Neo-antigen adjuvant and maintenance therapy

Abstract

Disclosed herein are compositions comprising antigen-encoding nucleic acid sequences and / or antigenic peptides. Also disclosed are nucleotides, cells, and methods relating to the compositions, including their use as vaccines, including vectors, and methods relating to heterologous prime / boost vaccination strategies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 245,663, filed September 17, 2021, No. 63 / 281,027, filed November 18, 2021, and No. 63 / 320,685, filed March 16, 2022, which are incorporated by reference in their entireties for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was made to XXX, is named XXX and is ###,### bytes in size. [Background technology]

[0003] Therapeutic vaccines based on tumor-specific antigens hold great promise as the next generation of personalized cancer immunotherapy. 1~3 For example, cancers with a high mutational burden, such as non-small cell lung cancer (NSCLC) and melanoma, are particularly suitable targets for such therapies due to their relative propensity to generate neoantigens. 4,5 Early evidence suggests that neoantigen-based vaccination induces T cell responses. 6 , showing that neoantigen-targeted cellular therapy can induce tumor regression under certain circumstances in selected patients. 7

[0004] One of the questions in neo-antigen vaccine design is which of the many coding mutations present in the tumor of interest can induce the "best" therapeutic antigen, e.g., an antigen that can induce anti-tumor immunity and cause tumor regression.

[0005] An initial method is presented that incorporates mutation-based analysis with next-generation sequencing, RNA gene expression, and prediction of MHC binding affinity of candidate neo-antigenic peptides. 8However, these presented methods may fail to model the entire epitope generation process, which contains many steps in addition to gene expression and MHC binding (e.g., TAP trafficking, proteasomal cleavage, and / or TCR recognition). 9 As a result, existing methods are likely to suffer from low positive predictive value (PPV).

[0006] Indeed, analyses of peptides presented by tumor cells performed by multiple groups have shown that less than 5% of predicted peptides can be presented using gene expression and MHC binding affinity found on tumor surface MHC. 10,11 This poor correlation between binding prediction and MHC presentation is further strengthened by the recent observation that the prediction accuracy of binding-restricted neoantigens to checkpoint inhibitor responses does not improve with the number of mutations alone. 12

[0007] This low positive predictive value (PPV) of existing methods for presenting predictions poses a problem for neoantigen-based vaccine design. If a vaccine is designed using a prediction with a low PPV, most patients will be unlikely to receive therapeutic neoantigens, and fewer patients will be likely to receive them more than once (even if all presented peptides are assumed to be immunogenic). Therefore, neoantigen vaccination with current methods is unlikely to be successful in a significant number of subjects with tumors.

[0008] In addition, previous approaches have generated candidate neoantigens using only cis-acting mutations, including mutations in splicing factors that occur in multiple tumor types and result in aberrant splicing of many genes. 13 , and have largely neglected to take into account additional sources of neo-ORFs, including mutations that create or remove protease cleavage sites.

[0009] Finally, standard approaches to tumor genome and transcriptome analysis may miss somatic mutations that give rise to candidate neoantigens due to suboptimal conditions in library construction, exome and transcriptome capture, sequencing, or data analysis. Similarly, standard tumor analysis approaches may inadvertently promote sequence artifacts or germline polymorphisms as neoantigens, leading to underutilized vaccine potential or autoimmune risk, respectively.

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

[0011] Furthermore, targeting antigens shared between patients with cancer, including targeting both mutated neo-antigens as well as non-mutated tumor antigens (e.g., inappropriately expressed tumor antigens), is a very promising vaccine strategy. Challenges with shared antigen vaccine strategies include at least those mentioned above. Summary of the Invention

[0012] A method for stimulating an immune response in a subject, comprising administering to the subject a composition for delivering a self-replicating alphavirus-based expression system and administering to the subject a composition for delivering a chimpanzee adenovirus (ChAdV)-based expression system, wherein the composition for delivering the ChAdV-based expression system is administered as a priming dose and the composition for delivering the self-replicating alphavirus-based expression system is administered as one or more booster doses, wherein the expression system encodes at least one antigen-encoding nucleic acid sequence, and wherein the self-replicating alphavirus-based expression system and the chimpanzee adenovirus (ChAdV)-based expression system are administered as maintenance therapy.

[0013] In some aspects, the maintenance therapy comprises combination therapy with chemotherapy, immune checkpoint inhibitor therapy, radiation therapy, or a combination thereof, optionally, the chemotherapy comprises a fluoropyrimidine and / or bevacizumab.

[0014] Also provided herein is a method for stimulating an immune response in a subject, comprising administering to the subject a composition for delivering a self-replicating alphavirus-based expression system and administering to the subject a composition for delivering a chimpanzee adenovirus (ChAdV)-based expression system, wherein the composition for delivering the ChAdV-based expression system is administered as a priming dose and the composition for delivering the self-replicating alphavirus-based expression system is administered as one or more booster doses, wherein the expression system encodes at least one antigen-encoding nucleic acid sequence, and wherein the self-replicating alphavirus-based expression system and the chimpanzee adenovirus (ChAdV)-based expression system are administered as adjuvant therapy.

[0015] In some aspects, the adjuvant therapy comprises combination therapy with chemotherapy, immune checkpoint inhibitor therapy, radiation therapy, or a combination thereof, optionally, the chemotherapy comprises a fluoropyrimidine and / or bevacizumab.

[0016] Also provided herein is a method for stimulating an immune response in a subject, comprising administering to the subject either (1) a composition for delivering a self-replicating alphavirus-based expression system, or (2) a composition for delivering a chimpanzee adenovirus (ChAdV)-based expression system, wherein the self-replicating alphavirus-based expression system or the chimpanzee adenovirus (ChAdV)-based expression system is administered as an adjunctive therapy, wherein the expression system encodes at least one antigen-encoding nucleic acid sequence, and wherein the adjunctive therapy comprises combination therapy with chemotherapy, immune checkpoint inhibitor therapy, radiation therapy, or a combination thereof, and optionally, the chemotherapy comprises a fluoropyrimidine and / or bevacizumab.

[0017] In some embodiments, one or more booster doses of the composition for delivering the autonomously replicating alphavirus-based expression system are administered.

[0018] In some embodiments, the combination therapy includes a fluoropyrimidine and / or bevacizumab. In some embodiments, the combination therapy includes a fluoropyrimidine and bevacizumab. In some embodiments, the combination therapy includes a fluoropyrimidine, bevacizumab, and immune checkpoint inhibitor therapy. In some embodiments, the immune checkpoint inhibitor includes (1) an anti-PD-1 antibody or an antigen-binding fragment thereof, (2) an anti-PD-L1 antibody or an antigen-binding fragment thereof, and / or (3) an anti-CTLA-4 antibody or an antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor therapy includes administration of an anti-CTLA-4 antibody or an antigen-binding fragment thereof with only a priming dose and a first booster dose. In some embodiments, the anti-CTLA-4 antibody includes ipilimumab. In some embodiments, the ipilimumab is administered subcutaneously at a dose of 30 mg. In some embodiments, the immune checkpoint inhibitor therapy includes administration of an anti-PD-L1 antibody or an antigen-binding fragment thereof every four weeks (Q4W). In some embodiments, the anti-PD-L1 antibody comprises atezolizumab or nivolumab. In some embodiments, atezolizumab is administered intravenously at a dose of 1680 mg. In some embodiments, nivolumab is administered intravenously at a dose of 480 mg.

[0019] In some embodiments, the immune checkpoint inhibitor therapy comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 administrations. In some embodiments, the administration of the anti-PD-L1 antibody or antigen-binding fragment thereof comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 administrations. In some embodiments, the immune checkpoint inhibitor therapy comprises at least 13 administrations. In some embodiments, the administration of the anti-PD-L1 antibody or antigen-binding fragment thereof comprises at least 13 administrations.

[0020] In some embodiments, the subject has previously undergone surgery to remove tumor and / or cancerous tissue, chemotherapy, immunotherapy (e.g., immune checkpoint inhibitor therapy), radiation therapy, or a combination thereof. In some embodiments, the previous chemotherapy comprises oxaliplatin, fluoropyrimidine, and / or bevacizumab. In some embodiments, the previous chemotherapy comprises oxaliplatin, fluoropyrimidine, and bevacizumab. In some embodiments, the previous chemotherapy was administered for up to 24 weeks prior to administration of the maintenance therapy.

[0021] 2. The method of any one of the preceding claims, wherein the subject has colorectal cancer (CRC). In some embodiments, the CRC is stage IV, microsatellite stable, and BRAF wt In some embodiments, the CRC is classified as stage II or III.

[0022] In some embodiments, the subject is classified as ctDNA positive.

[0023] In some embodiments, two or more booster doses are administered. In some embodiments, 1, 2, 3, 4, 5, 6, 7 or 8 booster doses are administered. In some embodiments, the ChAdV-based expression system is further administered as a booster dose. In some embodiments, the ChAdV-based booster dose is only administered as a single booster dose.

[0024] In some embodiments, the ChAdV-based expression system is administered as a booster dose at or about 140 days after the priming dose of the ChAdV-based expression system. In some embodiments, the ChAdV-based expression system is administered as a booster dose at or about 20 weeks after the priming dose of the ChAdV-based expression system. In some embodiments, the ChAdV-based expression system is administered as a booster dose at or about 5 months after the priming dose of the ChAdV-based expression system. In some embodiments, the ChAdV-based expression system is administered as a booster dose at or about 140 days after the priming dose of the ChAdV-based expression system. In some embodiments, the ChAdV-based expression system is administered as a booster dose at or about 20 weeks after the priming dose of the ChAdV-based expression system. In some embodiments, the ChAdV-based expression system is administered as a booster dose at or after 5 months following a priming dose of the ChAdV-based expression system.

[0025] In some embodiments, the composition for delivering the ChAdV-based expression system is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). In some embodiments, the composition for delivering the ChAdV-based expression system is administered (IM). In some embodiments, the intramuscular (IM) administration is performed at separate injection sites. In some embodiments, the separate injection sites are in opposing deltoid muscles. In some embodiments, the separate injection sites are in bilateral gluteus or rectus femoris sites.

[0026] In some embodiments, compositions for delivering a self-replicating alphavirus-based expression system are administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). In some embodiments, compositions for delivering a self-replicating alphavirus-based expression system are administered (IM).

[0027] In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two booster doses. In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two or more booster doses spaced at least 28 days apart. In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two or more booster doses spaced at least four weeks (Q4W). In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two or more booster doses spaced at least one month apart. In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two or more booster doses spaced at least 56 days apart. In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two or more booster doses spaced at least eight weeks (Q8W). In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two or more booster doses spaced at least two months apart.

[0028] In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two booster doses at, or about, days 28 and 84 after the priming dose of the ChAdV-based expression system. In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two booster doses at, or about, weeks 4 and 12 after the priming dose of the ChAdV-based expression system. In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two booster doses at, or about, months 1 and 3 after the priming dose of the ChAdV-based expression system.

[0029] In some embodiments, the self-replicating alphavirus-based expression system is administered as at least four booster doses. In some embodiments, the self-replicating alphavirus-based expression system is administered at or about days 28, 84, 224, and 308 relative to the priming dose of the ChAdV-based expression system. In some embodiments, the self-replicating alphavirus-based expression system is administered at or about weeks 4, 12, 32, and 44 relative to the priming dose of the ChAdV-based expression system. In some embodiments, the self-replicating alphavirus-based expression system is administered at or about months 1, 3, 8, and 11 relative to the priming dose of the ChAdV-based expression system.

[0030] In some embodiments, the methods further comprise determining, or having determined, the subject's HLA haplotype.

[0031] In some embodiments, stimulating an immune response comprises stimulating a molecular response. In some embodiments, the molecular response comprises a reduction in ctDNA.

[0032] In some embodiments, the reduction in ctDNA comprises at least a 20%, at least a 30%, at least a 40%, or at least a 50% reduction in ctDNA. In some embodiments, the reduction in ctDNA comprises at least a 30% reduction in ctDNA. In some embodiments, the reduction in ctDNA comprises at least a 50% reduction in ctDNA.

[0033] In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises a subject-specific neo-antigen-encoding nucleic acid sequence. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises 20 subject-specific neo-antigen-encoding nucleic acid sequences.

[0034] In some embodiments, a composition for delivering a self-replicating alphavirus-based expression system comprises: (A) A self-replicating alphavirus-based expression system, comprising: (a)(i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; an RNA alphavirus backbone comprising: (b) A cassette comprising: (i) a. at least one change that causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence; an epitope-encoding nucleic acid sequence, optionally comprising: b. optionally, a 5' linker sequence, and c. optionally, a 3' linker sequence At least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the at least one antigen-encoding nucleic acid sequence; and (iii) optionally, at least one second poly(A) sequence, wherein the second poly(A) sequence is a poly(A) sequence native to the alphavirus or an exogenous poly(A) sequence; and The cassette comprising: said self-replicating alphavirus-based expression system comprising one or more vectors comprising (B) Lipid nanoparticles (LNPs) encapsulating a self-replicating alphavirus-based expression system. Includes.

[0035] In some embodiments, a composition for delivering a self-replicating alphavirus-based expression system comprises: (A) A self-replicating alphavirus-based expression system, comprising: (a) an RNA alphavirus backbone, The RNA alphavirus backbone comprises the nucleic acid sequence set forth in SEQ ID NO:6, the RNA alphavirus backbone sequence comprises a 26S promoter nucleotide sequence and a poly(A) sequence; said 26S promoter sequence being exogenous to the RNA alphavirus backbone; the poly(A) sequence is exogenous to the RNA alphavirus backbone; said RNA alphavirus backbone, and (b) a cassette integrated between the 26S promoter nucleotide sequence and the poly(A) sequence, the cassette is operably linked to the 26S promoter nucleotide sequence; The cassette comprises: a. at least one change that causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence an epitope-encoding nucleic acid sequence, optionally comprising: b. optionally, a 5' linker sequence, and c. optionally, a 3' linker sequence At least one antigen-encoding nucleic acid sequence comprising Including, The cassette said self-replicating alphavirus-based expression system comprising one or more vectors comprising (B) A lipid nanoparticle (LNP) encapsulating the self-replicating alphavirus-based expression system; Includes.

[0036] In some embodiments, the ordered sequence of each element of the cassette in the composition for delivering a self-replicating alphavirus-based expression system is, in the 5' to 3' direction, P a -(L5 b -N c -L3 d )X-(G5 e -U f ) Y -G3 gwherein P comprises said second promoter nucleotide sequence, with the proviso that a=0 or 1; N comprises one of said epitope-encoding nucleic acid sequences, said epitope-encoding nucleic acid sequence being an MHC class I epitope-encoding nucleic acid sequence, with the proviso that c=1; L5 comprises a 5' linker sequence, with the proviso that b=0 or 1; L3 comprises a 3' linker sequence, with the proviso that d=0 or 1; G5 comprises one of said at least one nucleic acid sequence encoding a GPGPG amino acid linker, with the proviso that e=0 or 1; G3 comprises one of said at least one nucleic acid sequence encoding a GPGPG amino acid linker, with the proviso that g=0 or 1; U comprises one of said at least one MHC class II epitope-encoding nucleic acid sequences, with the proviso that f=1; X=1 to 400, with the proviso that for each X, c is an MHC class I epitope-encoding nucleic acid sequence, Y=0, 1, or 2, with the corresponding U f is an MHC class II epitope-encoding nucleic acid sequence. In some embodiments, for each X, a corresponding N c are distinct MHC class I epitope-encoding nucleic acid sequences. In some embodiments, for each Y, a corresponding U fare different MHC class II epitope-encoding nucleic acid sequences. In some embodiments, a=0, b=1, d=1, e=1, g=1, h=1, X=20, Y=2, said at least one promoter nucleotide sequence is a single 26S promoter nucleotide sequence provided by said RNA alphavirus backbone, said at least one polyadenylation poly(A) sequence is a poly(A) sequence of at least 100 consecutive A nucleotides provided by said RNA alphavirus backbone, said cassette is integrated between said 26S promoter nucleotide sequence and said poly(A) sequence, said cassette is operably linked to said 26S promoter nucleotide sequence and said 26S promoter nucleotide sequence, each N encodes an MHC class I epitope 7-15 amino acids in length, L5 is a native 5' linker sequence encoding a native N-terminal amino acid sequence of said MHC I epitope, said 5' linker sequence encodes a peptide at least 3 amino acids in length, and L3 is a native 5' linker sequence encoding a native N-terminal amino acid sequence of said MHC I epitope, and U is a native 3' linker sequence encoding the native C-terminal amino acid sequence of an MHC class I epitope, the 3' linker sequence encoding a peptide at least 3 amino acids in length, U is a PADRE class II sequence, and a tetanus toxoid MHC class II sequence, respectively, the RNA alphavirus backbone is the sequence set forth in SEQ ID NO:6, and each of the MHC class I epitope-encoding nucleic acid sequences encodes a polypeptide 13 to 25 amino acids in length.

[0037] In some embodiments, the LNP comprises a lipid selected from the group consisting of an ionizable amino lipid, a phosphatidylcholine, cholesterol, a PEG-based coated lipid, or a combination thereof. In some embodiments, the LNP comprises an ionizable amino lipid, a phosphatidylcholine, cholesterol, and a PEG-based coated lipid. In some embodiments, the ionizable amino lipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule. In some embodiments, the LNP encapsulated expression system has a diameter of about 100 nm.

[0038] In some embodiments, the cassette is integrated between at least one promoter nucleotide sequence and at least one poly(A) sequence.

[0039] In some embodiments, at least one promoter nucleotide sequence is operably linked to the cassette.

[0040] In some embodiments, the one or more vectors comprise one or more positive strand RNA vectors. In some embodiments, the one or more positive strand RNA vectors have a 5' 7-methylguanosine (m7g) cap. In some embodiments, the one or more positive strand RNA vectors are generated by in vitro transcription. In some embodiments, the one or more vectors are autonomously replicating in mammalian cells. In some embodiments, the RNA alphavirus backbone comprises at least one nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. In some embodiments, the RNA alphavirus backbone comprises at least one nucleotide sequence of Venezuelan equine encephalitis virus. In some embodiments, the RNA alphavirus backbone comprises at least sequences for nonstructural protein mediated amplification, a 26S promoter sequence, a poly(A) sequence, nonstructural protein 1 (nsP1) gene, nsP2 gene, nsP3 gene, and nsP4 gene encoded by a nucleotide sequence of an Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. In some embodiments, the RNA alphavirus backbone comprises at least sequences for nonstructural protein mediated amplification, a 26S promoter sequence, and a poly(A) sequence encoded by a nucleotide sequence of an Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. In some embodiments, the sequences for nonstructural protein mediated amplification are selected from the group consisting of an alphavirus 5' UTR, a 51 nt CSE, a 24 nt CSE, a 26S subgenomic promoter sequence, a 19 nt CSE, an alphavirus 3' UTR, or a combination thereof. In some embodiments, the RNA alphavirus backbone does not encode the structural virion proteins capsid E2 and E1.In some embodiments, the cassette is inserted in place of a structural virion protein in the nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. In some embodiments, the Venezuelan equine encephalitis virus comprises the sequence of SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, the Venezuelan equine encephalitis virus further comprises the sequence of SEQ ID NO:3 or SEQ ID NO:5 with a deletion between base pairs 7544 and 11175. In some embodiments, the RNA alphavirus backbone comprises the sequence shown in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the cassette is inserted at position 7544 to replace the deletion between base pairs 7544 and 11175 set forth in the sequence of SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, insertion of the cassette results in transcription of a polycistronic RNA comprising the nsP1-4 genes and the at least one nucleic acid sequence, wherein the nsP1-4 genes and the at least one nucleic acid sequence are in separate open reading frames.

[0041] In some embodiments, at least one promoter nucleotide sequence is a natural 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, the second promoter nucleotide sequence is a 26S promoter nucleotide sequence. In some embodiments, the second promoter nucleotide sequence comprises multiple 26S promoter nucleotide sequences, each 26S promoter nucleotide sequence directs the transcription of one or more of the separate open reading frames.

[0042] In some embodiments, the one or more vectors are each at least 300nt in size. In some embodiments, the one or more vectors are each at least 1kb in size. In some embodiments, the one or more vectors are each 2kb in size. In some embodiments, the one or more vectors are each less than 5kb in size.

[0043] In some embodiments, at least one antigen-encoding nucleic acid sequence comprises two or more antigen-encoding nucleic acid sequences. In some embodiments, each antigen-encoding nucleic acid sequence is directly linked to each other. In some embodiments, each antigen-encoding nucleic acid sequence is linked to a different antigen-encoding nucleic acid sequence by a nucleic acid sequence encoding a linker. In some embodiments, the linker links two MHC class I epitope-encoding nucleic acid sequences or an MHC class I epitope-encoding nucleic acid sequence to one MHC class II epitope-encoding nucleic acid sequence. In some embodiments, the linker is selected from the group consisting of: (1) a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive glycine residues in length; (2) a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive alanine residues in length; (3) two arginine residues (RR); (4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence that is efficiently processed by a mammalian protease and is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length; and (6) one or more naturally occurring sequences adjacent to an antigen derived from a protein of homologous origin and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length. In some embodiments, the linker links two MHC class II epitope-encoding nucleic acid sequences or one MHC class II sequence with one MHC class I epitope-encoding nucleic acid sequence. In some embodiments, the linker comprises the sequence GPGPG.

[0044] In some embodiments, the antigen-encoding nucleic acid sequence is operably or directly linked to a separate or contiguous sequence that enhances expression, stability, cellular trafficking, processing and presentation, and / or immunogenicity of the antigen-encoding nucleic acid sequence. In some embodiments, the separate or contiguous sequence includes at least one of a ubiquitin sequence, a ubiquitin sequence modified to enhance proteasome targeting (e.g., a ubiquitin sequence with a Gly or Ala substitution at position 76), an immunoglobulin signal sequence (e.g., IgK), a major histocompatibility class I sequence, a lysosomal-associated membrane protein (LAMP)-1, a human dendritic cell lysosomal-associated membrane protein, and a major histocompatibility class II sequence, optionally wherein the ubiquitin sequence modified to enhance proteasome targeting is A76.

[0045] In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen-encoding nucleic acid sequences, optionally where each antigen-encoding nucleic acid sequence encodes a different antigen-encoding nucleic acid sequence. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 antigen-encoding nucleic acid sequences, optionally where each antigen-encoding nucleic acid sequence encodes a different antigen-encoding nucleic acid sequence. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 antigen-encoding nucleic acid sequences. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 2-400 antigen-encoding nucleic acid sequences, and at least two of the antigen-encoding nucleic acid sequences encode a polypeptide sequence or a portion thereof that is presented by MHC class I at the cell surface. In some embodiments, at least two of the MHC class I epitopes are presented by MHC class I on the tumor cell surface.

[0046] In some embodiments, the epitope-encoding nucleic acid sequences comprise at least one MHC class I epitope-encoding nucleic acid sequence, where each antigen-encoding nucleic acid sequence encodes a polypeptide sequence that is 8-35 amino acids in length, optionally 9-17, 9-25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids in length.

[0047] In some embodiments, at least one MHC class II epitope-encoding nucleic acid sequence is present. In some embodiments, at least one MHC class II epitope-encoding nucleic acid sequence is present and includes at least one MHC class II epitope-encoding nucleic acid sequence that includes at least one change that causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence. In some embodiments, the epitope-encoding nucleic acid sequence includes an MHC class II epitope-encoding nucleic acid sequence, where each antigen-encoding nucleic acid sequence encodes a polypeptide sequence 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length. In some aspects, the epitope-encoding nucleic acid sequence comprises an MHC class II epitope-encoding nucleic acid sequence, wherein at least one MHC class II epitope-encoding nucleic acid sequence is present, and wherein the at least one MHC class II epitope-encoding nucleic acid sequence comprises at least one universal MHC class II epitope-encoding nucleic acid sequence, and optionally, the at least one universal sequence comprises at least one of tetanus toxoid and PADRE.

[0048] In some embodiments, at least one promoter nucleotide sequence or the second promoter nucleotide sequence is inducible. In some embodiments, at least one promoter nucleotide sequence or the second promoter nucleotide sequence is non-inducible. In some embodiments, the at least one poly(A) sequence comprises a naturally occurring alphavirus in the backbone. In some embodiments, at least one poly(A) sequence is operably linked to at least one of the at least one nucleic acid sequence. In some embodiments, the at least one poly(A) sequence is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 consecutive A nucleotides. In some embodiments, the at least one poly(A) sequence is at least 100 consecutive A nucleotides.

[0049] In some aspects, the epitope-encoding nucleic acid sequences comprise MHC class I epitope-encoding nucleic acid sequences, and the MHC class I epitope-encoding nucleic acid sequences are selected by: (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 a peptide sequence for each of a set of epitopes; (b) inputting the peptide sequence for each epitope into a presentation model to generate a set of numerical likelihoods that each of the epitopes is presented by one or more MHC alleles on the tumor cell surface of the tumor, wherein the set of numerical likelihoods have been identified based at least on the received mass spectrometry data; and (c) selecting a subset of the set of epitopes based on the set of numerical likelihoods to generate a set of selected epitopes, which are used to generate the MHC class I epitope-encoding nucleic acid sequences. In some embodiments, each of the MHC class I epitope-encoding nucleic acid sequences is selected by: (a) obtaining at least one of exome, transcriptome, or whole genome tumor nucleotide sequencing data from the tumor, wherein the tumor nucleotide sequencing data is used to obtain data representing a peptide sequence for each of a set of epitopes; (b) inputting the peptide sequence for each epitope into a presentation model to generate a set of numerical likelihoods that each of the epitopes is presented by one or more MHC alleles at the tumor cell surface of the tumor, wherein the set of numerical likelihoods have been identified based on at least the received mass spectrometry data; and (c) selecting a subset of the set of epitopes based on the set of numerical likelihoods to generate a set of selected epitopes, wherein the set of selected epitopes is used to generate at least 20 MHC class I epitope-encoding nucleic acid sequences. In some embodiments, the number of sets of selected epitopes is between 2 and 20.In some embodiments, the presentation model represents a dependency between (a) the presence of a pair of a particular one of the MHC alleles and a particular amino acid at a particular position of a peptide sequence, and (b) the likelihood of presentation of such a peptide sequence comprising the particular amino acid at the particular position on a tumor cell surface by the particular one of the MHC alleles of the pair. In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have an increased likelihood of being presented on a tumor cell surface compared to non-selected epitopes based on the presentation model. In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have an increased likelihood of being able to induce a tumor-specific immune response in a subject compared to non-selected epitopes based on the presentation model. In some embodiments, selecting the set of selected epitopes comprises selecting antigens that have an increased likelihood of being presented to naive T cells by professional epitope-presenting cells (APCs) relative to non-selected epitopes based on the presentation model, optionally the APCs are dendritic cells (DCs). In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have a reduced likelihood of being inhibited by central or peripheral tolerance relative to epitopes that are not selected based on the presentation model. In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have a reduced likelihood of inducing an autoimmune response against normal tissue in a subject relative to epitopes that are not selected based on the presentation model. In some embodiments, the exome or transcriptome nucleotide sequencing data is obtained by sequencing tumor tissue. In some embodiments, the sequencing is next generation sequencing (NGS) or any massively parallel sequencing approach.

[0050] In some embodiments, the ChAdV vector comprises: (a)(i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; A ChAdV backbone comprising: (b) A cassette comprising: (i) a. at least one change that causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence; an epitope-encoding nucleic acid sequence, optionally comprising: b. optionally, a 5' linker sequence; c. optionally, a 3' linker sequence At least one antigen-encoding nucleic acid sequence comprising: The cassette comprising: Including, The cassette is operably linked to the at least one promoter nucleotide sequence and to the at least one poly(A) sequence.

