KRAS neoantigen therapy

Abstract

Disclosed herein are compositions comprising antigen-encoding nucleic acid sequences that have multiple repeats of KRAS neoepitope-encoding sequences and / or lack immunodominant epitopes. Also disclosed are nucleotides, cells, and methods related to the compositions, including their use as vaccines, such as in subjects with cancer, including (1) solid tumors expressing KRAS-associated MHC class I neoepitopes, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 245,703, filed September 17, 2021, 63 / 281,029, filed November 18, 2021, 63 / 321,587, filed March 18, 2022, and 63 / 374,888, filed September 7, 2022, which are incorporated by reference in their entireties for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on XXX, 20XX, is named XXX, and is XXX 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 cell therapy can cause tumor regression under certain circumstances in selected patients. 7

[0004] One of the questions in antigen vaccine design in both cancer and infectious disease settings is which of the many coding mutations present gives rise to the "best" therapeutic antigen, i.e., the antigen capable of inducing immunity.

[0005] In addition to the challenges of current antigen prediction methods, existing vector systems that can be used for antigen delivery in humans, many of which are of human origin, also present certain challenges. For example, many humans have pre-existing immunity to human viruses as a result of previous natural exposure, and this immunity can pose a significant obstacle to the use of recombinant human viruses to deliver antigens in vaccination programs, such as for cancer treatment or vaccination against infectious diseases. Although some progress has been made in vaccination programs to address the above challenges, improvements are still needed, especially in clinical applications, such as improved vaccine potency and efficacy. Summary of the Invention

[0006] Disclosed herein is a method for treating a subject having a disease, wherein the disease is cancer, including (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen-encoding cassette, or a polypeptide sequence encoded by the cassette, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence described in a 5' to 3' direction by the following formula: (E x -(E N n ) y ) z [In the formula, E represents a nucleotide sequence comprising a different epitope-encoding nucleic acid sequence; n represents the number of distinct epitope-encoding nucleic acid sequences and is any integer, including 0; E N represents a nucleotide sequence comprising said distinct epitope-encoding nucleic acid sequence 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 a combination thereof, At least one of said different epitope-encoding nucleic acid sequences comprising said at least two repeats encodes a KRAS-associated MHC class I neoepitope.

[0007] Provided herein is a method for treating a subject having a disease, wherein the disease is cancer, including (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen expression system, the antigen expression system comprising one or more vectors, the one or more vectors comprising: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) optionally, at least one polyadenylation (poly(A)) sequence; the vector backbone comprising: (b) a cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) an epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope; wherein each of the epitope-encoding nucleic acid sequences comprises: (A) optionally, a 5' linker sequence; (B) optionally, a 3' linker sequence; and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II epitope-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; the cassette comprising: wherein, when the second promoter nucleotide sequence is absent, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, and the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of the epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope.

[0008] In some embodiments, the epitope-encoding nucleic acid sequence is derived from a tumor of a subject with cancer or from a cell or sample of an infected subject. In some embodiments, the epitope-encoding nucleic acid sequence is not derived from a tumor of a subject with cancer or from a cell or sample of an infected subject.

[0009] Also disclosed herein is a method for stimulating an immune response in a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen-encoding cassette, or a polypeptide sequence encoded by the cassette, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence described in the 5' to 3' direction by the following formula: (E x -(E N n ) y ) z [In the formula, E represents a nucleotide sequence comprising a different epitope-encoding nucleic acid sequence; n represents the number of distinct epitope-encoding nucleic acid sequences and is any integer, including 0; E Nrepresents a nucleotide sequence comprising said distinct epitope-encoding nucleic acid sequence 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 a combination thereof, At least one of said different epitope-encoding nucleic acid sequences comprising said at least two repeats encodes a KRAS-associated MHC class I neoepitope.

[0010] Provided herein is a method for stimulating an immune response in a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen expression system, the antigen expression system comprising one or more vectors, the one or more vectors comprising: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) optionally, at least one polyadenylation (poly(A)) sequence; the vector backbone comprising: (b) a cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) an epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope; wherein each of the epitope-encoding nucleic acid sequences comprises: (A) optionally, a 5' linker sequence; (B) optionally, a 3' linker sequence; and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II epitope-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; the cassette comprising: wherein, when the second promoter nucleotide sequence is absent, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, and the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of the epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope.

[0011] In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present at least one epitope sequence, and optionally, the at least one epitope sequence predicted or known to be presented comprises a KRAS-associated MHC class I neoepitope. In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present at least one epitope sequence, and the at least one epitope sequence comprises an epitope known or suspected to be presented by MHC class I on the surface of a cell, and optionally, the at least one epitope sequence predicted or known to be presented comprises a KRAS-associated MHC class I neoepitope. In some embodiments, the surface of a cell is a tumor cell surface.

[0012] Also disclosed herein is a method for inducing an immune response in a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen-encoding cassette, or a polypeptide sequence encoded by the cassette, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence described in the 5' to 3' direction by the following formula: (E x -(E N n ) y ) z [In the formula, E represents a nucleotide sequence comprising a different epitope-encoding nucleic acid sequence; n represents the number of distinct epitope-encoding nucleic acid sequences and is any integer, including 0; E N represents a nucleotide sequence comprising said distinct epitope-encoding nucleic acid sequence 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 a combination thereof, At least one of said different epitope-encoding nucleic acid sequences comprising said at least two repeats encodes a KRAS-associated MHC class I neoepitope.

[0013] Provided herein is a method for inducing an immune response in a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen expression system, the antigen expression system comprising one or more vectors, the one or more vectors comprising: a) a vector backbone, the backbone comprising (i) at least one promoter nucleotide sequence and (ii) optionally at least one polyadenylation (poly(A)) sequence; and (b) a cassette, the cassette comprising (i) at least one antigen-encoding nucleic acid sequence, (I) an epitope-encoding nucleic acid sequence encoding the KRAS-associated MHC class I neoepitope, each of the epitope-encoding nucleic acid sequences comprising (A) optionally a 5' linker sequence and (B) 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 a poly(A) sequence exogenous to the vector backbone, and the second promoter nucleotide sequence is Also disclosed are the methods, wherein, in the absence of a promoter nucleotide sequence, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of the epitope-encoding nucleic acid sequence encoding the KRAS-associated MHC class I neoepitope, and the subject expresses at least one HLA allele that is expected to or known to present the at least one KRAS-associated MHC class I neoepitope.

[0014] In some embodiments, the antigen-based vaccine is administered as a priming dose. In some embodiments, the antigen-based vaccine is administered as one or more booster doses. In some embodiments, the booster dose is different from the priming dose. In some embodiments, a) the priming dose comprises a chimpanzee adenovirus vector and the booster dose comprises an alphavirus vector; or b) the priming dose comprises an alphavirus vector and the booster dose comprises a chimpanzee adenovirus vector. In some embodiments, the booster dose is the same as the priming dose. In some embodiments, the injection site of the one or more booster doses is as close as possible to the injection site of the priming dose.

[0015] In some embodiments, the method further comprises determining or having determined the subject's HLA haplotype.

[0016] In some embodiments, the antigen-based vaccine is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). In some embodiments, the antigen-based vaccine is administered intramuscularly (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 muscles.

[0017] In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation. In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises any one of the amino acid sequences set forth in SEQ ID NOs: 75-82. In some embodiments, the antigen-encoding cassette comprises each of the amino acid sequences set forth in SEQ ID NOs: 75-82. In some embodiments, the antigen-encoding cassette comprises two or more repeats of each of the amino acid sequences set forth in SEQ ID NOs: 75-82, and optionally, four repeats of each of the amino acid sequences set forth in SEQ ID NOs: 75-82. In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises the amino acid sequence set forth in SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60. In some embodiments, the epitope-encoding nucleic acid sequence comprises two or more different epitope-encoding nucleic acid sequences that independently encode different KRAS-associated MHC class I neoepitopes or different KRAS mutations. In some embodiments, each of the epitope-encoding nucleic acid sequences independently encodes a different KRAS-associated MHC class I neoepitope or a different KRAS mutation. In some embodiments, the epitope-encoding nucleic acid sequence comprises two or more different epitope-encoding nucleic acid sequences that independently encode a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation. In some embodiments, the epitope-encoding nucleic acid sequence independently encodes each of a KRAS G12C mutation, a KRAS G12V mutation, and a KRAS G12D mutation, and optionally a KRAS Q61H mutation. In some embodiments, the antigen-encoding nucleic acid sequence encodes a peptide comprising the amino acid sequence set forth in SEQ ID NO:64 or SEQ ID NO:65.

[0018] In some embodiments, the cassette comprises: (1) stimulates an immune response that is 5-fold greater when administered to a subject in a vaccine composition than another MHC class I epitope encoded in the cassette and capable of stimulating an immune response in the subject; and / or (2) When administered to a subject in a vaccine composition, the immune response to another MHC class I epitope encoded within the cassette is reduced compared to the immune response when the other MHC class I epitope is administered in the absence of the immunodominant MHC class I epitope, and optionally the immune response is reduced below the limit of detection and / or the immune response is not a therapeutically effective response. Immunodominant MHC class I epitopes Do not code.

[0019] In some embodiments, the cancer comprises a solid tumor expressing a KRAS- and / or NRAS-associated MHC class I neoepitope. In some embodiments, the KRAS- and / or NRAS-associated MHC class I neoepitope comprises a mutation selected from the group consisting of KRAS_G12C, NRAS_G12C, KRAS_G12D, NRAS_G12D, KRAS_G12V, NRAS_G12V, KRAS_Q61H, and NRAS_Q61H.

[0020] In some embodiments, the cancer comprises colorectal cancer (CRC). In some embodiments, the cancer comprises non-small cell lung cancer (NSCLC). In some embodiments, the cancer comprises pancreatic ductal adenocarcinoma (PDA).

[0021] In some embodiments, the antigen-based vaccine, or one or more booster doses, are administered every four weeks (Q4W). In some embodiments, the antigen-based vaccine, or one or more booster doses, are administered every eight weeks (Q8W). In some embodiments, the antigen-based vaccine, or one or more booster doses, are administered monthly. In some embodiments, the antigen-based vaccine, or one or more booster doses, are administered every two months.

[0022] In some embodiments, the method includes administering to a 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.

[0023] In some embodiments, two or more booster doses are administered, hi some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 booster doses are administered.

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

[0025] 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. In some embodiments, the self-replicating alphavirus-based expression system is administered as at least two booster doses at, or about, 28 and 84 days 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, 4 and 12 weeks 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, 1 and 3 months after the priming dose of the ChAdV-based expression system.

[0026] 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 28, 84, 196, and 252 days after the priming dose of the ChAdV-based expression system. In some embodiments, the self-replicating alphavirus-based expression system is administered at or about 4, 12, 28, and 40 weeks after the priming dose of the ChAdV-based expression system. In some embodiments, the self-replicating alphavirus-based expression system is administered at or about 1, 3, 7, and 10 months after the priming dose of the ChAdV-based expression system.

[0027] In some embodiments, the method further comprises administering one or more immunomodulatory agents, optionally administered before, simultaneously with, or after administration of the composition or pharmaceutical composition. In some embodiments, the one or more immunomodulatory agents are selected from the group consisting of an anti-CTLA-4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof. In some embodiments, the immunomodulatory agent is administered intravenously (IV), intramuscularly (IM), intradermally (ID), or subcutaneously (SC). In some embodiments, the subcutaneous administration is near the site of administration of the composition or pharmaceutical composition or in close proximity to one or more vector or composition-draining lymph nodes. In some embodiments, the method comprises administering the anti-CTLA-4 antibody or antigen-binding fragment thereof with only a priming dose and a first booster dose. In some embodiments, the anti-CTLA-4 antibody comprises ipilimumab. In some embodiments, ipilimumab is administered subcutaneously at a dose of 30 mg. In some embodiments, the method optionally comprises administering an anti-PD-L1 antibody or 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, or nivolumab is administered intravenously at a dose of 480 mg.

[0028] In some embodiments, the methods comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 administrations of one or more immunomodulatory agents. In some embodiments, the methods comprise at least 13 administrations of an anti-PD-L1 antibody. In some embodiments, the one or more immunomodulatory agents are selected from the group consisting of atezolizumab, nivolumab, cemiplimab, and ipilimumab. In some aspects, the KRAS-associated MHC class I neoepitope comprising a KRAS G12C mutation is selected from the group consisting of VVVGACGVGK (SEQ ID NO: 75), KLVVVGACGV (SEQ ID NO: 76), and GACGVGKSAL; the KRAS-associated MHC class I neoepitope comprising a KRAS G12D mutation is selected from the group consisting of VVGADGVGK (SEQ ID NO: 77), VVVGADGVGK (SEQ ID NO: 78), KLVVVGADGV, and GADGVGKSAL; and the KRAS-associated MHC class I neoepitope comprising a KRAS G12V mutation is selected from the group consisting of VVGAVGVGK (SEQ ID NO: 79), VVVGAVGVGK (SEQ ID NO: 81), AVGVGKSAL (SEQ ID NO: 80), and GAVGVGKSAL. In some embodiments, the KRAS-associated MHC class I neoepitope comprising a KRAS G12C mutation is selected from the group consisting of VVVGACGVGK (SEQ ID NO:75), KLVVVGACGV (SEQ ID NO:76), and GACGVGKSAL. In some embodiments, the KRAS-associated MHC class I neoepitope comprising a KRAS G12D mutation is selected from the group consisting of VVGADGVGK (SEQ ID NO:77), VVVGADGVGK (SEQ ID NO:78), KLVVVGADGV, and GADGVGKSAL. In some embodiments, the KRAS-associated MHC class I neoepitope comprising a KRAS G12V mutation is selected from the group consisting of VVGAVGVGK (SEQ ID NO:79), VVVGAVGVGK (SEQ ID NO:81), AVGVGKSAL (SEQ ID NO:80), and GAVGVGKSAL.

[0029] In some embodiments, stimulating an immune response comprises stimulating a molecular response. In some embodiments, the molecular response comprises a reduction in ctDNA. In some embodiments, the reduction in ctDNA is a reduction of at least 20%, at least 30%, at least 40%, or at least 50% of ctDNA. In some embodiments, the reduction in ctDNA is a reduction of at least 30% of ctDNA.

[0030] In some embodiments, the antigen-encoding cassette, or the polypeptide sequence encoded by the cassette, comprises, in the 5' to 3' direction, at least one antigen-encoding nucleic acid sequence described by the formula: (E x -(E N n ) y ) z [In the formula, E represents a nucleotide sequence comprising a different epitope-encoding nucleic acid sequence; n represents the number of distinct epitope-encoding nucleic acid sequences and is any integer, including 0; E N represents a nucleotide sequence comprising said distinct epitope-encoding nucleic acid sequence 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 a combination thereof, At least one of said different epitope-encoding nucleic acid sequences comprising said at least two repeats encodes a KRAS-associated MHC class I neoepitope.

[0031] In some embodiments, the antigen-encoding cassette encodes at least four repeats of each of the amino acid sequences VVVGACGVGK (SEQ ID NO:75), VVVGADGVGK (SEQ ID NO:78), VVGAVGVGK (SEQ ID NO:79), and ILDTAGHEEY (SEQ ID NO:82). In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation. In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises any one of the amino acid sequences set forth in SEQ ID NOs:75-82. In some embodiments, the antigen-encoding cassette comprises each of the amino acid sequences set forth in SEQ ID NOs:75-82. In some embodiments, the antigen-encoding cassette comprises two or more repeats of each of the amino acid sequences set forth in SEQ ID NOs:75-82. In some embodiments, the antigen-encoding cassette comprises four repeats of each of the amino acid sequences set forth in SEQ ID NOs: 75-82. In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises the amino acid sequence set forth in SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60. In some embodiments, the epitope-encoding nucleic acid sequence comprises two or more different epitope-encoding nucleic acid sequences that independently encode different KRAS-associated MHC class I neoepitopes or different KRAS mutations. In some embodiments, each of the epitope-encoding nucleic acid sequences independently encodes a different KRAS-associated MHC class I neoepitope or different KRAS mutation. In some embodiments, the epitope-encoding nucleic acid sequence comprises two or more different epitope-encoding nucleic acid sequences that independently encode a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation. In some embodiments, the epitope-encoding nucleic acid sequence independently encodes each of the KRAS G12C, G12V, and G12D mutations, and optionally the KRAS Q61H mutation. In some embodiments, the antigen-encoding nucleic acid sequence encodes a peptide comprising the amino acid sequence set forth in SEQ ID NO:64 or SEQ ID NO:65.In some embodiments, the antigen-encoding nucleic acid sequence encodes a peptide comprising the amino acid sequence set forth in SEQ ID NO:65.

[0032] In some embodiments, at least two of the different epitope-encoding nucleic acid sequences comprising at least two repeats encode different KRAS-associated MHC class I neoepitopes. In some embodiments, at least three, at least four, at least five, at least six, at least seven, or at least eight of the different epitope-encoding nucleic acid sequences comprising at least two repeats encode different KRAS-associated MHC class I neoepitopes. In some embodiments, each of the different epitope-encoding nucleic acid sequences comprising at least two repeats encodes a different KRAS-associated MHC class I neoepitope. In some embodiments, one or more of the nucleic acid sequences encoding KRAS-associated MHC class I neoepitopes comprises at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight repeats. In some embodiments, each of the nucleic acid sequences encoding a KRAS-associated MHC class I neoepitope comprises at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight repeats. In some embodiments, one or more of the nucleic acid sequences encoding different KRAS-associated MHC class I neoepitopes comprises at least four repeats. In some embodiments, each of the nucleic acid sequences encoding different KRAS-associated MHC class I neoepitopes comprises at least four repeats. In some embodiments, one or more of the different KRAS-associated MHC class I neoepitopes independently comprise a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation.

[0033] In some embodiments, each E or E N is expressed in the 5' to 3' direction by the formula (L5 b -N c -L3 d), wherein N independently comprises a nucleotide sequence described by each E or E N wherein c=1, L5 comprises a 5' linker sequence, b=0 or 1, and L3 comprises a 3' linker sequence, d=0 or 1. In some embodiments, each N encodes an epitope 7-15 amino acids in length, L5 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the epitope, wherein the 5' linker sequence encodes a peptide at least 2 amino acids in length, and L3 is a native 3' linker sequence encoding the native C-terminal amino acid sequence of the epitope, wherein the 3' linker sequence encodes a peptide at least 32 amino acids in length. In some embodiments, the 5' and / or 3' linker sequence encodes a peptide at least 3 amino acids in length. In some embodiments, the 5' and / or 3' linker sequence encodes a peptide at least 4 amino acids in length. In some embodiments, the 5' and / or 3' linker sequence encodes a peptide at least 5 amino acids in length. In some embodiments, the 5' and / or 3' linker sequences encode peptides that are at least 8 amino acids in length. In some embodiments, the 5' and / or 3' linker sequences encode peptides that are at least 2-8 amino acids in length. In some embodiments, the 5' and / or 3' linker sequences encode peptides that are at least 2-10 amino acids in length.

[0034] In some embodiments, each E and E N encodes an epitope at least 7 amino acids in length. In some embodiments, each of E and E N encodes an epitope 7 to 15 amino acids in length. In some embodiments, each of E and E N is a nucleotide sequence at least 21 nucleotides in length. In some embodiments, each E and E N is a nucleotide sequence 75 nucleotides in length.

[0035] In some embodiments, the antigen expression system is one or more vectors, the one or more vectors comprising: (a) a vector backbone comprising (i) at least one promoter nucleotide sequence and (ii) optionally at least one polyadenylation (poly(A)) sequence; (b) a cassette comprising: (i) at least one antigen-encoding nucleic acid sequence, the at least one antigen-encoding nucleic acid sequence comprising (I) an epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope, each of the epitope-encoding nucleic acid sequences comprising: (A) optionally a 5' linker sequence and (B) optionally a 3' linker sequence; and (ii) optionally a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence. and (iii) optionally, at least one MHC class II 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 that is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone, wherein if the second promoter nucleotide sequence is not present, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, and the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of the epitope-encoding nucleic acid sequence encoding the KRAS-associated MHC class I neoepitope.

[0036] In some embodiments, the antigen expression system comprises one or more vectors, said one or more vectors comprising: (a) a vector backbone comprising (i) at least one promoter nucleotide sequence and (ii) at least one polyadenylation (poly(A)) sequence; (b) a cassette comprising: (i) at least one antigen-encoding nucleic acid sequence, said at least one antigen-encoding nucleic acid sequence comprising: (I) at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different epitope-encoding nucleic acid sequences linearly linked to one another, at least one of which encodes a KRAS-associated MHC class I neoepitope, each of said epitope-encoding nucleic acid sequences comprising: (A) an optional 5' linker sequence; and (B) an optional 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, which is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; wherein, if the second promoter nucleotide sequence is not present, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, and the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of at least one of the different epitope-encoding nucleic acid sequences encoding a KRAS-associated MHC class I neoepitope.

[0037] In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least three different epitope-encoding nucleic acid sequences.

[0038] In some embodiments, the antigen expression system comprises one or more vectors, wherein the one or more vectors comprise: (a) a vector backbone comprising a chimpanzee adenovirus vector, optionally a ChAdV68 vector, or an alphavirus vector, optionally a Venezuelan equine encephalitis virus vector; and (b) a cassette, wherein the cassette is optionally integrated between a native promoter nucleotide sequence and a poly(A) sequence naturally present in the vector backbone, wherein the poly(A) sequence is optionally naturally present in the vector backbone, the cassette comprising: (i) at least one antigen-encoding nucleic acid sequence, wherein (I) an epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope, the epitope-encoding nucleic acid sequence optionally comprising at least two different epitope-encoding nucleic acid sequences linearly linked to each other, wherein optionally each epitope-encoding nucleic acid sequence encodes: (A) an MHC class I epitope between 7 and 15 amino acids in length; (B) a 5' linker sequence encoding a peptide that is at least two amino acids long and that encodes the natural N-terminal amino acid sequence of the MHC class I epitope; and (C) a 3' linker sequence encoding a peptide that is at least two amino acids long and that encodes the natural C-terminal amino acid sequence of the MHC class I epitope, wherein the cassette is operably linked to the natural promoter nucleotide sequence, and wherein each of the epitope-encoding nucleic acid sequences encodes a polypeptide that is 13 to 25 amino acids long, and wherein each 3' end of each epitope-encoding nucleic acid sequence is linked to the 5' end of the subsequent epitope-encoding nucleic acid sequence, except for the last epitope-encoding nucleic acid sequence in the cassette; and (ii) at least two MHC class II epitope-encoding nucleic acid sequences, comprising: (I) a PADRE (II) a tetanus toxoid MHC class II sequence (SEQ ID NO: 46); and (III) the PADRE.(IV) a second nucleic acid sequence encoding a GGPPG amino acid linker sequence (SEQ ID NO: 56) linking the 5' ends of the at least two MHC class II epitope-encoding nucleic acid sequences to the epitope-encoding nucleic acid sequences; and (V) optionally, a third nucleic acid sequence encoding a GGPPG amino acid linker sequence (SEQ ID NO: 56) at the 3' ends of the at least two MHC class II epitope-encoding nucleic acid sequences. and (iii) the cassette comprising the at least two MHC class II epitope-encoding nucleic acid sequences; and optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequences, wherein if the second promoter nucleotide sequence is not present, the antigen-encoding nucleic acid sequences are operably linked to the native promoter nucleotide sequence, and the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of the epitope-encoding nucleic acid sequence encoding the KRAS-associated MHC class I neoepitope.

