EGFR vaccine cassette

Abstract

Disclosed herein are compositions comprising antigen-encoding nucleic acid sequences having multiple repeats of an EGFR neoepitope-encoding sequence, as well as methods related to the compositions, including nucleotides, cells, and their use as vaccines.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 392,072, filed July 25, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically and is incorporated herein by reference in its entirety. The .XML copy created on November 10, 2022 is named GSO-086WOC1 and is 400,000 bytes in size. [Background technology]

[0003] background 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 attractive targets for such therapies due to their relatively high potential for neoantigen generation. 4、5 Early evidence suggests that neoantigen-based vaccination can induce T cell responses. 6 and that neoantigen-targeted cell therapy can produce tumor regression in certain patients under certain circumstances. 7 is shown.

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

[0005] In addition to the challenges of current antigen prediction methods, certain challenges also exist with available vector systems that can be used for antigen delivery in humans, many of which are of human origin. For example, many humans have pre-existing immunity to human viruses as a result of previous natural exposure, and this immunity can pose a major obstacle to the use of recombinant human viruses for antigen delivery in vaccination strategies, such as cancer treatment or vaccination against infectious diseases. While some progress has been made in vaccination strategies that address the above issues, improvements are still needed, particularly for clinical application, e.g., improvements in vaccine potency and efficacy. Summary of the Invention

[0006] overview As used herein, an antigen-encoding cassette, or a polypeptide sequence encoded by a cassette, is defined as an antigen-encoding cassette comprising: (i) a nucleic acid sequence A(E A ); and (ii) nucleic acid sequence B(E B ), including E A and E B Each encodes one MHC epitope, and E A MHC epitopes encoded by and E B The MHC epitopes encoded by the cassettes are distinct and non-identical, and the cassettes are A and at least two repetitions of E B and E A and E B Each repeat of E contains the same nucleic acid sequence. A discloses antigen-encoding cassettes, or polypeptide sequences encoded by cassettes, that encode EGFR-associated MHC class I neoepitopes.

[0007] In some embodiments, E AThe EGFR-associated MHC class I neoepitope encoded by E is selected from the group consisting of an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, an EGFR E746-A750 deletion MHC class I epitope, and combinations thereof. A The EGFR-associated MHC class I neoepitope encoded by E comprises the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, ITDFGRAKL, STVQLIMQL, TVQLIMQL, LTSTVQLIM, PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK. A The EGFR-associated MHC class I neoepitope encoded by comprises the amino acid sequence KITDFGRAK, KITDFGRAKL, STVQLIMQL, or LSTVQLIM.

[0008] In some embodiments, E B is E A In some embodiments, the E B The EGFR-associated neoepitope encoded by E comprises an MHC class I neoepitope. B The EGFR-associated MHC class I neoepitope encoded by E is selected from the group consisting of an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, an EGFR E746-A750 deletion MHC class I epitope, and combinations thereof. B The EGFR-associated MHC class I neoepitope encoded by E comprises the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, ITDFGRAKL, STVQLIMQL, TVQLIMQL, LTSTVQLIM, PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK. BThe EGFR-associated MHC class I neoepitope encoded by comprises the amino acid sequence KITDFGRAK, KITDFGRAKL, STVQLIMQL, or LSTVQLIM.

[0009] In some embodiments, E A and E B collectively contain each of the EGFR_L858R and EGFR T790M MHC class I epitopes.

[0010] In some embodiments, the cassette comprises E A and E B In some embodiments, the cassette encodes at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 repeats of one or both of E A and E B The nucleic acid sequence encodes at least eight repeats of each of the following:

[0011] In some embodiments, the cassette comprises a unit E A -E B and optionally encoding two or more repeats of E A and E B are linked by a linker-encoding nucleic acid. In some embodiments, the cassette comprises a unit E A -E B In some embodiments, the E A The epitopes encoded by include the EGFR_L858R MHC class I epitope, B The epitope encoded by comprises the EGFR T790M MHC class I epitope. In some embodiments, the cassette encodes the amino acid sequence of SEQ ID NO:71.

[0012] In some embodiments, the antigen-encoding cassette comprises the nucleic acid sequence C(E C ), and E C encodes one MHC epitope, and E C The MHC epitopes encoded by E AMHC epitopes encoded by and E B The MHC epitopes encoded by the cassette are distinct and non-identical to those encoded by E C The cassette comprises at least two iterations of E C and, and)E C Each repeat of E comprises an identical nucleic acid sequence. C encodes an EGFR-associated MHC class I neoepitope. C is E A and E B In some embodiments, the E C The EGFR-associated neoepitopes encoded by include MHC class I neoepitopes.

[0013] In some embodiments, E C The EGFR-associated MHC class I neoepitope encoded by E is selected from the group consisting of an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, an EGFR E746-A750 deletion MHC class I epitope, and combinations thereof. C The EGFR-associated MHC class I neoepitope encoded by E comprises the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, ITDFGRAKL, STVQLIMQL, TVQLIMQL, LTSTVQLIM, PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK. C The EGFR-associated MHC class I neoepitope encoded by comprises the amino acid sequence KITDFGRAK, KITDFGRAKL, STVQLIMQL, or LSTVQLIM.

[0014] In some embodiments, E A , E B , and E Ccollectively comprise each of the EGFR_L858R MHC class I epitope, the EGFR T790M MHC class I epitope, and the EGFR E746-A750 deletion MHC class I epitope. A , E B , and E C In some embodiments, the cassette encodes at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 repeats of one or each of E A , E B , and E C In some embodiments, the cassette encodes at least eight repeats of each of the units E A -E B -E C and optionally encoding two or more repeats of E A , E B , and E C are linked by a linker-encoding nucleic acid. In some embodiments, the cassette comprises a unit E A -E B -E C In some embodiments, the E A The epitopes encoded by include the EGFR_L858R MHC class I epitope, B The epitope encoded by comprises the EGFR T790M MHC class I epitope. In some embodiments, the cassette encodes the amino acid sequence of SEQ ID NO:94.

