encodes an epitope that, when expressed and translated, can be presented on the target cell by MHC class II.

[0037] In some embodiments, at least one antigen-coding nucleic acid sequence comprises two or more antigen-coding nucleic acid sequences. In some embodiments, each antigen-coding nucleic acid sequence is directly concatenated with respect to another.

[0038] In some embodiments, each antigen-coding nucleic acid sequence is linked to a separate antigen-coding nucleic acid sequence by a nucleic acid sequence encoding a linker. In some embodiments, the linker links two epitope-coding nucleic acid sequences or links an epitope-coding nucleic acid sequence to an MHC class II epitope-coding nucleic acid sequence. In some embodiments, the linker is selected from the group consisting of (1) consecutive glycine residues having a length of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues, (2) consecutive alanine residues having 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 adjacent to an antigen derived from a cognate protein and having a length of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2–20 amino acid residues. In some embodiments, the linker either concatenates two MHC class II epitope-coding nucleic acid sequences or concatenates an MHC class II sequence to an epitope-coding nucleic acid sequence. In some embodiments, the linker comprises the sequence GPGPG.

[0039] In some embodiments, an antigen-coding nucleic acid sequence is functionally or directly ligated to a separate or contiguous sequence that enhances the expression, stability, cell transport, processing and presentation, and / or immunogenicity of the antigen-coding nucleic acid sequence. In some embodiments, the separate or contiguous sequence comprises at least one of the following: a ubiquitin sequence, a ubiquitin sequence modified to enhance proteasome targeting (e.g., the ubiquitin sequence contains 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 optionally, the ubiquitin sequence modified to enhance proteasome targeting is A76.

[0040] In some embodiments, the epitope-coding nucleic acid sequence includes at least one change, which gives the encoded epitope increased binding affinity to its corresponding MHC allele compared to the translated corresponding wild-type nucleic acid sequence. In some embodiments, the epitope-coding nucleic acid sequence includes at least one change, which gives the encoded epitope increased binding stability to its corresponding MHC allele compared to the translated corresponding wild-type nucleic acid sequence. In some embodiments, the epitope-coding nucleic acid sequence includes at least one change, which gives the encoded epitope increased presentation potential on its corresponding MHC allele compared to the translated corresponding wild-type nucleic acid sequence. In some embodiments, the at least one change 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 proteasome-generated splice antigen.

[0041] In some embodiments, the epitope-coding nucleic acid sequence encodes an epitope known or suspected to be expressed in subjects known or suspected to have cancer. In some embodiments, cancer includes solid tumors. In some embodiments, cancer 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 tumor, B-cell lymphoma, acute myeloid leukemia, adult acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0042] In some embodiments, at least one antigen-coding nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen-coding nucleic acid sequences, and optionally each antigen-coding nucleic acid sequence codes for a separate antigen-coding nucleic acid sequence. In some embodiments, 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 antigen-coding nucleic acid sequences, and optionally each antigen-coding nucleic acid sequence codes for a separate antigen-coding nucleic acid sequence. In some embodiments, 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 antigen-coding nucleic acid sequences.

[0043] In some embodiments, at least one antigen-coding nucleic acid sequence comprises at least 2 to 400 antigen-coding nucleic acid sequences, of which at least two encode epitope sequences or portions thereof that are presented on the cell surface by MHC class I. In some embodiments, at least two of the MHC class I epitopes are presented on the tumor cell surface by MHC class I.

[0044] In some embodiments, the epitope-coding nucleic acid sequence comprises at least one MHC class I epitope-coding nucleic acid sequence, each antigen-coding nucleic acid sequence encoding a polypeptide sequence of 8–35 amino acid length, optionally 9–17, 9–25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid length.

[0045] In some embodiments, at least one MHC class II epitope-coding nucleic acid sequence is present.

[0046] In some embodiments, at least one MHC class II epitope-coding nucleic acid sequence comprises at least one MHC class II epitope-coding nucleic acid sequence that is present and includes at least one change, the change causing the encoded epitope sequence to be different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence.

[0047] In some embodiments, the epitope-coding nucleic acid sequence comprises an MHC class II epitope-coding nucleic acid sequence, where each antigen-coding nucleic acid sequence encodes a polypeptide sequence of 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acid lengths. In some embodiments, the epitope-coding nucleic acid sequence comprises an MHC class II epitope-coding nucleic acid sequence, where at least one MHC class II epitope-coding nucleic acid sequence comprises at least one universal MHC class II epitope-coding nucleic acid sequence, and optionally, at least one universal sequence comprises at least one of tetanus toxoid and PADRE.

[0048] In some embodiments, at least one promoter nucleotide sequence is inducible. In some embodiments, at least one promoter nucleotide sequence is non-inducible. In some embodiments, at least one poly(A) sequence contains a bovine growth hormone (BGH) SV40 polyA sequence. In some embodiments, at least one poly(A) sequence consists of 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. In some embodiments, at least one poly(A) sequence consists of at least 100 consecutive A nucleotides.

[0049] In some embodiments, the cassette further comprises at least one of the following: an intron sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence, an intra-sequence ribosome entry sequence (IRES) sequence, a nucleotide sequence encoding a 2A self-cleaving peptide sequence, a nucleotide sequence encoding a furin cleavage site, or a sequence in a 5' or 3' non-coding region that is known to enhance mRNA nuclear export, stability, or translation efficiency and is functionally linked to at least one of at least one antigen-coding nucleic acid sequences. In some embodiments, the cassette further comprises a reporter gene, which comprises, but is not limited to, green fluorescent protein (GFP), a GFP variant, a 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.

[0050] In some embodiments, one or more vectors further include 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) that is single-specific or has multiple specificities linked together (e.g., an antibody domain from a camelid animal), or a full-length single-chain antibody (e.g., a full-length IgG in which the heavy and light chains are linked by a mobile linker). In some embodiments, the heavy and light chain sequences of the antibody are contiguous sequences delimited by either a self-cleaving sequence such as 2A or an IRES, or the heavy and light chain sequences of the antibody are linked by a mobile linker such as a contiguous glycine residue. 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 their variants.

[0051] In some embodiments, at least one epitope-coding nucleic acid sequence is selected by performing the following steps: (a) obtaining at least one of exome, transcriptome, or whole-genome nucleotide sequencing data from a tumor, infected cell, or infectious organism, wherein the nucleotide sequencing data is used to obtain data representing peptide sequences for each of a set of antigens; (b) generating a set of numerical likelihoods in which each of the antigens is presented on the cell surface by one or more MHC alleles, optionally on the surface of a tumor cell or an infected cell, wherein the set of numerical likelihoods is identified at least on the mass spectrometry data received; and (c) selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a selected set of antigens used to generate at least one epitope-coding nucleic acid sequence. In some embodiments, each epitope-coding nucleic acid sequence is selected by performing the following steps: (a) obtaining at least one of exome, transcriptome, or whole-genome nucleotide sequencing data from a tumor, infected cell, or infectious organism, wherein the nucleotide sequencing data is used to obtain data representing peptide sequences for each of the set of antigens; (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented on the cell surface by one or more MHC alleles, optionally on the surface of tumor cells or infected cells, wherein the set of numerical likelihoods is identified at least on the received mass spectrometry data; and (c) selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a selected set of antigens used to generate at least 20 epitope-coding nucleic acid sequences. In some embodiments, the number of selected sets of epitopes is 2 to 20.In some embodiments, the presentation model represents a dependence between (a) the presence of a pair of a specific MHC allele and a specific amino acid at a specific position in a peptide sequence, and (b) the likelihood of presentation of such a peptide sequence containing a specific amino acid at a specific position on the cell surface, optionally on the surface of tumor cells or infected cells, by the specific MHC allele of the pair. In some embodiments, selecting a set of selected antigens involves, based on the presentation model, selecting antigens that are more likely to be presented on the cell surface compared to unselected antigens, optionally confirmed to be presented by one or more specific HLA alleles. In some embodiments, selecting a set of selected epitopes involves, based on the presentation model, selecting epitopes that are more likely to induce a tumor-specific immune response in a subject compared to unselected epitopes. In some embodiments, selecting a set of selected antigens involves, based on the presentation model, selecting antigens that are more likely to induce a tumor-specific or infection-specific immune response in a subject compared to unselected antigens. In some embodiments, selecting a set of antigens involves, based on a presentation model, selecting antigens that are more likely to be presented to naive T cells by professional antigen-presenting cells (APCs) compared to unselected antigens, where APCs are optionally dendritic cells (DCs). In some embodiments, selecting a set of antigens involves, based on a presentation model, selecting antigens that are less likely to be inhibited via central or peripheral tolerance compared to unselected antigens. In some embodiments, selecting a set of antigens involves, based on a presentation model, selecting antigens that are less likely to induce an autoimmune response against normal tissue in a subject compared to unselected antigens. In some embodiments, exome or transcriptome nucleotide sequencing data are obtained by performing sequencing in tumor cells or tumor tissue, infected cells, or infectious organisms.In some embodiments, sequencing is next-generation sequencing (NGS) or any massively parallel sequencing method.

[0052] In some embodiments, the cassette includes junctional epitope sequences formed by adjacent sequences within the cassette. In some embodiments, at least one or each of the junctional epitope sequences has an affinity for MHC greater than 500 nM. In some embodiments, each junctional epitope sequence is non-self.

[0053] In some embodiments, each MHC class I epitope is predicted or confirmed 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 confirmed to be presentable by at least one HLA allele, and the antigen / HLA prevalence of each antigen / HLA pair in the population is at least 0.01%. In some embodiments, each MHC class I epitope is predicted or confirmed to be presentable by at least one HLA allele, and the antigen / HLA prevalence of each antigen / HLA pair in the population is at least 0.1%.

[0054] In some embodiments, the cassette codes neither a non-therapeutic MHC class I epitope nucleic acid sequence nor a class II epitope nucleic acid sequence, including a translated wild-type nucleic acid sequence, where the non-therapeutic epitope is predicted to be presented on the MHC allele of interest. In some embodiments, the predicted non-therapeutic MHC class I or class II epitope sequence is a junctional epitope sequence formed by adjacent sequences within the cassette. In some embodiments, the prediction is based on the presentation likelihood generated by inputting the sequences of the non-therapeutic epitopes into a presentation model. In some embodiments, the order of the antigen-coding nucleic acid sequences within the cassette is determined by a series of steps including (a) generating a set of candidate cassette sequences corresponding to various orders of antigen-coding nucleic acid sequences, (b) determining a presentation score for each candidate cassette sequence based on the presentation of non-therapeutic epitopes in the candidate cassette sequence, and (c) selecting a candidate cassette sequence associated with a presentation score below a predetermined threshold as the cassette sequence for the antigen vaccine.

[0055] In some embodiments, the composition is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a virus particle containing a ChAdV vector.

[0056] In some embodiments, one or more of the epitope-coding nucleic acid sequences originate from the tumor of the subject. In some embodiments, each of the epitope-coding nucleic acid sequences originates from the tumor of the subject. In some embodiments, one or more of the epitope-coding nucleic acid sequences do not originate from the tumor of the subject. In some embodiments, each of the epitope-coding nucleic acid sequences does not originate from the tumor of the subject.

[0057] In some embodiments, the epitope-coding nucleic acid sequence includes an epitope selected from the group consisting of SEQ ID NOs. 57 to 29,364. In some embodiments, at least one antigen-coding nucleic acid sequence is at least (A) a KRAS_G12C MHC class I epitope-coding nucleic acid sequence encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs. 14,954, 19,848, and 19,850; (B) a 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. 19,749, 19,865, and 19,863; and (C) a KRAS_G12V Each of the KRAS_G12V MHC class I epitope coding nucleic acid sequences comprises an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19,976, 19,779, 11,495, and 19,974.

[0058] In some embodiments, at least one antigen-coding nucleic acid sequence includes (A) KRAS_G12C MHC class I epitope-coding nucleic acid sequence, (B) KRAS_G12D MHC class I epitope-coding nucleic acid sequence, (C) KRAS_G12V MHC class I epitope-coding nucleic acid sequence, (D) KRAS Q61H MHC class I epitope-coding nucleic acid sequence, (E) TP53_R213L MHC class I epitope-coding nucleic acid sequence, (F) TP53_S127Y MHC class I epitope-coding nucleic acid sequence, (G) TP53_R249M MHC class I epitope-coding nucleic acid sequence, or a combination thereof.

[0059] In some embodiments, the method further includes administering an expression system based on an autoregenerated alphavirus. In some embodiments, the composition for delivery of the expression system based on the autoregenerated alphavirus is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). In some embodiments, the composition for delivery of the expression system based on the autoregenerated alphavirus is administered (IM). In some embodiments, the IM administration is performed at separate injection sites. In some embodiments, the separate injection sites are injection sites in the opposing deltoid muscles. In some embodiments, the separate injection sites are injection sites in the bilateral gluteal or rectus femoris muscles. In some embodiments, the injection sites for one or more boost doses are as close as possible to the injection sites for the priming dose.

[0060] In some embodiments, compositions for the delivery of an expression system based on a self-amplified alphavirus include: (A) an expression system based on a self-amplified alphavirus, wherein the expression system comprises one or more vectors, the one or more vectors comprising: (a) an RNA alphavirus skeleton comprising (i) at least one promoter nucleotide sequence and (ii) at least one polyadenylated (poly(A)) sequence; and (b) a cassette comprising (i) at least one antigen-coding nucleic acid sequence, a. an epitope-coding nucleic acid sequence, wherein the encoded epitope sequence is different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence. An expression system based on a self-amplifying alphavirus, comprising a cassette, comprising: (ii) an epitope-coding nucleic acid sequence having at least one optional change; b) an optional 5' linker sequence; and c) an optional 3' linker sequence; (ii) an optional second promoter nucleotide sequence functionally linked to at least one antigen-coding nucleic acid sequence; and (iii) an optional second poly(A) sequence having at least one second poly(A) sequence which is either a native poly(A) sequence for the alphavirus or an exogenous poly(A) sequence; and (B) a lipid nanoparticle (LNP) containing the expression system based on a self-amplifying alphavirus.

[0061] In some embodiments, a composition for delivery of an expression system based on a self-amplified alphavirus is (A) an expression system based on a self-amplified alphavirus, the expression system based on a self-amplified alphavirus comprising one or more vectors, the one or more vectors comprising (a) an RNA alphavirus skeleton, the RNA alphavirus skeleton comprising the nucleic acid sequence described in Sequence ID No. 6, the RNA alphavirus skeleton comprising a 26S promoter nucleotide sequence and a poly(A) sequence, the 26S promoter sequence being endogenous to the RNA alphavirus skeleton and the poly(A) sequence being endogenous to the RNA alphavirus skeleton, and (b) between the 26S promoter nucleotide sequence and the poly(A) sequence An expression system based on a self-amplifying alphavirus, comprising: (B) a lipid nanoparticle (LNP) containing the expression system based on a self-amplifying alphavirus, wherein the cassette is functionally linked to a 26S promoter nucleotide sequence, and the cassette comprises at least one antigen-coding nucleic acid sequence, the at least one antigen-coding nucleic acid sequence comprising: a. an epitope-coding nucleic acid sequence which optionally includes at least one change that makes the encoded epitope sequence different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence; b. optionally a 5' linker sequence; and c. optionally a 3' linker sequence.

[0062] In some embodiments, the ChAdV vector cassette is identical to the cassette of the composition for delivery of the self-amplifying alphavirus-based expression system.

[0063] In some embodiments, the sequence of elements in a cassette in a composition for delivery of an expression system based on a self-amplifying alphavirus is arranged in the 5' to 3' direction, P a -(L5 b -N c -L3 d ) X -(G5 e -U f )Y -G3 g It is represented by a formula including, where P is the second promoter nucleotide sequence, a=0 or 1, N contains one of the epitope-coding nucleic acid sequences, c=1, L5 contains the 5' linker sequence, b=0 or 1, L3 contains the 3' linker sequence, d=0 or 1, G5 contains one of the at least one nucleic acid sequences encoding the GPGPG amino acid linker, e=0 or 1, G3 contains one of the at least one nucleic acid sequences encoding the GPGPG amino acid linker, g=0 or 1, U contains one of the at least one MHC class II epitope-coding nucleic acid sequences, f=1, X=1 to 400, and for each X, the corresponding N c is an epitope-coding nucleic acid sequence, where Y = 0, 1, or 2, and for each Y, the corresponding U f This is an MHC class II epitope-coding nucleic acid sequence.

[0064] In some embodiments, for each X, the corresponding N c is a separate epitope-coding nucleic acid sequence. In some embodiments, for each Y, the corresponding U fis a distinct MHC class II epitope coding nucleic acid sequence. In some embodiments, a=0, b=1, d=1, e=1, g=1, h=1, X=20, Y=2, where at least one promoter nucleotide sequence is a single 26S promoter nucleotide sequence provided by the RNA alphavirus skeleton, and at least one polyadenylated poly(A) sequence is a poly(A) sequence of at least 100 consecutive A nucleotides provided by the RNA alphavirus skeleton, where a cassette is incorporated between the 26S promoter nucleotide sequence and the poly(A) sequence, where the cassette is functionally linked to the 26S promoter nucleotide sequence and the poly(A) sequence, and each N is an MHC class I epitope, MHC class II epitope of 7-15 amino acid length. The sequence codes for a pitope, an epitope capable of stimulating a B-cell response, or a combination thereof; L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of the epitope, where the 5' linker sequence codes for a peptide of at least 3 amino acids; L3 is a natural 3' linker sequence encoding the natural C-terminal amino acid sequence of the epitope, where the 3' linker sequence codes for a peptide of at least 3 amino acids; U is the PADRE class II sequence and the tetanus toxoid MHC class II sequence, respectively; the RNA alphavirus backbone is the sequence described in Sequence ID No. 6; and each of the MHC class I epitope-coding nucleic acid sequences codes for a polypeptide of 13-25 amino acids.

[0065] In some embodiments, the LNP comprises a lipid selected from the group consisting of ionizable aminolipids, phosphatidylcholine, cholesterol, PEG-based coating lipids, or combinations thereof. In some embodiments, the LNP comprises ionizable aminolipids, phosphatidylcholine, cholesterol, and PEG-based coating lipids. In some embodiments, the ionizable aminolipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule. In some embodiments, the expression system encapsulated in the LNP has a diameter of approximately 100 nm.

[0066] In some embodiments, the 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 ligated to the cassette.

[0067] 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.

[0068] In some embodiments, one or more vectors self-replicate within mammalian cells. In some embodiments, the RNA alphavirus backbone comprises at least one nucleotide sequence of Auravirus, Fort Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semryqui Forest virus, Sindbis virus, or Mayarovirus. In some embodiments, the RNA alphavirus backbone comprises at least one nucleotide sequence of Venezuelan encephalitis virus. In some embodiments, the RNA alphavirus backbone 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 Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semryqui Forest virus, Sindbis virus, or Mayarovirus. In some embodiments, the RNA alphavirus backbone comprises 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 Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semryqui Forest virus, Sindbis virus, 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 subgenome promoter sequence, 19nt CSE, alphavirus 3'UTR, or a combination thereof. In some embodiments, the RNA alphavirus backbone does not encode E2 and E1, which are structural virion protein capsids. In some embodiments, the cassette is inserted in place of the structural virion protein within the nucleotide sequence of Auravirus, Fort Morgan virus, Venezuelan encephalitis virus, Ross River virus, Semryqui Forest virus, Sindbis virus, or Mayarovirus. In some embodiments, the Venezuelan encephalitis virus contains the sequence of 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 including a deletion between base pairs 7544 and 11175. In some embodiments, the RNA alphavirus backbone comprises the sequence described in SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, a cassette is inserted at position 7544 to replace the deletion between base pairs 7544 and 11175 described in the sequence of SEQ ID NO: 3 or SEQ ID NO: 5. In some embodiments, the insertion of the cassette provides a transcription of polycistronic RNA comprising the nsP1-4 genes and at least one nucleic acid sequence, the nsP1-4 genes and at least one nucleic acid sequence residing in separate open reading frames.

[0069] In some embodiments, at least one promoter nucleotide sequence is a native 26S promoter nucleotide sequence encoded by an RNA alphavirus 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 a plurality of 26S promoter nucleotide sequences, each 26S promoter nucleotide sequence providing one or more transcriptions of separate open reading frames.

[0070] In some embodiments, one or more vectors are at least 300 nt in size. In some embodiments, one or more vectors are at least 1 kb in size. In some embodiments, one or more vectors are at least 2 kb in size. In some embodiments, one or more vectors are at least 5 kb in size.

[0071] In some embodiments, at least one antigen-coding nucleic acid sequence comprises two or more antigen-coding nucleic acid sequences. In some embodiments, each antigen-coding nucleic acid sequence is directly linked to one another. In some embodiments, each antigen-coding nucleic acid sequence is linked to a separate antigen-coding nucleic acid sequence by a nucleic acid sequence encoding a linker. In some embodiments, the linker links two MHC class I epitope-coding nucleic acid sequences, or links an MHC class I epitope-coding nucleic acid sequence to an MHC class II epitope-coding nucleic acid sequence. In some embodiments, the linker is selected from the group consisting of (1) consecutive glycine residues having a length of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues, (2) consecutive alanine residues having 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 adjacent to an antigen derived from a cognate protein and having a length of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2–20 amino acid residues. In some embodiments, the linker concatenates two MHC class II epitope-coding nucleic acid sequences, or concatenates an MHC class II sequence to an MHC class I epitope-coding nucleic acid sequence. In some embodiments, the linker comprises the sequence GPGPG.

[0072] In some embodiments, an antigen-coding nucleic acid sequence is functionally or directly ligated to a separate or contiguous sequence that enhances the expression, stability, cell transport, processing and presentation, and / or immunogenicity of the antigen-coding nucleic acid sequence. In some embodiments, the separate or contiguous sequence comprises at least one of the following: a ubiquitin sequence, a ubiquitin sequence modified to enhance proteasome targeting (e.g., the ubiquitin sequence contains 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 optionally, the ubiquitin sequence modified to enhance proteasome targeting is A76.

[0073] In some embodiments, at least one antigen-coding nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen-coding nucleic acid sequences, and optionally each antigen-coding nucleic acid sequence codes for a separate antigen-coding nucleic acid sequence. In some embodiments, 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 antigen-coding nucleic acid sequences, and optionally each antigen-coding nucleic acid sequence codes for a separate antigen-coding nucleic acid sequence. In some embodiments, 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 antigen-coding nucleic acid sequences. In some embodiments, at least one antigen-coding nucleic acid sequence comprises at least 2-400 antigen-coding nucleic acid sequences, of which at least two encode epitope sequences or portions thereof presented on the cell surface by MHC class I.

[0074] In some embodiments, at least two of the MHC class I epitopes are presented on the tumor cell surface by MHC class I. In some embodiments, the epitope-coding nucleic acid sequence comprises at least one MHC class I epitope-coding nucleic acid sequence, each antigen-coding nucleic acid sequence encoding a polypeptide sequence of 8–35 amino acid length, optionally 9–17, 9–25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid length.

[0075] In some embodiments, at least one MHC class II epitope-coding nucleic acid sequence is present. In some embodiments, at least one MHC class II epitope-coding nucleic acid sequence comprises at least one MHC class II epitope-coding nucleic acid sequence that is present and includes at least one change, the change causing the encoded epitope sequence to be different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence. In some embodiments, the epitope-coding nucleic acid sequence comprises an MHC class II epitope-coding nucleic acid sequence, where each antigen-coding nucleic acid sequence encodes a polypeptide sequence of 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acid length. In some embodiments, the epitope-coding nucleic acid sequence comprises an MHC class II epitope-coding nucleic acid sequence, wherein at least one MHC class II epitope-coding nucleic acid sequence is present, and at least one MHC class II epitope-coding nucleic acid sequence comprises at least one universal MHC class II epitope-coding nucleic acid sequence, and optionally, at least one universal sequence comprises at least one of tetanus toxoid and PADRE.

[0076] 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.

[0077] In some embodiments, at least one poly(A) sequence includes a poly(A) sequence native to the alphavirus. In some embodiments, at least one poly(A) sequence includes an exogenous poly(A) sequence for the alphavirus. In some embodiments, at least one poly(A) sequence is functionally ligated to at least one of at least one nucleic acid sequence. 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. In some embodiments, at least one poly(A) sequence is at least 100 consecutive A nucleotides.

[0078] In some embodiments, the epitope-coding nucleic acid sequence comprises an MHC class I epitope-coding nucleic acid sequence, which is selected by performing the following steps: (a) obtaining at least one of exome, transcriptome, or whole-genome tumor nucleotide sequencing data from a tumor, wherein the tumor nucleotide sequencing data is used to obtain data representing peptide sequences for each of the set of epitopes; (b) generating a set of numerical likelihoods in which each of the epitopes is presented on the surface of tumor cells of the tumor by one or more MHC alleles, wherein the set of numerical likelihoods is identified at least on received mass spectrometry data; and (c) selecting a subset of the set of epitopes based on the set of numerical likelihoods to generate a selected set of epitopes used to generate the MHC class I epitope-coding nucleic acid sequence. In some embodiments, each of the MHC class I epitope-coding nucleic acid sequences is selected by performing the following steps: (a) obtaining at least one of exome, transcriptome, or whole-genome tumor nucleotide sequencing data from a tumor, wherein the tumor nucleotide sequencing data is used to obtain data representing peptide sequences for each of the sets of epitopes; (b) inputting the peptide sequences of each epitope into a presentation model to generate a set of numerical likelihoods for each of the epitopes being presented on the tumor cell surface by one or more MHC alleles, wherein the set of numerical likelihoods is identified at least on received mass spectrometry data; and (c) selecting a subset of the set of epitopes based on the set of numerical likelihoods to generate a selected set of epitopes used to generate at least 20 MHC class I epitope-coding nucleic acid sequences. In some embodiments, the number of selected sets of epitopes is 2 to 20.In some embodiments, the presentation model represents a dependence between (a) the presence of a pair of a specific MHC allele and a specific amino acid at a specific position in a peptide sequence, and (b) the likelihood of the specific MHC allele of the pair presenting such a peptide sequence containing the specific amino acid at a specific position on the tumor cell surface. In some embodiments, selecting a set of selected epitopes involves, based on the presentation model, selecting epitopes that are more likely to be presented on the tumor cell surface compared to unselected epitopes. In some embodiments, selecting a set of selected epitopes involves, based on the presentation model, selecting epitopes that are more likely to induce a tumor-specific immune response in a target compared to unselected epitopes. In some embodiments, selecting a set of selected epitopes involves, based on the presentation model, selecting epitopes that are more likely to be presented to naive T cells by professional antigen-presenting cells (APCs) compared to unselected epitopes, where APCs are, optionally, dendritic cells (DCs). In some embodiments, selecting a set of selected epitopes involves, based on a presentation model, selecting epitopes that are less likely to be inhibited via central or peripheral tolerance compared to epitopes that were not selected. In some embodiments, selecting a set of selected epitopes involves, based on a presentation model, selecting epitopes that are less likely to induce an autoimmune response against normal tissue in the subject compared to epitopes that were not selected.