[0051] In some embodiments, the ChAdV vector comprises: (a) a ChAdV backbone, (i) a modified ChAdV68 sequence comprising at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1, wherein said nucleotides 2 to 36,518 are (1) nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, which corresponds to the E1 deletion; (2) nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO:1, which corresponds to the E3 deletion; and Optionally, (3) nucleotides 34,916 to 35,642 of the sequence set forth in SEQ ID NO:1, which corresponds to a partial E4 deletion. and (ii) a CMV promoter nucleotide sequence; and (iii) the SV40 polyadenylation (poly(A)) sequence; The ChAdV backbone, comprising: (b) A cassette comprising: (i) a. at least one change that causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence; and optionally an epitope-encoding nucleic acid sequence comprising: b. optionally, a 5' linker sequence; c. optionally, a 3' linker sequence; At least one antigen-encoding nucleic acid sequence comprising: The cassette comprising: Including, The cassette is inserted within the E1 deletion, the cassette being operably linked to the at least one promoter nucleotide sequence and the SV40 poly(A) sequence.

[0052] In some embodiments, the epitope-encoding nucleic acid sequence encodes an epitope known or suspected to be presented by MHC class I on the surface of a cell, optionally the surface of the cell is a tumor cell surface or an infected cell surface, and optionally the cell is a cell of the subject. 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 cancer, 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 a pathogen-infected cell, a virus-infected cell, a bacteria-infected cell, a fungus-infected cell, and a parasite-infected cell. In some embodiments, the virus-infected cell is an HIV-infected cell.

[0053] In some embodiments, the ordered sequence of each element of the cassette in the composition for delivering a ChAdV-based expression system is, in the 5' to 3' direction, P a -(L5 b -N c -L3 d )X-(G5 e -U f ) Y -G3 gwherein P is at least one promoter sequence operably linked to at least one of said at least one antigen-encoding nucleic acid sequences, with the proviso that a=1; N comprises one of said epitope-encoding nucleic acid sequences, said epitope-encoding nucleic acid sequence being an MHC class I epitope-encoding nucleic acid sequence, with the proviso that c=1; L5 comprises a 5' linker sequence, with the proviso that b=0 or 1; L3 comprises a 3' linker sequence, with the proviso that d=0 or 1; G5 comprises one of said at least one nucleic acid sequence encoding a GPGPG amino acid linker, with the proviso that e=0 or 1; G3 comprises one of said at least one nucleic acid sequence encoding a GPGPG amino acid linker, with the proviso that g=0 or 1; U comprises one of said at least one MHC class II epitope-encoding nucleic acid sequences, with the proviso that f=1; X=1-400, with the proviso that for each X, c is an MHC class I epitope-encoding nucleic acid sequence, Y=0, 1, or 2, with the corresponding U f is an MHC class II epitope-encoding nucleic acid sequence. In some embodiments, for each X, a corresponding N c are distinct MHC class I epitope-encoding nucleic acid sequences. In some embodiments, for each Y, a corresponding U fare distinct MHC class II epitope-encoding nucleic acid sequences. In some embodiments, b=1, d=1, e=1, g=1, h=1, X=16, and Y=2, P is a CMV promoter sequence, each N encodes an MHC class I epitope 7-15 amino acids in length, L5 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the MHC I epitope, the 5' linker sequence encodes a peptide at least 3 amino acids in length, L3 is a native 3' linker sequence encoding the native C-terminal amino acid sequence of the MHC I epitope, the 3' linker sequence encodes a peptide at least 3 amino acids in length, U is each of a PADRE class II sequence and a tetanus toxoid MHC class II sequence, and the ChAdV vector comprises an E1 (nt 577-3403) deletion and an E3 (nt 577-3403) deletion. The modified ChAdV68 sequence comprises the sequence of SEQ ID NO:1 comprising an E1 deletion, wherein the neoantigen cassette is inserted within the E1 deletion, and each of the MHC class I antigen-encoding nucleic acid sequences encodes a polypeptide that is 25 amino acids in length.

[0054] In some embodiments, the ChAdV-based expression system comprises a ChAdV68 vector backbone, the ChAdV68 vector backbone comprising: (1) nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, which corresponds to the E1 deletion; (2) nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO:1, which corresponds to the E3 deletion; and optionally (3) nucleotides 34,916 to 35,642, corresponding to the partially deleted E4 gene of ChAdV68; It contains at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1, except that it lacks:

[0055] In some embodiments, the cassette is integrated between at least one promoter nucleotide sequence and at least one poly(A) sequence. In some embodiments, the at least one promoter nucleotide sequence is operably linked to the cassette.

[0056] In some embodiments, the ChAdV backbone comprises a ChAdV68 vector backbone. In some embodiments, the ChAdV68 vector backbone comprises the sequence set forth in SEQ ID NO:1. In some embodiments, 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 relative to the ChAdV68 genome or relative to the sequence set forth in SEQ ID NO:1, and optionally the adenoviral backbone or modified ChAdV68 sequence is completely or functionally deleted in (1) E1A and E1b, or (2) E1A, E1b, and E3 relative to the adenoviral genome or relative to the sequence set forth in SEQ ID NO:1, and optionally the E1 gene is functionally deleted by an E1 deletion of at least nucleotides 577-3403 relative to the sequence set forth in SEQ ID NO:1, and optionally the E3 gene is functionally deleted by an E3 deletion of at least nucleotides 27,125-31,825 relative to the sequence set forth in SEQ ID NO:1. In some aspects, the ChAdV68 vector backbone comprises one or more genes or regulatory sequences associated with the ChAdV68 genome or associated with the sequence set forth in SEQ ID NO:1, and optionally, the one or more genes or regulatory sequences are selected from the group consisting of chimpanzee adenovirus inverted terminal repeat (ITR), E1A, E1b, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes.

[0057] In some embodiments, the ChAdV68 vector backbone comprises a truncated E4 gene. In some embodiments, the truncated E4 gene is selected from the group consisting of: A. an E4 gene sequence as set forth in SEQ ID NO:1 lacking at least nucleotides 34,916-35,642 of the sequence as set forth in SEQ ID NO:1; B. an E4 gene sequence as set forth in SEQ ID NO:1 lacking at least nucleotides 34,916-34,942, nucleotides 34,952-35,305, and nucleotides 35,302-35,642 of the sequence as set forth in SEQ ID NO:1, wherein the vector comprises at least nucleotides 2-36,518 of the sequence as set forth in SEQ ID NO:1; C. an E4 gene sequence as set forth in SEQ ID NO:1 lacking at least nucleotides 34,980-35,516 of the sequence as set forth in SEQ ID NO:1, wherein the vector comprises at least nucleotides 2-36,518 of the sequence as set forth in SEQ ID NO:1. an E4 gene sequence comprising at least one E4Orf2 gene, a completely deleted E4Orf3 gene, and at least one E4Orf4 gene; D. an E4 gene sequence as shown in SEQ ID NO: 1 lacking at least nucleotides 34,979 to 35,642 of the sequence as shown in SEQ ID NO: 1, and the vector comprising at least nucleotides 2 to 36,518 of the sequence as shown in SEQ ID NO: 1; E. an E4 deletion of at least a partial deletion of E4Orf2, a completely deleted E4Orf3, and at least a partial deletion of E4Orf4; F. an E4 deletion of 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 of at least a partial deletion of E4Orf1, a completely deleted E4Orf2 gene, and at least a partial deletion of E4Orf3; or H. an E4 deletion of at least a partial deletion of E4Orf2 and at least a partial deletion of E4Orf3. In some embodiments, the ChAdV68 vector backbone comprises a modified ChAdV68 sequence including at least nucleotides 2-36,518, wherein said nucleotides 2-36,518 lack (1) nucleotides 577-3403 of the sequence set forth in SEQ ID NO:1, which corresponds to an E1 deletion, (2) nucleotides 27,125-31,825 of the sequence set forth in SEQ ID NO:1, which corresponds to an E3 deletion, and (3) nucleotides 34,916-35,642 of the sequence set forth in SEQ ID NO:1, which corresponds to a partial E4 deletion, and optionally, the antigen cassette is inserted within the E1 deletion.In some embodiments, the ChAdV68 vector backbone comprises the sequence set forth in SEQ ID NO: 29369, and optionally, the antigen cassette is inserted within the E1 deletion. In some embodiments, the ChAdV68 vector backbone comprises a modified ChAdV68 sequence comprising at least nucleotides 2-36,518, said nucleotides 2-36,518 lacking: A. nucleotides 577-3403 of the sequence set forth in SEQ ID NO:1, corresponding to an E1 deletion; B. nucleotides 27,125-31,825 of the sequence set forth in SEQ ID NO:1, corresponding to an E3 deletion; C. nucleotides 34,916-35,642 of the sequence set forth in SEQ ID NO:1, corresponding to a partial E4 deletion; D. nucleotides 456-3014 of the sequence set forth in SEQ ID NO:1; E. nucleotides 27,816-31,333 of the sequence set forth in SEQ ID NO:1; F. nucleotides 3957-10346 of the sequence set forth in SEQ ID NO:1; G. nucleotides 21787-23370 of the sequence set forth in SEQ ID NO:1; H. nucleotides 33486-36193 of the sequence set forth in SEQ ID NO:1, or a combination thereof. In some embodiments, the ChAdV68 vector backbone comprises a modified ChAdV68 sequence including at least nucleotides 2 to 36,518, which lack (1) nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, which corresponds to an E1 deletion, and (2) nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO:1, which corresponds to an E3 deletion.

[0058] In some embodiments, the ChAdV68 vector backbone comprises the sequence set forth in SEQ ID NO:1, except that at least one gene selected from the group consisting of chimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence set forth in SEQ ID NO:1 is completely deleted or functionally deleted from the sequence, and optionally, the sequence is completely deleted or functionally deleted from (1) E1A and E1B, (2) E1A, E1B, and E3, or (3) E1A, E1B, E3, and E4 of the sequence set forth in SEQ ID NO:1. In some embodiments, the ChAdV68 vector backbone comprises genes or regulatory sequences derived from the sequence of SEQ ID NO: 1, and optionally the genes are selected from the group consisting of the inverted terminal repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of chimpanzee adenovirus of the sequence shown in SEQ ID NO: 1. In some embodiments, the ChAdV68 vector backbone comprises one or more deletions between base pairs 577 and 3403 or between base pairs 456 and 3014 of the sequence shown in SEQ ID NO: 1, and optionally the vector further comprises one or more deletions between base pairs 27,125 and 31,825 or between base pairs 27,816 and 31,333. In some embodiments, the ChAdV68 vector backbone contains one or more deletions between base pair numbers 3957 to 10346, base pair numbers 21787 to 23370, and base pair numbers 33486 to 36193 of the sequence set forth in SEQ ID NO:1.

[0059] In some embodiments, the cassette is inserted into the ChAdV backbone in the E1 region, the E3 region, and / or any deleted AdV region that allows for integration of the cassette. In some embodiments, the ChAdV backbone is generated from one of a first generation, second generation, or helper-dependent adenoviral vector.

[0060] In some embodiments, the at least one promoter nucleotide sequence is selected from the group consisting of CMV, SV40, EF-1, RSV, PGK, HSA, MCK, and EBV promoter sequences, hi some embodiments, the at least one promoter nucleotide sequence is a CMV promoter sequence.

[0061] In some embodiments, at least one of the epitope-encoding nucleic acid sequences, when expressed and translated, encodes an epitope capable of being presented on cells of a subject by MHC class I. In some embodiments, at least one of the epitope-encoding nucleic acid sequences, when expressed and translated, encodes an epitope capable of being presented on cells of a subject by MHC class II.

[0062] In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises two or more antigen-encoding nucleic acid sequences, hi some embodiments, each antigen-encoding nucleic acid sequence is directly linked to each other.

[0063] In some embodiments, each antigen-encoding nucleic acid sequence is linked to a different antigen-encoding nucleic acid sequence by a nucleic acid sequence encoding a linker, hi some embodiments, the linker links two MHC class I epitope-encoding nucleic acid sequences or an MHC class I epitope-encoding nucleic acid sequence to one MHC class II epitope-encoding nucleic acid sequence. In some embodiments, the linker is selected from the group consisting of: (1) a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive glycine residues in length; (2) a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive alanine residues in length; (3) two arginine residues (RR); (4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence that is efficiently processed by a mammalian protease and is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length; and (6) one or more naturally occurring sequences adjacent to an antigen derived from a protein of homologous origin and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length. In some embodiments, the linker links two MHC class II epitope-encoding nucleic acid sequences or one MHC class II sequence with one MHC class I epitope-encoding nucleic acid sequence. In some embodiments, the linker comprises the sequence GPGPG.

[0064] In some embodiments, the antigen-encoding nucleic acid sequence is operably or directly linked to a separate or contiguous sequence that enhances expression, stability, cellular trafficking, processing and presentation, and / or immunogenicity of the antigen-encoding nucleic acid sequence. In some embodiments, the separate or contiguous sequence includes at least one of a ubiquitin sequence, a ubiquitin sequence modified to enhance proteasome targeting (e.g., a ubiquitin sequence with a Gly or Ala substitution at position 76), an immunoglobulin signal sequence (e.g., IgK), a major histocompatibility class I sequence, a lysosomal-associated membrane protein (LAMP)-1, a human dendritic cell lysosomal-associated membrane protein, and a major histocompatibility class II sequence, optionally wherein the ubiquitin sequence modified to enhance proteasome targeting is A76.

[0065] In some embodiments, the epitope-encoding nucleic acid sequence comprises at least one change that increases the binding affinity of the encoded epitope to its corresponding MHC allele, compared to the translated corresponding wild-type nucleic acid sequence. In some embodiments, the epitope-encoding nucleic acid sequence comprises at least one change that increases the binding stability of the encoded epitope to its corresponding MHC allele, compared to the translated corresponding wild-type nucleic acid sequence. In some embodiments, the epitope-encoding nucleic acid sequence comprises at least one change that increases the likelihood of the encoded epitope being presented at its corresponding MHC allele, compared to the translated corresponding wild-type nucleic acid sequence. In some embodiments, the at least one change comprises a point mutation, a frameshift mutation, a non-frameshift mutation, a deletion mutation, an insertion mutation, a splice variant, a genomic rearrangement, or a splice antigen generated by the proteasome.

[0066] In some aspects, the epitope-encoding nucleic acid sequence encodes an epitope known or suspected to be expressed in a subject known or suspected to have cancer. In some aspects, the cancer comprises a solid tumor. In some aspects, 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.

[0067] In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen-encoding nucleic acid sequences, optionally where each antigen-encoding nucleic acid sequence encodes a different antigen-encoding nucleic acid sequence. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 antigen-encoding nucleic acid sequences, optionally where each antigen-encoding nucleic acid sequence encodes a different antigen-encoding nucleic acid sequence. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 antigen-encoding nucleic acid sequences. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 2-400 antigen-encoding nucleic acid sequences, and at least two of the antigen-encoding nucleic acid sequences encode a polypeptide sequence or a portion thereof that is presented by MHC class I at the cell surface. In some embodiments, at least two of the MHC class I epitopes are presented by MHC class I on the tumor cell surface.

[0068] In some embodiments, the epitope-encoding nucleic acid sequences comprise at least one MHC class I epitope-encoding nucleic acid sequence, wherein each antigen-encoding nucleic acid sequence encodes a polypeptide sequence that is 8-35 amino acids in length, optionally 9-17, 9-25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids in length.

[0069] In some embodiments, at least one MHC class II epitope-encoding nucleic acid sequence is present. In some embodiments, at least one MHC class II epitope-encoding nucleic acid sequence is present, comprising at least one MHC class II epitope-encoding nucleic acid sequence that includes at least one change that causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence. In some embodiments, the epitope-encoding nucleic acid sequence comprises an MHC class II epitope-encoding nucleic acid sequence, wherein each antigen-encoding nucleic acid sequence encodes a polypeptide sequence 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length. In some aspects, the epitope-encoding nucleic acid sequence comprises an MHC class II epitope-encoding nucleic acid sequence, wherein at least one MHC class II epitope-encoding nucleic acid sequence is present, and wherein the at least one MHC class II epitope-encoding nucleic acid sequence comprises at least one universal MHC class II epitope-encoding nucleic acid sequence, and optionally, the at least one universal sequence comprises at least one of tetanus toxoid and PADRE.

[0070] 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 comprises a bovine growth hormone (BGH) SV40 polyA 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.

[0071] In some embodiments, the cassette further comprises at least one of an intron sequence, a woodchuck hepatitis virus post-transcriptional regulator (WPRE) sequence, an internal ribosome entry sequence (IRES) sequence, a nucleotide sequence encoding a 2A self-cleaving peptide sequence, a nucleotide sequence encoding a furin cleavage site, or a sequence within a non-coding region at the 5' or 3' end known to improve the nuclear transport, stability, or translation efficiency of mRNA operably linked to at least one of the at least one antigen-encoding nucleic acid sequence. In some embodiments, the cassette further comprises a reporter gene, including but 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 an HA tag, a Flag tag, a His tag, or a V5 tag.

[0072] In some embodiments, the one or more vectors further comprise one or more nucleic acid sequences encoding at least one immunomodulator. In some embodiments, the immunomodulator is an anti-CTLA4 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-4-1BB antibody or an antigen-binding fragment thereof, or an anti-OX-40 antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a Fab fragment, a Fab' fragment, a single chain Fv (scFv), a single domain antibody (sdAb) (e.g., camelid antibody domains) as a monospecific antibody or a multispecific antibody linked together, or a full-length single chain antibody (e.g., a full-length IgG in which the heavy and light chains are linked by a flexible linker). In some embodiments, the heavy and light chain sequences of the antibody are contiguous sequences separated by a self-cleaving sequence such as 2A or IRES, or the heavy and light chain sequences of the antibody are linked by a flexible linker such as consecutive glycine residues. In some embodiments, the immune modulator is a cytokine, hi some embodiments, the cytokine is IL-2, IL-7, IL-12, IL-15, or IL-21, or each of its variants.

[0073] In some aspects, the epitope-encoding nucleic acid sequences comprise MHC class I epitope-encoding nucleic acid sequences, and the MHC class I epitope-encoding nucleic acid sequences are selected by: (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 a peptide sequence for each of a set of epitopes; (b) inputting the peptide sequence for each epitope into a presentation model to generate a set of numerical likelihoods that each of the epitopes is presented by one or more MHC alleles on the tumor cell surface of the tumor, wherein the set of numerical likelihoods have been identified based at least on the received mass spectrometry data; and (c) selecting a subset of the set of epitopes based on the set of numerical likelihoods to generate a set of selected epitopes, which are used to generate the MHC class I epitope-encoding nucleic acid sequences. In some embodiments, each of the MHC class I epitope-encoding nucleic acid sequences is selected by: (a) obtaining at least one of exome, transcriptome, or whole genome tumor nucleotide sequencing data from the tumor, wherein the tumor nucleotide sequencing data is used to obtain data representing a peptide sequence for each of a set of epitopes; (b) inputting the peptide sequence for each epitope into a presentation model to generate a set of numerical likelihoods that each of the epitopes is presented by one or more MHC alleles at the tumor cell surface of the tumor, wherein the set of numerical likelihoods have been identified based on at least the received mass spectrometry data; and (c) selecting a subset of the set of epitopes based on the set of numerical likelihoods to generate a set of selected epitopes, wherein the set of selected epitopes is used to generate at least 20 MHC class I epitope-encoding nucleic acid sequences. In some embodiments, the number of sets of selected epitopes is between 2 and 20.In some embodiments, the presentation model represents a dependency between (a) the presence of a pair of a particular one of the MHC alleles and a particular amino acid at a particular position of a peptide sequence, and (b) the likelihood of presentation of such a peptide sequence comprising the particular amino acid at the particular position on a tumor cell surface by the particular one of the MHC alleles of the pair. In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have an increased likelihood of being presented on a tumor cell surface compared to non-selected epitopes based on the presentation model. In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have an increased likelihood of being able to induce a tumor-specific immune response in a subject compared to non-selected epitopes based on the presentation model. In some embodiments, selecting the set of selected epitopes comprises selecting antigens that have an increased likelihood of being presented to naive T cells by professional epitope-presenting cells (APCs) relative to non-selected epitopes based on the presentation model, optionally the APCs are dendritic cells (DCs). In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have a reduced likelihood of being inhibited by central or peripheral tolerance relative to epitopes that are not selected based on the presentation model. In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have a reduced likelihood of inducing an autoimmune response against normal tissue in a subject relative to epitopes that are not selected based on the presentation model. In some embodiments, the exome or transcriptome nucleotide sequencing data is obtained by sequencing tumor tissue. In some embodiments, the sequencing is next generation sequencing (NGS) or any massively parallel sequencing approach.

[0074] In some embodiments, the cassette comprises a junction epitope sequence formed by adjacent sequences within the cassette. In some embodiments, at least one or each junction epitope sequence has an affinity for MHC greater than 500 nM. In some embodiments, each junction epitope sequence is non-self.

[0075] In some embodiments, the cassette does not encode a non-therapeutic MHC class I and MHC class II epitope nucleic acid sequence comprising a translated wild-type nucleic acid sequence, and the non-therapeutic epitope is predicted to be presented on the MHC allele of the subject. In some embodiments, the non-therapeutic predicted MHC class I or MHC class II epitope sequence is a junction epitope sequence formed by adjacent sequences in the cassette. In some embodiments, the prediction is based on a presentation likelihood generated by inputting the sequence of the non-therapeutic epitope into a presentation model. In some embodiments, the order of the antigen-encoding nucleic acid sequence in the cassette is determined by a series of steps including: (a) generating a set of candidate cassette sequences corresponding to different orders of the antigen-encoding nucleic acid sequence; (b) determining a presentation score for each of the candidate cassette sequences based on the presentation of the non-therapeutic epitope in the candidate cassette sequence; and (c) selecting the candidate cassette sequences associated with a presentation score below a predetermined threshold as the cassette sequence for the vaccine.

[0076] In some embodiments, the composition for delivering the ChAdV-based expression system is formulated in a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

[0077] In some embodiments, one or more of the epitope-encoding nucleic acid sequences are derived from the subject's tumor. In some embodiments, each of the epitope-encoding nucleic acid sequences are derived from the subject's tumor. In some embodiments, one or more of the epitope-encoding nucleic acid sequences are not derived from the subject's tumor. In some embodiments, each of the epitope-encoding nucleic acid sequences are not derived from the subject's tumor. In some embodiments, the epitope-encoding nucleic acid sequence comprises an epitope selected from the group consisting of SEQ ID NOs: 57-29,364.

[0078] In some embodiments, the cassette of the composition for delivering the ChAdV-based expression system is the same as the cassette of the composition for delivering the self-replicating alphavirus-based expression system, hi some embodiments, for each of the doses, the cassette of the composition for delivering the self-replicating alphavirus-based expression system is the same. [Brief description of the drawings]

[0079] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings.

[0080] [Figure 1] FIG. 1 depicts a schematic diagram showing a clinical approach to maintenance therapy.

[0081] [Diagram 2] FIG. 1 depicts a schematic diagram showing the clinical approach of adjuvant therapy.

[0082] [Diagram 3] Stands for clinical maintenance treatment dosing regimen. STS = Investigational Treatment Stage. VPS = Vaccine Production Stage. SOC = Standard of Care. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] Detailed Description I. Definition In general, the terms used in the claims and the specification shall be interpreted as having their ordinary meanings as understood by those skilled in the art. Certain terms are defined below to provide further clarity. If there is a discrepancy between the ordinary meaning and a given definition, the given definition shall prevail.

[0084] 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 "shared antigen," which is an antigen found among a particular population, for example, a particular population of cancer patients.

[0085] The term "neo-antigen" as used herein refers to an antigen that has at least one change that makes it different from the corresponding wild-type antigen, for example, due to a tumor cell mutation or a tumor cell-specific post-translational modification. A neo-antigen may include a polypeptide sequence or a nucleotide sequence. The mutation may include a frameshift or non-frameshift indel, a missense or nonsense substitution, a splice site change, a genomic rearrangement or gene fusion, or any genomic or expression change that results in a new ORF. The mutation may also include a splice variant. The tumor cell-specific post-translational modification may include aberrant phosphorylation. The tumor cell-specific post-translational modification may also include a splice antigen generated by the proteasome. See Liepe et al., A large fraction of HLAclass I ligands are proteasome-generated spliced ​​peptides; Science. 2016 Oct 21; 354(6310): 354-358. Exemplary shared antigens are shown in Table A and in the AACR GENIE results (SEQ ID NOs: 10,755-29,357), with the HLA allele(s) corresponding to each antigen also shown. Such shared neo-antigens are useful for inducing an immune response in a subject upon administration. Subjects for administration can be identified by the use of various diagnostic methods, such as the patient selection methods described further below.

[0086] As used herein, the term "tumor antigen" is an antigen that is present in tumor cells or tissues of a subject but not in the corresponding normal cells or tissues of the subject, or an antigen derived from a polypeptide that is known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues.

[0087] As used herein, the term "antigen-based vaccine" refers to a vaccine composition based on one or more antigens, e.g., a plurality of antigens. The vaccine may be nucleotide-based (e.g., viral-based, RNA-based, or DNA-based), protein-based (e.g., peptide-based), or a combination thereof.

[0088] As used herein, the term "candidate antigen" refers to a mutation or other abnormality that gives rise to a sequence that may represent an antigen.

[0089] As used herein, the term "coding region" refers to that portion or portions of a gene that encodes a protein.

[0090] As used herein, the term "coding mutation" refers to a mutation that occurs in a coding region.

[0091] As used herein, the term "ORF" means open reading frame.

[0092] As used herein, the term "neo-ORF" refers to a tumor-specific ORF that arises due to mutation or other abnormalities such as splicing.

[0093] As used herein, the term "missense mutation" is a mutation that results in the substitution of one amino acid for another.

[0094] As used herein, the term "nonsense mutation" is a mutation that results in the substitution of an amino acid with a stop codon or the removal of the canonical start codon.

[0095] As used herein, the term "frameshift mutation" is a mutation that causes an alteration in the frame of a protein.

[0096] As used herein, the term "indel" is an insertion or deletion of one or more nucleic acids.

[0097] As used herein, the term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences in which a certain percentage (%) of nucleotides or amino acid residues are the same when compared and aligned for maximum correspondence as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the "percent identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or over the full length of the two sequences being compared.

[0098] In sequence comparison, generally, one sequence serves as a reference sequence to which test sequences are compared.When using sequence comparison algorithm, test sequences and reference sequences are input into computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity (%) of test sequence to reference sequence based on designated program parameters.Alternatively, sequence similarity or difference can also be established by the combination of the presence or absence of a particular nucleotide at a selected sequence position (e.g., sequence motif) or an amino acid in a translated sequence.

[0099] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).

[0100] One example of an algorithm that is suitable for determining percent sequence identity and percent sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0101] As used herein, the term "non-stop or read-through" refers to a mutation that results in the removal of the natural stop codon.

[0102] As used herein, the term "epitope" refers to a specific portion of an antigen generally bound by an antibody or T-cell receptor.

[0103] As used herein, the term "immunogenic" refers to the ability to stimulate an immune response, for example, via T cells, B cells, or both.

[0104] As used herein, the terms "HLA binding affinity," "MHC binding affinity," refer to the affinity of binding between a specific antigen and a specific HLA or MHC allele.

[0105] As used herein, the term "bait" refers to a nucleic acid probe used to enrich a specific sequence of DNA or RNA from a sample.

[0106] As used herein, the term "mutation" is a difference between the nucleic acid of a subject and a reference human genome used as a control.

[0107] As used herein, the term "variant calling" is the algorithmic determination, typically from sequencing, of the presence of a variant.

[0108] As used herein, the term "polymorphism" refers to a germline mutation, ie, a mutation found in all DNA-bearing cells of an individual.

[0109] As used herein, the term "somatic mutation" is a mutation that occurs in a non-germline cell of an individual.

[0110] As used herein, the term "allele" refers to one version of a gene or one version of a gene sequence or one version of a protein.

[0111] As used herein, the term "HLA type" refers to the complement of HLA gene alleles.

[0112] As used herein, the term "nonsense-mediated decay" or "NMD" refers to the degradation of mRNA by a cell due to a premature stop codon.

[0113] As used herein, the term "truncal mutation" is a mutation that occurs early in the development of a tumor and is present in the majority of the cells of the tumor.

[0114] As used herein, the term "subclonal mutation" is a mutation that occurs late in the development of a tumor and is present in only a portion of the cells of the tumor.

[0115] As used herein, the term "exome" is the subset of the genome that encodes proteins. The exome can be the collection of exons of the genome.