[0015] In some embodiments, the antigen-encoding cassette encodes a peptide comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, the antigen-encoding cassette encodes a peptide comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the antigen-encoding cassette encodes a peptide comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antigen-encoding cassette encodes a peptide comprising the amino acid sequence of SEQ ID NO: 94.

[0016] Also disclosed herein are antigen-encoding cassettes, or polypeptide sequences encoded by cassettes, which comprise, from 5' to 3', the following formula: (E x -(E N n ) y ) z wherein E represents a nucleotide sequence comprising distinct epitope-encoding nucleic acid sequences, n represents the number of distinct epitope-encoding nucleic acid sequences, and is any integer including 0; N represents a nucleotide sequence constituting a separate and distinct epitope-encoding nucleic acid sequence for each corresponding n, where for every z repeats, x=0 or 1, y=0 or 1 for each n, and at least one of x or y=1, and z=2 or more, where the antigen-encoding nucleic acid sequence is E, a given E N or a combination thereof, wherein at least one of the distinct epitope-encoding nucleic acid sequences comprising at least two repeats encodes an EGFR-associated MHC class I neoepitope.

[0017] In some embodiments, at least two of the distinct epitope-encoding nucleic acid sequences comprising at least two repeats encode distinct EGFR-associated MHC class I neoepitopes. In some embodiments, at least three of the distinct epitope-encoding nucleic acid sequences comprising at least two repeats encode distinct EGFR-associated MHC class I neoepitopes. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight of the distinct epitope-encoding nucleic acid sequences comprising at least two repeats encode distinct EGFR-associated MHC class I neoepitopes.

[0018] In some embodiments, each of the distinct epitope-encoding nucleic acid sequences comprising at least two repeats encodes a distinct EGFR-associated MHC class I neoepitope. In some embodiments, one or more of the epitope-encoding nucleic acid sequences encoding an EGFR-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, each of the epitope-encoding nucleic acid sequences encoding an EGFR-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.

[0019] In some embodiments, one or more of the nucleic acid sequences encoding distinct EGFR-associated MHC class I neoepitopes comprises at least four repeats, hi some embodiments, each of the nucleic acid sequences encoding distinct EGFR-associated MHC class I neoepitopes comprises at least four repeats.

[0020] In some embodiments, the EGFR-associated MHC class I neoepitope is selected from the group consisting of an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, an EGFR E746-A750 deletion MHC class I epitope, and combinations thereof.

[0021] In some embodiments, the EGFR-associated MHC class I neoepitope comprises an EGFR_L858R MHC class I epitope. In some embodiments, the EGFR_L858R MHC class I epitope comprises the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, or ITDFGRAKL. In some embodiments, the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, or ITDFGRAKL. In some embodiments, the EGFR_L858R MHC class I epitope comprises the amino acid sequence KITDFGRAK. In some embodiments, the EGFR_L858R MHC class I epitope is KITDFGRAK or KITDFGRAKL. In some embodiments, the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAK. In some embodiments, the EGFR_L858R MHC class I epitope is KITDFGRAKL.

[0022] In some embodiments, the EGFR-associated MHC class I neoepitope comprises the T790M MHC class I epitope. In some embodiments, the T790M MHC class I epitope comprises the amino acid sequence STVQLIMQL, TVQLIMQL, or LTSTVQLIM. In some embodiments, the T790M MHC class I epitope is the amino acid sequence STVQLIMQL, TVQLIMQL, or LTSTVQLIM. In some embodiments, the T790M MHC class I epitope comprises the amino acid sequence STVQLIMQL or LTSTVQLIM. In some embodiments, the T790M MHC class I epitope is the amino acid sequence STVQLIMQL or LTSTVQLIM. In some embodiments, the T790M MHC class I epitope comprises the amino acid sequence STVQLIMQL. In some embodiments, the T790M MHC class I epitope comprises the amino acid sequence TVQLIMQL. In some embodiments, the T790M MHC class I epitope comprises the amino acid sequence LTSTVQLIM. In some embodiments, the T790M MHC class I epitope is the amino acid sequence STVQLIMQL. In some embodiments, the T790M MHC class I epitope is the amino acid sequence TVQLIMQL. In some embodiments, the T790M MHC class I epitope is the amino acid sequence LTSTVQLIM.

[0023] In some embodiments, the EGFR-associated MHC class I neoepitope comprises an EGFR E746-A750 deletion MHC class I epitope. In some embodiments, the E746-A750 deletion MHC class I epitope comprises the amino acid sequence PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK. In some embodiments, the E746-A750 deletion MHC class I epitope is the amino acid sequence PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK.

[0024] In some embodiments, the antigen-encoding cassette or at least one antigen-encoding nucleic acid sequence encodes at least each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. In some embodiments, the antigen-encoding cassette or at least one antigen-encoding nucleic acid sequence encodes at least four repeats of each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. In some embodiments, the antigen-encoding cassette or at least one antigen-encoding nucleic acid sequence encodes at least eight repeats of each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope.

[0025] In some embodiments, the antigen-encoding cassette or at least one antigen-encoding nucleic acid sequence encodes at least each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope. In some embodiments, the antigen-encoding cassette or at least one antigen-encoding nucleic acid sequence encodes at least four repeats of each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope. In some embodiments, the antigen-encoding cassette or at least one antigen-encoding nucleic acid sequence encodes at least eight repeats of each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope.

[0026] In some embodiments, the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. In some embodiments, the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least four repeats of each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. In some embodiments, the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least eight repeats of each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. In some embodiments, the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope. In some embodiments, the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least four repeats of each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope. In some embodiments, the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least eight repeats of each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope.

[0027] In some embodiments, x=1, n=0, and E xencodes an EGFR-associated MHC class I neoepitope. In some embodiments, one or more of the distinct epitope-encoding nucleic acid sequences encoding an EGFR-associated MHC class I neoepitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 repeats. In some embodiments, at least one distinct epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope comprises at least 8 repeats. In some embodiments, at least one distinct epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope comprises at least 4 repeats.