[0079] In some embodiments, exome or transcriptome nucleotide sequencing data are obtained by performing sequencing in tumor tissue. In some embodiments, sequencing is next-generation sequencing (NGS) or any massively parallel sequencing method. [Brief explanation of the drawing]

[0080] These and other features, aspects, and advantages of the present invention will be better understood with reference to the following description and the accompanying drawings. [Figure 1] The vaccination protocol is outlined below. [Figure 2A] This shows antigen-specific cellular immune responses measured using ELISpot. Antigen-specific IFN-gamma production was measured in PBMCs stimulated with six overlapping peptide pools (20 peptides, 15 amino acid length each). The mean for each pool across all animals (n=6) is shown. Background was corrected for each sample to match the DMSO control, and a count conversion factor was applied from weeks 0 to 4. The stacked bar graphs show the responses for pools 1-6 from top to bottom. [Figure 2B] This shows antigen-specific cellular immune responses measured using ELISpot. Antigen-specific IFN-gamma production was measured in PBMCs stimulated with six overlapping peptide pools (20 peptides, 15 amino acid length each). Responses from individual primates #1–3 are shown (from top panel to bottom panel, respectively). Background was corrected for each sample to match the DMSO control, and a count conversion factor was applied from weeks 0–4. The stacked bar graphs show the responses from pools 1–6, from top to bottom. [Figure 2C] This shows antigen-specific cellular immune responses measured using ELISpot. Antigen-specific IFN-gamma production was measured in PBMCs stimulated with six overlapping peptide pools (20 peptides, 15 amino acid length each). Responses from individual primates #4–6 are shown (from top panel to bottom panel, respectively). Background was corrected for each sample to match the DMSO control, and a count conversion factor was applied from weeks 0–4. The stacked bar graphs show the responses from pools 1–6, from top to bottom. [Figure 3A]This graph shows the antigen-specific cellular immune response of GRANITE subject G1 as measured using ELISpot. Antigen-specific IFN-gamma production was measured in PBMCs stimulated with a CD8 epitope pool. The graph shows the mean spot-forming units (SFU) + / - standard deviation (SD) per 10⁶ PBMCs in a triple ELISpot well. LOD = Assay detection limit. ULOQ = Upper limit of quantification (5,000 SFU / 10⁶ PBMC). [Figure 3B] This graph shows the antigen-specific cellular immune response of GRANITE subject G1 as measured using ELISpot. Antigen-specific IFN-gamma production was measured in PBMCs stimulated with a mini-CD8 epitope pool. The graph shows the mean spot-forming units (SFU) + / - standard deviation (SD) per 10⁶ PBMCs in a triple-well ELISpot array. LOD = assay detection limit. Changes over time are shown. [Figure 3C] This shows the antigen-specific cellular immune response of GRANITE subject G1 as measured using ELISpot. Antigen-specific IFN-gamma production was measured in PBMCs stimulated with a mini-CD8 epitope pool. The graph shows the mean spot-forming units (SFU) + / - standard deviation (SD) per 106 PBMCs in a triple ELISpot well. LOD = assay detection limit. Figures 3B show data from weeks 68 and 70. [Figure 4] This graph shows the antigen-specific cellular immune response of GRANITE subjects G3, measured using ELISpot. Antigen-specific IFN-gamma production was measured in PBMCs stimulated with a CD8 epitope pool. The graph shows the mean spot-forming units (SFU) + / - standard deviation (SD) per 10⁶ PBMCs in a triple ELISpot well. LOD = Limit of Detection of Assay. [Figure 5] This graph shows the antigen-specific cellular immune response of GRANITE subject G8, measured using ELISpot. Antigen-specific IFN-gamma production was measured in PBMCs stimulated with a CD8 epitope pool. The graph shows the mean spot-forming units (SFU) + / - standard deviation (SD) per 10⁶ PBMCs in a triple ELISpot well. LOD = Limit of Detection of Assay. [Figure 6]This graph shows the antigen-specific cellular immune response of GRANITE subject G11, measured using ELISpot. Antigen-specific IFN-gamma production was measured in PBMCs stimulated with a CD8 epitope pool. The graph shows the mean spot-forming units (SFU) + / - standard deviation (SD) per 10⁶ PBMCs in a triple ELISpot well. LOD = Limit of Detection of Assay. [Figure 7] This report presents immune cell characterization data for the allogeneic ChAdV prime-boost vaccine strategy. Left panel: ELISpot data before and after ChAdV boosting from PBMCs stimulated overnight with exovivo IFNg ELISpot in a patient-specific peptide pool for patients G1, G3, G8, and G11. The graph shows the mean spot-forming units (SFUs) per 106 PBMCs in a triple ELISpot well. Center panel: Supernatants from exovivo IFNg ELISpot assays (G1, G3, G8, and G11) before and after ChAdV boosting were analyzed by the MSD U-plex assay for granzyme B (GRZB) levels after stimulation in a patient-specific CD8 pool. The graph shows the mean GRZB levels from replication wells in pg / ml (background subtracted). Right panel: PBMCs from patient G11 were analyzed before and after ChAdV boosting using the exovivo-FluoroSpot assay to measure IFNγ, GRZB, and IL-2 expression after overnight stimulation with DMSO or a patient-specific CD8 pool. The graphs show the stacked mean SFU+ / - standard deviation (SD) per 106 PBMCs in replicated FluoroSpot wells for IFNγ, GRZB, and IL-2. [Figure 8A]Flow cytometry data for tetramer staining and memory phenotyping to evaluate the effector memory population are shown. A density plot shows the recognition of peptide-HLA (HLA-B*44:05-PE) in patient G1 before ChAd68 boosting. The flow plot shows the frequencies of naive (CD45RA+CCR7+) cells, central memory (CD45RA-CCR7+) cells, effector memory (CD45RA-CCR7-) cells, and T effector memory RA+ (CD45RA+CCR7-) cells in the CD8 tetramer+ population. [Figure 8B] Flow cytometry data for tetramer staining and memory phenotyping to evaluate the effector memory population are shown. A density plot shows the recognition of peptide-HLA (HLA-B*44:05-APC) after ChAd68 boosting in patient G1. The flow plot shows the frequencies of naive (CD45RA+CCR7+) cells, central memory (CD45RA-CCR7+) cells, effector memory (CD45RA-CCR7-) cells, and T effector memory RA+ (CD45RA+CCR7-) cells in the CD8 tetramer+ population. [Figure 8C] Flow cytometry data for tetramer staining and memory phenotyping to evaluate the effector memory population are shown. A density plot showing the recognition of peptide-HLA (HLA-A*02:01-BV421) before and after ChAd68 boosting in patient G1. A pie chart showing the frequencies of naive (CD45RA+CCR7+) cells, central memory (CD45RA-CCR7+) cells, effector memory (CD45RA-CCR7-) cells, and T effector memory RA+ (CD45RA+CCR7-) cells in the CD8 tetramer+ population from the corresponding dot plot. [Modes for carrying out the invention]

[0081] Detailed explanation I. Definition In general, terms used in the claims and specification are intended to be interpreted as having a plain meaning that will be understood by those skilled in the art. For further clarification, certain terms are defined below. In the event of any conflict between the plain meaning and the stated definition, the stated definition shall prevail.

[0082] As used herein, the term “antigen” refers to a substance that stimulates an immune response. An antigen may be a neoantigen. An antigen may be a “common antigen,” which is an antigen found among a particular population, for example, a particular population of cancer patients.

[0083] As used herein, the term “neoantigen” refers to an antigen having at least one alteration that makes it distinct from the corresponding wild-type antigen, for example, through mutation in tumor cells or tumor-cell-specific post-translational modifications. Neoantigens may include polypeptide sequences or nucleotide sequences. Mutations may include frameshift or non-frameshift indels, missense or nonsense substitutions, splice site alterations, genomic rearrangements or gene fusions, or any genomic or expression changes that result in neoORFs. 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 proteasome-generated spliced ​​antigens. 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. The target population can be identified for administration through the use of various diagnostic methods, such as the patient selection methods detailed below.

[0084] As used herein, the term “tumor antigen” means 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.

[0085] As used herein, the term “antigen-based vaccine” refers to a vaccine composition based on one or more antigens, e.g., multiple antigens. The vaccine may be nucleotide-based (e.g., virus-based, RNA-based, or DNA-based), protein-based (e.g., peptide-based), or a combination thereof.

[0086] As used herein, the term "candidate antigen" refers to a mutation or other abnormality that results in a sequence that may represent an antigen.

[0087] As used herein, the term “coding region” refers to the portion of a gene that codes for a protein.

[0088] As used herein, the term “coding mutation” refers to a mutation occurring in the coding region.

[0089] As used herein, the term "ORF" means Open Reading Frame.

[0090] As used herein, the term “NEO-ORF” refers to tumor-specific ORFs resulting from mutations or other abnormalities such as splicing.

[0091] As used herein, the term “missense mutation” refers to a mutation that results in the substitution of one amino acid with another.

[0092] 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 standard start codon.

[0093] As used herein, the term “frameshift mutation” refers to a mutation that causes a change in the frame of a protein.

[0094] As used herein, the term "indel" refers to the insertion or deletion of one or more nucleic acids.

[0095] As used herein, in relation to the sequences of two or more nucleic acids or polypeptides, the term “identity” percentage means that two or more sequences or subsequences that, when compared and aligned for maximum similarity 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, have the specified percentage of nucleotide or amino acid residues identical. Depending on the application, the “identity” percentage may exist across regions of the sequences being compared, for example, across functional domains, or in other cases, across the entire length of the two sequences being compared.

[0096] In sequence comparison, typically one sequence acts as a reference sequence, against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, subsequence coordinates are specified as needed, 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 dissimilarity can be established by the presence or absence of specific nucleotides, or, in the case of translated sequences, amino acids at selected sequence locations (e.g., sequence motifs).

[0097] Optimal alignment of sequences for comparison can be achieved, 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 computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, found in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see Ausubel et al. for an overview).

[0098] One example of a suitable algorithm for determining sequence identity percentage 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.

[0099] As used herein, the terms “nonstop” or “readthrough” refer to a mutation that results in the removal of a native stop codon.

[0100] As used herein, the term “epitope” refers to a specific portion of an antigen to which an antibody or T cell receptor typically binds.

[0101] As used herein, the term “immunogenicity” refers to the ability to stimulate an immune response, for example, through T cells, B cells, or both.

[0102] As used herein, the terms “HLA binding affinity” and “MHC binding affinity” mean the affinity for binding between a particular antigen and a particular MHC allele.

[0103] 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.

[0104] As used herein, the term “variant” refers to the difference between the nucleic acid in question and the reference human genome used as a control.

[0105] As used herein, the term “variant call” is typically the algorithmic determination of the presence of a variant from sequencing.

[0106] As used herein, the term “polymorphism” refers to germline variants, i.e., variants found in all DNA-containing cells of an individual.

[0107] As used herein, the term “somatic cell variant” refers to a variant that occurs in the non-germ cells of an individual.

[0108] As used herein, the term "allele" refers to a version of a gene, a version of a gene sequence, or a version of a protein.

[0109] As used herein, the term "HLA type" refers to a supplement to the HLA gene allele.

[0110] 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.

[0111] 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.

[0112] As used herein, the term “subclonal mutation” refers to a mutation that occurs in the later stages of tumor development and is present only in a subset of tumor cells.

[0113] 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.

[0114] As used herein, the term “logistic regression” is a regression model for binary data from statistics that models the logit, the probability that the dependent variable is equal to 1, as a linear function of the dependent variable.

[0115] As used herein, the term “neural network” refers to a machine learning model for classification or regression consisting of multiple layers of linear transformations followed by element-wise nonlinearities, typically trained by stochastic gradient descent and backpropagation.

[0116] As used herein, the term “proteome” refers to the total set of proteins expressed and / or translated by a cell, a group of cells, or an organism.

[0117] As used herein, the term “peptideome” refers to the total set of peptides presented by MHC-I or MHC-II on the surface of a cell. The peptideome may also refer to the characteristics of a cell or cell collection (e.g., tumor peptideome, meaning the combined peptideome of all cells constituting a tumor, or infectious peptideome, meaning the combined peptideome of all cells infected by an infection).

[0118] As used herein, the term "ELISPOT" means an enzyme-linked immunosorbent spot assay, which is a common method for monitoring immune responses in humans and animals.

[0119] As used herein, the term “dextramer” refers to a dextran-based peptide-MHC polymer used for antigen-specific T cell staining in flow cytometry.

[0120] 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.

[0121] As used herein, the term “central tolerance” refers to tolerance affected in the thymus by either removing autoreactive T cell clones or promoting autoreactive T cell clones to differentiate into immunosuppressive regulatory T cells (Tregs).

[0122] As used herein, the term “peripheral tolerance” refers to tolerance affected in the periphery by downregulating or anerating autoreactive T cells that survive central tolerance, or by promoting the differentiation of these T cells into Tregs.

[0123] The term “sample” may include aliquots of single or multiple cells or cell fragments or bodily fluids taken from a subject by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspiration, lavage, scraping, surgical incision, or other interventions or means known in the art.

[0124] The term "subject" includes cells, tissues, or organisms, human or non-human, regardless of sex, in vivo, exogenic, or in vitro. The term "subject" includes mammals such as humans.

[0125] 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.

[0126] The term “clinical factor” refers to a measure of a subject’s condition, e.g., disease activity or severity. “Clinical factors” encompass all markers of a subject’s health status, including non-sample markers, and / or other characteristics of the subject, e.g., age and sex, without limitation. Clinical factors may be scores, values, or sets of values ​​that can be obtained from a sample (or population of samples) from a subject or from an assessment of the subject under determined conditions. Clinical factors can also be predicted by markers and / or other parameters, e.g., gene expression substitutes. Clinical factors may include tumor type, tumor subtype, infection type, infection subtype, and smoking history.

[0127] The term “tumor-derived antigen-coding nucleic acid sequence” refers to nucleic acid sequences obtained from a tumor, for example via RT-PCR; or sequence data obtained by sequencing a tumor and then using the sequencing data to synthesize nucleic acid sequences, for example, via various synthetic or PCR-based methods known in the art. Derived sequences may include nucleic acid sequence variants, such as sequence-optimized nucleic acid sequence variants (e.g., codon optimization and / or other optimizations for expression), which encode the same polypeptide sequence as the corresponding native nucleic acid sequence obtained from the tumor.

[0128] The term "antigen-coding nucleic acid sequence derived from an infectious disease" refers to nucleic acid sequences obtained from infected cells or infectious organisms, for example, via RT-PCR; or sequence data obtained by sequencing infected cells or infectious organisms and then using the sequencing data to synthesize nucleic acid sequences, for example, via various synthetic or PCR-based methods known in the art. Derived sequences may include nucleic acid sequence variants, such as sequence-optimized nucleic acid sequence variants (e.g., codon optimization and / or other optimizations for expression), which encode the same polypeptide sequence as the corresponding naturally occurring infectious disease organism nucleic acid sequence. Derived sequences may include nucleic acid sequence variants encoding modified infectious disease organism polypeptide sequences that have one or more (e.g., 1, 2, 3, 4, or 5) mutations relative to the naturally occurring infectious disease organism polypeptide sequence. For example, a modified polypeptide sequence may have one or more missense mutations relative to the naturally occurring polypeptide sequence of the infectious disease organism protein.

[0129] The term "alphavirus" refers to members of the Togaviridae family and is a single-stranded positive-sense RNA virus. Alphaviruses are typically classified into Old World viruses such as Sindbis virus, Ross River virus, Mayarovirus, Chikungunya virus, and Semlik Forest virus, or New World viruses such as Eastern Equine Encephalitis virus, Aura virus, Fort Morgan virus, or Venezuelan Equine Encephalitis virus and its derivative TC-83. Alphaviruses are typically self-replicating RNA viruses.

[0130] The term "alphaviral skeleton" refers to the minimal sequence of an alphavirus that enables the self-replication of the viral genome. This minimal sequence may include conserved sequences for non-structural protein-mediated amplification, non-structural protein 1 (nsP1) genes, nsP2 genes, nsP3 genes, nsP4 genes, and poly(A) sequences, as well as sequences for the expression of viral subgenomic RNAs, such as subgenome (e.g., 26S) promoter elements.

[0131] The term "sequence for non-structural protein-mediated amplification" includes alphavirus conserved sequence elements (CSEs) well known to those skilled in the art. Examples of CSEs include, but are not limited to, alphavirus 5'UTR, 51nt CSE, 24nt CSE, subgenome promoter sequences (e.g., 26S subgenome promoter sequences), 19nt CSE, and alphavirus 3'UTR.

[0132] The term "RNA polymerase" includes polymerases that catalyze the production of RNA polynucleotides from DNA templates. Examples of RNA polymerases include, but are not limited to, those derived from bacteriophages such as T3, T7, and SP6.

[0133] The term "lipid" includes hydrophobic and / or amphiphilic molecules. Lipids can be cationic, anionic, or neutral. Lipids can be synthetic or naturally occurring and may be biodegradable. Lipids may include, but are not limited to, cholesterol, phospholipids, and lipid complexes, including 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.

[0134] The term "lipid nanoparticles" or "LNPs" refers to 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 salts (sodium taurocholate), and sterols (cholesterol) can be used as stabilizers. Lipid nanoparticles can be formed using defined ratios of different lipid molecules, including, but not limited to, defined ratios of one or more cationic, anionic, or neutral lipids. Lipid nanoparticles can encapsulate molecules within their outer membrane shell, and then, upon contact with target cells, the encapsulated molecules can be delivered to the host cell cytosol. Lipid nanoparticles can be modified or functionalized with non-lipid molecules, including their surface. Lipid nanoparticles can be single-layer (unilamellar) or multi-layer (multilamellar). Lipid nanoparticles can form complexes with nucleic acids. Monolayer lipid nanoparticles can form complexes with nucleic acids, where the nucleic acids are located within the aqueous layer. Multilayer lipid nanoparticles can also form complexes with nucleic acids, where the nucleic acids are located within, form, or are sandwiched between the aqueous layers.

[0135] Abbreviations: MHC: Major histocompatibility complex; HLA: Human leukocyte antigen, or human MHC gene locus; NGS: Next-generation sequencing; PPV: Positive predictive value; TSNA: Tumor-specific neoantigen; FFPE: Formalin-fixed, paraffin-embedded; NMD: Nonsense-mediated decay; NSCLC: Non-small-cell lung cancer; DC: Dendritic cell.

[0136] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless specifically indicated by the context.

[0137] Unless otherwise specifically stated or evident from the context, the term “about” as used herein is understood to mean within the normal tolerance range of the prior art, for example, within two standard deviations of the mean. “About” may be understood to mean 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 ​​provided herein are modified by the term “about.”

[0138] Any terms not directly defined herein should be understood to have a common meaning as understood within the art of the present invention. Certain terms are discussed herein to provide additional guidance to practitioners when describing compositions, devices, methods, etc., of aspects of the present invention and how to prepare or use them. It should be recognized that the same thing may be expressed in multiple ways. Therefore, alternative languages ​​and synonyms may be used for any one or more of the terms discussed herein. Whether a term is detailed or discussed herein is irrelevant. Several synonyms or alternative methods, materials, etc., are provided. Unless expressly stated otherwise, the description of one or more synonyms or equivalents does not preclude the use of other synonyms or equivalents. The use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of aspects of the present invention herein.

[0139] All references, published patents, and patent applications cited herein are incorporated herein in their entirety by reference for any purpose.

[0140] II. Antigen Identification Research methods for NGS analysis of tumor and normal exomes and transcriptomes have been reported and are being applied to the antigen identification field. 6、14、15Specific optimizations can be considered to increase the sensitivity and specificity of antigen identification in clinical settings. These optimizations can be classified into two areas: those related to laboratory processes and those related to NGS data analysis. The described research methods can also be applied to the identification of antigens in other contexts, e.g., infectious organisms, infection in a subject, or identification of antigens from infected cells of a subject. Examples of optimizations are known to those skilled in the art, and the methods are detailed in, for example, U.S. Patent No. 10,055,540, U.S. Patent Publication No. US20200010849A1, U.S. Patent Publication No. 16 / 606,577, and International Patent Publication Nos. WO2020181240A1, WO / 2018 / 195357, and WO / 2018 / 208856, respectively, which are incorporated herein by reference in their entirety for any purpose.

[0141] Methods for identifying antigens (e.g., antigens derived from tumors or infectious organisms) include identifying antigens that are likely to be presented on the cell surface (e.g., presented by MHC on tumor cells, infected cells, or immune cells, including professional antigen-presenting cells such as dendritic cells), and / or antigens that are likely to be immunogenic. As an example, one such method may include the steps of: obtaining at least one of exome, transcriptome, or whole-genome nucleotide sequencing and / or expression data from a tumor, infected cell, or infectious organism, wherein the nucleotide sequencing and / or expression data are used to obtain data representing each peptide sequence of a set of antigens (e.g., antigens derived from a tumor or infectious organism); inputting the peptide sequences of each antigen into one or more presentation models to generate a set of numerical likelihoods in which each of the antigens is presented on the cell surface, for example, on the surface of a tumor cell or infected cell of interest, by one or more MHC alleles, wherein the set of numerical likelihoods is identified at least on the mass spectrometry data received; and selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a selected set of antigens.

[0142] III. Identification of Tumor-Specific Mutations in Neoantigens This specification also discloses methods for identifying certain mutations (e.g., variants or alleles present in cancer cells). In particular, these mutations may be present in the genome, transcriptome, proteome, or exome of cancer cells of a subject having cancer, but not in the normal tissue of the subject. Certain methods for identifying tumor-specific neoantigens, including common neoantigens, are known to those skilled in the art, for example, in U.S. Patent No. 10,055,540, U.S. Patent Publication No. US20200010849A1, and International Patent Publication Nos. WO / 2018 / 195357 and WO / 2018 / 208856, which are incorporated herein by reference in their entirety for any purpose. Examples of tumor-specific common neoantigens are described in International Patent Publication No. WO2019226941A1, which is incorporated herein by reference in its entirety for any purpose. Common neoantigens include, but are not limited to, KRAS-related mutations (e.g., KRAS G12C, KRAS G12V, KRAS G12D, and / or KRAS Q61H mutations). For example, KRAS-related MHC class I neoepitopes may include mutations related to wild-type (WT) human KRAS, such as the following exemplary amino acid sequence: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM.

[0143] Gene mutations in tumors can be considered useful for immunological tumor targeting if they result in changes to the amino acid sequence of a protein only in the tumor. Useful mutations include: (1) non-synonymous mutations that result in different amino acids in a protein; (2) read-through mutations that alter or delete a stop codon, resulting in the translation of a longer protein with a novel tumor-specific sequence at the C-terminus; (3) splice site mutations that result in the inclusion of an intron in mature mRNA, thus resulting in a unique tumor-specific protein sequence; (4) chromosomal rearrangements (i.e., gene fusions) that result in a chimeric protein with a tumor-specific sequence at the junction of two proteins; and (5) frameshift mutations or deletions that result in a new open reading frame with a novel tumor-specific protein sequence. Mutations may also include one or more non-frameshift indels, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic or expression changes that result in neo-ORFs.

[0144] In tumor cells, peptides or mutant polypeptides with mutations resulting from, for example, splice site, frameshift, readthrough, or gene fusion mutations can be identified by sequencing DNA, RNA, or proteins in comparison to tumor and normal cells.

[0145] 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.

[0146] 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 led to accurate, easy, and inexpensive large-scale SNP genotyping. For example, several techniques have been reported, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and various DNA "chip" technologies such as the Affymetrix SNP chip. These methods utilize amplification of the target gene region, typically by PCR. Further methods are based on invasive cleavage followed by mass spectrometry or the generation of small signal molecules by immobilized padlock probes and rolling circle amplification. Some of the methods known in the art for detecting specific mutations are summarized below.

[0147] PCR-based detection methods may include the simultaneous multiplex amplification of multiple 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 using differentially labeled primers, and thus each can be detected differentially. Naturally, hybridization-based detection methods enable the differential detection of multiple PCR products in a sample. Other techniques that enable the multiplex analysis of multiple markers are known in the art.

[0148] Several methods have been developed to facilitate the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA or cellular RNA. For example, SNPs can be detected by using specialized exonuclease-resistant nucleotides, such as those disclosed in Mundy, CR (U.S. Patent No. 4,656,127). This method allows a primer complementary to the allele sequence adjacent to the 3' of the polymorphic site to hybridize 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. Because the identity of the exonuclease-resistant derivative of the sample is known, the finding that the primer has become exonuclease-resistant reveals that the nucleotide present in the polymorphic site of the target molecule is complementary to the nucleotide 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.

[0149] A solution-based method can be used to determine the identity of nucleotides at polymorphic sites. (Cohen, D. et al., French Patent No. 2,650,840, PCT Application No. WO91 / 02087). Similar to Mundy's method (U.S. Patent No. 4,656,127), a primer complementary to the allele sequence adjacent to the 3' of the polymorphic site is used. In this method, the identity of the nucleotide at the polymorphic site is determined using a labeled dideoxynucleotide derivative incorporated at the end of the primer if complementary to the nucleotide at that site.

[0150] An alternative method known as Genetic Bit Analysis or GBA has been reported 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 sequence at 3' relative to the polymorphic site. Thus, 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, where the primer or target molecule is immobilized on a solid phase.

