Alphavirus neoantigen vectors and interferon inhibitors
Disclosed herein are vectors that include alphavirus-based expression platforms. Also disclosed are methods associated with the alphavirus-based expression platforms and co-administration of an inhibitor of Type I interferon signaling.
1 . A method for stimulating an immune response in a subject, the method comprising administering to the subject a composition for delivery of an expression system and administering to the subject an inhibitor of Type I interferon signaling,
wherein the composition for delivery of the expression system comprises the expression system,
wherein the expression system comprises one or more vectors,
the one or more vectors comprising:
(a) an RNA alphavirus backbone, wherein the RNA alphavirus backbone comprises the nucleic acid sequence set forth in SEQ ID NO: 6, comprising:
(i) at least one promoter nucleotide sequence, and
(ii) at least one polyadenylation (poly(A)) sequence; and
(b) a cassette, wherein the cassette comprises:
(i) at least one nucleic acid sequence, optionally wherein the at least one nucleic acid sequence comprises a polypeptide-encoding nucleic acid sequence, optionally wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence comprising:
a. an epitope-encoding nucleic acid sequence, optionally comprising at least one alteration that makes the encoded epitope sequence distinct from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence,
b. optionally a 5′ linker sequence, and
c. optionally a 3′ linker sequence;
(ii) optionally, a second promoter nucleotide sequence operably linked to the at least one nucleic acid sequence; and
(iii) optionally, at least one second poly(A) sequence, wherein the second poly(A) sequence is a native poly(A) sequence or an exogenous poly(A) sequence to the alphavirus.
2 . A method for stimulating a tumor specific immune response in a subject, the method comprising administering to the subject a composition for delivery of an expression system and administering to the subject an inhibitor of Type I interferon signaling,
wherein the composition for delivery of the expression system comprises the expression system,
wherein the expression system comprises one or more vectors,
the one or more vectors comprising:
(a) an RNA alphavirus backbone, wherein the RNA alphavirus backbone comprises the nucleic acid sequence set forth in SEQ ID NO: 6, comprising:
(i) at least one promoter nucleotide sequence, and
(ii) at least one polyadenylation (poly(A)) sequence; and
(b) a cassette, wherein the cassette comprises:
(i) at least one nucleic acid sequence, optionally wherein the at least one nucleic acid sequence comprises a polypeptide-encoding nucleic acid sequence,
optionally wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence comprising:
a. an epitope-encoding nucleic acid sequence, optionally comprising at least one alteration that makes the encoded epitope sequence distinct from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence,
b. optionally a 5′ linker sequence, and
c. optionally a 3′ linker sequence;
(ii) optionally, a second promoter nucleotide sequence operably linked to the at least one nucleic acid sequence; and
(iii) optionally, at least one second poly(A) sequence, wherein the second poly(A) sequence is a native poly(A) sequence or an exogenous poly(A) sequence to the alphavirus.
3 . A method of enhancing delivery of an alphavirus-based expression system, the method comprising administering to the subject a composition for delivery of an expression system and administering to the subject an inhibitor of Type I interferon signaling,
wherein the composition for delivery of the expression system comprises the expression system,
wherein the expression system comprises one or more vectors,
the one or more vectors comprising:
(a) an RNA alphavirus backbone, wherein the RNA alphavirus backbone comprises the nucleic acid sequence set forth in SEQ ID NO: 6, comprising:
(i) at least one promoter nucleotide sequence, and
(ii) at least one polyadenylation (poly(A)) sequence; and
(b) a cassette, wherein the cassette comprises:
(i) at least one nucleic acid sequence, optionally wherein the at least one nucleic acid sequence comprises a polypeptide-encoding nucleic acid sequence, optionally wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence comprising:
a. an epitope-encoding nucleic acid sequence, optionally comprising at least one alteration that makes the encoded epitope sequence distinct from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence,
b. optionally a 5′ linker sequence, and
c. optionally a 3′ linker sequence;
(ii) optionally, a second promoter nucleotide sequence operably linked to the at least one nucleic acid sequence; and
(iii) optionally, at least one second poly(A) sequence, wherein the second poly(A) sequence is a native poly(A) sequence or an exogenous poly(A) sequence to the alphavirus.