[0028] In some embodiments, x=1, n=0, z=4, and E x encodes an EGFR-associated MHC class I neoepitope, the EGFR-associated MHC class I neoepitope comprising the EGFR_L858R MHC class I epitope comprising the amino acid sequence KITDFGRAK. In some embodiments, x=1, n=0, z=8, and E x encodes an EGFR-associated MHC class I neoepitope, and the EGFR-associated MHC class I neoepitope includes the EGFR_L858R MHC class I epitope, which includes the amino acid sequence KITDFGRAK.

[0029] In some embodiments, E A and E B and optionally E C From 5' to 3', the formula (L5 b -N c -L3 d ) in which N is E A , E B , and / or E Cwherein c=1, L5 comprises a 5' linker sequence, where b=0 or 1, and L3 comprises a 3' linker sequence, where d=0 or 1. In some embodiments, each N encodes an epitope 7-15 amino acids in length, L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of the epitope, where the 5' linker sequence encodes a peptide that is at least 2 amino acids in length, and optionally 2-20 amino acids in length, and L3 is a natural 3' linker sequence encoding the natural C-terminal amino acid sequence of the epitope, where the 3' linker sequence encodes a peptide that is at least 2 amino acids in length, and optionally 2-20 amino acids in length, and optionally A , E B , and / or E C Each of the E encodes a polypeptide that is 12 to 35 amino acids in length. A and E B , and optionally E C Each of E encodes an epitope at least 7 amino acids in length. A and E B , and optionally E C Each of the E encodes an epitope of 7 to 15 amino acids in length. A and E B , and optionally E C Each of E is a nucleotide sequence at least 21 nucleotides in length. A and E B , and optionally E C Each of these is a nucleotide sequence of 75 nucleotides.

[0030] In some embodiments, each E or E N are independently converted from 5' to 3' into the formula (L5 b -N c -L3 d ), wherein N is each E or E Nwherein c=1, L5 comprises a 5' linker sequence, where b=0 or 1, and L3 comprises a 3' linker sequence, where d=0 or 1. In some embodiments, each N encodes an epitope 7-15 amino acids in length, L5 is a native 5' linker sequence that encodes the native N-terminal amino acid sequence of the epitope, where the 5' linker sequence encodes a peptide that is at least 2 amino acids in length, and L3 is a native 3' linker sequence that encodes the native C-terminal amino acid sequence of the epitope, where the 3' linker sequence encodes a peptide that is at least 2 amino acids in length.

[0031] In some embodiments, each of the epitope-encoding nucleic acid sequences encodes a polypeptide that is between 12 and 35 amino acids in length.

[0032] 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 of 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.

[0033] Also provided herein is a composition for delivering an antigen expression system, comprising the 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, the epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope, the EGFR-associated MHC class I neoepitope being selected from the group consisting of an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, an EGFR_L858R MHC class I epitope, an EGFR_T790M ... and (iii) optionally, at least one MHC class II epitope-encoding nucleic acid sequence; and (iv) optionally, 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 an exogenous poly(A) sequence 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 an epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope.

[0034] Also provided herein is a composition for delivering an antigen expression system, comprising the 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) at least one polyadenylation (poly(A)) sequence; and (b) a cassette, the cassette comprising: (i) at least one antigen-encoding nucleic acid sequence, the epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope, wherein the EGFR-associated MHC class I neoepitope is EGFR_L858R. (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; 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 an exogenous poly(A) sequence 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 an epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope.

[0035] In some embodiments, the EGFR_L858R MHC class I epitope comprises the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, or ITDFGRAKL. In some embodiments, the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, or ITDFGRAKL. In some embodiments, the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAK or KITDFGRAKL. In some embodiments, the EGFR_L858R MHC class I epitope comprises the amino acid sequence KITDFGRAK. In some embodiments, the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAK. In some embodiments, the EGFR_L858R MHC class I epitope comprises the amino acid sequence KITDFGRAKL. In some embodiments, the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAKL.

[0036] In some embodiments, the T790M MHC class I epitope comprises the amino acid sequence STVQLIMQL, TVQLIMQL, or LTSTVQLIM. In some embodiments, the T790M MHC class I epitope is the amino acid sequence STVQLIMQL, TVQLIMQL, or LTSTVQLIM.

[0037] In some embodiments, the T790M MHC class I epitope comprises the amino acid sequence STVQLIMQL or LTSTVQLIM. In some embodiments, the T790M MHC class I epitope is the amino acid sequence STVQLIMQL or LTSTVQLIM. In some embodiments, the T790M MHC class I epitope comprises the amino acid sequence STVQLIMQL. In some embodiments, the T790M MHC class I epitope is the amino acid sequence STVQLIMQL. In some embodiments, the T790M MHC class I epitope comprises the amino acid sequence LTSTVQLIM. In some embodiments, the T790M MHC class I epitope is the amino acid sequence LTSTVQLIM.

[0038] In some embodiments, the E746-A750 deletion MHC class I epitope comprises the amino acid sequence PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK. In some embodiments, the E746-A750 deletion MHC class I epitope is the amino acid sequence PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK. In some embodiments, the E746-A750 deletion MHC class I epitope comprises the amino acid sequence PVAIKTSPK. In some embodiments, the E746-A750 deletion MHC class I epitope is the amino acid sequence PVAIKTSPK. In some embodiments, the E746-A750 deletion MHC class I epitope comprises the amino acid sequence AIKTSPKANK. In some embodiments, the E746-A750 deletion MHC class I epitope is the amino acid sequence AIKTSPKANK. In some embodiments, the E746-A750 deletion MHC class I epitope comprises the amino acid sequence VAIKTSPK. In some embodiments, the E746-A750 deletion MHC class I epitope is the amino acid sequence VAIKTSPK. In some embodiments, the E746-A750 deletion MHC class I epitope comprises the amino acid sequence KIPVAIKTSPK. In some embodiments, the E746-A750 deletion MHC class I epitope is the amino acid sequence KIPVAIKTSPK.