[0151] Several primer-induced nucleotide insertion procedures have been reported for evaluating polymorphic sites in DNA (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. al., Analyst. Biochem. 208:171-175 (1993). Unlike GBA, these methods utilize the incorporation of labeled deoxynucleotides to distinguish between bases at polymorphic sites. In such a format, the signal is proportional to the number of incorporation of deoxynucleotides, so polymorphisms occurring in the same nucleotide run can produce a signal proportional to the length of the run (Syvanen, A.-C., et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0152] Numerous initiatives are obtaining sequence information directly and in parallel from millions of individual molecules of DNA or RNA. Synthetic real-time single-molecule sequencing technology relies on the detection of fluorescent nucleotides, which are incorporated into a nascent strand of DNA complementary to the template to be sequenced. In one method, oligonucleotides 30–50 nucleotides long are covalently anchored to a coverslip at their 5' ends. These anchoring strands serve two functions. Firstly, if the template consists of oligonucleotides bound to its surface with complementary capture tails, their anchoring strands act as capture sites on the target template strand. They also act as primers for template-directed primer extension, forming the basis for sequence reading. The capture primers function as situational sites for sequencing using chemical cleavage of a dye-linker for synthesis, detection, and dye removal over multiple cycles. Each cycle involves the addition of 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, and each nucleotide is color-coded by the acceptor fluorescence moiety bound to gamma phosphate. The system detects the interaction between the fluorescently tagged polymerase and the fluorescently modified nucleotide as it is incorporated into a new chain. Other synthesis-time sequencing techniques also exist.

[0153] Any suitable synthetic sequencing platform can be used to identify mutations. As mentioned above, four major synthetic sequencing platforms are currently available: Roche / 454 Life Sciences' Genome Sequencers, Illumina / Solexa's 1G Analyzer, Applied BioSystems' SOLiD system, and Helicos Biosciences' Heliscope system. Synthetic sequencing platforms have also been reported by Pacific BioSciences and VisiGen Biotechnologies. In some embodiments, multiple nucleic acid molecules to be sequenced are bound to a support (e.g., a solid support). To immobilize the nucleic acid to the support, a capture sequence / universal priming site can be added to the 3' and / or 5' ends of the template. Nucleic acids can be immobilized to the support by hybridizing the capture sequence to a complementary sequence covalently bound to the support. The capture sequence (also called a universal capture sequence) is a nucleic acid sequence complementary to the sequence bound to the support, which can serve a dual role as a universal primer.

[0154] As an alternative to capture sequences, members of a binding pair (e.g., an antibody / antigen, receptor / ligand, or avidin-biotin pair, as described in U.S. Patent Application No. 2006 / 0252077) can be linked to each fragment to be captured on a surface coated with the second member of each binding pair.

[0155] After 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 sequencing. In sequencing, a surface-bound molecule is exposed to multiple labeled nucleotide triphosphates in the presence of polymerase. The sequence of the template is determined by the order in which the labeled nucleotides are incorporated into the 3' end of the growth chain. This can be performed in real time or in a step-and-repeat manner. In real-time analysis, different optical labels can be incorporated into each nucleotide, and multiple lasers can be used to stimulate the incorporated nucleotides.

[0156] Sequencing may also include other massively parallel sequencing or next-generation sequencing (NGS) technologies and platforms. Further examples of massively parallel sequencing technologies and platforms include Illumina HiSeq or MiSeq, Thermo PGM or Proton, Pac Bio RS II or Sequel, Qiagen's Gene Reader, and Oxford Nanopore MinION. In addition, similar current massively parallel sequencing technologies, and future generations of these technologies, may be used.

[0157] Any cell type or tissue can be used to obtain nucleic acid samples for use in the methods described herein. For example, DNA or RNA samples can be obtained from a tumor or from bodily fluids obtained by known techniques (e.g., venipuncture), such as blood or saliva. Alternatively, nucleic acid testing can be performed on dry samples (e.g., hair or skin). Furthermore, some samples can be obtained from a tumor for sequencing, and other samples can be obtained from normal tissue of the same histological type as the tumor for sequencing. Some samples can be obtained from a tumor for sequencing, and other samples can be obtained from normal tissue of a different histological type compared to the tumor for sequencing.

[0158] Tumors may include one or more of the following: lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain tumor, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and T-cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0159] Alternatively, protein mass spectrometry can be used to identify or verify the presence of mutant peptides bound to MHC proteins on tumor cells. These peptides can be identified by acid elution from tumor cells or HLA molecules immunoprecipitated from tumors, followed by mass spectrometry.

[0160] IV. Antigen Antigens can contain nucleotides or polypeptides. For example, an antigen may be an RNA sequence encoding a polypeptide sequence. Therefore, antigens useful for vaccines may contain nucleotide sequences or polypeptide sequences.

[0161] This specification discloses 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. Neoantigen peptides can be described in relation to their coding sequence, in which case the neoantigen includes a nucleotide sequence (e.g., DNA or RNA) encoding the relevant polypeptide sequence.

[0162] 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, such as 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 antigen peptides may be derived can be found, for example, in the COSMIC database, which maintains comprehensive information on somatic mutations in human cancers. The peptides contain tumor-specific mutations. Tumor antigens (e.g., common tumor antigens and tumor neoantigens) may include, but are not limited to, those described in U.S. Patent Application No. 17 / 058,128, which is incorporated herein by reference for any purpose. Antigen peptides can be described in relation to their coding sequences, in which case the antigen includes a nucleotide sequence (e.g., DNA or RNA) encoding the relevant polypeptide sequence.

[0163] This specification also discloses peptides derived from infectious organisms, infections in subjects, or any polypeptides associated with infected cells of subjects. Antigens may be derived from nucleotide or polypeptide sequences of infectious organisms. Polypeptide sequences of infectious organisms include, but are not limited to, pathogen-derived peptides, virus-derived peptides, bacterial-derived peptides, fungal-derived peptides, and / or parasitic-derived peptides. Infectious organisms include, but are not limited to, severe acute respiratory syndrome-associated coronaviruses (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola, HIV, hepatitis B virus (HBV), influenza, hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), chikungunya virus, polynuclear respiratory virus (RSV), dengue virus, orthymyxoviridae viruses, and tuberculosis.

[0164] This specification discloses isolated peptides containing infectious disease organism-specific antigens or epitopes identified by the methods disclosed herein, peptides containing known infectious disease organism-specific antigens or epitopes, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. Antigen peptides can be described in relation to their coding sequences, in which case the antigen includes a nucleotide sequence (e.g., DNA or RNA) encoding the relevant polypeptide sequence.

[0165] The vectors and related compositions described herein can be used to deliver antigens from any organism, such as their toxins or other by-products, to prevent and / or treat infections or other adverse reactions associated with the organism or its by-products.

[0166] Antigens that can be incorporated into vaccines (e.g., encoded as cassettes) include immunogens useful for immunizing humans or non-human animals against viruses, such as pathogenic viruses that infect humans and non-human vertebrates. Antigens can be selected from various viridae. Examples of desirable viridae for which a counteracting immune response is desired include the Picornaviridae family, which includes rhinoviruses, which account for about 50% of cases of the common cold; enteroviruses, which include human enteroviruses such as poliovirus, coxsackievirus, echovirus, and hepatitis A virus; and apthoviruses, which cause foot-and-mouth disease, mainly in non-human animals. Among the viruses in the Picornaviridae family, target antigens include VP1, VP2, VP3, VP4, and VPG. Another viridae family is the Calcivirus family, which includes viruses of the Norwalk group, which are important pathogens of epidemic gastroenteritis. Another viridae family preferred for use as target antigens to stimulate immune responses in humans and non-human animals is the Togaviridae family, which includes the alphavirus genus, including Sindbis virus, Ross River virus, and rubiviruses such as Venezuelan horse encephalitis, eastern and western horse encephalitis, and rubella virus. The Flaviviridae family includes dengue virus, yellow fever virus, Japanese encephalitis virus, St. Louis encephalitis virus, and tick-borne encephalitis virus. Other target antigens may be generated from hepatitis C or coronavirus families, which include numerous non-human viruses such as infectious bronchitis virus (poultry), porcine gastroenteritis virus (pig), porcine hemagglutinating encephalomyelitis virus (pig), feline infectious peritonitis virus (cat), feline enteric coronavirus (cat), canine coronavirus (dog), and human respiratory coronaviruses, which may cause the common cold and / or non-type A, B, or C hepatitis.Within the Coronaviridae family, target antigens include glycoproteins (not present in all coronaviruses) such as E1 (also called the M protein or matrix protein), E2 (also called the S protein or spike protein), and E3 (also called HE or hemagglutin-elterose), or N (nucleocapsid). Further antigens may target the Rhabdoviridae family, which includes the Becyclovirus genus (e.g., vesicular stomatitis virus) and lyssaviruses in general (e.g., rabies). Within the Rhabdoviridae family, suitable antigens may be derived from the G protein or N protein. The Filoviridae family, which includes hemorrhagic fever viruses such as Marburg virus and Ebola virus, may be suitable antigen sources. The Paramyxoviridae family includes parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, Ubravirus (mumps virus), parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), rinderpest, morbillivirus (including measles and canine distemper), and pneumoviruses (e.g., glyco(G) proteins and fusion(F) proteins whose sequences are available from GenBank), which include respiratory multinucleated viruses. Influenza viruses are classified within the Orthomyxoviridae family and can be a suitable antigen source (e.g., HA protein, N1 protein). The Bunyaviridae family includes the Bunyavirus genera (California encephalitis, Lacrosse), Phlebovirus genera (Rift Valley fever), Hantavirus genera (puremala is the hemahagin fever virus), Nairovirus genera (Nairobi sheep disease), and various unclassified bungaviruses. The Arenaviridae family provides antigen sources for LCM and Lassa fever virus. The Reoviridae family includes the genera Reovirus, Rotavirus (which causes acute gastroenteritis in children), Orbivirus, and Cultivirus (which includes Colorado tick fever, Lebombo (human), equine encephalopathy, and Bluetongue).The Retroviridae family includes the oncorivirinal subfamily, which encompasses human and animal diseases such as feline leukemia virus, HTLV-VI and HTLV-II, and lentivirals (including human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus, and spumavirals). Many suitable antigens have been reported from lentiviruses and can be easily selected. Examples of suitable HIV and SIV antigens include, non-limitingly, proteins such as gag, pol, Vif, Vpx, VPR, Env, Tat, Nef, and Rev, as well as various fragments thereof. For example, suitable fragments of the Env protein may include its subunits such as gp120, gp160, gp41, or smaller fragments thereof, e.g., those with a length of at least approximately 8 amino acids. Similarly, fragments of the tat protein can be selected. [See U.S. Patent Nos. 5,891,994 and 6,193,981.] Also see HIV and SIV proteins described in DHBarouch et al, J. Virol., 75(5):2462-2467 (March 2001) and RRAmara, et al, Science, 292:69-74 (6 Apr. 2001). In another example, fusion proteins or other immunogenic molecules may be formed using immunogenic proteins or peptides of HIV and / or SIV. See, for example, WO01 / 54719, published August 2, 2001, and WO99 / 16884, published April 8, 1999, for HIV-1 Tat and / or Nef fusion proteins and immunization regimens. The present invention is not limited to the immunogenic proteins or peptides of HIV and / or SIV described herein. Furthermore, various modifications to these proteins have been reported or are readily available to those skilled in the art. For example, see the modified gag protein described in U.S. Patent No. 5,972,596.Furthermore, any desired HIV immunogen and / or SIV immunogen may be delivered alone or in combination. Such combinations may include expression from a single vector or from multiple vectors. The Papovaviridae family includes the Polyomavirinae subfamily (BKU virus and JCU virus) and the Papillomavirinae subfamily (associated with the malignant progression of cancer or papillomas). The Adenoviridae family includes viruses that cause respiratory diseases and / or enteritis (EX, AD7, ARD, OB). The Parvoviridae family includes feline parvovirus (feline enteritis), feline panleukopenia virus, canine parvovirus, and porcine parvovirus. The Herpesviridae family includes the alphaherpesvirinae, which encompasses the genera *Simplevirus* (HSVI, HSVII) and *Varicerovirus* (pseudorabies, varicella-zoster virus); the betaherpesvirinae, which includes the genera *Cytomegalovirus* (human CMV) and *Muro-megalovirus*; and the gammaherpesvirinae, which includes the genera *Lymphocryptovirus*, *EBV* (Burkitt lymphoma), infectious rhinotracheitis, Marek's disease virus, and *Razinovirus*. The Poxviridae family includes the Chordopoxyvirinae subfamily, which encompasses the Orthopoxvirus (smallpox and Vaccinia (cowpox)), Parapoxvirus, Avipoxvirus, Capripoxvirus, Lepolipoxvirus, and Porcipoxvirus genera, as well as the Entomopoxvirinae subfamily. The Hepadnaviridae family includes hepatitis B virus. One unclassified virus that may be a suitable antigenic source is hepatitis delta virus. Further viral sources may include avian bursal disease virus and porcine reproductive and respiratory syndrome virus. The Alphaviridae family includes equine arteritis virus and various encephalitis viruses.

[0167] Antigens that can be incorporated into vaccines (for example, coded as a cassette) include immunogens useful for immunizing humans or non-human animals against pathogens that infect humans and non-human vertebrates, including bacteria, fungi, parasitic microorganisms, or multicellular parasites. Examples of bacterial pathogens include pathogenic Gram-positive cocci, such as Streptococcus pneumoniae, Staphylococcus staphylococci, and Streptococcus streptococci. Pathogenic Gram-negative cocci include Neisseria meningitidis and Neisseria gonorrhoeae. Pathogenic enteric Gram-negative bacilli include: Enterobacteriaceae; Pseudomonas, Acidobacteria, and Eikenella; Melioidosis; Salmonella; Shigella; Haemophilus (Haemophilus influenzae, Haemophilus somnus); Moraxella; H. ducreyi (causes chancroid); Brucella; Franisella tularensis (causes tularemia); Yersinia (Pasteurella); Streptobacillus moniliformis This includes *Moniliformis* and the genus *Spirillum*. Gram-positive bacilli include *Listeria monocytogenes*, *Erysipelothrix rhusiopathiae*, and *Corynebacterium*.Diseases caused by pathogenic anaerobic bacteria include diphtheria, cholera, anthrax (B. anthracis), Donovan disease (inguinal granuloma), and bartonellosis. Diseases caused by pathogenic anaerobic bacteria include tetanus, botulism, other Clostridium species, tuberculosis, leprosy, and other mycobacteria. Examples of specific bacterial species include, without limitation, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Moraxella catarrhalis, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis, Salmonella typhi, Salmonella typhi * Mycobacterium typhimurium*, *Salmonella choleraesuis*, *Escherichia coli*, *Shigella*, *Vibrio cholerae*, *Corynebacterium diphtheriae*, *Mycobacterium tuberculosis*, *Mycobacterium avium*, *Mycobacterium intracellulare* complex, *Proteus mirabilis*, *Proteus vulgaris*, *Staphylococcus aureus*, *Clostridium tetani*, *Leptospira interlogans*These include *Mycoplasma interrogans*, *Borrelia burgdorferi* (Lyme disease), *Pasteurella haemolytica*, *Pasteurella multocida*, *Actinobacillus pleuropneumoniae*, and *Mycoplasma gallisepticum*. Diseases caused by pathogenic spirochetes include syphilis; treponemic diseases: strawberry syphilis, pinta and endemic syphilis; and leptospirosis. Other infections caused by highly pathogenic bacteria and pathogenic fungi include actinomycosis / actinomycosis; nocardiosis; cryptococcosis (Cryptococcus), fermentomycosis (Blastomyces), histoplasmosis (Histoplasma), and coccidioidomycosis (Coccidiodes); candidiasis (Candida), aspergillosis (Aspergilli), and mucormycosis; sporotrichumosis; paracoccidioidomycosis, petriellidiosis, torulopsis, mycomas, and chromomycosis; and dermatophytosis. Rickettsial infections include typhus fever, Rocky Mountain spotted fever, Q fever, and rickettsial pox. Examples of mycoplasma and chlamydia infections include mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydia infection. Pathogenic eukaryotes include pathogenic protozoa and helminths, and infections caused by them include amoebiasis; malaria; leishmaniasis (e.g., caused by Leishmania major); trypanosomiasis; toxoplasmosis (e.g., caused by Toxoplasma gondii); and Pneumocystis carinii.Carinii infections; Trican infections; Toxoplasmosis (Toxoplasma gondii); Babesiosis; Giardiasis (e.g., caused by Giardia); Trichomonas infections (e.g., caused by Trichomonas); Filariasis; Schistosomiasis (e.g., caused by Schistosoma); Nematode infections; Trematode or fluke infections; and Tapeworm infections. Other parasitic infections may be caused in particular by Ascaris, Trichuris, Cryptosporidium, and Pneumocystis carinii.

[0168] This specification also discloses peptides derived from infectious organisms, infections in subjects, or any polypeptides associated with infected cells in subjects. Antigens may be derived from nucleic acid sequences or polypeptide sequences of infectious organisms. Polypeptide sequences of infectious organisms include, but are not limited to, pathogen-derived peptides, virus-derived peptides, bacterial-derived peptides, fungal-derived peptides, and / or parasitic-derived peptides. Infectious organisms include, but are not limited to, severe acute respiratory syndrome-associated coronaviruses (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola, HIV, hepatitis B virus (HBV), influenza, hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), chikungunya virus, polynuclear respiratory virus (RSV), dengue virus, orthymyxoviridae viruses, and tuberculosis.

[0169] Antigens can be selected from those expected to be presented on the cell surface of cells such as tumor cells, infected cells, or immune cells, including professional antigen-presenting cells such as dendritic cells. Antigens can also be selected from those expected to be immunogenic.

[0170] 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; a length of 8–15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids for MHC class I peptides; the presence of a sequence motif inside or near a peptide that promotes proteasome cleavage; and the presence of a sequence motif that promotes TAP transport. For MHC class II peptides, this may include a length of 6–30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids; and the presence of a sequence motif inside or near a peptide that promotes HLA binding catalyzed by proteases (e.g., cathepsin) extracellularly or lysosomes, or by HLA-DM.

[0171] One or more antigens may be presented on the surface of the tumor. One or more antigens may be presented on the surface of infected cells.

[0172] One or more antigens may be immunogenic in subjects with tumors, for example, having the ability to stimulate T cell and / or B cell responses in the subject. One or more antigens may be immunogenic in subjects with or suspected of having an infection, for example, having the ability to stimulate T cell and / or B cell responses in the subject. One or more antigens may be immunogenic in subjects at risk of infection, for example, having the ability to stimulate T cell and / or B cell responses in the subject that provide immunological protection (i.e., immunity) against the infection, such as stimulating the production of memory T cells, memory B cells, or antibodies specific to that infection.

[0173] One or more antigens may have the ability to stimulate a B cell response, such as the production of antibodies that recognize one or more antigens (e.g., antibodies that recognize tumor or infectious disease antigens). Antibodies may recognize linear polypeptide sequences or secondary and tertiary structures. Therefore, B cell antigens may include, but are not limited to, linear polypeptide sequences or polypeptides having secondary and tertiary structures, such as full-length proteins, protein subunits, protein domains, or any polypeptide sequence known or predicted to have secondary and tertiary structures. Each antigen capable of stimulating a B cell response to a tumor or infectious disease antigen may be an antigen found on the surface of tumor cells or infectious organisms. Each antigen capable of inducing a B cell response to a tumor or infectious disease antigen may be an intracellular neoantigen expressed in tumors or infectious organisms.

[0174] One or more antigens may include a combination of antigens capable of stimulating a T cell response (e.g., peptides such as predicted T cell epitope sequences) and distinct antigens capable of stimulating a B cell response (e.g., full-length proteins, protein subunits, protein domains).

[0175] One or more antigens that stimulate an autoimmune response in a target can be excluded from consideration in relation to vaccine development for that target.

[0176] The size of at least one antigen peptide molecule (e.g., epitope sequence) is not limited, but 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. It may include approximately 32, 33, 34, 35, 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 that can be derived therefrom. In specific embodiments, the antigen peptide molecule has 50 amino acids or less.

[0177] Antigen peptides and polypeptides, in the case of MHC class I, consist of 15 residues or less in length, usually about 8 to 11 residues, particularly 9 or 10 residues, while in the case of MHC class II, they may consist of 6 to 30 residues.

[0178] If necessary, longer peptides can be designed in several ways. In some cases, where the potential for peptide presentation on an HLA allele is predicted or known, longer peptides may consist of either (1) each presented peptide having an extension of 2-5 amino acids toward the N-terminus and C-terminus of its corresponding gene product, or (2) a linkage of some or all of the presented peptide with the extended sequence for each. In other cases, where sequencing reveals long (more than 10 residues) neoepitope sequences present in the tumor (e.g., by frameshift, read-through, or intron inclusion resulting in a novel peptide sequence), longer peptides may consist of (3) an entire region of novel tumor-specific or infection-specific amino acids, thus eliminating the need to select the shortest peptide with the most potent HLA presentation based on calculation or in vitro testing. In either case, the use of longer peptides may enable endogenous processing by patient cells, potentially leading to more effective antigen presentation and stimulation of T cell responses. Longer peptides may also include full-length proteins, protein subunits, protein domains, and combinations thereof, such as those expressed in tumors or infectious organisms, respectively. Longer peptides (e.g., full-length proteins, protein subunits, or protein domains) and combinations thereof may be included to stimulate a B-cell response.

[0179] Antigen peptides and polypeptides can be presented on HLA proteins. In some embodiments, antigen peptides and polypeptides are presented on HLA proteins with higher affinity than wild-type peptides. In some embodiments, antigen 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 lower.

[0180] In some embodiments, the antigenic peptides and polypeptides do not induce an autoimmune response and / or induce immune tolerance when administered to a subject.

[0181] Compositions comprising at least two antigenic peptides are also provided. In some embodiments, the composition contains at least two distinct peptides. The at least two distinct peptides may originate from the same polypeptide. Distinct polypeptides mean that the peptides differ in length, amino acid sequence, or both. The peptides may include tumor-specific mutations. Tumor-specific peptides may originate from any polypeptide known or found to contain tumor-specific mutations, or from any polypeptide known or found to have altered expression in tumor cells or cancerous tissue compared to normal cells or tissues, for example, peptides derived from any polypeptide known or found to be abnormally expressed in tumor cells or cancerous tissue compared to normal cells or tissues. The peptides may originate from any polypeptide known or suspected to be associated with infectious organisms, or from any polypeptide known or found to have altered expression in infected cells compared to normal cells or tissues (for example, infectious polynucleotides or polypeptides, which include infectious polynucleotides or polypeptides whose expression is restricted to host cells). Suitable polypeptides from which antigen peptides may be derived can be found, for example, in the COSMIC database or the AACR Genomics Evidence Neoplasia Information Exchange (GENIE) database. COSMIC manages comprehensive information on somatic mutations in human cancers. AACR GENIE aggregates clinical-grade cancer genomic data and correlates them with clinical outcomes from tens of thousands of cancer patients. In some embodiments, tumor-specific mutations are driver mutations specific to certain cancer types. Peptides may include KRAS mutations (e.g., mutations in KRAS G12C, KRAS G12V, KRAS G12D, and / or KRAS Q61H).

[0182] Antigen peptides and polypeptides can be modified to possess desired activity or properties, thereby providing improvements to certain desired attributes, such as pharmacological properties, while simultaneously increasing or at least retaining the biological activity of the unmodified peptide in binding to desired MHC molecules and activating appropriate T cells. For example, antigen peptides and polypeptides can undergo various modifications, such as conservative or non-conservative substitutions, in which case certain advantages in their use may be provided, such as improved MHC binding, stability, or presentation. A conservative substitution means replacing one amino acid residue with another that is biologically and / or chemically similar, for example, replacing one hydrophobic residue with another, or one polar residue with another. Substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effects of single amino acid substitutions can also be probed 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).

[0183] Modification of peptides and polypeptides with various amino acid mimes or non-natural amino acids can be particularly useful in increasing the stability of peptides and polypeptides in vivo. Stability can be assayed in numerous ways. For example, peptidases and 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 delipidated by centrifugation before use. The serum is then diluted to 25% in RPMI tissue culture medium and used to test peptide stability. Small amounts of the reaction solution are taken out at predetermined time intervals and added to an aqueous solution of 6% trichloroacetic acid or ethanol. The turbidity reaction sample is cooled for 15 minutes (4°C), and then the precipitated serum protein is centrifuged to form a pellet. Subsequently, the presence of peptides is determined by reverse-phase HPLC using stability-specific chromatographic conditions.

[0184] Peptides and polypeptides can be modified to obtain desired attributes other than improved serum half-life. For example, the ability of a peptide to stimulate CTL activity can be enhanced by binding to a sequence containing at least one epitope capable of stimulating a T helper cell response. Immunogenic peptide / T helper complexes can be linked by spacer molecules. Spacers typically consist of relatively small neutral molecules, such as amino acids or amino acid mimes, that 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. Spacers present by choice do not need to consist of the same residues and can therefore be heterooligomers or homooligomers. If present, spacers generally consist of at least one or two residues, more commonly three to six residues. Alternatively, peptides can be linked to T helper peptides without the use of spacers.

[0185] Antigen peptides can be directly linked to T helper peptides, or they can be linked via a spacer at either the amino or carboxyl terminus of the peptide. Both the amino terminus of the antigen peptide and the T helper peptide are acylated. Exemplary T helper peptides include tetanus toxoids 830-843, influenza 307-319, and malaria sporozoites 382-398 and 378-389.

