MULTIEPITOPE VACCINE CASSETTES

1 .- 20 . (canceled)

21 . A composition for delivery of an antigen expression system, comprising:

the antigen expression system, wherein the antigen expression system comprises one or more vectors,

the one or more vectors comprising:

(a) a vector backbone, wherein the backbone comprises:

(i) at least one promoter nucleotide sequence, and

(ii) optionally, at least one polyadenylation (poly(A)) sequence; and

(b) a cassette, wherein the cassette comprises:

(i) at least one antigen-encoding nucleic acid sequence, comprising:

(I) an epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope, and

wherein each of the epitope-encoding nucleic acid sequences comprises;

(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; 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, wherein the second poly(A) sequence is a native poly(A) sequence or an exogenous poly(A) sequence to the vector backbone,

wherein 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, and

wherein the at least one antigen-encoding nucleic acid sequence comprises at least two iterations of the epitope-encoding nucleic acid sequence encoding the KRAS-associated MHC class I neoepitope,

wherein at least one of the at least two KRAS-associated MHC class I neoepitopes is characterized by each of the following characteristics:

(i) the KRAS-associated MHC class I neoepitope is expressed in a tumor cell of a subject according to exome, transcriptome, or whole genome nucleotide sequencing data obtained from the subject;

(ii) the KRAS-associated MHC class I neoepitope comprises at least one alteration that makes the KRAS-associated MHC class I neoepitope distinct from a corresponding wild-type peptide expressed in a normal cell of the subject according to the exome, transcriptome, or whole genome sequencing data obtained from the subject; and

(iii) the KRAS-associated MHC class I neoepitope is predicted to presented by an MHC allele on the surface of the tumor cell of the subject according to a presentation model trained on peptide-KRAS binding data.

22 . The composition of claim 21 , wherein each of the epitope-encoding nucleic acid sequences further encodes a 5′ linker sequence that encodes an N-terminal amino acid sequence that flanks the respective KRAS-associated MHC class I neoepitope and/or a 3′ linker sequence that encodes a C-terminal amino acid sequence that flanks the respective KRAS-associated MHC class I neoepitope.

23 . The composition of claim 22 , wherein the N-terminal amino acid sequence that flanks the respective KRAS-associated MHC class I neoepitope is between 2-20 amino acids in length, and/or the C-terminal amino acid sequence that flanks the respective KRAS neoepitope is between 2-20 amino acids in length.

24 . The composition of claim 22 , wherein the N-terminal amino acid sequence that flanks the respective KRAS-associated MHC class I neoepitope and the C-terminal amino acid sequence that flanks the respective KRAS-associated MHC class I neoepitope are both present, and wherein the N-terminal amino acid sequence that flanks the respective KRAS-associated MHC class I neoepitope is a native N-terminal flanking amino acid sequence from the respective KRAS protein, and the C-terminal amino acid sequence that flanks the respective KRAS-associated MHC class I neoepitope is a native C-terminal flanking amino acid sequence from the respective KRAS protein.

25 . The composition of claim 24 , wherein each of the encoded KRAS-associated MHC class I neoepitopes when expressed are linked directly together through the native N-terminal flanking amino acid sequences and the native C-terminal flanking amino acid sequences without intervening non-native amino acids.

26 . The composition of claim 21 , wherein each of the epitope-encoding nucleic acid sequences encodes a polypeptide between 8 and 35 amino acids in length.

27 . The composition of claim 26 , wherein:

(i) the epitope-encoding nucleic acid sequence encodes a KRAS G12C neoepitope-encoding polypeptide less than 35 amino acids in length having the amino acid sequence MTEYKLVVVGACGVGKSALTIQLIQ (SEQ ID NO: 57);

(ii) the epitope-encoding nucleic acid sequence encodes a KRAS G12D neoepitope-encoding polypeptide less than 35 amino acids in length having the amino acid sequence MTEYKLVVVGADGVGKSALTIQLIQ (SEQ ID NO: 58);

(iii) the epitope-encoding nucleic acid sequence encodes a KRAS G12V neoepitope-encoding polypeptide less than 35 amino acids in length having the amino acid sequence MTEYKLVVVGAVGVGKSALTIQLIQ (SEQ ID NO: 59); or

(iv) the epitope-encoding nucleic acid sequence encodes a KRAS Q61H neoepitope-encoding polypeptide less than 35 amino acids in length having the amino acid sequence ETCLLDILDTAGHEEYSAMRDQYMR (SEQ ID NO: 60).