[0039] Also provided herein is a composition for delivering an antigen expression system, comprising the antigen expression system, the antigen expression system comprising one or more vectors, the one or more vectors comprising: (a) a vector backbone, comprising a chimpanzee adenovirus vector, optionally wherein the chimpanzee adenovirus vector is a ChAdV68 vector or an alphavirus vector, optionally wherein the alphavirus vector is a Venezuelan equine encephalitis virus vector; and (b) a cassette, optionally wherein the cassette is incorporated between a native promoter nucleotide sequence and a poly(A) sequence native to the vector backbone, optionally wherein the poly(A) sequence is native to the vector backbone, and comprising: (i) at least one antigen-encoding nucleic acid sequence, wherein the cassette comprises: (I) an epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope, optionally comprising: (A) an MHC class I epitope coding sequence encoding an EGFR-associated MHC class I neoepitope, at least one antigen-encoding nucleic acid sequence, wherein the cassette comprises epitope-encoding nucleic acid sequences comprising: (A) an MHC class I epitope-encoding nucleic acid sequence encoding an MHC class I epitope of 7 to 15 amino acids in length; (B) a 5' linker sequence encoding the native N-terminal amino acid sequence of the MHC class I epitope, wherein the 5' linker sequence encodes a peptide that is at least 3 amino acids in length; and (C) a 3' linker sequence encoding the native C-terminal amino acid sequence of the MHC class I epitope, wherein the 3' linker sequence encodes a peptide that is at least 3 amino acids in length; and the cassette is operably linked to a native promoter nucleotide sequence, wherein each of the epitope-encoding nucleic acid sequences encodes a polypeptide that is 12 to 35 amino acids in length, and wherein each 3' end of each epitope-encoding nucleic acid sequence is linked to the 5' end of the next 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, including: (I) the PADRE MHC class II sequence (SEQ ID NO: 48), (II) the tetanus toxoid MHC class II sequence (SEQ ID NO: 46), (III) the PADRE (IV) a second nucleic acid sequence encoding a GGPPG amino acid linker sequence linking the 5' ends of the at least two MHC Class II epitope-encoding nucleic acid sequences to the epitope-encoding nucleic acid sequences; (V) optionally, a third nucleic acid sequence encoding a GGPPG amino acid linker sequence at the 3' ends of the at least two MHC Class II epitope-encoding nucleic acid sequences; (iii) optionally, a second promoter nucleotide sequence operably linked to the antigen-encoding nucleic acid sequences; and, if the second promoter nucleotide sequence is not present, the antigen-encoding nucleic acid sequence is operably linked to a native promoter nucleotide sequence, and at least one antigen-encoding nucleic acid sequence comprises at least two repeats of an epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC Class I neoepitope.

[0040] In some embodiments, the ordered sequence of each element of the cassette comprises, from 5' to 3', the formula: P a -(L5 b -N c -L3 d ) X -(G5 e -U f ) Y -G3 g wherein P comprises a second promoter nucleotide sequence, a=0 or 1, N comprises one of the distinct epitope-encoding nucleic acid sequences, c=1, L5 comprises a 5' linker sequence, b=0 or 1, L3 comprises a 3' linker sequence, d=0 or 1, G5 comprises one of at least one nucleic acid sequences encoding a GPGPG amino acid linker, e=0 or 1, G3 comprises one of at least one nucleic acid sequences encoding a GPGPG amino acid linker, g=0 or 1, U comprises one of at least one MHC class II epitope-encoding nucleic acid sequences, f=1, X=1 to 400, and for each X, a corresponding N c is an epitope-encoding nucleic acid sequence, Y=0, 1, or 2, and for each Y, a corresponding U f is an MHC class II epitope-encoding nucleic acid sequence.

[0041] In some embodiments, for each X, a corresponding N c is an epitope-encoding nucleic acid sequence that encodes an EGFR-associated MHC class I neoepitope. In some embodiments, for each Y, a corresponding U fare distinct MHC class II epitope-encoding nucleic acid sequences. In some embodiments, a=0, b=1, d=1, e=1, g=1, h=1, X=10, and Y=2, the at least one promoter nucleotide sequence is a single native promoter nucleotide sequence native to the vector backbone, the at least one polyadenylation poly(A) sequence is a poly(A) sequence of at least 100 contiguous A nucleotides provided by the vector 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, where the 5' linker sequence encodes a peptide that is at least 3 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 that is at least 3 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 adenoviral vector, optionally the chimpanzee adenoviral vector is a ChAdV68 vector or an alphavirus vector, optionally the alphavirus vector is a Venezuelan equine encephalitis virus vector; and optionally, if the vector backbone comprises an alphavirus vector, the native promoter nucleotide sequence is a 26S promoter; and each of the MHC class I epitope-encoding nucleic acid sequences encodes a polypeptide that is 12 to 35 amino acids in length.

[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 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, 6 or fewer, 5 or fewer, 4 or fewer, or 3 or fewer repeats. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least two repeats of at least two distinct epitope-encoding nucleic acid sequences. In some embodiments, the 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 distinct epitope-encoding nucleic acid sequences. In some embodiments, the at least two repeats are separated by at least one separate distinct epitope-encoding nucleic acid sequence. In some embodiments, the at least two repeats are separated by at least two separate and distinct epitope-encoding nucleic acid sequences. 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.

[0043] In some embodiments, the at least two repeats comprising the optional 5' linker sequence and / or the 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 comprising 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, at least one antigen-encoding nucleic acid sequence has the formula, from 5' to 3': (E x -(E N n ) y ) z wherein E represents a nucleotide sequence comprising at least one of the at least one distinct epitope-encoding nucleic acid sequence, n represents the number of separate distinct epitope-encoding nucleic acid sequences and is any integer including 0, and E N represents a nucleotide sequence constituting a separate and distinct epitope-encoding nucleic acid sequence for each corresponding n, where for every z repeats, x=0 or 1, y=0 or 1 for each n, and at least one of x or y=1, and z=2 or more, where the antigen-encoding nucleic acid sequence is E, a given E N or a combination thereof.