[0186] Proteins or peptides can be prepared by any technique known to those skilled in the art, such as the expression of proteins, polypeptides, or peptides by standard molecular biological techniques, the isolation of proteins or peptides from natural products, 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 National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or techniques known to those skilled in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0187] In further embodiments, the antigen includes nucleic acids (e.g., polynucleotides) encoding an antigenic peptide or a portion thereof. The polynucleotides may be single-stranded and / or double-stranded, or natural polynucleotides or stabilized forms of polynucleotides, such as polynucleotides having a thiophosphate backbone, or combinations thereof, such as DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), and may or may not contain introns. The polynucleotide sequence encoding the antigen can be sequence-optimized through transcription, translation, post-transcriptional processing, and / or RNA stability improvements to enhance expression. For example, the polynucleotide sequence encoding the antigen may be codon-optimized. As used herein, “codon optimization” means replacing less frequently used codons with more frequently used synonymous codons with respect to the codon bias of a given organism. Polynucleotide sequences can be optimized to improve post-transcriptional processing, for example, by removing splicing motifs (e.g., standard and / or potential / non-standard splice donor sequences, branched sequences, and / or acceptor sequences) to reduce unintended splicing, and / or by introducing exogenous splicing motifs (e.g., splice donor sequences, branched sequences, and / or acceptor sequences) to favor splicing phenomena over bias. Exogenous intron sequences include, but are not limited to, those derived from SV40 (e.g., SV40 miniintrons) and those derived from immunoglobulins (e.g., human β-globin genes). Exogenous intron sequences can be incorporated between promoter / enhancer sequences and antigen sequences. Exogenous intron sequences for use in expression vectors are detailed in Callendret et al. (Virology. 2007 Jul 5;363(2):288-302), which are incorporated herein by reference for all purposes. Polynucleotide sequences can be optimized to improve transcriptional stability, for example, by removing RNA instability motifs (e.g., AU-rich elements and 3'UTR motifs) and / or repeating nucleotide sequences.Polynucleotide sequences can be optimized to improve accurate transcription, for example, by removing potential transcription initiators and / or terminators. Polynucleotide sequences can also be optimized to improve translation and translation accuracy, for example, by removing potential AUG start codons, early polyA sequences, and / or secondary structure motifs. Polynucleotide sequences can also be optimized to improve nuclear export of transcripts, for example, by adding constitutive transport elements (CTEs), RNA transport factors (RTEs), or Woodchuck post-transcriptional regulatory elements (WPREs). Nuclear export signals for use in expression vectors are detailed in Callendret et al. (Virology. 2007 Jul 5;363(2):288-302), which are incorporated herein by reference for all purposes. Polynucleotide sequences can also be optimized with respect to GC content, for example, to reflect the average GC content of a given organism. Sequence optimization can balance one or more sequence properties, such as transcription, translation, post-transcriptional processing, and / or RNA stability. Sequence optimization can generate optimal sequences that balance transcription, translation, post-transcription processing, and RNA stability. Sequence optimization algorithms such as GeneArt (Thermo Fisher), Codon Optimization Tool (IDT), Cool Tool (University of Singapore), and SGI-DNA (La Jolla, California) are known to those skilled in the art. One or more regions of an antigen-coding protein can be sequence-optimized separately.

[0188] In yet another embodiment, an expression vector capable of expressing a polypeptide or a portion thereof is provided. 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, with the appropriate orientation and correct reading frame for expression. If necessary, the DNA can be ligated to appropriate transcriptional and translational regulatory nucleotide sequences that are recognized by the desired host, although such regulation is generally available in the expression vector. The vector is then introduced into the host using standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0189] V. Vaccine composition This specification also discloses immunogenic compositions, such as vaccine compositions, that have the ability to induce a specific immune response, for example, a tumor-specific immune response or an infectious disease organism-specific immune response. A vaccine composition typically comprises one or more antigens, selected, for example, using the methods described herein, or selected from pathogen-derived peptides, virus-derived peptides, bacterial-derived peptides, fungal-derived peptides, and / or parasite-derived peptides. A vaccine composition may also be called a vaccine.

[0190] The vaccine may contain 1 to 30 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. Peptides may include post-translational modifications. The vaccine contains 1 to 100 or more nucleotide sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 It may contain 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different nucleotide sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different nucleotide sequences, or 12, 13, or 14 different nucleotide sequences.The vaccine contains 1 to 30 antigen sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 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 antigen sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different antigen sequences, or 12, 13, or 14 different antigen sequences.

[0191] The vaccine contains 1 to 30 antigen-coding nucleic acid sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 It may contain 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-coding nucleic acid sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different antigen-coding nucleic acid sequences, or 12, 13, or 14 different antigen-coding nucleic acid sequences. An antigen-coding nucleic acid sequence may refer to an antigen that codes for a portion of the "antigen cassette". The characteristics of the antigen cassette are described in detail herein. An antigen-coding nucleic acid sequence may contain one or more epitope-coding nucleic acid sequences (for example, antigen-coding nucleic acid sequences that code for linked T cell epitopes).

[0192] The vaccine contains 1 to 30 distinct epitope-coding nucleic acid sequences: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 6 It may contain 7, 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 distinct epitope-coding nucleic acid sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 distinct epitope-coding nucleic acid sequences, or 12, 13, or 14 distinct epitope-coding nucleic acid sequences. An epitope-coding nucleic acid sequence may refer to a sequence for an individual epitope sequence, for example, each T cell epitope in an antigen-coding nucleic acid sequence encoding a linked T cell epitope.

[0193] The vaccine may contain at least two repeats of the epitope-coding nucleic acid sequence. As used herein, “repeat” (or interchangeably “repeat”) means two or more repeats of a nucleic acid sequence that codes for the same nucleic acid epitope within an antigen-coding nucleic acid sequence (including any optional 5' linker sequence and / or optional 3' linker sequence as described herein). In one example, the antigen-coding nucleic acid sequence portion of the cassette codes for at least two repeats of the epitope-coding nucleic acid sequence. In a further non-limiting example, the antigen-coding nucleic acid sequence portion of the cassette codes for several distinct epitopes, at least one of which is coded by at least two repeats of the nucleic acid sequence that codes for the distinct epitope (i.e., at least two distinct epitope-coding nucleic acid sequences). In an exemplary non-limiting example, the antigen-coding nucleic acid sequence codes for epitopes A, B, and C, which are coded by the epitope-coding nucleic acid sequence A(E A ), epitope code sequence B(E B ), and epitope code sequence C(E C Exemplary antigen-coding nucleic acid sequences having, as well as at least one repeat of distinct epitopes, are exemplified by, but are not limited to, the following formulas: - Repetition of a distinct epitope (repetition of epitope A): E A -E B -E C -E A ,or E A -E A -E B -E C - Repeats of multiple distinct epitopes (repetitions of epitopes A, B, and C): E A -E B -E C -E A -E B -E C ,or E A -E A -E B -E B -EC -E C - Multiple repetitions of distinct epitopes (repetitions of epitopes A, B, and C): E A -E B -E C -E A -E B -E C -E A -E B -E C ,or E A -E A -E A -E B -E B -E B -E C -E C -E C

[0194] The above examples are not limiting, and an antigen-coding nucleic acid sequence having at least one repeat of distinct epitopes can encode each of the distinct epitopes in any order or frequency. For example, the order and frequency can be a random arrangement of distinct epitopes, e.g., formula E in the example using epitopes A, B, and C. A -E B -E C -E C -E A -E B -E A -E C -E A -E C -E C -E B This is possible.

[0195] Furthermore, this specification also provides an antigen code cassette, which is configured as follows in the 5' to 3' direction: (E x -(E N n ) y ) z It has at least one antigen-coding nucleic acid sequence represented by In the formula, E represents a nucleotide sequence such as a separate epitope-coding nucleic acid sequence. n represents the number of distinct and separate epitope-encoding nucleic acid sequences, and is any integer including 0, E N represents the nucleotide sequence that constitutes each corresponding distinct and separate epitope-encoding nucleic acid sequence of n, for each z-fold repetition, x = 0 or 1, for each n, y = 0 or 1, and at least one of x or y = 1, and z ≥ 2, where the antigen-encoding nucleic acid sequence includes at least two repetitions of E, a given E N , or a combination thereof.

[0196] Each E or E N can independently include any epitope-encoding nucleic acid sequence described herein (e.g., a peptide encoding an infectious disease T cell epitope and / or a neoantigen epitope). For example, each E or E N can independently include a nucleotide sequence represented by formula (L5 b -N c -L3 d ), where N includes a distinct epitope-encoding nucleic acid sequence associated with each E or E N , c = 1, L5 includes a 5' linker sequence, b = 0 or 1, L3 includes a 3' linker sequence, and d = 0 or 1. Epitopes and linkers that can be used are detailed herein, see, for example, the V.A. antigen cassette.

[0197] Repetitions of the epitope-encoding nucleic acid sequence (including optional 5' linker sequence and / or optional 3' linker sequence) can be directly linked linearly to each other (e.g., E A -E A-···). Repeats of an epitope-coding nucleic acid sequence may be delimited by one or more additional nucleotide sequences. In general, repeats of an epitope-coding nucleic acid sequence may be delimited by nucleotide sequences of any size applicable to the compositions described herein. For example, repeats of an epitope-coding nucleic acid sequence may be delimited by separate, distinct epitope-coding nucleic acid sequences (e.g., E A -E B -E C -E A ..., as exemplified above) can be delimited by. In an example where the repeats are delimited by a single separate epitope-coding nucleic acid sequence, and each epitope-coding nucleic acid sequence (including an optional 5' linker sequence and / or an optional 3' linker sequence) codes for a 25-amino acid peptide, the repeats may be delimited by 75 nucleotides, for example, E A -E B -E A In the case of antigen-coding nucleic acids represented by ..., E A It is separated by 75 nucleotides. To illustrate with an example, in an antigen-coding nucleic acid having sequences VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT which encode repeats of the 25-mer antigens Trp1 (VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDTVTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT, the Trp1 repeats are separated by the 25-mer Trp2, and therefore the repeats of the Trp1 epitope-coding nucleic acid sequence are separated by the 75-nucleotide Trp2 epitope-coding nucleic acid sequence. In an example where each repeat is delimited by 2, 3, 4, 5, 6, 7, 8, or 9 separate epitope-coding nucleic acid sequences, and each epitope-coding nucleic acid sequence (including an optional 5' linker sequence and / or an optional 3' linker sequence) codes for a 25-amino acid peptide, each repeat may be delimited by 150, 225, 300, 375, 450, 525, 600, or 675 nucleotides.

[0198] In one embodiment, different peptides and / or polypeptides, or encoding nucleotide sequences, are selected such that the peptides and / or polypeptides have the ability to associate with different MHC molecules, such as different MHC class I molecules and / or different MHC class II molecules. In some embodiments, a single vaccine composition includes a encoding sequence for a peptide and / or polypeptide that has the ability to associate with the most frequently occurring MHC class I molecules and / or different MHC class II molecules. Thus, a vaccine composition may contain different fragments that have the ability to associate with at least two preferred, at least three preferred, or at least four preferred MHC class I molecules and / or different MHC class II molecules.

[0199] The vaccine composition may have the ability to stimulate a specific cytotoxic T cell response and / or a specific helper T cell response.

[0200] The vaccine composition may have the ability to stimulate a specific B cell response (e.g., an antibody response).

[0201] The vaccine composition may have the ability to stimulate a specific cytotoxic T cell response, a specific helper T cell response, and / or a specific B cell response. The vaccine composition may have the ability to stimulate a specific cytotoxic T cell response and a specific B cell response. The vaccine composition may have the ability to stimulate a specific helper T cell response and a specific B cell response. The vaccine composition may have the ability to stimulate a specific cytotoxic T cell response, a specific helper T cell response, and a specific B cell response.

[0202] The vaccine composition may further contain adjuvants and / or carriers. Examples of useful adjuvants and carriers are described below herein. The composition may associate with carriers, such as protein or antigen-presenting cells, such as dendritic cells (DCs) capable of presenting peptides to T cells.

[0203] An adjuvant is any substance that, when mixed into a vaccine composition, enhances or otherwise modifies the immune response to an antigen. The carrier may be a scaffold structure, such as a polypeptide or polysaccharide to which the antigen can be associated. Optionally, the adjuvant is bound by covalent or non-covalent bonds.

[0204] 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, cytotoxicity, or cytokine secretion. Adjuvants can also alter the immune response, for example, by changing a response that is primarily humoral or Th-mediated to a response that is primarily cellular or Th-mediated.

[0205] 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 ISA 206, Montanide ISA 50V, Montanide This list includes, but is not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector systems, PLG microparticles, regiquimod, SRL172, virosoms and other virus-like particles, YF-17D, VEGF traps, R848, beta-glucans, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimes, as well as 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 immunoadjuvants (e.g., MF59) and their preparations have been previously reported (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison AC; Dev Biol Stand. 1998;92:3-11). Cytokines may also be used. Some cytokines are directly related to influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-alpha), promoting the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, which is incorporated specifically herein by reference in whole), and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0206] CpG immunostimulatory oligonucleotides have also been reported to enhance the adjuvant effect in vaccine contexts. Other TLR-binding molecules, such as RNA that binds to TLR7, TLR8, and / or TLR9, may also be used.

[0207] Other 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 can act as therapeutic and / or 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).

[0208] The vaccine composition may contain several different adjuvants. Furthermore, the therapeutic composition may contain any adjuvant substance, including any of the above or any combination thereof. It is also intended that the vaccine and adjuvants may be administered together or separately in any appropriate order.

[0209] The carrier (or excipient) can exist independently of the adjuvant. The function of the carrier may be, for example, to increase the molecular weight of the variant in particular to enhance activity or immunogenicity, to confer stability, to enhance biological activity, or to extend the serum half-life. Furthermore, the carrier can assist in the presentation of the peptide to T cells. The carrier may 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, keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones such as insulin or palmitic acid. In the case of 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.

[0210] Cytotoxic T cells (CTLs) recognize antigens not as intact exogenous antigens themselves, but in the form of peptides bound to MHC molecules. MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, CTL activation is possible when a trimer complex of peptide antigen, MHC molecule, and APC is present. In line with this, the immune response can be enhanced not only when peptides are used to activate CTLs, but also when additional APCs are added along with their respective MHC molecules. Therefore, in some embodiments, the vaccine composition further contains at least one antigen-presenting cell.

[0211] Antigens also include, but are not limited to, lentiviruses, including, second-generation, third-generation, or second / third-generation hybrid lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors). It can also be included in such materials (see Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72(12): 9873-9880, etc.). Depending on the packaging capacity of the above viral vector-based vaccine platform, this method can deliver one or more nucleotide sequences encoding one or more 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 stimulating a host immune response (e.g., CTLs) to the peptide. Vaccine vectors and methods useful for immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). 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.

[0212] VA antigen cassette The selection of one or more antigens, the cloning and construction of "antigen cassettes," and the methods used for their insertion into viral vectors are within the scope of the skill of those skilled in the art, given the teachings provided herein. "Antigen cassette" or "cassette" means a combination of a selected antigen or a group of antigens (e.g., an antigen-coding nucleic acid sequence) and other regulatory elements necessary to transcribe the antigen and express the transcribed product. The selected antigen or a group of antigens may refer to distinct epitope sequences; for example, the antigen-coding nucleic acid sequence in the cassette may encode an epitope-coding nucleic acid sequence (or a group of epitope-coding nucleic acid sequences) such that the epitope is transcribed and expressed. The antigen or a group of antigens may be functionally linked to regulatory components in a transcribable manner. Such components include conventional regulatory elements that can induce antigen expression in cells transfected with a viral vector. Thus, an antigen cassette may also contain a selected promoter linked to the antigen and positioned within the selected viral sequence of the recombinant vector together with other optional regulatory elements. The cassette may contain one or more antigens, such as pathogen-derived peptides, virus-derived peptides, bacterial-derived peptides, fungal-derived peptides, parasite-derived peptides, and / or tumor-derived peptides. The cassette may have one or more antigen-coding nucleic acid sequences, for example, cassettes containing multiple antigen-coding nucleic acid sequences may be functionally linked to separate promoters independently and / or linked together using other multicistonic systems, such as 2A ribosome skipping sequence elements (e.g., E2A, P2A, F2A, or T2A sequences) or intra-sequence ribosome entry site (IRES) sequence elements. The linker may also have cleavage sites, such as TEV or furin cleavage sites. A linker having a cleavage site can be used in combination with other elements, such as those in a multicistronic system.To illustrate with a non-limiting example, a furin protease cleavage site can be used in combination with a 2A ribosome skipping sequence element so that the furin protease cleavage site is configured to facilitate the removal of the post-translational 2A sequence. In a cassette containing multiple antigen-coding nucleic acid sequences, each antigen-coding nucleic acid sequence may contain one or more epitope-coding nucleic acid sequences (e.g., antigen-coding nucleic acid sequences encoding linked T cell epitopes).

[0213] Useful promoters can be constitutive or regulatory (inducible) promoters, allowing control over the amount of antigen 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 cytoplasmic beta-actin promoter [TAKost et al, Nucl. Acids Res., 11(23):8287 (1983)]. Other suitable or preferred promoters may be selected by those skilled in the art.

[0214] The antigen cassette may also contain nucleic acid sequences heterogeneous to the viral vector sequence, including sequences that provide a signal for efficient polyadenylation of the transcript (poly(A), polyA, or pA), and introns having functional splice donor and splice acceptor sites. A common polyA sequence used in the exemplary vector of the present invention is derived from papovavirus SV-40. The polyA sequence can generally 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 called SV-40 T intron sequences. The antigen cassette may also contain introns positioned between the promoter / enhancer sequence and the antigen. 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 therein], and many such sequences are available from commercial and industrial sources as well as from Genbank.

[0215] An antigen cassette can have one or more antigens. For example, a given cassette may 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 can be linked to each other directly. Antigens can also be linked to each other with linkers. Antigens can be oriented in any direction relative to each other, such as N to C or C to N.

[0216] As described elsewhere in this specification, the antigen cassette may be located at any selective deletion site within a viral vector, such as a structural protein with a VEE backbone deleted, or at the site of an E1 gene region deletion or an E3 gene region deletion in a ChAd-based vector, among other selectable sites.

[0217] The antigen cassette is represented by the following formula to show an arrangement of each element in the order 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 expressed using the formula, where P and P2 comprise promoter nucleotide sequences, N comprises an MHC class I epitope coding nucleic acid sequence, L5 comprises a 5' linker sequence, L3 comprises a 3' linker sequence, G5 comprises a nucleic acid sequence encoding an amino acid linker, G3 comprises one of at least one nucleic acid sequences encoding an amino acid linker, U comprises an MHC class II antigen coding nucleic acid sequence, for each X, the corresponding Nc is an epitope encoding a nucleic acid sequence, and for each Y, the corresponding Uf is an MHC class II epitope coding nucleic acid sequence (e.g., a universal MHC class II epitope coding nucleic acid sequence). The universal sequence may contain at least one of tetanus toxoid and PADRE. The universal sequence may contain a tetanus toxoid peptide. The universal sequence may contain a PADRE peptide. The universal sequence may contain tetanus toxoid and PADRE peptide. The composition and regular 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.

[0218] In one example, the elements present include a=0, b=1, d=1, e=1, g=1, h=0, X=10, Y=2, Z=1, and W=1, which indicates that there are no additional promoters (only promoter nucleotide sequences provided by a vector skeleton such as the RNA alphavirus or ChAdV skeleton are present), there are 10 MHC class I epitopes, a 5' linker is present on each N, a 3' linker is present on each N, there are 2 MHC class II epitopes, there is a linker linking the 2 MHC class II epitopes, there is a linker linking the 5' ends of the 2 MHC class II epitopes to the 3' linker of the final MHC class I epitope, and there is a linker linking the 3' ends of the 2 MHC class II epitopes to the vector skeleton (e.g., the ChAdV or RNA alphavirus skeleton).

[0219] Examples of ligating the 3' end of the antigen cassette to a vector backbone (e.g., an RNA alphavirus backbone) include direct ligation to a 3'UTR element provided by the vector backbone, such as a 3' 19nt CSE. Examples of ligating the 5' end of the antigen cassette to a vector backbone (e.g., an RNA alphavirus backbone) include direct ligation to a promoter or 5'UTR element of the vector backbone, such as a subgenome promoter sequence (e.g., a 26S subgenome promoter sequence), an alphavirus 5'UTR, a 51nt CSE, or a 24nt CSE.

[0220] Other examples include: a = 1, indicating that there is a promoter other than the promoter nucleotide sequence provided by the vector backbone (e.g., the backbone of ChAdV or RNA alphavirus); when a = 1 and Z is greater than 1, there are multiple promoters other than the promoter nucleotide sequence provided by the vector backbone, each inducing the expression of one or more distinct MHC class I epitopes encoding nucleic acid sequences; h = 1, indicating that there is a separate promoter for inducing the expression of the MHC class II epitope-encoding nucleic acid sequence; and g = 0, indicating that the MHC class II epitope-encoding nucleic acid sequence, if present, is directly linked to the vector backbone (e.g., the backbone of ChAdV or RNA alphavirus). For example, the ChAdV vector backbone can have an antigen cassette under the control of a CMV promoter / enhancer.

[0221] Other examples include that each MHC class I epitope present may have a 3' linker if it can have a 5' linker, may not have either, or may have both. In examples where multiple MHC class I epitopes are present within the same antigen cassette, some MHC class I epitopes may have both a 5' and a 3' linker, while other MHC class I epitopes may have a 5' linker, a 3' linker, or neither. In other examples where multiple MHC class I epitopes are present within the same antigen cassette, some MHC class I epitopes may have either a 5' or a 3' linker, while other MHC class I epitopes may have a 5' linker, a 3' linker, or neither.

[0222] In examples where multiple MHC class II epitopes are present within the same antigen cassette, some MHC class II epitopes may have both a 5' linker and a 3' linker, while other MHC class II epitopes may have a 5' linker, a 3' linker, or neither. In other examples where multiple MHC class II epitopes are present within the same antigen cassette, some MHC class II epitopes may have either a 5' linker or a 3' linker, while other MHC class II epitopes may have a 5' linker, a 3' linker, or neither.

[0223] Other examples include cases where each antigen present may have a 5' linker, may have a 3' linker, may have neither, or may have both. In examples where multiple antigens are present within the same antigen cassette, some antigens may have both a 5' linker and a 3' linker, while other antigens may have a 5' linker, a 3' linker, or neither. In other examples where multiple antigens are present within the same antigen cassette, some antigens may have either a 5' linker or a 3' linker, while other antigens may have a 5' linker, a 3' linker, or neither.

[0224] The promoter nucleotide sequences P and / or P2 can be the same as the promoter nucleotide sequences provided by a vector backbone such as an RNA alphavirus backbone. For example, Pn and P2, which are promoter sequences provided by an RNA alphavirus backbone, can each include a subgenomic promoter sequence (e.g., a 26S subgenomic promoter sequence) or a CMV promoter. The promoter nucleotide sequences P and / or P2 can be different from the promoter nucleotide sequences provided by a vector backbone (e.g., the backbone of a ChAdV or an RNA alphavirus) and can also be different from each other.

[0225] The 5' linker, L5, can be a natural or non-natural sequence. Non-natural sequences include, but are not limited to, AAY, RR, and DPP. The 3' linker, L3, can also be a natural or non-natural sequence. Furthermore, both L5 and L3 can be natural sequences, both can be non-natural sequences, or one can be natural and the other non-natural. For each X, the amino acid linkers are 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, 5 The amino acid lengths may be 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more. For each X, the amino acid linker may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid lengths.

[0226] For each Y, the amino acid linker G5 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, The amino acid lengths 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. For each Y, the amino acid linker may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid lengths.

[0227] Amino acid linker G3 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, The amino acid lengths may be 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 can also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid lengths.

[0228] For each X, each N can encode an MHC class I epitope, an MHC class II epitope, an epitope / antigen capable of stimulating a B cell response, or a combination thereof. For each X, each N can encode an MHC class I epitope, an MHC class II epitope, and a combination of epitopes / antigens capable of stimulating a B cell response. For each X, each N can encode a combination of an MHC class I epitope and an MHC class II epitope. For each X, each N can encode an MHC class I epitope and a combination of epitopes / antigens capable of stimulating a B cell response. For each X, each N can encode an MHC class II epitope. For each X, each N can encode an epitope / antigen capable of stimulating a B cell response. For each X, each N can encode an MHC class I epitope of 7–15 amino acid length. For each X, each N can also encode an MHC class I epitope 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 acid length. For each X, each N can also encode an MHC class I epitope 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 acid length.

[0229] A cassette encoding one or more antigens may be 700 nucleotides or less in length. A cassette encoding one or more antigens may be 700 nucleotides or less in length and may encode two distinct epitope-coding nucleic acid sequences (for example, it may encode two distinct nucleic acid sequences derived from an immunogenic polypeptide, derived from an infection or tumor). A cassette encoding one or more antigens may be 700 nucleotides or less in length and may encode at least two distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 700 nucleotides or less in length and may encode three distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 700 nucleotides or less in length and may encode at least three distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 700 nucleotides or less in length and may contain 1 to 10, 1 to 5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0230] A cassette encoding one or more antigens may be 375–700 nucleotides long. A cassette encoding one or more antigens may be 375–700 nucleotides long and may encode two distinct epitope-coding nucleic acid sequences (for example, it may encode two distinct infection or tumor-derived nucleic acid sequences encoding immunogenic polypeptides). A cassette encoding one or more antigens may be 375–700 nucleotides long and may encode at least two distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 375–700 nucleotides long and may encode three distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 375–700 nucleotides long and may encode at least three distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 375–700 nucleotides long and may contain 1–10, 1–5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0231] A cassette encoding one or more antigens may be 600, 500, 400, 300, 200, or 100 nucleotides long or less. A cassette encoding one or more antigens may be 600, 500, 400, 300, 200, or 100 nucleotides long or less and may encode two distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 600, 500, 400, 300, 200, or 100 nucleotides long or less and may encode at least two distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 600, 500, 400, 300, 200, or 100 nucleotides long or less and may encode three distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 600, 500, 400, 300, 200, or 100 nucleotides long or less, and may encode at least three distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 600, 500, 400, 300, 200, or 100 nucleotides long or less, and may contain 1 to 10, 1 to 5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0232] A cassette encoding one or more antigens may be 375–600, 375–500, or 375–400 nucleotides long. A cassette encoding one or more antigens may be 375–600, 375–500, or 375–400 nucleotides long and can encode two distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 375–600, 375–500, or 375–400 nucleotides long and can encode at least two distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 375–600, 375–500, or 375–400 nucleotides long and can encode three distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 375–600, 375–500, or 375–400 nucleotides long and may encode at least three distinct epitope-coding nucleic acid sequences. A cassette encoding one or more antigens may be 375–600, 375–500, or 375–400 nucleotides long and may contain 1–10, 1–5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0233] In some cases, an antigen or epitope within a cassette encoding additional antigens and / or epitopes may be an immunodominant epitope to others it encodes. Immunodominance is generally a bias of the immune response to only one or a few specific immunogenic peptides. Immunodominance can be assessed as part of an immune monitoring protocol. For example, immunodominance can be assessed by evaluating the T cell and / or B cell response to the encoded antigen.