[0116] As used herein, the term "logistic regression" is a regression model for binary data from statistics in which the logit of the probability that the dependent variable is equal to 1 is modeled as a linear function of the dependent variable.

[0117] As used herein, the term "neural network" is a machine learning model for classification or regression that consists of multiple layers of linear transformations followed by element-wise nonlinear transformations typically trained by stochastic gradient descent and backpropagation.

[0118] As used herein, the term "proteome" refers to the set of all proteins expressed and / or translated by a cell, a group of cells, or an individual.

[0119] As used herein, the term "peptidome" refers to the set of all peptides presented by MHC-I or MHC-II on the cell surface. Peptidome can also refer to the properties of a cell or a collection of cells (e.g., a tumor peptidome refers to the collection of all cellular peptidomes contained in a tumor).

[0120] As used herein, the term "ELISpot" refers to enzyme-linked immunosorbent spot assay, a common method for monitoring immune responses in humans and animals.

[0121] As used herein, the term "dextramer" is a dextran-based peptide-MHC multimer used for antigen-specific T cell staining in flow cytometry.

[0122] As used herein, the term "tolerance or immune tolerance" refers to a state of immune unresponsiveness to one or more antigens, eg, self-antigens.

[0123] As used herein, the term "central tolerance" is tolerance conferred in the thymus by either deleting autoreactive T cell clones or promoting the differentiation of autoreactive T cell clones into immunosuppressive regulatory T cells (Tregs).

[0124] As used herein, the term "peripheral tolerance" is tolerance conferred in the peripheral system by downregulating or anergizing autoreactive T cells that survived central tolerance or by promoting the differentiation of these T cells into Tregs.

[0125] The term "sample" can include a single cell, or multiple cells, or fragments of cells, or an aliquot of bodily fluid obtained from a subject by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage sample, scraping, surgical incision, or intervention, or other means known in the art.

[0126] The term "subject" includes cells, tissues, or organisms, either male or female, human or non-human, whether in vivo, ex vivo, or in vitro. The term subject includes mammals, including humans.

[0127] The term "mammal" encompasses both humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0128] The term "clinical factor" refers to a measurement of a subject's condition, e.g., disease activity or severity. "Clinical factor" encompasses all markers of a subject's health status, including non-sample markers, and / or other characteristics of the subject, such as, but not limited to, age and sex. A clinical factor can be a score, value, or set of values ​​that can be obtained from the assessment of a subject or a sample (or a population of samples) from a subject under a given condition. A clinical factor can also be predicted by other parameters, such as markers and / or gene expression surrogates. Clinical factors can include tumor type, tumor subtype, and smoking history.

[0129] "Tumor-derived antigen-encoding nucleic acid sequence" refers to nucleic acid sequence obtained from a tumor, e.g., by RT-PCR, or sequence data obtained by sequencing the tumor and then using the sequencing data to synthesize nucleic acid sequence, e.g., by various synthetic or PCR-based methods known in the art. The resulting sequences can include nucleic acid sequence variants, such as sequence-optimized nucleic acid sequence variants (e.g., codon-optimized and / or otherwise expression-optimized), that encode the same polypeptide sequence as the corresponding native nucleic acid sequence obtained from the tumor.

[0130] The term "alphavirus" refers to members of the Togaviridae family and are single-stranded positive-sense RNA viruses. Alphaviruses are generally classified as Old World types, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World types, such as Eastern equine encephalitis virus, Aula, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83. Alphaviruses are generally self-replicating RNA viruses.

[0131] The term "alphavirus backbone" refers to the minimal sequence(s) of an alphavirus that allows for autonomous replication of the viral genome. Minimal sequences can include conserved sequences for nonstructural protein-mediated amplification, the nonstructural protein 1 (nsP1), nsP2, nsP3, nsP4 genes, and polyA sequences, as well as sequences for expression of subgenomic viral RNA, including subgenomic (e.g., 26S) promoter elements.

[0132] The term "conserved sequence for nonstructural protein-mediated amplification" includes alphavirus conserved sequence elements (CSEs) that are well known in the art. CSEs include, but are not limited to, alphavirus 5'UTR, 51-nt CSE, 24-nt CSE, subgenomic promoter sequences (e.g., 26S subgenomic promoter sequences), 19-nt CSE, and alphavirus 3'UTR. The term "RNA polymerase" includes polymerases that catalyze the production of RNA polynucleotides from a DNA template. RNA polymerases include, but are not limited to, bacteriophage-derived polymerases, including T3, T7, and SP6.

[0133] The term "lipid" includes hydrophobic and / or amphipathic molecules. Lipids may be cationic, anionic, or neutral. Lipids may be synthetic or naturally derived, and in certain instances may be biodegradable. Lipids may include cholesterol, phospholipids, lipid complexes including, but not limited to, 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 nanoparticle" or "LNP" includes vesicle-like structures formed with a lipid-containing membrane surrounding an aqueous interior, also called liposomes. Lipid nanoparticles include lipid-based compositions with a solid lipid core stabilized by surfactants. The core lipids can be fatty acids, acylglycerols, waxes, and mixtures of these surfactants. Biological membrane lipids such as phospholipids, sphingomyelin, bile acids (taurocholic acid), and sterols (cholesterol) can be used as stabilizers. Lipid nanoparticles can be formed with 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 can then be contacted with target cells to deliver the encapsulated molecules to the cytosol of the host cell. Lipid nanoparticles can be modified or functionalized with non-lipid molecules, such as their surfaces. Lipid nanoparticles can be single-layered (unilamellar) or multi-layered (multilamellar). Lipid nanoparticles can be complexed with nucleic acids. Unilamellar lipid nanoparticles can be complexed with nucleic acid, where the nucleic acid is in the aqueous interior. Multilamellar lipid nanoparticles can be complexed with nucleic acid, where the nucleic acid is in the aqueous interior or forms or is sandwiched between the aqueous interior.

[0135] The term "pharmacologically effective amount" is that amount of vaccine components (peptides, recombinant vectors, and / or adjuvants) effective in a given route of administration to confer sufficient levels of protein, protein expression, and / or cell signaling activity (e.g., adjuvant-mediated activation) in cells to confer a vaccine effect, i.e., some measurable level of immunity.

[0136] Abbreviations: MHC: major histocompatibility complex; HLA: human leukocyte antigen, or human MHC locus; NGS: next generation sequencing; PPV: positive predictive value; TSNA: tumor-specific neoantigens; FFPE: formalin-fixed paraffin-embedded; NMD: nonsense-mediated decay; NSCLC: non-small cell lung cancer; DC: dendritic cell.

[0137] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0138] Unless otherwise specified or clear from the context, the term "about" used herein is understood to be within the range of normal tolerance in the art, for example, within 2 standard deviations from the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the word "about".

[0139] Terms not directly defined herein should be understood to have the meanings generally associated with them as understood within the technical field of the present invention. Certain terms are discussed herein for the purpose of providing further guidance to the practitioner in describing the compositions, devices, methods, etc. of the present invention's embodiments, as well as their manufacture or use. It will be recognized that multiple ways of saying the same thing can be used. Thus, alternative terms and synonyms may be used for any one or more of the terms discussed herein. No weight should be placed on whether a term is detailed or discussed herein. Some synonyms or substitute methods, materials, etc. are provided. The description of one or several synonyms or equivalents does not exclude the use of other synonyms or equivalents unless expressly stated. The use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the inventive embodiments herein.

[0140] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0141] II. Identification of antigens Research models for NGS analysis of tumor and normal exomes and transcriptomes have been described previously and applied in the antigen-specific space. 6,14,15 Specific optimizations can be considered to increase the sensitivity and specificity of antigen identification in clinical situations. These optimizations can be divided into two areas: those related to laboratory processes and those related to NGS data analysis. The described approaches can also be applied to antigen identification in other contexts, such as identification from infectious disease organisms, infectious diseases in subjects, or infected cells in subjects. Examples of optimizations are well known to those skilled in the art, and such methods are described in more detail in, for example, U.S. Patent No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1, U.S. Patent Application No. 16 / 606,577, and International Patent Application Publication Nos. WO2020181240A1, WO / 2018 / 195357, and WO2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.

[0142] Methods for identifying shared antigens (e.g., neo-antigens) include identifying antigens derived from the subject's tumor that are likely to be presented on the surface of tumor or immune cells, including professional antigen-presenting cells such as dendritic cells, and / or that are likely to be immunogenic. By way of example, one such method may include obtaining at least one of exome, transcriptome, or whole genome tumor nucleotide sequencing and / or expression data from tumor cells of a subject, using the tumor nucleotide sequencing data and / or expression data to obtain data representing peptide sequences for each of a set of antigens (e.g., in the case of neo-antigens that include at least one alteration that causes the peptide sequence of each neo-antigen to differ from a corresponding wild-type peptide sequence, or in the case of shared antigens where the peptide does not have a mutation derived from any polypeptide known or discovered to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues); inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical likelihoods that each of the antigens will be presented by one or more MHC alleles on the tumor cell surface of tumor cells or cells present in the tumor of the subject, wherein the set of numerical likelihoods have been determined based at least on the received mass spectrometry data; and selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens.

[0143] III. Identification of tumor-specific mutations of neoantigens Also disclosed herein are methods for identifying specific mutations (e.g., variants or alleles present in cancer cells). In particular, these mutations may be present in the genome, transcriptome, proteome, or exome of cancer cells of a subject with cancer, but not present in normal tissue from the subject. Specific methods for identifying neoantigens, including tumor-specific shared neoantigens, are known to those skilled in the art, and are described in more detail, for example, in U.S. Pat. No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1, and International Patent Application Publication Nos. WO / 2018 / 195357 and WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes. Examples of tumor-specific shared neoantigens are described in more detail in International Patent Application Publication No. WO2019226941A1, each of which is incorporated herein by reference in its entirety for all purposes.

[0144] Genetic mutations in tumors may be useful for tumor immunological targeting when such mutations result in changes in the amino acid sequence of proteins only in tumors. Useful mutations include: (1) nonsynonymous mutations that result in different amino acids in proteins; (2) read-through mutations that alter or delete stop codons, resulting in the translation of longer proteins with new tumor-specific sequences at the C-terminus; (3) splice site mutations that introduce introns into mature mRNAs, thereby resulting in unique tumor-specific protein sequences; (4) chromosomal rearrangements (i.e., gene fusions) that result in chimeric proteins with tumor-specific sequences at the junction of two proteins; (5) frameshift mutations or deletions that result in new open reading frames with new tumor-specific protein sequences. Mutations also include one or more of nonframeshift indels, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic or expression changes that result in new ORFs.

[0145] For example, peptides or mutant polypeptides having mutations resulting from splice site, frameshift, readthrough, or gene fusion mutations in tumor cells can be identified by sequencing DNA, RNA, or proteins of tumors versus normal cells.

[0146] Mutations may also include previously identified tumor-specific mutations. Known tumor mutations can be found in the COSMIC (Catalogue of Somatic Mutations in Cancer) database.

[0147] A variety of methods are available for detecting the presence of specific mutations or alleles in an individual's DNA or RNA. Advances in this field have made large-scale SNP genotyping possible that is accurate, easy, and inexpensive. Several techniques have been described, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and various DNA "chip" technologies such as the Affymetrix SNP chip. These methods generally rely on the amplification of target gene regions by PCR. Still other methods are based on the generation of small signal molecules by invasive cleavage, followed by mass spectrometry or immobilized padlock probes and rolling circle amplification. Some of the methods well known in the art for detecting specific mutations are summarized below.

[0148] PCR-based detection means can include multiplex amplification of multiple markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously. Alternatively, different markers can be amplified with primers that are differentially labeled and therefore can be differentially detected. Of course, hybridization-based detection means allow differential detection of multiple PCR products in a sample. Other techniques that allow multiplex analysis of multiple markers are also known in the art.

[0149] Several methods have been developed to facilitate the analysis of single nucleotide polymorphisms in genomic DNA or cellular RNA. For example, single nucleotide polymorphisms can be detected by using special exonuclease-resistant nucleotides, as disclosed, for example, in Mundy, CR (US Pat. No. 4,656,127). According to this method, a primer that is complementary to the allele sequence immediately 3' from the polymorphic site is hybridized to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the specific exonuclease-resistant nucleotide derivative present, the derivative is incorporated at the end of the hybridized primer. This incorporation makes the primer resistant to exonucleases, allowing its detection. Since the nature of the exonuclease-resistant derivative of the sample is known, the fact that the primer has become resistant to exonucleases indicates that the nucleotide(s) present at the polymorphic site of the target molecule are complementary to the nucleotide of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of exogenous sequence data.

[0150] Solution-based methods can be used to determine the type of nucleotide at a polymorphic site (Cohen, D. et al. (French Patent No. 2,650,840; PCT Application No. WO 91 / 02087)). As in the method of Mundy, U.S. Pat. No. 4,656,127, a primer is used that is complementary to the allelic sequence immediately 3' to the polymorphic site. This method uses a labeled dideoxynucleotide derivative that is incorporated onto the end of the primer when it is complementary to the nucleotide at the polymorphic site to determine the nature of the nucleotide at that site.

[0151] An alternative method known as Genetic Bit Analysis or GBA has been described by Goelet, P. et al. (PCT Application No. 92 / 15712). The method of Goelet, P. et al. uses a mixture of labeled terminators and primers that are complementary to the sequence 3' of the polymorphic site. The labeled terminators incorporated are therefore determined by and complementary to the nucleotides present at the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent No. 2,650,840; PCT Application No. WO91 / 02087), the method of Goelet, P. et al. can be a heterogeneous phase assay in which the primers or the target molecule are immobilized on a solid phase.

[0152] Several primer-guided nucleotide incorporation methods for assaying polymorphic sites in DNA have been described (Komher, J. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990); Kuppuswamy, M. et al., Proc. Natl. Acad. Sci. (USA) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al., GATA 9:107-112 (1992); al., Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA in that they use the incorporation of labeled deoxynucleotides to distinguish between bases at polymorphic sites. In such formats, the signal is proportional to the number of deoxynucleotides incorporated, so that polymorphisms occurring in multiple runs of the same nucleotide can produce a signal proportional to the length of the run (Syvanen, A.-C., et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0153] A number of techniques obtain sequence information directly from millions of individual molecules of DNA or RNA in parallel. Real-time single molecule sequencing-by-synthesis techniques are based on the detection of fluorescent nucleotides as they are incorporated into nascent strands of DNA complementary to the template to be sequenced. In one method, the 5' ends of 30-50 base long oligonucleotides are covalently anchored to a coverslip. These anchored strands serve two functions. First, they serve as capture strands for the target template strands when the template strands are configured to have capture tails complementary to the surface-bound oligonucleotides. They also serve as primers for template-guided primer extension that forms the basis of sequence reading. The capture primers serve as fixed-position sites for sequencing using multiple cycles of dye-linker synthesis, detection, and chemical cleavage to remove the dye. Each cycle involves the addition of a polymerase / labeled nucleotide mix, washing, imaging, and cleavage of the dye. In another method, the polymerase is modified with a fluorescent donor molecule and immobilized on a coverslip, while each nucleotide is color coded with an acceptor fluorescent moiety attached to the gamma phosphate. This system detects the interaction of a fluorescently labeled polymerase with a fluorescently modified nucleotide as the nucleotide is incorporated into the nascent strand. Other sequencing by synthesis techniques also exist.

[0154] Any suitable sequencing by synthesis platform can be used to identify the mutations. As mentioned above, four major sequencing by synthesis platforms are currently available: the Genome Sequencer from Roche / 454 Life Sciences, the 1G Analyzer from Illumina / Solexa, the SOLiD system from Applied BioSystems, and the Heliscope system from Helicos Biosciences. Sequencing by synthesis platforms have also been described by Pacific BioSciences and VisiGen Biotechnologies. In some embodiments, multiple nucleic acid molecules to be sequenced are attached to a support (solid support). To immobilize the nucleic acid on the support, a capture sequence / universal priming site can be added to the 3' and / or 5' end of the template. The nucleic acid can be attached to the support by hybridizing the capture sequence to a complementary sequence covalently attached to the support. The capture sequence (also called the universal capture sequence) is a nucleic acid sequence complementary to the support-attached sequence that can double as a universal primer.

[0155] As an alternative to a capture sequence, a member of a coupling pair (e.g., an antibody / antigen, receptor / ligand, or avidin / biotin pair, as described in U.S. Patent Application Publication No. 2006 / 0252077) can be attached to each fragment to be captured onto a surface coated with the corresponding second member of the coupling pair.

[0156] After capture, the sequence can be analyzed, for example, by single molecule detection / sequencing as described in the Examples and / or U.S. Pat. No. 7,283,337, including template-dependent sequencing by stepwise synthesis. In sequencing by stepwise synthesis, the surface-bound molecules are exposed to multiple labeled nucleotide triphosphates in the presence of a polymerase. The sequence of the template is determined by the order of labeled nucleotides incorporated at the 3' end of the growing strand. This can be done in real-time and in step-and-repeat mode. For real-time analysis, a different optical label for each nucleotide is incorporated and multiple lasers can be used to stimulate the incorporated nucleotides.

[0157] Sequencing may 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. Further similar current massively parallel sequencing technologies, as well as future generations of these technologies, can be used.

[0158] 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 tumors or bodily fluids such as blood or saliva obtained by known methods (e.g., venipuncture). Alternatively, nucleic acid testing can be performed on dry samples (e.g., hair or skin). Furthermore, a sample for sequencing can be obtained from a tumor, and another sample for sequencing can be obtained from normal tissue of the same tissue type as the tumor. A sample for sequencing can be obtained from a tumor, and another sample for sequencing can be obtained from normal tissue of a different tissue type than the tumor.

[0159] The tumor may include one or more of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and T-cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0160] Alternatively, protein mass spectrometry can be used to identify or verify the presence of mutant peptides bound to MHC proteins on tumor cells. Peptides can be acid eluted from tumor cells or from HLA molecules immunoprecipitated from tumors and then identified using mass spectrometry.

[0161] IV. Antigen Antigens may include nucleotides or polypeptides. For example, antigens may be RNA sequences that code for a polypeptide sequence. Antigens useful in vaccines therefore include nucleotide sequences or polypeptide sequences. Shared neo-antigens are shown in Table A (see SEQ ID NOs: 10,755-21,015) and AACR GENIE results (SEQ ID NOs: 21,016-29.357). Shared antigens are shown in Table 1.2 (see SEQ ID NOs: 57-10,754).

[0162] Disclosed herein are isolated peptides comprising a tumor-specific mutation identified by the methods disclosed herein, peptides comprising a known tumor-specific mutation, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. Neo-antigen peptides can be described in terms of their coding sequences, and neo-antigens include nucleotide sequences (e.g., DNA or RNA) that encode the relevant polypeptide sequence.

[0163] The present specification also discloses peptides derived from any polypeptide known or found to be altered in expression in tumor cells or cancerous tissues compared to normal cells or tissues, for example, any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissues compared to normal cells or tissues. Suitable polypeptides from which antigenic peptides may be derived can be found, for example, in the COSMIC database. COSMIC is a curated comprehensive collection of information on somatic mutations in human cancers. Peptides include tumor-specific mutations. Tumor antigens (e.g., shared tumor antigens and tumor neo-antigens) include, but are not limited to, those described in U.S. Patent Application No. 17 / 058,128, which is incorporated by reference herein for all purposes. Antigenic peptides can be described in terms of their coding sequences, and antigens include nucleotide sequences (e.g., DNA or RNA) that code for the relevant polypeptide sequences. Antigens can be selected that are predicted to be presented on the cell surface of cells, such as tumor cells or immune cells, including professional antigen-presenting cells, such as dendritic cells. Antigens can be selected that are predicted to be immunogenic.

[0164] The one or more polypeptides encoded by the antigen nucleotide sequence can include at least one of the following: binding affinity to MHC with an IC50 value of less than 1000 nM; for MHC class I peptides, 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length; the presence of a sequence motif within or near the peptide that promotes proteasomal cleavage; and the presence of a sequence motif that promotes TAP transport; for MHC class II polypeptides, 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 in length; the presence of a sequence motif within or near the peptide-promoted cleavage site by extracellular or lysosomal proteases (e.g., cathepsins) or the HLA binding site catalyzed by HLA-DM.

[0165] The one or more antigens can be present on the surface of the tumor.

[0166] One or more antigens may be immunogenic in a subject with a tumor (e.g., capable of stimulating a T cell response and / or a B cell response in a subject). One or more antigens may be capable of stimulating a B cell response, such as the production of an antibody that recognizes one or more antigens (e.g., an antibody that recognizes a tumor). An antibody may recognize a linear polypeptide sequence or may recognize a secondary and tertiary structure. Thus, antigens for B cells may include linear polypeptide sequences or polypeptides having secondary and tertiary structure, including, but not limited to, full-length proteins, protein subunits, protein domains, or any polypeptide known or predicted to have secondary and tertiary structure. An antigen capable of stimulating a B cell response against a tumor may be an antigen found on the surface of a tumor cell. An antigen capable of inducing a B cell response against a tumor may be an intracellular neo-antigen expressed in a tumor.

[0167] The one or more antigens can include a combination of an antigen capable of stimulating a T cell response (e.g., a peptide containing a predicted T cell epitope sequence) and a different antigen capable of stimulating a B cell response (e.g., a full-length protein, a protein subunit, a protein domain).

[0168] One or more antigens that stimulate an autoimmune response in a subject can be excluded from consideration in the context of generating a vaccine for a subject.

[0169] The size of the at least one antigenic peptide molecule (e.g., epitope sequence) can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102 In a specific embodiment, the antigenic peptide molecule is 50 or less amino acid residues.

[0170] Antigenic peptides and polypeptides can be 15 residues or less in length, usually between about 8 and about 11 residues, particularly 9 or 10 residues, for MHC class I; and can be 6 to 30 residues for MHC class II.

[0171] If desired, longer peptides can be designed in several ways. In one example, where the presentation likelihood of a peptide on an HLA allele is predicted or known, the longer peptide can consist of either (1) an individual presented peptide with an extension of 2-5 amino acids toward the N-terminus and C-terminus of each corresponding gene product; (2) a concatenation of some or all of the presented peptides, each with an extended sequence. In another case, where sequencing reveals the presence of long (more than 10 residues) neoepitope sequences in the tumor (e.g., due to frameshift, read-through, or intron introduction resulting in a novel peptide sequence), the longer peptide can consist of (3) the entire stretch of novel tumor-specific amino acids (which avoids the need to select the most strongly HLA-presented shorter peptide based on computational or in vitro test selection). In either case, the longer peptide can allow for endogenous processing by the patient's cells, resulting in more effective antigen presentation and stimulation of T cell responses. Longer peptides may include full-length proteins, protein subunits, protein domains, and combinations thereof, such as those expressed in tumors. Longer peptides (e.g., full-length proteins, protein subunits, or protein domains) and combinations thereof may be included to stimulate a B cell response.

[0172] Antigenic peptides and polypeptides can be presented on HLA proteins. In some embodiments, antigenic peptides or polypeptides are presented on HLA proteins with higher affinity than wild-type peptides. In some embodiments, antigenic peptides or polypeptides can have an IC50 of at least 5000nM or less, at least 1000nM or less, at least 500nM or less, at least 250nM or less, at least 200nM or less, at least 150nM or less, at least 100nM or less, at least 50nM or less.

[0173] In some embodiments, the antigenic peptides and polypeptides do not induce an autoimmune response and / or do not cause immune tolerance when administered to a subject.

[0174] Compositions comprising at least two or more antigenic peptides are also provided. In some embodiments, the composition comprises at least two different peptides. The at least two different peptides may be derived from the same polypeptide. Different peptides means that the peptides differ in length, amino acid sequence, or both. The peptides may comprise tumor-specific mutations. The tumor-specific peptides may be derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues, for example, any polypeptide known or found to contain tumor-specific mutations or peptides derived from any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissues compared to normal cells or tissues. The peptides may be derived from any polypeptide known or suspected to be associated with infectious disease organisms, or the peptides may be derived from any polypeptide known or found to have altered expression in infected cells compared to normal cells or tissues (e.g., infectious disease polynucleotides or polypeptides including infectious disease polynucleotides or polypeptides whose expression is restricted to host cells). Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database or the AACR GENIE (Genomics Evidence Neoplasia Information Exchange) database. COSMIC curates comprehensive information on somatic mutations in human cancers. AACR GENIE aggregates and links clinical-grade cancer genomic data with clinical outcomes from tens of thousands of cancer patients. In some embodiments, the tumor-specific mutation is a driver mutation for a particular cancer type.

[0175] Antigenic peptides and polypeptides with desired activities or properties can be modified to confer certain desired attributes, e.g., improved pharmacological characteristics, while at least retaining, or increasing, substantially all of the biological activity of the unmodified peptides that bind to desired MHC molecules and activate appropriate T cells. By way of example, antigenic peptides and polypeptides can be further subjected to various modifications, such as either conservative or non-conservative substitutions, which may provide certain advantages in their use, such as improved MHC binding, stability, or presentation. Conservative substitution means replacing an amino acid residue with another that is biologically and / or chemically similar, e.g., one hydrophobic residue with another hydrophobic residue, or one polar residue with another polar residue. Substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions may also be explored using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984).

[0176] Modification of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful in increasing the stability of peptides and polypeptides in vivo. Stability can be assayed in a number of ways. For example, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, for example, Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Peptide half-life can be conveniently determined using a 25% human serum (v / v) assay. The protocol is generally as follows: Pooled human serum (type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, small aliquots of the reaction solution are removed and added to either 6% aqueous trichloroacetic acid or ethanol. The cloudy reaction sample is cooled (4° C.) for 15 minutes and then spun to precipitate the precipitated serum proteins. The presence of the peptide is then determined by reverse-phase HPLC using stability-specific chromatographic conditions.

[0177] Peptides and polypeptides can be modified to provide desirable attributes other than improved serum half-life. As an example, the ability of a peptide to stimulate CTL activity can be enhanced by linkage to a sequence containing at least one epitope capable of stimulating a T helper cell response. The immunogenic peptide / T helper conjugate can be linked by a spacer molecule. The spacer is typically composed of relatively small neutral molecules, such as amino acids or amino acid mimetics, that are substantially uncharged under physiological conditions. The spacer is typically selected, for example, from Ala, Gly, or other neutral spacers of non-polar amino acids or neutral polar amino acids. It will be understood that the spacer, if present, need not be composed of the same residues and thus may be a hetero- or homo-oligomer. If present, the spacer will usually be at least 1 or 2 residues, more usually 3-6 residues. Alternatively, the peptide can be linked to the T helper peptide without a spacer.

[0178] The antigenic peptide can be linked to the T helper peptide either directly or via a spacer at either the amino or carboxy terminus of the peptide. The amino terminus of either the antigenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include 830-843 of tetanus toxoid, 307-319 of influenza, and around 382-398 and 378-389 of malaria sporozoites.