[0039] In some embodiments, the ordered sequence of each element of the cassette is, in a 5' to 3' direction: P a -(L5 b -N c -L3 d ) X -(G5 e- U f ) Y -G3 g wherein P comprises the second promoter nucleotide sequence, with the proviso that a=0 or 1; N comprises one of the different epitope-encoding nucleic acid sequences, 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 the at least one nucleic acid sequence encoding a GPGPG amino acid linker (SEQ ID NO: 56), with the proviso that e=0 or 1; G3 comprises one of the at least one nucleic acid sequence encoding a GPGPG amino acid linker (SEQ ID NO: 56), with the proviso that g=0 or 1; U comprises one of the 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 epitope-encoding nucleic acid sequence, where Y=0, 1, or 2, with the proviso that for each Y, the corresponding U f is an MHC class II epitope-encoding nucleic acid sequence].

[0040] In some embodiments, for each X, a corresponding N c N corresponding to the at least two repeats of the different epitope-encoding nucleic acid sequences c 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=16, and Y=2, the at least one promoter nucleotide sequence is a single native promoter nucleotide sequence naturally present in the vector backbone, the at least one polyadenylation poly(A) sequence is a poly(A) sequence of at least 80 consecutive A nucleotides provided by the vector viral backbone, each N encodes an epitope 7 to 15 amino acids in length, L5 is a native 5' linker sequence encoding the native N-terminal amino acid sequence of the epitope, the 5' linker sequence encodes a peptide at least 2 amino acids in length, and L3 is the native C-terminal amino acid sequence of the epitope. and U is a native 3' linker sequence encoding a sequence, wherein the 3' linker sequence encodes a peptide at least 2 amino acids in length; U is each of a PADRE class II sequence and a tetanus toxoid MHC class II sequence; the vector backbone comprises a chimpanzee adenovirus vector, optionally a ChAdV68 vector, or an alphavirus vector, optionally a Venezuelan equine encephalitis virus vector; and when the vector backbone comprises an alphavirus vector, optionally the native promoter nucleotide sequence is a subgenomic (e.g., 26S) promoter; and each of the MHC class II epitope-encoding nucleic acid sequences encodes a polypeptide between 13 and 25 amino acids in length.

[0041] In some embodiments, the antigen-encoding cassette encodes at least four repeats of each of the amino acid sequences VVVGACGVGK (SEQ ID NO:75), VVVGADGVGK (SEQ ID NO:78), VVGAVGVGK (SEQ ID NO:79), and ILDTAGHEEY (SEQ ID NO:82). In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation. In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises any one of the amino acid sequences set forth in SEQ ID NOs:75-82. In some embodiments, the antigen-encoding cassette comprises each of the amino acid sequences set forth in SEQ ID NOs:75-82. In some embodiments, the antigen-encoding cassette comprises two or more repeats of each of the amino acid sequences set forth in SEQ ID NOs:75-82. In some embodiments, the antigen-encoding cassette comprises four repeats of each of the amino acid sequences set forth in SEQ ID NOs: 75-82. In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises the amino acid sequence set forth in SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60. In some embodiments, the epitope-encoding nucleic acid sequence comprises two or more different epitope-encoding nucleic acid sequences that independently encode different KRAS-associated MHC class I neoepitopes or different KRAS mutations. In some embodiments, each of the epitope-encoding nucleic acid sequences independently encodes a different KRAS-associated MHC class I neoepitope or different KRAS mutation. In some embodiments, the epitope-encoding nucleic acid sequence comprises two or more different epitope-encoding nucleic acid sequences that independently encode a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation. In some embodiments, the epitope-encoding nucleic acid sequence independently encodes each of the KRAS G12C, G12V, and G12D mutations, and optionally the KRAS Q61H mutation. In some embodiments, the antigen-encoding nucleic acid sequence encodes a peptide comprising the amino acid sequence set forth in SEQ ID NO:64 or SEQ ID NO:65.In some embodiments, the antigen-encoding nucleic acid sequence encodes a peptide comprising the amino acid sequence set forth in SEQ ID NO:65.

[0042] In some embodiments, the at least two repeats are at least three, at least four, at least five, at least six, at least seven, or at least eight repeats. In some embodiments, the at least two repeats are at least eight repeats. In some embodiments, the at least two repeats are at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least four, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 repeats. In some embodiments, the at least two repeats are 2-3, 2-4, 2-5, 2-6, 2-7, or 2-8 repeats. In some embodiments, the at least two repeats are 7 or fewer repeats, 6 or fewer repeats, 5 or fewer repeats, 4 or fewer repeats, or 3 or fewer repeats.

[0043] In some embodiments, at least one antigen-encoding nucleic acid sequence comprises at least two repeats of at least two different epitope-encoding nucleic acid sequences. In some embodiments, at least one antigen-encoding nucleic acid sequence comprises at least two repeats of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different epitope-encoding nucleic acid sequences. In some embodiments, the at least two repeats are separated by at least one distinct, different epitope-encoding nucleic acid sequence. In some embodiments, the at least two repeats, including the optional 5' linker sequence and / or the optional 3' linker sequence, are separated by at least 75 nucleotides. In some embodiments, the at least two repeats, including the optional 5' linker sequence and / or the optional 3' linker sequence, are separated by at least 150 nucleotides, at least 300 nucleotides, or at least 675 nucleotides. In some embodiments, the at least two repeats, including an optional 5' linker sequence and / or an optional 3' linker sequence, are separated by at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 700 nucleotides, at least 700 nucleotides, at least 750 nucleotides, at least 800 nucleotides, at least 900 nucleotides, or at least 1000 nucleotides.In some embodiments, the at least two repeats, including the optional 5' linker sequence and / or the optional 3' linker sequence, are separated by at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, or at least 70 nucleotides.

[0044] In some embodiments, the at least one antigen-encoding nucleic acid sequence is described in the 5' to 3' direction by the formula: (E x -(E N n ) y ) z [In the formula, E represents a nucleotide sequence comprising at least one of the 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 comprising said distinct epitope-encoding nucleic acid sequence 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 two repetitions of any combination thereof].

[0045] In some embodiments, the antigen-encoding cassette encodes at least four repeats of each of the amino acid sequences VVVGACGVGK (SEQ ID NO: 75), VVVGADGVGK (SEQ ID NO: 78), VVGAVGVGK (SEQ ID NO: 79), and ILDTAGHEEY (SEQ ID NO: 82).

[0046] In some embodiments, the different epitope-encoding nucleic acid sequences include at least two different epitope-encoding nucleic acid sequences, each encoding a different KRAS-associated MHC class I neoepitope. In some embodiments, the different epitope-encoding nucleic acid sequences include at least three, at least four, at least five, at least six, at least seven, or at least eight different epitope-encoding nucleic acid sequences, each encoding a different KRAS-associated MHC class I neoepitope. In some embodiments, each of the epitope-encoding nucleic acid sequences of at least one antigen-encoding nucleic acid sequence encodes a different KRAS-associated MHC class I neoepitope. In some embodiments, one or more of the nucleic acid sequences encoding the different KRAS-associated MHC class I neoepitopes include at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight repeats. In some embodiments, each of the nucleic acid sequences encoding the different KRAS-associated MHC class I neoepitopes comprises at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight repeats. In some embodiments, one or more of the nucleic acid sequences encoding the different KRAS-associated MHC class I neoepitopes comprises at least four repeats. In some embodiments, each of the nucleic acid sequences encoding the different KRAS-associated MHC class I neoepitopes comprises at least four repeats. In some embodiments, one or more of the different KRAS-associated MHC class I neoepitopes independently comprise a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation.

[0047] In some embodiments, the at least two repeats comprise a sufficient number of repeats, or z comprises such a sufficient number, to stimulate a higher immune response compared to an antigen-encoding nucleic acid sequence comprising a single repeat of the epitope-encoding nucleic acid sequence. In some embodiments, the at least two repeats comprise a sufficient number of repeats to stimulate an immune response, or z comprises such a sufficient number, and a single repeat of the epitope-encoding nucleic acid sequence is insufficient to stimulate the immune response or is insufficient to stimulate a detectable immune response. In some embodiments, the immune response is the proliferation of epitope-specific T cells following in vivo immunization with the composition for delivering the antigen expression system. In some embodiments, the immune response is increased activation of epitope-specific T cells and / or increased epitope-specific killing by epitope-specific T cells following in vivo immunization with the composition for delivering the antigen expression system.

[0048] Also provided herein is a composition for delivering an antigen expression system, the antigen expression system comprising one or more vectors, the one or more vectors comprising: (a) a vector backbone comprising (i) at least one promoter nucleotide sequence and (ii) optionally at least one polyadenylation (poly(A)) sequence; and (b) a cassette comprising: (i) at least one antigen-encoding nucleic acid sequence, the at least one antigen-encoding nucleic acid sequence comprising: (I) at least two different epitope-encoding nucleic acid sequences, optionally comprising: (1) at least one alteration that renders the encoded epitope sequence different from a corresponding peptide sequence encoded by a wild-type nucleic acid sequence, the at least one alteration being optionally a KRAS mutation; or (2) a nucleic acid sequence encoding an infectious disease organism peptide selected from the group consisting of a pathogen-derived peptide, a virus-derived peptide, a bacteria-derived peptide, a fungus-derived peptide, and a parasite-derived peptide, each of the epitope-encoding nucleic acid sequences comprising: (A) optionally a 5' linker sequence; and (B) 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 that is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone. A) a cassette comprising a sequence; and A) a sequence; wherein when the second promoter nucleotide sequence is absent, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, the cassette does not encode an immunodominant MHC class I epitope, and the immunodominant MHC class I epitope (1) stimulates an immune response in a subject that is at least five times greater than another MHC class I epitope encoded within the cassette when administered to the subject in a vaccine composition; orand / or (2) a composition that, when administered to a subject in a vaccine composition, reduces an immune response to another MHC class I epitope encoded within the cassette compared to the immune response when the other MHC class I epitope is administered in the absence of the immunodominant MHC class I epitope, and optionally, the immune response is reduced to below detection limits and / or the immune response is not a therapeutically effective response.

[0049] Also provided herein is a composition for delivering an antigen expression system, comprising the antigen expression system comprising one or more vectors, the one or more vectors comprising: (a) a vector backbone comprising (i) at least one promoter nucleotide sequence and (ii) optionally at least one polyadenylation (poly(A)) sequence; and (b) a cassette comprising (i) at least one antigen-encoding nucleic acid sequence, the nucleic acid sequence comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different epitope-encoding nucleic acids linearly linked to each other. a nucleic acid sequence, optionally encoding (1) at least one alteration that causes the encoded epitope sequence to differ from a corresponding peptide sequence encoded by a wild-type nucleic acid sequence, the at least one alteration being optionally a KRAS mutation; or (2) a nucleic acid sequence encoding an infectious disease organism peptide selected from the group consisting of a pathogen-derived peptide, a virus-derived peptide, a bacteria-derived peptide, a fungus-derived peptide, and a parasite-derived peptide, each of the epitope-encoding nucleic acid sequences comprising: (A) optionally a 5' linker sequence; and (B) optionally a , and 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 poly(A) sequence that is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone. and a second poly(A) sequence, wherein when the second promoter nucleotide sequence is absent, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, and the cassette does not encode an immunodominant MHC class I epitope, which immunodominant MHC class I epitope (1) stimulates an immune response in a subject that is at least five times greater than another MHC class I epitope encoded within the cassette when administered to the subject in a vaccine composition, and is capable of stimulating an immune response in the subject;and / or (2) a composition that, when administered to a subject in a vaccine composition, reduces an immune response to another MHC class I epitope encoded within the cassette compared to the immune response when the other MHC class I epitope is administered in the absence of the immunodominant MHC class I epitope, and optionally, the immune response is reduced to below detection limits and / or the immune response is not a therapeutically effective response.

[0050] In some embodiments, at least one of the different epitope-encoding nucleic acid sequences encodes a KRAS-associated MHC class I neoepitope.

[0051] Also provided herein is a composition for delivering an antigen expression system, comprising the antigen expression system comprising one or more vectors, wherein the one or more vectors comprise: (a) a vector backbone comprising a chimpanzee adenovirus vector, optionally a ChAdV68 vector, or an alphavirus vector, optionally a Venezuelan equine encephalitis virus vector; and (b) a cassette, wherein the cassette is optionally incorporated between a native promoter nucleotide sequence and a poly(A) sequence naturally present in the vector backbone, and wherein the poly(A) sequence is optionally naturally present in the vector backbone, the cassette comprising: (i) at least one antigen-encoding nucleic acid sequence, (I) at least one epitope-encoding nucleic acid sequence, optionally comprising at least two different epitope-encoding nucleic acid sequences linearly linked to each other, each epitope-encoding nucleic acid sequence optionally encoding (A) an MHC class I epitope of 7 to 15 amino acids in length; (B) a 5' linker sequence encoding a peptide that is at least two amino acids long and that encodes the natural N-terminal amino acid sequence of the MHC class I epitope; and (C) a 3' linker sequence encoding a peptide that is at least two amino acids long and that encodes the natural C-terminal amino acid sequence of the MHC class I epitope, wherein the cassette is operably linked to the natural promoter nucleotide sequence, and wherein each of the epitope-encoding nucleic acid sequences encodes a polypeptide that is 13 to 25 amino acids long, and wherein each 3' end of each epitope-encoding nucleic acid sequence is linked to the 5' end of the subsequent epitope-encoding nucleic acid sequence, except for the last epitope-encoding nucleic acid sequence in the cassette; and (ii) at least two MHC class II epitope-encoding nucleic acid sequences, comprising: (I) a PADRE (II) a tetanus toxoid MHC class II sequence (SEQ ID NO: 46); and (III) the PADRE.(IV) a second nucleic acid sequence encoding a GGPPG amino acid linker sequence (SEQ ID NO: 56) linking the at least two MHC class II epitope-encoding nucleic acid sequences to the 5' ends of the at least two MHC class II epitope-encoding nucleic acid sequences; and (V) optionally, a third nucleic acid sequence encoding a GGPPG amino acid linker sequence (SEQ ID NO: 56) at the 3' ends of the at least two MHC class II epitope-encoding nucleic acid sequences; and (iii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; wherein if the second promoter nucleotide sequence is absent, Also provided are compositions wherein the antigen-encoding nucleic acid sequence is operably linked to the native promoter nucleotide sequence, wherein the cassette does not encode an immunodominant MHC class I epitope, and wherein the immunodominant MHC class I epitope (1) when administered to a subject in a vaccine composition stimulates an immune response in the subject that is five-fold or greater than another MHC class I epitope encoded within the cassette, and / or (2) when administered to a subject in a vaccine composition reduces an immune response to another MHC class I epitope encoded within the cassette compared to the immune response when the other MHC class I epitope is administered in the absence of the immunodominant MHC class I epitope, and optionally the immune response is reduced to below detection limits and / or the immune response is not a therapeutically effective response.

[0052] In some embodiments, the immunodominant MHC class I epitope, when administered to a subject in a vaccine composition, stimulates an immune response in the subject that is 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or 10,000-fold or more greater than another MHC class I epitope encoded within the cassette. In some embodiments, the immunodominant MHC class I epitope reduces the immune response of the other MHC class I epitope to below detection limits and / or does not stimulate a therapeutically effective response. In some embodiments, the subject expresses at least one HLA allele that is known or predicted to present both the immunodominant MHC class I epitope and the other MHC class I epitope encoded within the cassette.

[0053] In some embodiments, one or more of the epitope-encoding nucleic acid sequences are derived from a tumor. In some embodiments, each of the epitope-encoding nucleic acid sequences is derived from a tumor, infection, or infected cells of the subject. In some embodiments, one or more of the epitope-encoding nucleic acid sequences are not derived from a tumor, infection, or infected cells of the subject. In some embodiments, each of the epitope-encoding nucleic acid sequences is not derived from a tumor, infection, or infected cells of the subject.

[0054] 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 or an infected cell, and optionally the cell is a cell of a 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 cells are selected from the group consisting of HIV-infected cells, severe acute respiratory syndrome-associated coronavirus (SARS)-infected cells, severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2)-infected cells, Ebola-infected cells, hepatitis B virus (HBV)-infected cells, influenza-infected cells, orthomyxoviridae-infected cells, human papillomavirus (HPV)-infected cells, cytomegalovirus (CMV)-infected cells, chikungunya virus-infected cells, respiratory syncytial virus (RSV)-infected cells, dengue virus-infected cells, and hepatitis C virus (HCV)-infected cells.

[0055] In some embodiments, the composition further comprises a nanoparticulate delivery vehicle. In some embodiments, the nanoparticulate delivery vehicle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable amino lipid. In some embodiments, the ionizable amino lipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule. In some embodiments, the nanoparticle delivery vehicle encapsulates an antigen expression system.

[0056] In some embodiments, the cassette is integrated between at least one promoter nucleotide sequence and at least one poly(A) sequence, hi some embodiments, a second promoter is not present and the at least one promoter nucleotide sequence is operably linked to the antigen-encoding nucleic acid sequence.

[0057] 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 backbone comprises at least one nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. In some embodiments, the backbone comprises at least one nucleotide sequence of Venezuelan equine encephalitis virus. In some embodiments, the backbone comprises at least sequences for nonstructural protein-mediated amplification 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, a 26S promoter sequence, a poly(A) sequence, nonstructural protein 1 (nsP1) gene, an nsP2 gene, an nsP3 gene, and an nsP4 gene. In some embodiments, the backbone comprises at least sequences for nonstructural protein-mediated amplification 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, a 26S promoter sequence, and a poly(A) sequence. In some embodiments, the sequence for nonstructural protein-mediated amplification is selected from the group consisting of an alphavirus 5' UTR, a 51 nt CSE, a 24 nt CSE, a 26S subgenomic promoter sequence, a 19 nt CSE, an alphavirus 3' UTR, or a combination thereof. In some embodiments, the scaffold 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 within the nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus.

[0058] 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 backbone comprises the sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, a cassette is inserted at position 7544 to replace the deletion between base pairs 7544 and 11175 set forth in the sequence of SEQ ID NO:3 or SEQ ID NO:5.

[0059] In some embodiments, insertion of the cassette results in transcription of a polycistronic RNA comprising the nsP1-4 genes and the at least one antigen-encoding nucleic acid sequence, wherein the nsP1-4 genes and the at least one antigen-encoding nucleic acid sequence are in separate open reading frames.

[0060] In some embodiments, the backbone comprises at least one nucleotide sequence of a chimpanzee adenoviral vector. In some embodiments, the chimpanzee adenoviral vector is a ChAdV68 vector. In some embodiments, the ChAdV68 backbone comprises a ChAdV68 vector backbone comprising the sequence set forth in SEQ ID NO: 1. In some embodiments, the ChAdV68 vector comprises a ChAdV68 vector backbone comprising 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 has been completely or functionally deleted from the sequence, optionally with the sequence being completely 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 comprises a ChAdV68 vector backbone comprising a gene or regulatory sequence derived from the sequence of SEQ ID NO: 1, and optionally the gene is selected from the group consisting of the inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of a chimpanzee adenovirus of the sequence set forth in SEQ ID NO: 1. In some embodiments, the ChAdV68 vector comprises a ChAdV68 vector backbone comprising a 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. In some embodiments, the ChAdV68 vector comprises a ChAdV68 vector backbone comprising at least nucleotides 2-36,518 of the sequence set forth in SEQ ID NO:1, and further comprising: (1) an E1 deletion of at least nucleotides 577-3403 of the sequence set forth in SEQ ID NO:1; (2) an E3 deletion of at least nucleotides 27,125-31,825 of the sequence set forth in SEQ ID NO:1; and (3) an E4 deletion of at least nucleotides 34,916-35,642 of the sequence set forth in SEQ ID NO:1, and optionally, the antigen cassette is inserted within the E1 deletion.In some embodiments, the ChAdV68 vector comprises a ChAdV68 vector backbone comprising the sequence set forth in SEQ ID NO: 68, and optionally, the antigen cassette is inserted within the E1 deletion. In some embodiments, the ChAdV68 vector comprises a ChAdV68 vector backbone with one or more deletions between base pairs 577-3403 or between base pairs 456-3014 of the sequence set forth in SEQ ID NO: 1, and optionally, the vector further comprises one or more deletions between base pairs 27,125-31,825 or between base pairs 27,816-31,333. In some embodiments, the ChAdV68 vector comprises a ChAdV68 vector backbone with one or more deletions between base pairs 3957-10346, base pairs 21787-23370, and base pairs 33486-36193 of the sequence set forth in SEQ ID NO: 1. In some embodiments, the cassette is inserted into the ChAdV vector backbone in the E1 region, the E3 region, and / or any deleted AdV region that allows for integration of the cassette.

[0061] In some embodiments, at least one promoter nucleotide sequence is a natural 26S promoter nucleotide sequence encoded by the backbone. In some embodiments, at least one promoter nucleotide sequence is an exogenous RNA promoter. In some embodiments, the second promoter nucleotide sequence is a 26S promoter nucleotide sequence. In some embodiments, the second promoter nucleotide sequence comprises multiple 26S promoter nucleotide sequences, each of which causes transcription of one or more of the separate open reading frames.

[0062] In some embodiments, one or more vectors are each at least 300 nt in size. In some embodiments, one or more vectors are each at least 1 kb in size. In some embodiments, one or more vectors are each 2 kb in size. In some embodiments, one or more vectors are each less than 5 kb in size.

[0063] In some embodiments, at least one of the at least one antigen-encoding nucleic acid sequence encodes a polypeptide sequence or a portion thereof that is presented by MHC class I on a cell surface, optionally on a tumor cell surface or an infected cell.