[0234] Immunodominance can be assessed as the effect that the presence of an immunodominant antigen has on the immune response to one or more other antigens. For example, an immunodominant antigen and its respective immune response (e.g., an immunodominant MHC class I epitope) can reduce the immune response to another antigen compared to the immune response in the absence of the immunodominant antigen. This reduction may be such that the immune response in the presence of the immunodominant antigen is no longer considered a therapeutically effective response. For example, an MHC class I epitope is generally considered immunodominant if the T cell response to another antigen is no longer considered a therapeutically effective response compared to the response stimulated in the absence of the immunodominant MHC class I epitope. The immune response may also be reduced to below or near the detection limit compared to the response in the absence of the immunodominant antigen. For example, an MHC class I epitope is generally considered immunodominant if the T cell response to another antigen is below the detection limit compared to the response stimulated in the absence of the immunodominant MHC class I epitope. Generally, immunodominance is assessed between two antigens capable of stimulating an immune response, for example, between two T cell epitopes in a vaccine composition administered to a subject having a congeneral MHC allele known or predicted to present each epitope. Immunodominance can be assessed by evaluating the relative immune response to other antigens in the presence and absence of a suspected immunodominant antigen.

[0235] Immunodominance can be assessed as the relative difference in immune responses between two or more antigens. Immunodominance may refer to an immune response to a particular antigen that is 5, 10, 20, 30, 40, or 50 times stronger than another antigen encoded in the same cassette. Immunodominance may refer to an immune response to a particular antigen that is 100, 200, 300, 400, or 500 times stronger than another antigen encoded in the same cassette. Immunodominance may refer to an immune response to a particular antigen that is 1000, 2000, 3000, 4000, or 5000 times stronger than another antigen encoded in the same cassette. Immunodominance may refer to an immune response to a particular antigen that is 10,000 times stronger than another antigen encoded in the same cassette.

[0236] In some cases, it may be desirable to avoid vaccine compositions containing immunodominant epitopes. For example, it may be desirable to avoid designing vaccine cassettes that encode immunodominant epitopes. While we do not wish to be bound by theory, administering and / or encoding immunodominant epitopes together with additional epitopes may reduce the immune response to those additional epitopes, including the possibility that it may ultimately reduce the vaccine's effectiveness against those additional epitopes. As an exemplary, non-limiting example, vaccine compositions such as those containing TP53-related neoepitopes may bias the immune response, e.g., the T-cell response, towards the TP53-related neoepitope, negatively impacting the immune response to other antigens or epitopes in the vaccine composition (e.g., one or more KRAS-related neoepitopes in the vaccine composition) (e.g., reducing the immune response to a point where it is not a therapeutically effective response and / or below the detection limit). Therefore, vaccine compositions can be designed to not contain immunodominant epitopes, for example, vaccine cassettes (e.g., (neo)antigen-coding cassettes) can be designed not to encode immunodominant epitopes. For example, a cassette does not encode an epitope that, when administered to a subject as part of a vaccine composition, would reduce the immune response to another epitope encoded within the cassette compared to the immune response when that other epitope is administered in the absence of an immunodominant MHC class I epitope. In another example, a cassette does not encode an epitope that, when administered to a subject as part of a vaccine composition, would reduce the immune response to another epitope encoded within the cassette to below the detection limit compared to the immune response when that other epitope is administered in the absence of an immunodominant MHC class I epitope. In another example, the cassette, when administered to a subject as part of a vaccine composition, reduces the immune response to another epitope encoded within the cassette compared to the immune response when that other epitope is administered in the absence of an immunodominant MHC class I epitope, and does not encode an epitope whose immune response is not therapeutically effective.In another example, the cassette does not encode an epitope that stimulates an immune response 5, 10, 20, 30, 40, or 50 times or more compared to another epitope encoded within the same cassette in the vaccine composition administered to the target, in which case each antigen has the ability to stimulate an immune response in the target. In yet another example, the cassette does not encode an epitope that stimulates an immune response 100, 200, 300, 400, or 500 times or more compared to another epitope encoded within the same cassette in the vaccine composition administered to the target, in which case each antigen has the ability to stimulate an immune response in the target. In another example, the cassette does not encode an epitope that stimulates an immune response 1,000, 2,000, 3,000, 4,000, or 5,000 times or more compared to another epitope encoded within the same cassette in the vaccine composition administered to the target, in which case each antigen has the ability to stimulate an immune response in the target. In yet another example, the cassette does not encode an epitope that results in an immune response 10,000 times or more compared to another epitope encoded within the same cassette in the vaccine composition administered to the target, in which case each antigen has the ability to stimulate an immune response in the target.

[0237] VB immunomodulator The vectors described herein, such as the ChAdV vector or the alphavirus vector described herein, may contain nucleic acids encoding at least one antigen, and the same or different vectors may contain nucleic acids encoding at least one immunomodulator. Immunomodulators may include binding molecules (e.g., antibodies such as scFv) that bind to immune checkpoint molecules and block their activity. Immunomodulators may include cytokines such as IL-2, IL-7, IL-12 (including p35, p40, p70, and / or p70 fusion constructs of IL-12), IL-15, or IL-21. Immunomodulators may include modified cytokines (e.g., pegIL-2). A vector may contain an antigen cassette and one or more nucleic acid molecules encoding immunomodulators.

[0238] Exemplary 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 on all NK, γδ, and memory CD8+(αβ) T cells), CD160 (also known as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, their antigen-binding fragments, or other binding proteins that bind to one or more of the following: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN-15049, thereby blocking or inhibiting their activity. Exemplary 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 sequences encoding the antibodies can be manipulated within vectors such as C68 using common skills in the art. Exemplary methods are 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 are incorporated herein by reference for all purposes.

[0239] Additional considerations for the design and manufacture of VC vaccines VC1. Determination of the peptide set targeting all tumor subclones. Truncal peptides, which are peptides presented by all or most tumor subclones, can be prioritized for inclusion in the vaccine. If, by choice, no trunk peptides are presented and predicted to be highly immunogenic, or if the number of presented and predicted to be highly immunogenic trunk peptides is small enough to allow for the inclusion of additional non-truncal peptides 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.

[0240] VC2. Antigen prioritization Even after all of the above antigen filters have been applied, there may still be more candidate antigens available for inclusion in a vaccine than can be supported by vaccine technology. Furthermore, various aspects of antigen analysis may still be uncertain, and trade-offs may exist between different properties of vaccine antigen candidates. Therefore, instead of predetermined filters at each step of the selection process, an integrated multidimensional model can be considered, i.e., a model that places candidate antigens in a space having at least the following axes and optimizes selection using an integrated method. 1. Risk of autoimmunity or tolerance (germline risk) (typically, a lower risk of autoimmunity is preferable) 2. Probability of sequencing artifacts (typically, a lower probability of artifacts is preferable) 3. Probability of immunogenicity (typically, a higher probability of immunogenicity is preferable) 4. Probability of presentation (typically, a higher probability of presentation is preferable) 5. Gene expression (typically, higher expression is preferable) 6. Target range of HLA genes (The larger the number of HLA molecules involved in the presentation of a set of antigens, the lower the probability that tumors, infectious diseases, and / or infected cells can escape immune attack through downregulation or mutation of HLA molecules) 7. Target range of HLA classes (By targeting both HLA-I and HLA-II, the probability of therapeutic effect can be increased and the probability of escape of tumors or infectious diseases can be decreased)

[0241] Furthermore, optionally, if an antigen is predicted to be presented by an HLA allele that is lost or inactivated in all or part of the patient's tumor or infected cells, those antigens can be removed (e.g., excluded) from the vaccination priority. Loss of HLA alleles occurs by any of somatic mutations, loss of heterozygosity, or deletion of homozygosity at the locus. Methods for detecting somatic mutations of HLA alleles are well known in the art (e.g., Shukla et al., 2015). Methods for detecting somatic LOH (loss of heterozygosity) and deletion of homozygosity (including HLA loci) have also been well reported. (Carter et al., 2012, McGranahan et al., 2017; Van Loo et al., 2010). Antigens can also be removed from the priority if it is shown from mass spectrometry data that the predicted antigen is not presented by the predicted HLA allele.

[0242] V.D. Self-amplifying RNA vector Generally, all self-amplifying RNA (SAM) vectors contain a self-amplifying backbone derived from a self-replicating virus. The term “self-amplifying backbone” refers to the minimal sequence of a self-replicating virus that enables the self-replication of the viral genome. For example, the minimal sequence that enables the self-replication of alphaviruses may include conserved sequences for non-structural protein-mediated amplification (e.g., non-structural protein 1 (nsP1) gene, nsP2 gene, nsP3 gene, nsP4 gene, and / or poly-A sequence). The self-amplifying backbone may also include sequences for the expression of subgenomic RNAs of the virus (e.g., the 26S promoter element in alphaviruses). SAM vectors can be positive-strand RNA polynucleotides or negative-strand RNA polynucleotides, such as vectors with a backbone derived from a positive-strand or negative-strand self-replicating virus. Self-replicating viruses include, but are not limited to, alphaviruses, flaviviruses (e.g., Kunzin virus), measles virus, and rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus). Examples of SAM vector systems derived from self-replicating viruses are detailed in Lundstrom (Molecules.2018 Dec 13;23(12).pii:E3310.doi:10.3390 / molecules23123310), which are incorporated herein by reference for all purposes.

[0243] VD1. Alphavirus Biology Alphaviruses are members of the Togaviridae family and are single-stranded positive-sense RNA viruses. Their members are typically classified into Old World viruses such as Sindbis virus, Ross River virus, Mayarovirus, Chikungunya virus, and Semlik Forest virus, or New World viruses such as Eastern Equine Encephalitis virus, Aura virus, Fort Morgan virus, or Venezuelan Equine Encephalitis virus and its derivative TC-83 (Strauss Microbial Review 1994). Natural alphavirus genomes are typically about 12 kb in length, with the first two-thirds containing genes encoding non-structural proteins (nsPs) that form RNA replication complexes for viral genome self-replication, and the last third containing subgenome expression cassettes encoding structural proteins for virion production (Frolov RNA 2001).

[0244] The model life cycle of alphaviruses involves several distinct stages (Strauss Microbial Review 1994, Jose Future Microbiol 2009). Following viral adsorption to host cells, virions fuse with the membrane within the intracellular compartment, ultimately leading to the release of genomic RNA into the cytosol. The genomic RNA, oriented in a positive-straight orientation and containing a 5' methylguanylate cap and a 3' poly-A tail, is translated to produce non-structural proteins nsP1-4, which form a replication complex. In the early stages of infection, the positive-straight strand is then replicated by the complex to a negative-straight strand template. In current models, the replication complex undergoes further processing as infection progresses, and the resulting processed complex switches to transcription of the negative-straight strand into both the full-length positive-straight genomic RNA and the positive-straight RNA of the 26S subgenome containing structural genes. Several conserved sequence elements (CSEs) of alphaviruses have been shown to play roles in various RNA replication steps, including the complementary 5'UTR in the replication of positive-strand RNA from a negative-strand template, the 51nt CSE in the replication of negative-strand synthesis from a genomic template, the 24nt CSE in the junction region between nsP and 26S RNA in the transcription of subgenomic RNA from the negative-strand, and the 3' 19nt CSE in the synthesis of negative-strand synthesis from a positive-strand template.

[0245] Following the replication of various RNA species, viral particles are typically constructed in the natural life cycle of the virus. 26S RNA is translated, and the resulting proteins undergo further processing to produce structural proteins such as the capsid protein, glycoproteins E1 and E2, and two small polypeptides, E3 and 6K (Strauss 1994). Capsid formation of the viral RNA occurs, usually with a capsid protein specific only to the genomic RNA that is packaged, after which the virion is assembled and budding onto the membrane surface.

[0246] VD2. Alphavirus as a delivery vector Alphaviruses (including alphavirus sequences, characteristics, and other elements) can be used to generate alphavirus-based delivery vectors (also known as alphavirus vectors, alphavirus viral vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying mRNA (SAM) vectors). Alphaviruses have long been engineered for use as expression vector systems (Pushko 1997, Rheme 2004). Alphaviruses offer several advantages, particularly in vaccine situations where heterologous antigen expression may be desired. Due to their ability to self-replicate in the host cytosol, alphavirus vectors can generally generate many copy number expression cassettes within cells, resulting in high levels of heterologous antigen generation. Furthermore, because vectors are generally transient, this leads to improved biosafety and reduced induction of immune tolerance to the vector. Generally, the public also lacks pre-existing immunity to alphavirus vectors compared to other standard viral vectors such as human adenovirus. Alphavirus-based vectors also generally produce a cytotoxic response to infected cells. Cytotoxicity can be important to some extent in the context of vaccines, in order to appropriately stimulate an immune response to the expressed heterologous antigen. However, the desired degree of cytotoxicity can be delicate, so several attenuated alphaviruses have been developed, including the TC-83 strain of VEE. Therefore, an example of an antigen expression vector described herein can utilize an alphaviral scaffold that enables high levels of antigen expression, stimulates a strong immune response to the antigen, does not stimulate an immune response to the vector itself, and is usable in a safe manner. Furthermore, the antigen expression cassette can be designed to stimulate different levels of immune response through optimization of the alphaviral sequence used by the vector, including but not limited to sequences derived from VEE or its attenuated derivative TC-83.

[0247] Several expression vector design strategies have been designed using alphavirus sequences (Pushko 1997). One strategy involves inserting a second copy of the 26S promoter sequence element downstream of a structural protein gene, followed by the insertion of a heterologous gene (Frolov 1993). Thus, in addition to native non-structural and structural proteins, an additional subgenomic RNA expressing the heterologous protein is generated. In this system, since all elements for the generation of infectious virions are present, repeated infection of the expression vector in uninfected cells can occur.

[0248] Another expression vector design utilizes a helper virus system (Pushko 1997). In this strategy, the structural protein is replaced by a heterologous gene. Thus, following the self-replication of viral RNA mediated by the still intact non-structural gene, the expression of the heterologous protein is provided by the 26S subgenomic RNA. Traditionally, the additional vector expressing the structural protein is then supplied trans-transfected by co-transfection of the cell line, etc., to generate an infectious virus. The system is detailed in USPN 8,093,021, which is incorporated herein by reference in its entirety for all purposes. The helper vector system offers the benefit of limiting the possibility of infectious particle formation, and therefore improves biosafety. Furthermore, the helper vector system shortens the overall vector length and improves replication and expression efficiency. Thus, an example of an antigen expression vector described herein can utilize an alphaviral backbone, where the structural protein is replaced by an antigen cassette, and the resulting vector reduces biosafety issues while promoting efficient expression by reducing the overall size of the expression vector.

[0249] VD3. In vitro generation of self-amplifying viruses A well-known and convenient technique in the art for RNA generation is in vitro transcription (IVT). In this technique, a DNA template of the desired vector is first generated by techniques well known to those skilled in the art, such as cloning, restriction enzyme digestion, ligation, gene synthesis (e.g., chemical synthesis and / or enzymatic synthesis), and polymerase chain reaction (PCR).

[0250] The DNA template contains an RNA polymerase promoter at the 5' end of the sequence to be transcribed into RNA (e.g., SAM). Promoter options include, but are not limited to, bacteriophage polymerase promoters such as T3, T7, K11, or SP6. Depending on the specific RNA polymerase promoter sequence selected, additional 5' nucleotides can be transcribed in addition to the desired sequence. For example, the standard T7 promoter may be referenced by the sequence TAATACGACTCACTATAGG, where an IVT reaction using the DNA template TAATACGACTCACTATAGGN yields the mRNA sequence GG-N. While we do not wish to be constrained by theory, generally, T7 polymerase more efficiently transcribes RNA transcripts beginning with guanosine. If additional 5' nucleotides are not desired (e.g., no additional GG), the RNA polymerase promoter contained in the DNA template may be a sequence that yields a transcript containing only the 5' nucleotide of the desired sequence (e.g., SAM with the natural 5' sequence of the self-replicating virus from which the SAM vector originates). For example, the minimal T7 promoter may be referenced by the sequence TAATACGACTCACTATA, where the mRNA sequence N is obtained by an IVT reaction using the DNA template TAATACGACTCACTATAN for the generation of the desired sequence N. Similarly, the minimal SP6 promoter referenced by the sequence ATTTAGGTGACACTATA can be used to generate a transcript that does not contain an additional 5' nucleotide. In a typical IVT reaction, the DNA template is incubated with the appropriate RNA polymerase enzyme, buffer, and nucleotides (NTPs).

[0251] The resulting RNA polynucleotides can be further modified optionally, including, but not limited to, the addition of a 5' cap structure such as 7-methylguanosine or related structures, and optionally, modification of the 3' end to include a polyadenylated (poly-A) tail. In the modified IVT reaction, the RNA is capped with a 5' cap structure by co-transcription via the addition of a cap analog during IVT. The cap analogs include dinucleotides (m 7 G-ppp-N) cap analog or trinucleotide (m 7 The G-ppp-NN) cap analogue may be included, where N represents a nucleotide or modified nucleotide (e.g., ribonucleosides such as adenosine, guanosine, cytidine, and uridine, but not limited to these). Exemplary cap analogues and their use in IVT reactions are also detailed in U.S. Patent No. 10,519,189, which is incorporated herein by reference for all purposes. As discussed, T7 polymerase more efficiently transcribes RNA transcripts that begin with guanosine. To improve transcription efficiency with templates that do not begin with guanosine, trinucleotide cap analogues (m 7 G-ppp-NN) can be used. Trinucleotide cap analogs can be dinucleotide cap analogs (m 7 Compared to IVT reactions using G-ppp-N), the transcription efficiency can be increased by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more.

[0252] The 5' cap structure can also be added after transcription using a vaccinia capping system containing mRNA 2'-O-methyltransferase and S-adenosylmethionine (e.g., NEB catalog number M2080).

[0253] The resulting RNA polynucleotides may be further modified, at the discretion of the authorities, in addition to or apart from the described capping techniques, including, but not limited to, modifying the 3' end to include a polyadenylated (poly-A) tail.

[0254] Subsequently, the RNA can be purified using techniques well known in the art, such as phenol-chloroform extraction or column purification (e.g., purification by chromatography).

[0255] VD4. Delivery by lipid nanoparticles An important aspect to consider in vaccine vector design is immunity to the vector itself (Riley 2017). This can be in the form of pre-existing immunity to the vector itself, as in the case of certain human adenovirus strains, or it can be in the form of immunity to the vector developing after vaccine administration. The latter is an important consideration when multiple doses of the same vaccine are administered, such as when priming and booster doses are used, or when the same vaccine vector system is used to deliver different antigen cassettes.

[0256] In the case of alphaviral vectors, the standard delivery method is the previously discussed helper virus system, which provides the capsid protein, E1 protein, and E2 protein in trans to generate infectious viral particles. However, it is important to note that the E1 and E2 proteins are often the primary targets of neutralizing antibodies (Strauss 1994). Therefore, if the infectious particles are targeted by neutralizing antibodies, the effectiveness of using alphaviral vectors to deliver the target antigen to target cells may be reduced.

[0257] An alternative to virus particle-mediated gene delivery is the use of nanomaterials for delivering expression vectors (Riley 2017). Importantly, nanomaterial vehicles can be fabricated from non-immunogenic materials and generally avoid the stimulation of immunity to the delivery vector itself. These materials may include, but are not limited to, lipids, inorganic nanomaterials, and other polymer materials. Lipids may be cationic, anionic, or neutral. Materials may be synthetic or naturally occurring and may be biodegradable in some cases. Lipids may include, but are not limited to, fats, cholesterol, phospholipids, and lipid complexes, including polyethylene glycol (PEG) complexes (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins.

[0258] Lipid nanoparticles (LNPs) are an attractive delivery system because the amphiphilic nature of lipids allows for the formation of membrane and vesicle-like structures (Riley 2017). Generally, these vesicles deliver expression vectors by absorption into the membrane of target cells and release of 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 cases, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that facilitate the binding of additional parts. The lipid composition can affect the overall size and stability of the LNP. For example, the lipid composition may include dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. MC3 and MC3-like lipid compositions may be formulated to include one or more other lipids, such as PEG or PEG-bound lipids, sterols, or neutral lipids.

[0259] Nucleic acid vectors, such as expression vectors, that are directly exposed to serum can result in several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target immune system stimulation by free nucleic acids. Therefore, encapsulation of alphaviral vectors can be used to avoid degradation and potential off-target effects. In certain examples, the alphaviral vector is completely encapsulated within a delivery vehicle, for example, within the aqueous interior of an LNP. Intra-LNP encapsulation of alphaviral vectors can be carried out by techniques well known to those skilled in the art, such as microfluidic mixing and droplet generation in microfluidic droplet generation devices. Such devices include, but are not limited to, standard T-shaped devices or flow-focus devices. In one example, a desired lipid formulation, such as a composition containing MC3 or an MC3-like substance, is supplied to the droplet generation device in parallel with the alphaviral delivery vector and other desired substances, and the delivery vector and desired substances are completely encapsulated within an LNP using MC3 or an MC3-like substance. In one example, the droplet generation device can control the size range and particle size distribution of the produced LNP. For example, LNPs may have diameters ranging from 1 to 1000 nanometers, e.g., in the range of 1, 10, 50, 100, 500, or 1000 nanometers. After droplet generation, the delivery vehicles encapsulating the expression vectors can be further processed or modified to prepare them for administration.

[0260] VE Chimpanzee Adenovirus (ChAd) VE1. Virus delivery by chimpanzee adenovirus Vaccine compositions for the delivery of one or more antigens (e.g., via an antigen cassette) can be prepared 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) (see Sequence ID No. 1) can be used in vaccine compositions for antigen delivery. The C68 adenovirus-derived vector is detailed in USPN 6,083,716, which is incorporated herein by reference in its entirety for all purposes. Vectors and delivery systems using ChAdV68 are detailed in U.S. Patent Application Publication US20200197500A1 and International Patent Application Publication WO2020243719A1, each of which is incorporated herein by reference for all purposes.

[0261] In a further embodiment, recombinant adenoviruses containing the DNA sequence of a chimpanzee adenovirus such as C68, and antigen cassettes functionally linked to a regulatory sequence that leads to the expression of the recombinant virus, are provided herein. The recombinant virus is capable of infecting mammalian cells, preferably human cells, and of expressing an antigen cassette product in the cells. The vector can be used to delete the native chimpanzee E1 gene and / or the E3 gene and / or the E4 gene. An antigen cassette can be inserted into any of these gene deletion sites. The antigen cassette may contain an antigen of which an antigen-stimulated immune response is desired.

[0262] In another embodiment, mammalian cells infected with chimpanzee adenovirus such as C68 are provided herein.

[0263] 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.

[0264] In yet another embodiment, a method for delivering an antigen cassette to a mammalian cell is provided herein, comprising the step of introducing an effective amount of a chimpanzee adenovirus, such as C68, which has been engineered to express the antigen cassette, into the cell.

[0265] In yet another embodiment, a method is provided for stimulating an immune response in a mammalian host to treat cancer. This method may include administering to the host an effective dose of a recombinant chimpanzee adenovirus, such as C68, which contains an antigen cassette encoding one or more antigens from a tumor that is the target of the immune response.

[0266] In yet another embodiment, a method is provided for stimulating an immune response in a mammalian host to treat or prevent a disease in an infectious disease or the like. This method may include administering to a host an effective dose of a recombinant chimpanzee adenovirus, such as C68, which contains an antigen cassette encoding one or more antigens from an infectious disease or the like that is the target of the immune response.

[0267] We also disclose non-monkey mammalian cells expressing the chimpanzee adenovirus gene obtained from the sequence of Sequence ID No. 1. The gene may 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.

[0268] Also disclosed are nucleic acid molecules containing a chimpanzee adenovirus DNA sequence that includes a gene obtained from the sequence of Sequence ID No. 1. The gene may 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 includes Sequence ID No. 1. In some embodiments, the nucleic acid molecule includes the sequence of Sequence ID No. 1 and lacks 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.

[0269] Also disclosed are a vector 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 lead to the expression of such cassette in a heterologous host cell, wherein the chimpanzee adenovirus DNA sequence optionally comprises at least cis elements necessary for replication and virion capsid formation, the cis elements being adjacent to the antigen cassette and regulatory sequences. In some embodiments, the chimpanzee adenovirus DNA sequence comprises genes selected from the group consisting of 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 lack the E1A and / or E1B genes.

[0270] This specification also discloses adenovirus vectors comprising a partially deleted E4 gene, comprising a deleted or partially deleted E4orf2 region, a deleted or partially deleted E4orf3 region, and optionally, a deleted or partially deleted E4orf4 region. The partially deleted E4 may comprise an E4 deletion of at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1, wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence described in SEQ ID NO: 1. A partially deleted E4 can include E4 deletions of at least nucleotides 34,916-34,942, at least nucleotides 34,952-35,305, and at least nucleotides 35,302-35,642 of the sequence shown in SEQ ID NO: 1, where the vector contains at least nucleotides 2-36,518 of the sequence described in SEQ ID NO: 1. A partially deleted E4 can include E4 deletions of at least nucleotides 34,980-36,516 of the sequence shown in SEQ ID NO: 1, where the vector contains at least nucleotides 2-36,518 of the sequence described in SEQ ID NO: 1. A partially deleted E4 can include E4 deletions of at least nucleotides 34,979-35,642 of the sequence shown in SEQ ID NO: 1, where the vector contains at least nucleotides 2-36,518 of the sequence described in SEQ ID NO: 1. A partially deleted E4 can include E4 deletions that are at least a partial deletion of E4Orf2, a complete deletion of E4Orf3, and at least a partial deletion of E4Orf4. A partially deleted E4 can include E4 deletions that are at least a partial deletion of E4Orf2, at least a partial deletion of E4Orf3, and at least a partial deletion of E4Orf4. A partially deleted E4 can include E4 deletions that are at least a partial deletion of E4Orf1, a complete deletion of E4Orf2, and at least a partial deletion of E4Orf3. A partially deleted E4 can include E4 deletions that are at least a partial deletion of E4Orf2 and at least a partial deletion of E4Orf3.A partially deleted E4 can include E4 deletions from the start site of E4Orf1 to the start site of E4Orf5. A partially deleted E4 may be an E4 deletion adjacent to the start site of E4Orf1. A partially deleted E4 may be an E4 deletion adjacent to the start site of E4Orf2. A partially deleted E4 may be an E4 deletion adjacent to the start site of E4Orf3. A partially deleted E4 may be an E4 deletion adjacent to the start site of E4Orf4. An E4 deletion may consist of at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, at least 1100 nucleotides, at least 1200 nucleotides, at least 1300 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1600 nucleotides, at least 1700 nucleotides, at least 1800 nucleotides, at least 1900 nucleotides, or at least 2000 nucleotides. An E4 deletion may consist of at least 700 nucleotides. An E4 deletion may consist of at least 1500 nucleotides. E4 deletions may be 50 nucleotides or less, 100 nucleotides or less, 200 nucleotides or less, 300 nucleotides or less, 400 nucleotides or less, 500 nucleotides or less, 600 nucleotides or less, 700 nucleotides or less, 800 nucleotides or less, 900 nucleotides or less, 1000 nucleotides or less, 1100 nucleotides or less, 1200 nucleotides or less, 1300 nucleotides or less, 1400 nucleotides or less, 1500 nucleotides or less, 1600 nucleotides or less, 1700 nucleotides or less, 1800 nucleotides or less, 1900 nucleotides or less, or 2000 nucleotides or less. E4 deletions may be 750 nucleotides or less. E4 deletions may be at least 1550 nucleotides or less.