[0179] Proteins or peptides can be produced by any technique known to those skilled in the art, including expressing the protein, polypeptide, or peptide through standard molecular biology techniques, isolating the protein or peptide from a natural source, or chemically synthesizing the protein or peptide. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed and can be found in computerized databases known to those skilled in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, located at the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art. Alternatively, various commercial preparations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0180] In further embodiments, the antigen comprises a nucleic acid (e.g., polynucleotide) encoding an antigenic peptide or a portion thereof. The polynucleotide can be, for example, a single-stranded and / or double-stranded polynucleotide, such as DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), e.g., a polynucleotide having a phosphorothioate backbone, or in either a natural or stabilized form, or a combination thereof, and may or may not contain introns. The polynucleotide sequence encoding the antigen can be sequence-optimized to improve expression, such as by improving transcription, translation, post-transcriptional processing, and / or RNA stability. For example, the polynucleotide sequence encoding the antigen can be codon-optimized. As used herein, "codon optimization" refers to the replacement of low-frequency codons with high-frequency synonymous codons with respect to the codon bias of a particular organism. The polynucleotide sequence can be optimized to improve post-transcriptional processing, for example, by removing splicing motifs (e.g., canonical and / or cryptic / non-canonical splice donor, branch, and / or acceptor sequences) and / or by introducing exogenous splicing motifs (splice donor, branch, and / or acceptor sequences) to bias toward favorable splicing events, to reduce unintended splicing. Exogenous intron sequences include, but are not limited to, those derived from SV40 (e.g., SV40 mini-intron) and / or immunoglobulins (e.g., human β-globulin gene). Exogenous intron sequences can be incorporated between the promoter / enhancer sequence and the antigen(s) sequence. Exogenous intron sequences for use in expression vectors are described in detail in Callendret et al. (Virology. 2007 Jul. 5;363(2):288-302), each of which is incorporated by reference in its entirety for all purposes.The polynucleotide sequence can be optimized to improve transcript stability, for example, by removing RNA stability motifs (e.g., AU-rich elements and / or 3'UTR motifs) and / or repetitive nucleotide sequences. The polynucleotide sequence can be optimized to improve accurate transcription, for example, by removing cryptic transcription initiators and / or terminators. The polynucleotide sequence can be optimized to improve translation and translation accuracy, for example, by removing cryptic AUG start codons, premature polyA sequences, and / or secondary structure motifs. The polynucleotide sequence can be optimized to improve nuclear export of the transcript, such as by adding a constitutive transport element (CTE), an RNA transport element (RTE), or a woodchuck posttranscriptional regulatory element (WPRE). Nuclear export signals for use in expression vectors are described in detail by Callendret et al. (Virology. 2007 Jul 5;363(2):288-302), each of which is incorporated herein by reference in its entirety for all purposes. Polynucleotide sequences can be optimized for GC content, for example, to reflect the average GC content of a particular 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 each of transcription, translation, post-transcriptional processing, and RNA stability. Sequence optimization algorithms are well known to those skilled in the art, such as GeneArt (Thermo Fisher), Codon Optimization Tool (IDT), Cool Tool (University of Singapore), SGI-DNA (La Jolla California), etc. One or more regions of the antigen-encoding protein can be sequence-optimized separately.

[0181] Yet further aspects provide expression vectors capable of expressing the polypeptide or a portion thereof. Expression vectors for various cell types are well known in the art and can be selected without undue experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, in the proper direction and correct reading frame for expression. If necessary, DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, but such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0182] V. Vaccine Compositions Also disclosed herein is an immunogenic composition, e.g., a vaccine composition, that can generate a specific immune response, e.g., a tumor-specific immune response. A vaccine composition typically comprises one or more antigens selected using the methods described herein or as described in Table A, Table 1.2, or AACR GENIE results. A vaccine composition can also be called a vaccine.

[0183] The vaccine can include 1-30 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. The peptides may have post-translational modifications. The vaccines contain 1 to 100 or more nucleotide sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, , 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 or more different nucleotide sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different antigen-encoding nucleic acid sequences, or 12, 13, or 14 different antigen-encoding nucleic acid sequences.The vaccine contains antigen sequences 1-30, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, The antigen sequences may include 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 or more different antigen sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different antigen sequences, or 12, 13, or 14 different antigen sequences.

[0184] The vaccines comprise 1-30 antigen-encoding 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, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 109, The nucleic acid sequence may include 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 or more different antigen-encoding nucleic acid sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different antigen-encoding nucleic acid sequences, or 12, 13, or 14 different antigen-encoding nucleic acid sequences. The antigen-encoding nucleic acid sequence may also be referred to as the antigen-encoding portion of an "antigen cassette." The characteristics of the antigen cassette are described in more detail herein. An antigen-encoding nucleic acid sequence can include one or more epitope-encoding nucleic acid sequences (eg, an antigen-encoding nucleic acid sequence encoding linked T cell epitopes).

[0185] The vaccines may comprise one to 30 different epitope-encoding 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, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 102, 103, 104, 105, 106, 107, 108, 109, , 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 or more different epitope-encoding nucleic acid sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different epitope-encoding nucleic acid sequences, or 12, 13, or 14 different epitope-encoding nucleic acid sequences. An epitope-encoding nucleic acid sequence may also refer to a sequence of individual epitope sequences, such as each of the T cell epitopes in an antigen-encoding nucleic acid sequence that encodes linked T cell epitope-encoding nucleic acid sequences.

[0186] The vaccine can include at least two repeats of an epitope-encoding nucleic acid sequence. As used herein, "repeat" (or interchangeably "repeat") refers to two or more of the same nucleic acid epitope-encoding nucleic acid sequences (including any 5' linker sequence and / or any 3' linker sequence described herein) within an antigen-encoding nucleic acid sequence. In one example, the antigen-encoding nucleic acid sequence portion of the cassette encodes at least two repeats of an epitope-encoding nucleic acid sequence. In a further non-limiting example, the antigen-encoding nucleic acid sequence portion of the cassette encodes a plurality of different epitopes, at least one of the different epitopes being encoded by at least two repeats of a nucleic acid sequence encoding a different epitope (i.e., at least two different epitope-encoding nucleic acid sequences). In an illustrative non-limiting example, the antigen-encoding nucleic acid sequence comprises an epitope-encoding nucleic acid sequence A(E A ), epitope coding sequence B (EB ), and epitope coding sequence C (E C ) and having at least one repeat of the different epitopes is shown, without limitation, by the formula: - Repeat of one different epitope (repeat of epitope A): E A -E B -E C -E A ,or E A -E A -E B -E C - Repeat of multiple different epitopes (repeats 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 -E C -E C - multiple repeats of multiple different epitopes (repeats 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

[0187] The above examples are not limiting, and an antigen-encoding nucleic acid sequence having at least one repeat of a different epitope can encode each of the different epitopes in any order or frequency. For example, the order and frequency can be, for example, a sequence of the formula E A -E B -E C -E C -E A -E B -E A -E C -E A -E C -E C -E B It can be a random arrangement of different epitopes, such as in the example with epitopes A, B, and C according to the formula:

[0188] Provided herein is an antigen-encoding cassette having, in a 5' to 3' direction, at least one antigen-encoding nucleic acid sequence described by the formula: (E x -(E N n ) y )z wherein E represents a nucleotide sequence comprising different epitope-encoding nucleic acid sequences; n represents the number of distinct epitope-encoding nucleic acid sequences and is any integer including 0; E N represents a nucleotide sequence that contains a distinct epitope-encoding nucleic acid sequence that is separate at each corresponding n, For each repetition of z, for each n, x=0 or 1, y=0 or 1, and at least one of x or y is 1; z=2 or more, and the antigen-encoding nucleic acid sequence is E, a given E N or at least one of these combinations repeated two times.

[0189] Each E or E N can independently comprise an epitope-encoding nucleic acid sequence described herein (e.g., a peptide encoding an infectious disease T cell epitope and / or a neo-antigen epitope). For example, each E or EN is a 5' to 3' sequence of the formula (L5 b -Nc-L3 d ), wherein N is each E or E N where c=1, L5 comprises a 5' linker sequence where b=0 or 1, and L3 comprises a 3' linker sequence where d=0 or 1. Epitopes and linkers that can be used are further described herein.

[0190] Repeats of the epitope-encoding nucleic acid sequence (including any 5' linker sequence and / or any 3' linker sequence) may be directly linked to one another (e.g., as shown above, E A -E A -...). The repeats of the epitope-encoding nucleic acid sequence may be separated by one or more additional nucleotide sequences. Generally, the repeats of the epitope-encoding nucleic acid sequence may be separated by a nucleotide sequence of any size applicable to the compositions described herein. In one example, the repeats of the epitope-encoding nucleic acid sequence may be separated by separate and distinct epitope-encoding nucleic acid sequences (e.g., as shown above, E A -E B -E C -E A ...). In examples where the repeats are separated by single distinct and different epitope-encoding nucleic acid sequences, and each epitope-encoding nucleic acid sequence (including any 5' linker sequence and / or any 3' linker sequence) encodes a peptide 25 amino acids in length, the repeats can be, for example, A -E B -E A...(EA is separated by 75 nucleotides). In one illustrative example, an antigen-encoding nucleic acid having the sequence VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDTVTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT (SEQ ID NO: 85) encoding repeats of 25-mer antigens Trp1 (VTNTEMFVTAPDNLGYMYEVQWPGQ) [SEQ ID NO: 86] and Trp2 (TQPQIANCSVYDFFVWLHYYSVRDT) [SEQ ID NO: 87], where the repeats of Trp1 are separated by the 25-mer Trp2, and thus the repeats of the Trp1 epitope-encoding nucleic acid sequence are separated by 75 nucleotides of the Trp2 epitope-encoding nucleic acid sequence. In examples where the repeats are separated by 2, 3, 4, 5, 6, 7, 8, or 9 separate and distinct epitope-encoding nucleic acid sequences, each epitope-encoding nucleic acid sequence (including any 5' linker sequence and / or any 3' linker sequence) encoding a peptide 25 amino acids in length, the repeats can be separated by 150, 225, 300, 375, 450, 525, 600, or 675 nucleotides, respectively.

[0191] In some cases, the antigen or epitope in the cassette that encodes the additional antigen and / or epitope may be an epitope that is immunodominant to the other epitopes encoded. In general, immunodominance is the bias of the immune response to only one or a few specific immunogenic peptides. Immunodominance can be evaluated as part of an immune monitoring protocol. For example, immunodominance can be evaluated by evaluating the T cell and / or B cell response to the encoded antigen.

[0192] In some cases, it may be desirable to avoid vaccine compositions that contain immunodominant epitopes. For example, it may be desirable to avoid designing vaccine cassettes that code for immunodominant epitopes. Without wishing to be bound by theory, administering and / or coding an immunodominant epitope together with an additional epitope may reduce the immune response to the additional epitope, which may ultimately reduce vaccine efficacy against the additional epitope. As an illustrative, non-limiting example, a vaccine composition that contains a TP53-related neoepitope may have an immune response, e.g., a T cell response, biased toward the TP53-related neoepitope to negatively impact other antigens or epitopes in the vaccine composition.

[0193] In one embodiment, the different peptides and / or polypeptides, or the nucleotide sequences encoding them, are selected such that the peptides and / or polypeptides can bind to different MHC molecules, such as different MHC class I molecules and / or different MHC class II molecules. In some embodiments, one vaccine composition comprises coding sequences for peptides and / or polypeptides capable of binding to the most frequently occurring MHC class I molecules and / or different MHC class II molecules. Thus, the vaccine composition can comprise different fragments capable of binding to at least two preferred, at least three preferred, or at least four preferred MHC class I molecules and / or different MHC class II molecules.

[0194] The vaccine composition can stimulate a specific cytotoxic T cell response and / or a specific helper T cell response. The vaccine composition can stimulate a specific cytotoxic T cell response and a specific helper T cell response.

[0195] The vaccine composition is capable of stimulating a specific B cell response (eg, an antibody response).

[0196] The vaccine composition can stimulate a specific cytotoxic T cell response, a specific helper T cell response, and / or a specific B cell response (e.g., an antibody response). The vaccine composition can stimulate a specific cytotoxic T cell response and a specific B cell response. The vaccine composition can stimulate a specific helper T cell response and a specific B cell response. The vaccine composition can stimulate a specific cytotoxic T cell response, a specific helper T cell response, and a specific B cell response.

[0197] The vaccine composition may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are provided herein below. The composition may be accompanied by a carrier, such as an antigen-presenting cell, such as a dendritic cell (DC), capable of presenting the protein or peptide to a T cell.

[0198] An adjuvant is any substance that enhances or otherwise modifies the immune response to an antigen when mixed into a vaccine composition. The carrier may be a scaffold structure, such as a polypeptide or polysaccharide, to which the antigen can associate. Optionally, the adjuvant is attached by covalent or non-covalent bonds.

[0199] The ability of adjuvants to increase immune response to antigens is generally manifested as a significant or large increase in immune-mediated reactions or a reduction in disease symptoms.For example, the increase in humoral immunity is generally manifested as a significant increase in the titer of antibodies produced against antigens, and the increase in T cell activity is generally manifested as an increase in cell proliferation, or cytotoxicity, or cytokine secretion.Adjuvants can also change immune response, for example, by changing a predominantly humoral or Th response to a predominantly cellular or Th response.

[0200] Suitable adjuvants include, but are not limited to, 1018 ISS, alum, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA206, Montanide ISA 50V, Montanide Adjuvants include ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon derived from saponin (Aquila Biotech, Worcester, Mass., USA), mycobacterium extracts and synthetic bacterial wall mimics, as well as other proprietary adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are useful. Several immunological adjuvants specific for dendritic cells (e.g., MF59) and their formulations have been described (Dupuis M, et al., Cell Immunol. 1998; 186(1):18-27; Allison AC; Dev Biol Stand. 1998; 92:3-11). Cytokines can also be used.Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated by reference herein in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996 (6):414-418).

[0201] CpG immunostimulatory oligonucleotides have also been reported to enhance the effect of adjuvants in a vaccine environment. Other TLR binding molecules, such as RNA binding TLR 7, TLR 8 and / or TLR 9, can also be used.

[0202] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), Poly(I:C) (e.g., polyi:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies that may have a therapeutic effect and / or act as an adjuvant, such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175. The amounts and concentrations of adjuvants and additives can be readily determined by one of skill in the art without undue experimentation. Further adjuvants include colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0203] Vaccine compositions can include multiple different adjuvants. Additionally, therapeutic compositions can include any adjuvant material, including any of the above or a combination thereof. Vaccines and adjuvants can be administered together or separately in any suitable order.

[0204] A carrier (or excipient) may be present independent of the adjuvant. The function of the carrier may be to enhance activity or immunogenicity, confer stability, enhance biological activity, or extend serum half-life, for example by increasing the molecular weight of a particular variant. Additionally, the carrier may aid in the presentation of the peptide to T cells. The carrier may be any suitable carrier known to those skilled in the art, for example, a protein or an 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; hormones such as insulin or palmitic acid. For human immunization, the carrier is generally a safe, physiologically acceptable carrier that is tolerated by humans. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier may be a dextran, for example, sepharose.

[0205] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules, rather than intact foreign antigens themselves. The MHC molecules themselves are located on the cell surface of antigen-presenting cells. Thus, activation of CTLs is possible when a trimeric complex of peptide antigen, MHC molecule and APC is present. Correspondingly, CTLs can enhance immune responses when APCs bearing the corresponding MHC molecules are further added, rather than when only peptides are used to activate CTLs. Thus, in certain embodiments, the vaccine composition further comprises at least one antigen-presenting cell.

[0206] Antigens may be derived from vaccinia, fowlpox, self-replicating alphaviruses, Maraba viruses, adenoviruses (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or from second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C ( promoter, Nucl. Acids Res. (2015) 43(1): 682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72(12): 9873-9880) may be included in a vaccine platform based on a viral vector. Depending on the packaging capacity of the viral vector-based vaccine platform described above, this approach can deliver one or more nucleotide sequences encoding one or more antigenic peptides.The sequence may be flanked by non-mutated sequences, separated by a linker, or preceded by one or more sequences that target intracellular compartments (see, e.g., Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22(4): 433-8, Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science. (2016) 352(6291): 1337-41, Lu et al., Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20(13): 3401-10). Upon introduction into the host, the infected cells express the antigen, thereby stimulating a host immune (e.g., CTL) response against the peptide. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus Calmette-Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful for therapeutic administration of antigens or immunization, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the description herein.

[0207] VA antigen cassette The methods used to select one or more antigens, the cloning and construction of an "antigen cassette" and its insertion into a viral vector are within the skill of the art in view of the teachings provided herein. By "antigen cassette" or "cassette" is meant a combination of a selected antigen or antigens (e.g., an antigen-encoding nucleic acid sequence) with other regulatory elements required to transcribe the antigen(s) and express the transcript. The selected antigen or antigens may refer to different epitope sequences (e.g., an antigen-encoding nucleic acid sequence in a cassette may encode an epitope-encoding nucleic acid sequence (or epitope-encoding nucleic acid sequences) such that the epitopes are transcribed and expressed). The antigen or antigens may be operably linked to regulatory elements in a manner that allows transcription. Such elements include conventional regulatory elements that can induce expression of the antigen(s) in cells transfected with the viral vector. Thus, the antigen cassette may also include a selected promoter linked to the antigen(s) and located with other optional regulatory elements within the selected viral sequence of the recombinant vector. The cassette may include one or more neo-antigens shown in Table A and / or the AACR GENIE results, and / or one or more antigens shown in Table 1.2.

[0208] A cassette can have one or more antigen-encoding nucleic acid sequences, such as a cassette containing multiple antigen-encoding nucleic acid sequences, each independently operably linked to a separate promoter and / or linked to each other using 2A ribosomal skipping sequence elements (e.g., E2A, P2A, F2A, or T2A sequences) or other multicistronic systems such as internal ribosome entry site (IRES) sequence elements. The linker can also have a cleavage site, such as a TEV or furin cleavage site. Linkers with cleavage sites can also be combined with other elements, such as elements in multicistronic systems. In a non-limiting illustrative example, a furin protease cleavage site can be used in combination with a 2A ribosomal skipping sequence element to configure a furin protease cleavage site to facilitate removal of the 2A sequence after translation. In a cassette containing multiple antigen-encoding nucleic acid sequences, each antigen-encoding nucleic acid sequence can include one or more epitope-encoding nucleic acid sequences (e.g., antigen-encoding nucleic acid sequences encoding linked T cell epitopes).

[0209] Useful promoters may be constitutive promoters or regulated (inducible) promoters that allow the amount of antigen(s) to be expressed to be controlled. For example, a desirable promoter is the cytomegalovirus immediate early promoter / enhancer [see, for example, Boshart et al, Cell, 41:521-530 (1985)]. Another desirable promoter is the Rous sarcoma virus LTR promoter / enhancer. Yet another promoter / enhancer sequence is the chicken β-actin promoter [TAKost et al, Nucl. Acids Res., 11 (23): 8287 (1983)]. Those skilled in the art can also select other suitable or desirable promoters.

[0210] The antigen cassette may also contain nucleic acid sequences heterologous to the viral vector sequence, including sequences that provide signals for efficient polyadenylation of the transcript (poly(A), polyA or pA) and introns containing functional splice donor and acceptor sites. A common polyA sequence used in the exemplary vectors herein is derived from the papovavirus SV-40. The polyA sequence can be inserted into the cassette after the antigen-based sequence and before the viral vector sequence. A common intron sequence may also be derived from SV-40 and is referred to as the SV-40T intron sequence. The antigen cassette may contain an intron located between the promoter / enhancer sequence and the antigen(s). The selection of these and other common vector elements is conventional [see, for example, Sambrook et al, "Molecular Cloning. A Laboratory Manual.", 2d edit., Cold Spring Harbor Laboratory, New York (1989) and references cited therein], and many such sequences are available from commercial and industrial sources, as well as Genbank.

[0211] An antigen cassette can have one or more antigens. For example, a particular cassette can include 1-10, 1-20, 1-30, 10-20, 15-25, 15-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens. The antigens may be directly linked to each other. The antigens may be linked to each other by a linker. The antigens can be in any orientation relative to each other, including N-C or C-N.

[0212] As noted elsewhere herein, the antigen cassette can be placed within any selected deletion site within the viral vector, such as, for example, the site of the deleted structural proteins of the VEE backbone or the deleted E1 gene region or the deleted E3 gene region of a ChAd-based vector, among other choices.

[0213] The antigen cassette can be described using the following formula, which describes the ordered sequence of each element in the 5' to 3' direction: (P a -(L5 b -N c -L3 d ) X ) Z -(P2 h -(G5 e -U f ) Y ) W -G3 g [wherein P and P2 comprise a promoter nucleotide sequence, N comprises an MHC class I epitope-encoding nucleic acid sequence, L5 comprises a 5' linker sequence, L3 comprises a 3' linker sequence, G5 comprises a nucleic acid sequence encoding an amino acid linker, G3 comprises one of at least one nucleic acid sequence encoding an amino acid linker, and U comprises an MHC class II antigen-encoding nucleic acid sequence, where for each X, the corresponding Nc is an epitope-encoding nucleic acid sequence, and for each Y, the corresponding Uf is an MHC class II epitope-encoding nucleic acid sequence (e.g., a universal MHC class II epitope-encoding nucleic acid sequence). The universal sequence may comprise at least one of tetanus toxoid and PADRE. The universal sequence may comprise a tetanus toxoid peptide. The universal sequence may comprise a PADRE peptide. The universal sequence may comprise a tetanus toxoid and a PADRE peptide.] The compositions and ordered sequences can be further defined by selecting the number of elements present, for example, a=0 or 1, b=0 or 1, c=1, d=0 or 1, e=0 or 1, f=1, g=0 or 1, h=0 or 1, X=1-400, Y=0, 1, 2, 3, 4 or 5, Z=1-400, and W=0, 1, 2, 3, 4 or 5.

[0214] In one example, the elements present are a=0, b=1, d=1, e=1, g=1, h=0, X=10, Y=2, Z=1, and W=1, where the case is described where there is no additional promoter (e.g., only the promoter nucleotide sequence provided by the vector backbone, such as an RNA alphavirus backbone, is present), there are 10 MHC class I epitopes, there is a 5' linker at each N, there is a 3' linker at each N, there are 2 MHC class II epitopes, there is a linker connecting the 2 MHC class II epitopes, there is a linker connecting the 5' ends of the 2 MHC class II epitopes to the 3' linker of the last MHC class I epitope, and there is a linker connecting the 3' ends of the 2 MHC class II epitopes to the vector backbone (e.g., an RNA alphavirus backbone). Examples of linking the 3' end of the antigen cassette to a vector backbone (e.g., an RNA alphavirus backbone) include linking directly to a 3'UTR element provided by the vector backbone, such as the 3' 19 nt CSE. Examples of linking the 5' end of the antigen cassette to a vector backbone (e.g., an RNA alphavirus backbone) include linking directly to a promoter or 5'UTR element of the vector backbone, such as a subgenomic promoter sequence (e.g., a 26S subgenomic promoter sequence), an alphavirus 5'UTR, a 51 nt CSE, or a 24 nt CSE.

[0215] Other examples include when a=1, describing the presence of a promoter other than the promoter nucleotide sequence provided by the vector backbone (e.g., an RNA alphavirus backbone); when a=1 and Z is greater than 1, describing the presence of multiple promoters other than the promoter nucleotide sequence provided by the vector backbone, each of which results in expression of one or more different MHC Class I epitope-encoding nucleic acid sequences; when h=1, describing the presence of another promoter that results in expression of an MHC Class II epitope-encoding nucleic acid sequence; and when g=0, describing the presence of an MHC Class II epitope-encoding nucleic acid sequence (if present) that is directly linked to the vector backbone (e.g., an RNA alphavirus backbone).

[0216] Other examples include where each MHC class I epitope present has a 5' linker, a 3' linker, neither, or both. In examples where multiple MHC class I epitopes are present in the same antigen cassette, some MHC class I epitopes may have both a 5' linker and a 3' linker, while other MHC class I epitopes may have a 5' linker or a 3' linker, or neither. In other examples where multiple MHC class I epitopes are present in the same antigen cassette, some MHC class I epitopes may have either a 5' linker or a 3' linker, while other MHC class I epitopes may have a 5' linker or a 3' linker, or neither.

[0217] In instances where multiple MHC class II epitopes are present within the same antigen cassette, some MHC class II epitopes may have both a 5' linker and a 3' linker, while other MHC class II epitopes may have a 5' linker, a 3' linker, or neither. In other instances where multiple MHC class II epitopes are present within the same antigen cassette, some MHC class II epitopes may have either a 5' linker or a 3' linker, while other MHC class II epitopes may have a 5' linker, a 3' linker, or neither.

[0218] Other examples include cases where each antigen present has a 5' linker, a 3' linker, neither, or both. In examples where multiple antigens are present in the same antigen cassette, some antigens may have both a 5' linker and a 3' linker, while other antigens may have a 5' linker or a 3' linker, or neither. In other examples where multiple antigens are present in the same antigen cassette, some antigens may have either a 5' linker or a 3' linker, while other antigens may have a 5' linker or a 3' linker, or neither.

[0219] The promoter nucleotide sequence P and / or P2 may be the same as the promoter nucleotide sequence provided by the vector backbone, such as the RNA alphavirus backbone. For example, the promoter sequences Pn and P2 provided by the vector backbone may each comprise a subgenomic promoter sequence (e.g., a 26S subgenomic promoter sequence) or a CMV promoter. The promoter nucleotide sequence P and / or P2 may be different from the promoter nucleotide sequence provided by the vector backbone (e.g., the RNA alphavirus backbone) and may be different from each other.

[0220] The 5' linker L5 may be a natural sequence or a non-natural sequence. Non-natural sequences include, but are not limited to, AAY, RR, and DPP. The 3' linker L3 may also be a natural sequence or a non-natural sequence. Furthermore, L5 and L3 may both be natural sequences, both may be non-natural sequences, or one may be natural and the other non-natural. For each X, the amino acid linker may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, The lengths may be 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more. For each X, the amino acid linker may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0221] For each Y, the amino acid linker G5 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more lengths. For each Y, the amino acid linker may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0222] The amino acid linker G3 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 amino acids. , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more lengths. G3 may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0223] For each X, each N can code for 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 code for a combination of an MHC class I epitope, an MHC class II epitope, and an epitope / antigen capable of stimulating a B cell response. For each X, each N can code for a combination of an MHC class I epitope and an MHC class II epitope. For each X, each N can code for a combination of an MHC class I epitope and an epitope / antigen capable of stimulating a B cell response. For each X, each N can code for a combination of an MHC class II epitope and an epitope / antigen capable of stimulating a B cell response. For each X, each N can code for an MHC class II epitope. For each X, each N can code for an epitope / antigen capable of stimulating a B cell response. For each X, each N can encode an MHC class I epitope 7-15 amino acids in length. For each X, each N may encode an MHC class I epitope 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. For each X, each N may also encode an MHC class I epitope at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0224] The cassette encoding one or more antigens may be 700 nucleotides or less. The cassette encoding one or more antigens may be 700 nucleotides or less and may encode two different epitope-encoding nucleic acid sequences (e.g., encoding two different infectious disease- or tumor-derived nucleic acid sequences encoding immunogenic polypeptides). The cassette encoding one or more antigens may be 700 nucleotides or less and may encode at least two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 700 nucleotides or less and may encode three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 700 nucleotides or less and may encode at least three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 700 nucleotides or less and may include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0225] The cassette encoding one or more antigens may be 375-700 nucleotides in length. The cassette encoding one or more antigens may be 375-700 nucleotides in length and may encode two different epitope-encoding nucleic acid sequences (e.g., encoding nucleic acid sequences from two different infectious diseases or tumors that encode immunogenic polypeptides). The cassette encoding one or more antigens may be 375-700 nucleotides in length and may encode at least two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-700 nucleotides in length and may encode three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-700 nucleotides in length and may encode at least three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-700 nucleotides in length and may include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0226] The cassette encoding one or more antigens may be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length. The cassette encoding one or more antigens may be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length and may encode two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length and may encode at least two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length and may encode three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens can be up to 600, 500, 400, 300, 200, or 100 nucleotides in length and can encode at least three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens can be up to 600, 500, 400, 300, 200, or 100 nucleotides in length and can include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0227] The cassette encoding one or more antigens may be 375-600, 375-500, or 375-400 nucleotides in length. The cassette encoding one or more antigens may be 375-600, 375-500, or 375-400 nucleotides in length and may encode two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-600, 375-500, or 375-400 nucleotides in length and may encode at least two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-600, 375-500, or 375-400 nucleotides in length and may encode three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-600, 375-500, or 375-400 nucleotides in length and may encode at least three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens can be 375-600, 375-500, or 375-400 nucleotides in length and can contain 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0228] In some cases, the antigen or epitope in the cassette that encodes the additional antigen and / or epitope may be an epitope that is immunodominant to the other epitopes encoded. In general, immunodominance is the bias of the immune response to only one or a few specific immunogenic peptides. Immunodominance can be evaluated as part of an immune monitoring protocol. For example, immunodominance can be evaluated by evaluating the T cell and / or B cell response to the encoded antigen.