4 . The method of claim 1 , wherein:
(a) the RNA alphavirus backbone sequence comprises a 26S promoter nucleotide sequence and a poly(A) sequence, wherein the 26S promoter sequence is endogenous to the RNA alphavirus backbone, and wherein the poly(A) sequence is endogenous to the RNA alphavirus backbone; and
(b) the cassette is integrated between the 26S promoter nucleotide sequence and the poly(A) sequence, wherein the cassette is operably linked to the 26S promoter nucleotide sequence; and
wherein the inhibitor of Type I interferon signaling comprises an anti-IFNαβ receptor (IFNAR) blocking antibody.
5 . The method of claim 1 , wherein:
the antigen-encoding nucleic acid sequence encodes a polypeptide sequence capable of undergoing antigen processing into the encoded epitope, and/or the epitope-encoding nucleic acid sequence encodes an epitope known or suspected to be presented by MHC class I on a surface of a cell, optionally wherein the surface of the cell is a tumor cell surface or an infected cell surface, and optionally wherein the cell is the subject's cell, optionally wherein 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 myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer, or
optionally wherein the cell is an infected cell selected from the group consisting of: a pathogen infected cell, a virally infected cell optionally an HIV infected cell, a bacterially infected cell, a fungally infected cell, and a parasitically infected cell; or
the polypeptide-encoding nucleic acid sequence encodes a full-length protein or functional portion thereof, optionally wherein the full-length protein or functional portion thereof is selected from the group consisting of: an antibody, a cytokine, a chimeric antigen receptor (CAR), a T-cell receptor, and a genome-editing system nuclease; or
the at least one nucleic acid sequence comprises a non-coding nucleic acid sequence, optionally wherein the non-coding nucleic acid sequence is an RNA interference (RNAi) polynucleotide or genome-editing system polynucleotide.
6 . The method of claim 1 , wherein the cassette comprises:
i) the at least one nucleic acid sequence comprising the polypeptide-encoding nucleic acid sequence, wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence comprising:
a. an epitope-encoding nucleic acid sequence, optionally comprising at least one alteration that makes the encoded epitope sequence distinct from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence
b. optionally a 5′ linker sequence, and
c. optionally a 3′ linker sequence;
(ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence;
(iii) optionally, at least one MHC class II epitope-encoding nucleic acid sequence;
(iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and
(v) optionally, at least one second poly(A) sequence, wherein the second poly(A) sequence is a native poly(A) sequence or an exogenous poly(A) sequence to the alphavirus;
optionally wherein an ordered sequence of each element of the cassette is described in the formula, from 5′ to 3′, comprising
Pa-(L5b-Nc-L3d)X-(G5e-Uf)Y-G3g
wherein P comprises the second promoter nucleotide sequence, where a=0 or 1,
N comprises one of the epitope-encoding nucleic acid sequences, wherein the epitope-encoding nucleic acid sequence comprises an MHC class I epitope-encoding nucleic acid sequence, where c=1,
L5 comprises the 5′ linker sequence, where b=0 or 1,
L3 comprises the 3′ linker sequence, where d=0 or 1,
G5 comprises one of the at least one nucleic acid sequences encoding a GPGPG amino acid linker, where e=0 or 1,
G3 comprises one of the at least one nucleic acid sequences encoding a GPGPG amino acid linker, where g=0 or 1,
U comprises one of the at least one MHC class II epitope-encoding nucleic acid sequence, where f=1,
X=1 to 400, where for each X the corresponding Nc is an epitope-encoding nucleic acid sequence, optionally wherein for each X the corresponding Nc is a distinct MHC class I epitope-encoding nucleic acid sequence, and
Y=0, 1, or 2, where for each Y the corresponding U f is an epitope-encoding nucleic acid sequence, optionally wherein for each Y the corresponding U f is a distinct MHC class II epitope-encoding nucleic acid sequence.