[0064] In some embodiments, each epitope-encoding nucleic acid sequence is directly linked to another. In some embodiments, at least one of the antigen-encoding nucleic acid sequences is linked to a different epitope-encoding nucleic acid sequence by a nucleic acid sequence encoding a linker. In some embodiments, the linker links two MHC class I sequences or one MHC class I sequence to one MHC class II sequence. In some embodiments, the linker is selected from the group consisting of: (1) a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive glycine residues in length; (2) a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive alanine residues in length; (3) two arginine residues (RR); (4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length that is efficiently processed by mammalian proteases; and (6) one or more naturally occurring sequences adjacent to an antigen derived from a homologous protein 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 sequences or one MHC class II sequence to one MHC class I sequence. In some embodiments, the linker comprises the sequence GPGPG (SEQ ID NO: 56). In some embodiments, at least one of the epitope-encoding nucleic acid sequences is operably or directly linked to a separate or contiguous sequence that enhances expression, stability, cellular trafficking, processing and presentation, and / or immunogenicity of the epitope-encoding nucleic acid sequence encoded thereby.In some embodiments, the separated or contiguous sequences comprise at least one of a ubiquitin sequence, a ubiquitin sequence modified to enhance proteasome targeting (e.g., a ubiquitin sequence having a Gly or Ala substitution at position 76), an immunoglobulin signal sequence (e.g., IgK), 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 enhance proteasome targeting is A76.

[0065] In some embodiments, at least one of the epitope-encoding nucleic acid sequences encodes a polypeptide sequence or a portion thereof that has increased binding affinity for its corresponding MHC allele relative to the corresponding wild-type nucleic acid sequence after translation. In some embodiments, at least one of the epitope-encoding nucleic acid sequences encodes a polypeptide sequence or a portion thereof that has increased binding stability for its corresponding MHC allele relative to the corresponding wild-type nucleic acid sequence after translation. In some embodiments, at least one of the epitope-encoding nucleic acid sequences encodes a polypeptide sequence or a portion thereof that has increased likelihood of presentation on its corresponding MHC allele relative to the corresponding wild-type nucleic acid sequence after translation. In some embodiments, the at least one alteration comprises a point mutation, a frameshift mutation, a non-frameshift mutation, a deletion mutation, an insertion mutation, a splice variant, a genomic rearrangement, or a splice antigen generated by the proteasome.

[0066] In some embodiments, the tumor is selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, bladder cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, adult acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer, or the infectious disease organism is selected from the group consisting of severe acute respiratory syndrome-associated coronavirus (SARS), severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), Ebola, HIV, hepatitis B virus (HBV), influenza, hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), chikungunya virus, respiratory syncytial virus (RSV), dengue virus, orthomyxoviridae virus, and tuberculosis.

[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 epitope-encoding nucleic acid sequences. 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 epitope-encoding nucleic acid sequences. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 2-400 epitope-encoding nucleic acid sequences, and at least two of the epitope-encoding nucleic acid sequences encode polypeptide sequences or portions thereof that are presented by MHC class I on a cell surface, optionally a tumor cell surface or an infected cell surface. 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. 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 polypeptide sequences or portions thereof that are presented by MHC class I on a cell surface, optionally a tumor cell surface or an infected cell surface. In some embodiments, at least two of said epitope-encoding nucleic acid sequences encode polypeptide sequences or portions thereof that are presented by MHC class I on a cell surface, optionally on a tumor cell surface or an infected cell surface.

[0068] In some embodiments, when administered to a subject and translated, at least one of the epitopes encoded by the epitope-encoding nucleic acid sequence is presented on antigen-presenting cells, resulting in an immune response that targets at least one of the antigens on the surface of the tumor cell or infected cell. In some embodiments, when administered to a subject and translated, the at least one antigen-encoding nucleic acid sequence is presented on antigen-presenting cells, resulting in an immune response that targets at least one of the epitopes on the surface of the tumor cell or infected cell, and optionally, expression of each of the at least one antigen-encoding nucleic acid sequence is directed by the at least one promoter nucleotide sequence.

[0069] In some embodiments, each epitope-encoding nucleic acid sequence encodes a polypeptide sequence that is 8 to 35 amino acids in length, optionally 9 to 17, 9 to 25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length.

[0070] In some embodiments, at least one MHC class II epitope-encoding nucleic acid sequence is present. In some embodiments, 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 having at least one alteration that causes the encoded peptide sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence. In some embodiments, the at least one MHC class II epitope-encoding nucleic acid sequence is 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length. In some embodiments, the at least one MHC class II epitope-encoding nucleic acid sequence is present and comprises at least one universal MHC class II antigen-encoding nucleic acid sequence, optionally, the at least one universal sequence comprises at least one of tetanus toxoid and PADRE.

[0071] In some embodiments, at least one of the promoter nucleotide sequences or the second promoter nucleotide sequence is inducible. In some embodiments, at least one of the promoter nucleotide sequences or the second promoter nucleotide sequence is non-inducible.

[0072] In some embodiments, at least one poly(A) sequence comprises a naturally occurring poly(A) sequence in the backbone. In some embodiments, at least one poly(A) sequence comprises an exogenous poly(A) sequence in the backbone. In some embodiments, at least one poly(A) sequence is operably linked to at least one of the at least one antigen-encoding nucleic acid sequences. In some embodiments, at least one poly(A) sequence is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 consecutive A nucleotides. In some embodiments, at least one poly(A) sequence is at least 80 consecutive A nucleotides.

[0073] 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 the 5' or 3' non-coding region 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, 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.

[0074] In some embodiments, the one or more vectors further comprise one or more nucleic acid sequences encoding at least one immunomodulatory agent. In some embodiments, the immunomodulatory agent is an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a Fab fragment, a Fab' fragment, a single-chain Fv (scFv), a single domain antibody (sdAb) (e.g., camelid antibody domains) as a monospecific antibody or linked multispecific antibody, or a full-length single-chain antibody (e.g., a full-length IgG in which the heavy and light chains are linked by a flexible linker). In some embodiments, the heavy and light chain sequences of the antibody are contiguous sequences separated by a self-cleaving sequence such as 2A or an IRES, or the heavy and light chain sequences of the antibody are linked by a flexible linker such as consecutive glycine residues. In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the cytokine is IL-2, IL-7, IL-12, IL-15, or IL-21, or each of its variants.

[0075] In some embodiments, at least one epitope-encoding nucleic acid sequence is selected by performing the following steps: (a) obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing data from a tumor, an infected cell, or an infectious disease organism, wherein the nucleotide sequencing data is used to obtain data representing the peptide sequences of each of a set of antigens; (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented by one or more MHC alleles on a cell surface, optionally on a tumor cell surface or an infected cell, wherein the set of numerical likelihoods was determined based at least on the received mass spectrometry data; and (c) selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens used to generate the epitope-encoding nucleic acid sequence.

[0076] In some embodiments, each of the epitope-encoding nucleic acid sequences is selected by performing the following steps: (a) obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing data from a tumor, an infected cell, or an infectious disease organism, wherein the nucleotide sequencing data is used to obtain data representing each peptide sequence of a set of antigens; (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented by one or more MHC alleles on a cell surface, optionally on a tumor cell surface or an infected cell, wherein the set of numerical likelihoods is determined based at least on the received mass spectrometry data; and (c) selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens used to generate the at least 20 epitope-encoding nucleic acid sequences. In some embodiments, the number of sets of selected antigens 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 in 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 cell surface, optionally on a tumor cell surface or an infected cell surface, by the particular one of the MHC alleles of the pair. In some embodiments, selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of being presented on the cell surface relative to antigens not selected based on the presentation model, and optionally, the selected antigens have been validated as being presented by one or more specific HLA alleles. In some embodiments, selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of being able to induce a tumor-specific or infection-specific immune response in the subject relative to antigens not selected based on the presentation model.In some embodiments, selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of being presented to naive T cells by professional antigen-presenting cells (APCs) relative to antigens not selected based on the presentation model, where optionally the APCs are dendritic cells (DCs). In some embodiments, selecting the set of selected antigens comprises selecting antigens that have a decreased likelihood of being inhibited by central or peripheral tolerance relative to antigens not selected based on the presentation model. In some embodiments, selecting the set of selected antigens comprises selecting antigens that have a decreased likelihood of inducing an autoimmune response against normal tissue in a subject relative to antigens not selected based on the presentation model. In some embodiments, exome or transcriptome nucleotide sequencing data is obtained by sequencing tumor cells or tissues, infected cells, or infectious disease organisms. In some embodiments, sequencing is next-generation sequencing (NGS) or any massively parallel sequencing approach.

[0077] In some embodiments, the cassette comprises a junction epitope sequence formed by adjacent sequences within the antigen 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.

[0078] In some embodiments, each of the MHC class I epitopes is predicted or verified to be capable of presentation by at least one HLA allele that is present in at least 5% of the population. In some embodiments, each of the MHC class I epitopes is predicted or verified to be capable of presentation by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA frequency of at least 0.01% in the population. In some embodiments, each of the MHC class I epitopes is predicted or verified to be capable of presentation by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA frequency of at least 0.1% in the population.

[0079] In some embodiments, the cassette does not encode a non-therapeutic MHC class I or class II epitope nucleic acid sequence that comprises a wild-type nucleic acid sequence after translation, and the non-therapeutic epitope is predicted to be presented on an MHC allele of interest. In some embodiments, the predicted non-therapeutic MHC class I or class II epitope sequence is a junction epitope sequence formed by adjacent sequences within the cassette.

[0080] In some aspects, the prediction is based on a presentation likelihood generated by inputting the sequence of said non-therapeutic epitope into a presentation model.

[0081] In some embodiments, the order of at least one antigen-encoding nucleic acid sequence within a cassette is determined by a series of steps including: (a) generating a set of candidate cassette sequences corresponding to different orders of the at least one antigen-encoding nucleic acid sequence; (b) determining, for each candidate cassette sequence, a presentation score based on the presentation of non-therapeutic epitopes within the candidate cassette sequence; and (c) selecting candidate cassette sequences associated with a presentation score below a predetermined threshold as cassette sequences for an antigen vaccine.

[0082] Also provided herein are pharmaceutical compositions comprising any of the compositions described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the composition further comprises an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof.

[0083] Also provided herein is an isolated nucleotide sequence or set of isolated nucleotide sequences comprising a cassette of any of the compositions described herein and one or more elements derived from the sequence of SEQ ID NO:3 or SEQ ID NO:5, wherein optionally the one or more elements are selected from the group consisting of sequences required for nonstructural protein-mediated amplification, a 26S promoter nucleotide sequence, a poly(A) sequence, and the nsP1-4 genes of the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, and optionally the nucleotide sequences are cDNA. In some embodiments, the isolated nucleotide sequence or set of sequences comprises a cassette of any of the preceding composition claims inserted at position 7544 of the sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the composition further comprises: a) a nucleotide sequence for a T7 or SP6 RNA polymerase promoter located 5' to the one or more elements derived from the sequence of SEQ ID NO:3 or SEQ ID NO:5; and b) optionally, one or more restriction sites located 3' to the poly(A) sequence. In some embodiments, the cassette of any of the preceding composition claims is inserted at position 7563 of SEQ ID NO:8 or SEQ ID NO:9.

[0084] Also provided herein is a vector or set of vectors comprising any of the nucleotide sequences described herein.

[0085] Also provided herein is an isolated cell comprising a nucleotide sequence or set of isolated nucleotide sequences described herein, optionally wherein the cell is a BHK-21, CHO, HEK293 or a variant thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a cell.

[0086] Also provided herein are kits comprising any of the compositions described herein and instructions for use.

[0087] In some embodiments, any of the above compositions further comprises a nanoparticle delivery vehicle. In some embodiments, the nanoparticle delivery vehicle can be a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable amino lipid. In some embodiments, the ionizable amino lipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule. In some embodiments, the nanoparticle delivery vehicle encapsulates an antigen expression system.

[0088] In some embodiments, any of the above compositions further comprise a plurality of LNPs, the LNPs comprising an antigen expression system, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of the LNPs, wherein at least about 95% of the LNPs have either a non-lamellar morphology or are electron-dense.

[0089] In some embodiments, the non-cationic lipid is a mixture of (1) a phospholipid and (2) cholesterol or a cholesterol derivative.

[0090] In some embodiments, the conjugated lipid that inhibits aggregation of LNPs is a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate is selected from the group consisting of PEG-diacylglycerol (PEG-DAG) conjugates, PEG-dialkyloxypropyl (PEG-DAA) conjugates, PEG-phospholipid conjugates, PEG-ceramide (PEG-Cer) conjugates, and mixtures thereof. In some embodiments, the PEG-DAA conjugate is PEG-didecyloxypropyl (C 10 ) conjugate, PEG-dilauryloxypropyl (C 12 ) conjugate, PEG-dimyristyloxypropyl (C 14 ) conjugate, PEG-dipalmityloxypropyl (C 16 ) conjugate, PEG-distearyloxypropyl (C 18 ) complexes, and mixtures thereof.

[0091] In some embodiments, the antigen expression system is fully encapsulated in the LNP.

[0092] In some embodiments, the non-lamellar morphology of the LNPs is an inverted hexagonal (H II ) or cubic phase structure.

[0093] In some embodiments, the cationic lipids are present in an LNP at about 10 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the cationic lipids are present in an LNP at about 20 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the cationic lipids are present in an LNP at about 20 mol% to about 40 mol% of the total lipids present in the LNP.

[0094] In some embodiments, the non-cationic lipids comprise about 10 mol% to about 60 mol% of the total lipids present in the LNP. In some embodiments, the non-cationic lipids comprise about 20 mol% to about 55 mol% of the total lipids present in the LNP. In some embodiments, the non-cationic lipids comprise about 25 mol% to about 50 mol% of the total lipids present in the LNP.

[0095] In some embodiments, complex lipids comprise about 0.5 mol% to about 20 mol% of the total lipids present in the LNP. In some embodiments, complex lipids comprise about 2 mol% to about 20 mol% of the total lipids present in the LNP. In some embodiments, complex lipids comprise about 1.5 mol% to about 18 mol% of the total lipids present in the LNP.

[0096] In some embodiments, greater than 95% of the LNPs have a non-lamellar morphology. In some embodiments, greater than 95% of the LNPs are electron-dense.

[0097] In some embodiments, any of the above compositions further comprises a plurality of LNPs, the LNPs comprising a cationic lipid present in the LNP at 50 mol% to 85 mol% of the total lipids present in the LNP, a complex lipid that inhibits aggregation of the LNP at 0.5 mol% to 2 mol% of the total lipids present in the LNP, and a non-cationic lipid, the mixture being a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid is present at 4 mol% to 10 mol% of the total lipids present in the LNP, and the cholesterol or a derivative thereof is present at 30 mol% to 40 mol% of the total lipids present in the LNP. The non-cationic lipid comprises either a mixture of phospholipids and cholesterol or a derivative thereof, wherein the phospholipid is contained in 3 mol% to 15 mol% of the total lipids present in the LNP and the cholesterol or a derivative thereof is contained in 30 mol% to 40 mol% of the total lipids present in the LNP, or a mixture comprising a mixture of phospholipids and cholesterol or a derivative thereof that is 49.5 mol% or less of the total lipids present in the LNP and wherein the cholesterol or a derivative thereof is contained in 30 mol% to 40 mol% of the total lipids present in the LNP.

[0098] In some embodiments, any of the above compositions further comprises a plurality of LNPs, the LNPs comprising a cationic lipid present in an amount of 50 mol% to 65 mol% of the total lipids present in the LNPs, a complex lipid that inhibits aggregation of the LNPs present in an amount of 0.5 mol% to 2 mol% of the total lipids present in the LNPs, and a non-cationic lipid present in an amount of 13 mol% to 49.5 mol% of the total lipids present in the LNPs.

[0099] In some embodiments, the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.

[0100] In some embodiments, the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate comprises a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or a mixture thereof. In some embodiments, the PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate, a PEG-distearoyloxypropyl (PEG-DSA) conjugate, or a mixture thereof. In some embodiments, the PEG portion of the conjugate has an average molecular weight of about 2000 daltons.

[0101] In some embodiments, complex lipids comprise between 1 mol% and 2 mol% of the total lipids present in the LNP.

[0102] In some embodiments, the LNP has the structure of Formula I: TIFF2024535855000002.tif51128[where L 1 and L 2 are each independently -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) x -, -SS-, -C(=0)S-, -SC(=0)-, -R a C(=0)-, -C(=0)R a -, -R a C(=0)R a -, -OC(=0)Ra -, -R a C(=0)0- or a direct bond, and G1 is C i ~C2 alkylene, -(C=0)-, -0(C=0)-, -SC(=0)-, -R a C(=0)-, or a direct bond, -C(=0)-, -(C=0)0-, -C(=0)S-, -C(=0)R a - or a direct bond, and G is C i ~C6 alkylene, and R a is H or C1-C12 alkyl, and R 1a and R 1b is, for each occurrence, independently (a) H or C1-C 12 alkyl, or (b) R 1a is H or C1~C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 2a and R 2b is, for each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 2a is H or C1~C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 3a and R 3b is, for each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 3a is H or C1~C 12 alkyl, and R 3b together with the carbon atom to which it is attached, form a carbon-carbon double bond with the adjacent R and the carbon atom to which it is attached, and R4a and R4b, for each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 4a is H or C1~C 12 alkyl, and R4b together with the carbon atom to which it is attached, the adjacent R 4b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 5 and R 6 are each independently H or methyl, and R 7 is C4~C 20 alkyl, and R 8 and R 9 are C1 to C 12 alkyl or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle; a, b, c, and d are each independently an integer from 1 to 24; and x is 0, 1, or 2; or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0103] In some embodiments, the LNP has the structure of Formula II: TIFF2024535855000003.tif42128[where L 1 and L 2 are each independently -0(C=0)-, -(C=0)0-, or a carbon-carbon double bond; R 1a and R 1b is, for each occurrence, independently: (a) H or C 1 ~C 12 alkyl, or (b) R 1a is H or C1~C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 2a and R 2b is, for each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 2a is H or C1~C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2band form a carbon-carbon double bond together with the carbon atom to which it is attached, R 3a and R 3b is, for each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 3a is H or C1~C 12 alkyl, and R 3b together with the carbon atom to which it is attached, the adjacent R 3b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 4a and R 4b is, for each occurrence, independently: (a) H or C1-C 12 alkyl, or (b) R 4a is H or C1~C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 5 and R 6 are each independently methyl or cycloalkyl, and R 7 is independently H or C1-C for each occurrence 12 alkyl, and R 8 and R 9 are each independently unsubstituted C1 to C 12 alkyl or R 8 and R 9 and together with the nitrogen atom to which they are attached, form a 5-, 6-, or 7-membered heterocycle containing one nitrogen atom, a and d each independently represent an integer of 0 to 24, b and c each independently represent an integer of 1 to 24, and e is 1 or 2, with the proviso that R 1a , R 2a , R 3a , or R 4a At least one of C1 to C 12 alkyl or L 1 or L 2 At least one of is -0(C=0)- or -(C=0)0-, and R 1a and R 1bis not isopropyl when a is 6 and is not n-butyl when a is 8; or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0104] In some embodiments, any of the above compositions further comprises one or more excipients including a neutral lipid, a steroid, and a polymer-bound lipid. In some embodiments, the neutral lipid comprises at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the neutral lipid is DSPC.

[0105] In some embodiments, the molar ratio of compound to neutral lipid ranges from about 2:1 to about 8:1.

[0106] In some embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to cholesterol ranges from about 2:1 to 1:1.

[0107] In some embodiments, the polymer-bound lipid is a PEGylated lipid. In some embodiments, the molar ratio of the compound to the PEGylated lipid ranges from about 100:1 to about 25:1. In some embodiments, the PEGylated lipid is PEG-DAG, PEG polyethylene (PEG-PE), PEG-succinoyl-diacylglycerol (PEG-S-DAG), PEG-cer, or PEG dialkyloxypropylcarbamate. In some embodiments, the PEGylated lipid has the following structure III: TIFF2024535855000004.tif29128[where, R 10 and R 11are each independently a straight or branched, saturated or unsaturated alkyl chain having from 10 to 30 carbon atoms, said alkyl chain optionally interrupted by one or more ester bonds, and z has an average value in the range of 30 to 60, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. 10 and R 11 are each independently a linear saturated alkyl chain having 12 to 16 carbon atoms. In some embodiments, the average z is about 45.

[0108] In some embodiments, the LNPs self-assemble into non-bilayer structures when mixed with polyanionic nucleic acids. In some embodiments, the non-bilayer structures have diameters of 60 nm to 120 nm. In some embodiments, the non-bilayer structures have diameters of about 70 nm, about 80 nm, about 90 nm, or about 100 nm. In some embodiments, the nanoparticulate delivery vehicles have diameters of about 100 nm.

[0109] Also provided herein are methods for treating a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject any of the compositions described herein or any of the pharmaceutical compositions described herein. In some embodiments, the epitope-encoding nucleic acid sequence is derived from a tumor of the subject having cancer or from a cell or sample of the infected subject. In some embodiments, the epitope-encoding nucleic acid sequence is not derived from a tumor of the subject having cancer or from a cell or sample of the infected subject.

[0110] Also provided herein are methods of stimulating an immune response in a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject any of the compositions described herein or any of the pharmaceutical compositions described herein.

[0111] In some embodiments, the subject expresses at least one HLA allele predicted or known to present MHC class I epitopes. In some embodiments, the HLA alleles predicted or known to present MHC class I epitopes are A*03:01, A*11:01, A*02:01, A*68:01, B*07:02, C*01:02, C*03:04, C*08:02, and / or A*01:01. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is A*03:01. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is A*11:01. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is A*02:01. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is C*01:02. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is A*68:01. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is B*07:02. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is C*03:04. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is C*08:02. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is A*01:01.

[0112] In some embodiments, the composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). In some embodiments, the composition is administered intramuscularly. In some embodiments, the method further comprises administering one or more immunomodulatory agents, optionally administered before, simultaneously with, or after administration of the composition or pharmaceutical composition. In some embodiments, the one or more immunomodulatory agents are selected from the group consisting of an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof. In some embodiments, the immunomodulatory agent is administered intravenously (IV), intramuscularly (IM), intradermally (ID), or subcutaneously (SC). In some embodiments, the subcutaneous administration is near the site of administration of the composition or pharmaceutical composition or in close proximity to one or more vector or composition-draining lymph nodes. In some embodiments, the one or more immunomodulatory agents are selected from the group consisting of nivolumab, cemiplimab, and ipilimumab. In some embodiments, the one or more immunomodulatory agents include nivolumab. In some embodiments, the one or more immunomodulatory agents include cemiplimab. In some embodiments, the one or more immunomodulatory agents include ipilimumab.

[0113] In some embodiments, the method further comprises administering a second vaccine composition to the subject. In some embodiments, the second vaccine composition is administered before administration of any of the compositions or pharmaceutical compositions described herein. In some embodiments, the second vaccine composition is administered after administration of any of the compositions or pharmaceutical compositions described herein. In some embodiments, the second vaccine composition is the same as any of the compositions or pharmaceutical compositions described herein. In some embodiments, the second vaccine composition is different from any of the compositions or pharmaceutical compositions described herein. In some embodiments, the second vaccine composition comprises a chimpanzee adenoviral vector encoding at least one antigen-encoding nucleic acid sequence. In some embodiments, the at least one antigen-encoding nucleic acid sequence encoded by the chimpanzee adenoviral vector is the same as at least one antigen-encoding nucleic acid sequence described in any of the preceding composition claims.