[0229] Immunodominance can be evaluated 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 corresponding immune response (e.g., an immunodominant MHC class I epitope) can reduce the immune response of another antigen compared to the immune response in the absence of the immunodominant antigen. This reduction can be such that the immune response in the presence of the immunodominant antigen is not considered a therapeutically effective response. For example, an MHC class I epitope is generally considered immunodominant when the T cell response to the other antigen is no longer considered a therapeutically effective response compared to the response induced in the absence of the immunodominant MHC class I epitope. The immune response may be reduced to below or near the limit of detection compared to the response in the absence of the immunodominant antigen. For example, an MHC class I epitope is generally considered immunodominant when the T cell response to the other antigen is below the limit of detection compared to the response induced in the absence of the immunodominant MHC class I epitope. Generally, immunodominance is assessed between two antigens, e.g., two T cell epitopes, both capable of stimulating an immune response, in a vaccine composition administered to a subject bearing cognate MHC alleles known or predicted to present each epitope, respectively. Immunodominance can be assessed by the relative immunodominance to other antigens in the presence and absence of the suspected immunodominant antigen.

[0230] Immunodominance can be evaluated as the relative difference in immune response between two or more antigens. Immunodominance can refer to a 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold increase in the immune response of a particular antigen over another antigen encoded in the same cassette. Immunodominance can refer to a 100-fold, 200-fold, 300-fold, 400-fold, or 500-fold increase in the immune response of a particular antigen over another antigen encoded in the same cassette. Immunodominance can refer to a 100-fold, 200-fold, 300-fold, 400-fold, or 500-fold increase in the immune response of a particular antigen over another antigen encoded in the same cassette. Immunodominance can refer to a 10,000-fold increase in the immune response of a particular antigen over another antigen encoded in the same cassette.

[0231] In some cases, it may be desirable to avoid vaccine compositions that include immunodominant epitopes. For example, it may be desirable to avoid designing vaccine cassettes that encode immunodominant epitopes. Without wishing to be bound by theory, administering and / or encoding an immunodominant epitope together with an additional epitope may reduce the immune response to the additional epitope, which may ultimately reduce vaccine efficacy against the additional epitope. As an illustrative non-limiting example, a vaccine composition that includes a TP53-related neoepitope may have an immune response, e.g., a T cell response, that is biased toward the TP53-related neoepitope (e.g., reducing the immune response to the point where the immune response is no longer a therapeutically effective response and / or below detection limits) to negatively affect other antigens or epitopes in the vaccine composition (e.g., one or more KRAS-related neoepitopes in the vaccine composition, such as any of the KRAS-related MHC class I neoepitopes set forth in SEQ ID NOs: 75-82). Thus, the vaccine composition can be designed to not include an immunodominant epitope, such as by designing a vaccine cassette (e.g., a (neo)antigen-encoding cassette) that does not encode an immunodominant epitope. For example, the cassette does not encode an epitope that, when administered to a subject in a vaccine composition, reduces the immune response to another epitope encoded within the cassette, compared to the immune response when the other epitope is administered in the absence of the immunodominant MHC class I epitope. In another example, the cassette does not encode an epitope that, when administered to a subject in a vaccine composition, reduces the immune response to another epitope encoded within the cassette to below the limit of detection, compared to the immune response when the other epitope is administered in the absence of the immunodominant MHC class I epitope. In another example, the cassette does not encode an epitope that, when administered to a subject in a vaccine composition, reduces the immune response to another epitope encoded within the cassette to the point where the immune response is not therapeutically effective, compared to the immune response when the other epitope is administered in the absence of the immunodominant MHC class I epitope.In another example, the cassette does not encode an epitope that stimulates a 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold or more greater immune response relative to another epitope encoded within the same cassette administered to a subject in a vaccine composition when each antigen is capable of stimulating an immune response in the subject. In another example, the cassette does not encode an epitope that stimulates a 100-fold, 200-fold, 300-fold, 400-fold, or 500-fold or more greater immune response relative to another epitope encoded within the same cassette administered to a subject in a vaccine composition when each antigen is capable of stimulating an immune response in the subject. In another example, the cassette does not encode an epitope that stimulates a 1000-fold, 2000-fold, 3000-fold, 4000-fold, or 5000-fold or more greater immune response relative to another epitope encoded within the same cassette administered to a subject in a vaccine composition when each antigen is capable of stimulating an immune response in the subject. In another example, the cassette does not encode an epitope that confers a 10,000-fold or greater immune response relative to another epitope encoded within the same cassette that is administered to a subject in a vaccine composition where each antigen is capable of stimulating an immune response in the subject.

[0232] VB immunomodulator A vector described herein, such as a C68 vector described herein, or an alphavirus vector described herein, can include a nucleic acid encoding at least one antigen, and the same or another vector can include a nucleic acid encoding at least one immunomodulator. The immunomodulator can include a binding molecule (e.g., an antibody such as an scFv) that binds to and blocks the activity of an immune checkpoint molecule. The immunomodulator can include a cytokine such as IL-2, IL-7, IL-12 (including IL-12 p35, p40, p70, and / or p70 fusion constructs), IL-15, or IL-21. The immunomodulator can include a modified cytokine (e.g., pegylated IL-2). The vector can include an antigen cassette and one or more nucleic acid molecules encoding an immunomodulator.

[0233] Exemplary immune checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (which belongs to the CD2 family of molecules and is expressed on all NK (gamma delta) and memory CD8+ (alpha beta) T cells), CD160 (also called BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, or antigen-binding fragments thereof, or other binding proteins that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), ipilimumab, MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), cemiplimab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A / atezolizumab (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody), and yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor). The antibody coding sequence can be engineered into a vector using ordinary skill in the art. One exemplary method is described in Fang et al., Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol. 2005 May;23(5):584-90. Epub 2005 Apr 17, incorporated herein by reference for all purposes.

[0234] Further considerations in VC vaccine design and manufacturing Determination of a set of peptides covering all VC1 tumor subclones Truncal peptides, meaning peptides presented by all or most of the tumor subclones, can be prioritized for inclusion in the vaccine. 53 Optionally, if there are no stem peptides that are predicted to be highly presented and immunogenic, or if the number of stem peptides that are predicted to be highly presented and immunogenic is small enough that additional non-stem peptides can be included in the vaccine, additional peptides can be ranked by estimating the number and type of tumor subclones and selecting peptides that maximize the number of tumor subclones encompassed by the vaccine. 54

[0235] VC2. Antigen prioritization After all of the above antigen filters have been applied, there may still be more candidate antigens available for vaccine inclusion than vaccine technology can accommodate. Additionally, there may remain uncertainties about various aspects of antigen analysis, and trade-offs may exist between various properties of candidate vaccine antigens. Therefore, instead of pre-determined filters at each stage of the selection process, an integral multidimensional model can be considered, placing candidate antigens in a space with at least the following axes, and optimizing the selection using an integral approach: 1. Risk of autoimmunity or tolerance (germline risk) (lower autoimmunity risk is typically favorable) 2. Probability of sequencing artifacts (lower artifact probability is typically preferred) 3. Probability of immunogenicity (higher probability of immunogenicity is typically preferred) 4. Probability of presentation (higher probability of presentation is typically preferred) 5. Gene Expression (higher expression is typically preferred) 6. HLA gene coverage (a higher number of HLA molecules involved in the presentation of a set of antigens may reduce the probability that a tumor will evade immune attack through downregulation or mutation of HLA molecules) 7. HLA class coverage (covering both HLA-I and HLA-II may increase the chance of treatment response and decrease the chance of tumor avoidance)

[0236] Furthermore, in some cases, an antigen can be deprioritized (e.g., excluded) from vaccination if it is expected to be presented by HLA alleles that are lost or inactivated in all or part of the patient's tumor. Loss of HLA alleles can occur either by somatic mutation, loss of heterozygosity, or homozygous deletion of the locus. Methods for detecting somatic mutations of HLA alleles are well known in the art (e.g., Shukla et al., 2015). Methods for detecting somatic LOH and homozygous deletion (including HLA loci) have also been described (Carter et al., 2012; McGranahan et al., 2017; Van Loo et al., 2010). An antigen may be deprioritized if mass spectrometry data indicates that the predicted antigen is not presented by the predicted HLA allele.

[0237] VD alphavirus VD1. Alphavirus Biology Alphaviruses are members of the Togaviridae family and are single-stranded positive-sense RNA viruses. Members are generally classified as Old World types, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World types, such as Eastern equine encephalitis virus, Aula, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83 (Strauss Microbial Review 1994). Natural alphavirus genomes are usually about 12 kb in length, the first two-thirds of which contain genes encoding nonstructural proteins (nsPs) that form an RNA replication complex for autonomous replication of the viral genome, and the last third contains subgenomic expression cassettes encoding structural proteins for the production of virions (Frolov RNA 2001).

[0238] The model life cycle of alphaviruses involves several distinct steps (Strauss Microbial Review 1994, Jose Future Microbiol 2009). After viral adsorption to the host cell, the virion fuses with the membrane in the intracellular compartment, eventually releasing the genomic RNA into the cytosol. The genomic RNA, which has a positive-strand orientation, a methylguanylic acid cap at the 5' end and a polyA tail at the 3' end, is translated to generate the nonstructural proteins nsP1-4, which form a replication complex. Early in infection, the positive strand is replicated by this complex onto the negative strand template. In the current model, the replication complex is further processed as infection progresses, and the resulting processed complex switches to transcribe the negative strand into the full-length positive strand genomic RNA and the 26S subgenomic positive strand RNA that contains the structural genes. Several conserved sequence elements (CSEs) in alphaviruses have been identified with potential roles in various steps of RNA synthesis, including the complement of the 5' UTR in replication of positive-strand RNA from a negative-strand template, a 51-nt CSE in replication of negative-strand synthesis from a genomic template, a 24-nt CSE within the junction region of nsP and 26S RNA in transcription of subgenomic RNA from the negative strand, and a 19-nt CSE at the 3' end in negative-strand synthesis from a positive-strand template.

[0239] In the natural life cycle of viruses, after replication of different RNA species, viral particles are usually assembled. The 26S RNA is translated and the resulting proteins are further processed to generate structural proteins including capsid proteins, glycoproteins E1 and E2, and two small polypeptides E3 and 6K (Strauss 1994). After encapsidation of the viral particles occurs and capsid proteins, usually specific only to the genomic RNA, are packaged, the virions are assembled and budded onto the membrane surface.

[0240] VD2. Alphaviruses as Delivery Vectors Alphaviruses (alphavirus sequences, characteristics, and other elements) can be used to generate alphavirus-based delivery vectors (also called alphavirus vectors, alphavirus virus vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying mRNA (SAM) or samRNA vectors). Alphaviruses have traditionally been genetically engineered for use as expression vector systems (Pushko 1997, Rheme 2004). Alphaviruses have several advantages in vaccine settings where expression of heterologous antigens may be desirable. Due to their ability to replicate autonomously in the host cytosol, alphaviruses can generally achieve high copy numbers of the expression cassette within the cell, thus achieving high levels of heterologous antigen production. Furthermore, the vectors are generally transient, resulting in high biosafety and low induction of immune tolerance to the vector. Additionally, the general public generally does not have pre-existing immunity to alphaviruses compared to other standard viral vectors such as human adenoviruses. Alphavirus-based vectors also generally generate cytotoxic responses to infected cells. Cytotoxicity may have some importance in a vaccine setting to adequately stimulate an immune response to the expressed heterologous antigen. However, the degree of desired cytotoxicity is a matter of balance, and therefore several attenuated alphaviruses have been developed, including the TC-83 strain of VEE. Thus, one example of an antigen expression vector described herein uses an alphavirus backbone that allows high levels of antigen expression, stimulates a strong immune response to the antigen, does not stimulate an immune response to the vector itself, and can be used safely. Furthermore, antigen expression cassettes can be designed to stimulate different levels of immune response through optimizing which alphavirus sequences the vector uses, including, but not limited to, sequences derived from VEE or its attenuated derivative TC-83.

[0241] Several expression vector design strategies using alphavirus sequences have been developed (Pushko 1997). In one strategy, the design of the alphavirus vector involves inserting a second copy of the 26S promoter sequence element downstream of the structural protein gene followed by insertion of the heterologous gene (Frolov 1993). This results in the production of a subgenomic RNA that expresses an additional heterologous protein in addition to the native nonstructural and structural proteins. In this system, all the factors are present to produce infectious virions, and therefore repeated rounds of infection of the expression vector in uninfected cells can be carried out.

[0242] Another expression vector design utilizes a helper virus system (Pushko 1997). In this strategy, structural proteins are replaced by heterologous genes. Thus, after autonomous replication of the viral RNA mediated by the still intact nonstructural genes, the 26S subgenomic RNA provides expression of the heterologous protein. Traditionally, an additional vector expressing the structural proteins is given in trans, for example by co-transfection of a cell line, resulting in an infectious virus. One system is described in U.S. Pat. No. 8,093,021, the entire contents of which are incorporated herein by reference for all purposes. Helper vector systems offer the advantage of limiting the possibility of forming infectious particles, thus improving biosafety. In addition, helper vector systems may reduce the overall vector length and improve the efficiency of replication and expression. Thus, in one example of an antigen expression vector described herein, an alphavirus backbone may be used in which the structural proteins are replaced by an antigen cassette, and the resulting vector promotes efficient expression due to reduced biosafety concerns as well as reduced overall expression vector size.

[0243] VD3. In vitro generation of alphaviruses Alphavirus delivery vectors are generally positive-sense RNA polynucleotides. A conventional method known in the art for RNA production is in vitro translation (IVT). In this method, a DNA template of the desired vector is first generated by methods well known in the art, including standard molecular biology methods such as cloning, restriction digestion, ligation, gene synthesis (e.g., chemical and / or enzymatic synthesis), and polymerase chain reaction (PCR). The DNA template has a promoter for an RNA polymerase at the 5' end of the sequence desired to be transcribed into RNA. Promoters include, but are not limited to, promoters for bacteriophage polymerases such as T3, T7, or SP6. The DNA template is then incubated with the appropriate RNA polymerase enzyme, buffering agents, and nucleotides (NTPs). The resulting RNA polynucleotide can be optionally further modified by methods including, but not limited to, the addition of a 5' cap structure, such as 7-methylguanosine or related structures, and optionally modifying the 3' end to have a polyadenylated (polyA) tail. The RNA can then be purified using methods well known in the art, such as phenol-chloroform extraction or column purification (eg, chromatography-based purification).

[0244] VD4. Delivery via lipid nanoparticles One important aspect to consider in the design of vaccine vectors is immunity to the vector itself (Riley 2017). This can be in the form of pre-existing immunity to the vector itself, for example certain human adenovirus systems, or immunity to the vector that arises after administration of the vaccine. The latter is an important consideration when multiple doses of the same vaccine are administered, for example separate priming and booster doses, or when the same vaccine vector system is used to deliver different antigen cassettes.

[0245] In the case of alphavirus vectors, the standard delivery method is the helper virus system mentioned above, which generates infectious viral particles by delivering the capsid, E1, and E2 proteins in trans. However, it is important to note that E1 and E2 proteins are often the primary targets of neutralizing antibodies (Strauss 1994). Therefore, the efficacy of using alphavirus vectors to deliver antigens of interest to target cells may be reduced if the infectious particles are targeted by neutralizing antibodies.

[0246] An alternative to viral particle-mediated gene delivery is the delivery of expression vectors using nanoparticles (Riley 2017). Importantly, nanomaterial carriers can be formed of non-immunogenic materials, generally avoiding the induction of immunity against the delivery vector itself. These materials can include, but are not limited to, lipids, inorganic nanomaterials, and other polymeric materials. Lipids can be cationic, anionic, or neutral. Such materials can be synthetic or naturally derived, and in certain instances, biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid complexes including, but not limited to, polyethylene glycol (PEG) complexes (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins.

[0247] Lipid nanoparticles (LNPs) are attractive delivery systems due to the amphiphilic nature of lipids that allows for the formation of membranes and vesicle-like structures (Riley 2017). Typically, these vesicles deliver expression vectors by absorbing into the membrane of target cells and releasing the nucleic acid into the cytosol. Additionally, LNPs can be further modified or functionalized to facilitate targeting of specific cell types. Another consideration in the design of LNPs is the balance between efficiency of targeting and cytotoxicity. The lipid composition generally includes a defined mixture of cationic, neutral, anionic, and amphipathic lipids. In some instances, specific lipids are included to prevent aggregation of the LNP, prevent lipid oxidation, or provide chemical functional groups that facilitate attachment of additional moieties. The lipid composition can affect the size and stability of the overall LNP. In one example, the lipid composition includes dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or MC3-like molecules. The MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as, for example, PEG or PEG-conjugated lipids, sterols, or neutral lipids.

[0248] Nucleic acid vectors, such as expression vectors, directly exposed to serum can have several undesirable effects, including degradation of the nucleic acid by nucleases in serum, or stimulation of off-targets of the immune system by free nucleic acid. Thus, encapsulation of the alphavirus vector can be used to prevent degradation while also preventing potential off-target effects. In certain examples, the alphavirus vector is fully encapsulated within the delivery vehicle, such as the aqueous interior of the LNP. Encapsulation of the alphavirus vector within the LNP can be performed by methods well known in the art, such as microfluidic mixing and droplet generation performed in a microfluidic droplet generator. Such devices include, but are not limited to, standard T-junction devices or flow focusing devices. In one example, a desired lipid formulation, such as an MC3 or MC3-like molecule-containing composition, is fed into the droplet generator in parallel with the alphavirus delivery vector and other desired substances, resulting in the delivery vector and desired substances being fully encapsulated within the MC3 or MC3-like molecule-based LNP. In one example, the droplet generator can control the particle size range and size distribution of the generated LNP. For example, LNPs can have particle sizes ranging from 1 to 1000 nm in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nm in diameter. After droplet generation, the delivery vector encapsulating the expression vector can be further processed or modified in preparation for administration.

[0249] VE chimpanzee adenovirus (ChAd) VE1. Viral delivery by chimpanzee adenovirus Vaccine compositions for delivering one or more antigens (e.g., by antigen cassettes containing one or more neo-antigens shown in Table A and / or AACR GENIE results and / or one or more antigens shown in Table 1.2) can be made by providing chimpanzee-derived adenovirus nucleotide sequences, various novel vectors, and cell lines expressing chimpanzee adenovirus genes. The nucleotide sequence of chimpanzee C68 adenovirus (also referred to herein as ChAdV68) can be used in vaccine compositions for delivering antigens (see SEQ ID NO:1). The use of C68 adenovirus-derived vectors is described in more detail in U.S. Patent Application Publication No. US20200197500A1 and International Patent Application Publication No. WO2020243719A1, each of which is incorporated herein by reference in its entirety for all purposes. ChAdV68-based vectors and delivery systems are described in detail in U.S. Patent Application Publication No. US20200197500A1 and International Patent Application Publication No. WO2020243719A1, each of which is incorporated herein by reference for all purposes.

[0250] In a further aspect, provided herein is a recombinant adenovirus comprising a DNA sequence of a chimpanzee adenovirus, such as C68, and an antigen cassette operably linked to a regulatory sequence that induces expression. The recombinant virus is capable of infecting mammalian, preferably human, cells and expressing the product of the neo-antigen cassette in the cells. The vector can be deleted of the native chimpanzee E1 gene, and / or E3 gene, and / or E4 gene. An antigen cassette can be inserted into any of these gene deletion sites. The antigen cassette can include an antigen against which a primed immune response is desired.

[0251] In another embodiment, provided herein is a mammalian cell infected with a chimpanzee adenovirus, such as C68.

[0252] In yet another embodiment, novel mammalian cell lines are provided that express chimpanzee adenovirus genes (eg, from C68) or functional fragments thereof.

[0253] In an even further aspect, provided herein is a method for delivering an antigen cassette into a mammalian cell, comprising introducing into the cell an effective amount of a chimpanzee adenovirus, such as C68, engineered to express the antigen cassette.

[0254] Yet another embodiment provides a method of stimulating an immune response in a mammalian host to treat cancer, comprising administering to the host an effective amount of a recombinant chimpanzee adenovirus, such as C68, that includes an antigen cassette encoding one or more antigens derived from the tumor against which the immune response is targeted.

[0255] Yet another embodiment provides a method for stimulating an immune response in a mammalian host to treat or prevent a disease in a subject, comprising administering to the host an effective amount of a recombinant chimpanzee adenovirus, such as C68, that includes an antigen cassette encoding one or more antigens, such as antigens from a disease to which the immune response is targeted.

[0256] Also disclosed is a non-simian mammalian cell expressing a chimpanzee adenovirus gene obtained from the sequence of SEQ ID NO: 1. The gene can be selected from the group consisting of the adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 of SEQ ID NO: 1.

[0257] Also disclosed are nucleic acid molecules comprising a DNA sequence of a chimpanzee adenovirus comprising a gene obtained from the sequence of SEQ ID NO: 1. The gene can be selected from the group consisting of said chimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genes of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 1. In some embodiments, the nucleic acid molecule comprises the sequence of SEQ ID NO: 1, with at least one gene deleted selected from the group consisting of E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genes of SEQ ID NO: 1.

[0258] Also disclosed is a vector comprising a chimpanzee adenovirus DNA sequence taken from SEQ ID NO:1 and an antigen cassette operably linked to one or more regulatory sequences directing expression of the cassette in a heterologous host cell, optionally wherein the chimpanzee adenovirus DNA sequence comprises at least cis elements required for replication and encapsidation, the cis elements flanking the antigen cassette and the regulatory sequences. In some embodiments, the chimpanzee adenovirus DNA sequence comprises genes selected from the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 gene sequences of SEQ ID NO:1. In some embodiments, the vector may be deleted for the E1A and / or E1B genes.

[0259] Also disclosed herein is an adenoviral vector comprising a partially deleted E4 gene, comprising a deleted or partially deleted E4orf2 region and a deleted or partially deleted E4orf3 region, and optionally a deleted or partially deleted E4orf4 region. The partially deleted E4 can comprise an E4 deletion of at least nucleotides 34,916-35,642 of the sequence set forth in SEQ ID NO:1, the vector comprising at least nucleotides 2-36,518 of the sequence set forth in SEQ ID NO:1. The partially deleted E4 can comprise an E4 deletion of at least a partial deletion of nucleotides 34,916-34,942 of the sequence set forth in SEQ ID NO:1, at least a partial deletion of nucleotides 34,952-35,305 of the sequence set forth in SEQ ID NO:1, and at least a partial deletion of nucleotides 35,302-35,642 of the sequence set forth in SEQ ID NO:1, the vector comprising at least nucleotides 2-36,518 of the sequence set forth in SEQ ID NO:1. The partially deleted E4 can include an E4 deletion of at least nucleotides 34,980-36,516 of the sequence shown in SEQ ID NO:1, and the vector includes at least nucleotides 2-36,518 of the sequence shown in SEQ ID NO:1. The partially deleted E4 can include an E4 deletion of at least nucleotides 34,979-35,642 of the sequence shown in SEQ ID NO:1, and the vector includes at least nucleotides 2-36,518 of the sequence shown in SEQ ID NO:1. The partially deleted E4 can include an E4 deletion of at least a partial deletion of E4Orf2, a completely deleted E4Orf3, and at least a partial deletion of E4Orf4. The partially deleted E4 can include an E4 deletion of at least a partial deletion of E4Orf2, at least a partial deletion of E4Orf3, and at least a partial deletion of E4Orf4. The partially deleted E4 may comprise an E4 deletion of at least a partial deletion of E4Orf1, a completely deleted E4Orf2, and at least a partial deletion of E4Orf3. The partially deleted E4 may comprise an E4 deletion of at least a partial deletion of E4Orf2 and at least a partial deletion of E4Orf3. The partially deleted E4 may comprise an E4 deletion from the start of E4Orf1 to the start of E4Orf5. The partially deleted E4 may be an E4 deletion adjacent to the start of E4Orf1. The partially deleted E4 may be an E4 deletion adjacent to the start of E4Orf2.The partial deletion E4 may be an E4 deletion adjacent to the start site of E4Orf3. The partial deletion E4 may be an E4 deletion adjacent to the start site of E4Orf4. The E4 deletion may be at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 nucleotides. The E4 deletion may be at least 700 nucleotides. The E4 deletion may be at least 1500 nucleotides. The E4 deletion may be 50 or less, 100 or less, 200 or less, 300 or less, 400 or less, 500 or less, 600 or less, 700 or less, 800 or less, 900 or less, 1000 or less, 1100 or less, 1200 or less, 1300 or less, 1400 or less, 1500 or less, 1600 or less, 1700 or less, 1800 or less, 1900 or less, or 2000 or less nucleotides. The E4 deletion may be 750 or less nucleotides. The E4 deletion may be at least 1550 nucleotides or less.

[0260] The 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. The 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. The partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1 lacking at least nucleotides 34,980 to 36,516 of the sequence shown in SEQ ID NO: 1. The partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1 lacking at least nucleotides 34,979 to 35,642 of the sequence shown in SEQ ID NO: 1. The adenoviral vector having a partially deleted E4 gene can include a cassette, the cassette includes at least one payload nucleic acid sequence, and the cassette further includes at least one promoter sequence operably linked to the at least one payload nucleic acid sequence. The adenoviral vector having a partially deleted E4 gene can include one or more genes or regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO: 1, and optionally, the one or more genes or regulatory sequences include at least one of the chimpanzee adenovirus inverted terminal repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence shown in SEQ ID NO: 1. An adenoviral vector having a partially deleted E4 gene can have nucleotides 2 to 34,915 of the sequence set forth in SEQ ID NO:1, where the partially deleted E4 gene is the 3' end of nucleotides 2 to 34,915, and optionally nucleotides 2 to 34,915 further lacking nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, which corresponds to an E1 deletion, and / or nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO:1, which corresponds to an E3 deletion.An adenoviral vector having a partially deleted E4 gene can have nucleotides 35,643 to 36,518 as set forth in SEQ ID NO: 1, the partially deleted E4 gene being at the 5' end of nucleotides 35,643 to 36,518. An adenoviral vector having a partially deleted E4 gene can have nucleotides 2 to 34,916 of the sequence as set forth in SEQ ID NO: 1, the partially deleted E4 gene being at the 3' end of nucleotides 2 to 34,916, the nucleotides 2 to 34,916 further lacking nucleotides 577 to 3403 of the sequence as set forth in SEQ ID NO: 1, which corresponds to an E1 deletion, and lacking nucleotides 27,125 to 31,825 of the sequence as set forth in SEQ ID NO: 1, which corresponds to an E3 deletion. An adenoviral vector having a partially deleted E4 gene can have nucleotides 2 to 34,915 of the sequence shown in SEQ ID NO:1, where the partially deleted E4 gene is at the 3' end of nucleotides 2 to 34,916, where nucleotides 2 to 34,915 further lack nucleotides 577 to 3403 of the sequence shown in SEQ ID NO:1, which corresponds to an E1 deletion, and also lack nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO:1, which corresponds to an E3 deletion, and has nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO:1, where the partially deleted E4 gene is at the 5' end of nucleotides 35,643 to 36,518.

[0261] An adenoviral vector having a partially deleted E4 gene may be an 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 34,915 of the sequence shown in SEQ ID NO: 1, wherein the partially deleted E4 gene is the 3' end of nucleotides 2 to 34,915, which further lacks nucleotides 577 to 3403 of the sequence shown in SEQ ID NO: 1, which corresponds to an E1 deletion, and further lacks nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO: 1, which corresponds to an E3 deletion, and has nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO: 1, wherein the partially deleted E4 gene is the 5' end of nucleotides 35,643 to 36,518.

[0262] Also disclosed are host cells transfected with a vector disclosed herein, such as the C68 vector engineered to express an antigen cassette. Also disclosed are human cells that express a selected gene introduced into the cell by introducing into the cell a vector disclosed herein.

[0263] Also provided is a method for delivering an antigen cassette to a mammalian cell, comprising introducing into said cell an effective amount of a vector disclosed herein, such as a ChAd vector or a self-replicating RNA vector engineered to express the antigen cassette.

[0264] Further disclosed is a method for producing an antigen comprising introducing a vector disclosed herein into a mammalian cell, culturing the cell under appropriate conditions, and producing the antigen.

[0265] Complementation cell lines expressing VE2.E1 To generate recombinant chimpanzee adenoviruses (Ad) with deletions in any of the genes described herein, the function of the deleted gene region (if essential for viral replication and infectivity) can be provided to the recombinant virus by a helper virus or cell line (i.e., complementation or packaging cell line). For example, to generate replication-defective chimpanzee adenovirus vectors, cell lines expressing the E1 gene product of human or chimpanzee adenovirus can be used, and such cell lines can include HEK293 or variants thereof. Cell lines expressing any selected chimpanzee adenovirus gene can be generated according to the protocol for generating cell lines expressing chimpanzee E1 gene (Examples 3 and 4 of U.S. Patent No. 6,083,716).