28 . The composition of claim 26 , wherein:

(i) the epitope-encoding nucleic acid sequence encodes a KRAS G12C neoepitope-encoding polypeptide consisting of the amino acid sequence MTEYKLVVVGACGVGKSALTIQLIQ (SEQ ID NO: 57);

(ii) the epitope-encoding nucleic acid sequence encodes a KRAS G12D neoepitope-encoding polypeptide consisting of the amino acid sequence MTEYKLVVVGADGVGKSALTIQLIQ (SEQ ID NO: 58);

(iii) the epitope-encoding nucleic acid sequence encodes a KRAS G12V neoepitope-encoding polypeptide consisting of the amino acid sequence MTEYKLVVVGAVGVGKSALTIQLIQ (SEQ ID NO: 59); or

(iv) the epitope-encoding nucleic acid sequence encodes a KRAS Q61H neoepitope-encoding polypeptide consisting of the amino acid sequence ETCLLDILDTAGHEEYSAMRDQYMR (SEQ ID NO: 60).

29 . The composition of claim 24 , wherein each of the epitope-encoding nucleic acid sequences encode the respective KRAS neoepitope, the native N-terminal amino acid sequence, and the native C-terminal amino acid sequence, wherein each of the epitope-encoding nucleic acid sequences encode a polypeptide consisting of an amino acid sequence 25 amino acids in length.

30 . The composition of claim 21 , wherein each of the epitope-encoding nucleic acid sequences are linearly linked to one another in any order.

31 . The composition of claim 21 , wherein each of the epitope-encoding nucleic acid sequences are linearly linked to one another in an order configured to minimize generation of non-therapeutic MHC class I or class II junctional epitopes.

32 . The composition of claim 21 , wherein each of the epitope-encoding nucleic acid sequences further encodes a 5′ linker sequence that encodes a native N-terminal amino flanking acid sequence that flanks the respective KRAS neoepitope and a 3′ linker sequence that encodes a native C-terminal flanking amino acid sequence that flanks the respective KRAS-associated MHC class I neoepitope, wherein each of the encoded KRAS-associated MHC class neoepitopes when expressed are linked directly together through the native N-terminal flanking amino acid sequences and the native C-terminal amino acid flanking sequences without intervening non-native amino acids, and wherein each of the epitope-encoding nucleic acid sequence encodes a polypeptide less than 35 amino acids in length.

33 . The composition of claim 21 , wherein the cassette comprises a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequence.

34 . The composition of claim 21 , wherein the cassette comprises at least one MHC class II epitope-encoding nucleic acid sequence.

35 . The composition of claim 34 , wherein the at least one MHC class II epitope-encoding nucleic acid sequence comprises a universal MHC class II epitope-encoding nucleic acid sequence.

36 . The composition of claim 21 , wherein the vector backbone comprises either:

a chimpanzee adenovirus vector, or

a self-amplifying RNA (samRNA) vector.

37 . The composition of claim 36 , wherein the samRNA vector is a Venezuelan equine encephalitis virus vector.

38 . The composition of claim 36 , wherein the samRNA vector comprises the sequence of SEQ ID NO:6.

39 . The composition of claim 38 , wherein the cassette is inserted at position 7544 of the sequence of SEQ ID NO:6.

40 . The composition of claim 36 , wherein the chimpanzee adenovirus vector is a ChAdV68 vector comprising:

the sequence of SEQ ID NO:1;

the sequence of SEQ ID NO:1, except that the sequence is fully deleted or functionally deleted in at least one gene selected from the group consisting of the chimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence of SEQ ID NO:1;

a gene or regulatory sequence obtained from the sequence of SEQ ID NO:1;

a partially deleted E4 gene comprising a deleted or partially-deleted E4orf2 region and a deleted or partially-deleted E4orf3 region;

at least nucleotides 2 to 36,518 of the sequence of SEQ ID NO:1 and further comprising: (1) an E1 deletion of at least nucleotides 577 to 3403 of the sequence of SEQ ID NO:1, (2) an E3 deletion of at least nucleotides 27,125 to 31,825 of the sequence of SEQ ID NO:1, and (3) a partial deletion of at least nucleotides 34,916 to 35,642 of the sequence of SEQ ID NO:1;

the sequence of SEQ ID NO:68;

one or more deletions between base pair number 577 and 3403 or between base pair 456 and 3014; or

one or more deletions between base pair number 3957 and 10346, base pair number 21787 and 23370, and base pair number 33486 and 36193 of the sequence of SEQ ID NO: 1.

41 . The composition of claim 40 , wherein the cassette is inserted in the ChAdV68 vector at any one of the deleted E1 regions, any one of the deleted E3 regions, or any one of the deleted ChAdV68 regions that allows incorporation of the cassette.