[0114] Also provided herein are methods for producing one or more vectors of any of the preceding composition claims, the methods comprising: (a) obtaining a linearized DNA sequence comprising a backbone and a cassette; (b) in vitro transcribing the linearized DNA sequence by adding the linearized DNA sequence to an in vitro transcription reaction containing all components necessary for transcribing the linearized DNA sequence into RNA, optionally including in vitro addition of an m7g cap to the resulting RNA; and (c) isolating one or more vectors from the in vitro transcription reaction. In some embodiments, the linearized DNA sequence is generated by linearizing a DNA plasmid sequence or by amplification using PCR. In some embodiments, the DNA plasmid sequence is generated using one of bacterial recombination or total genome DNA synthesis or total genome DNA synthesis involving amplification of DNA synthesized in bacterial cells. In some embodiments, isolating one or more vectors from the in vitro transcription reaction comprises one or more of phenol-chloroform extraction, purification using a silica column, or similar RNA purification methods.

[0115] Also provided herein is a method of making a composition according to any of the preceding composition claims for delivering an antigen expression system, the method comprising: (a) providing components of a nanoparticulate delivery vehicle; (b) providing an antigen expression system; and (c) providing conditions sufficient for the nanoparticulate delivery vehicle and the antigen expression system to form a composition for delivering the antigen expression system. In some aspects, such conditions are provided by microfluidic mixing.

[0116] Also provided herein is a method for treating a subject having a disease, wherein the disease is cancer, the cancer comprising (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen-encoding cassette, or a polypeptide sequence encoded by the cassette, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence described in a 5' to 3' direction by the following formula: (E x -(E N n ) y ) z [In the formula, E represents a nucleotide sequence comprising a different epitope-encoding nucleic acid sequence; n represents the number of distinct epitope-encoding nucleic acid sequences and is any integer, including 0; E N represents a nucleotide sequence comprising said distinct epitope-encoding nucleic acid sequence 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 a combination thereof, At least one of said different epitope-encoding nucleic acid sequences comprising said at least two repeats encodes a KRAS-associated MHC class I neoepitope.

[0117] Provided herein is a method for treating a subject having a disease, wherein the disease is cancer, and the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen expression system, the antigen expression system comprising one or more vectors, the one or more vectors comprising: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) optionally, at least one polyadenylation (poly(A)) sequence; the vector backbone comprising: (b) a cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) an epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope, wherein each of the epitope-encoding nucleic acid sequences comprises: (A) optionally, a 5' linker sequence; (B) optionally, a 3' linker sequence; and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II epitope-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which may be a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; the cassette comprising: wherein, when the second promoter nucleotide sequence is absent, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, and the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of the epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope.

[0118] In some embodiments, the antigen-encoding cassette encodes at least four repeats of each of the amino acid sequences VVVGACGVGK (SEQ ID NO: 75), VVVGADGVGK (SEQ ID NO: 78), VVGAVGVGK (SEQ ID NO: 79), and ILDTAGHEEY (SEQ ID NO: 82).

[0119] In some embodiments, the epitope-encoding nucleic acid sequence is derived from a tumor of a subject with cancer or from a cell or sample of an infected subject. In some embodiments, the epitope-encoding nucleic acid sequence is not derived from a tumor of a subject with cancer or from a cell or sample of an infected subject.

[0120] Also provided herein are methods for stimulating an immune response in a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen-encoding cassette, or a polypeptide sequence encoded by the cassette, the antigen-encoding cassette comprising, in a 5' to 3' direction, at least one antigen-encoding nucleic acid sequence described by the following formula: (E x -(E N n ) y ) z [In the formula, E represents a nucleotide sequence comprising a different epitope-encoding nucleic acid sequence; n represents the number of distinct epitope-encoding nucleic acid sequences and is any integer, including 0; E N represents a nucleotide sequence comprising said distinct epitope-encoding nucleic acid sequence 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 a combination thereof, At least one of said different epitope-encoding nucleic acid sequences comprising said at least two repeats encodes a KRAS-associated MHC class I neoepitope.

[0121] Provided herein is a method for stimulating an immune response in a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen expression system, the antigen expression system comprising one or more vectors, the one or more vectors comprising: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) optionally, at least one polyadenylation (poly(A)) sequence; the vector backbone comprising: (b) a cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) an epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope, wherein each of the epitope-encoding nucleic acid sequences comprises: (A) optionally, a 5' linker sequence; (B) optionally, a 3' linker sequence; and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II epitope-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which may be a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; the cassette comprising: wherein, when the second promoter nucleotide sequence is absent, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, and the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of at least one of the epitope-encoding nucleic acid sequences encoding a KRAS-associated MHC class I neoepitope.

[0122] Provided herein is a method for treating a subject having a disease, wherein the disease is cancer, and the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen expression system, the antigen expression system comprising one or more vectors, the one or more vectors comprising: (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) optionally, at least one polyadenylation (poly(A)) sequence; the vector backbone comprising: (b) a cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) at least two different epitope-encoding nucleic acid sequences, optionally (1) at least one alteration that causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence, wherein the at least one alteration is optionally a KRAS mutation; or (2) a nucleic acid sequence encoding an infectious disease organism peptide selected from the group consisting of a pathogen-derived peptide, a virus-derived peptide, a bacteria-derived peptide, a fungus-derived peptide, and a parasite-derived peptide; wherein each of the epitope-encoding nucleic acid sequences comprises: (A) optionally, a 5' linker sequence; (B) optionally, a 3' linker sequence; the at least two different epitope-encoding nucleic acid sequences comprising and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II epitope-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which may be a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; the cassette comprising: wherein, when the second promoter nucleotide sequence is absent, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, and the cassette does not encode an immunodominant MHC class I epitope, which (1) when administered to a subject in a vaccine composition, stimulates an immune response that is five-fold or greater than that of another MHC class I epitope encoded within the cassette, and is capable of stimulating an immune response in the subject, and / or (2) when administered to a subject in a vaccine composition, reduces an immune response to another MHC class I epitope encoded within the cassette compared to the immune response when the other MHC class I epitope is administered in the absence of the immunodominant MHC class I epitope, and optionally, the immune response is reduced to below detection limits and / or the immune response is not a therapeutically effective response.

[0123] In some embodiments, the antigen-encoding cassette encodes at least four repeats of each of the amino acid sequences VVVGACGVGK (SEQ ID NO: 75), VVVGADGVGK (SEQ ID NO: 78), VVGAVGVGK (SEQ ID NO: 79), and ILDTAGHEEY (SEQ ID NO: 82).

[0124] In some aspects, the subject expresses at least one HLA allele predicted or known to present said at least one epitope sequence, and optionally, said at least one epitope sequence predicted or known to be presented comprises (1) a KRAS-associated MHC class I neoepitope, and / or (2) said immunodominant MHC class I epitope and other MHC class I epitopes encoded within said cassette. In some embodiments, the subject expresses at least one HLA allele predicted or known to present said at least one epitope sequence, wherein said at least one epitope sequence comprises an epitope known or suspected to be presented by MHC class I on the surface of a cell, and optionally, said at least one epitope sequence predicted or known to be presented comprises (1) a KRAS-associated MHC class I neoepitope, and / or (2) said immunodominant MHC class I epitope and other MHC class I epitopes encoded within said cassette. In some embodiments, the surface of a cell is a tumor cell surface. 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. In some embodiments, the surface of the cell is an infected cell surface. In some embodiments, 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 cells are selected from the group consisting of HIV-infected cells, severe acute respiratory syndrome-associated coronavirus (SARS)-infected cells, severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2)-infected cells, Ebola-infected cells, hepatitis B virus (HBV)-infected cells, influenza-infected cells, orthomyxoviridae-infected cells, human papillomavirus (HPV)-infected cells, cytomegalovirus (CMV)-infected cells, chikungunya virus-infected cells, respiratory syncytial virus (RSV)-infected cells, dengue virus-infected cells, and hepatitis C virus (HCV)-infected cells.

[0125] Also provided herein is a method for inducing an immune response in a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen-encoding cassette, or a polypeptide sequence encoded by the cassette, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence described in the 5' to 3' direction by the following formula: (E x -(E N n ) y ) z [In the formula, E represents a nucleotide sequence comprising a different epitope-encoding nucleic acid sequence; n represents the number of distinct epitope-encoding nucleic acid sequences and is any integer, including 0; E N represents a nucleotide sequence comprising said distinct epitope-encoding nucleic acid sequence 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 Nor a combination thereof, At least one of said different epitope-encoding nucleic acid sequences comprising said at least two repeats encodes a KRAS-associated MHC class I neoepitope.

[0126] Provided herein is a method for inducing an immune response in a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen expression system, the antigen expression system comprising one or more vectors, the one or more vectors comprising: a) a vector backbone, the backbone comprising (i) at least one promoter nucleotide sequence and (ii) optionally at least one polyadenylation (poly(A)) sequence; and (b) a cassette, the cassette comprising (i) at least one antigen-encoding nucleic acid sequence, (I) an epitope-encoding nucleic acid sequence encoding the KRAS-associated MHC class I neoepitope, each of the epitope-encoding nucleic acid sequences comprising (A) optionally a 5' linker sequence and (B) 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 a poly(A) sequence exogenous to the vector backbone, and the second promoter nucleotide sequence is Also provided is the method, wherein, in the absence of a promoter nucleotide sequence, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of the epitope-encoding nucleic acid sequence encoding the KRAS-associated MHC class I neoepitope, and the subject expresses at least one HLA allele that is expected to or known to present the at least one KRAS-associated MHC class I neoepitope.

[0127] Provided herein is a method for inducing an immune response in a subject having cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), wherein the antigen-based vaccine comprises an antigen expression system, wherein the antigen expression system comprises one or more vectors, wherein the one or more vectors (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) optionally, at least one polyadenylation (poly(A)) sequence; the vector backbone comprising: (b) a cassette, (i) at least one antigen-encoding nucleic acid sequence, (I) at least two epitope-encoding nucleic acid sequences, optionally (1) at least one alteration that causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence, wherein the at least one alteration is optionally a KRAS mutation; or (2) a nucleic acid sequence encoding an infectious disease organism peptide selected from the group consisting of a pathogen-derived peptide, a virus-derived peptide, a bacteria-derived peptide, a fungus-derived peptide, and a parasite-derived peptide; and optionally wherein the epitope-encoding nucleic acid sequences encode MHC class I epitopes, and each of the epitope-encoding nucleic acid sequences comprises: (A) optionally, a 5' linker sequence; (B) optionally, a 3' linker sequence; the at least two different epitope-encoding nucleic acid sequences comprising and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II epitope-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which may be a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; the cassette comprising: and when the second promoter nucleotide sequence is absent, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence, and the cassette does not encode an immunodominant MHC class I epitope, the immunodominant MHC class I epitope (1) when administered to a subject in a vaccine composition stimulates an immune response in the subject that is 5-fold greater than another MHC class I epitope encoded within the cassette, and / or (2) stimulates an immune response in the subject that is not 5-fold greater than another MHC class I epitope in the absence of the immunodominant MHC class I epitope. The method also provides a method for reducing an immune response to another MHC class I epitope encoded within the cassette when administered to a subject in a vaccine composition compared to the immune response when the other MHC class I epitope is administered, and optionally, the immune response is reduced to below detection limits and / or the immune response is not a therapeutically effective response, wherein the subject expresses at least one HLA allele predicted or known to present both the immunodominant MHC class I epitope encoded within the cassette and the other MHC class I epitope.

[0128] In some embodiments, the antigen-encoding cassette encodes at least four repeats of each of the amino acid sequences VVVGACGVGK (SEQ ID NO:75), VVVGADGVGK (SEQ ID NO:78), VVGAVGVGK (SEQ ID NO:79), and ILDTAGHEEY (SEQ ID NO:82). In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation. In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises any one of the amino acid sequences set forth in SEQ ID NOs:75-82. In some embodiments, the antigen-encoding cassette comprises each of the amino acid sequences set forth in SEQ ID NOs:75-82. In some embodiments, the antigen-encoding cassette comprises two or more repeats of each of the amino acid sequences set forth in SEQ ID NOs:75-82. In some embodiments, the antigen-encoding cassette comprises four repeats of each of the amino acid sequences set forth in SEQ ID NOs: 75-82. In some embodiments, the KRAS-associated MHC class I neoepitope or KRAS mutation comprises the amino acid sequence set forth in SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60. In some embodiments, the epitope-encoding nucleic acid sequence comprises two or more different epitope-encoding nucleic acid sequences that independently encode different KRAS-associated MHC class I neoepitopes or different KRAS mutations. In some embodiments, each of the epitope-encoding nucleic acid sequences independently encodes a different KRAS-associated MHC class I neoepitope or different KRAS mutation. In some embodiments, the epitope-encoding nucleic acid sequence comprises two or more different epitope-encoding nucleic acid sequences that independently encode a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation. In some embodiments, the epitope-encoding nucleic acid sequence independently encodes each of the KRAS G12C, G12V, and G12D mutations, and optionally the KRAS Q61H mutation. In some embodiments, the antigen-encoding nucleic acid sequence encodes a peptide comprising the amino acid sequence set forth in SEQ ID NO:64 or SEQ ID NO:65.In some embodiments, the antigen-encoding nucleic acid sequence encodes a peptide comprising the amino acid sequence set forth in SEQ ID NO:65.

[0129] In some embodiments, the KRAS-associated MHC class I neoepitope comprising a KRAS G12C mutation is selected from the group consisting of VVVGACGVGK (SEQ ID NO:75), KLVVVGACGV (SEQ ID NO:76), and GACGVGKSAL. In some embodiments, the KRAS-associated MHC class I neoepitope comprising a KRAS G12D mutation is selected from the group consisting of VVGADGVGK (SEQ ID NO:77), VVVGADGVGK (SEQ ID NO:78), KLVVVGADGV, and GADGVGKSAL. In some embodiments, the KRAS-associated MHC class I neoepitope comprising a KRAS G12V mutation is selected from the group consisting of VVGAVGVGK (SEQ ID NO:79), VVVGAVGVGK (SEQ ID NO:81), AVGVGKSAL (SEQ ID NO:80), and GAVGVGKSAL.

[0130] In some embodiments, the cassette comprises: (1) stimulates an immune response that is 5-fold or greater when administered to a subject in a vaccine composition compared to another MHC class I epitope encoded in the cassette and capable of stimulating an immune response in the subject; and / or (2) When administered to a subject in a vaccine composition, the immune response to another MHC class I epitope encoded within the cassette is reduced compared to the immune response when the other MHC class I epitope is administered in the absence of the immunodominant MHC class I epitope, and optionally the immune response is reduced below the limit of detection and / or the immune response is not a therapeutically effective response. Immunodominant MHC class I epitopes In some embodiments, the cassette does not encode an immunodominant MHC class I epitope that, when administered to a subject in a vaccine composition, stimulates an immune response five-fold or more relative to a KRAS-associated neoepitope encoded within the cassette that is capable of stimulating an immune response in the subject. In some embodiments, the cassette does not encode an immunodominant MHC class I epitope that, when administered to a subject in a vaccine composition, reduces an immune response to another MHC class I epitope encoded within the cassette compared to the immune response when the other MHC class I epitope is administered in the absence of the immunodominant MHC class I epitope. In some embodiments, the cassette does not encode an immunodominant MHC class I epitope that, when administered to a subject in a vaccine composition, reduces an immune response to another MHC class I epitope encoded within the cassette to below the limit of detection compared to the immune response when the other MHC class I epitope is administered in the absence of the immunodominant MHC class I epitope. In some embodiments, the cassette does not encode an immunodominant MHC class I epitope that, when administered to a subject in a vaccine composition, reduces an immune response to another MHC class I epitope encoded within the cassette compared to the immune response when the other MHC class I epitope is administered in the absence of the immunodominant MHC class I epitope, and the immune response to the other MHC class I epitope is not a therapeutically effective response.

[0131] In some embodiments, the immunodominant epitope is a TP53-associated MHC class I neoepitope, and optionally, the TP53-associated MHC class I neoepitope comprises a S127Y mutation.

[0132] In some embodiments, the antigen expression system comprises any one of the antigen expression systems described herein. In some embodiments, the antigen-based vaccine comprises any one of the pharmaceutical compositions described herein.

[0133] In some embodiments, the antigen-based vaccine is administered as a priming dose. In some embodiments, the antigen-based vaccine is administered as one or more booster doses. In some embodiments, the booster dose is different from the priming dose. In some embodiments, a) the priming dose comprises a chimpanzee adenovirus vector and the booster dose comprises an alphavirus vector, or b) the priming dose comprises an alphavirus vector and the booster dose comprises a chimpanzee adenovirus vector. In some embodiments, the booster dose is the same as the priming dose. In some embodiments, the injection site of one or more booster doses is as close as possible to the injection site of the priming dose.

[0134] In some embodiments, the method further comprises determining or having determined the subject's HLA haplotype.

[0135] In some embodiments, the antigen-based vaccine is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). In some embodiments, the antigen-based vaccine is administered intramuscularly (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 muscles.

[0136] Also disclosed herein are pharmaceutical compositions comprising any of the compositions disclosed herein (such as an alphavirus-based or ChAd-based vector disclosed herein) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises an adjuvant. In some embodiments, the pharmaceutical composition further comprises an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof.

[0137] Also disclosed herein are vectors that include the isolated nucleotide sequences disclosed herein.

[0138] Also disclosed herein are kits comprising a vector or composition disclosed herein and instructions for use.

[0139] Also disclosed herein are methods for treating a subject with cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering the vector disclosed herein or the pharmaceutical composition disclosed herein. Also disclosed herein are methods for inducing an immune response in a subject with cancer, wherein the cancer comprises (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject any of the compositions, vectors, or pharmaceutical compositions described herein.

[0140] In some embodiments, the subject expresses at least one HLA allele predicted or known to present MHC class I epitopes. In some embodiments, the HLA alleles predicted or known to present MHC class I epitopes are A*03:01, A*11:01, A*02:01, A*68:01, B*07:02, C*01:02, C*03:04, C*08:02, and / or A*01:01. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is A*03:01. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is A*11:01. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is A*02:01. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is C*01:02. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is A*68:01. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is B*07:02. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is C*03:04. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is C*08:02. In some embodiments, the HLA allele predicted or known to present MHC class I epitopes is A*01:01.

[0141] In some embodiments, the vector or composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV).

[0142] In some embodiments, the cancer comprises a solid tumor expressing a KRAS- and / or NRAS-associated MHC class I neoepitope. In some embodiments, the KRAS- and / or NRAS-associated MHC class I neoepitope comprises a mutation selected from the group consisting of KRAS_G12C, NRAS_G12C, KRAS_G12D, NRAS_G12D, KRAS_G12V, NRAS_G12V, KRAS_Q61H, and NRAS_Q61H.

[0143] In some embodiments, the cancer comprises colorectal cancer (CRC). In some embodiments, the cancer comprises non-small cell lung cancer (NSCLC). In some embodiments, the cancer comprises pancreatic ductal adenocarcinoma (PDA).

[0144] In some embodiments, any one of the compositions, pharmaceutical compositions, antigen-based vaccines, or one or more booster doses described herein is administered every four weeks (Q4W). In some embodiments, any one of the compositions, pharmaceutical compositions, antigen-based vaccines, or one or more booster doses described herein is administered every eight weeks (Q8W). In some embodiments, any one of the compositions, pharmaceutical compositions, antigen-based vaccines, or one or more booster doses described herein is administered monthly. In some embodiments, any one of the compositions, pharmaceutical compositions, antigen-based vaccines, or one or more booster doses described herein is administered every two months.

[0145] In some embodiments, stimulating an immune response comprises stimulating a molecular response. In some embodiments, the molecular response comprises a reduction in ctDNA. In some embodiments, the reduction in ctDNA is a reduction of at least 20%, at least 30%, at least 40%, or at least 50% of ctDNA. In some embodiments, the reduction in ctDNA is a reduction of at least 30% of ctDNA.

[0146] Also provided herein are methods for producing one or more vectors of any of the above compositions, the methods comprising: obtaining a linearized DNA sequence comprising a backbone and an antigen cassette; in vitro transcribing the linearized DNA sequence by adding the linearized DNA sequence to an in vitro transcription reaction containing all components necessary for transcribing the linearized DNA sequence into RNA, optionally including in vitro addition of an m7g cap to the resulting RNA; and isolating one or more vectors from the in vitro transcription reaction. In some embodiments, the linearized DNA sequence is generated by linearizing a DNA plasmid sequence or by amplification using PCR. In some embodiments, the DNA plasmid sequence is generated using one of bacterial recombination or total genome DNA synthesis or total genome DNA synthesis involving amplification of DNA synthesized in bacterial cells. In some embodiments, isolating one or more vectors from the in vitro transcription reaction comprises one or more of phenol-chloroform extraction, purification using a silica column, or similar RNA purification methods.

[0147] Also provided herein are methods of making any of the compositions disclosed herein, comprising providing components of a nanoparticulate delivery vehicle, providing an antigen expression system, and providing conditions sufficient for the nanoparticulate delivery vehicle and the antigen expression system to form a composition for delivering the antigen expression system. In some aspects, such conditions are provided by microfluidic mixing.

[0148] Also disclosed herein is a method of producing an adenoviral vector disclosed herein, comprising obtaining a plasmid sequence comprising at least one promoter sequence and an antigen cassette, transfecting the plasmid sequence into one or more host cells, and isolating the adenoviral vector from the one or more host cells.

[0149] In some embodiments, the isolating comprises lysing the host cells to obtain a cell lysate comprising the adenoviral vector, and purifying the adenoviral vector from the cell lysate.

[0150] In some embodiments, the plasmid sequences are generated using one of bacterial recombination or total genomic DNA synthesis or total genomic DNA synthesis with amplification of DNA synthesized in bacterial cells. In some embodiments, the one or more host cells are at least one of CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, and AE1-2a cells. In some embodiments, purifying the adenoviral vector from the cell lysate involves one or more of chromatographic separation, centrifugation, virus precipitation, and filtration. [Brief explanation of the drawings]

[0151] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and accompanying drawings.

[0152] [Figure 1A] Diagrams of cassettes with either a single copy of the KRAS neoepitopes G12C, G12V, G12D, and Q61H ("KRAS 1X(20x1)", cassette = SEQ ID NO: 63), two repeats of the KRAS G12C, G12V, G12D, and Q61H neoepitopes and two repeats of an additional KRAS neoepitope ("KRAS 2X(8x2)", cassette = SEQ ID NO: 64), or four repeats of each KRAS neoepitope ("KRAS 4X(4x4)", cassette = SEQ ID NO: 65). Numeric identifiers are relative to the epitope "slot" for each cassette, not across cassette designs (e.g., the epitope in slot "3" of a 20x1 cassette is not the same as the epitope in slot 3 of an 8x2 cassette).