[0266] AAV enrichment assay can be used to identify chimpanzee adenovirus E1 expressing cell lines. This assay is useful for identifying E1 function in cell lines made using E1 genes of other uncharacterized adenoviruses (e.g., from other species). This assay is described in Example 4B of US Patent No. 6,083,716.

[0267] The selected chimpanzee adenovirus gene (e.g., E1) may be under the transcriptional control of a promoter for expression in the selected parent cell line. Inducible or constitutive promoters can be used for this purpose. Inducible promoters include the sheep metallothionine promoter, which is induced by zinc, or the mouse mammary tumor virus (MMTV) promoter, which is induced by glucocorticoids, particularly dexamethasone. Other inducible promoters, such as those identified in International Application No. WO 95 / 13392, which is incorporated herein by reference, can also be used to generate the packaging cell line. Constitutive promoters can also be used to control the expression of chimpanzee adenovirus genes.

[0268] Parental cells can be selected to generate new cell lines expressing any desired C68 gene. Without being limited thereto, such parental cell lines can be HeLa [ATCC deposit number CCL2], A549 [ATCC deposit number CCL185], KB [CCL17], Detroit [e.g. Detroit510, CCL72], and WI-38 [CCL75] cells. Other suitable parental cell lines can be obtained from other sources. Parental cell lines can include CHO, HEK293 or its variants, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a.

[0269] E1 expressing cell lines can be useful in producing recombinant chimpanzee adenovirus E1 deleted vectors. Cell lines constructed using essentially the same procedures that express one or more other chimpanzee adenovirus gene products are useful in producing recombinant chimpanzee adenovirus vectors with deletions in the genes encoding these products. In addition, cell lines expressing other human Ad E1 gene products are also useful in producing chimpanzee recombinant Ad.

[0270] VE3. Recombinant viral particles as vectors The compositions disclosed herein can include a viral vector that delivers at least one antigen to a cell. Such a vector includes a chimpanzee adenovirus DNA sequence, such as C68, and an antigen cassette operably linked to a regulatory sequence for directing expression of the cassette. The C68 vector is capable of expressing the cassette in an infected mammalian cell. The C68 vector can have a functional deletion in one or more viral genes. The antigen cassette includes at least one antigen under the control of one or more regulatory sequences, such as a promoter. Optional helper viruses and / or packaging cell lines can provide the chimpanzee viral vector with any necessary products of the deleted adenovirus genes.

[0271] The term "functionally deleted" means that a sufficient amount of the gene region has been removed or otherwise altered, for example by mutation or modification, such that the gene region is unable to produce one or more functional products of gene expression. Mutations or modifications that can lead to functional deletions include, but are not limited to, nonsense mutations such as the introduction of premature stop codons and removal of canonical and non-canonical start codons, mutations that alter mRNA splicing or other transcriptional processing, or combinations thereof. If necessary, the entire gene region can be removed.

[0272] Modifications of the nucleic acid sequences forming the vectors disclosed herein, including sequence deletions, insertions, and other mutations, can be produced using standard molecular biology techniques and are within the scope of the present invention.

[0273] VE4. Construction of viral plasmid vectors Chimpanzee adenovirus C68 vectors useful in the present invention include recombinant defective adenoviruses, i.e., chimpanzee adenovirus sequences that have functional deletions in the E1a or E1b genes and optionally other mutations, such as temperature-sensitive mutations or deletions in other genes. These chimpanzee sequences are also expected to be useful in forming hybrid vectors from other adenovirus and / or adeno-associated virus sequences. Homologous adenovirus vectors prepared from human adenoviruses are described in the published literature [see, for example, Kozarsky I and II, cited above, and references cited therein, U.S. Patent No. 5,240,846].

[0274] In constructing useful chimpanzee adenovirus C68 vectors for delivery of antigen cassettes to human (or other mammalian) cells, a wide range of adenovirus nucleic acid sequences can be used in the vector. Vectors containing minimal chimpanzee C68 adenovirus sequences can be used with helper viruses to generate infectious recombinant viral particles. The helper viruses provide the basic gene products required for viral infectivity and propagation of the minimal chimpanzee adenovirus vector. When only one or more selected deletions of chimpanzee adenovirus genes are introduced into an otherwise functional viral vector, the deleted gene products can be supplied in the viral vector production process by propagating the virus that provides the deleted gene function in trans in a selected packaging cell line.

[0275] VE5. Recombinant Minimal Adenovirus The minimal chimpanzee Ad C68 virus is a viral particle that contains only the adenoviral cis elements necessary for replication and virion encapsidation. That is, the vector contains the adenoviral cis-acting 5' and 3' inverted terminal repeats (ITRs) (which function as origins of replication) and the native 5' packaging / enhancer domain (which contains the sequences necessary for packaging the linear Ad genome and the enhancer element of the E1 promoter). See, for example, the methods described for the preparation of "minimal" human Ad vectors in International Application WO 96 / 13597, incorporated herein by reference.

[0276] VE6. Other defective adenoviruses Recombinant replication-deficient adenoviruses may contain more than the minimal chimpanzee adenovirus sequences. These other Ad vectors can be characterized by the deletion of different portions of the viral gene regions and the infectious viral particles formed by the use of helper viruses and / or packaging cell lines as needed.

[0277] As an example, suitable vectors can be made by deleting all or a sufficient portion of the immediate early gene E1a and the delayed early gene E1b of C68 adenovirus, thereby eliminating their normal biological functions. Replication-deficient E1-deleted viruses are capable of replicating and generating infectious viruses when grown in chimpanzee adenovirus-transformed complementation cell lines that contain functional adenovirus E1a and E1b genes that provide the corresponding gene products in trans. Based on homology to known adenovirus sequences, the resulting recombinant chimpanzee adenovirus is expected to be capable of infecting many cell types and expressing antigen(s), as are human recombinant E1-deleted adenoviruses in the art, but will not be able to replicate in many cells that do not carry chimpanzee E1 region DNA, unless the cells are infected at a very high multiplicity of infection.

[0278] As another example, all or part of the C68 adenovirus immediate early gene E3 can be removed from the chimpanzee adenovirus sequences that form part of the recombinant virus.

[0279] Chimpanzee adenovirus C68 vectors can also be constructed to have a deletion of the E4 gene. Yet another vector can have a deletion in the delayed early gene E2a.

[0280] Deletions can be introduced into any of the late genes L1-L5 of the chimpanzee C68 adenovirus genome. Similarly, deletions in intermediate genes IX and IVa2 may be useful for certain purposes. Other deletions can be introduced into other structural or nonstructural adenovirus genes.

[0281] The deletions mentioned above can be used individually. That is, the adenovirus sequence can have only E1 deletion. Any combination of whole genes or parts thereof that are effective in destroying or reducing their biological activity can also be used. For example, in one exemplary vector, the adenovirus C68 sequence can have E1 and E4 genes, or E1, E2a, and E3 genes, or E1 and E3 genes, or E1, E2a, and E4 genes with or without E3 deletion. As mentioned above, such deletions can be used in combination with other mutations, such as temperature-sensitive mutations, to achieve the desired results.

[0282] A cassette containing the antigen(s) is optionally inserted into any deleted region of the chimpanzee C68Ad virus, or, if desired, the function of an existing gene region can be disrupted by inserting the cassette within that region.

[0283] VE7.Helper virus Depending on the chimpanzee adenovirus gene content of the viral vector used to deliver the antigen cassette, helper adenovirus or non-replicating viral fragments can be used to provide sufficient chimpanzee adenovirus gene sequences to generate infectious recombinant viral particles containing the cassette.

[0284] Useful helper viruses contain selected adenoviral gene sequences that are not present in the adenoviral vector construct and / or are not expressed by the packaging cell line transfected with the vector. The helper virus may be replication-deficient and may contain a variety of adenoviral genes other than those mentioned above. Helper viruses may be used in combination with the E1-expressing cell lines described herein.

[0285] For C68, the "helper" virus can be a fragment formed by truncating the C-terminus of the C68 genome with SspI, which removes approximately 1300 bp from the left end of the virus. This truncated virus is then co-transfected with plasmid DNA into an E1-expressing cell line to form recombinant virus by homologous recombination with the C68 sequences in the plasmid.

[0286] Helper viruses can also be formulated as polycation complexes as described in Wu et al, J. Biol. Chem., 264:16985-16987 (1989); KJ Fisher and JM Wilson, Biochem. J., 299:49 (Apr. 1, 1994). Helper viruses can optionally contain a reporter gene. Many such reporter genes are known in the art. The presence of a reporter gene on the helper virus that is different from the antigen cassette on the adenovirus vector allows the Ad vector and the helper virus to be monitored independently. This second reporter gene allows the separation of the resulting recombinant virus and the helper virus during purification.

[0287] VE8. Assembly of Viral Particles and Infection of Cell Lines The assembly of selected DNA sequences of adenovirus, antigen cassettes, and other vector elements into various intermediate plasmids and shuttle vectors, and the use of the plasmids and shuttle vectors to generate recombinant viral particles can all be achieved using conventional techniques, including conventional cDNA cloning methods, in vitro recombination methods (e.g., Gibson assembly), the use of overlapping oligonucleotide sequences of the adenovirus genome, polymerase chain reaction, and any suitable method that provides the desired nucleotide sequence. Standard transfection and co-transfection techniques are used, such as CaPO4 precipitation or liposome-mediated transfection methods such as lipofectamine. Other conventional methods that are used include homologous recombination of the viral genome, plaque formation of the virus in an agar overlay, and signal generation measurement methods.

[0288] For example, following construction and assembly of a viral vector containing a desired antigen, the vector can be transfected in vitro into a packaging cell line in the presence of a helper virus, whereby homologous recombination occurs between the helper and vector sequences, allowing the adenovirus-antigen sequences within the vector to be replicated and packaged into virion capsids, resulting in recombinant viral vector particles.

[0289] The resulting recombinant chimpanzee C68 adenovirus is useful for transferring antigen cassettes into selected cells. In vivo experiments using recombinant viruses grown in packaging cell lines demonstrate the utility of the E1 deleted recombinant chimpanzee adenovirus for transferring cassettes into non-chimpanzee, preferably human, cells.

[0290] VE9. Use of Recombinant Viral Vectors Thus, the resulting recombinant chimpanzee C68 adenovirus containing the antigen cassette (produced by the integration of an adenoviral vector and a helper virus, or an adenoviral vector and a packaging cell line as described above) provides an efficient gene transfer vehicle capable of delivering antigen(s) to a subject in vivo or ex vivo.

[0291] The recombinant vector described above is administered to humans according to published methods for gene therapy. The chimpanzee virus vector carrying the antigen cassette can be administered to patients preferably suspended in a biocompatible solution or pharmaceutically acceptable delivery solvent. Suitable solvents include sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known as pharmaceutically acceptable carriers and well known to those skilled in the art can also be used for this purpose.

[0292] The chimpanzee adenoviral vector is administered in an amount sufficient to transform human cells and result in the introduction and expression of sufficient levels of antigens to provide a therapeutic effect without adverse effects or with medically acceptable physiological effects that can be determined by those skilled in the medical field. Conventional pharmacologic acceptable routes of administration include, but are not limited to, intrahepatic, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parenteral routes of administration. Routes of administration can be combined as necessary.

[0293] The dosage of the viral vector depends mainly on factors such as the condition to be treated, the age, weight, and health of the patient, and therefore may vary between patients. The dosage is adjusted to balance the therapeutic effect against any side effects, and such dosage may vary depending on the therapeutic application for which the recombinant vector is used. The frequency of administration can be determined by observing the expression level of the antigen(s).

[0294] The recombinant replication-deficient adenovirus can be administered in a "pharmacologically effective amount", i.e., an amount of recombinant adenovirus effective by a given route of administration to transfect the desired cells and provide a sufficient level of expression of the selected gene to provide a vaccine effect, i.e., some measurable level of protective immunity. The C68 vector containing the antigen can be co-administered with an adjuvant. The adjuvant can be a monomer separate from the vector (e.g., alum) or encoded within the vector, particularly if the adjuvant is a protein. Adjuvants are well known in the art.

[0295] Conventional pharma- ceutically acceptable routes of administration include, but are not limited to, intranasal, intramuscular, intratracheal, subcutaneous, intradermal, intrarectal, oral, and other parenteral routes of administration. Routes of administration can be combined as necessary or adjusted according to the immunogen or disease. For example, for the prophylaxis of rabies, subcutaneous, intratracheal, and intranasal routes are preferred. The route of administration is determined primarily by the nature of the disease to be treated.

[0296] By observing the expression level of the antigen(s), the need for boosters (if any) can be determined. For example, after evaluation of antibody titers in serum, booster immunizations may be desirable, if necessary.

[0297] VI. METHODS OF TREATMENT AND MANUFACTURING Also provided are methods of stimulating a tumor-specific immune response in a subject, vaccinating against a tumor, and treating and / or alleviating a symptom of cancer in a subject by administering to the subject one or more antigens, such as a plurality of antigens identified using the methods disclosed herein.

[0298] In some embodiments, the subject has been diagnosed with cancer or is at risk of developing cancer. The subject may have previously undergone cancer treatment, for example, may have previously undergone surgery to remove tumor and / or cancer tissue, chemotherapy, immunotherapy (e.g., immune checkpoint inhibitor therapy), radiation therapy, or a combination thereof. The subject may be a human, dog, cat, horse, or any animal in which a tumor-specific immune response is desired. The tumor may be any solid tumor, such as breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck tumors, pancreatic cancer, brain tumors, melanoma, and tumors of other tissue organs, as well as hematological tumors, such as lymphomas and leukemias, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma.

[0299] The antigen can be administered in an amount sufficient to stimulate a CTL response. The antigen can be administered in an amount sufficient to stimulate a T cell response. The antigen can be administered in an amount sufficient to stimulate a B cell response. The antigen can be administered in an amount sufficient to stimulate both a T cell response and a B cell response.

[0300] The antigen can be administered alone or in combination with other therapeutic agents. The therapeutic agent can be, for example, a chemotherapeutic agent, radiation, or immunotherapy. Any suitable therapeutic treatment for a particular cancer can be administered. A therapeutically effective amount of the therapeutic agent can be administered. An amount of a therapeutic agent that is not generally considered a therapeutically effective amount alone, but that exhibits beneficial properties when co-administered with any of the compositions described herein, can be administered.

[0301] In addition, the subject can be further administered with an anti-immunosuppressant / immunostimulatory agent, such as a checkpoint inhibitor.For example, the subject can be further administered with an anti-CTLA antibody or anti-PD-1 or anti-PD-L1.The blockade of CTLA4 or PD-L1 can enhance the immune response of the patient to cancerous cells.In particular, the blockade of CTLA4 has been shown to be effective when following a vaccination protocol.

[0302] The optimal amount and optimal administration regimen of each antigen included in the vaccine composition can be determined. For example, the antigen or its variants can be formulated for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Injection methods include subcutaneous (sc), intradermal (id), intraperitoneal (ip), intramuscular (im), and intravenous (iv). Injection methods for DNA or RNA include intradermal (id), intramuscular (im), subcutaneous (sc), intraperitoneal (ip), and intravenous (iv). Other methods of administration of the vaccine composition are known to those skilled in the art.

[0303] Vaccines can be adapted so that the selection, number and / or amount of antigens present in the composition are tissue, cancer and / or patient specific. For example, the exact selection of peptides can be guided by the expression pattern of a particular parent protein or by the mutational status of the patient. This selection can depend on the specific cancer type, disease state, initial treatment regimen, immune status of the patient, and of course the HLA haplotype of the patient. Furthermore, vaccines can contain components that are personalized according to the personal needs of a particular patient. Examples include changing the selection of antigens according to the expression of antigens in a particular patient, or adjusting secondary treatments after a first round or scheme of treatment.

[0304] Patients for administering antigen vaccines can be identified by using various diagnostic methods, such as the patient selection methods described further below. Patient selection can include identifying mutations or expression patterns of one or more genes. In some cases, patient selection can include identifying the patient's haplotype. Various patient selection methods can be performed in parallel, for example, sequencing diagnostics can identify both the patient's mutation and haplotype. Various patient selection methods can also be performed sequentially, for example, one diagnostic test can identify mutations and another diagnostic test can identify the patient's haplotype, where each test can be the same (e.g., both high-throughput sequencing) or different (e.g., one high-throughput sequencing and the other Sanger sequencing) diagnostic method.

[0305] For compositions to be used as cancer vaccines, antigens including similar normal self-peptides that are expressed in high amounts in normal tissues may be avoided or present in low amounts in the compositions described herein. In contrast, if a patient's tumor is known to express a particular antigen in high amounts, the respective pharmaceutical composition for treating this cancer may be present in high amounts and / or may include multiple antigens specific for this particular antigen or this antigen's pathway.

[0306] The composition comprising the antigen can be administered to an individual already suffering from cancer. In therapeutic applications, the composition is administered to a subject in an amount sufficient to stimulate an immune response, such as stimulating effective CTL against the tumor antigen, and to cure or at least partially prevent symptoms and / or complications. The immune response can include a reduction in tumor size or volume. The reduction in tumor size or volume can include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% reduction. The reduction in tumor size or volume can include at least a 15% reduction. The reduction in tumor size or volume can include at least a 20% reduction. The immune response can include a stabilization of tumor size or volume. The immune response can result in the amelioration of the subject's disease, such as complete response (CR), partial response (PR), or stable disease (SD) (e.g., as examined by criteria described in clinical studies). The amount suitable for achieving this is defined as a "therapeutically effective dose". The amount effective for this purpose depends, for example, on the composition, the mode of administration, the stage and severity of the disease being treated, the weight and general condition of the patient, and the judgment of the prescribing physician. It should be noted that the compositions can generally be used in severe disease conditions, i.e., life-threatening or potentially life-threatening situations, especially when the cancer has metastasized. In such cases, it is believed that, given the minimization of the relative non-toxic properties of the foreign substances and antigens, it is possible and may be felt desirable by the treating physician to administer significant excesses of these compositions.

[0307] In therapeutic applications, administration can begin upon detection or surgical removal of a tumor, followed by booster doses at least until symptoms have substantially disappeared and for a predetermined period thereafter, or until immunity is deemed achieved (e.g., memory B or T cell populations, or antigen-specific B cells or antibodies are produced).

[0308] A composition comprising an antigen (e.g., any composition for delivering a self-replicating alphavirus-based expression system or a chimpanzee adenovirus (ChAdV)-based expression system described herein) can be administered as an adjuvant therapy to a subject who has already undergone a primary therapy. The composition comprising an antigen can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days after the primary therapy, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more weeks after the primary therapy. For example, a composition comprising an antigen can be administered as an adjuvant therapy after surgery to remove tumor and / or cancerous tissue, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days after surgery, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more weeks after surgery.

[0309] A composition comprising an antigen (e.g., any composition for delivering a self-replicating alphavirus-based expression system or chimpanzee adenovirus (ChAdV)-based expression system described herein) can be administered as maintenance therapy to a subject who has already undergone a primary therapy, such as whose cancer is in remission (e.g., complete remission) after the primary therapy.

[0310] The primary therapy may include surgery to remove tumors and / or cancerous tissue, chemotherapy, immunotherapy (e.g., immune checkpoint inhibitor therapy), radiation therapy, or a combination thereof. The primary therapy may include surgery. The primary therapy may include chemotherapy, such as oxaliplatin, fluoropyrimidine, and / or bevacizumab. The primary therapy may include a combination of oxaliplatin, fluoropyrimidine, and bevacizumab.

[0311] The composition comprising the antigen can be administered as an adjuvant or maintenance therapy in combination with additional therapies, such as administered in combination with chemotherapy, immune checkpoint inhibitor therapy, radiation therapy, or a combination thereof. The combination therapy can include fluoropyrimidines, bevacizumab, and / or immune checkpoint inhibitor therapy. The combination therapy can include fluoropyrimidines and bevacizumab. The combination therapy can include fluoropyrimidines, bevacizumab, and immune checkpoint inhibitor therapy (e.g., anti-PD-1 or anti-PD-L1 antibodies).

[0312] The immune checkpoint inhibitor may include (1) an anti-PD-1 antibody or an antigen-binding fragment thereof, (2) an anti-PD-L1 antibody or an antigen-binding fragment thereof, and / or (3) an anti-CTLA-4 antibody or an antigen-binding fragment thereof. The immune checkpoint inhibitor therapy may include administration of an anti-CTLA-4 antibody or an antigen-binding fragment thereof with only a priming dose and a first booster dose. The immune checkpoint inhibitor therapy may include the anti-CTLA-4 antibody ipilimumab. The immune checkpoint inhibitor therapy may include ipilimumab administered subcutaneously at a dose of 30 mg. The immune checkpoint inhibitor therapy may include administration of an anti-PD-L1 antibody or an antigen-binding fragment thereof every four weeks (Q4W). The immune checkpoint inhibitor therapy may include the anti-PD-L1 antibody atezolizumab or nivolumab. Atezolizumab may be administered intravenously at a dose of 1680 mg. Nivolumab may be administered intravenously at a dose of 480 mg.

[0313] Immune checkpoint inhibitor therapy can include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 separate administrations, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 separate administrations, e.g., at or about every 28 days (alternatively, at or about every 4 weeks, and / or at or about every month).Immune checkpoint inhibitor therapy can include administration of an anti-PD-L1 antibody or antigen-binding fragment thereof, e.g., including at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 administrations, e.g., at or about every 28 days (alternatively, at or about every 4 weeks, and / or at or about every month). The immune checkpoint inhibitor therapy can include at least 13 separate administrations, e.g., at or about every 28 days (alternatively, at or about every 4 weeks, and / or at or about every month). In some embodiments, the administration of the anti-PD-L1 antibody or antigen-binding fragment thereof includes at least 13 administrations, e.g., at or about every 28 days (alternatively, at or about every 4 weeks, and / or at or about every month).

[0314] The ChAdV-based expression system can be administered as a booster dose at or about 140 days after the priming dose of the ChAdV-based expression system. The ChAdV-based expression system can be administered as a booster dose at or about 20 weeks after the priming dose of the ChAdV-based expression system. The ChAdV-based expression system can be administered as a booster dose at or about 5 months after the priming dose of the ChAdV-based expression system. The ChAdV-based expression system can be administered as a booster dose at or about 140 days after the priming dose of the ChAdV-based expression system. The ChAdV-based expression system can be administered as a booster dose at or about 140 days after the priming dose of the ChAdV-based expression system. The ChAdV-based expression system can be administered as a booster dose at or about 20 weeks after the priming dose of the ChAdV-based expression system. The ChAdV-based expression system can be administered as a booster dose at or about 5 months after the priming dose of the ChAdV-based expression system.

[0315] The self-replicating alphavirus-based expression system may be administered as at least two booster doses. The self-replicating alphavirus-based expression system may be administered as at least two or more booster doses spaced at least 28 days apart. The self-replicating alphavirus-based expression system may be administered as at least two or more booster doses spaced at least four weeks (Q4W). The self-replicating alphavirus-based expression system may be administered as at least two or more booster doses spaced at least one month apart. The self-replicating alphavirus-based expression system may be administered as at least two or more booster doses spaced at least 56 days apart. The self-replicating alphavirus-based expression system may be administered as at least two or more booster doses spaced at least eight weeks (Q8W). The self-replicating alphavirus-based expression system may be administered as at least two or more booster doses spaced at least two months apart.

[0316] The self-replicating alphavirus-based expression system can be administered as at least two booster doses at, or about, days 28 and 84 after the priming dose of the ChAdV-based expression system. The self-replicating alphavirus-based expression system can be administered as at least two booster doses at, or about, weeks 4 and 12 after the priming dose of the ChAdV-based expression system. The self-replicating alphavirus-based expression system can be administered as at least two booster doses at, or about, months 1 and 3 after the priming dose of the ChAdV-based expression system.

[0317] The self-replicating alphavirus-based expression system can be administered as at least four booster doses. The self-replicating alphavirus-based expression system can be administered at or about days 28, 84, 224, and 308 relative to the priming dose of the ChAdV-based expression system. The self-replicating alphavirus-based expression system can be administered at or about weeks 4, 12, 32, and 44 relative to the priming dose of the ChAdV-based expression system. The self-replicating alphavirus-based expression system can be administered at or about months 1, 3, 8, and 11 relative to the priming dose of the ChAdV-based expression system.

[0318] The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (alternatively, at or about 20 weeks or more and / or at or about 5 months or more) after a priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least two booster doses. The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (alternatively, at or about 20 weeks or more and / or at or about 5 months or more) after a priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least two or more booster doses spaced at least 28 days apart. The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (alternatively, at or about 20 weeks or more and / or at or about 5 months or more) after a priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least two or more booster doses spaced at least four weeks apart (Q4W). The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (alternatively, at or about 20 weeks or more and / or at or about 5 months or more) after a priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least two or more booster doses spaced at least one month apart.The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (alternatively, at or about 20 weeks or more and / or at or about 5 months or more) after a priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least two or more booster doses spaced at least 56 days apart. The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (alternatively, at or about 20 weeks or more and / or at or about 5 months or more) after a priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least two or more booster doses spaced at least 8 weeks (Q8W). The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (or at or about 20 weeks or more and / or at or about 5 months or more) after the priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least two or more booster doses spaced at least two months apart.

[0319] The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (or at or about 20 weeks or more and / or at or about 5 months or more) after the priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least two booster doses at or about 28 and 84 days after the priming dose of the ChAdV-based expression system. The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (or at or about 20 weeks or more and / or at or about 5 months or more) after the priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least two booster doses at or about 4 and 12 weeks after the priming dose of the ChAdV-based expression system. The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (or at or about 20 weeks or more and / or at or about 5 months or more) after the priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least two booster doses at or about 1 and 3 months after the priming dose of the ChAdV-based expression system.

[0320] The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (or at or about 20 weeks or more and / or at or about 5 months or more) after the priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered as at least four booster doses. The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (alternatively, at or about 20 weeks or more and / or at or about 5 months or more) after the priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered at or about 28, 84, 224, and 308 days based on the priming dose of the ChAdV-based expression system. The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (or at or about 20 weeks or more and / or at or about 5 months or more) after the priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered at or about 4, 12, 32, and 44 weeks based on the priming dose of the ChAdV-based expression system. The ChAdV-based expression system can be administered as a booster dose at or about 140 days or more (or at or about 20 weeks or more and / or at or about 5 months or more) after the priming dose of the ChAdV-based expression system, and the self-replicating alphavirus-based expression system can be administered at or about 1, 3, 8, and 11 months based on the priming dose of the ChAdV-based expression system.

[0321] Pharmaceutical compositions for therapeutic treatment (e.g., vaccine compositions) are intended for parenteral, topical, nasal, oral, or local administration. Pharmaceutical compositions can be administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. The compositions can be administered at the site of surgical resection to stimulate a local immune response against the tumor. The compositions can be administered to target specific tissues, organs, and / or cells of the subject. Disclosed herein are compositions for parenteral administration that include a solution of the antigen, the vaccine composition dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. For example, a variety of aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional, well-known sterilization techniques, or sterile filtered. The resulting aqueous solutions can be packaged for use as is, or can be lyophilized, and the lyophilized formulation can be combined with a sterile solution prior to administration. The compositions may contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0322] Antigens can also be administered via liposomes, which target the antigen to specific cellular tissues, such as lymphoid tissues. Liposomes are also useful for increasing half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these formulations, the antigen to be delivered is incorporated as part of the liposome, either alone or in combination with a molecule that binds to a receptor prevalent on lymphoid cells, such as a monoclonal antibody that binds to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes loaded with the desired antigen can be directed to the site of lymphoid cells, where they deliver the selected therapeutic / immunogenic composition. Liposomes can be formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid is typically guided by considerations, for example, of the size of the liposome, acid lability, and stability of the liposome in the bloodstream. There are various methods for preparing liposomes, for example, as described in Szoka et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

[0323] For targeting to immune cells, ligands incorporated into the liposomes can include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. The liposomal suspensions can be administered intravenously, locally, or topically in different doses depending, inter alia, on the method 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 one or more of the peptides described herein may be administered to a patient. Many methods for administering nucleic acids to a patient are conveniently used. For example, the nucleic acid can be administered directly as "naked DNA". This approach is described, for example, in Wolff et al., Science 247:1465-1468 (1990), and in U.S. Pat. Nos. 5,580,859 and 5,589,466. The nucleic acid can also be administered using ballistic delivery, for example, as described in U.S. Pat. No. 5,204,253. Particles composed of DNA alone can also be administered. Alternatively, the DNA can be attached to particles such as gold particles. Approaches for delivering nucleic acid sequences include viral vectors, mRNA vectors, and DNA vectors, with or without electroporation.