[0045] In some embodiments, the EGFR-associated MHC class I neoepitope comprises an EGFR_L858R MHC class I epitope. In some embodiments, the EGFR_L858R MHC class I epitope comprises the amino acid sequence KITDFGRAK. In some embodiments, the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAK. In some embodiments, the EGFR-associated MHC class I neoepitope comprises a T790M MHC class I epitope. In some embodiments, the T790M MHC class I epitope comprises the amino acid sequence STVQLIMQL or LTSTVQLIM. In some embodiments, the T790M MHC class I epitope is the amino acid sequence STVQLIMQL or LTSTVQLIM. In some embodiments, the EGFR-associated MHC class I neoepitope comprises an EGFR E746-A750 deletion MHC class I epitope. In some embodiments, the E746-A750 deleted MHC class I epitope comprises the amino acid sequence PVAIKTSPK, VAIKTSPK, or KIPVAIKTSPK. In some embodiments, the E746-A750 deleted MHC class I epitope is the amino acid sequence PVAIKTSPK, VAIKTSPK, or KIPVAIKTSPK.

[0046] In some embodiments, at least one antigen-encoding nucleic acid sequence encodes at least each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. In some embodiments, at least one antigen-encoding nucleic acid sequence encodes at least four repeats of each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. In some embodiments, at least one antigen-encoding nucleic acid sequence encodes at least eight repeats of each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. In some embodiments, at least one antigen-encoding nucleic acid sequence encodes at least each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope. In some embodiments, the at least one antigen-encoding nucleic acid sequence encodes at least four repeats of each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope. In some embodiments, the at least one antigen-encoding nucleic acid sequence encodes at least eight repeats of each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope.

[0047] In some embodiments, x=1, n=0, and E x encodes an EGFR-associated MHC class I neoepitope. In some embodiments, the epitope-encoding nucleic acid sequence encoding the EGFR-associated MHC class I neoepitope comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 repeats. In some embodiments, the epitope-encoding nucleic acid sequence encoding the EGFR-associated MHC class I neoepitope comprises at least 8 repeats. In some embodiments, the epitope-encoding nucleic acid sequence encoding the EGFR-associated MHC class I neoepitope comprises at least 4 repeats. In some embodiments, x=1, n=0, z=4, and Ex encodes an EGFR-associated MHC class I neoepitope, the EGFR-associated MHC class I neoepitope comprising the EGFR_L858R MHC class I epitope comprising the amino acid sequence KITDFGRAK. In some embodiments, x=1, n=0, z=8, and E x encodes an EGFR-associated MHC class I neoepitope, and the EGFR-associated MHC class I neoepitope includes the EGFR_L858R MHC class I epitope, which includes the amino acid sequence KITDFGRAK.

[0048] In some embodiments, at least two repeats comprise a plurality of repeats, or z comprises a number sufficient to stimulate a greater immune response compared to an antigen-encoding nucleic acid sequence comprising a single repeat of at least one epitope-encoding nucleic acid sequence. In some embodiments, at least two repeats comprise a plurality of repeats, or z comprises a number sufficient to stimulate an immune response, where a single repeat of at least one epitope-encoding nucleic acid sequence is insufficient to stimulate an immune response or is insufficient to stimulate a detectable immune response. In some embodiments, the immune response is proliferation of epitope-specific T cells following in vivo immunization with a composition for delivery of an 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 a composition for delivery of an antigen expression system.

[0049] 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 nanoparticulate delivery vehicle encapsulates an antigen expression system.

[0050] In some embodiments, the nanoparticulate delivery vehicle encapsulates an antigen expression system. In some embodiments, the cassette is incorporated between at least one promoter nucleotide sequence and at least one poly(A) sequence. In some embodiments, the second promoter is absent, and the at least one promoter nucleotide sequence is operably linked to the antigen-encoding nucleic acid sequence. 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 comprise 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, nsP2 gene, nsP3 gene, and 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, and an alphavirus 3' UTR, or a combination thereof. In some embodiments, the scaffold does not encode the structural virion protein capsids E2 and E1. In some embodiments, the cassette is inserted into the nucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus in place of the structural virion proteins.

[0051] 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 comprises the sequence of SEQ ID NO:3 or SEQ ID NO:5 further comprising 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, the cassette is inserted at position 7544 to replace the deletion between base pairs 7544 and 11175 set forth in the sequence of SEQ ID NO:3 or SEQ ID NO:5.

[0052] In some embodiments, insertion of the cassette provides for transcription of a polycistronic RNA comprising the nsP1-4 genes and 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.

[0053] In some embodiments, the chimpanzee adenovirus vector is a ChAdV68 vector, optionally wherein the ChAdV68 vector comprises a ChAdV68 vector backbone, the ChAdV68 vector backbone having a sequence set forth in SEQ ID NO:1; with the proviso that the sequence is completely or functionally deleted in 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, optionally wherein the sequence is completely or functionally deleted in: (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; and a gene or regulatory sequence derived from the sequence of SEQ ID NO:1, optionally wherein the gene is not a gene set forth in SEQ ID NO:1. a gene or regulatory sequence obtained from the sequence of SEQ ID NO: 1 selected from the group consisting of chimpanzee adenovirus inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence set forth in 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, and optionally a deleted or partially deleted E4orf4 region; at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1, and further comprising: (1) an E1 deletion of at least nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, (2) an E3 deletion of at least nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO:1, and (3) an E4 deletion of at least nucleotides 34,916 to 35,642 of the sequence set forth in SEQ ID NO:1; optionally, an antigen cassette is inserted within the E1 deletion;There are one or more deletions between base pairs 577 and 3403 or between base pairs 456 and 3014, and optionally the vector further comprises one or more deletions between base pairs 27,125 and 31,825 or between base pairs 27,816 and 31,333 of the sequence set forth in SEQ ID NO:1, or one or more deletions between base pairs 3957 and 10346, between base pairs 21787 and 23370, and between base pairs 33486 and 36193 of the sequence set forth in SEQ ID NO:1, and optionally the cassette is inserted into the E1 region, E3 region, and / or any deleted AdV region that allows for integration of the cassette of the ChAdV vector backbone;

[0054] 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 26S promoter nucleotide sequence providing transcription of one or more separate open reading frames.