[0153] [Figure 1B]This figure shows that repeated epitopes increase vaccine-induced antigen-specific T cell responses. ELISpot results for the repeated neoepitope KRAS G12C are shown. Mice engineered to express human HLA-A11:01 were immunized with 8x10 VP using the indicated ChAdV68 delivery vector, and splenocytes were isolated 14 days after immunization. After overnight stimulation with VVVGACGVGK (SEQ ID NO: 75), the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x10 splenocytes for each animal. Bars indicate median values.

[0154] [Figure 1C] Figure 1 shows that repeating epitopes increase vaccine-induced antigen-specific T cell responses. ELISpot results for the repeating neoepitope KRAS G12V are shown. Mice engineered to express human HLA-A11:01 were immunized with 8x10 VP using the indicated ChAdV68 delivery vector, and splenocytes were isolated 14 days after immunization. After overnight stimulation with VVVGAVGVGK (SEQ ID NO: 81), the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x10 splenocytes for each animal. Bars indicate median values. Dashed lines represent non-measurable majority (TNTC) samples.

[0155] [Figure 1D] Figure 1 shows that repeated epitopes increase vaccine-induced antigen-specific T cell responses. ELISpot results for the repeated neoepitope KRAS G12D are shown. Mice engineered to express human HLA-A11:01 were immunized with 8x10 VP using the indicated ChAdV68 delivery vector, and splenocytes were isolated 14 days after immunization. After overnight stimulation with VVVGADGVGK (SEQ ID NO: 78), the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x10 splenocytes for each animal. Bars indicate median values.

[0156] [Figure 2A] 1 shows a diagram of ChAdV68 delivery vectors designed to assess the immunodominance of TP53 epitopes, specifically vectors containing KRAS neoepitopes G12C, G12V, G12D, and Q61H alone ("KRAS 4x1", cassette = SEQ ID NO: 66), each KRAS neoepitope in combination with the TP53 R213L neoepitope ("KRAS 4x1+R213L", cassette = SEQ ID NO: 67), and each KRAS neoepitope in combination with the TP53 S127Y neoepitope ("KRAS 4x1+S127Y", cassette = SEQ ID NO: 68).

[0157] [Figure 2B] Figure 1 shows that removal of immunodominant epitopes increases antigen-specific T cell responses to vaccine-induced KRAS neoepitopes. ELISpot results for the neoepitope KRAS G12C are shown. Mice engineered to express human HLA-A11:01 were immunized with 5x10 VP using the indicated ChAdV68 delivery vector, and splenocytes were isolated 14 days after immunization. After overnight stimulation with VVVGACGVGK (SEQ ID NO: 75), the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x10 splenocytes for each animal. Bars indicate median values.

[0158] [Figure 2C] Figure 1 shows that removal of immunodominant epitopes increases antigen-specific T cell responses to vaccine-induced KRAS neoepitopes. ELISpot results for the neoepitope KRAS G12D are shown. Mice engineered to express human HLA-A11:01 were immunized with 5x10 VP using the indicated ChAdV68 delivery vector, and splenocytes were isolated 14 days after immunization. After overnight stimulation with VVVGADGVGK (SEQ ID NO: 78), the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x10 splenocytes for each animal. Bars indicate median values.

[0159] [Figure 2D] Figure 1 shows that removal of immunodominant epitopes increases antigen-specific T cell responses to vaccine-induced KRAS neoepitopes. ELISpot results for the neoepitope KRAS G12V are shown. Mice engineered to express human HLA-A11:01 were immunized with 5x10 VP using the indicated ChAdV68 delivery vector, and splenocytes were isolated 14 days after immunization. After overnight stimulation with VVVGAVGVGK (SEQ ID NO: 81), the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x10 splenocytes for each animal. Bars indicate median values.

[0160] [Figure 2E] Immune responses of immunodominant epitopes and relevant control epitopes are shown. ELISpot results are shown for the TP53 neoepitope pool of R213L and S127Y neoepitopes. Mice engineered to express human HLA-A11:01 were immunized with 5x1010 VP using the indicated ChAdV68 delivery vector, and splenocytes were isolated 14 days post-immunization. After overnight stimulation, the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x106 splenocytes for each animal. Bars indicate median values. Dashed lines represent non-measurable majority (TNTC) samples.

[0161] [Figure 3]This figure shows that repeated epitopes increase vaccine-induced antigen-specific T cell responses. ELISpot results for the repeated neoepitopes KRAS G12V (left panel) or KRAS G12D (right panel) are shown. Mice engineered to express human HLA-A11:01 were immunized with 5x10 VP using the indicated ChAdV68 delivery vector, and splenocytes were isolated 14 days after immunization. After overnight stimulation with VVVGAVGVGK (SEQ ID NO: 81) or VVVGADGVGK (SEQ ID NO: 78), respectively, the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x10 splenocytes for each animal. Bars indicate median values.

[0162] [Figure 4] This figure shows that repeated epitopes increase vaccine-induced antigen-specific T cell responses. ELISpot results for the repeated neoepitopes KRAS G12V (left panel) or KRAS G12D (right panel) are shown. Mice engineered to express human HLA-A11:01 were immunized with 7x10 VP using the indicated ChAdV68 delivery vector, and splenocytes were isolated 14 days after immunization. After overnight stimulation with VVVGAVGVGK (SEQ ID NO: 81) or VVVGADGVGK (SEQ ID NO: 78), respectively, the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x10 splenocytes for each animal. Bars indicate median values.

[0163] [Figure 5]Figure 1 shows that repeated epitopes increase vaccine-induced antigen-specific T cell responses to KRAS Q61H. ELISpot results for the repeated neoepitope KRAS Q61H are shown for each cassette format indicated. Mice engineered to express human HLA-A01:01 were immunized with 5x10 VP using the indicated ChAdV68 delivery vector, and splenocytes were isolated 12 days post-immunization. After overnight stimulation with ILDTAGHEEY (SEQ ID NO: 82), the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x10 splenocytes for each animal. Bars indicate median values. Dashed lines represent non-measurable majority (TNTC) samples.

[0164] [Figure 6] This figure shows that repeated epitopes increase vaccine-induced antigen-specific T cell responses to both ChAdV68 and SAM vector formats. ELISpot results for the repeated neoepitopes KRAS G12V (left panel) or KRAS G12D (right panel) are shown. Mice engineered to express human HLA-A11:01 were immunized with 5 x 10 VPs using the indicated ChAdV68 delivery vector or 10 μg of the indicated SAM vector, and splenocytes were isolated 14 days post-immunization. After overnight stimulation with peptide pools encompassing all 38 possible minimal epitopes across 25-mers, the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1 x 10 splenocytes for each animal. Bars indicate median values. The pillars, from left to right, are ChAdV68 20x1, SAM 20x1, ChAdV68 4x4, and SAM 4x4.

[0165] [Figure 7]Figure 1 shows that repeated epitopes increase vaccine-induced antigen-specific T cell responses to both ChAdV68 and SAM vector formats. ELISpot results for the repeated neoepitopes KRAS G12V (left panel) or KRAS G12D (right panel) are shown. Mice engineered to express human HLA-A11:01 were immunized with 5x10 VP using the indicated ChAdV68 delivery vector or 10 μg of the indicated SAM vector, and splenocytes were isolated 14 days after immunization. After overnight stimulation with VVGAVGVGK (SEQ ID NO: 79) or VVVGADGVGK (SEQ ID NO: 78), the number of antigen-specific T cells was measured by IFNg ELISpot. Data are presented as spot-forming colonies (SFC) per 1x10 splenocytes for each animal. Bars indicate median values. The pillars, from left to right, are ChAdV68 20x1, SAM 20x1, ChAdV68 4x4, and SAM 4x4.

[0166] [Figure 8]

[0016] A Phase 1 / 2 study is presented that is designed to investigate the dosing, safety and tolerability, immunogenicity, and early clinical activity of a cancer vaccine encoding a repetitive KRAS neoepitope cassette ("SLATE v2") described herein administered in combination with an immune checkpoint blockade in patients with advanced cancer.

[0167] [Figure 9] ELISpot CD8+ T cell responses are shown for SLATE patient S21, who received the SLATE "version 1" (v1) cassette, and patient S31, who received the optimized SLATE "version 2" (v2) cassette containing a repeating KRAS neoepitope. Overnight stimulation with a peptide pool containing 38 minimal epitopes is shown. Time points were collected after the second SAM administration.

[0168] [Figure 10]Clinical response for patient S31 treated with the SLATE v2 cassette containing a repeating KRAS neoepitope. Top panel: Radiology CT scan of tumor site. Bottom left panel: Quantification of radiology CT scan. Bottom right panel: Response assessment by monitoring neoantigen ctDNA.

[0169] [Figure 11]

[0016] A Phase 1 / 2 study is presented that is designed to investigate the dosing, safety and tolerability, immunogenicity, and early clinical activity of a cancer vaccine encoding a repetitive KRAS neoepitope cassette ("SLATE v2") described herein administered in combination with an immune checkpoint blockade in patients with advanced cancer.

[0170] [Figure 12] A summary of T cell responses investigated by IFNγ ELISpot against the various G12 mutations, alleles, and cassettes indicated is shown.

[0171] [Figure 13] Molecular responses are shown as monitored by monitoring neoantigen ctDNA (top left panel) and standard serum tumor markers CEA and CA 19-9 (bottom left panel), as well as radiology CT scans of the tumor site (right panel).

[0172] [Figure 14] Overall survival probability is shown for subjects with and without molecular response (ctDNA reduction > 30%).

[0173] [Figure 15] Clinical outcomes for patients with NSCLC are listed.

[0174] [Figure 16] Clinical outcomes for patients with late-stage CRC are tabulated.

[0175] [Figure 17] The two-month treatment schedule for the phase 2 clinical trial is shown. DETAILED DESCRIPTION OF THE INVENTION

[0176] Detailed Description I. Definition In general, terms used in the claims and the specification shall be interpreted as having their ordinary meaning as understood by one of ordinary skill in the art. Certain terms are defined below to provide further clarity. If there is a conflict between the ordinary meaning and a given definition, the given definition shall control.

[0177] As used herein, the term "antigen" refers to a substance that stimulates an immune response. An antigen may be a neoantigen. An antigen may be a "common antigen," which is an antigen found among a particular population, for example, a particular population of cancer patients.

[0178] As used herein, the term "neoantigen" refers to an antigen that has at least one alteration that makes it different from the corresponding wild-type antigen, for example, due to a tumor cell mutation or tumor cell-specific post-translational modification. Neoantigens may include polypeptide or nucleotide sequences. Mutations can include frameshift or non-frameshift indels, missense or nonsense substitutions, splice site alterations, genomic rearrangements or gene fusions, or any genomic or expression alteration that results in a neo-ORF. Mutations can also include splice variants. Tumor cell-specific post-translational modifications can include aberrant phosphorylation. Tumor cell-specific post-translational modifications can also include splice antigens generated by the proteasome. See Liepe et al., A large fraction of HLA class I ligands are proteasome-generated spliced ​​peptides; Science. 2016 Oct 21;354(6310):354-358. Subjects for administration can be identified through the use of various diagnostic methods, such as the patient selection methods described further below.

[0179] 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.

[0180] As used herein, the term "antigen-based vaccine" refers to a vaccine composition based on one or more antigens, e.g., multiple antigens. The vaccine may be nucleotide-based (e.g., virus-based, RNA-based, or DNA-based), protein-based (e.g., peptide-based), or a combination thereof.

[0181] As used herein, the term "candidate antigen" refers to a mutation or other abnormality that results in a sequence that may represent an antigen.

[0182] As used herein, the term "coding region" refers to the portion or portions of a gene that encode a protein.

[0183] As used herein, the term "coding mutation" refers to a mutation that occurs in the coding region.

[0184] As used herein, the term "ORF" means open reading frame.

[0185] As used herein, the term "neo-ORF" refers to a tumor-specific ORF that arises due to mutation or other abnormalities such as splicing.

[0186] As used herein, the term "missense mutation" is a mutation that results in the substitution of one amino acid for another.

[0187] 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.

[0188] As used herein, the term "frameshift mutation" is a mutation that causes an alteration in the frame of a protein.

[0189] As used herein, the term "indel" is an insertion or deletion of one or more nucleic acids.

[0190] As used herein, the term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences in which a certain percentage of nucleotides or amino acid residues are the same when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to those of skill in the art), or by visual inspection. Depending on the application, the "percent identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or over the full length of the two sequences being compared.

[0191] In sequence comparison, generally, one sequence serves as a reference sequence to which test sequences are compared.When using a sequence comparison algorithm, test sequences and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity (%) of the test sequence to the reference sequence based on the designated program parameters.Alternatively, sequence similarity or difference can also be established by the combination of the presence or absence of a specific nucleotide at a selected sequence position (e.g., sequence motif) or an amino acid in a translated sequence.

[0192] 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).

[0193] 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.

[0194] As used herein, the term "non-stop or read-through" refers to a mutation that results in the removal of the natural stop codon.

[0195] As used herein, the term "epitope" refers to a specific portion of an antigen that is typically bound by an antibody or T-cell receptor.

[0196] As used herein, the term "immunogenic" refers to the ability to stimulate an immune response, for example, via T cells, B cells, or both.

[0197] As used herein, the terms "HLA binding affinity" and "MHC binding affinity" refer to the affinity of binding between a specific antigen and a specific HLA or MHC allele.

[0198] 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.

[0199] 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.

[0200] As used herein, the term "variant calling" is the algorithmic determination, typically from sequencing, of the presence of a mutation.

[0201] As used herein, the term "polymorphism" refers to a germline mutation, ie, a mutation found in all DNA-bearing cells of an individual.

[0202] As used herein, the term "somatic mutation" is a mutation that occurs in a non-germline cell of an individual.

[0203] 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.

[0204] As used herein, the term "HLA type" refers to the complement of HLA gene alleles.

[0205] As used herein, the term "nonsense-mediated decay" or "NMD" refers to the degradation of mRNA by the cell due to a premature stop codon.

[0206] 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.

[0207] 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.

[0208] As used herein, the term "exome" refers to the subset of the genome that encodes proteins. The exome can be the collection of exons of the genome.

[0209] 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.

[0210] As used herein, the term "neural network" refers to a machine learning model for classification or regression that consists of multiple layers of linear transformations followed by element-wise nonlinear transformations typically trained by stochastic gradient descent and backpropagation.

[0211] 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.

[0212] 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., tumor peptidome refers to the collection of all cellular peptidomes contained in a tumor).

[0213] As used herein, the term "ELISPOT" refers to enzyme-linked immunosorbent spot assay, a common method for monitoring immune responses in humans and animals.

[0214] As used herein, the term "dextramer" refers to a dextran-based peptide-MHC multimer used for antigen-specific T cell staining in flow cytometry.

[0215] As used herein, the term "tolerance or immune tolerance" refers to a state of immune unresponsiveness to one or more antigens, eg, self-antigens.

[0216] As used herein, the term "central tolerance" is tolerance conferred in the thymus by either deleting autoreactive T cell clones or promoting their differentiation into immunosuppressive regulatory T cells (Tregs).

[0217] As used herein, the term "peripheral tolerance" refers to tolerance conferred in the peripheral system by downregulating or anergizing autoreactive T cells that survive central tolerance or by promoting the differentiation of these T cells into Tregs.

[0218] The term "sample" can include a single cell, or multiple cells, or fragments of cells, or an aliquot of bodily fluid obtained from a subject by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspiration, lavage sample, scraping, surgical incision, or intervention, or other means known in the art.

[0219] The term "subject" includes cells, tissues, or organisms, human or non-human, whether male or female, in vivo, ex vivo, or in vitro. The term subject includes mammals, including humans.

[0220] 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.

[0221] The term "clinical factor" refers to a measurement of a subject's condition, e.g., disease activity or severity. "Clinical factor" encompasses all markers of a subject's health status, including non-sample markers, and / or other characteristics of the subject, such as, but not limited to, age and sex. A clinical factor can be a score, value, or set of values ​​that can be obtained from assessing a subject or a sample (or a population of samples) from a subject under a given condition. A clinical factor can also be predicted by other parameters, such as markers and / or gene expression surrogates. Clinical factors can include tumor type, tumor subtype, and smoking history.

[0222] "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.

[0223] The term "alphavirus" refers to members of the Togaviridae family, which are single-stranded, positive-sense RNA viruses. Alphaviruses are generally classified as Old World types, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World types, such as Eastern equine encephalitis virus, Aura, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83. Alphaviruses are generally self-replicating RNA viruses.

[0224] The term "alphavirus backbone" refers to the minimal sequence(s) of an alphavirus that allows for autonomous replication of the viral genome. The minimal sequences can include conserved sequences for nonstructural protein-mediated amplification, the nonstructural protein 1 (nsP1), nsP2, nsP3, nsP4 genes, and polyA sequences, as well as sequences for expression of subgenomic viral RNA, including subgenomic (e.g., 26S) promoter elements.

[0225] The term "conserved sequence for nonstructural protein-mediated amplification" includes alphavirus conserved sequence elements (CSEs) well known in the art, including, but not limited to, 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.

[0226] The term "RNA polymerase" includes polymerases that catalyze the production of an RNA polynucleotide from a DNA template, including, but not limited to, bacteriophage-derived polymerases, including T3, T7, and SP6.

[0227] The term "lipid" includes hydrophobic and / or amphipathic molecules. Lipids may be cationic, anionic, or neutral. Lipids may be synthetic or naturally derived, and in certain instances may be biodegradable. Lipids may include cholesterol, phospholipids, lipid conjugates, including but not limited to polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, fats, and fat-soluble vitamins. Lipids may also include dilinoleylmethyl-4-dimethylaminobutyrate (MC3) and MC3-like molecules.

[0228] The term "lipid nanoparticle" or "LNP" includes vesicle-like structures, also known as liposomes, formed with a lipid-containing membrane surrounding an aqueous interior. Lipid nanoparticles include lipid-based compositions with a solid lipid core stabilized by surfactants. The core lipid can be fatty acids, acylglycerols, waxes, and mixtures of these surfactants. Biological membrane lipids, such as phospholipids, sphingomyelin, bile acids (taurocholate), and sterols (cholesterol), can be used as stabilizers. Lipid nanoparticles can be formed using defined ratios of different lipid molecules, including, but not limited to, defined ratios of one or more cationic, anionic, or neutral lipids. Lipid nanoparticles can encapsulate molecules within their outer membrane shell and then be contacted with target cells to deliver the encapsulated molecules to the host cell cytosol. Lipid nanoparticles can be modified or functionalized with non-lipid molecules, such as their surface. Lipid nanoparticles can be monolamellar (unilamellar) or multilamellar (multilamellar). Lipid nanoparticles can be complexed with nucleic acids. Unilamellar lipid nanoparticles can be complexed with nucleic acids, where the nucleic acid is in the aqueous interior. Multilamellar lipid nanoparticles can be complexed with nucleic acids, where the nucleic acid is in the aqueous interior, or forming or sandwiched between them.

[0229] Abbreviations: MHC: major histocompatibility complex; HLA: human leukocyte antigen, or human MHC locus; NGS: next-generation sequencing; PPV: positive predictive value; TSNA: tumor-specific neoantigen; FFPE: formalin-fixed, paraffin-embedded; NMD: nonsense-mediated decay; NSCLC: non-small cell lung cancer; DC: dendritic cell.

[0230] 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.

[0231] Unless otherwise specified or clear from the context, the term "about" used herein is understood to be within the range of normal tolerance in the art, for example, within two standard deviations from the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​set forth herein are modified by the word "about."

[0232] Terms not directly defined herein should be understood to have the meanings generally associated with them as understood within the technical field of the present invention. Certain terms are discussed herein to provide further guidance to the practitioner in describing the compositions, devices, methods, etc. of embodiments of the present invention, as well as how to make or use them. It will be recognized that multiple ways of saying the same thing may be used. Accordingly, alternative terms and synonyms may be used for any one or more of the terms discussed herein. No weight should be placed on whether a term is detailed or discussed herein. Several synonyms or alternative methods, materials, etc. are provided. The recitation of one or more synonyms or equivalents does not exclude the use of other synonyms or equivalents, unless expressly stated. The use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the inventive embodiments herein.

[0233] 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.

[0234] II. Antigen Identification Research models for NGS analysis of tumor and normal exomes and transcriptomes have been previously described and applied in the antigen-specific space. 6,14,15Specific optimizations can be considered to enhance the sensitivity and specificity of antigen identification in clinical settings. 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; for example, such methods are described in more detail in U.S. Pat. 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 by reference in its entirety for all purposes.

[0235] Methods for identifying common antigens (e.g., neoantigens) include identifying antigens derived from a 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 and / or expression data to obtain data representing each peptide sequence of a set of antigens (e.g., in the case of neo-antigens that include at least one mutation that causes the peptide sequence of each neo-antigen to differ from a corresponding wild-type peptide sequence, or in the case of common antigens that do not have mutations 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 has 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.

[0236] III. Identification of tumor-specific mutations in neoantigens Also disclosed herein are methods for identifying specific mutations (e.g., variants or alleles present in cancer cells). Specifically, 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 common neoantigens, are well 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 common 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. Common neoantigens include, but are not limited to, KRAS-associated mutations (e.g., KRAS G12C, KRAS G12V, KRAS G12D, and / or KRAS Q61H mutations). For example, KRAS-associated MHC class I neoepitopes can include these mutations relative to wild-type (WT) human KRAS, such as relative to the following exemplary amino acid sequence: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM (SEQ ID NO: 84).

[0237] Genetic mutations in tumors are considered useful for immunological targeting of tumors when they 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 the protein; (2) read-through mutations in which a stop codon is altered or deleted, resulting in the translation of a longer protein with a new tumor-specific sequence at the C-terminus; (3) splice site mutations that introduce an intron into mature mRNA, thereby generating a unique tumor-specific protein sequence; (4) chromosomal rearrangements (i.e., gene fusions) that result in chimeric proteins with tumor-specific sequences at the junction of two proteins; and (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 non-frameshift indels, missense or nonsense substitutions, splice site changes, genome rearrangements or gene fusions, or any genome or expression changes that result in neo-ORFs.

[0238] For example, peptides or mutant polypeptides with mutations resulting from splice site, frameshift, readthrough, or gene fusion mutations in tumor cells can be identified by sequencing tumor DNA, RNA, or proteins relative to normal cells.

[0239] Mutations can also include previously identified tumor-specific mutations. Known tumor mutations can be found in the COSMIC (Catalogue of Somatic Mutations in Cancer) database.

[0240] Various methods are available for detecting the presence of specific mutations or alleles in an individual's DNA or RNA. Advances in this field have enabled accurate, easy, and inexpensive large-scale SNP genotyping. Several techniques have been described, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and various DNA "chip" technologies such as the Affymetrix SNP chip. These methods generally rely on PCR amplification of target gene regions. 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 known in the art for detecting specific mutations are summarized below.