7 . The method of claim 6 , wherein
a=0, b=1, d=1, e=1, g=1, X=20, Y=2,
the at least one promoter nucleotide sequence is a single 26S promoter nucleotide sequence provided by the RNA alphavirus backbone,
the at least one polyadenylation poly(A) sequence is a poly(A) sequence of at least 100 consecutive A nucleotides provided by the RNA alphavirus backbone,
each N encodes a MHC class I epitope 7-15 amino acids in length,
L5 is a native 5′ linker sequence that encodes a native N-terminal amino acid sequence of the MHC I epitope, and wherein the 5′ linker sequence encodes a peptide that is at least 2 amino acids in length,
L3 is a native 3′ linker sequence that encodes a native C-terminal amino acid sequence of the MHC I epitope, and wherein the 3′ linker sequence encodes a peptide that is at least 2 amino acids in length,
U is each of a PADRE class II sequence and a Tetanus toxoid MHC class II sequence,
each of the MHC class I epitope-encoding nucleic acid sequences encodes a polypeptide that is between 13 and 25 amino acids in length.
8 . The method of claim 1 , wherein:
the composition for delivery of the expression system further comprises a nanoparticulate delivery vehicle that optionally encapsulates the expression system and optionally has a diameter of about 100 nm,
optionally wherein the nanoparticulate delivery vehicle is a lipid nanoparticle (LNP), optionally wherein the LNP comprises ionizable amino lipids, optionally wherein the ionizable amino lipids comprise MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecules; and/or
the inhibitor of Type I interferon signaling is selected from the group consisting of: an IFNα inhibitor, an IFNβ inhibitor, an IFNAR inhibitor, and a Type I interferon signaling pathway inhibitor, optionally wherein the inhibitor of Type I interferon signaling is selected from the group consisting of:
an antibody or an antigen-binding fragment thereof, a small molecule inhibitor, a RNAi polynucleotide, a genome-editing system, and an Fc-fusion protein, optionally wherein the antibody is selected from the group consisting of: an anti-IFNα antibody, an anti-IFNβ antibody, an anti-IFNαβ receptor (IFNAR) blocking antibody, optionally wherein the anti-IFNα antibody is selected from the group consisting of: Sifalimumab, Rontalizumab, and ASG-009, or optionally wherein the anti-IFNAR blocking antibody is selected from the group consisting of: MAR1-5A3, Anifrolumab, AmS3A5-1, 64G12, H2K6, H2K1, H3K6, H3K1 3F11, 4G5, 11E2, and 9D4, or
wherein the Type I interferon signaling pathway inhibitor comprises a JAK kinase inhibitor optionally a JAK1/2 inhibitor or a JAK1/3 inhibitor, optionally wherein the JAK kinase inhibitor comprises a small molecule, optionally wherein the JAK1/3 inhibitor is Tofacitinib; and/or
the inhibitor of Type I interferon signaling is administered before, concurrently with, or after administration of the composition for delivery of the expression system,
optionally wherein:
the inhibitor of Type I interferon signaling is administered 24 hours or less before administration of the composition for delivery of the expression system, or
the inhibitor of Type I interferon signaling is administered less than 12 hours after administration of the composition for delivery of the expression system, or
the inhibitor of Type I interferon signaling is administered 6 hours or less after administration of the composition for delivery of the expression system, or
the inhibitor of Type I interferon signaling is administered between 24 hours before and 6 hours or less after administration of the composition for delivery of the expression system;
and/or optionally wherein:
the composition for delivery of the expression system is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV); and/or
the inhibitor of Type I interferon signaling is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV); and/or
a single administration of the inhibitor of Type I interferon signaling is administered.