[0271] A partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, lacking at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1. A partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, lacking the E4 gene sequence shown in SEQ ID NO: 1, and lacking at least nucleotides 34,916 to 34,942, nucleotides 34,952 to 35,305, and nucleotides 35,302 to 35,642 of the sequence shown in SEQ ID NO: 1. A partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, and lacking at least nucleotides 34,980 to 36,516 of the sequence shown in SEQ ID NO: 1. A partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, and lacking at least nucleotides 34,979 to 35,642 of the sequence shown in SEQ ID NO: 1. An adenovirus vector having a partially deleted E4 gene may have a cassette, wherein the cassette comprises at least one payload nucleic acid sequence and the cassette comprises at least one promoter sequence functionally linked to at least one payload nucleic acid sequence. An adenovirus vector having a partially deleted E4 gene may have one or more genes or control sequences of the ChAdV68 sequence shown in Sequence ID No. 1, which optionally comprises at least one of the chimpanzee adenovirus terminal inverted repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence shown in Sequence ID No. 1. An adenovirus vector having a partially deleted E4 gene may have nucleotides 2-34,916 of the sequence shown in SEQ ID NO: 1, where the partially deleted E4 gene is the 3' of nucleotides 2-34,916, and optionally, nucleotides 2-34,916 further lack nucleotides 577-3403 of the sequence shown in SEQ ID NO: 1, corresponding to an E1 deletion, and / or lack nucleotides 27,125-31,825 of the sequence shown in SEQ ID NO: 1, corresponding to an E3 deletion.An adenovirus vector having a partially deleted E4 gene may have nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO: 1, where the partially deleted E4 gene is the 5' of nucleotides 35,643 to 36,518. An adenovirus vector having a partially deleted E4 gene may have nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO: 1, where the partially deleted E4 gene is the 3' of nucleotides 2 to 34,916, and nucleotides 2 to 34,916 further lack nucleotides 577 to 3403 of the sequence shown in SEQ ID NO: 1, corresponding to the E1 deletion, and nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO: 1, corresponding to the E3 deletion. An adenovirus vector having a partially deleted E4 gene may have nucleotides 2-34,916 of the sequence shown in SEQ ID NO: 1, where the partially deleted E4 gene is the 3' of nucleotides 2-34,916, and nucleotides 2-34,916 further lack nucleotides 577-3403 of the sequence shown in SEQ ID NO: 1, corresponding to the E1 deletion, and nucleotides 27,125-31,825 of the sequence shown in SEQ ID NO: 1, corresponding to the E3 deletion, and also have nucleotides 35,643-36,518 of the sequence shown in SEQ ID NO: 1, where the partially deleted E4 gene is the 5' of nucleotides 35,643-36,518.

[0272] A partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, lacking at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1, and nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO: 1, where the partially deleted E4 gene is the 3' of nucleotides 2 to 34,916, which further lack nucleotides 577 to 3403 of the sequence shown in SEQ ID NO: 1, corresponding to the E1 deletion, and nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO: 1, corresponding to the E3 deletion, and also has nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO: 1, where the partially deleted E4 gene is the 5' of nucleotides 35,643 to 36,518.

[0273] This specification also discloses host cells transfected with vectors disclosed herein, such as C68 vectors engineered to express antigen cassettes. This specification also discloses human cells expressing selected genes introduced via the introduction of vectors disclosed herein into human cells.

[0274] This specification also discloses a method for delivering an antigen cassette to mammalian cells, which includes introducing an effective amount of a vector disclosed herein, such as a C68 vector engineered to express an antigen cassette, into the cells.

[0275] This specification also discloses a method for generating an antigen, which includes introducing the vector disclosed herein into mammalian cells, culturing the cells under suitable conditions, and generating the antigen.

[0276] Complementary cell lines expressing VE2.E1 To produce recombinant chimpanzee adenovirus (Ad) lacking any of the genes described herein, the function of the deleted gene region can be supplied to the recombinant virus by a helper virus or cell line, i.e., a complementary cell line or a packaging cell line, provided that the function of the deleted gene region is essential for viral replication and infectivity. 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 a variant thereof. 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 product (Examples 3 and 4 of USPN 6,083,716).

[0277] To identify chimpanzee adenovirus E1-expressing cell lines, an AAV enhancement assay can be used. This assay is useful for confirming E1 function in cell lines created using the E1 gene of other uncharacterized adenoviruses (e.g., from other species). The assay is described in Example 4B of USPN 6,083,716.

[0278] A selected chimpanzee adenovirus gene, e.g., E1, may be under the transcriptional regulation of a promoter for expression in a selected parental cell line. For this purpose, inductive or constitutive promoters can be used. Inductive promoters include the zinc-inducible sheep metallothionine promoter, or the glucocorticoid-inducible mouse mammary cancer virus (MMTV) promoter, particularly the dexamethasone-inducible promoter. Other inductive promoters, such as the promoter identified in International Patent Application WO95 / 13392, incorporated herein by reference, can also be used in the production of packaging cell lines. Constitutive promoters can also be used to control the expression of chimpanzee adenovirus genes.

[0279] Parental cells can be selected for the creation of novel cell lines expressing any desired C68 gene. Without limitation, such parental cell lines may be HeLa [ATCC accession number CCL2], A549 [ATCC accession number CCL185], KB [CCL17], Detroit [e.g., Detroit510, CCL72], and WI-38 [CCL75] cells. Other suitable parental cell lines are available from other sources. Parental cell lines may include CHO, HEK293 or its variants, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a.

[0280] E1-expressing cell lines may be useful for constructing recombinant chimpanzee adenovirus E1 deletion vectors. Cell lines expressing the gene products of one or more other chimpanzee adenoviruses, constructed using essentially the same procedure, are useful in constructing recombinant chimpanzee adenovirus vectors in which the genes encoding those products are deleted. Furthermore, cell lines expressing other human Ad E1 gene products are also useful for constructing recombinant chimpanzee Ad.

[0281] 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, which is functionally linked to a regulatory sequence that leads to the expression of the cassette. The C68 vector has the ability to express the cassette in infected mammalian cells. The C68 vector may have one or more viral genes functionally deleted. The antigen cassette contains at least one antigen under the control of one or more regulatory sequences, such as a promoter. Optional helper viruses and / or packaging cell lines can supply any required products of the deleted adenovirus gene to the chimpanzee virus vector.

[0282] The term "functionally deleted" means that a sufficient amount of a gene region is removed, or otherwise modified, such that the gene region can no longer produce one or more functional products of gene expression. Mutations or modifications that can result in a 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 desired, an entire gene region may be removed.

[0283] Modifications of nucleic acid sequences forming vectors disclosed herein, such as sequence deletions, insertions, and other mutations, can be generated using standard molecular biological techniques and are within the scope of the present invention.

[0284] VE4. Construction of a viral plasmid vector The chimpanzee adenovirus C68 vectors useful in this invention include recombinant knockout adenoviruses, i.e., chimpanzee adenovirus sequences in which the E1a or E1b gene is functionally deleted, and optionally, chimpanzee adenovirus sequences with other mutations, e.g., temperature-sensitive mutations or deletions of other genes. These chimpanzee sequences are also expected to be useful in forming hybrid vectors from other adenoviruses and / or adeno-associated virus sequences. Homologous adenovirus vectors prepared from human adenoviruses are described in the published literature [see, for example, Kozarsky I and II above and the references cited therein, and U.S. Patent No. 5,240,846].

[0285] In the construction of chimpanzee adenovirus C68 vectors useful for delivering antigen cassettes to human (or other mammalian) cells, a series of adenovirus nucleic acid sequences can be used in the vector. A vector containing a minimal chimpanzee C68 adenovirus sequence can be used in combination with a helper virus to generate infectious recombinant viral particles. The helper virus provides essential gene products required for viral infectivity and replication of the minimal chimpanzee adenovirus vector. If only one or more selective deletions of chimpanzee adenovirus genes are performed on an otherwise functional viral vector, the deleted gene product can be supplied in the viral vector generation process by growing the virus in a selective packaging cell line that provides the deleted gene function in trans.

[0286] VE5. Recombinant Minimal Adenovirus The minimal chimpanzee Ad C68 virus is a viral particle containing only the adenovirus cis elements necessary for replication and virion capsid formation. Specifically, the vector contains the cis-active 5' and 3' terminal inverted repeat (ITR) sequences of the adenovirus (functioning as the origin of replication) as well as the natural 5' packaging / enhancer domain (containing sequences necessary for packaging the linear Ad genome and the enhancer element of the E1 promoter). See, for example, the technique described in International Patent Application WO96 / 13597, incorporated herein by reference, for the preparation of the “minimal” human Ad vector.

[0287] VE6. Other deficient adenoviruses Recombinant replication-deficient adenoviruses may also contain more than the smallest chimpanzee adenovirus sequence. These other Ad vectors may feature infectious viral particles formed by deletions of various parts of the viral gene region, as well as the use of optional helper viruses and / or packaging cell lines.

[0288] As an example, a suitable vector may be formed by deleting all or a significant portion of the C68 adenovirus early gene E1a and delayed early gene E1b in such a way that their normal biological function is eliminated. The replication-deficient E1 deletion virus, when grown in a complementary cell line transformed with chimpanzee adenovirus that provides the corresponding gene product in trans containing the functional adenovirus E1a and E1b genes, is capable of replicating and producing infectious viruses. Based on homology with known adenovirus sequences, and as is also true for human recombinant E1 deletion adenoviruses in the art, the resulting recombinant chimpanzee adenovirus is expected to be capable of infecting many cell types and expressing antigens, but will not be able to replicate in most cells lacking chimpanzee E1 region DNA unless the cells are infected with a very high degree of infection multiplicity.

[0289] As another example, all or part of the C68 adenovirus late early gene E3 can be excluded from chimpanzee adenovirus sequences that form part of a recombinant virus.

[0290] The chimpanzee adenovirus C68 vector can also be constructed with a deletion in the E4 gene. Yet another vector may contain a deletion in the late early gene E2a.

[0291] Deletions can also be made in any of the late-stage genes L1-L5 of the chimpanzee C68 adenovirus genome. Similarly, deletions in the intermediate genes IX and IVa2 may be useful for certain purposes. Other deletions can be made in other structural or non-structural adenovirus genes.

[0292] The deletions discussed above can be used individually; that is, the adenovirus sequence may contain only the E1 deletion. Alternatively, deletions of entire genes or parts thereof that are effective in disrupting or reducing biological activity can be used in any combination. For example, in one exemplary vector, the adenovirus C68 sequence may have deletions of the E1 and E4 genes, or deletions of the E1, E2a, and E3 genes, or deletions of the E1 and E3 genes, or deletions of the E1, E2a, and E4 genes, etc. As mentioned above, such deletions can be used in combination with other mutations, such as temperature-sensitive mutations, to achieve the desired results.

[0293] The antigen-containing cassette can be optionally inserted into any deletion region of the chimpanzee C68 Ad virus. Alternatively, if desired, the cassette can be inserted into an existing gene region to disrupt its function.

[0294] VE7. Helper virus Depending on the chimpanzee adenovirus gene content of the viral vector used to carry the antigen cassette, a helper adenovirus or non-replicating viral fragment can be used to provide a chimpanzee adenovirus gene sequence sufficient to generate infectious recombinant viral particles containing the cassette.

[0295] Useful helper viruses contain a selected adenovirus gene sequence that is not present in the adenovirus vector construct and / or not expressed by the packaging cell line to which the vector is transfected. Helper viruses may be replication-deficient and may contain various adenovirus genes in addition to the above sequence. Helper viruses can be used in combination with the E1-expressing cell lines described herein.

[0296] In the case of C68, the "helper" virus can be a fragment formed by clipping the C-terminus and SspI of the C68 genome, in which case approximately 1300 bp is removed from the left end of the virus. This clipped virus is then co-transfected with plasmid DNA into an E1-expressing cell line, thereby forming a recombinant virus through homologous recombination with the C68 sequence in the plasmid.

[0297] Helper viruses can also form polycation complexes, as described in Wu et al, J. Biol. Chem., 264:16985-16987 (1989) and K.J. Fisher and J.M. Wilson, Biochem. J., 299:49 (Apr. 1, 1994). Helper viruses may optionally contain a reporter gene. Numerous 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 monitor both the adenovirus vector and the helper virus independently. This second reporter is used to enable isolation between the resulting recombinant virus and the helper virus during purification.

[0298] VE8. Assembly of viral particles and infection of cell lines The assembly of selected adenovirus DNA sequences, antigen cassettes, and other vector elements into various intermediate plasmids and shuttle vectors, as well as the use of plasmids and vectors to generate recombinant viral particles, can all be achieved using conventional techniques. Such techniques include conventional cDNA cloning techniques, in vitro recombination techniques (e.g., Gibson assembly), the use of duplicate oligonucleotide sequences of the adenovirus genome, polymerase chain reactions, and any suitable method to yield the desired nucleotide sequence. Standard transfection and cotransfection techniques, such as CaPO4 precipitation or liposome-mediated transfection methods like lipofectamine, are used. Other conventional methods used include homologous recombination of the viral genome, viral plaque formation on agar stratification, and methods for measuring signal generation.

[0299] For example, after constructing and assembling a viral vector containing the desired antigen cassette, 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 in the vector and packaging it within a virion capsid, resulting in recombinant viral vector particles.

[0300] The resulting recombinant chimpanzee C68 adenovirus is useful for introducing antigen cassettes into selected cells. In in vivo studies using recombinant viruses grown in packaging cell lines, the E1 deletion recombinant chimpanzee adenovirus has shown usefulness in introducing cassettes into non-chimpanzee cells, preferably human cells.

[0301] VE9. Use of Recombinant Viral Vectors Thus, the recombinant chimpanzee C68 adenovirus obtained containing an antigen cassette (generated by the cooperation of an adenovirus vector and a helper virus or an adenovirus vector and a packaging cell line as described above) provides an efficient gene delivery vehicle that can deliver the antigen to the target in vivo or ex vivo.

[0302] The recombinant vectors described above are administered to humans according to publicly available gene therapies. The chimpanzee virus vector carrying the antigen cassette may be administered to the patient, preferably suspended in a biocompatible solution or a pharmaceutically acceptable delivery vehicle. Suitable vehicles include sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions, which are known to be pharmaceutically acceptable carriers and are well known to those skilled in the art, may be used for this purpose.

[0303] The chimpanzee adenovirus vector is administered in an amount sufficient to transduce human cells and to produce a level of antigen transduction and expression sufficient to provide therapeutic benefits with no excessive adverse effects or medically acceptable physiological effects, which can be determined by those skilled in the art of medicine. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the liver, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parenteral routes of administration. Routes of administration may be combined as needed.

[0304] The dosage of the viral vector may vary from patient to patient, primarily depending on factors such as the condition being treated, the patient's age, weight, and health. The dosage is adjusted to balance the therapeutic benefits with any potential side effects, and such dosages may vary depending on the therapeutic application in which the recombinant vector is used. The frequency of dose administration can be determined by monitoring antigen expression levels.

[0305] Recombinant replication-deficient adenoviruses may be administered in a "pharmaceutically effective amount," i.e., an amount of recombinant adenovirus effective in the route of administration for transfecting the desired cells and providing a vaccine effect, i.e., a level of expression of a select gene sufficient to obtain a measurable level of protective immunity of some degree. The C68 vector containing the antigen cassette may be administered co-administered with an adjuvant. The adjuvant may be encoded within the vector, or separately from the vector (e.g., alum), in particular, if the adjuvant is a protein. Adjuvants are well known in the art.

[0306] Conventional pharmaceutically acceptable routes of administration include, but are not limited to, intranasal, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral, and other parenteral routes. Routes of administration may be combined as needed, or modified depending on the immunogen or disease. For example, in rabies prevention, subcutaneous, intratracheal, and intranasal routes are preferred. The route of administration primarily depends on the nature of the disease being treated.

[0307] By monitoring the level of immunity to the antigen, the need for a booster, if present, can be determined. For example, after evaluating serum antibody titers, optional booster immunization may be desirable.

[0308] VI. Treatment Methods and Manufacturing Methods Methods are also provided for stimulating a tumor-specific immune response in a subject, vaccinating against a tumor, and treating and / or mitigating cancer symptoms in a subject by administering one or more antigens, for example, multiple antigens identified using the methods disclosed herein, to the subject.

[0309] Methods are also provided for stimulating an infectious organism-specific immune response in a subject, vaccinating against an infectious organism, and treating and / or mitigating symptoms of an infectious disease associated with an infectious organism in a subject by administering one or more antigens, for example, a group of antigens identified using the methods disclosed herein, to the subject.

[0310] 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 those of the mammary gland, ovary, prostate, lung, kidney, stomach, colon, testis, head and neck, pancreas, brain, melanoma, and other tumors of tissue organs, as well as hematological malignancies such as lymphoma and leukemia, e.g., acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma.

[0311] In some aspects, subjects are diagnosed with an infectious disease or are at risk of infection (e.g., they are at high risk or predisposed to infection due to age, geography / travel, and / or occupation, or they are at risk of infection due to seasonal and / or novel diseases).

[0312] The antigen may be administered in an amount sufficient to stimulate a CTL response. The antigen may be administered in an amount sufficient to stimulate a T cell response. The antigen may be administered in an amount sufficient to stimulate a B cell response. The antigen may be administered in an amount sufficient to stimulate both a T cell response and a B cell response.

[0313] Antigens may be administered alone or in combination with other therapeutic agents. Therapeutic agents may include those that target infectious organisms, such as antivirals or antibiotics.

[0314] Furthermore, immunosuppressants / immunostimulants such as checkpoint inhibitors can be administered to target specific targets. For example, anti-CTLA antibodies or anti-PD-1 or anti-PD-L1 antibodies can be administered to target specific targets. Blocking CTLA-4 or PD-L1 with antibodies can enhance the immune response against cancer cells in patients. In particular, blocking CTLA-4 has been shown to be effective when administered according to vaccination protocols.

[0315] The optimal amount of each antigen to be included in the vaccine composition and the optimal administration regimen can be determined. For example, the antigen or its variant can be prepared for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), 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 administration of the vaccine composition are known to those skilled in the art.

[0316] Vaccines can be edited so that the selection, number, and / or amount of antigens present in the composition are specific to the tissue, cancer, infection, and / or patient. For example, the precise selection of peptides can be guided by the expression pattern of the parent protein in a given tissue, and also by the patient's mutation or disease state. The selection may vary depending on the type of cancer, the specific infection (e.g., isolate / strain of a specific infection in which the subject is infected or at risk of infection), the disease state, the purpose of vaccination (e.g., prophylactic or targeting of an ongoing disease), prior treatment regimen, the patient's immune status, and, of course, the patient's HLA haplotype. Furthermore, vaccines can contain individualized components according to the specific needs of a particular patient. Examples include modifications to antigen selection according to antigen expression in a particular patient, or adjustments to secondary treatment following primary or secondary treatment schemes.

[0317] Patients may be identified for antigen vaccine administration through the use of various diagnostic methods, such as the patient selection methods detailed below. Patient selection may involve identifying mutations in one or more genes, or expression patterns of one or more genes. Patient selection may involve identifying an ongoing infection. Patient selection may involve identifying the risk of infection due to an infection. In some cases, patient selection may involve identifying the patient's haplotype. Various patient selection methods can be performed in parallel; for example, sequencing diagnostics can identify both the patient's mutations and haplotype. Various patient selection methods can be performed sequentially; for example, one diagnostic test can identify mutations and another diagnostic test can identify the patient's haplotype, in which case each test can be the same diagnostic method (e.g., both high-throughput sequencing) or different diagnostic methods (e.g., one high-throughput sequencing and the other Sanger sequencing).

[0318] In the case of compositions used as vaccines for cancer or infectious diseases, antigens having normal self-peptides similar to those highly expressed in normal tissues may be avoided or present in low amounts in the compositions described herein. On the other hand, if a patient's tumor or infected cells are known to express a particular antigen in high amounts, each pharmaceutical composition for treating this cancer or infectious disease may contain a high amount of this particular antigen and / or multiple antigens specific to the pathway of this antigen.

[0319] Compositions containing antigens may be administered to individuals already suffering from cancer or infection. For therapeutic use, the composition is administered to the subject in an amount sufficient to stimulate an effective CTL response to the tumor antigen or infectious organism antigen, thereby curing or at least partially cessating symptoms and / or complications. The appropriate amount to achieve this is defined as the “therapeutic effective dose.” The effective dose for this use varies, for example, depending on the composition, mode of administration, stage and severity of the disease being treated, the patient’s weight and overall health, and the prescribing physician’s judgment. It should be noted that compositions may generally be used in serious medical conditions, i.e., life-threatening or potentially life-threatening situations, particularly when cancer has metastasized or when an infectious organism is inducing organ damage and / or other immunopathologies. In such cases, considering the minimization of exogenous substances and the relative non-toxicity of the antigen, it may be possible, and even desirable, for the attending physician to perceive a substantially excessive dose of these compositions.

[0320] For therapeutic use, administration may be initiated at the time of tumor detection or surgical removal, or at the time of detection or treatment of infection. Thereafter, the dose may be boosted until at least the symptoms are substantially relieved, and thereafter, or until immunity is considered to be provided (e.g., memory B cells or memory T cell populations, or antigen-specific B cells or antibodies are produced).

[0321] 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 surgical excision sites to stimulate a local immune response against a tumor. Compositions may be administered to target specific infected tissues and / or cells of a subject. This specification discloses compositions for parenteral administration, which comprise a solution of an antigen, and vaccine compositions, which are 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 filter sterilization. The resulting aqueous solutions may be packaged for immediate use or lyophilized, and the lyophilized preparations may be 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, triethanolamine oleate, etc.

[0322] Antigens can also be administered by liposomes that target them to specific cell tissues, such as lymphoid tissue. Liposomes are also useful for extending half-lives. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. In these preparations, the antigen to be delivered is incorporated as part of the liposome, either alone or in combination with molecules that bind to receptors frequently found among lymphoid cells, such as monoclonal antibodies that bind to the CD45 antigen, or in combination with other therapeutic or immunogenic compositions. Thus, liposomes filled with the desired antigen can be oriented to sites on lymphoid cells, where the liposomes deliver the selected therapeutic / immunogenic composition. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols such as cholesterol. Lipid selection is generally guided by considering, for example, 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. Patent Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

[0323] In the case of targeting immune cells, the ligands incorporated into the liposomes may include, for example, antibodies or fragments of antibodies specific to the cell surface determinants of the desired immune system cells. The liposome suspension can be administered intravenously, locally, or topically, in doses that vary depending on the mode of administration, the peptide being delivered, and the stage of the disease being treated.

[0324] For therapeutic or immunization purposes, nucleic acids encoding peptides, and optionally encoding one or more of the peptides described herein, may also be administered to the patient. Numerous methods are conveniently used to deliver nucleic acids to the patient. For example, nucleic acids may be delivered directly as "naked DNA." This method is described, for example, in Wolff et al., Science 247:1465-1468 (1990) and U.S. Patents 5,580,859 and 5,589,466. Nucleic acids may also be delivered using ballistic delivery, for example, as described in U.S. Patent 5,204,253. Particles consisting solely of DNA may be delivered. Alternatively, DNA can be attached to particles such as gold particles. Methods for delivering nucleic acid sequences may include viral vectors, mRNA vectors, and DNA vectors, with or without electroporation.

[0325] Nucleic acids can also be delivered in complex with cationic compounds such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in 9618372WOAWO96 / 18372, 9324640WOAWO93 / 24640, Mannino & Gould-Fogerite, BioTechniques 6(7):682-691 (1988), U.S. Patent No. 5,279,833 (Rose), U.S. Patent No. 5,279,833, 9106309WOAWO91 / 06309, and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987).

[0326] Antigens also include, but are not limited to, lentiviruses, including, second-generation, third-generation, or second / third-generation hybrid lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors). It can also be included in such materials (see *containing the human ubiquitin C promoter*, Nucl. Acids Res. (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 capacity of the above viral vector-based vaccine platform, this method can deliver one or more nucleotide sequences encoding one or more 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 stimulating a host immune response (e.g., CTLs) to the peptide. Vaccine vectors and methods useful for immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). 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.

[0327] The means of administering nucleic acids utilize minigene constructs that encode one or more epitopes. The amino acid sequence of an epitope is back-translated to create a DNA sequence encoding a selected CTL epitope (minigene) for expression in human cells. A human codon usage table is used to derive codon selection for each amino acid. These epitope-coding DNA sequences are directly adjacent to create a continuous polypeptide sequence. Additional elements can be incorporated into the minigene design to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be back-translated and included in the minigene sequence include helper T lymphocytes, epitopes, leader (signal) sequences, and endoplasmic reticulum retained signals. Furthermore, including synthetic (e.g., polyalanine) or naturally occurring flanking sequences adjacent to the CTL epitope can improve the presentation of the CTL epitope in MHC. The minigene sequence is converted to DNA by assembling oligonucleotides encoding the positive and negative strands of the minigene. Duplicate oligonucleotides (30–100 nucleotides in length) are synthesized, phosphorylated, purified, and annealed under appropriate conditions using known techniques. The ends of the oligonucleotides are ligated using T4 DNA ligase. This synthetic minigene encoding a CTL epitope polypeptide can then be cloned into the desired expression vector.

[0328] Purified plasmid DNA can be prepared for injection using various formulations. The simplest of these is the reconstitution of lyophilized DNA in sterile phosphate-buffered saline (PBS). Various methods have been reported, and new technologies may become available. As described above, nucleic acids are conveniently formulated using cationic lipids. Furthermore, glycolipids, membrane-fused liposomes, peptides, and compounds collectively known as protective interaction non-condensable (PINCs) can also be complexed with purified plasmid DNA to influence variable factors such as stability, intramuscular dispersion, or transport to specific organs or cell types.

[0329] Furthermore, a method for producing a vaccine is disclosed, which includes carrying out steps of the method disclosed herein and generating a vaccine comprising multiple antigens or a subset of multiple antigens.