[0241] PCR-based detection methods can involve simultaneous multiplex amplification of multiple markers. 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 methods allow for differential detection of multiple PCR products in a sample. Other techniques that allow for multiplex analysis of multiple markers are also known in the art.

[0242] 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 (U.S. Pat. No. 4,656,127). According to this method, a primer complementary to the allele sequence immediately 3' from the polymorphic site is hybridized to a target molecule obtained from a specific animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to a specific exonuclease-resistant nucleotide derivative present, the derivative is incorporated at the end of the hybridized primer. This incorporation makes the primer resistant to exonucleases, thereby enabling 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(s) 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.

[0243] Solution-based methods can be used to determine the identity of the nucleotide at a polymorphic site (Cohen, D. et al. (French Patent No. 2,650,840; PCT Application No. WO 91 / 02087)). As in the method of Mundy, U.S. Pat. No. 4,656,127, a primer is used that is complementary to the allelic sequence immediately 3' to the polymorphic site. This method uses a labeled dideoxynucleotide derivative that is incorporated onto the end of the primer when it is complementary to the nucleotide at the polymorphic site to determine the identity of the nucleotide at that site.

[0244] An alternative method known as Genetic Bit Analysis (GBA) is described by Goelet, P. et al. (PCT Application No. 92 / 15712). The Goelet, P. et al. method uses a mixture of labeled terminators and primers complementary to the sequence 3' of the polymorphic site. Thus, the incorporated labeled terminators are determined by and complementary to the nucleotides present at the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (FR 2,650,840; PCT Application No. WO91 / 02087), the Goelet, P. et al. method can be a heterogeneous phase assay in which the primers or target molecules are immobilized on a solid phase.

[0245] 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. 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, signal is proportional to the number of incorporated deoxynucleotides, so polymorphisms occurring in multiple runs of the same nucleotide can produce signals proportional to the length of the run (Syvanen, A.-C., et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0246] Many techniques directly obtain sequence information from millions of individual molecules of DNA or RNA in parallel. Real-time single molecule sequencing-by-synthesis techniques rely on the detection of fluorescent nucleotides as they are incorporated into nascent strands of DNA complementary to the template being sequenced. In one method, 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 with capture tails complementary to the surface-bound oligonucleotides. They also serve as primers for template-guided primer extension, which 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 mixture, washing, imaging, and dye cleavage. 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 the fluorescently labeled polymerase with the fluorescently modified nucleotide as the nucleotide is incorporated into the nascent strand. Other sequencing-by-synthesis techniques also exist.

[0247] Any suitable sequencing-by-synthesis platform can be used to identify mutations. As mentioned above, four major sequencing-by-synthesis platforms are currently available: the Genome Sequencer 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 acids on the support, capture sequences / universal priming sites can be added to the 3' and / or 5' ends of the template. Nucleic acids 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 dually function as a universal primer.

[0248] As an alternative to capture sequences, 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 on a surface coated with the corresponding second member of the coupling pair.

[0249] After capture, the sequence can be analyzed by single-molecule detection / sequencing, for example, as described in the Examples and / or U.S. Patent No. 7,283,337, including template-dependent sequencing by stepwise synthesis. In stepwise synthesis sequencing, 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 or in a 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.

[0250] 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 Gene Reader, and Oxford Nanopore MinION. Other similar current massively parallel sequencing technologies, as well as future generations of these technologies, can be used.

[0251] 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 body fluids such as blood or saliva obtained by known methods (e.g., venipuncture). Alternatively, nucleic acid testing can be performed on dried samples (e.g., hair or skin). Furthermore, a sample for sequencing can be obtained from a tumor, and another sample can be obtained from normal tissue of the same tissue type as the tumor for sequencing. A sample for sequencing can be obtained from a tumor, and another sample can be obtained from normal tissue of a different tissue type from the tumor for sequencing.

[0252] The tumor may include one or more of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and T-cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0253] 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.

[0254] IV. Antigen Antigens can include nucleotides or polypeptides. For example, antigens can be RNA sequences that encode polypeptide sequences. Thus, antigens useful in vaccines include nucleotide sequences or polypeptide sequences.

[0255] Disclosed herein are isolated peptides comprising tumor-specific mutations identified by the methods disclosed herein, peptides comprising known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. 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.

[0256] Specifically, the present specification discloses a cassette comprising repeats of KRAS-associated MHC class I neoepitopes. Examples of KRAS-associated MHC class I neoepitopes include, but are not limited to, neoepitopes with a KRAS G12 mutation and / or a KRAS Q61 mutation. The cassette can comprise repeats of KRAS-associated MHC class I neoepitopes with a KRAS G12 mutation. The cassette can comprise repeats of KRAS-associated MHC class I neoepitopes with a KRAS Q61 mutation. The cassette can comprise repeats of KRAS-associated MHC class I neoepitopes with a KRAS G12C, KRAS G12V, KRAS G12D, and / or KRAS Q61H mutation. The cassette can comprise repeats of KRAS-associated MHC class I neoepitopes with a KRAS G12C mutation. The cassette can comprise repeats of a KRAS-associated MHC class I neoepitope having a KRAS G12V mutation. The cassette can comprise repeats of a KRAS-associated MHC class I neoepitope having a KRAS G12D mutation. The cassette can comprise repeats of a KRAS-associated MHC class I neoepitope having a KRAS Q61H mutation. The cassette can comprise repeats of each of KRAS-associated MHC class I neoepitopes having KRAS G12C, KRAS G12V, KRAS G12D, and KRAS Q61H mutations. The cassette can comprise repeats of at least two different KRAS-associated MHC class I neoepitopes selected from the group consisting of KRAS G12C, KRAS G12V, KRAS G12D, and / or KRAS Q61H mutations. The cassette can comprise repeats of at least three different KRAS-associated MHC class I neoepitopes selected from the group consisting of KRAS G12C, KRAS G12V, KRAS G12D, and / or KRAS Q61H mutations. The cassette can comprise repeats of only a single different KRAS-associated MHC class I neoepitope. The cassette can comprise repeats of only a single different KRAS-associated MHC class I neoepitope with a KRAS G12C mutation.The cassette can comprise repeats of only a single, different KRAS-associated MHC class I neoepitope with a KRAS G12D mutation. The cassette can comprise repeats of only a single, different KRAS-associated MHC class I neoepitope with a KRAS G12V mutation. The cassette can comprise repeats of only a single, different KRAS-associated MHC class I neoepitope with a KRAS Q61H mutation.

[0257] KRAS-associated MHC class I neoepitopes with KRAS G12C mutations include VVVGACGVGK (SEQ ID NO: 75), KLVVVGACGV (SEQ ID NO: 76), or GACGVGKSAL. KRAS-associated MHC class I neoepitopes with KRAS G12D mutations include VVGADGVGK (SEQ ID NO: 77), VVVGADGVGK (SEQ ID NO: 78), KLVVVGADG, or GADGVGKSAL. KRAS-associated MHC class I neoepitopes with KRAS G12V mutations include VVGAVGVGK (SEQ ID NO: 79), VVVGAVGVGK (SEQ ID NO: 81), AVGVGKSAL (SEQ ID NO: 80), or GAVGVGKSAL.

[0258] The cassette can comprise repeats of each of the KRAS-associated MHC class I neoepitopes having the amino acid sequences VVVGACGVGK (SEQ ID NO:75), VVVGADGVGK (SEQ ID NO:78), VVGAVGVGK (SEQ ID NO:79), and ILDTAGHEEY (SEQ ID NO:82). The cassette can comprise repeats of at least two different KRAS-associated MHC class I neoepitopes having amino acid sequences selected from the amino acid sequences VVVGACGVGK (SEQ ID NO:75), VVVGADGVGK (SEQ ID NO:78), VVGAVGVGK (SEQ ID NO:79), and ILDTAGHEEY (SEQ ID NO:82). The cassette can comprise repeats of at least three different KRAS-associated MHC class I neoepitopes having amino acid sequences selected from the amino acid sequences VVVGACGVGK (SEQ ID NO:75), VVVGADGVGK (SEQ ID NO:78), VVGAVGVGK (SEQ ID NO:79), and ILDTAGHEEY (SEQ ID NO:82). The cassette can include repeats of at least one of the KRAS-associated MHC class I neoepitopes having the amino acid sequences VVVGACGVGK (SEQ ID NO: 75), VVVGADGVGK (SEQ ID NO: 78), VVGAVGVGK (SEQ ID NO: 79), and ILDTAGHEEY (SEQ ID NO: 82).

[0259] KRAS-associated MHC class I neoepitopes can include the native N- and / or C-terminal flanking sequences of a therapeutic vaccine epitope in the context of the native KRAS protein. Illustrative, non-limiting examples of KRAS-associated MHC class I neoepitopes include the 25-mer MTEYKLVVVGACGVGKSALTIQLIQ (SEQ ID NO: 57) for KRAS G12C, MTEYKLVVVGADGVGKSALTIQLIQ (SEQ ID NO: 58) for KRAS G12D, MTEYKLVVVGAVGVGKSALTIQLIQ (SEQ ID NO: 59) for KRAS G12V, and ETCLLDILDTAGHEEYSAMRDQYMR (SEQ ID NO: 60) for KRAS Q61H. A KRAS-associated MHC class I neoepitope comprising a natural flanking sequence can be linked (concatenated) with other neoepitopes encoded within the cassette, including other neoepitopes (e.g., other KRAS-associated MHC class I neoepitopes) comprising their respective natural flanking sequences. An exemplary, non-limiting cassette of linked KRAS-associated MHC class I neoepitopes comprising four repeats each of KRAS neoepitopes with mutations KRAS G12C, KRAS G12D, KRAS G12V, and KRAS Q61H, linked by natural flanking sequences, is represented by the amino acid sequence set forth in SEQ ID NO:65.

[0260] Epitope-encoding nucleic acid sequences encoding KRAS-associated MHC class I neoepitopes, including those including naturally occurring N- and / or C-terminal flanking sequences, can encode multiple known and / or predicted KRAS-associated MHC class I neoepitopes. As an illustrative example, the KRAS G12V 25-mer MTEYKLVVVGAVGVGKSALTIQLIQ (SEQ ID NO:59) encodes each of the known and / or predicted KRAS-associated MHC class I neoepitopes VVGAVGVGK (SEQ ID NO:79), VVVGAVGVGK (SEQ ID NO:81), and AVGVGKSAL (SEQ ID NO:80).

[0261] Epitope-encoding nucleic acid sequences, including those encoding KRAS-associated MHC class I neoepitopes, can be arranged in any order within the cassette. Epitope-encoding nucleic acid sequences, including those encoding KRAS-associated MHC class I neoepitopes, can be arranged in an order that minimizes junction epitopes, as further described herein. As an illustrative, non-limiting example, a linked KRAS-associated MHC class I neoepitopes linked together to minimize junction epitopes is represented by the amino acid sequence set forth in SEQ ID NO: 65, and has the order G12C G12D Q61H G12D G12V G12C Q61H G12D G12V G12C Q61H G12D G12V Q61H G12V G12C.

[0262] The present specification also discloses peptides derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues, e.g., any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissues compared to normal cells or tissues. Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database. COSMIC curates comprehensive information on somatic mutations in human cancers. Peptides include tumor-specific mutations. Tumor antigens (e.g., common tumor antigens and tumor neoantigens) 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 encode the relevant polypeptide sequence.

[0263] 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.

[0264] The one or more polypeptides encoded by the antigen nucleotide sequence can comprise at least one of the following: a binding affinity to MHC with an IC50 value of less than 1000 nM; a length of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids for MHC class I peptides; the presence of a sequence motif within or near the peptide that promotes proteasomal cleavage and the presence of a sequence motif that promotes TAP transport; a length of 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids for MHC class II polypeptides; the presence of a sequence motif within or near the peptide-promoted cleavage site by an extracellular or lysosomal protease (e.g., cathepsin) or the HLA binding site catalyzed by HLA-DM.

[0265] The one or more antigens can be present on the surface of the tumor.

[0266] 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 the subject). One or more antigens may be capable of stimulating a B cell response, such as the production of antibodies that recognize one or more antigens (e.g., antibodies that recognize the tumor). Antibodies can recognize linear polypeptide sequences or secondary and tertiary structures. Thus, B cell antigens can include linear polypeptide sequences or polypeptides having secondary and tertiary structures, including, but not limited to, full-length proteins, protein subunits, protein domains, or any polypeptide known or predicted to have secondary and tertiary structures. An antigen capable of stimulating a B cell response against a tumor may be an antigen found on the surface of tumor cells. An antigen capable of inducing a B cell response against a tumor may be an intracellular neoantigen expressed in the tumor.

[0267] 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).

[0268] 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.

[0269] 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 The antigenic peptide molecule can include, but is not limited to, 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 60, about 70, about 80, about 90, about 100, about 110, about 120, or more amino acid residues, and any range derivable therein. In a specific embodiment, the antigenic peptide molecule is 50 or fewer amino acids.

[0270] Antigenic peptides and polypeptides can be 15 residues or less in length, typically between about 8 and about 11 residues, particularly 9 or 10 residues, for MHC class I; and 6 to 30 residues for MHC class II.

[0271] If desired, longer peptides can be designed in several ways. In one example, when the likelihood of peptide presentation on HLA alleles is predicted or known, the longer peptides can consist of either (1) individual presented peptides with extensions of 2-5 amino acids toward the N- and C-termini of each corresponding gene product; or (2) a concatenation of some or all of the presented peptides, each with its extended sequence. In another case, when sequencing reveals the presence of long (more than 10 residues) neoepitope sequences in the tumor (e.g., due to frameshifts, readthrough, or intron introduction resulting in novel peptide sequences), the longer peptides can consist of the entire novel tumor-specific stretch of amino acids (thus eliminating the need to select the most strongly HLA-presented shorter peptides based on computational or in vitro selection). In either case, longer peptides may allow for endogenous processing by patient 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.

[0272] Antigenic peptides and polypeptides can be presented on HLA proteins. In some embodiments, the antigenic peptide or polypeptide is presented on HLA proteins with higher affinity than the wild-type peptide. In some embodiments, the antigenic peptide or polypeptide can have an IC50 of at least 5000 nM or less, at least 1000 nM or less, at least 500 nM or less, at least 250 nM or less, at least 200 nM or less, at least 150 nM or less, at least 100 nM or less, at least 50 nM or less, or less.

[0273] In some embodiments, the antigenic peptides and polypeptides do not stimulate an autoimmune response and / or do not induce immune tolerance when administered to a subject.

[0274] Compositions comprising at least two or more antigenic peptides are also provided. In some embodiments, the compositions comprise at least two different peptides. At least two different peptides may be derived from the same polypeptide. Different peptides mean that the peptides differ in length, amino acid sequence, or both. The peptide may comprise a tumor-specific mutation. The tumor-specific peptide can be derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissue compared to normal cells or tissues, e.g., any polypeptide known or found to contain a tumor-specific mutation or peptide derived from any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissue compared to normal cells or tissues. The peptide can be derived from any polypeptide known or suspected to be associated with an infectious disease organism, or the peptide can be derived from any polypeptide known or found to have altered expression in infected cells compared to normal cells or tissues (e.g., an infectious disease polynucleotide or polypeptide including an infectious disease polynucleotide or polypeptide 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. The peptide can include a KRAS mutation (e.g., a KRAS G12C, KRAS G12V, KRAS G12D, and / or KRAS Q61H mutation).

[0275] Antigenic peptides and polypeptides with desired activities or properties can be modified to confer certain desirable attributes, e.g., improved pharmacological characteristics, while enhancing or at least retaining substantially all of the biological activity of the unmodified peptide, which binds to desired MHC molecules and activates appropriate T cells. For example, antigenic peptides and polypeptides can be further subjected to various modifications, such as conservative or non-conservative substitutions, which may provide certain advantages in their use, such as improved MHC binding, stability, or presentation. Conservative substitutions refer to the replacement of an amino acid residue with another that is biologically and / or chemically similar, e.g., one hydrophobic residue with another hydrophobic residue, or one polar residue with another polar residue. Substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effects of single amino acid substitutions can also be explored using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2nd Ed. (1984).

[0276] Modification of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful for increasing peptide and polypeptide stability in vivo. Stability can be assayed in a number of ways. For example, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Peptide half-life can be conveniently determined using a 25% human serum (v / v) assay. The protocol generally follows: Pooled human serum (type AB, non-heat-inactivated) is defatted by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, small aliquots of the reaction solution are removed and added to either 6% aqueous trichloroacetic acid or ethanol. The cloudy reaction sample is cooled (4°C) for 15 minutes and then spun to precipitate the precipitated serum proteins. The presence of the peptide is then determined by reverse-phase HPLC using stability-specific chromatography conditions.

[0277] Peptides and polypeptides can be modified to provide desirable attributes other than improved serum half-life. For example, the ability of a peptide to stimulate CTL activity can be enhanced by linking it 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 from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that the optional spacer need not be composed of the same residues and can therefore be a hetero- or homo-oligomer. If present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the peptide can be linked to the T helper peptide without a spacer.

[0278] The antigenic peptide can be linked to the T helper peptide at either the amino or carboxy terminus of the peptide, either directly or via a spacer. The amino terminus of either the antigenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid at 830-843, influenza at 307-319, and malaria sporozoites at around 382-398 and 378-389.

[0279] Proteins or peptides can be produced by any technique known to those of skill in the art, including expressing proteins, polypeptides, or peptides through standard molecular biology techniques, isolating proteins or peptides from natural sources, or chemically synthesizing proteins or peptides. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed and can be found in computerized databases known to those of skill in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, located on the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as known to those of skill in the art. Alternatively, various commercial preparations of proteins, polypeptides, and peptides are known to those of skill in the art.

[0280] In a further embodiment, the antigen comprises a nucleic acid (e.g., a polynucleotide) encoding an antigenic peptide or a portion thereof. The polynucleotide can be, for example, a single-stranded and / or double-stranded polynucleotide, such as DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), or a polynucleotide having a phosphorothioate backbone, in either a native 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 replacing low-frequency codons with high-frequency synonymous codons with respect to the codon bias of a given organism. Polynucleotide sequences can be optimized to improve post-transcriptional processing, for example, by removing splicing motifs (e.g., canonical and / or cryptic / non-canonical splice donor, branch, and / or acceptor sequences) to bias favorable splicing events and / or by introducing exogenous splicing motifs (splice donor, branch, and / or acceptor sequences). 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-globin 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.Polynucleotide sequences 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. Polynucleotide sequences can be optimized to improve accurate transcription, for example, by removing cryptic transcription initiators and / or terminators. Polynucleotide sequences 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. Polynucleotide sequences can be optimized to improve nuclear export of transcripts, for example, 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 given organism. Sequence optimization can balance one or more sequence properties, such as transcription, translation, post-transcriptional processing, and / or RNA stability. Sequence optimization can generate optimal sequences that balance 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), CoolTool (University of Singapore), and SGI-DNA (La Jolla California). One or more regions of an antigen-encoding protein can be sequence-optimized separately.

[0281] A further aspect provides an expression vector capable of expressing a polypeptide or a portion thereof. Expression vectors for various cell types are well known in the art and can be selected without undue experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, in the proper orientation and correct reading frame for expression. If necessary, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host; such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0282] V. Vaccine Compositions Also disclosed herein are immunogenic compositions, e.g., vaccine compositions, that can generate specific immune responses, e.g., tumor-specific immune responses. Vaccine compositions typically contain one or more antigens selected using the methods described herein. Vaccine compositions can also be referred to as vaccines.

[0283] The vaccine can comprise 1 to 30 peptides, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different peptides, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different peptides, or 12, 13, or 14 different peptides. 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 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 1–30 antigen sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 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.

[0284] The vaccines contain 1 to 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, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 The antigen-encoding 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. An antigen-encoding nucleic acid sequence may also be referred to as the antigen-encoding portion of an "antigen cassette." The characteristics of an 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).

[0285] The vaccines contain 1 to 30 different epitope-encoding nucleic acid sequences: , 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 a linked T cell epitope-encoding nucleic acid sequence.

[0286] The vaccine can comprise 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 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 are shown by, but not limited to, the following 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 - Repeats 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

[0287] 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 determined by, for example, 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, as in the example with epitopes A, B, and C according to the formula:

[0288] Provided herein is an antigen-encoding cassette having at least one antigen-encoding nucleic acid sequence described in the 5' to 3' direction by the formula: (E x -(E N n ) y ) z During the ceremony, E represents a nucleotide sequence comprising a different epitope-encoding nucleic acid sequence; n represents the number of distinct epitope-encoding nucleic acid sequences and is any integer, including 0; E N represents a nucleotide sequence comprising said distinct epitope-encoding nucleic acid sequence 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, given E N or a combination thereof. In some embodiments, at least one of the different epitope-encoding nucleic acid sequences comprising at least two repeats encodes a KRAS-associated MHC class I neoepitope.

[0289] Each E or EN 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-antigenic epitope). For example, each E or E N is expressed in the 5' to 3' direction by the formula (L5 b -N c -L3 d ), wherein N is each E or E N wherein c=1, L5 comprises a 5' linker sequence wherein b=0 or 1, and L3 comprises a 3' linker sequence wherein d=0 or 1. Epitopes and linkers that can be used are further described herein.

[0290] 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 -...). Repeats of an epitope-encoding nucleic acid sequence may be separated by one or more additional nucleotide sequences. Generally, repeats of an epitope-encoding nucleic acid sequence may be separated by a nucleotide sequence of any size applicable to the compositions described herein. In one example, repeats of an epitope-encoding nucleic acid sequence may be separated by distinct and different 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 may be, for example, E A -E B -E A...(EA are separated by 75 nucleotides). One illustrative example is an antigen-encoding nucleic acid having the sequence VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDTVTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT (SEQ ID NO: 85) that encodes repeats of the 25-mer antigens Trp1 (VTNTEMFVTAPDNLGYMYEVQWPGQ) [SEQ ID NO: 86] and Trp2 (TQPQIANCSVYDFFVWLHYYSVRDT) [SEQ ID NO: 87], where the Trp1 repeats are separated by a 25-mer Trp2, and therefore 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, 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 separated by 150, 225, 300, 375, 450, 525, 600, or 675 nucleotides, respectively.

[0291] In one embodiment, the different peptides and / or polypeptides, or the nucleotide sequences encoding them, are selected such that the peptides and / or polypeptides are capable of binding to different MHC molecules, such as different MHC class I molecules and / or different MHC class II molecules. In some embodiments, a vaccine composition comprises coding sequences for peptides and / or polypeptides capable of binding to the most frequently occurring MHC class I molecules and / or different MHC class II molecules. Thus, the vaccine composition can comprise different fragments capable of binding to at least two preferred, at least three preferred, or at least four preferred MHC class I molecules and / or different MHC class II molecules.

[0292] The vaccine composition is capable of stimulating a specific cytotoxic T cell response and / or a specific helper T cell response.The vaccine composition is capable of stimulating a specific cytotoxic T cell response and a specific helper T cell response.