[0325] Nucleic acids can also be delivered by complexing with cationic compounds, such as cationic lipids.Lipid-mediated gene delivery methods are described, for example, in 9618372 WOAWO 96 / 18372, 9324640 WOAWO 93 / 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, 9106309 WOAWO 91 / 06309, and Felgner et al., Proc.Natl.Acad.Sci.USA 84:7413-7414 (1987).

[0326] Antigens may be derived from vaccinia, fowlpox, self-replicating alphaviruses, Maraba viruses, adenoviruses (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or from second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C ( promoter, Nucl. Acids Res. (2015) 43(1): 682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72(12): 9873-9880) may be included in a vaccine platform based on a viral vector. Depending on the packaging capacity of the viral vector-based vaccine platform described above, this approach can deliver one or more nucleotide sequences encoding one or more antigenic peptides.The sequence may be flanked by non-mutated sequences, separated by a linker, or preceded by one or more sequences that target intracellular compartments (see, e.g., Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22(4): 433-8, Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science. (2016) 352(6291): 1337-41, Lu et al., Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20(13): 3401-10). Upon introduction into the host, the infected cells express the antigen, thereby stimulating a host immune (e.g., CTL) response against the peptide. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus Calmette-Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful for therapeutic administration of antigens or immunization, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the description herein.

[0327] One means of administering nucleic acids is to use a minigene construct that encodes one or more epitope-encoding nucleic acid sequences. To generate a DNA sequence (minigene) encoding a selected CTL epitope for expression in human cells, the amino acid sequence of the epitope is reverse translated. A human codon usage table is used to guide the codon selection for each amino acid. These epitope-encoding DNA sequences are directly linked to generate a contiguous 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 reverse translated and incorporated into the minigene sequence include helper T lymphocytes, epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. Additionally, MHC presentation of CTL epitopes can be improved by incorporating synthetic (e.g., polyalanine) or natural flanking sequences adjacent to the CTL epitope. The minigene sequence is converted to DNA by assembling oligonucleotides encoding the + and - strands of the minigene. Overlapping oligonucleotides (30-100 bases in length) are synthesized, phosphorylated, purified using well-known methods, and annealed under appropriate conditions. The ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene, encoding the CTL epitope polypeptide, is then cloned into the desired expression vector.

[0328] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is to reconstitute lyophilized DNA with sterile phosphate-buffered saline (PBS). A variety of methods have been described and new techniques may become available. As mentioned above, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and compounds collectively referred to as PINCs (protective, interactive, non-condensing) can be complexed with purified plasmid DNA to affect variables such as stability, distribution in muscle, or trafficking to specific organs or cell types.

[0329] Also disclosed is a method of manufacturing a vaccine, e.g., a tumor vaccine, comprising performing each of the steps of the methods disclosed herein and producing a vaccine, e.g., a tumor vaccine, comprising multiple antigens or a subset of multiple antigens.

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

[0331] The host cell may comprise a Chinese Hamster Ovary (CHO) cell, an NS0 cell, yeast, or a HEK293 cell. The host cell may be transformed with one or more polynucleotides comprising at least one nucleic acid sequence encoding an antigen or vector disclosed herein, and optionally, the isolated polynucleotide comprises a promoter sequence operably linked to at least one nucleic acid sequence encoding the antigen or vector. In certain embodiments, the isolated polypeptide may be a cDNA.

[0332] VII. Use and Administration of Antigens Vaccination protocols can be used to administer one or more antigens to subjects.Vaccination methods, protocols, and schedules that can be used include, but are not limited to, those described in International Publication No. WO2021092095, each of which is incorporated herein by reference in its entirety for all purposes.

[0333] The priming vaccine can be based on C68 (e.g., the sequence shown in SEQ ID NO: 1 or 2) or SamRNA (e.g., the sequence shown in SEQ ID NO: 3 or 4). The booster vaccine can also be based on C68 (e.g., the sequence shown in SEQ ID NO: 1 or 2) or SamRNA (e.g., the sequence shown in SEQ ID NO: 3 or 4).

[0334] Each vector in a prime / boost regimen typically includes a cassette containing antigens. The cassette can include about 1-50 antigens separated by spacers, such as the natural sequences that typically surround each antigen, or other non-natural spacer sequences, such as AAY. The cassette can include about 20 neo-antigens, e.g., 20, subject and / or tumor specific antigens. The cassette can include an MHCII antigen, such as a tetanus toxoid antigen, and a PADRE antigen, which is considered a universal class II antigen. The cassette can include a targeting sequence, such as a ubiquitin targeting sequence. Additionally, each vaccine dose can be administered to the subject in combination with (e.g., simultaneously with, prior to, or after) an immune modulator. Each vaccine dose can be administered to the subject in combination with (e.g., simultaneously with, prior to, or after) a checkpoint inhibitor (CPI). The CPI can include an antibody or antigen-binding portion thereof that inhibits CTLA4, PD1, and / or PDL1. Such antibodies can include atezolizumab, ipilimumab, nivolumab, cemiplimab, tremelimumab, or durvalumab. Each vaccine dose can be administered to a subject in combination with (e.g., simultaneously with, prior to, or after) a cytokine, such as IL-2, IL-7, IL-12 (including IL-12 p35, p40, p70, and / or p70 fusion constructs), IL-15, or IL-21. Each vaccine dose can be administered to a subject in combination with (e.g., simultaneously with, prior to, or after) a modified cytokine (e.g., pegIL-2).

[0335] The priming vaccine can be injected (e.g., intramuscularly) into the subject. Bilateral injections can be used for each dose. For example, one or more injections of ChAdV68 (C68) can be used (e.g., a total dose of 1×10 1210 virus particles), a low vaccine dose selected from the range of 0.001-1 ug of RNA, particularly one or more injections of 0.1 or 1 ug of samRNA vector, or a high vaccine dose selected from the range of 1-1000 ug of RNA, particularly one or more injections of 30 μg, 100 μg, or 300 μg of RNA, can be used. For ChAdV68 priming, 12 For ChAdV68 priming, up to 3 × 10 viral particles can be administered. 11 For ChAdV68 priming, at least 1 × 10 viral particles can be administered. 11 For ChAdV68 priming, 1 × 10 viral particles can be administered. 11 pieces~1×10 12 pieces, 3×10 11 ~1×10 12 Pieces or 1×10 11 pieces~3×10 11 For ChAdV68 priming, 1 × 10 viral particles can be administered. 11 pieces, 3×10 11 pcs or 1×10 12 For ChAdV68 priming, viral particles can be administered at 5×10 11 It can be at a concentration of vp / mL.

[0336] A vaccine boost (booster vaccine) can be injected (e.g., intramuscularly) after the prime vaccination. The booster vaccine can be administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the prime, e.g., every 4 weeks and / or every 8 weeks. Bilateral injections can be used for each dose. For example, one or more injections of ChAdV68 (C68) can be used (e.g., a total dose of 1×10 12100μg、10-30μg、10-100μg、10-300μg、30-100μg、30-300μg、100-300μg。 10-300μg, 10-1000μg, 10-3 ... A SAM booster of at least 400 μg, at least 500 μg, at least 600 μg, at least 700 μg, at least 800 μg, at least 900 μg, at least 1000 μg of RNA may be administered. A SAM booster of 10 μg, 30 μg, 100 μg, or 300 μg of RNA may be administered. A SAM booster of 300 μg of RNA may be administered. A SAM booster of 100 μg of RNA may be administered. A SAM booster of 30 μg of RNA may be administered. A SAM booster of 10 μg of RNA may be administered. A SAM booster of at least 300 μg of RNA may be administered. A SAM booster of at least 100 μg of RNA may be administered. A SAM booster of at least 30 μg of RNA may be administered. A SAM booster of at least 10 μg of RNA may be administered. A SAM booster of up to 300 μg of RNA can be administered.

[0337] Anti-CTLA-4 (e.g., tremelimumab) can also be administered to the subject. For example, anti-CTLA4 can be administered subcutaneously near the site of intramuscular vaccine injection (ChAdV68 prime or SamRNA low dose) to ensure delivery to the same lymph nodes. Tremelimumab is a selective human IgG2 mAb inhibitor of CTLA-4. Targeted anti-CTLA-4 (tremelimumab) subcutaneous doses are typically 70-75 mg (specifically 75 mg), e.g., in the dose range of 1-100 mg or 5-420 mg.

[0338] 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 typically administered intravenously at 20 mg / kg every 4 weeks.

[0339] Immune monitoring can be performed before, during, and / or after vaccine administration. Such monitoring can provide information about safety and efficacy, among other parameters.

[0340] PBMCs are generally used for immune monitoring. PBMCs can be isolated before and after prime vaccination (e.g., 4 weeks and 8 weeks). PBMCs can be collected immediately before and after each boost vaccination (e.g., 4 weeks and 8 weeks).

[0341] Immune responses, such as T cell responses 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, "stimulating an immune response" refers to any increase in an immune response, such as initiating an immune response (e.g., a priming vaccine that stimulates the initiation of an immune response in a naive subject) or enhancing an immune response (e.g., a booster vaccine that stimulates the enhancement of an immune response in a subject with a pre-existing immune response to an antigen, such as a pre-existing immune response initiated by a priming vaccine). T cell responses can be measured using one or more methods well known to those skilled in the art, such as 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 induction of cytokines, such as IFN-γ, 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 intracellularly or extracellularly captured cytokines such as IFN-γ using flow cytometry. Specific CD4 or CD8 T cell responses to vaccine-encoded epitopes can be monitored from PBMCs by measuring T cell populations expressing specific T cell receptors for the epitope / MHC class I complex using MHC multimer staining. Specific CD4 or CD8 T cell responses to vaccine-encoded epitopes can be monitored from PBMCs by measuring ex vivo proliferation of T cell populations following incorporation of 3H-thymidine, bromodeoxyuridine, and carboxyfluorescein diacetate succinimidyl ester (CFSE). Antigen recognition and lytic activity of PBMC-derived T cells specific for vaccine-encoded epitopes can be functionally assessed by chromium release assays or alternative colorimetric cytotoxicity assays.

[0342] B cell responses can be measured using one or more methods well known in the art, such as assays used to determine B cell differentiation (e.g., differentiation into plasma cells), B cell or plasma cell proliferation, B cell or plasma cell activation (e.g., increase in costimulatory markers such as CD80 or CD86), antibody class switching, and / or antibody production (e.g., ELISA). Antibodies can also be assessed for functionality.

[0343] The disease state of a subject can be monitored after administration of any of the vaccine compositions described herein. For example, the disease state can be monitored using cell-free DNA (cfDNA) (also referred to as circular tumor DNA "ctDNA") isolated from a subject. In addition, the effectiveness of a vaccine therapy can be monitored using cfDNA isolated from a subject. cfDNA monitoring can include the steps of: a. isolating or has been isolated from a subject; b. sequencing or has been sequenced the isolated cfDNA; c. determining or has been determined the frequency of one or more mutations in the cfDNA relative to the wild-type germline nucleic acid sequence of the subject; and d. assessing or has been assessed the disease state of the subject from step (c). Following the above step (c), the method can include the steps of: d. repeating steps (a)-(c) multiple times for a particular subject and comparing the frequency of one or more mutations determined in the multiple repeats; and f. assessing or has been assessed the disease state of the subject from step (d). The multiple repetitions can be performed at different times, such as a first repetition of steps (a)-(c) performed before administration of the vaccine composition, and a second repetition of steps (a)-(c) performed after administration of the vaccine composition. Step (c) can include comparing the frequency of the one or more mutations determined in the multiple repetitions, or comparing the frequency of the one or more mutations determined in the first repetition with the frequency of the one or more mutations determined in the second repetition. An increase in the frequency of the one or more mutations determined in the subsequent or second repetition can be evaluated as disease progression. A decrease in the frequency of the one or more mutations determined in the subsequent or second repetition can be evaluated as a response. In some embodiments, the response is a complete response (CR) or a partial response (PR). The therapy can be administered to the subject after the evaluation step, such as when evaluation of the frequency of the one or more mutations in the cfDNA indicates that the subject has the disease. In the cfDNA isolation step, centrifugation can be used to separate the cfDNA from the cells and cell debris.cfDNA can be isolated from whole blood, such as by separating the plasma layer, buffy coat, red blood. Sequencing of cfDNA can use next-generation sequencing (NGS), Sanger sequencing, duplex sequencing, whole exome sequencing, whole genome sequencing, de novo sequencing, phased sequencing, targeted amplicon sequencing, shotgun sequencing, or combinations thereof, and cfDNA can be enriched for one or more polynucleotide regions of interest (e.g., polynucleotides known or suspected to encode one or more mutations, coding regions, and / or polynucleotides of the tumor exome) prior to sequencing. Enriching cfDNA can include hybridizing one or more polynucleotide probes, which can be modified (e.g., biotinylated), to one or more polynucleotide regions of interest. Generally, any number of mutations can be monitored simultaneously or in parallel.

[0344] Response to treatment (i.e., treatment response) can be measured by radiological surveillance and / or molecular response, e.g., by monitoring neo-antigen ctDNA (e.g., variant allele frequency "VAF"), e.g., as described in Zhang et al. Cancer Discov. 2020;10:1842-1853, Parikh et al. Clin Cancer Res. 2020;26:1877-1885, and Vega et al. JCO Precision Oncology 2022;6:e2100372, or neo-antigen variant haploid genomic equivalents (variant hGE), e.g., as described in Palmer et al. Nat. Med. 2022;28:1619-1629 and Chabon et al. Nature 2020;580(7802): 245-251. Molecular response can be defined as a reduction in ctDNA relative to a reference ctDNA, for example, a reduction of ctDNA of 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more relative to a reference ctDNA. In some embodiments, molecular response is defined as a reduction of ctDNA of 30% or more relative to a reference ctDNA. In some embodiments, molecular response is defined as a reduction of ctDNA of 30% or more relative to a reference ctDNA, where the reduction in ctDNA occurs by 24 months, by 18 months, by 12 months, or by 6 months after treatment. In some embodiments, molecular response is defined as a reduction of ctDNA of 30% or more relative to a reference ctDNA, where the reduction in ctDNA occurs by 12 weeks, 8 weeks, 4 weeks, or 2 weeks after treatment. In other embodiments, molecular response is defined as a reduction of ctDNA of 50% or more relative to a reference ctDNA, where the reduction in ctDNA occurs by 24 months, by 18 months, by 12 months, or by 6 months after treatment. In other embodiments, a molecular response is defined as a 50% or greater decrease in ctDNA relative to a baseline ctDNA. In some embodiments, a molecular response is defined as a 50% or greater decrease in ctDNA relative to a baseline ctDNA, the decrease in ctDNA occurring by 12 weeks, 8 weeks, 4 weeks, or 2 weeks of treatment.Molecular responses can be observed during initial treatment with radiologically stable disease (SD) and / or in patients with radiologically progressive disease (PD).

[0345] VIII. Antigen Identification VIII.A Identification of candidate antigens Research models for NGS analysis of tumor and normal exomes and transcriptomes have been described previously and applied in the antigen-specific space. 6,14,15 Certain optimizations can be considered to improve the sensitivity and specificity of antigen identification in clinical situations.These optimizations can be divided into two areas: those related to laboratory processes and those related to NGS data analysis.Examples of optimization are well known to those skilled in the art, and such methods are described in more detail in, for example, International Patent Application Publication Nos. WO / 2017 / 106638, WO / 2018 / 195357 and WO2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.

[0346] VIII.B HLA peptide isolation and detection Isolation of HLA peptide molecules was performed using classical immunoprecipitation (IP) techniques after lysis and solubilization of tissue samples (55-58). The cleared lysates were used for HLA-specific IP.

[0347] Immunoprecipitation was performed using antibodies coupled to beads, where the antibody is specific for the HLA molecule. For pan-class I HLA immunoprecipitation, a pan-class I CR antibody is used, and for class II HLA-DR, an HLA-DR antibody is used. The antibody is covalently attached to the NHS-Sepharose beads during an overnight incubation. After covalent attachment, the beads were washed and aliquoted for IP (59,60). Immunoprecipitation can also be performed with antibodies that are not covalently attached to beads. Typically, this is done using Sepharose or magnetic beads coated with Protein A and / or Protein G to retain the antibody on the column. Below are some antibodies that can be used to selectively enrich for MHC / peptide complexes. TIFF2024534442000002.tif47161

[0348] The clarified tissue lysate is added to the antibody beads for immunoprecipitation. After immunoprecipitation, the beads are removed from the lysate and the lysate is saved for further experiments, including additional IPs. The IP beads are washed to remove non-specific binding and the HLA / peptide complexes are eluted from the beads using standard techniques. Protein components are removed from the peptides using molecular weight spin columns or C18 fractionation. The resulting peptides are dried by SpeedVac evaporation and in some cases stored at -20°C prior to MS analysis. HLA IP can also be performed in 96-well plates using plates containing filter bottoms. Using plates allows multiple IPs to be performed in tandem.

[0349] The dried peptides are reconstituted in an HPLC buffer suitable for reversed-phase chromatography and loaded onto a C-18 microcapillary HPLC column for gradient elution on a Fusion Lumos mass spectrometer (Thermo). MS1 spectra of peptide mass / charge (m / z) were collected at high resolution on an Orbitrap detector, followed by MS2 low-resolution scans collected on an ion trap detector after HCD fragmentation of selected ions. Additionally, MS2 spectra can be acquired using either CID or ETD fragmentation methods, or any combination of the three techniques to obtain greater amino acid coverage of the peptide. MS2 spectra can also be measured at high-resolution mass accuracy on an Orbitrap detector by a targeted method known as parallel reaction monitoring (PRM). In targeted PRM, specific peptide precursor ions are isolated in the Orbitrap detector, and all resulting HCD fragmentation ions are scanned throughout the elution of the peptide peak. This allows both peptide identification and quantification of endogenous peptides in the presence of co-injected stable isotope-labeled peptide standards.

[0350] MS2 spectra from each run are searched against protein databases using Comet (61,62) and peptide identifications are scored using Percolator (63-65). Further sequencing is performed using PEAKS studio (Bioinformatics Solutions Inc.) and other search engines, or sequencing methods including spectral matching and de novo sequencing (97) can be used.

[0351] VIII.B.1. MS Detection Limit Studies Supporting Comprehensive HLA Peptide Sequencing The peptide YVYVADVAAK (SEQ ID NO: 29364) was used to determine the limit of detection using different amounts of peptide loaded onto the LC column. The amounts of peptide tested were 1 pmol, 100 fmol, 10 fmol, 1 fmol, and 100 amol (Table 1). These results show that the lowest limit of detection (LoD) was in the attomolar range (10 -18 ), a dynamic range spanning five orders of magnitude, and a signal-to-noise ratio in the low femtomole range (10 -15 ) is considered sufficient for sequencing.

[0352] [Table 1]

[0353] IX. Presented Model Presentation models can be used to identify the likelihood of peptide presentation in patients.Different presentation models are known to those skilled in the art, and such presentation models are described in more detail in, for example, U.S. Patent No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1 and U.S. Patent Application Publication No. US20110293637, and International Patent Application Publication No. WO / 2018 / 195357, WO / 2018 / 208856, and WO2016187508, each of which is incorporated herein by reference in its entirety for all purposes.

[0354] X. Training Module The training module can be used to build one or more presentation models based on the training data set, which generate the likelihood that a peptide sequence will be presented by the MHC allele associated with the peptide sequence.Various training modules are known to those skilled in the art, and such presentation models are described in detail in, for example, U.S. Patent No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1, and International Patent Application Publication Nos. WO / 2018 / 195357 and WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.The training module can build a predictive model for predicting the presentation likelihood of a peptide on an allele-by-allele basis.The training module can also build a presentation model for predicting the presentation likelihood of a peptide in a multi-allele context where two or more MHC alleles are present.

[0355] XI. Prediction Module The prediction module can be used to receive sequence data and select candidate antigens in the sequence data using the proposed model. Specifically, the sequence data can be DNA sequences, RNA sequences, and / or protein sequences extracted from the patient's tumor tissue cells. The prediction module can identify candidate neo-antigens that are mutated peptide sequences by comparing the sequence data extracted from the patient's normal tissue cells with the sequence data extracted from the patient's tumor tissue cells to identify portions that contain one or more mutations. The prediction module can identify candidate antigens that have altered expression in tumor cells or cancer tissue compared to normal cells or tissues by comparing the sequence data extracted from the patient's normal tissue cells with the sequence data extracted from the patient's tumor tissue cells to identify candidate antigens that are inappropriately expressed.

[0356] The presentation module can apply one or more presentation models to the processed peptide sequences to estimate the presentation likelihood of the peptide sequences. Specifically, the prediction module can select one or more candidate antigen peptide sequences that are likely to be presented on the HLA molecules of the tumor by applying the presentation model to the candidate antigens. In one implementation, the presentation module selects the candidate antigen sequences that have an estimated presentation likelihood above a predetermined threshold. In another implementation, the presentation model selects the N candidate antigen sequences that have the highest estimated presentation likelihood (N is generally the maximum number of epitopes that can be delivered in the vaccine). The vaccine containing the selected candidate antigens for a particular patient can be injected into the subject to stimulate an immune response.

[0357] XI.B. Cassette Design Module XI.B.1 Overview The cassette design module can be used to generate vaccine cassette sequences based on the selected candidate peptides for injection into patients.Various cassette design modules are known to those skilled in the art, and such cassette design training modules are described in more detail in, for example, U.S. Patent No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1, and International Patent Application Publication Nos. WO / 2018 / 195357 and WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.

[0358] The set of therapeutic epitopes can be generated based on selected peptides determined by the prediction module that are associated with a presentation likelihood above a predefined threshold (the presentation likelihood being determined by the presentation model). However, in other embodiments, the set of therapeutic epitopes can be generated based on any one or more of a number of methods (single or in combination), such as, for example, based on binding affinity or predicted binding affinity to the patient's HLA class I or HLA class II alleles, binding stability or predicted binding stability to the patient's HLA class I or HLA class II alleles, random sampling, etc.

[0359] The therapeutic epitope may itself correspond to the selected peptide. The therapeutic epitope may also comprise C- and / or N-terminal flanking sequences in addition to the selected peptide. The N- and C-terminal flanking sequences may be the natural N- and C-terminal flanking sequences of the therapeutic vaccine epitope in the context of its source protein. The therapeutic epitope may represent an epitope of fixed length. The therapeutic epitope may represent an epitope of variable length, where the length of the epitope may vary, for example, depending on the length of the C- or N-terminal flanking sequences. For example, the C- and N-terminal flanking sequences may each have different lengths of 2-5 residues, giving 16 possible choices of epitopes.

[0360] The cassette design module can also generate cassette sequences by considering the representation of junction epitopes that span the junction between two therapeutic epitopes in the cassette. Junction epitopes are novel non-self but unrelated epitope sequences that arise within the cassette by the process of linking therapeutic epitopes and linker sequences within the cassette. The novel sequence of the junction epitope is different from the therapeutic epitopes of the cassette itself.

[0361] The cassette design module can generate cassette sequences that reduce the likelihood that the junction epitope will be presented in the patient. Specifically, when the cassette is injected into a patient, the junction epitope has the potential to be presented by the patient's HLA class I or HLA class II alleles, stimulating a CD8 or CD4 T cell response, respectively. Such responses are undesirable since T cells with reactivity against the junction epitope have no therapeutic effect, and may extinguish the immune response against the selected therapeutic epitope in the cassette due to antigen competition. 76 .

[0362] The cassette design module can iteratively process one or more candidate cassettes to determine cassette sequences for which the presentation score of the junction epitope associated with the cassette sequence is below a numerical threshold. The junction epitope presentation score is a quantity associated with the likelihood of presentation of the junction epitope in the cassette, with higher values ​​of the junction epitope presentation score indicating that the junction epitope of the cassette is more likely to be presented by HLA class I or HLA class II or both.

[0363] In one embodiment, the cassette design module can determine the cassette sequence associated with the lowest junction epitope presentation score among the candidate cassette sequences.

[0364] The cassette design module can iteratively process one or more candidate cassette sequences, determine a junction epitope presentation score for each candidate cassette, and identify an optimal cassette sequence associated with a junction epitope presentation score below a threshold.

[0365] The cassette design module can further check one or more candidate cassette sequences to identify whether any of the junction epitopes in the candidate cassette sequences are self-epitopes for the particular patient for whom the vaccine is being designed. To do this, the cassette design module checks the junction epitopes against a known database, such as BLAST. In one embodiment, the cassette design module can be configured to design cassettes that prevent junction self-epitopes.

[0366] The cassette design module can implement a brute force approach to iterate through all or most of the possible candidate cassette sequences to select the sequence with the minimum junction epitope presentation score. However, the number of such candidate cassettes can become prohibitively large as the vaccine volume becomes larger. For example, for a vaccine volume of 20 epitopes, the cassette design module may select approximately 10 18 possible candidate cassettes. This determination can be computationally intensive (in terms of required computing resources) and sometimes intractable for the cassette design module to generate a vaccine for a patient within a reasonable length of time. Furthermore, processing possible junction epitopes for each candidate cassette can be even more intensive. Therefore, the cassette design module can select cassette sequences based on a method that iterates through a number of candidate cassette sequences that is significantly smaller than the number of candidate cassette sequences in the brute force approach.

[0367] The cassette design module can generate randomly or at least pseudo-randomly generated candidate cassettes and select as cassette sequences those candidate cassettes associated with a junction epitope presentation score below a predefined threshold. Furthermore, the cassette design module can select as cassette sequences those candidate cassettes from the subset with the lowest junction epitope presentation score. For example, the cassette design module can generate a subset of about 1 million candidate cassettes for a set of 20 selected epitopes and select the candidate cassette with the lowest junction epitope presentation score. Although generating a subset of random cassette sequences and selecting from this subset those cassette sequences with low junction epitope presentation scores is suboptimal compared to the brute force approach, it requires significantly less computational resources and is therefore technically feasible to implement. Furthermore, performing brute force methods against this more efficient approach may only result in a slight or even negligible improvement in the junction epitope presentation score, making it less worthwhile in terms of resource allocation. The cassette design module can determine improved cassette configurations by formulating the cassette epitope sequences as an asymmetric traveling salesman problem (TSP). Given a list of nodes and the distance between each pair of nodes, the TSP determines the sequence of nodes associated with the minimum total distance to visit each node exactly once and return to the original node. For example, given cities A, B, and C with known distances between each other, a solution to the TSP generates a closed sequence of cities such that the total distance traveled to visit each city exactly once among the possible tours is minimized. An asymmetric version of the TSP determines the optimal sequence of nodes when the distances between pairs of nodes 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 asymmetric TSP, the cassette design module can find a cassette sequence that gives a low presentation score across the junction between each epitope of the cassette. The solution of the asymmetric TSP indicates the sequence of therapeutic epitopes corresponding to the order in which each epitope must be linked to minimize the junction epitope presentation score across each junction of the cassette. The cassette sequence determined by this approach may give sequences with significantly lower presentation of junction epitopes while requiring significantly less computational resources than the random sampling approach, especially when the number of candidate cassette sequences generated is large. Illustrative examples of the comparison of different computational approaches and optimized cassette designs are described in more detail in U.S. Pat. No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1, and International Patent Application Publication Nos. WO / 2018 / 195357 and WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.