[0330] The antigens disclosed herein may be produced using methods known in the art. For example, a method for producing an antigen or vector disclosed herein (e.g., a vector comprising at least one sequence encoding one or more antigens) may include culturing host cells containing at least one polynucleotide encoding the antigen or vector under conditions suitable for the expression of such antigen or vector, and purifying the antigen or vector. Standard purification methods include chromatography, electrophoresis, immunological techniques, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques.

[0331] Host cells may include Chinese hamster ovary (CHO) cells, NS0 cells, yeast, or HEK293 cells. Host cells can be transformed with one or more polynucleotides comprising at least one nucleic acid sequence encoding an antigen or vector disclosed herein, where optionally, the isolated polynucleotide further comprises a promoter sequence functionally ligated to at least one nucleic acid sequence encoding the antigen or vector. In certain embodiments, the isolated polynucleotide may be a cDNA.

[0332] VII. Use and Administration of Antigens Other available vaccination methods, protocols, and schedules include, but are not limited to, those described in International Patent Application Publication WO2021092095, which is incorporated herein by reference for all purposes.

[0333] Each vector in a prime / boost strategy typically contains a cassette containing antigens. The cassette may contain approximately 1 to 50 antigens, usually separated by spacers such as native sequences or other non-native spacer sequences such as AAY. The cassette may also contain MHCII antigens, such as tetanus toxoid antigen and PADRE antigen, which can be considered universal class II antigens. The cassette may also contain targeting sequences, such as ubiquitin targeting sequences. Furthermore, each vaccine dose may be administered to the subject in conjunction with an immunomodulator (e.g., simultaneously, before, or after). Each vaccine dose may also be administered to the subject in conjunction with a checkpoint inhibitor (CPI) (e.g., simultaneously, before, or after). CPIs may include those that inhibit CTLA4, PD1, and / or PDL1, such as antibodies or their antigen-binding moieties. Such antibodies may include tremelimumab or durvalumab. Each vaccine dose may be administered to the subject in combination with cytokines such as IL-2, IL-7, IL-12 (including p35, p40, p70, and / or p70 fusion constructs of IL-12), IL-15, or IL-21 (e.g., simultaneously, before, or after). Each vaccine dose may also be administered to the subject in combination with modified cytokines (e.g., pegIL-2) (e.g., simultaneously, before, or after).

[0334] A vaccination protocol can be used to administer one or more antigens to a subject. A priming vaccine and a boosting vaccine can be used for administration to the subject. The priming vaccine may be one of the antigen-coding vectors described herein, for example, a vector based on ChAdV68 (e.g., the sequence shown in SEQ ID NO: 1 or 2). The booster dose may be one of the antigen-coding vectors described herein, for example, a vector based on ChAdV68 (e.g., the sequence shown in SEQ ID NO: 1 or 2) or a SAM-based vector (e.g., the sequence shown in SEQ ID NO: 3 or 4). One or more booster doses can be administered, and sequential administration of the same boosting vaccine (e.g., sequential administration of the same ChAdV68-based vector or sequential administration of the same SAM-based vector) or sequential administration of different boosting vaccines (e.g., administration of a SAM-based vector followed by administration of a ChAdV68-based vector) is possible. Sequential administration of different vaccines may include any combination of different vaccines. For example, a vaccine strategy could use a ChAdV68-based prime, followed by one or more SAM-based boosters, and then a ChAdV68-based booster following a SAM-based booster. Exemplary, non-limiting vaccine strategies include, but are not limited to, ChAdV prime-SAM booster-SAM booster-ChAdV booster, or ChAdV prime-SAM booster-SAM booster-SAM booster-SAM booster-ChAdV booster.

[0335] The vaccine based on ChAdV68 is 1 x 10 11 Virus particles ~1 x 10 12 It can be administered in doses ranging from 1 to 10⁶ of viral particles. A vaccine based on ChAdV68 is 1 × 10⁶ 11 It can be administered in doses of viral particles. A vaccine based on ChAdV68 is 5 × 10 11 It can be administered in doses of viral particles. A vaccine based on ChAdV68 is 1 × 10⁶ 12It may be administered in doses of viral particles. The selected dosage for a ChAdV68-based vaccine varies depending, for example, the composition, mode of administration, stage and severity of the disease being treated, the patient's weight and overall health, and the prescribing physician's judgment.

[0336] SAM-based vaccines may be administered in doses ranging from 10 to 300 μg of RNA. SAM-based vaccines may be administered in doses ranging from 100 to 300 μg of RNA. SAM-based vaccines may be administered in doses of 100 μg of RNA. SAM-based vaccines may be administered in doses of 300 μg of RNA. The selected dose for a SAM-based vaccine may vary depending, for example, the composition, mode of administration, the stage and severity of the disease being treated, the patient's weight and overall health, and the prescribing physician's judgment.

[0337] The priming vaccine may be injected into the target (e.g., intramuscularly). Bilateral injections per dose may be used. For example, one or more injections of ChAdV68(C68) may be used (e.g., total dose of 1 × 10⁶). 12 Viral particles; low vaccine doses selected from the RNA range of 0.001 to 1 ug, particularly one or more injections of a SAM vector at 0.1 ug or 1 ug, may be used; or high vaccine doses selected from the RNA range of 1 to 100 ug, particularly one or more injections of a SAM vector at 10 ug, 100 ug, or 300 ug, may be used.

[0338] Vaccine boosters (boosting vaccines) can be injected (e.g., intramuscularly) after prime vaccination. Bilateral injections per dose can be used. For example, one or more injections of ChAdV68 (C68) can be used (e.g., total dose of 1 × 10⁶). 12Viral particles; low vaccine doses selected from the RNA range of 0.001 to 1 ug, particularly one or more injections of a SAM vector at 0.1 ug or 1 ug, may be used; or high vaccine doses selected from the RNA range of 1 to 100 ug, particularly one or more injections of a SAM vector at 10 ug, 100 ug, or 300 ug, may be used.

[0339] The boosting vaccine may be administered approximately every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, or every ten weeks after the prime dose, for example, every four weeks and / or every eight weeks. The boosting vaccine may be administered every four weeks after the prime dose. The boosting vaccine may be administered every six weeks after the prime dose. The boosting vaccine may be administered every twelve weeks after the prime dose. The boost dose may be administered at different intervals throughout the duration of the vaccination protocol. For example, exemplary non-limiting examples include prime-4w-boost-12w-boost-12w-boost; or prime-4w-boost-6w-boost-6w-boost-6w-boost-6w-boost (where "w" represents weeks).

[0340] One or more doses of the vaccine may include the co-administration of one or more checkpoint inhibitors. Exemplary 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). Exemplary, non-limiting examples include nivolumab, Yervoy / ipilimumab, or combinations thereof.

[0341] Anti-CTLA-4 (e.g., tremelimumab) may also be administered to the target population. For example, anti-CTLA4 may be subcutaneously administered near the site of intramuscular vaccine injection (ChAdV68 prime or SAM low dose) to ensure that it flows into the same lymph nodes. Tremelimumab is a selective human IgG2 mAb inhibitor of CTLA-4. The target subcutaneous dose of anti-CTLA-4 (tremelimumab) is typically 70–75 mg (especially 75 mg), with a dose range of, for example, 1–100 mg or 5–420 mg.

[0342] In certain cases, anti-PD-L1 antibodies such as durvalumab (MEDI4736) can be used. Durvalumab is a selective high-affinity human IgG1 mAb that blocks the binding of PD-L1 to PD-1 and CD80. Durvalumab is generally administered intravenously at a dose of 20 mg / kg every four weeks.

[0343] Immunotherapy monitoring may be performed before, during, and / or after vaccine administration. Such monitoring can provide information on parameters, particularly safety and efficacy.

[0344] PBMCs are commonly used for performing immune monitoring. PBMCs can be isolated before prime vaccination and after prime vaccination (e.g., 4 weeks and 8 weeks later). PBMCs can also be collected immediately before booster vaccination and after each booster vaccination (e.g., 4 weeks and 8 weeks later).

[0345] Immune responses, such as T-cell and B-cell responses, can be evaluated as part of an immune monitoring protocol. For example, the ability of the vaccine compositions described herein to stimulate an immune response can be monitored and / or evaluated. As used herein, “stimulates an immune response” means any increase in an immune response, e.g., initiation of an immune response (e.g., a priming vaccine stimulating the initiation of an immune response in an untreated subject), or any enhancement of an immune response (e.g., a boosting vaccine stimulating an enhancement of an immune response in a subject with an existing immune response to an antigen, such as an existing immune response initiated by a priming vaccine). T-cell responses can be measured using one or more methods known in the art, e.g., by ELISpot, intracellular cytokine staining, cytokine secretion and cell surface capture, T-cell proliferation, MHC multimer staining, or cytotoxicity assays. T-cell responses to vaccine-encoded epitopes can be monitored from PBMCs by measuring the induction of cytokines such as IFN-gamma using an ELISpot assay. Specific CD4 or CD8 T cell responses to vaccine-encoded epitopes can be monitored from PBMCs by measuring the induction of cytokines such as IFN-gamma, captured intracellularly or extracellularly, using flow cytometry. Specific CD4 or CD8 T cell responses to vaccine-encoded epitopes can also be monitored from PBMCs by measuring T cell populations expressing T cell receptors specific to the epitope / MHC class I complex, using MHC multimer staining. Specific CD4 or CD8 T cell responses to vaccine-encoded epitopes can also be monitored from PBMCs by measuring ex vivo proliferation of T cell populations after 3H-thymidine, bromodeoxyuridine, and carboxyfluorescein diacetatosuccinimidyl ester (CFSE) uptake. Antigen recognition and lytic activity of PBMC-derived T cells specific to vaccine-encoded epitopes can be functionally evaluated by chromium release assays or alternative colorimetric cytotoxicity assays.

[0346] B cell responses may be measured using one or more methods known in the art, for example, assays used to determine B cell differentiation (e.g., differentiation into plasma cells), proliferation of B cells or plasma cells, activation of B cells or plasma cells (e.g., upregulation of costimulatory markers such as CD80 or CD86), antibody class switching, and / or antibody production (e.g., ELISA).

[0347] Vaccine regimens may include evaluating neutralizing antibody titers to the target vaccine composition, such as a ChAdV68-based vaccine. For example, a ChAdV68-based vaccine may be administered as a priming dose, and after determining that the ChAdV-specific neutralizing antibody titer is below the neutralization threshold, a booster dose of the ChAdV68-based vaccine may be re-administered. The neutralizing antibody titer may be the NT50 value, calculated as the minimum dilution of serum from an immunized subject that neutralizes 50% of the ChAdV virus. Determining the neutralizing antibody titer may include (1) contacting one or more dilutions of serum from an immunized subject with the ChAdV virus under conditions sufficient to neutralize the ChAdV virus, and (2) evaluating the neutralization of the ChAdV virus compared to an unneutralized virus.

[0348] The disease status of the subject can be monitored after administration of any of the vaccine compositions described herein. For example, the disease status may be monitored using cell-free DNA (cfDNA) isolated from the subject. Furthermore, the efficacy of vaccine therapy may be monitored using cfDNA isolated from the subject. Monitoring of cfDNA may include the steps of a. isolating or keeping cfDNA isolated from the subject, b. sequencing or keeping the isolated cfDNA, c. determining or keeping the frequency of one or more mutations in the cfDNA compared to the wild-type germline nucleic acid sequence of the subject, and d. evaluating or keeping the disease status of the subject from step (c). The method may also include, following step (c) above, d. performing multiple iterations of steps (a) to (c) for a given subject and comparing the frequencies of one or more mutations determined in the multiple iterations, and f. evaluating or keeping the disease status of the subject from step (d). Multiple iterations may be performed at different time points; for example, the first iteration of steps (a) to (c) may be performed before administration of the vaccine composition, and the second iteration of steps (a) to (c) may be performed after administration of the vaccine composition. Step (c) may include comparing the frequency of one or more mutations determined in multiple iterations, or the frequency of one or more mutations determined in the first iteration with the frequency of one or more mutations determined in the second iteration. An increase in the frequency of one or more mutations determined in subsequent or second iterations may be evaluated as disease progression. A decrease in the frequency of one or more mutations determined in subsequent or second iterations may be evaluated as a response. In some embodiments, the response is a complete response (CR) or a partial response (PR). Following the evaluation step, if, for example, evaluation of the frequency of one or more mutations in cfDNA indicates that the subject has the disease, therapy may be administered to the subject. In the cfDNA isolation step, centrifugation may be used to separate cfDNA from cells or cell fragments. cfDNA can be isolated from whole blood by separating the plasma layer, buffy coat, and red blood cells, among other methods.cfDNA sequencing may utilize next-generation sequencing (NGS), Sanger sequencing, duplex sequencing, whole-exome sequencing, whole-genome sequencing, de novo sequencing, stepwise sequencing, targeted amplicon sequencing, shotgun sequencing, or a combination thereof, and may involve enriching the cfDNA for one or more target polynucleotide regions before sequencing (e.g., polynucleotides, coding regions, and / or tumor exome polynucleotides known or suspected to encode one or more mutations). Enriching the cfDNA may involve hybridizing one or more target polynucleotide regions with one or more polynucleotide probes that can be modified (e.g., biotinylation). In general, any number of mutations may be monitored simultaneously or in parallel.

[0349] Homogeneous vaccination regimens may include intervals between homogeneous doses to improve the efficacy of the second dose. For example, a ChAdV68-based vaccine can be administered as the initial dose, and an interval may be included before re-administration of a ChAdV68-based vaccine as a booster dose to improve efficacy, for example, to reduce the impact of ChAdV-specific neutralizing antibody titers on the efficacy of the booster dose. For example, the initial dose may induce the production of neutralizing antibodies, which then decrease over time. In the exemplary, non-limiting examples of ChAdV68-based vaccines described herein, the interval is at least 27 weeks. The interval may be 27 weeks. The interval may be 28 weeks. The interval may be 29 weeks. The interval may be 30 weeks. The interval may be 31 weeks. The interval may be 32 weeks. The interval may be 33 weeks. The interval may be at least 27 weeks. The interval may be at least 28 weeks. The interval may be at least 29 weeks. The interval may be at least 30 weeks. The interval may be at least 31 weeks. The interval may be at least 32 weeks. The interval may be at least 33 weeks.

[0350] The interval between ChAdV68-based vaccine doses in the same prime-boost strategy can be as short as 8 weeks. The interval can be 8 weeks. The interval can be 9 weeks. The interval can be 10 weeks. The interval can be 11 weeks. The interval can be 12 weeks. The interval can be 13 weeks. The interval can be 14 weeks. The interval can be 15 weeks. The interval can be 16 weeks. The interval can be 17 weeks. The interval can be 18 weeks. The interval can be 19 weeks. The interval can be 20 weeks. The interval can be 21 weeks. The interval can be 23 weeks. The interval can be 24 weeks. The interval can be 25 weeks. The interval can be 26 weeks.

[0351] The interval between ChAdV68-based vaccine doses in the same prime-boost strategy may be at least 8 weeks. The interval may be at least 9 weeks. The interval may be at least 10 weeks. The interval may be at least 11 weeks. The interval may be at least 12 weeks. The interval may be at least 13 weeks. The interval may be at least 14 weeks. The interval may be at least 15 weeks. The interval may be at least 16 weeks. The interval may be at least 17 weeks. The interval may be at least 18 weeks. The interval may be at least 19 weeks. The interval may be at least 20 weeks. The interval may be at least 21 weeks. The interval may be at least 23 weeks. The interval may be at least 24 weeks. The interval may be at least 25 weeks. The interval may be at least 26 weeks.

[0352] The interval between ChAdV68-based vaccine doses in the same prime-boost strategy may be 2 months. The interval may be 2.5 months. The interval may be 3 months. The interval may be 3.5 months. The interval may be 4 months. The interval may be 4.5 months. The interval may be 5 months. The interval may be 5.5 months. The interval may be 6 months. The interval may be 6.5 months. The interval may be 7 months. The interval may be 7.5 months. The interval may be 8 months. The interval may be 8.5 months. The interval may be at least 2 months. The interval may be at least 2.5 months. The interval may be at least 3 months. The interval may be at least 3.5 months. The interval may be at least 4 months. The interval may be at least 4.5 months. The interval may be at least 5 months. The interval may be at least 5.5 months. The interval may be at least 6 months. The interval may be at least 6.5 months. The interval can be at least 7 months. The interval can be at least 7.5 months. The interval can be at least 8 months. The interval can be at least 8.5 months.

[0353] VIII. Isolation and Detection of HLA Peptides Following the lysis and solubilization of tissue samples, HLA peptide molecules were isolated using classical immunoprecipitation (IP) (55-58). Clarified lysates were used for HLA-specific IP. Examples and methods are detailed in International Patent Application Publication WO / 2018 / 208856, which is incorporated herein by reference in its entirety for all purposes.

[0354] IX. Proposed Model Presentation models can be used to identify the potential for peptide presentation in patients. Various presentation models are known to those skilled in the art, for example, in U.S. Patent No. 10,055,540, U.S. Patent Publication Nos. US20200010849A1 and US20110293637, and in International Patent Publication Nos. WO / 2018 / 195357, WO / 2018 / 208856 and WO2016187508, which are incorporated herein by reference in their entirety for all purposes.

[0355] X. Training Module Training modules can be used to construct one or more presentation models based on a training dataset that generates the probability of whether a peptide sequence is presented by an MHC allele associated with that peptide sequence. Various training modules are known to those skilled in the art, and presentation models are detailed, for example, in U.S. Patent No. 10,055,540, U.S. Patent Publication No. US20200010849A1, and International Patent Publications WO / 2018 / 195357 and WO / 2018 / 208856, respectively, which are incorporated herein by reference in their entirety for all purposes. Training modules can be used to construct presentation models for predicting the presentation probability of a peptide on an allele-by-allele basis. Training modules can also be used to construct presentation models for predicting the presentation probability of a peptide in a multi-allelic setting where two or more MHC alleles exist.

[0356] XI. Prediction Module A prediction module can be used to receive sequence data and select candidate antigens in the sequence data using a presentation model. Specifically, the sequence data may be DNA sequences, RNA sequences, and / or protein sequences extracted from the patient's tumor tissue cells, the patient's infected cells, or the infectious organism itself. The prediction module can identify candidate neoantigens, which are mutant peptide sequences, by comparing sequence data extracted from the patient's normal tissue cells with sequence data extracted from the patient's tumor tissue cells and identifying portions containing one or more mutations. The prediction module can identify candidate antigens, which are pathogen-derived peptides, virus-derived peptides, bacterial-derived peptides, fungal-derived peptides, and parasite-derived peptides, by comparing sequence data extracted from the patient's normal tissue cells with sequence data extracted from the patient's infected cells and identifying portions containing one or more antigens associated with infectious organisms. The prediction module can identify candidate antigens whose expression is altered in tumor cells or cancerous tissue compared to normal cells or tissues, by comparing sequence data extracted from the patient's normal tissue cells with sequence data extracted from the patient's tumor tissue cells and identifying inappropriately expressed candidate antigens. The prediction module can identify candidate antigens expressed in infected cells or tissues compared to normal cells or tissues by comparing sequence data extracted from the patient's normal tissue cells with sequence data extracted from the patient's infected tissue cells and identifying expressed candidate antigens (e.g., identifying expressed polynucleotides and / or polypeptides specific to the infection).

[0357] The presentation module can estimate the presentation potential of a peptide sequence by applying one or more presentation models to a processed peptide sequence. Specifically, the prediction module can select one or more candidate antigen peptide sequences that are likely to be presented to tumor HLA molecules or infected cell HLA molecules by applying presentation models to candidate antigens. In one embodiment, the presentation module selects candidate antigen sequences whose estimated presentation potential exceeds a predetermined threshold. In another embodiment, the presentation model selects the N candidate antigen sequences with the highest estimated presentation potential (where N is generally the maximum number of epitopes that can be delivered by the vaccine). For a given patient, a vaccine containing the selected candidate antigens can be injected to stimulate an immune response.

[0358] XI.B. Cassette Design Module XI.B.1 Overview Cassette design modules can be used to generate vaccine cassette sequences based on candidate peptides selected for injection into patients. For example, a cassette design module can be used to generate sequences encoding linked epitope sequences, such as linked T cell epitopes. Various cassette design modules are known to those skilled in the art, and are described in detail, for example, U.S. Patent No. 10,055,540, U.S. Patent Publication No. US20200010849A1, and International Patent Publication Nos. WO / 2018 / 195357 and WO / 2018 / 208856, respectively, which are incorporated herein by reference in their entirety for all purposes.

[0359] A set of therapeutic epitopes may be generated based on selected peptides determined by a predictive module associated with presentation potential exceeding a predetermined threshold, where presentation potential is determined by a presentation model. However, in other embodiments, it is understood that a set of therapeutic epitopes may be generated based on one or more of a number of methods (single or in combination), such as binding affinity or predicted binding affinity to a patient's HLA class I or HLA class II allele, binding stability or predicted binding stability to a patient's HLA class I or HLA class II allele, random sampling, etc.

[0360] A therapeutic epitope may correspond to the selected peptide itself. In addition to the selected peptide, a therapeutic epitope may also include C-terminal and / or N-terminal flanking sequences. These N-terminal and C-terminal flanking sequences may be the native N-terminal and C-terminal flanking sequences of the therapeutic vaccine epitope in relation to its source protein. A therapeutic epitope may represent an epitope of fixed length. A therapeutic epitope may represent an epitope of variable length, where the epitope length may vary, for example, depending on the length of the C-flanking sequence or the N-flanking sequence. For example, the C-terminal and N-terminal flanking sequences can each have different lengths of 2 to 5 residues, resulting in 16 possible choices for the epitope.

[0361] The cassette design module can also generate cassette sequences that take into account the presentation of junctional epitopes spanning the junction between a pair of therapeutic epitopes within the cassette. Junctional epitopes are novel, non-self but unrelated epitope sequences that arise in the cassette as a result of the process of linking therapeutic epitopes and linker sequences within the cassette. The novel sequences of junctional epitopes are different from the therapeutic epitopes of the cassette itself.

[0362] The cassette design module can generate cassette sequences that reduce the likelihood of junctional epitopes being presented in a patient. Specifically, when a cassette is injected into a patient, junctional epitopes may be presented by the patient's HLA class I or HLA class II alleles, stimulating a CD8 or CD4 T cell response, respectively. Such a response is often undesirable because it offers no therapeutic benefit to the T cells reactive to that junctional epitope and can attenuate the immune response to the selected therapeutic epitopes within the cassette through antigen competition. 76

[0363] The cassette design module can iterate through one or more candidate cassettes and determine which cassette sequences have a presentation score for the associated junctional epitope that falls below a numerical threshold. The junctional epitope presentation score is a quantity related to the presentation likelihood of a junctional epitope within a cassette; a higher score indicates a higher likelihood that the cassette's junctional epitope is presented by HLA class I, HLA class II, or both.

[0364] In one embodiment, the cassette design module may determine the cassette sequence associated with the candidate cassette sequence that has the lowest junction epitope presentation score.

[0365] The cassette design module can iterate through one or more candidate cassette sequences, determine the junction epitope presentation score of the candidate cassettes, and identify the optimal cassette sequence associated with a junction epitope presentation score below a threshold.

[0366] The cassette design module may further check one or more candidate cassette sequences to identify whether any of the junctional epitopes within the candidate cassette sequences are autoepitopes of a given patient for whom the vaccine is being designed. To achieve this, the cassette design module checks junctional epitopes against a known database such as BLAST. In one embodiment, the cassette design module is configured to design cassettes that avoid junctional autoepitopes.

[0367] The cassette design module can employ a brute-force method to iterate through all or most possible candidate cassette sequences and select the sequence with the lowest junction epitope presentation score. However, the number of such candidate cassettes can become enormous as vaccine volume increases. For example, if the vaccine volume is 20 epitopes, the cassette design module would need to iterate through approximately 10 to determine the cassette with the lowest junction epitope presentation score. 18 The number of possible candidate cassettes must be iterated over. Such decisions are computationally burdensome (in terms of the computational resources required) and sometimes difficult to resolve for the cassette design module to complete vaccine production for patients within a reasonable timeframe. Furthermore, considering possible junctional epitopes for each candidate cassette can be an even greater burden. Therefore, the cassette design module may select cassette sequences based on a method that iterates over a large number of candidate cassette sequences, significantly fewer than the number of candidate cassette sequences in the brute-force method.

[0368] The cassette design module can generate a subset of candidate cassettes that are randomly or at least quasi-randomly generated, and select candidate cassettes associated with junction epitope presentation scores below a predetermined threshold as the cassette sequence. Furthermore, the cassette design module may select candidate cassettes from the subset with the lowest junction epitope presentation scores as the cassette sequence. For example, the cassette design module may generate a subset of approximately 1 million candidate cassettes for a set of 20 selected epitopes and select the candidate cassette with the smallest junction epitope presentation score. Generating a subset of random cassette sequences and selecting a cassette sequence with a low junction epitope presentation score from that subset may be suboptimal compared to brute force, but it requires significantly fewer computational resources, making its implementation technically feasible. Moreover, in contrast to this more efficient method, brute force may only yield a small, or even negligible, improvement in junction epitope presentation scores, making it worthless from a resource allocation perspective. The Cassette Design Module can determine an improved cassette configuration by formulating the cassette's epitope array as an asymmetric traveling salesman problem (TSP). Given a list of nodes and the distances between each node pair, the TSP determines the array of nodes associated with the minimum total distance traveled to each node only once and returning to the original node. For example, given cities A, B, and C whose distances to each other are known, the TSP solution generates a closed city array where the total distance traveled to visit each city only once is the minimum among the possible paths. The asymmetric version of the TSP determines the optimal node array when the distances between node pairs are asymmetric. For example, the "distance" to travel from node A to node B may be different from the "distance" to travel from node B to node A. By solving for the improved optimal cassette using the asymmetric TSP, the Cassette Design Module can traverse the junctions between the cassette's epitopes to find a cassette array that results in a reduction in presentation score.The solution for the asymmetric TSP presents a therapeutic epitope sequence corresponding to the order in which epitopes should be linked within the cassette, in order to minimize the junctional epitope presentation score across the entire cassette junction. Cassette sequences determined by this method may result in sequences with significantly less junctional epitope presentation, while requiring significantly fewer computational resources than random sampling methods, especially when the number of candidate cassette sequences generated is large. Exemplary examples of various computational methods and comparisons for cassette design optimization are detailed in U.S. Patent No. 10,055,540, U.S. Patent Publication No. US20200010849A1, and International Patent Publications WO / 2018 / 195357 and WO / 2018 / 208856, respectively, which are incorporated herein by reference in their entirety for all purposes.