42 . The composition of claim 36 , wherein the chimpanzee adenovirus vector is a ChAdV68 vector comprising at least nucleotides 2 to 36,518 of the sequence of SEQ ID NO: 1 and further comprising: (1) an E1 deletion of nucleotides 577 to 3403 of the sequence of SEQ ID NO:1, (2) an E3 deletion of nucleotides 27,125 to 31,825 of the sequence of SEQ ID NO: 1, and (3) a partial E4 deletion of nucleotides 34,916 to 35,642 of the sequence of SEQ ID NO:1; wherein the antigen cassette is inserted within the E1 deletion.

43 . A method for treating a subject with cancer, the method comprising administering to the subject an immunotherapy comprising an antigen-based vaccine to the subject, wherein the antigen-based vaccine comprises an antigen expression system, comprising:

the antigen expression system,

wherein the antigen expression system comprises one or more vectors,

the one or more vectors comprising:

(a) a vector backbone, wherein the backbone comprises:

(i) at least one promoter nucleotide sequence, and

(ii) optionally, at least one polyadenylation (poly(A)) sequence; and

(b) a cassette, wherein the cassette comprises:

(i) at least one antigen-encoding nucleic acid sequence, comprising:

(I) an epitope-encoding nucleic acid sequence encoding a KRAS-associated MHC class I neoepitope, and

wherein each of the epitope-encoding nucleic acid sequences comprises;

(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; 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, wherein the second poly(A) sequence is a native poly(A) sequence or an exogenous poly(A) sequence to the vector backbone,

wherein 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, and wherein the at least one antigen-encoding nucleic acid sequence comprises at least two iterations of the epitope-encoding nucleic acid sequence encoding the KRAS-associated MHC class I neoepitope,

wherein at least one of the at least two KRAS-associated MHC class I neoepitopes is characterized by each of the following characteristics:

(i) the KRAS-associated MHC class I neoepitope is expressed in a tumor cell of a subject according to exome, transcriptome, or whole genome nucleotide sequencing data obtained from the subject;

(ii) the KRAS-associated MHC class I neoepitope comprises at least one alteration that makes the KRAS-associated MHC class I neoepitope distinct from a corresponding wild-type peptide expressed in a normal cell of the subject according to the exome, transcriptome, or whole genome sequencing data obtained from the subject; and

(iii) the KRAS-associated MHC class I neoepitope is predicted to presented by an MHC allele on the surface of the tumor cell of the subject according to a presentation model trained on peptide-KRAS binding data.

44 . An antigen-based vaccine comprising:

an antigen expression system,

wherein the antigen expression system comprises one or more vectors,

the one or more vectors comprising:

(a) a vector backbone, wherein the backbone comprises:

(i) at least one promoter nucleotide sequence, and

(ii) at least one polyadenylation (poly(A)) sequence; and

(b) a cassette, wherein the cassette comprises an antigen-encoding nucleic acid sequence encoding:

(i) at least 4 iterations of an epitope-encoding nucleic acid sequence encoding a KRAS G12C neoepitope having the amino acid sequence VVVGACGVGK (SEQ ID NO: 75);

(ii) at least 4 iterations of a epitope-encoding nucleic acid sequence encoding a KRAS G12D neoepitope having the amino acid sequence VVVGADGVGK (SEQ ID NO: 78);

(iii) at least 4 iterations of a epitope-encoding nucleic acid sequence encoding a KRAS G12V neoepitope having the amino acid sequence VVGAVGVGK (SEQ ID NO: 79); and

(iv) at least 4 iterations of a epitope-encoding nucleic acid sequence encoding a KRAS Q61H neoepitope having the amino acid sequence ILDTAGHEEY (SEQ ID NO: 82),

and wherein each of the epitope-encoding nucleic acid sequences are linearly linked to one another and configured such that translation of the antigen-encoding nucleic acid sequence expresses each of the encoded KRAS neoepitopes together as a single polypeptide.

45 . The composition of claim 44 , wherein each of the epitope-encoding nucleic acid sequences are linearly linked to one another such that the KRAS neoepitopes are encoded in the following order: KRAS G12C, KRAS G12D, KRAS Q61H, KRAS G12D, KRAS G12V, KRAS G12C, KRAS Q61H, KRAS G12D, KRAS G12V, KRAS G12C, KRAS Q61H, KRAS G12D, KRAS G12V, KRAS Q61H, KRAS G12V, KRAS G12C.

46 . A method for treating a subject with cancer, the method comprising administering the composition of claim 44 to the subject.