9 . The method of claim 1 , wherein:
the cassette is integrated between the at least one promoter nucleotide sequence and the at least one poly(A) sequence; and/or
the at least one promoter nucleotide sequence is operably linked to the cassette; and/or
the one or more vectors are self-replicating within a mammalian cell; and/or
the RNA alphavirus backbone comprises at least sequences for nonstructural protein-mediated amplification, a 26S promoter sequence, a poly(A) sequence, a nonstructural protein 1 (nsP1) gene, a nsP2 gene, a nsP3 gene, and a nsP4 gene encoded by the nucleotide sequence of the Venezuelan equine encephalitis virus, or
the RNA alphavirus backbone comprises at least sequences for nonstructural protein-mediated amplification, a 26S promoter sequence, and a poly(A) sequence encoded by the nucleotide sequence of the Venezuelan equine encephalitis virus,
optionally wherein 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 combinations thereof, and/or wherein the RNA alphavirus backbone does not encode structural virion proteins capsid, E2 and E1 optionally wherein the cassette is inserted in place of structural virion proteins within the nucleotide sequence of the Venezuelan equine encephalitis virus, optionally wherein the insertion of the cassette provides for 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; and/or
the at least one promoter nucleotide sequence is the native 26S promoter nucleotide sequence encoded by the RNA alphavirus backbone or is an exogenous RNA promoter; and/or
wherein the second promoter nucleotide sequence is a 26S promoter nucleotide sequence or the second promoter nucleotide sequence comprises multiple 26S promoter nucleotide sequences, wherein each 26S promoter nucleotide sequence provides for transcription of one or more of the separate open reading frames.
10 . The method of claim 1 , wherein:
the one or more vectors are each at least 300 nt in size, each at least 1 kb in size, each 2 kb in size and/or each less than 5 kb in size; and/or
at least one of the epitope-encoding nucleic acid sequences encodes an epitope that, when expressed and translated, is capable of being presented by MHC class I on a cell of the subject; and/or
at least one of the epitope-encoding nucleic acid sequences encodes an epitope that, when expressed and translated, is capable of being presented by MHC class II on a cell of the subject.
11 . The method of claim 1 , wherein:
the at least one nucleic acid sequence comprises two or more nucleic acid sequences, or the at least one nucleic acid sequence comprises two or more polypeptide-encoding nucleic acid sequences optionally wherein each polypeptide-encoding nucleic acid sequence is linked directly to one another; and/or
each polypeptide-encoding nucleic acid sequence is linked to a distinct polypeptide-encoding nucleic acid sequence with a nucleic acid sequence encoding a linker, optionally
wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence, and wherein the linker links two MHC class I epitope-encoding nucleic acid sequences or an MHC class I epitope-encoding nucleic acid sequence to an MHC class II epitope-encoding nucleic acid sequence, optionally wherein the linker is selected from the group consisting of: (1) consecutive glycine residues, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues in length; (2) consecutive alanine residues, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues in length; (3) two arginine residues (RR); (4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length that is processed efficiently by a mammalian proteasome; and (6) one or more native sequences flanking the antigen derived from the cognate protein of origin and that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length, or
wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence, and wherein the linker links two MHC class II epitope-encoding nucleic acid sequences or an MHC class II sequence to an MHC class I epitope-encoding nucleic acid sequence, optionally wherein the linker comprises the sequence GPGPG (SEQ ID NO: 56); and/or
the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence, and wherein the antigen-encoding nucleic acid sequences is linked, operably or directly, to a separate or contiguous sequence that enhances the expression, stability, cell trafficking, processing and presentation, and/or immunogenicity of the antigen-encoding nucleic acid sequence,
optionally wherein the separate or contiguous sequence comprises at least one of: a ubiquitin sequence, a ubiquitin sequence modified to increase proteasome targeting, an immunoglobulin signal sequence, a major histocompatibility class I sequence, lysosomal-associated membrane protein (LAMP)-1, human dendritic cell lysosomal-associated membrane protein, and a major histocompatibility class II sequence; optionally wherein the ubiquitin sequence modified to increase proteasome targeting is A76.
12 . The method of claim 1 , wherein:
the epitope-encoding nucleic acid sequence comprises at least one alteration that makes the encoded epitope have increased binding affinity to its corresponding MHC allele relative to the translated, corresponding wild-type nucleic acid sequence; and/or
the epitope-encoding nucleic acid sequence comprises at least one alteration that makes the encoded epitope have increased binding stability to its corresponding MHC allele relative to the translated, corresponding wild-type nucleic acid sequence; and/or
the epitope-encoding nucleic acid sequence comprises at least one alteration that makes the encoded epitope have an increased likelihood of presentation on its corresponding MHC allele relative to the translated, corresponding wild-type nucleic acid sequence;
and/or 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 spliced antigen; and/or
the subject is known or suspected to have cancer, optionally wherein stimulating the immune response treats the cancer and/or wherein 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 myelogenous leukemia, adult acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer; and/or
wherein the subject has one or more tumors, optionally wherein stimulating the immune response reduces tumor volume of the one or more tumors.