[0055] In some embodiments, one or more of the cassettes are at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides in length. In some embodiments, one or more of the cassettes are at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nucleotides in length. In some embodiments, one or more of the cassettes are at least 3500 nucleotides in length. In some embodiments, one or more of the cassettes are at least 6000 nucleotides in length.

[0056] 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.

[0057] In some embodiments, each epitope-encoding nucleic acid sequence is directly linked to one another. In some embodiments, at least one of the at least one epitope-encoding nucleic acid sequence is linked to another 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 an MHC class II sequence. In some embodiments, the linker is selected from the group consisting of: (1) a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive glycine residues in length; (2) a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive alanine residues in length; (3) two arginine residues (RR); (4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length that is efficiently processed by the mammalian proteasome; and (6) one or more naturally occurring sequences flanking the antigen from the original cognate 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 an MHC class I sequence. In some embodiments, the linker comprises the sequence GPGPG. In some embodiments, at least one of the at least one epitope-encoding nucleic acid sequences is operably or directly linked to a separate or consecutive sequence that enhances expression, stability, cellular trafficking, processing and presentation, and / or immunogenicity of the at least one epitope-encoding nucleic acid sequence of the epitope encoded therefrom.In some aspects, the separate or consecutive sequences comprise at least one of a ubiquitin sequence, a ubiquitin sequence modified to increase proteasome targeting (e.g., the ubiquitin sequence contains a Gly to 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, and optionally, the ubiquitin sequence modified to increase proteasome targeting is A76.

[0058] In some embodiments, at least one of the at least one epitope-encoding nucleic acid sequences encodes a polypeptide sequence or a portion thereof that has increased binding affinity to a corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence when translated. In some embodiments, at least one of the at least one epitope-encoding nucleic acid sequences encodes a polypeptide sequence or a portion thereof that has increased binding stability to a corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence when translated. In some embodiments, at least one of the at least one epitope-encoding nucleic acid sequences encodes a polypeptide sequence or a portion thereof that has increased presentation potential on a corresponding MHC allele compared to the corresponding wild-type nucleic acid sequence when translated. 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.

[0059] In some embodiments, the tumor is selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, bladder cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, adult acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0060] 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 on a tumor 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, wherein at least two of the antigen-encoding nucleic acid sequences encode polypeptide sequences or portions thereof that are presented by MHC class I on a cell surface, optionally on a tumor cell surface. In some embodiments, 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 on a tumor cell surface.

[0061] In some embodiments, when administered to a subject and translated, the EGFR-associated MHC class I neoepitopes are presented on antigen-presenting cells, generating an immune response that targets the EGFR-associated MHC class I neoepitopes on the surface of tumor cells. In some embodiments, at least one antigen-encoding nucleic acid sequence is administered to a subject and translated, when the EGFR-associated MHC class I neoepitopes are presented on antigen-presenting cells, generating an immune response that targets the EGFR-associated MHC class I neoepitopes on the surface of tumor cells, and optionally, expression of each of the at least one antigen-encoding nucleic acid sequence is driven by at least one promoter nucleotide sequence.

[0062] In some embodiments, each epitope-encoding nucleic acid sequence encodes a polypeptide sequence between 8 and 35 amino acids in length, optionally between 9 and 17, 9 and 25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids in length.

[0063] 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 that includes at least one alteration that renders the encoded peptide sequence distinct 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, 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 wherein the at least one universal sequence comprises at least one of tetanus toxoid and PADRE.

[0064] 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.

[0065] In some embodiments, at least one poly(A) sequence comprises a poly(A) sequence native to the backbone. In some embodiments, at least one poly(A) sequence comprises a poly(A) sequence exogenous to 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, or at least 90 consecutive A nucleotides. In some embodiments, at least one poly(A) sequence is at least 100 consecutive A nucleotides.

[0066] In some embodiments, the cassette further comprises at least one of an intron sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence, an internal ribosome entry sequence (IRES) sequence, a nucleotide sequence encoding a 2A self-cleaving peptide sequence, a nucleotide sequence encoding a Furin cleavage site, or a sequence within a 5' or 3' non-coding region known to enhance nuclear export, stability, or translation efficiency of an mRNA operably linked to at least one of the at least one antigen-encoding nucleic acid sequences. 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.

[0067] In some embodiments, the one or more vectors further comprise one or more nucleic acid sequences encoding at least one immunomodulator. In some embodiments, the immunomodulator is an anti-CTLA4 antibody or 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) that is monospecific or has multiple specificities linked together, or a full-length single-chain antibody (e.g., a full-length IgG in which the heavy and light chains are linked by a flexible linker). In some embodiments, the antibody heavy and light chain sequences are contiguous sequences separated by either a self-cleaving sequence such as 2A or an IRES, or the antibody heavy and light chain sequences are linked by a flexible linker such as consecutive glycine residues. In some embodiments, the immunomodulator is a cytokine, hi some embodiments, the cytokine is at least one of IL-2, IL-7, IL-12, IL-15, or IL-21, or a variant of each thereof.

[0068] In some aspects, the 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 the tumor, 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 sequences of each antigen into a presentation model to generate a set of numerical possibilities that each of the antigens is presented by one or more MHC alleles on a cell surface, optionally on the tumor cell surface, wherein the set of numerical possibilities has been identified 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 possibilities to generate a set of selected antigens to be used to generate the at least one epitope-encoding nucleic acid sequence.