[0369] Common (neo)antigen sequences for inclusion in common antigen vaccines and patients suitable for treatment with such vaccines can be selected by those skilled in the art, for example, as described in U.S. Patent Application No. 17 / 058,128, which is incorporated herein by reference for all purposes. Confirmation of common (neo)antigen candidates by mass spectrometry (MS) can be carried out as part of the selection process.

[0370] The cassette design module can also generate cassette sequences that take into account additional protein sequences encoded by the vaccine. For example, a cassette design module used to generate sequences encoding linked T cell epitopes can take into account T cell epitopes already encoded by additional protein sequences (e.g., full-length protein sequences) present in the vaccine, for example, by removing T cell epitopes already encoded by additional protein sequences from the list of candidate sequences.

[0371] The cassette design module can also generate cassette sequences while taking sequence size into account. While we do not wish to be constrained by theory, generally, increasing cassette size can negatively impact aspects of the vaccine, such as vaccine generation and / or vaccine efficacy. For example, the cassette design module can take into account duplicate sequences, such as duplicate T cell epitope sequences. Generally, a single sequence containing duplicate T cell epitope sequences (also called a "frame") is more efficient than sequences that separately link individual T cell epitopes, as the sequence size required to encode multiple peptides is reduced. Therefore, in an exemplary example, the cassette design module used to generate a sequence encoding linked T cell epitopes can take into account the cost / benefit of extending the T cell epitope candidate to encode one or more additional T cell epitopes, for example, determining the benefit obtained by targeting a further population with MHCs presenting additional T cell epitopes in comparison to the cost of increasing sequence size.

[0372] The cassette design module can also generate cassette sequences that take into account the degree of stimulation of the immune response triggered by the identified epitope.

[0373] The cassette design module can also generate cassette sequences that take into account the presentation of an encoded epitope across a population, for example, that at least one immunogenic epitope is presented by at least one HLA (e.g., HLA-A, HLA-B, and HLA-C genes across four major ethnic groups, namely Europeans (EUR), African Americans (AFA), Asian Pacific Islanders (APA), and Hispanics (HIS)). As an exemplary, non-limiting example, the cassette design module can also generate cassette sequences such that at least one HLA presents across at least 85%, 90%, or 95% of the population, either presenting at least one confirmed epitope or at least four, five, six, or seven predicted epitopes.

[0374] The cassette design module can also generate cassette sequences that take into account other aspects of improving potential safety, such as encoding or limiting the encoding of functional proteins, functional protein domains, functional protein subunits, or functional protein fragments that may pose a safety risk. In some cases, the cassette design module can limit the sequence size of the encoded peptide to less than 50%, less than 49%, less than 48%, less than 47%, less than 46%, less than 45%, less than 45%, less than 43%, less than 42%, less than 41%, less than 40%, less than 39%, less than 38%, less than 37%, less than 36%, less than 35%, less than 34%, or less than 33% of the translated corresponding full-length protein. In some cases, the cassette design module can limit the sequence size of the encoded peptide to less than 50% of the translated corresponding full-length protein, although multiple sequences may originate from the same translated corresponding full-length protein and together encode more than 50%. To illustrate with an example, if a single sequence containing a duplicated T cell epitope sequence ("frame") is larger than 50% of the translated corresponding full-length protein, the frame can be split into multiple frames (e.g., f1, f2, etc.) so that each frame is less than 50% of the translated corresponding full-length protein. The cassette design module can also restrict the sequence size of the encoded peptide so that a single consecutive sequence is less than 49%, less than 48%, less than 47%, less than 46%, less than 45%, less than 45%, less than 43%, less than 42%, less than 41%, less than 40%, less than 39%, less than 38%, less than 37%, less than 36%, less than 35%, less than 34%, or less than 33% of the translated corresponding full-length protein. If multiple frames are encoded from the same gene, the multiple frames can have sequences that overlap with each other; in other words, each can separately encode the same sequence.When multiple frames are encoded from the same gene, two or more nucleic acid sequences derived from the same gene can be arranged such that the first nucleic acid sequence is not immediately preceding or ligated to the second nucleic acid sequence, provided that in the corresponding gene, the second nucleic acid sequence follows the first, regardless of whether it is immediately following or not. For example, if there are three frames within the same gene (f1, f2, f3 in increasing order of amino acid position): - The following cassette order is not allowed: ○ f2 immediately after f1 ○ f3 immediately after f2 ○ f3 immediately after f1 - The following cassette order is acceptable: ○ f2 immediately after f3 ○ f1 immediately after f2

[0375] XIII. Examples of Computers A computer may be used in any of the calculation methods described herein. A person skilled in the art will recognize that computers may have different architectures. Examples of computers are known to those skilled in the art, for example, in U.S. Patent No. 10,055,540, U.S. Patent Publication No. US20200010849A1, and International Patent Publications WO / 2018 / 195357 and WO / 2018 / 208856, which are incorporated herein by reference in their entirety for all purposes. [Examples]

[0376] XIV. Examples The following are examples of specific embodiments for carrying out the present invention. These examples are merely illustrative and do not limit the scope of the present invention in any way. Although attempts have been made to ensure accuracy regarding the numbers used (e.g., quantities, temperatures, etc.), some experimental errors and deviations should naturally be accepted.

[0377] Unless otherwise specified, the implementation of this invention will utilize conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the scope of the art. 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), and Carey and Sundberg Advanced Organic Chemistry 3. rd See Ed. (Plenum Press) Vols A and B (1992).

[0378] XIV.A.ChAd antigen cassette delivery vector In one example, chimpanzee adenovirus (ChAdV) was modified to serve as an antigen cassette delivery vector. In another example, a full-length ChAdV68 vector was synthesized based on AC_000011.1 (sequence 2 from patent US6083716), with deletions in the E1 (nt457~3014) and E3 (nt27,816~31,332) sequences. A reporter gene under the control of a CMV promoter / enhancer was inserted in place of the deleted E1 sequence. Transfection of this clone into HEK293 cells did not yield an infectious virus. To confirm the sequence of the wild-type ChAdV68 virus, isolate VR-594 was obtained from ATCC, passaged, and then independently sequenced (SEQ ID NO: 10). When the AC_000011.1 sequence was compared with the ATCC VR-594 sequence of the wild-type ChAdV68 virus (SEQ ID NO: 10), six nucleotide differences were identified. In one example, an improved ChAdV68 vector was generated based on AC_000011.1, with the corresponding ATCC VR-594 nucleotide substituted at five positions (ChAdV68.5WTnt SEQ ID NO: 1).

[0379] In another example, an improved ChAdV68 vector was generated based on AC_000011.1, with deletions of the E1(nt577~3403) and E3(nt27,816-31,332) sequences, and substitutions of the corresponding ATCC VR-594 nucleotides at four positions. A GFP reporter (ChAdV68.4WTnt.GFP; SEQ ID NO: 11) or a model neoantigen cassette (ChAdV68.4WTnt.MAG25mer; SEQ ID NO: 12) under the control of a CMV promoter / enhancer was inserted in place of the deleted E1 sequence.

[0380] In another example, an improved ChAdV68 vector was generated based on AC_000011.1, with deletions of the E1 (nt577~3403) and E3 (nt27,125~31,825) sequences, and substitutions of the corresponding ATCC VR-594 nucleotides at five positions. A GFP reporter (ChAdV68.5WTnt.GFP; SEQ ID NO: 13) or a model neoantigen cassette (ChAdV68.5WTnt.MAG25mer; SEQ ID NO: 2) under the control of a CMV promoter / enhancer was inserted in place of the deleted E1 sequence.

[0381] In another example, an improved ChAdV68 vector for antigen expression ("chAd68-empty-E4 deletion" SEQ ID NO: 29369) was generated based on AC_000011.1, with deletions in the E1 (nt577~3403), E3 (nt27,125~31,825), and E4 region (nt34,916~35,642) sequences, and substitutions of the corresponding ATCC VR-594 (independently sequenced full-length VR-594 C68 SEQ ID NO: 10) nucleotides at five positions. The full-length ChAdV68 AC_000011.1 sequence with the corresponding ATCC VR-594 nucleotides substituted at five positions is called "ChAdV68.5WTnt" (SEQ ID NO: 1). An antigen cassette under the control of a CMV promoter / enhancer is inserted in place of the deleted E1 sequence.

[0382] Related vectors are listed below: - Full-length ChAdVC68 sequence "ChAdV68.5WTnt" (SEQ ID NO: 1); AC_000011.1 sequence in which the corresponding ATCC VR-594 nucleotide is substituted at five positions. - ATCC VR-594 C68 (Sequence ID 10); Sequence determined independently; Total length C68, - ChAdV68.4WTnt.GFP (SEQ ID NO: 11); AC_000011.1 with deletions of E1 (nt577~3403) and E3 (nt27,816-31,332) sequences; corresponding ATCC VR-594 nucleotides substituted at four positions; GFP reporter under the control of a CMV promoter / enhancer inserted in place of the deleted E1. - ChAdV68.4WTnt.MAG25mer (SEQ ID NO: 12); AC_000011.1 with deletions of E1 (nt577~3403) and E3 (nt27,816-31,332) sequences; substitution of the corresponding ATCC VR-594 nucleotide at four positions; insertion of a model neoantigen cassette under the control of a CMV promoter / enhancer in place of the deleted E1. - ChAdV68.5WTnt.GFP (SEQ ID NO: 13); AC_000011.1 with deletions of E1 (nt577~3403) and E3 (nt27,125~31,825) sequences; substitution of the corresponding ATCC VR-594 nucleotides at five positions; insertion of a GFP reporter under the control of a CMV promoter / enhancer in place of the deleted E1. - ChAd68-empty-E4 deletion (SEQ ID NO: 29369): AC_000011.1, in which the E1 (nt577~3403) sequence, E3 (nt27,125~31,825) sequence, and E4 region (nt34,916~35,642) sequence are deleted and the corresponding ATCC VR-594 (independently sequenced full-length VR-594 C68 SEQ ID NO: 10) nucleotides are substituted at five positions. - ChAdV68-GAG (full-length ChAd68-CMV-SIVGag-SV40 polyA; SEQ ID NO: 29371); AC_000011.1 with deletions of E1 (nt577~3403) and E3 (nt27,125~31,825) sequences; substitution of the corresponding ATCC VR-594 nucleotide at five positions; full-length codon-optimized SIV under the control of the CMV promoter / enhancer. SME543 Insert GAG in place of the deleted E1.

[0383] Adenovirus generation in 293F cells ChAdV68 virus generation was performed in 293F cells grown in 293FreeStyle™ (ThermoFisher) medium at 8% CO2 in an incubator. On the day of infection, cells were 10 with a 98% viability rate. 6The solution was diluted to cells / mL and 400 mL was used per production run in a 1 L Corning Erlenmeyer flask. 4 mL of tertiary virus stock solution with a target MOI greater than 3.3 was used for each infection. Cells were incubated for 48-72 hours until the viability, as measured by trypan blue, fell below 70%. Infected cells were then collected by centrifugation (high-speed benchtop centrifuge), washed with 1×PBS, centrifuged again, and resuspended in 20 mL of 10 mM Tris pH 7.4. The cell pellet was thawed three times and clarified by centrifugation at 4,300×g for 5 minutes.

[0384] Purification of adenovirus by CsCl centrifugation Viral DNA was purified by CsCl centrifugation. Two discontinuous gradient runs were performed. In the first run, the virus was purified from cellular components, and in the second run, it was further separated from the cellular components, and defective particles were separated from infectious particles.

[0385] 10 mL of 1.2 CsCl (26.8 g CsCl dissolved in 92 mL of 10 mM Tris pH 8.0) was added to a polyalomer tube. Then, 8 mL of 1.4 CsCl (53 g CsCl dissolved in 87 mL of 10 mM Tris pH 8.0) was carefully added to the bottom layer of the tube using a pipette. The clarified virus was carefully overlaid on top of the 1.2 layer. If necessary, an additional 10 mM Tris was added to balance the tube. The tube was then placed in an SW-32Ti rotor and centrifuged at 10°C for 2 hours and 30 minutes. The tube was then transferred to a laminar flow cabinet, and the virus band was aspirated using an 18 gauge needle and a 10 mL syringe. Care was taken not to extract any contaminating host cell DNA and proteins. The band was then diluted at least twice with 10 mM Tris pH 8.0 and overlaid with a discontinuous gradient as described above. The centrifugal run was performed as described above, but this time the run was performed overnight. The next day, the bands were aspirated, taking care not to aspirate any defective particle bands. Subsequently, the virus was dialyzed against ARM buffer (20mM Tris pH8.0, 25mM NaCl, 2.5% glycerol) using a Slide-a-Lyzer® Cassette (Pierce). This was done for one hour with each buffer change, for a total of three times. After that, the virus was divided into equal volumes and stored at -80°C.

[0386] Adenovirus virus assay The VP concentration is 1.1 × 10⁻⁶. 12 The assay was performed using the OD260 assay, which is based on the fact that the extinction coefficient of viral particles (VP) is equal to an absorbance value of 1 at OD260 nm. Two dilutions of adenovirus (1:5 and 1:10) were prepared in viral lysis buffer (0.1% SDS, 10 mM Tris pH 7.4, 1 mM EDTA). The OD was measured in two series for both dilutions, and the result was calculated as OD260 value × dilution factor × 1.1 × 10⁻¹⁰. 12 The VP solution was applied, and the VP concentration / mL was measured.

[0387] Infectious unit (IU) titers were calculated by limiting dilution assay of the viral stock solution. The virus was first diluted 100-fold with DMEM / 5%NS / 1×PS, and then 1×10⁻¹ dilutions were used to calculate the titer. -7 The solution was diluted to [value missing]. Then, 100 μL of these dilutions was added to 293A cells in a 24-well plate that had been seeded at least 1 hour earlier at a rate of 3e5 cells / well. This was repeated in two sets. The plates were incubated in a CO2 (5%) incubator at 37°C for 48 hours. After that, the cells were washed with 1×PBS and fixed with 100% cold methanol (-20°C). The plates were then incubated at -20°C for at least 20 minutes. After washing the wells with 1×PBS, they were blocked in 1×PBS / 0.1% BSA at room temperature for 1 hour. Rabbit anti-Ad antibody (Abcam, Cambridge, MA) was added to blocking buffer at a dilution of 1:8,000 (0.25 ml per well) and incubated at room temperature for 1 hour. The wells were washed four times with 0.5 mL of PBS per well. A 1000-fold dilution of HRP-conjugated goat anti-rabbit antibody (Bethyl Labs, Montgomery Texas) was added to each well and incubated for 1 hour before the final round of washing. Five washes were performed using PBS, and plates were prepared using Tris-buffered saline solution with 0.01% H2O2 added DAB (diaminobenzidine tetrahydrochloride) substrate (50 mM Tris pH 7.5, 0.67 mg / mL DAB in 150 mM NaCl). Wells were prepared 5 minutes before counting. Cells were counted under a 10x objective lens using dilutions that yielded 4–40 stained cells per field of view. The field of view used was 0.32 mm, corresponding to 625 grids per field of view in a 24-well plate. 2 The grid was as follows. The infectious virus count / mL can be determined by multiplying the number of stained cells per grid by the number of grids per field of view and then multiplying by a dilution factor of 10. Similarly, when using GFP-expressing cells, the GFP-expressing virion coun...

Claims

1. 1. A composition comprising a chimpanzee adenovirus (ChAdV) vector for use in a method of treatment, the method comprising administering multiple doses of the composition to a subject, the multiple doses comprising at least a first dose and a second dose, wherein the period between the first dose and the second dose is at least 27 weeks.

2. 1. A composition comprising a chimpanzee adenovirus (ChAdV) vector for use in a method of treatment, the method comprising administering multiple doses of the composition to a subject, the multiple doses comprising at least a first dose and a second dose, and wherein a ChAdV-specific neutralizing antibody titer is determined to be below a neutralization threshold prior to administration of the second dose.

3. 1. A composition for use in a method of treatment comprising a chimpanzee adenovirus (ChAdV) vector, said method comprising: (a) administering to a subject an initial dose of the composition; (b) determining or having determined a ChAdV-specific neutralizing antibody titer; and (c) if the ChAdV-specific neutralizing antibody titer is determined to be below the neutralization threshold, administering a second dose of the composition to the subject. The composition comprising: (i) the period between the first dose and the second dose is at least 27 weeks, at least 28 weeks, at least 29 weeks, at least 30 weeks, at least 31 weeks, or at least 32 weeks; and / or (ii) the first dose is a priming dose; and / or (iii) the plurality of doses comprises three or more doses, and optionally one or more of the plurality of doses is administered prior to the first dose; and / or (iv) no additional dose of the composition is administered between the first dose and the second dose; and / or (v) the neutralizing antibody titer is NT50 value calculated as the lowest dilution of serum from the immunized subject that neutralizes the ChAdV virus by 50% and optionally, the neutralization threshold is an NT50 value of 900 or less. A composition for use according to any one of claims 1 to 3.

5. The composition for use according to any one of claims 1 to 4, wherein the period between the first dose and the second dose is at least 32 weeks. (i) determining the neutralizing antibody titer, (1) contacting one or more dilutions of serum from the immunized subject with the ChAdV virus under conditions sufficient to neutralize the ChAdV virus; and (2) assessing neutralization of the ChAdV virus relative to non-neutralized virus. and / or wherein the step of assessing neutralization comprises assessing expression of a reporter construct expressed by the ChAdV virus. (ii) the neutralization threshold is the minimum neutralizing antibody titer for complete neutralization of the ChAdV virus at the second dose; and / or (iii) the neutralization threshold is the minimum neutralizing antibody titer at which the second dose induces an immune response in the subject; A composition for use according to any one of claims 2 to 5.

7. The ChAdV vector encodes at least one antigen, and optionally (i) the at least one antigen is a non-autologous peptide; and / or (ii) the at least one antigen is a non-autologous peptide, and the non-autologous peptide is not encoded by a wild-type gene of the subject; and / or (iii) the at least one antigen is a tumor-associated antigen, and / or (iv) the tumor-associated antigen is a neoantigen; and / or (v) the at least one antigen is a foreign antigen, optionally wherein the foreign antigen is from a pathogen, a virus, a bacterium, a fungus, or a parasite; 7. The composition for use according to any one of claims 1 to 6, optionally wherein each of said multiple doses comprises the same antigen. (i) the composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV); Optionally, said IM administration is performed at separate injection sites; Further optionally, the separate injection sites are injection sites in opposing deltoid muscles, or the separate injection sites are injection sites in bilateral gluteus or rectus femoris muscle sites; and / or (ii) the method does not include administration of an immunomodulatory agent or the method is performed in the absence of an immunomodulatory agent, optionally wherein the immunomodulatory agent is a checkpoint inhibitor; and / or (iii) the method further comprises determining or having determined the HLA haplotype of the subject; A composition for use according to any one of claims 1 to 7.

9. The ChAdV vector, (a) a ChAdV backbone, (i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; The ChAdV backbone comprising: (b) a cassette comprising: (i) at least one antigen-encoding nucleic acid sequence, a. an epitope-encoding nucleic acid sequence, optionally including at least one change that causes the encoded epitope sequence to differ from a corresponding peptide sequence encoded by a wild-type nucleic acid sequence; b. optionally a 5' linker sequence; c. optionally a 3' linker sequence; The at least one antigen-encoding nucleic acid sequence optionally comprises The cassette comprising: Including, 9. The composition for use according to any one of claims 1 to 8, wherein said cassette is operably linked to said at least one promoter nucleotide sequence and to said at least one poly(A) sequence, and optionally said at least one promoter nucleotide sequence is operably linked to said cassette.

10. The ChAdV backbone comprises a ChAdV68 vector backbone, and optionally (i) the ChAdV68 vector backbone comprises a functional deletion in at least one gene selected from the group consisting of the adenoviral E1A, E1B, E2A, E2B, E3, L1, L2, L3, L4, and L5 genes with respect to the ChAdV68 genome or with respect to the sequence shown in SEQ ID NO:1, and optionally the adenoviral backbone or modified ChAdV68 sequence is completely or functionally deleted in (A) E1A and E1B, or (B) E1A, E1B, and E3 with respect to the adenoviral genome or with respect to the sequence shown in SEQ ID NO:1, and optionally the E1 gene is functionally deleted via an E1 deletion of at least nucleotides 577-3403 with respect to the sequence shown in SEQ ID NO:1, and optionally the E3 gene is functionally deleted via an E3 deletion of at least nucleotides 27,125-31,825 with respect to the sequence shown in SEQ ID NO:1; and / or (ii) the ChAdV68 vector backbone comprises a partially deleted E4 gene, and optionally the partially deleted E4 gene comprises: A. The E4 gene sequence set forth in SEQ ID NO:1, and lacking at least nucleotides 34,916 to 35,642 of the sequence set forth in SEQ ID NO:1; B. The E4 gene sequence shown in SEQ ID NO:1 and lacking at least nucleotides 34,916-34,942, nucleotides 34,952-35,305, and nucleotides 35,302-35,642 of the sequence shown in SEQ ID NO:1, wherein the vector comprises at least nucleotides 2-36,518 of the sequence shown in SEQ ID NO:1; C. The E4 gene sequence set forth in SEQ ID NO:1 and lacking at least nucleotides 34,980-36,516 of the sequence set forth in SEQ ID NO:1, wherein said vector comprises at least nucleotides 2-36,518 of the sequence set forth in SEQ ID NO:1; D. The E4 gene sequence set forth in SEQ ID NO:1 and lacking at least nucleotides 34,979-35,642 of the sequence set forth in SEQ ID NO:1, wherein said vector comprises at least nucleotides 2-36,518 of the sequence set forth in SEQ ID NO:1; E. E4 deletions, which are at least a partial deletion of E4Orf2, a complete deletion of E4Orf3, and at least a partial deletion of E4Orf4; F. An E4 deletion, which is at least a partial deletion of E4Orf2, at least a partial deletion of E4Orf3, and at least a partial deletion of E4Orf4; G. An E4 deletion, which is at least a partial deletion of E4Orf1, a complete deletion of E4Orf2, and at least a partial deletion of E4Orf3, or H. An E4 deletion that is at least a partial deletion of E4Orf2 and at least a partial deletion of E4Orf3 and / or (iii) the ChAdV68 vector backbone comprises the sequence set forth in SEQ ID NO: 29369, and optionally an antigen cassette is inserted within the E1 deletion; A composition for use according to claim 9. (i) at least one of the epitope-encoding nucleic acid sequences encodes an epitope that, when expressed and translated, can be presented by MHC class I on cells of the subject; and / or (ii) at least one of the epitope-encoding nucleic acid sequences encodes an epitope that, when expressed and translated, can be presented by MHC class II on cells of the subject; and / or (iii) the epitope-encoding nucleic acid sequence contains at least one alteration that causes the encoded epitope to have increased binding affinity for its corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence translated; and / or (iv) the epitope-encoding nucleic acid sequence contains at least one alteration that causes the encoded epitope to have increased binding stability to its corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence translated; and / or (v) the epitope-encoding nucleic acid sequence contains at least one alteration that causes the encoded epitope to have an increased likelihood of presentation on its corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence translated; and / or (vi) the epitope-encoding nucleic acid sequences comprise at least one MHC class I epitope-encoding nucleic acid sequence, each antigen-encoding nucleic acid sequence encoding a polypeptide sequence between 8 and 35 amino acids in length, optionally between 9 and 17, 9 and 25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length; A composition for use according to any one of claims 9 to 10.

12. each antigen-encoding nucleic acid sequence is linked to a separate antigen-encoding nucleic acid sequence by a nucleic acid sequence encoding a linker; Optionally, the linker comprises one or more naturally occurring sequences flanking the antigen from the original cognate protein and being at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length; A composition for use according to any one of claims 9 to 11.

13. the at least one alteration comprises a point mutation, a frameshift mutation, a non-frameshift mutation, a deletion mutation, an insertion mutation, a splice variant, a genomic rearrangement, or a proteasome-generated splice antigen; and optionally (i) the epitope-encoding nucleic acid sequence encodes an epitope that is known or suspected to be expressed in a subject known or suspected to have cancer; and / or (ii) the cancer comprises a solid tumor; and / or (iii) the cancer 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-cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer; A composition for use according to any one of claims 9 to 12.

14. The at least one antigen-encoding nucleic acid sequence is (i) at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen-encoding nucleic acid sequences, optionally each antigen-encoding nucleic acid sequence encoding a separate antigen-encoding nucleic acid sequence; or (ii) 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 antigen-encoding nucleic acid sequences, optionally each antigen-encoding nucleic acid sequence encoding a distinct antigen-encoding nucleic acid sequence; or (iii) 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 antigen-encoding nucleic acid sequences; A composition for use according to any one of claims 9 to 13. (i) one or more of the epitope-encoding nucleic acid sequences and / or each of the epitope-encoding nucleic acid sequences is derived from a tumor in the subject; and / or (ii) one or more of the epitope-encoding nucleic acid sequences and / or each of the epitope-encoding nucleic acid sequences is not derived from a tumor in the subject; and / or (iii) the epitope-encoding nucleic acid sequence comprises an epitope selected from the group consisting of SEQ ID NOs: 57 to 29,364; A composition for use according to any one of claims 9 to 14.

16. The at least one antigen-encoding nucleic acid sequence comprises at least: (A) a KRAS_G12C MHC class I epitope-encoding nucleic acid sequence, optionally encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 14,954, 19,848, and 19,850; (B) a KRAS_G12D MHC class I epitope-encoding nucleic acid sequence, optionally encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19,749, 19,865, and 19,863; and (C) a KRAS_G12V MHC class I epitope-encoding nucleic acid sequence, optionally encoding an MHC class I epitope selected from the group consisting of SEQ ID NOs: 19,976, 19,779, 11,495, and 19,974. A composition for use according to any one of claims 9 to 15, comprising one and / or each of: (i) the composition is formulated as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier; and / or (ii) the composition comprises a viral particle comprising the ChAdV vector; and / or (iii) the method further comprises administering a self-amplifying alphavirus-based expression system, and optionally, the cassette of the ChAdV vector is identical to a cassette encoded in the self-amplifying alphavirus-based expression system. A composition for use according to any one of claims 1 to 16.

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