13 . The method of claim 1 , wherein:
the at least one nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleic acid sequences, optionally wherein each nucleic acid sequence encodes a distinct non-coding nucleic acid sequence, a distinct polypeptide-encoding nucleic acid sequence, or a combination thereof; or
the at least one nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or up to 400 nucleic acid sequences, optionally wherein each nucleic acid sequence encodes a distinct non-coding nucleic acid sequence, a distinct polypeptide-encoding nucleic acid sequence, or a combination thereof; or
the at least one nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptide-encoding nucleic acid sequences; or
the at least one 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 polypeptide-encoding nucleic acid sequences; or
the at least one nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen-encoding nucleic acid sequences; or
the at least one 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; or
the at least one nucleic acid sequence comprises at least 2-400 antigen-encoding nucleic acid sequences and wherein at least two of the antigen-encoding nucleic acid sequences encode polypeptide sequences or portions thereof that are presented by MHC class I on a cell surface.
14 . The method of claim 1 , wherein:
when administered to the subject and translated, at least one of the epitopes encoded by the epitope-encoding nucleic acid sequence are presented on antigen presenting cells resulting in an immune response targeting a cell presenting at least one of the epitopes on the cell surface; and/or
the epitope-encoding nucleic acid sequences comprises at least one MHC class I epitope-encoding nucleic acid sequence or MHC class II epitope-encoding nucleic acid sequence, and, when administered to the subject and translated, at least one of the MHC class I or class II epitopes are presented on antigen presenting cells resulting in an immune response targeting a cell presenting at least one of the epitopes on the cell surface, and optionally wherein the expression of each of the MHC class I and/or class II epitope-encoding nucleic acid sequences is driven by the at least one promoter nucleotide sequence.
15 . The method of claim 1 , wherein the epitope-encoding nucleic acid sequences comprises at least one MHC class I epitope-encoding nucleic acid sequence, and wherein each antigen-encoding nucleic acid sequence encodes a polypeptide sequence between 8 and 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; and/or
the at least one MHC class II epitope-encoding nucleic acid sequence is present, optionally wherein the at least one MHC class II epitope-encoding nucleic acid sequence is present and comprises at least one MHC class II epitope-encoding nucleic acid sequence that comprises at least one alteration that makes the encoded epitope sequence distinct from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence; and/or
the epitope-encoding nucleic acid sequence comprises an MHC class II epitope-encoding nucleic acid sequence and wherein each antigen-encoding nucleic acid sequence encodes a polypeptide sequence that is 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length; and/or
the epitope-encoding nucleic acid sequences comprises an MHC class II epitope-encoding nucleic acid sequence, wherein the 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, optionally wherein the at least one universal sequence comprises at least one of Tetanus toxoid and PADRE; and/or
the at least one promoter nucleotide sequence or the second promoter nucleotide sequence is inducible or non-inducible; and/or
the at least one poly(A) sequence comprises a poly(A) sequence native to the alphavirus or exogenous to the alphavirus; and/or
the at least one poly(A) sequence is operably linked to at least one of the at least one nucleic acid sequences; and/or
wherein the at least one poly(A) sequence is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 consecutive A nucleotides, or is at least 100 consecutive A nucleotides.
16 . The method of claim 1 , wherein:
the cassette further comprises at least one of: an intron sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (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 in the 5′ or 3′ non-coding region known to enhance the nuclear export, stability, or translation efficiency of mRNA that is operably linked to at least one of the at least one nucleic acid sequences; and/or
the cassette further comprises a reporter gene, optionally, green fluorescent protein (GFP), a GFP variant, secreted alkaline phosphatase, luciferase, a luciferase variant, or a detectable peptide or epitope, optionally wherein 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; and/or
the one or more vectors further comprises one or more nucleic acid sequences encoding at least one immune modulator, optionally
wherein the immune modulator 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, optionally
wherein the antibody or antigen-binding fragment thereof is a Fab fragment, a Fab′ fragment, a single chain Fv (scFv), a single domain antibody (sdAb) either as single specific or multiple specificities linked together, or full-length single-chain antibody, and/or
wherein the heavy and light chain sequences of the antibody are a contiguous sequence separated by either a self-cleaving sequence optionally 2A or IRES; or the heavy and light chain sequences of the antibody are linked by a flexible linker optionally consecutive glycine residues,
or wherein the immune modulator is a cytokine, optionally wherein the cytokine is at least one of IL-2, IL-7, IL-12, IL-15, or IL-21 or variants thereof of each.