[0368] XI.B.2 Shared antigen vaccine sequence selection The sequence of the shared antigen for inclusion in the shared antigen vaccine and the patients suitable for treatment with such a vaccine can be selected by the skilled artisan using the detailed disclosure provided herein. For example, Table: A, 1.2, additional MS recognized neo-antigens, or AACR GENIE results can be used for sequence selection. In certain cases, a combination of a particular mutation and HLA allele may be preferred (e.g., based on available sequencing data from a given subject showing that each is present in the subject), and then shared neo-antigen sequences can be identified using Table A, additional MS recognized neo-antigens, or AACR GENIE results for inclusion together in the vaccine for use in combination.

[0369] XIII. Exemplary Computer Any of the computational methods described herein can use a computer.Those skilled in the art will recognize that the computer can have different architectures.Examples of computers known to those skilled in the art are described in more detail in, for example, U.S. Patent No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1, and International Patent Application Publication Nos. WO / 2017 / 106638, WO / 2018 / 195357, and WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.

[0370] XIV. Clinical evaluation of neoantigen vaccines in adjuvant and maintenance therapy A personalized cancer vaccine encoding a neoepitope cassette (as described throughout this specification) is administered in combination with immune checkpoint blockade in patients with advanced cancer. The heterologous prime / boost vaccine regime involves (1) a ChAdV used as a prime vaccination, and (2) a SAM formulated in LNP used to boost the vaccination after the ChAdV vector. Both the ChAdV and SAM vectors encode the same personalized neoepitope cassette specific for each subject, which also encodes two universal CD4 T cell epitopes (PADRE and tetanus toxoid). To include subjects, tumors are used for whole exome and transcriptome sequencing to detect somatic mutations, and blood is used for HLA typing.

[0371] The ChAdV vector is a replication-deficient E1, E3, E4 open reading frame 2-4 (ORF2-4) deleted adenoviral vector based on the subgroup E adenovirus, chimpanzee adenovirus 68 (C68, 68 / SAdV-25, originally designated as Pan9) [ChAdV68-Empty-E4deleted; see SEQ ID NO: 29365, which represents SEQ ID NO: 1 with an E1 deletion (577-3403), an E3 deletion (27,125-31,825), and a partial E4 deletion spanning ORF2-4 (34,916-35,642)]. The ChAdV vector can be expressed in a volume of 5×10 11 It is formulated in solution at vp / mL and 1.0 mL is injected IM at two bilateral vaccine injection sites in each of the opposing deltoid muscles (the deltoid is preferred, the gluteal muscles [dorsal or ventral], or the rectus femoris on each side may be used).

[0372] The SAM vector (GRT-R902) is derived from an alphavirus. The SAM vector encodes viral proteins and 5' and 3' RNA sequences required for RNA amplification, but not structural proteins. The SAM vector is formulated in an LNP composed of four types of lipids: ionic amino lipid, phosphatidylcholine, cholesterol, and a PEG-based coat lipid, which encapsulates the SAM to form the LNP. The SAM vector contains the same neoantigen expression cassettes used in the ChAdV vector. The SAM vector is formulated in solution at mg / mL and injected IM at two bilateral vaccine injection sites in opposite deltoid muscles (deltoid is preferred, gluteal muscles [dorsal or ventral], or rectus femoris on each side may be used). The booster vaccination site is as close as possible to the prime vaccination site. The injection volume is based on the dose administered. The dose level volume refers explicitly to the amount of SAM vector, i.e., not other components such as the LNP. The LNP:SAM ratio is approximately 24:1.

[0373] Ipilimumab is a human monoclonal IgG1 antibody that binds to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). Ipilimumab is formulated in solution at 5 mg / mL and injected proximal (within approximately 2 cm) SC at each of the bilateral vaccination sites. The SC route of ipilimumab differs from the approved IV route of administration. Ipilimumab is administered at a dose of 30 mg in one of two ways: 1. Four 1.5 mL (7.5 mg) injections proximal to the vaccine-draining LN at each of the bilateral vaccination sites (i.e., 1.5 mL below the vaccination site and 1.5 mL above the vaccination site in each deltoid, ventral gluteal muscle of the lower back, dorsal gluteal muscle of the buttocks, or rectus femoris muscle on each side [deltoid muscle is preferred, but dependent on clinical site and patient preference]). 2. Six 1 mL (5 mg) injections proximal to the vaccine-draining LN at each of the bilateral vaccination sites (i.e., 1 mL below the vaccination site, 1 mL lateral to the vaccination site, and 1.5 mL below and above the vaccination site [deltoid preferred, but dependent on clinical site and patient preference] in each deltoid, ventral gluteal muscle of the lower back, dorsal gluteal muscle of the buttocks, or rectus femoris muscle on each side).

[0374] Nivolumab is a human monoclonal IgG4 antibody that blocks the interaction of PD-1 with its ligands PD-L1 and PD-L2. Nivolumab is formulated in solution at 10 mg / mL and administered as an IV infusion at the dose prescribed by protocol through a 0.2-1.2 micron pore size, low protein binding, in-line filter. It is not administered as an IV push or bolus injection. When the dose is fixed (e.g., 240 mg flat dose), nivolumab injections are infused undiluted or diluted so that the total infusion volume does not exceed 160 mL. Nivolumab infusion is followed immediately by flushing the diluent to clear the line. Nivolumab is administered after each vaccination (i.e., each of the SAM or ChAdV vaccines) with or without ipilimumab on the same day. The dose and route of nivolumab will be based on the dose and route approved by the Food and Drug Administration. The dose of nivolumab can be interrupted, delayed, or stopped depending on how well the participant tolerates the treatment. Administration visits will not be skipped, only delayed. Vaccination will not be performed without nivolumab unless the investigator and sponsor believe that it is in the patient's best interest to treat with the SAM vector without nivolumab. For example, atezolizumab or cemiplimab can be administered instead of nivolumab, according to the manufacturer's instructions and / or the appropriate measured dose as recognized by those skilled in the art. Other PD1 and / or PD-L1 checkpoint inhibitors may be used instead. For example, atezolizumab can be administered by intravenous injection at a dose of 1680 mg every 4 weeks.

[0375] A phase 2 study will be performed to characterize the clinical activity of maintenance therapy with fluoropyrimidine / bevacizumab and fluoropyrimidine / bevacizumab alone plus GRT-C901 / GRT-R902 in combination with checkpoint inhibitors, as assessed by changes in circulating tumor (ct) RNA. A phase 3 study will be performed to demonstrate the clinical efficacy of the treatment regimen, as assessed by progression-free survival. Tumors with nonsynonymous deoxyribonucleic acid (DNA) mutations can present peptides containing these mutations as self-antigens on the tumor cell surface, in the context of human leukocyte antigens (HLA). A fraction of the mutated peptides provide neo-antigens capable of generating T-cell responses that exclusively target tumor cells. Selective detection of these mutations allows for the identification of neo-antigens specific to each patient's tumor for inclusion in a personalized cancer vaccine that targets these neo-antigens. This vaccine regimen uses two vaccine vectors (first GRT-C901, followed by GRT-C902) in a heterologous prime / boost approach to stimulate immune responses. This study explores the antitumor activity of this patient-specific immunotherapy in combination with checkpoint inhibitors in addition to fluoropyrimidine / bevacizumab. Study arms are shown in Table 2. Schematic diagrams showing maintenance and adjuvant therapy are shown in Figures 1 and 2, respectively.

[0376] The study has two stages. In the vaccine production stage, neoantigen prediction will be performed using tumor biopsies and Gritstone's EDGE™ neoantigen production model while patients undergo FOLFOX / bev induction therapy. For patients randomized to the vaccine arm, the top 20 predicted neoantigens will be included in the vaccine vector. After completion of oxaliplatin, patients will enter the study treatment stage. Patients in the control arm will continue their maintenance therapy, while patients in the vaccine arm will add the vaccine regimen to their maintenance therapy. The vaccine regimen will consist of GRT-C901 / GRT-R902, plus SC ipilimumab (30 mg), and IV atezolizumab (1680 mg). Six vaccinations will occur over the first year of treatment. Ipilimumab will be administered SC along with the first dose of GRT-C901 and GRT-R902. Atezolizumab will be administered every 4 weeks for up to 2 years.

[0377] Inclusion criteria: - Patients with histologically confirmed metastatic colorectal cancer (CRC) who will receive or have received one or less cycles of first-line treatment in the metastatic setting with fluoropyrimidine and oxaliplatin in combination with bevacizumab - Measurable and unresectable disease per RECIST v1.1 -Availability of formalin-fixed paraffin-embedded (FFPE) tumor specimens. - Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, or equivalent, for patients aged 12 to 17 years - The patient has sufficient organ function in the opinion of the investigator - If female of childbearing potential (WCBP), must be willing to undergo pregnancy testing and agree to use highly effective contraception at least once during the treatment period and for 5 months after the last investigational study treatment.

[0378] Exclusion criteria: -Patients with microsatellite instability-high disease - Patient has a known tumor mutational burden of less than 1 nonsynonymous substitution / million bases -Known DNA polymerase ε mutation - Patients with a known BRAFV600E mutation - Bleeding disorder or history of significant purpura or bleeding after IM injection or transfusion - Anticipated immunosuppression at the time of study treatment - History of allogeneic tissue / solid organ transplant - Active or history of autoimmune disease or immune deficiency - Patient has symptomatic or actively progressing central nervous system (CNS) metastases, carcinoma meningitis, or has been treated with whole brain irradiation - History of other cancers within 2 years, except for tumors that have received potentially curative therapy - Any serious, concurrent non-cancer illness that, in the investigator's judgment, makes the patient unsuitable for the current study - Evidence of active tuberculosis or recent (<2 weeks), clinically significant infection, active hepatitis B or C; or known history of a positive test for HIV History of pneumonia (except for previously resolved in-field irradiation pneumonitis) requiring systemic steroids for treatment - History of myocardial infarction, unstable angina, serious uncontrolled cardiac arrhythmias, myocarditis, or congestive heart failure within the past 3 months or prior to any therapeutic procedure - Pregnant, planning to become pregnant, or nursing.

[0379] [Table 2] TIFF2024534442000005.tif47163

[0380] XV. Clinical evaluation of neo-antigen vaccines in combination with immune checkpoint blockade for patients with colorectal cancer Tumors with nonsynonymous deoxyribonucleic acid (DNA) mutations present peptides containing these mutations as self-antigens on the tumor cell surface, in the context of human leukocyte antigens (HLA). A fraction of mutated peptides result in neo-antigens capable of generating T-cell responses that exclusively target tumor cells. By selectively detecting these mutations, it becomes possible to identify neo-antigens specific to each patient's tumor for inclusion in a personalized cancer vaccine that targets these neo-antigens.

[0381] The vaccine regimen will be evaluated to stimulate immune responses using two vaccine vectors in a heterologous prime / boost approach (GRT-C901 as priming and GRT-R902 as booster). The study will explore the antitumor activity of this patient-specific immunotherapy in combination with checkpoint inhibitors. The study will evaluate and characterize the antitumor activity of adjuvant treatment with a vaccine regimen, commonly referred to as GRANITE (GRT-C901 / GRT-R902), in combination with checkpoint inhibitors based on molecular responses, and evaluate the safety and tolerability of GRT-C901 / GRT-R902 in combination with checkpoint blockade in patients with colorectal cancer who are circulating tumor deoxyribonucleic acid (ctDNA) positive after surgical resection.

[0382] A personalized cancer vaccine encoding a neoepitope cassette (as described throughout this specification) is administered in combination with immune checkpoint blockade in patients with advanced cancer. The heterologous prime / boost vaccine regime involves (1) a ChAdV used as a prime vaccination, and (2) a SAM formulated in LNP used to boost the vaccination after the ChAdV vector. Both the ChAdV and SAM vectors encode the same personalized neoepitope cassette specific for each subject, which also encodes two universal CD4 T cell epitopes (PADRE and tetanus toxoid). To include subjects, tumors are used for whole exome and transcriptome sequencing to detect somatic mutations, and blood is used for HLA typing.

[0383] The ChAdV vector is a replication-deficient E1, E3, E4 open reading frame 2-4 (ORF2-4) deleted adenoviral vector based on the subgroup E adenovirus, chimpanzee adenovirus 68 (C68, 68 / SAdV-25, originally designated as Pan9) [ChAdV68-Empty-E4deleted; see SEQ ID NO: 29365, which represents SEQ ID NO: 1 with an E1 deletion (577-3403), an E3 deletion (27,125-31,825), and a partial E4 deletion spanning ORF2-4 (34,916-35,642)]. The ChAdV vector can be expressed in a volume of 5×10 11 It is formulated in solution at vp / mL and 1.0 mL is injected IM at two bilateral vaccine injection sites in each of the opposing deltoid muscles (the deltoid is preferred, the gluteal muscles [dorsal or ventral], or the rectus femoris on each side may be used).

[0384] The SAM vector (GRT-R902) is derived from an alphavirus. The SAM vector encodes viral proteins and 5' and 3' RNA sequences required for RNA amplification, but not structural proteins. The SAM vector is formulated in an LNP composed of four types of lipids: ionic amino lipid, phosphatidylcholine, cholesterol, and a PEG-based coat lipid, which encapsulates the SAM to form the LNP. The SAM vector contains the same neoantigen expression cassettes used in the ChAdV vector. The SAM vector is formulated in solution at mg / mL and injected IM at two bilateral vaccine injection sites in opposite deltoid muscles (deltoid is preferred, gluteal muscles [dorsal or ventral], or rectus femoris on each side may be used). The booster vaccination site is as close as possible to the prime vaccination site. The injection volume is based on the dose administered. The dose level volume refers explicitly to the amount of SAM vector, i.e., not other components such as the LNP. The LNP:SAM ratio is approximately 24:1.

[0385] Ipilimumab is a human monoclonal IgG1 antibody that binds to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). Ipilimumab is formulated in solution at 5 mg / mL and injected proximal (within approximately 2 cm) SC at each of the bilateral vaccination sites. The SC route of ipilimumab differs from the approved IV route of administration. Ipilimumab is administered at a dose of 30 mg in one of two ways: 1. Four 1.5 mL (7.5 mg) injections proximal to the vaccine-draining LN at each of the bilateral vaccination sites (i.e., 1.5 mL below the vaccination site and 1.5 mL above the vaccination site in each deltoid, ventral gluteal muscle of the lower back, dorsal gluteal muscle of the buttocks, or rectus femoris muscle on each side [deltoid muscle is preferred, but dependent on clinical site and patient preference]). 2. Six 1 mL (5 mg) injections proximal to the vaccine-draining LN at each of the bilateral vaccination sites (i.e., 1 mL below the vaccination site, 1 mL lateral to the vaccination site, and 1.5 mL below and above the vaccination site [deltoid preferred, but dependent on clinical site and patient preference] in each deltoid, ventral gluteal muscle of the lower back, dorsal gluteal muscle of the buttocks, or rectus femoris muscle on each side).

[0386] Nivolumab is a human monoclonal IgG4 antibody that blocks the interaction of PD-1 with its ligands PD-L1 and PD-L2. Nivolumab is formulated in solution at 10 mg / mL and administered as an IV infusion at the dose prescribed by protocol through a 0.2-1.2 micron pore size, low protein binding, in-line filter. It is not administered as an IV push or bolus injection. When the dose is fixed (e.g., 240 mg flat dose), nivolumab injections are infused undiluted or diluted so that the total infusion volume does not exceed 160 mL. Nivolumab infusion is followed immediately by flushing the diluent to clear the line. Nivolumab is administered after each vaccination (i.e., each of the SAM or ChAdV vaccines) with or without ipilimumab on the same day. The dose and route of nivolumab will be based on the dose and route approved by the Food and Drug Administration. The dose of nivolumab can be interrupted, delayed, or stopped depending on how well the participant tolerates the treatment. Administration visits will not be skipped, only delayed. Vaccination will not be performed without nivolumab unless the investigator and sponsor believe that it is in the patient's best interest to treat with the SAM vector without nivolumab. For example, atezolizumab or cemiplimab can be administered instead of nivolumab, according to the manufacturer's instructions and / or the appropriate measured dose as recognized by those skilled in the art. Other PD1 and / or PD-L1 checkpoint inhibitors may be used instead. For example, atezolizumab can be administered by intravenous injection at a dose of 1680 mg every 4 w...

Claims

1. 1. A composition for use in a method for stimulating an immune response in a subject, the method comprising administering to said subject: (A) a composition for delivering a self-replicating alphavirus-based expression system, wherein the self-replicating alphavirus-based expression system encodes at least one antigen-encoding nucleic acid sequence, wherein the adjuvant therapy comprises combination therapy with chemotherapy, immune checkpoint inhibitor therapy, radiation therapy, or a combination thereof, optionally wherein the chemotherapy comprises a fluoropyrimidine and / or bevacizumab, and optionally wherein one or more booster doses of the composition for delivering the self-replicating alphavirus-based expression system are administered; or (B) a composition for delivering a chimpanzee adenovirus (ChAdV)-based expression system, wherein the ChAdV-based expression system is administered as an adjuvant therapy, wherein the expression system encodes at least one antigen-encoding nucleic acid sequence, wherein the adjuvant therapy comprises combination therapy with chemotherapy, immune checkpoint inhibitor therapy, radiation therapy, or a combination thereof, optionally wherein the chemotherapy comprises a fluoropyrimidine and / or bevacizumab; or (C) a composition for delivering a self-replicating alphavirus-based expression system, and for administering to said subject a composition for delivery of a chimpanzee adenovirus (ChAdV)-based expression system, comprising: the composition for delivering the ChAdV-based expression system is administered as a priming dose, and the composition for delivering the self-replicating alphavirus-based expression system is administered as one or more booster doses, the expression systems encoding at least one antigen-encoding nucleic acid sequence, and the self-replicating alphavirus-based expression system and the chimpanzee adenovirus (ChAdV)-based expression system are administered as maintenance or adjuvant therapy. The composition.

2. 2. The composition for use of claim 1, wherein the maintenance or adjuvant therapy comprises combination therapy with chemotherapy, immune checkpoint inhibitor therapy, radiation therapy, or a combination thereof, and optionally the chemotherapy comprises a fluoropyrimidine and / or bevacizumab.

3. the combination therapy comprising a fluoropyrimidine, bevacizumab, and an immune checkpoint inhibitor therapy, and optionally (A) The immune checkpoint inhibitor is (1) an anti-PD-1 antibody or an antigen-binding fragment thereof; (2) an anti-PD-L1 antibody or antigen-binding fragment thereof; optionally, the immune checkpoint inhibitor therapy comprises administration of an anti-PD-L1 antibody or antigen-binding fragment thereof every four weeks (Q4W), optionally wherein the anti-PD-L1 antibody comprises atezolizumab or nivolumab, optionally wherein the atezolizumab is administered intravenously at a dose of 1680 mg or the nivolumab is administered intravenously at a dose of 480 mg; and / or (3) an anti-CTLA-4 antibody or antigen-binding fragment thereof; optionally, the immune checkpoint inhibitor therapy comprises administration of an anti-CTLA-4 antibody or antigen-binding fragment thereof with only the priming dose and the first booster dose, optionally, the anti-CTLA-4 antibody comprises ipilimumab, and optionally, the ipilimumab is administered subcutaneously at a dose of 30 mg; and / or (B) the immune checkpoint inhibitor therapy (1) at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 administrations, optionally wherein the administration of the anti-PD-L1 antibody or antigen-binding fragment thereof comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 administrations; or (2) at least 13 administrations, optionally wherein the administration of the anti-PD-L1 antibody or antigen-binding fragment thereof comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 administrations; A composition for use according to claim 2.

4. The object is (A) has previously received surgery, chemotherapy, immunotherapy, immune checkpoint inhibitor therapy, radiation therapy, or a combination thereof to remove tumor and / or cancerous tissue, optionally wherein the previous chemotherapy included oxaliplatin, fluoropyrimidine, and / or bevacizumab, and optionally wherein the previous chemotherapy was administered for up to 24 weeks prior to administration of said maintenance therapy; and / or (B) has colorectal cancer (CRC), optionally wherein the CRC is classified as stage IV, microsatellite stable, and BRAF wt , optionally wherein the CRC is classified as stage II or III; and / or (C) classified as ctDNA positive; A composition for use according to claim 1.

5. two or more booster doses are administered, or one, two, three, four, five, six, seven, or eight booster doses are administered; optionally, the self-replicating alphavirus-based expression system is administered as at least two booster doses; optionally, the two or more booster doses are administered at least 28 days apart, at least four weeks apart (Q4W), at least one month apart, at least 56 days apart, at least eight weeks apart (Q8W), at least 8 weeks apart, at or about 28 and 84 days after the priming dose of the ChAdV-based expression system, at or about 4 and 12 weeks after the priming dose of the ChAdV-based expression system, and / or at or about 1 and 3 months after the priming dose of the ChAdV-based expression system. A composition for use according to claim 1.

6. the ChAdV-based expression system is further administered as a booster dose, optionally, the ChAdV-based booster dose is only administered as a single booster dose; and optionally, (A) the ChAdV-based expression system is administered as the booster dose at or about day 140 after the priming dose of the ChAdV-based expression system; (B) the ChAdV-based expression system is administered as the booster dose at or about 20 weeks after the priming dose of the ChAdV-based expression system; (C) the ChAdV-based expression system is administered as the booster dose at or about 5 months after the priming dose of the ChAdV-based expression system; (D) the ChAdV-based expression system is administered as the booster dose at or after 140 days following the priming dose of the ChAdV-based expression system; (E) the ChAdV-based expression system is administered as the booster dose at or after 20 weeks following the priming dose of the ChAdV-based expression system; or (F) the ChAdV-based expression system is administered as the booster dose at or after 5 months following the priming dose of the ChAdV-based expression system; A composition for use according to claim 1.

7. the composition for delivering the ChAdV-based expression system and / or the self-replicating alphavirus-based expression system is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV); optionally, the composition for delivering the ChAdV-based expression system and / or the self-replicating alphavirus-based expression system is administered IM; optionally, the IM administration is performed at separate injection sites; optionally, the separate injection sites are in opposing deltoid muscles, or the separate injection sites are in bilateral gluteus or rectus femoris muscle sites; optionally, the injection site of the one or more booster doses is as close as possible to the injection site of the priming dose; A composition for use according to claim 1.

8. the self-replicating alphavirus-based expression system is administered as at least four booster doses, and optionally (A) the self-replicating alphavirus-based expression system is administered at or about days 28, 84, 224, and 308 relative to the priming dose of the ChAdV-based expression system; (B) the self-replicating alphavirus-based expression system is administered at or about weeks 4, 12, 32, and 44 relative to the priming dose of the ChAdV-based expression system; or (C) the self-replicating alphavirus-based expression system is administered at or about months 1, 3, 8, and 11 relative to the priming dose of the ChAdV-based expression system. A composition for use according to claim 1.

9. further comprising determining or having determined the HLA haplotype of said subject; and / or wherein said stimulating said immune response comprises stimulating a molecular response, optionally wherein said molecular response comprises a reduction in ctDNA, optionally wherein said reduction in ctDNA comprises at least a 20%, at least a 30%, at least a 40%, or at least a 50% reduction in ctDNA, optionally wherein said reduction in ctDNA is at least a 30% or at least a 50% reduction in ctDNA; and / or wherein the at least one antigen-encoding nucleic acid sequence comprises a subject-specific neoantigen-encoding nucleic acid sequence, and / or the at least one antigen-encoding nucleic acid sequence comprises 20 subject-specific neoantigen-encoding nucleic acid sequences; A composition for use according to claim 1.

10. A composition for use according to claim 1, which is: (1) The composition for delivering the self-replicating alphavirus-based expression system comprises: (1A) The self-replicating alphavirus-based expression system comprises one or more vectors, (a)(i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; and an RNA alphavirus backbone comprising: (b) a cassette, (i) a. at least one change that causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence; an epitope-encoding nucleic acid sequence optionally comprising: b. optionally, a 5' linker sequence, and c. optionally, a 3' linker sequence and at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the at least one antigen-encoding nucleic acid sequence; and (iii) optionally, at least one second poly(A) sequence, wherein said second poly(A) sequence is a poly(A) sequence native to said alphavirus or an exogenous poly(A) sequence; and The cassette comprising: the self-replicating alphavirus-based expression system comprising one or more vectors comprising: (1B) a lipid nanoparticle (LNP) encapsulating the self-replicating alphavirus-based expression system; and Including, Optionally, the self-replicating alphavirus-based expression system comprises: Alphavirus backbone containing Venezuelan equine encephalitis virus comprising the sequence of SEQ ID NO:3 or SEQ ID NO:5, except that it lacks nucletics 7544 and 11175. Including, Optionally, the RNA alphavirus backbone comprises the sequence set forth in SEQ ID NO:6 or SEQ ID NO:7, and optionally, the cassette is inserted at position 7544 to replace the deletion between base pairs 7544 and 11175 set forth in the sequence of SEQ ID NO:3 or SEQ ID NO:5; and / or (2) Wherein the ChAdV vector is (2A)(i) at least one promoter nucleotide sequence; (ii) at least one polyadenylation (poly(A)) sequence; and a ChAdV backbone comprising: (2B) A cassette, (i) a. at least one change that causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence; an epitope-encoding nucleic acid sequence optionally comprising: b. optionally, a 5' linker sequence; c. optionally, a 3' linker sequence at least one antigen-encoding nucleic acid sequence comprising The cassette comprising: Including, the cassette is operably linked to the at least one promoter nucleotide sequence and the at least one poly(A) sequence; optionally, the cassette of the ChAdV vector is identical to the cassette of one or more vectors of the composition for delivering the self-replicating alphavirus-based expression system; Optionally, the ChAdV-based expression system comprises a ChAdV68 vector backbone; The ChAdV68 vector backbone (1) nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, corresponding to the E1 deletion; (2) nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO: 1, which corresponds to the E3 deletion; and optionally (3) nucleotides 34,916 to 35,642, corresponding to the partially deleted E4 gene of ChAdV68; comprising at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1, except that it lacks Optionally, the cassette is inserted into the ChAdV backbone in the E1 region, E3 region, and / or any deleted AdV region that allows integration of the cassette. A composition for use according to claim 1.

11. 2. The composition for use of claim 1, wherein for each of said doses, the cassette of said composition for delivering said self-replicating alphavirus-based expression system is identical.

12. 2. The composition for use of claim 1, wherein the epitope-encoding nucleic acid sequence contains at least one alteration that causes the encoded epitope to: (A) increasing the binding affinity of the encoded epitope to its corresponding MHC allele compared to the translated corresponding wild-type nucleic acid sequence; (B) increasing the binding stability of the encoded epitope to its corresponding MHC allele compared to the translated corresponding wild-type nucleic acid sequence; and / or (C) increasing the likelihood of presentation of the encoded epitope on its corresponding MHC allele, compared to the translated corresponding wild-type nucleic acid sequence; Optionally, 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.

13. the epitope-encoding nucleic acid sequence encodes an epitope known or suspected to be expressed in subjects known or suspected to have cancer; optionally, the cancer comprises a solid tumor, and optionally, the cancer is selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, bladder cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, adult acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer; and / or wherein one or more of the epitope-encoding nucleic acid sequences are derived from a tumor of the subject or are not derived from a tumor of the subject. Optionally, the epitope-encoding nucleic acid sequence comprises an epitope selected from the group consisting of SEQ ID NOs: 57-29,364. A composition for use according to claim 1.

14. the at least one antigen-encoding nucleic acid sequence is (A) at least 2 to 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 different antigen-encoding nucleic acid sequence; (B) 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 different antigen-encoding nucleic acid sequence; and / or (C) comprising 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 claim 1.

15. The cassette comprises: (A) a junction epitope sequence formed by adjacent sequences in the cassette; At least one or each junction epitope sequence has an affinity for MHC greater than 500 nM and / or each junction epitope sequence is non-self; and / or (B) does not encode a non-therapeutic MHC class I or MHC class II epitope nucleic acid sequence comprising a translated wild-type nucleic acid sequence, wherein the non-therapeutic epitope is predicted to be presented on an MHC allele of the subject; optionally, the predicted non-therapeutic MHC class I or MHC class II epitope sequence is a junction epitope sequence formed by adjacent sequences within the cassette; and optionally, the prediction is based on a presentation likelihood generated by inputting the sequence of the non-therapeutic epitope into a presentation model. A composition for use according to claim 1.

16. The composition for use described in claim 1, wherein the composition for delivering the ChAdV-based expression system and / or the self-replicating alphavirus-based expression system is formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

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