[0069] 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 the tumor, where the nucleotide sequencing data is used to obtain data representing the peptide sequence 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 possibilities that each of the antigens is presented by one or more MHC alleles on a cell surface, optionally on the tumor cell surface, where the set of numerical possibilities is identified 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 possibilities to generate a set of selected antigens that are used to generate 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 amino acid with a particular one of the MHC alleles at a particular position in a peptide sequence and (b) the likelihood of presentation of such a peptide sequence containing a particular amino acid at a particular position on a cell surface, optionally on a tumor cell surface, by a particular one of the paired MHC alleles. In some embodiments, selecting the set of selected antigens includes selecting antigens that are more likely to be presented on a cell surface than antigens not selected based on the presentation model, and optionally, the selected antigens are verified to be presented by one or more particular HLA alleles. In some embodiments, selecting the set of selected antigens includes selecting antigens that are more likely to be capable of inducing a tumor-specific or infectious disease-specific immune response in a subject than antigens not selected based on the presentation model. In some embodiments, selecting the set of selected antigens includes selecting antigens that are more likely to be capable of being presented to naive T cells by professional antigen-presenting cells (APCs) than antigens not selected based on the presentation model, and optionally, the APCs are dendritic cells (DCs).In some embodiments, selecting the set of selected antigens comprises selecting antigens that are less likely to be inhibited by central tolerance or peripheral tolerance than antigens that are not selected based on the presentation model. In some embodiments, selecting the set of selected antigens comprises selecting antigens that are less likely to induce an autoimmune response against normal tissues in a subject than antigens that are not selected based on the presentation model. In some embodiments, the nucleotide sequencing data of the exome or transcriptome is obtained by performing sequencing on tumor cells or tissues. In some embodiments, the sequencing is next-generation sequencing (NGS) or any massively parallel sequencing method.

[0070] In some embodiments, the cassette comprises a junctional epitope sequence formed by adjacent sequences within the cassette. In some embodiments, at least one or each junctional epitope sequence has an affinity for MHC greater than 500 nM. In some embodiments, each junctional epitope sequence is non-self.

[0071] 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 prevalence in the population of at least 0.01%. 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 prevalence in the population of at least 0.1%. In some embodiments, the EGFR-associated MHC class I neoepitope is verified to be capable of presentation by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA prevalence in the population of at least 1%. In some embodiments, the EGFR-associated MHC class I neoepitope has been validated for presentation by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA prevalence in the population of at least 10%. In some embodiments, the EGFR-associated MHC class I neoepitope has been validated for presentation by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA prevalence in the population of at least 14%. In some embodiments, the at least one HLA allele is HLA A * 03:01, HLA C * 15:02, and / or HLA A * In some embodiments, at least one HLA allele is HLA A * In some embodiments, at least one HLA allele is HLA C 03:01. * In some embodiments, at least one HLA allele is HLA A 15:02. * 11:01. In some embodiments, the population is the Chinese Hubei Han population.

[0072] In some embodiments, the cassette does not encode a non-therapeutic MHC class I or class II epitope nucleic acid sequence, including a translated wild-type nucleic acid sequence, where the non-therapeutic epitope is predicted to be presented on an MHC allele of interest. In some embodiments, the non-therapeutic predicted MHC class I or class II epitope sequence is a junctional epitope sequence formed by flanking sequences within the cassette.

[0073] In some embodiments, the prediction is based on presentation probabilities generated by inputting the sequence of the non-therapeutic epitope into a presentation model.

[0074] In some aspects, the order of at least one antigen-encoding nucleic acid sequence in the cassette is determined by a series of steps including: (a) generating a set of candidate cassette sequences corresponding to various orders of the at least one antigen-encoding nucleic acid sequence; (b) for each candidate cassette sequence, determining a presentation score based on the presentation of non-therapeutic epitopes in the candidate cassette sequence; and (c) selecting candidate cassette sequences associated with a presentation score below a predetermined threshold as cassette sequences for the antigen vaccine.

[0075] 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 immunomodulator. In some embodiments, the immunomodulator 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.

[0076] 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, where 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 sequence or set of isolated nucleotide sequences comprises the cassette of any of the above 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 T7 or SP6 RNA polymerase promoter nucleotide sequence 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 3' to the poly(A) sequence. In some embodiments, the cassette of any of the above composition claims is inserted at position 7563 of SEQ ID NO:8 or SEQ ID NO:9.

[0077] Also provided herein are vectors or vector sets comprising any of the nucleotide sequences described herein.

[0078] Also provided herein is an isolated cell comprising any of the nucleotide sequences or sets 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.

[0079] Also provided herein are kits comprising any of the compositions described herein and instructions for use.

[0080] In some embodiments, any of the above compositions further comprises a nanoparticulate delivery vehicle. In some embodiments, the nanoparticulate delivery vehicle may 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 nanoparticulate delivery vehicle encapsulates an antigen expression system.

[0081] 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 in the plurality of LNPs have a non-lamellar morphology or are electron dense.

[0082] In some embodiments, the non-cationic lipid is a mixture of (1) a phospholipid and (2) cholesterol or a cholesterol derivative.

[0083] 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 ) conjugates, and mixtures thereof.

[0084] In some embodiments, the antigen expression system is fully encapsulated in the LNP.

[0085] In some embodiments, the non-lamellar morphology of the LNPs is an inverted hexagonal (H II ) or cubic phase structure.

[0086] In some embodiments, the cationic lipids comprise about 10 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the cationic lipids comprise about 20 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the cationic lipids comprise about 20 mol% to about 40 mol% of the total lipids present in the LNP.

[0087] 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.

[0088] In some embodiments, the conjugated lipids comprise between about 0.5 mol% and about 20 mol% of the total lipids present in the LNP. In some embodiments, the conjugated lipids comprise between about 2 mol% and about 20 mol% of the total lipids present in the LNP. In some embodiments, the conjugated lipids comprise between about 1.5 mol% and about 18 mol% of the total lipids present in the LNP.

[0089] 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.