17 . The method of claim 1 , wherein the epitope-encoding nucleic acid sequence comprises a MHC class I epitope-encoding nucleic acid sequence, and wherein the MHC class I epitope-encoding nucleic acid sequence is selected by performing the steps of:
(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 peptide sequences of each of a set of epitopes;
(b) inputting the peptide sequence of each epitope into a presentation model to generate a set of numerical likelihoods that each of the epitopes is presented by one or more of the MHC alleles on the tumor cell surface of the tumor, the set of numerical likelihoods having been identified at least based on received mass spectrometry data; and
(c) selecting a subset of the set of epitopes based on the set of numerical likelihoods to generate a set of selected epitopes which are used to generate the MHC class I epitope-encoding nucleic acid sequence,
optionally wherein
a number of the set of selected epitopes is 2-20; and/or
the presentation model represents dependence between:
(1) 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
(2) likelihood of presentation on the tumor cell surface, by the particular one of the MHC alleles of the pair, of such a peptide sequence comprising the particular amino acid at the particular position; and/or
selecting the set of selected epitopes comprises selecting epitopes that have an increased likelihood of being presented on the tumor cell surface relative to unselected epitopes based on the presentation model; and/or
selecting the set of selected epitopes comprises selecting epitopes that have an increased likelihood of being capable of inducing a tumor-specific immune response in the subject relative to unselected epitopes based on the presentation model; and/or
selecting the set of selected epitopes comprises selecting epitopes that have an increased likelihood of being capable of being presented to naïve T cells by professional antigen presenting cells (APCs) relative to unselected epitopes based on the presentation model, optionally wherein the APC is a dendritic cell (DC); and/or
selecting the set of selected epitopes comprises selecting epitopes that have a decreased likelihood of being subject to inhibition via central or peripheral tolerance relative to unselected epitopes based on the presentation model; and/or
the set of selected epitopes comprises selecting epitopes that have a decreased likelihood of being capable of inducing an autoimmune response to normal tissue in the subject relative to unselected epitopes based on the presentation model; and/or
exome or transcriptome nucleotide sequencing data is obtained by performing sequencing on the tumor tissue, optionally wherein the sequencing is next generation sequencing (NGS) or any massively parallel sequencing approach.
18 . The method of claim 1 , wherein:
the cassette comprises junctional epitope sequences formed by adjacent sequences in the cassette, optionally wherein
at least one or each junctional epitope sequence has an affinity of greater than 500 nM for MHC and/or wherein each junctional epitope sequence is non-self; and/or
the cassette does not encode a non-therapeutic MHC class I or class II epitope nucleic acid sequence comprising a translated, wild-type nucleic acid sequence, wherein the non-therapeutic epitope is predicted to be displayed on an MHC allele of the subject, optionally wherein the non-therapeutic predicted MHC class I or class II epitope sequence is a junctional epitope sequence formed by adjacent sequences in the cassette; and/or
the prediction is based on presentation likelihoods generated by inputting sequences of the non-therapeutic epitopes into a presentation model; and/or
an order of the antigen-encoding nucleic acid sequences in the cassette is determined by a series of steps comprising:
(a) generating a set of candidate cassette sequences corresponding to different orders of the antigen-encoding nucleic acid sequences;
(b) determining, for each candidate cassette sequence, a presentation score based on presentation of non-therapeutic epitopes in the candidate cassette sequence; and
(c) selecting a candidate cassette sequence associated with a presentation score below a predetermined threshold as the cassette sequence for a vaccine; and/or
the composition for delivery of the expression system and/or the inhibitor of Type I interferon signaling are formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier; and/or
the method further comprises administering an adjuvant.