[0293] The vaccine composition can stimulate a specific B cell response (eg, an antibody response).

[0294] 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.

[0295] 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, for example, an antigen-presenting cell, such as a dendritic cell (DC), capable of presenting a protein or peptide to a T cell.

[0296] An adjuvant is any substance that, when mixed into a vaccine composition, enhances or otherwise modifies the immune response to an antigen. A carrier can be a scaffold structure, such as a polypeptide or polysaccharide, to which an antigen can associate. Optionally, the adjuvant is attached by covalent or non-covalent bonds.

[0297] The ability of adjuvants to increase the immune response to antigens is generally manifested as a significant or substantial increase in immune-mediated reactions or a reduction in disease symptoms.For example, an increase in humoral immunity is generally manifested as a significant increase in the titer of antibodies produced against antigens, and an increase in T cell activity is generally manifested as an increase in cell proliferation, cytotoxicity, or cytokine secretion.Adjuvants can also change immune responses, for example, by changing a primarily humoral or Th response to a primarily cellular or Th response.

[0298] 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 (Aquila Biotech, Worcester, Mass., USA), which is derived from saponin, mycobacterium extracts and synthetic bacterial wall mimics, and other proprietary adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are also useful. Several immunological adjuvants specific for dendritic cells (e.g., MF59) and their 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 herein by reference 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).

[0299] CpG immunostimulatory oligonucleotides have also been reported to enhance the effectiveness of adjuvants in a vaccine setting. Other TLR-binding molecules, such as RNA-binding TLR 7, TLR 8, and / or TLR 9, can also be used.

[0300] 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 adjuvants, 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 ordinary skill in the art without undue experimentation. Further adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0301] A vaccine composition can include multiple different adjuvants. Additionally, a therapeutic composition can include any adjuvant material, including any of the above or a combination thereof. The vaccine and adjuvant can be administered together or separately in any suitable order.

[0302] A carrier (or excipient) may be present independently 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. Furthermore, 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, such as 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; or 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 dextran, such as Sepharose.

[0303] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules, rather than intact foreign antigens themselves. MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, CTL activation is possible in the presence of a trimeric complex of peptide antigen, MHC molecule, and APC. Correspondingly, CTLs can enhance immune responses not only when peptides alone are used to activate CTLs, but also when APCs bearing the corresponding MHC molecules are added. Thus, in certain embodiments, the vaccine composition further comprises at least one antigen-presenting cell.

[0304] Antigens can also be derived from vaccinia, fowlpox, self-replicating alphaviruses, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including, but not limited to, second, third, or hybrid second / third generation lentiviruses, and any generation of recombinant lentiviruses designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human They can also be included in viral vector-based vaccine platforms, such as the ubiquitin C promoter, Nucl. Acids Res. (2015) 43(1):682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72(12):9873-9880. 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 linkers, or preceded by one or more sequences that target intracellular compartments (see, e.g., Gros et al., Prospective identification of antigen-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 to the peptide. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette-Guerin). BCG vectors are described by Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful for therapeutic administration of antigens or immunization, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the description herein.

[0305] VA antigen cassette The methods used to select one or more antigens, cloning and construction of an "antigen cassette," and its insertion into a viral vector are within the skill of the art in light of the teachings provided herein. An "antigen cassette" or "cassette" refers to 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 transcripts. The selected antigen or antigens may refer to different epitope sequences (e.g., an antigen-encoding nucleic acid sequence within a cassette can encode an epitope-encoding nucleic acid sequence (or multiple epitope-encoding nucleic acid sequences) such that the epitopes are transcribed and expressed). The antigen or antigens can be operably linked to regulatory elements in a manner that allows transcription. Such elements include conventional regulatory elements capable of directing expression of the antigen(s) in cells transfected with the viral vector. Thus, the antigen cassette can also include a selected promoter linked to the antigen(s) and positioned, along with other optional regulatory elements, within the selected viral sequence of the recombinant vector. The cassette can contain one or more antigens (e.g., one or more KRAS-associated neoepitopes in a vaccine composition, such as any of the KRAS-associated neoepitopes set forth in SEQ ID NOS: 75-82). The cassette can contain one or more antigen-encoding nucleic acid sequences, each operably linked independently to a separate promoter and / or linked together using other multicistronic systems, such as 2A ribosomal skipping sequence elements (e.g., E2A, P2A, F2A, or T2A sequences) or internal ribosome entry site (IRES) sequence elements. The linker can also contain a cleavage site, such as a TEV or furin cleavage site. Linkers containing 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 the furin protease cleavage site to facilitate removal of the 2A sequence after translation. In a cassette comprising multiple antigen-encoding nucleic acid sequences, each antigen-encoding nucleic acid sequence can comprise one or more epitope-encoding nucleic acid sequences (e.g., antigen-encoding nucleic acid sequences encoding linked T cell epitopes).

[0306] Useful promoters may be constitutive promoters or regulated (inducible) promoters that allow for control of the amount of antigen(s) expressed. For example, a desirable promoter is the cytomegalovirus immediate early promoter / enhancer [see, e.g., Boshart et al., Cell, 41:521-530 (1985)]. Another desirable promoter is the Rous sarcoma virus LTR promoter / enhancer. Yet another promoter / enhancer sequence is the chicken β-actin promoter [TAKost et al., Nucl. Acids Res., 11(23):8287 (1983)]. Those skilled in the art can also select other suitable or desirable promoters.

[0307] Also disclosed herein is a viral vector comprising a cassette having at least one payload sequence operably linked to a regulatable promoter, such as a TET promoter system, such as a TET-On system or a TET-Off system. Without wishing to be bound by theory, the use of a TET promoter system can minimize transcription of the payload nucleic acid encoded in the cassette, such as an antigen encoded in the vaccine cassette, during virus production. The TET promoter system is described in more detail in International Patent Application Publication No. WO2020 / 243719, the entire contents of which are incorporated herein by reference for all purposes.

[0308] The TET promoter system can include a promoter controlled by the tetracycline (TET) repressor protein (TETr). Accordingly, a viral vector is also disclosed herein that includes a cassette having at least one payload sequence operably linked to a promoter controlled by the tetracycline (TET) repressor protein (TETr). The TETr-controlled promoter can include the 19-bp TET operator (TETo) sequence TCCCTATCAGTGATAGAGA (SEQ ID NO: 83). The TETr-controlled promoter can include 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more TETo nucleic acid sequences. In a TETr-controlled promoter having two or more TETo nucleic acid sequences, the TETo sequences can be linked to each other. In a TETr-controlled promoter having two or more TETo nucleic acid sequences, the TETo sequences can be directly linked to each other. In a TETr-controlled promoter having two or more TETo nucleic acid sequences, the TETo sequences may be linked to each other by a linker sequence, such as a linker sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides. Generally, the TETr-controlled promoter can use any desired promoter sequence, such as an SV40, EF-1, RSV, PGK, HSA, MCK, or EBV promoter sequence. The TETr-controlled promoter can use a CMV promoter sequence. The TETr-controlled promoter can use a minimal CMV promoter sequence. The TETo sequence can be located upstream (5') of the promoter sequence region to which RNA polymerase binds. In one illustrative example, seven TETo sequences are located upstream (5') of the promoter sequence. A TETr-controlled promoter operably linked to at least one payload nucleic acid sequence, with the TETo sequence upstream of the promoter sequence region, can have, in the 5' to 3' direction, an ordered sequence described by the formula: (TL Y ) X -PN wherein N is a payload nucleic acid sequence, P is an RNA polymerase binding sequence of a promoter sequence operably linked to the payload nucleic acid sequence, T is a TETo nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO: 66, and L is a linker sequence, with the proviso that for each X, Y=0 or 1, and X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one illustrative example, X=7 and Y=1 for each X describes the case where seven TETo sequences are upstream (5') of the promoter sequence, with each TETo sequence separated by a linker.

[0309] The TETo sequence may be downstream (3') of the promoter sequence region to which RNA polymerase binds. In another illustrative example, two TETo sequences are downstream (3') of the promoter sequence. A TETr-controlled promoter operably linked to at least one payload nucleic acid sequence, with a TETo sequence downstream of the promoter sequence region, can have an ordered sequence described by the following formula in the 5' to 3' direction: P-(TL Y ) X -N wherein N is a payload nucleic acid sequence, P is an RNA polymerase binding sequence of a promoter sequence operably linked to the payload nucleic acid sequence, T is a TETo nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO: 66, and L is a linker sequence, with the proviso that for each X, Y=0 or 1, and X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one illustrative example, X=2 and Y=1 for each X describes the case where two TETo sequences are downstream (3') of the promoter sequence, and each TETo sequence is separated by a linker.

[0310] For viral production of vectors having a TETr-regulated promoter, any viral producer cell line engineered to express a TETr sequence (tTS), such as the 293 cell line or its derivatives (e.g., the 293F cell line) engineered to express tTS, can be used. Viral production of vectors having a TETr-regulated promoter in tTS-expressing cells can improve viral production. Viral production of vectors having a TETr-regulated promoter in tTS-expressing cells can improve viral infectivity, defined as viral particles (VP) per infectious unit (IU). Viral production of vectors having a TETr-regulated promoter in tTS-expressing cells can improve viral production and / or viral infectivity by at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10-fold compared to production in non-tTS-expressing cells. Viral production of vectors having a TETr-regulated promoter in tTS-expressing cells can improve viral production and / or viral infectivity by 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 60, at least 70, at least 80, at least 90, or at least 100-fold compared to production in non-tTS-expressing cells. Viral production of vectors having a TETr-regulated promoter in tTS-expressing cells can improve viral production and / or viral infectivity by at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10-fold compared to production of vectors without a TETr-regulated promoter.Viral production of vectors with a TETr-regulated promoter in tTS-expressing cells can improve viral production and / or viral infectivity by 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 60, at least 70, at least 80, at least 90, or at least 100-fold compared to production of vectors without a TETr-regulated promoter.

[0311] The antigen cassette may also contain nucleic acid sequences heterologous to the viral vector sequence, including sequences providing signals for efficient polyadenylation of the transcript (poly(A), polyA, or pA) and introns containing functional splice donor and acceptor sites. A common polyA sequence used in the exemplary vectors herein is derived from the papovavirus SV-40. The polyA sequence can be inserted into the cassette after the antigen-based sequence and before the viral vector sequence. A common intron sequence may also be derived from SV-40 and is referred to as the SV-40T intron sequence. The antigen cassette may also contain an intron located between the promoter / enhancer sequence and the antigen(s). The selection of these and other common vector elements is conventional (see, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual," 2d ed., Cold Spring Harbor Laboratory, New York (1989) and references cited therein), and many such sequences are available from commercial and industrial sources, as well as Genbank.

[0312] An antigen cassette can have one or more antigens. For example, a given cassette can contain 1-10, 1-20, 1-30, 10-20, 15-25, 15-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens. The antigens can be directly linked to each other. The antigens can be linked to each other by a linker. The antigens can be in any orientation relative to each other, including N to C or C to N.

[0313] 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 deleted E3 gene region of a ChAd-based vector, among other choices.

[0314] 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 [Where 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, wherein 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 can comprise at least one of tetanus toxoid and PADRE. The universal sequence can comprise a tetanus toxoid peptide. The universal sequence can comprise a PADRE peptide. The universal sequence can 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.

[0315] In one example, the elements present are a=0, b=1, d=1, e=1, g=1, h=0, X=10, Y=2, Z=1, and W=1, and when the case is described where there is no additional promoter (e.g., only promoter nucleotide sequences provided by a vector backbone such as an RNA alphavirus backbone are present), there are 10 MHC class I epitopes, there is a 5' linker for each N, there is a 3' linker for each N, there are two MHC class II epitopes, there is a linker connecting the two MHC class II epitopes, there is a linker connecting the 5' ends of the two MHC class II epitopes to the 3' linker of the last MHC class I epitope, and there is a linker connecting the 3' ends of the two MHC class II epitopes to the 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 direct linkage to a 3' UTR element provided by the vector backbone, such as a 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 direct linkage 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.

[0316] 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 drives expression of one or more different MHC Class I epitope-encoding nucleic acid sequences; when h=1, describing the presence of another promoter that drives 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) directly linked to the vector backbone (e.g., an RNA alphavirus backbone).

[0317] Other examples include cases where each MHC class I epitope present has a 5' linker, a 3' linker, neither, or both. In examples where multiple MHC class I epitopes are present in the same antigen cassette, some MHC class I epitopes may have both a 5' linker and a 3' linker, while other MHC class I epitopes may have a 5' linker, a 3' linker, or neither. In other examples where multiple MHC class I epitopes are present in the same antigen cassette, some MHC class I epitopes may have either a 5' linker or a 3' linker, while other MHC class I epitopes may have a 5' linker, a 3' linker, or neither.

[0318] 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.

[0319] 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, 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, a 3' linker, or neither.

[0320] The promoter nucleotide sequences P and / or P2 can be the same as the promoter nucleotide sequences provided by the vector backbone, such as the RNA alphavirus backbone. For example, the promoter sequences Pn and P2 provided by the vector backbone can each comprise a subgenomic promoter sequence (e.g., a 26S subgenomic promoter sequence) or a CMV promoter. The promoter nucleotide sequences P and / or P2 can be different from the promoter nucleotide sequences provided by the vector backbone (e.g., the RNA alphavirus backbone) and can also be different from each other.

[0321] The 5' linker L5 can be a natural or non-natural sequence. Non-natural sequences include, but are not limited to, AAY, RR, and DPP. The 3' linker L3 can also be a natural or non-natural sequence. Furthermore, L5 and L3 can both be natural sequences, both can be non-natural sequences, or one can be natural and the other can be non-natural. For each X, the amino acid linker may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, The lengths may be 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more. For each X, the amino acid linker may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0322] 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.

[0323] The amino acid linker G3 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more lengths. G3 may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0324] For each X, each N can encode an MHC class I epitope, an MHC class II epitope, an epitope / antigen capable of stimulating a B cell response, or a combination thereof. For each X, each N can encode 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 encode a combination of an MHC class I epitope and an MHC class II epitope. For each X, each N can encode 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 encode 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 encode an MHC class II epitope. For each X, each N can encode an epitope / antigen capable of stimulating a B cell response. For each X, each N can encode an MHC class I epitope 7 to 15 amino acids in length. For each X, each N may encode an MHC class I epitope 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. For each X, each N may also encode an MHC class I epitope at least 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.

[0325] A cassette encoding one or more antigens may be 700 nucleotides or less. A 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). A cassette encoding one or more antigens may be 700 nucleotides or less and may encode at least two different epitope-encoding nucleic acid sequences. A cassette encoding one or more antigens may be 700 nucleotides or less and may encode three different epitope-encoding nucleic acid sequences. A cassette encoding one or more antigens may be 700 nucleotides or less and may encode at least three different epitope-encoding nucleic acid sequences. A 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.

[0326] 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.

[0327] 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 the 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 the 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.

[0328] 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.

[0329] In some cases, the antigen or epitope in the cassette encoding the additional antigen and / or epitope may be an epitope that is immunodominant relative to the other encoded epitopes. Generally, immunodominance is the bias of the immune response toward only one or a few specific immunogenic peptides. Immunodominance can be assessed as part of an immune monitoring protocol. For example, immunodominance can be assessed by assessing T cell and / or B cell responses to the encoded antigen.

[0330] Immunodominance can be assessed as the effect that the presence of an immunodominant antigen has on the immune response to one or more other antigens. For example, an immunodominant antigen and its corresponding immune response (e.g., an immunodominant MHC class I epitope) may reduce the immune response to another antigen compared to the immune response in the absence of the immunodominant antigen. This reduction may be such that the immune response in the presence of the immunodominant antigen is not considered a therapeutically effective response. For example, an MHC class I epitope is generally considered immunodominant if T cell responses to other antigens are no longer considered therapeutically effective compared to responses 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 responses in the absence of the immunodominant antigen. For example, an MHC class I epitope is generally considered immunodominant if T cell responses to other antigens are below the limit of detection compared to responses 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. Immunodominance can be assessed by the relative immunodominance of the suspected immunodominant antigen relative to other antigens in the presence and absence of the suspected immunodominant antigen.

[0331] Immunodominance can be measured as the relative difference in immune response between two or more antigens. Immunodominance can refer to an immune response of a specific antigen that is 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than another antigen encoded within the same cassette. Immunodominance can refer to an immune response of a specific antigen that is 100-fold, 200-fold, 300-fold, 400-fold, or 500-fold greater than another antigen encoded within the same cassette. Immunodominance can refer to an immune response of a specific antigen that is 1000-fold, 2000-fold, 3000-fold, 4000-fold, or 5000-fold greater than another antigen encoded within the same cassette. Immunodominance can refer to an immune response of a specific antigen that is 10,000-fold greater than another antigen encoded within the same cassette.

[0332] In some cases, it may be desirable to avoid vaccine compositions containing immunodominant epitopes. For example, it may be desirable to avoid designing vaccine cassettes that encode immunodominant epitopes. Without wishing to be bound by theory, co-administration of and / or co-encoding an immunodominant epitope with an additional epitope may reduce the immune response to the additional epitope, ultimately reducing vaccine efficacy against the additional epitope. As an illustrative, non-limiting example, a vaccine composition comprising a TP53-associated neoepitope may have an immune response, e.g., a T cell response, biased toward the TP53-associated neoepitope (e.g., to the point where the immune response is no longer therapeutically effective and / or reduces the immune response below the limit of detection) in a manner that negatively impacts other antigens or epitopes in the vaccine composition (e.g., one or more KRAS-associated neoepitopes in the vaccine composition, such as any of the KRAS-associated MHC class I neoepitopes set forth in SEQ ID NOS: 75-82). Thus, a vaccine composition can be designed to not contain 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 than 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 than 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 than 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 provokes a 10,000-fold or greater immune response relative to another epitope encoded within the same cassette administered to a subject in a vaccine composition, where each antigen is capable of stimulating an immune response in the subject.

[0333] VB immunomodulator A vector described herein, such as a C68 vector described herein, or an alphavirus vector described herein, can contain a nucleic acid encoding at least one antigen, and the same or a separate vector can contain a nucleic acid encoding at least one 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 the immunomodulator.

[0334] Exemplary immune checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (a member of the CD2 family of molecules expressed on all NK (gamma delta) and memory CD8+ (alpha beta) T cells), CD160 (also known as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, or antigen-binding fragments thereof, or other binding proteins that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (a CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), ipilimumab, MK-3475 (a PD-1 blocker), nivolumab (an anti-PD1 antibody), cemiplimab (an anti-PD1 antibody), CT-011 (an anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (an anti-PDL1 antibody), BMS-936559 (an anti-PDL1 antibody), MPLDL3280A / atezolizumab (an anti-PDL1 antibody), MSB0010718C (an anti-PDL1 antibody), and yervoy / ipilimumab (an anti-CTLA-4 checkpoint inhibitor). The antibody-encoding sequence can be engineered into a vector using ordinary skill in the art. One exemplary method is described in Fang et al., Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol. 2005 May;23(5):584-90. Epub 2005 Apr 17, which is incorporated herein by reference for all purposes.

[0335] Further considerations for VC vaccine design and manufacturing VC1. Determining a set of peptides covering all tumor subclones Truncal peptides, meaning peptides presented by all or most tumor subclones, can be prioritized for inclusion in the vaccine. Optionally, if there are no trunk peptides predicted to be highly presented and immunogenic, or if the number of trunk peptides predicted to be highly presented and immunogenic is small enough to allow additional non-trunk peptides to be included in the vaccine, additional peptides can be prioritized by estimating the number and types of tumor subclones and selecting peptides that maximize the number of tumor subclones encompassed by the vaccine.

[0336] 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 predetermined filters at each stage of the selection process, one can consider an integral multidimensional model, in which candidate antigens are placed in a space with at least the following axes, and an integral approach is used to optimize selection: 1. Risk of autoimmunity or tolerance (germline risk) (lower autoimmune risk is typically preferred) 2. Probability of sequencing artifacts (lower artifact probabilities are typically preferred) 3. Probability of immunogenicity (higher probability of immunogenicity is typically preferred) 4. Probability of presentation (higher probability of presentation is typically preferred) 5. Gene Expression (higher expression is typically preferred) 6. HLA gene coverage (a greater number of HLA molecules involved in presenting a set of antigens may decrease the probability that a tumor will evade immune attack through downregulation or mutation of HLA molecules) 7. HLA class coverage (covering both HLA-I and HLA-II may increase the probability of treatment response and decrease the probability of tumor avoidance)

[0337] Furthermore, in some cases, antigens can be deprioritized (e.g., excluded) from vaccination if they are predicted to be presented by HLA alleles that are lost or inactivated in all or part of a patient's tumor. Loss of HLA alleles can occur through somatic mutation, loss of heterozygosity, or homozygous deletion of the locus. Methods for detecting somatic mutations of HLA alleles are well known in the art (e.g., Shukla et al., 2015). Methods for detecting somatic loss of heterozygosity and homozygous deletion (including HLA loci) have also been described (Carter et al., 2012; McGranahan et al., 2017; Van Loo et al., 2010). Antigens may be deprioritized if mass spectrometry data indicates that the predicted antigen is not presented by the predicted HLA allele.

[0338] VD alphavirus VD1. Alphavirus Biology Alphaviruses are members of the Togaviridae family and are single-stranded, positive-sense RNA viruses. Members are generally classified as either Old World types, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World types, such as Eastern equine encephalitis virus, Aura, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83 (Strauss Microbial Review 1994). Natural alphavirus genomes are typically approximately 12 kb in length, with the first two-thirds containing genes encoding nonstructural proteins (nsPs) that form the RNA replication complex for autonomous replication of the viral genome, and the last one-third containing subgenomic expression cassettes encoding structural proteins for virion production (Frolov RNA 2001).

[0339] The model life cycle of alphaviruses involves multiple distinct steps (Strauss Microbial Review 1994, Jose Future Microbiol 2009). After viral adsorption to the host cell, the virion fuses with membranes within the intracellular compartment, ultimately releasing the genomic RNA into the cytosol. The genomic RNA, which has a positive-strand orientation, a 5'-methylguanylate cap, and a 3'-poly(A) tail, is translated to generate nonstructural proteins nsP1-4, which form a replication complex. Early in infection, the positive-strand RNA is replicated onto a negative-strand template by this complex. In the current model, the replication complex undergoes further processing as infection progresses, and the resulting processed complex switches to transcribing the negative-strand full-length positive-strand genomic RNA and the 26S subgenomic positive-strand RNA containing the structural genes. Several conserved sequence elements (CSEs) in alphaviruses have been identified as potentially playing roles in various steps of RNA synthesis, including the complement of the 5' UTR in replication of plus-strand RNA from a minus-strand template, a 51-nt CSE in replication of minus-strand synthesis from a genomic template, a 24-nt CSE within the junction region between nsP and 26S RNA in transcription of subgenomic RNA from the minus strand, and a 19-nt CSE at the 3' end in minus-strand synthesis from a plus-strand template.

[0340] In the natural life cycle of viruses, viral particles are typically assembled after replication of different RNA species. The 26S RNA is translated, and the resulting proteins are further processed to generate structural proteins, including capsid proteins, glycoproteins E1 and E2, and two small polypeptides, E3 and 6K (Strauss 1994). After encapsidation of the viral particle occurs and capsid proteins, typically specific only to the genomic RNA, are packaged, the virion assembles and buds onto the membrane surface.

[0341] VD2. Alphaviruses as Delivery Vectors Alphaviruses (alphavirus sequences, characteristics, and other elements) can be used to generate alphavirus-based delivery vectors (also referred to as alphavirus vectors, alphavirus viral vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying mRNA (SAM) or samRNA vectors). Alphaviruses have traditionally been genetically engineered for use as expression vector systems (Pushko 1997, Rheme 2004). Alphaviruses offer several advantages in vaccine settings, where expression of heterologous antigens may be desirable. Because of their ability to replicate autonomously in the host cytosol, alphaviruses generally achieve high intracellular copy numbers of the expression cassette, thereby enabling high levels of heterologous antigen production. Furthermore, vectors are generally transient, resulting in high biosafety and low induction of immune tolerance to the vector. The general public generally lacks pre-existing immunity to alphaviruses compared to other standard viral vectors, such as human adenoviruses. Alphavirus-based vectors also generally elicit cytotoxic responses against infected cells. Cytotoxicity may be of some importance in vaccine settings to adequately stimulate an immune response to the expressed heterologous antigen. However, the desired degree of cytotoxicity is a matter of balance, and for this reason, several attenuated alphaviruses have been developed, including the TC-83 strain of VEE. Thus, one example of an antigen expression vector described herein utilizes an alphavirus backbone that allows for high levels of antigen expression, stimulates a strong immune response to the antigen, does not stimulate an immune response to the vector itself, and is safe to use. Furthermore, antigen expression cassettes can be designed to stimulate different levels of immune response through optimization of which alphavirus sequences the vector uses, including, but not limited to, sequences derived from VEE or its attenuated derivative, TC-83.

[0342] Several strategies for designing expression vectors using alphavirus sequences have been developed (Pushko 1997). In one strategy, alphavirus vector design involves inserting a second copy of the 26S promoter sequence element downstream of the structural protein gene followed by the insertion of a heterologous gene (Frolov 1993). This results in the production of subgenomic RNA that expresses additional heterologous proteins in addition to the native nonstructural and structural proteins. In this system, all the factors for producing infectious virions are present, and therefore repeated rounds of infection of the expression vector in uninfected cells can be carried out.

[0343] Another expression vector design utilizes a helper virus system (Pushko 1997). In this strategy, structural proteins are replaced by heterologous genes. Thus, after autonomous replication of viral RNA mediated by the still-intact nonstructural genes, the 26S subgenomic RNA drives expression of the heterologous protein. Traditionally, an additional vector expressing the structural proteins is provided in trans, for example, by cotransfection of a cell line, to generate infectious virus. One system is described in U.S. Patent No. 8,093,021, the entire contents of which are incorporated herein by reference for all purposes. Helper vector systems offer the advantage of limiting the potential for infectious particle formation, thus improving biosafety. Furthermore, helper vector systems may shorten the overall vector length and improve replication and expression efficiency. Thus, one example of an antigen expression vector described herein can use an alphavirus backbone in which structural proteins are replaced with an antigen cassette, and the resulting vector promotes efficient expression by reducing biosafety concerns while simultaneously reducing the overall expression vector size.

[0344] VD3. In vitro generation of alphavirus Alphavirus delivery vectors are generally positive-sense RNA polynucleotides. A conventional method for RNA generation known in the art is in vitro translation (IVT). In this method, a DNA template of the desired vector is first generated by standard molecular biology methods well known in the art, including cloning, restriction digestion, ligation, gene synthesis (e.g., chemical and / or enzymatic synthesis), and polymerase chain reaction (PCR). This DNA template carries an RNA polymerase promoter at the 5' end of the sequence desired to be transcribed into RNA. Promoters include, but are not limited to, promoters for bacteriophage polymerases such as T3, T7, or SP6. The DNA template is then incubated with an appropriate RNA polymerase enzyme, buffering agents, and nucleotides (NTPs). The resulting RNA polynucleotide can optionally be further modified by methods including, but not limited to, the addition of a 5' cap structure, such as 7-methylguanosine or a related structure, and optionally modifying the 3' end to include a polyadenylated (polyA) tail. The RNA can then be purified using methods well known in the art, such as phenol-chloroform extraction or column purification (eg, chromatography-based purification).

[0345] VD4. Delivery via lipid nanoparticles One important aspect to consider in vaccine vector design is immunity to the vector itself (Riley 2017). This can be in the form of pre-existing immunity to the vector itself, for example, certain human adenovirus systems, or immunity to the vector that develops after administration of the vaccine. The latter is an important consideration when multiple doses of the same vaccine are administered, for example, separate priming and booster doses, or when the same vaccine vector system is used to deliver different antigen cassettes.

[0346] In the case of alphavirus vectors, the standard delivery method is the helper virus system described above, which generates infectious viral particles by delivering the capsid, E1, and E2 proteins in trans. However, it is important to note that E1 and E2 proteins are often the primary targets of neutralizing antibodies (Strauss 1994). Therefore, the effectiveness of using alphavirus vectors to deliver antigens of interest to target cells may be reduced if the infectious particles are targeted by neutralizing antibodies.

[0347] An alternative to viral particle-mediated gene delivery is the delivery of expression vectors using nanoparticles (Riley 2017). Importantly, nanomaterial carriers can be made 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 conjugates including, but not limited to, polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins.

[0348] Lipid nanoparticles (LNPs) are attractive delivery systems due to the amphiphilic nature of lipids, which allows them to form membrane- and vesicular structures (Riley 2017). These vesicles typically deliver expression vectors by absorbing into the membrane of target cells and releasing the nucleic acid into the cytosol. Furthermore, LNPs can be further modified or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity. Lipid compositions typically contain defined mixtures of cationic, neutral, anionic, and amphiphilic lipids. In some instances, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide chemical functional groups that facilitate the attachment of additional moieties. The lipid composition can affect the size and stability of the overall LNP. In one example, the lipid composition includes dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. Compositions of MC3 and MC3-like lipids can be formulated to include one or more other lipids, such as, for example, PEG or PEG-conjugated lipids, sterols, or neutral lipids.

[0349] Nucleic acid vectors, such as expression vectors, directly exposed to serum can have several undesirable effects, including degradation of the nucleic acid by nucleases in serum or stimulation of off-targets of the immune system by free nucleic acids. Therefore, encapsulation of alphavirus vectors can be used to prevent degradation while also preventing potential off-target effects. In certain examples, the alphavirus vector is fully encapsulated within the delivery vehicle, such as the aqueous interior of an LNP. Encapsulation of the alphavirus vector within the LNP can be achieved by methods well known in the art, such as microfluidic mixing and droplet generation performed in a microfluidic droplet generator. Such devices include, but are not limited to, standard T-junction or flow-focusing devices. In one example, a desired lipid formulation, such as an MC3- or MC3-like molecule-containing composition, is fed into the droplet generator in parallel with the alphavirus delivery vector and other desired substances, thereby fully encapsulating the delivery vector and desired substances within the MC3- or MC3-like molecule-based LNP. In one example, the droplet generator can control the particle size range and size distribution of the generated LNPs. For example, LNPs can have particle sizes ranging from 1 to 1000 nm in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nm. After droplet generation, the delivery vector encapsulating the expression vector can be further processed or modified in preparation for administration.

[0350] VE chimpanzee adenovirus (ChAd) VE1. Viral delivery by chimpanzee adenovirus Vaccine compositions for delivering one or more antigens (e.g., antigen cassettes and including one or more KRAS-associated neoepitopes, such as any of the KRAS-associated neoepitopes set forth in SEQ ID NOS: 75-82) can be generated by providing chimpanzee-derived adenovirus nucleotide sequences, various novel vectors, and cell lines expressing chimpanzee adenovirus genes. The nucleotide sequence of chimpanzee C68 adenovirus (also referred to herein as ChAdV68) can be used in vaccine compositions for delivering antigens (see SEQ ID NOS: 1). The use of C68 adenovirus-derived vectors is described in further detail in U.S. Patent No. 6,083,716, the entire contents of which are incorporated herein by reference for all purposes. 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.

[0351] In a further aspect, provided herein is a recombinant adenovirus comprising a DNA sequence of a chimpanzee adenovirus, such as C68, and an antigen cassette operably linked to a regulatory sequence that directs expression. The recombinant virus is capable of infecting mammalian, preferably human, cells and expressing the product of the antigen cassette in the cells. The vector can be deleted for the native chimpanzee E1 gene, E3 gene, and / or E4 gene. An antigen cassette can be inserted into any of these gene deletion sites. The antigen cassette can include an antigen against which a primed immune response is desired.

[0352] In another aspect, provided herein are mammalian cells infected with a chimpanzee adenovirus, such as C68.

[0353] In yet another embodiment, novel mammalian cell lines are provided that express chimpanzee adenovirus genes (eg, from C68) or functional fragments thereof.

[0354] In an even further aspect, provided herein is a method for delivering an antigen cassette into a mammalian cell, the method comprising introducing into the cell an effective amount of a chimpanzee adenovirus, such as C68, engineered to express the antigen cassette.

[0355] Yet another embodiment provides a method of stimulating an immune response in a mammalian host to treat cancer, the method comprising administering to the host an effective amount of a recombinant chimpanzee adenovirus, such as C68, that includes an antigen cassette encoding one or more antigens derived from the tumor to which the immune response is targeted.

[0356] Yet another embodiment provides a method for stimulating an immune response in a mammalian host to treat or prevent a disease in a subject, such as 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, such as antigens derived from a cancer / tumor, to which the immune response is targeted.

[0357] Also disclosed are non-simian mammalian cells expressing chimpanzee adenovirus genes obtained from the sequence of SEQ ID NO: 1. The genes can be selected from the group consisting of adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 of SEQ ID NO: 1.

[0358] Also disclosed are nucleic acid molecules comprising a DNA sequence of a chimpanzee adenovirus comprising a gene obtained from the sequence of SEQ ID NO: 1. The gene can be selected from the group consisting of the chimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 1. In some embodiments, the nucleic acid molecule comprises the sequence of SEQ ID NO: 1, deleted for at least one gene selected from the group consisting of the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of SEQ ID NO: 1.

[0359] Also disclosed are vectors comprising a chimpanzee adenoviral DNA sequence derived from SEQ ID NO: 1 and an antigen cassette operably linked to one or more regulatory sequences that direct expression of the cassette in a heterologous host cell, optionally wherein the chimpanzee adenoviral DNA sequence comprises at least cis elements required for replication and encapsidation, the cis elements flanking the antigen cassette and regulatory sequences. In some embodiments, the chimpanzee adenoviral DNA sequence comprises genes selected from the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 gene sequences of SEQ ID NO: 1. In some embodiments, the vector may be deleted for the E1A and / or E1B genes.

[0360] Also disclosed herein are adenoviral vectors comprising a partially deleted E4 gene, including 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 include an E4 deletion of at least nucleotides 34,916-35,642 of the sequence set forth in SEQ ID NO: 1, wherein the vector comprises 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 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, wherein the vector comprises 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 nucleotides 34,980-36,516 of the sequence set forth in SEQ ID NO: 1, and the vector comprises 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 nucleotides 34,979-35,642 of the sequence set forth in SEQ ID NO: 1, and the vector comprises 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 E4Orf2, a completely deleted E4Orf3, and at least a partial deletion of E4Orf4. The partially deleted E4 can comprise 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 include 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 include an E4 deletion of at least a partial deletion of E4Orf2 and at least a partial deletion of E4Orf3. The partially deleted E4 may include an E4 deletion from the start site of E4Orf1 to the start site of E4Orf5. The partially deleted E4 may be an E4 deletion adjacent to the start site of E4Orf1. The partially deleted E4 may be an E4 deletion adjacent to the start site of E4Orf2.The partially deleted E4 may be an E4 deletion adjacent to the start site of E4Orf3. The partially deleted 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.

[0361] 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 deleted E4 gene can include a cassette, the cassette including at least one payload nucleic acid sequence, and the cassette further including at least one promoter sequence operably linked to the at least one payload nucleic acid sequence. The adenoviral vector having a 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 repeats (ITRs), 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, wherein the partially deleted E4 gene is at the 3' end of nucleotides 2 to 34,915, and optionally, nucleotides 2 to 34,915 further lack 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 set forth in SEQ ID NO: 1, where the partially deleted E4 gene is 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 34,915 of the sequence set forth in SEQ ID NO: 1, where the partially deleted E4 gene is at the 3' end of nucleotides 2 to 34,915, where nucleotides 2 to 34,915 further lack nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO: 1, which corresponds to an E1 deletion, and 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 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, and 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 also have 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.

[0362] An adenoviral vector having a partially deleted E4 gene may have the E4 gene sequence shown in SEQ ID NO: 1, lacking at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1 and nucleotides 2 to 34,915 of the sequence shown in SEQ ID NO: 1, wherein the partially deleted E4 gene is at the 3' end of nucleotides 2 to 34,915, and 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, wherein the partially deleted E4 gene is at the 5' end of nucleotide...

Claims

1. 1. An antigen-based vaccine for use in a method for treating a subject having a disease or for stimulating an immune response in a subject having cancer, wherein the disease is cancer, including (1) a solid tumor expressing a KRAS-associated MHC class I neoepitope, (2) colorectal cancer (CRC), (3) non-small cell lung cancer (NSCLC), and / or (4) pancreatic ductal adenocarcinoma (PDA), the method comprising administering to the subject an antigen-based vaccine, the antigen-based vaccine comprising an antigen expression system, the antigen expression system comprising one or more vectors; the one or more vectors (a) a vector backbone, (i) at least one promoter nucleotide sequence; (ii) optionally, at least one polyadenylation (poly(A)) sequence; the vector backbone comprising: (b) a cassette comprising: (i) at least one antigen-encoding nucleic acid sequence, (I) an epitope-encoding nucleic acid sequence encoding the KRAS-associated MHC class I neoepitope; wherein each of the epitope-encoding nucleic acid sequences comprises: (A) optionally, a 5' linker sequence; and (B) optionally, a 3' linker sequence; and the at least one antigen-encoding nucleic acid sequence comprising: (ii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence; and (iii) optionally, at least one MHC class II epitope-encoding nucleic acid sequence; and (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO: 56); and (v) optionally, at least one second poly(A) sequence, which is a native poly(A) sequence or a poly(A) sequence exogenous to the vector backbone; the cassette comprising: Including, if the second promoter nucleotide sequence is absent, the antigen-encoding nucleic acid sequence is operably linked to the at least one promoter nucleotide sequence; The antigen-based vaccine, wherein the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of the epitope-encoding nucleic acid sequence encoding the KRAS-associated MHC class I neoepitope.

2. 2. The antigen-based vaccine for use according to claim 1, wherein the epitope-encoding nucleic acid sequence is derived from a tumor of the subject with cancer or from a cell or sample of the subject that is infected, and wherein the epitope-encoding nucleic acid sequence is not derived from a tumor of the subject with cancer or from a cell or sample of the subject that is infected.

3. 2. The antigen-based vaccine for use according to claim 1, wherein the subject expresses at least one HLA allele that is expected or known to present the at least one epitope sequence, and optionally the at least one epitope sequence comprises an epitope that is known or suspected to be presented by MHC class I on the surface of a cell, and optionally the at least one epitope sequence that is expected or known to be presented comprises the KRAS-associated MHC class I neoepitope, and optionally the surface of the cell is a tumor cell surface, and optionally the method further comprises determining or having determined the HLA haplotype of the subject. (a) the antigen-based vaccine is administered as a priming dose, and / or (b) the antigen-based vaccine is administered as one or more booster doses; optionally, (i) the booster dose is different from the priming dose; optionally, (I) the priming dose comprises a chimpanzee adenovirus vector and the booster dose comprises an alphavirus vector; or (II) the priming dose comprises an alphavirus vector and the booster dose comprises a chimpanzee adenovirus vector; or (ii) the booster dose is the same as the priming dose; Optionally, the injection site of said one or more booster doses is as close as possible to the injection site of said priming dose. An antigen-based vaccine for use according to claim 1.

5. the antigen-based vaccine is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV); optionally, the antigen-based vaccine is administered intramuscularly (IM); optionally, the IM administration is given at separate injection sites; optionally, the separate injection sites are in opposing deltoid muscles; optionally, the separate injection sites are in bilateral gluteus or rectus femoris sites; An antigen-based vaccine for use according to claim 1.

6. the KRAS-associated MHC class I neoepitope or the KRAS mutation, (a) a KRAS G12C mutation; optionally, the KRAS G12C mutation is selected from the group consisting of VVVGACGVGK (SEQ ID NO: 75), KLVVVGACGV (SEQ ID NO: 76), and GACGVGKSAL (SEQ ID NO: 93); a KRAS G12V mutation; optionally, the KRAS G12V mutation is selected from the group consisting of VVGAVGVGK (SEQ ID NO: 79), VVVGAVGVGK (SEQ ID NO: 81), AVGVGKSAL (SEQ ID NO: 80), and GAVGVGKSAL (SEQ ID NO: 96); a KRAS G12D mutation; optionally, said KRAS G12D mutation is selected from the group consisting of VVGADGVGK (SEQ ID NO: 77), VVVGADGVGK (SEQ ID NO: 78), KLVVVGADGV (SEQ ID NO: 94), and GADGVGKSAL (SEQ ID NO: 95); or KRAS Q61H mutation; (b) the KRAS-associated MHC class I neoepitope or the KRAS mutation comprises any one of the amino acid sequences set forth in SEQ ID NOs: 75-82; or (c) an amino acid sequence set forth in SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60; 2. An antigen-based vaccine for use according to claim 1, comprising:

7. the antigen-encoding cassette comprises each of the amino acid sequences set forth in SEQ ID NOs: 75-82, and / or the antigen-encoding cassette comprises two or more repeats of each of the amino acid sequences set forth in SEQ ID NOs: 75-82, and optionally four repeats of each of the amino acid sequences set forth in SEQ ID NOs: 75-82; An antigen-based vaccine for use according to claim 1.

8. An antigen-based vaccine for use according to claim 1, which is: (a) the epitope-encoding nucleic acid sequences comprise two or more different epitope-encoding nucleic acid sequences that independently encode different KRAS-associated MHC class I neoepitopes or different KRAS mutations; (b) each of said epitope-encoding nucleic acid sequences independently encodes a different KRAS-associated MHC class I neoepitope or a different KRAS mutation; (c) the epitope-encoding nucleic acid sequence comprises two or more different epitope-encoding nucleic acid sequences that independently encode a KRAS G12C mutation, a KRAS G12V mutation, a KRAS G12D mutation, or a KRAS Q61H mutation; (d) the epitope-encoding nucleic acid sequence independently encodes each of the KRAS G12C mutation, the KRAS G12V mutation, and the KRAS G12D mutation, and optionally the KRAS Q61H mutation; and / or (e) the antigen-encoding nucleic acid sequence encodes a peptide comprising the amino acid sequence set forth in SEQ ID NO:64 or SEQ ID NO:

65.

9. the cancer comprises a solid tumor expressing a KRAS-related and / or NRAS-related MHC class I neoepitope, optionally wherein the KRAS-related and / or NRAS-related MHC class I neoepitope comprises a mutation selected from the group consisting of KRAS_G12C, NRAS_G12C, KRAS_G12D, NRAS_G12D, KRAS_G12V, NRAS_G12V, KRAS_Q61H, and NRAS_Q61H; An antigen-based vaccine for use according to claim 1.

10. 2. The antigen-based vaccine for use according to claim 1, wherein the antigen-based vaccine or the one or more booster doses are administered every 4 weeks (Q4W), every 8 weeks (Q8W), every month, or every two months.

11. The method comprises: administering to said subject a composition for delivering a self-replicating alphavirus-based expression system; administering to said subject a composition for delivering a chimpanzee adenovirus (ChAdV)-based expression system; Including, 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; and / or 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 at least two booster doses are at least 28 days apart, at least 4 weeks (Q4W) apart, at least 1 month apart, at least 56 days apart, at least 8 weeks (Q8W) apart, at least 2 months apart, 28 and 84 days or about 28 and 84 days after the priming dose of the ChAdV-based expression system, 4 and 12 weeks or about 4 and 12 weeks after the priming dose of the ChAdV-based expression system, and / or 1 and 3 months or about 1 and 3 months after the priming dose of the ChAdV-based expression system. be administered, An antigen-based vaccine for use according to claim 1.

12. 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 140 days 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 140 days or more after the priming dose of the ChAdV-based expression system; (E) the ChAdV-based expression system is administered as the booster dose 20 weeks or more after the priming dose of the ChAdV-based expression system; (F) the ChAdV-based expression system is administered as the booster dose 5 months or more after the priming dose of the ChAdV-based expression system. An antigen-based vaccine for use according to claim 1.

13. Optionally, the self-replicating alphavirus-based expression system is administered as at least four booster doses; (A) the self-replicating alphavirus-based expression system is administered at or about 28, 84, 196, and 252 days relative to the priming dose of the ChAdV-based expression system; (B) the self-replicating alphavirus-based expression system is administered at or about 4, 12, 28, and 40 weeks later 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 1, 3, 7, and 10 months relative to the priming dose of the ChAdV-based expression system. An antigen-based vaccine for use according to claim 1.

14. The method further comprising administering one or more immunomodulatory agents, optionally administered before, simultaneously with, or after administration of the composition or pharmaceutical composition; optionally, the method comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 administrations of one or more immunomodulatory agents, optionally, the one or more immunomodulatory agents being selected from the group consisting of an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, or an anti-OX-40 antibody or antigen-binding fragment thereof, atezolizumab, nivolumab, cemiplimab, and ipilimumab; Optionally, the immunomodulatory agent is administered intravenously (IV), intramuscularly (IM), intradermally (ID), or subcutaneously (SC); Optionally, the subcutaneous administration is near the site of administration of the composition or pharmaceutical composition or in close proximity to one or more vector or composition-draining lymph nodes; and / or the method comprises administering an anti-CTLA-4 antibody or antigen-binding fragment thereof with only the priming dose and a first booster dose, optionally wherein the anti-CTLA-4 antibody comprises ipilimumab, and optionally wherein the ipilimumab is administered subcutaneously at a dose of 30 mg; and / or the method comprises administering 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, and 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 optionally wherein the method comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 administrations of the immunomodulatory agent of one or more of the anti-PD-L1 antibodies, and optionally comprises at least 13 administrations of the anti-PD-L1 antibody. An antigen-based vaccine for use according to claim 1.

15. 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 is 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% reduction in ctDNA. An antigen-based vaccine for use according to claim 1.

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