[0090] In some embodiments, any of the above compositions further comprise a plurality of LNPs, the LNPs comprising: a cationic lipid comprising 50 mol% to 65 mol% of the total lipids present in the LNP; a conjugated lipid that inhibits aggregation of the LNPs comprising 0.5 mol% to 2 mol% of the total lipids present in the LNP; and a non-cationic lipid, which is either a mixture of phospholipids and cholesterol or a derivative thereof, wherein the phospholipid comprises 4 mol% to 10 mol% of the total lipids present in the LNP and the cholesterol or a derivative thereof comprises 30 mol% to 40 mol% of the total lipids present in the LNP. a mixture of phospholipids and cholesterol or a derivative thereof, wherein the phospholipid constitutes 3 mol% to 15 mol% of the total lipids present in the LNP and the cholesterol or derivative thereof constitutes 30 mol% to 40 mol% of the total lipids present in the LNP; or ... cholesterol or derivative thereof constitutes 30 mol% to 40 mol% of the total lipids present in the LNP; and a mixture of phospholipids and cholesterol or a derivative thereof, wherein the cholesterol or derivative thereof constitutes 30 mol% to 40 mol% of the total lipids present in the LNP.

[0091] In some embodiments, any of the above compositions further comprise a plurality of LNPs, wherein the LNPs comprise a cationic lipid comprising 50 mol% to 85 mol% of the total lipids present in the LNPs; a conjugated lipid that inhibits aggregation of the LNPs comprising 0.5 mol% to 2 mol% of the total lipids present in the LNPs; and a non-cationic lipid comprising 13 mol% to 49.5 mol% of the total lipids present in the LNPs.

[0092] In some embodiments, the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.

[0093] 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-distearyloxypropyl (PEG-DSA) conjugate, or a mixture thereof. In some embodiments, the PEG portion of the conjugate has an average molecular weight of about 2,000 daltons.

[0094] In some embodiments, the conjugated lipid comprises 1 mol% to 2 mol% of the total lipid present in the LNP.

[0095] In some embodiments, the LNP has Formula I: TIFF2025534986000002.tif52165, or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, or -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -R a C(=O)-, -C(=O)R a -, -R a C(=O)R a -, -OC(=O)R a -, -R a C(=O)O-, or a direct bond; G 1 is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -R a C(=O)- or direct bond: -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)R a - or a direct bond; G is C1-C6 alkylene; R ais H or C1-C12 alkyl; R 1a and R 1b is, independently at each occurrence, either: (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, form an adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 2a and R 2b is, independently at each occurrence, either: (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, form an adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 3a and R 3b is, independently at each occurrence, either: (a) H or C1-C 12 alkyl; or (b) R 3a is H or C1-C 12 alkyl, and R 3b forms a carbon-carbon double bond together with the carbon atom to which it is attached and the adjacent R and the carbon atom to which it is attached; R 4a and R 4b is, independently at each occurrence, either: (a) H or C1-C12 alkyl; or (b) R 4a is H or C1-C12 alkyl, and R 4b together with the carbon atom to which it is attached, form an adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 5 and R 6 are each independently H or methyl; R 7 is C4-C20 alkyl; R 8 and R 9are each independently C1-C12 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.

[0096] In some embodiments, the LNP has Formula II: TIFF2025534986000003.tif37165, or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein L 1 and L 2 are each independently —O(C═O)—, —(C═O)O—, or a carbon-carbon double bond; R 1a and R 1b is, independently at each occurrence, either: (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, form an adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 2a and R 2b is, independently at each occurrence, either: (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, form an adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 3a and R 3b is, independently at each occurrence, either: (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 an adjacent R3b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 4a and R 4b is, independently at each occurrence, either: (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, form an adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 5 and R 6 are each independently methyl or cycloalkyl; R 7 is independently H or C1-C for each occurrence 12 alkyl; R 8 and R 9 are each independently an unsubstituted C1-C12 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 containing one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; e is 1 or 2, with the proviso that R 1a , R 2a , R 3a Or R 4a at least one of L is C1-C12 alkyl; 1 Or L 2 at least one of is -O(C=O)- or -(C=O)O-; R 1a and R 1b is not isopropyl when a is 6 or n-butyl when a is 8.

[0097] In some embodiments, any of the above compositions further comprise one or more excipients, including a neutral lipid, a steroid, and a polymer-conjugated 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.

[0098] In some embodiments, the molar ratio of compound to neutral lipid ranges from about 2:1 to about 8:1.

[0099] In some embodiments, the steroid is cholesterol, hi some embodiments, the molar ratio of the compound to cholesterol ranges from about 2:1 to 1:1.

[0100] In some embodiments, the polymer-conjugated 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 dialkyoxypropylcarbamate. In some embodiments, the PEGylated lipid has the following structure III: TIFF2025534986000004.tif27165 or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R 10 and R 11are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally interrupted by one or more ester linkages; and z has an average value in the range of 30 to 60. In some embodiments, R 10 and R 11 are each independently a straight saturated alkyl chain having 12 to 16 carbon atoms. In some embodiments, the average z is about 45. start here

[0101] 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.

[0102] Also provided herein are methods for treating a subject with cancer, comprising administering to the subject any of the compositions or any of the pharmaceutical compositions described herein. In some embodiments, at least one epitope-encoding nucleic acid sequence is derived from a tumor of a subject with cancer, or a cell or sample of an infected subject. In some embodiments, at least one epitope-encoding nucleic acid sequence is not derived from a tumor of a subject with cancer, or a cell or sample of an infected subject.

[0103] Also provided herein is a method for stimulating an immune response in a subject, the method comprising administering to the subject any of the compositions or any of the pharmaceutical compositions described herein.

[0104] In some embodiments, the subject expresses at least one HLA allele predicted or known to present an MHC class I epitope. 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, subcutaneous administration is near the site of administration of the composition or pharmaceutical composition or in proximity to lymph nodes that drain one or more vectors or compositions.

[0105] In some embodiments, the method further comprises administering a second vaccine composition to the subject. In some embodiments, the second vaccine composition is administered prior to 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 the antigen-encoding nucleic acid sequence of any of the compositions described herein. In some embodiments, the at least one antigen-encoding nucleic acid sequence encoded by the chimpanzee adenoviral vector is the same as the at least one antigen-encoding nucleic acid sequence of any of the compositions described herein.

[0106] Also provided herein are methods for producing one or more vectors according to any of the above composition claims, 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 further comprising adding an m7g cap to the resulting RNA in vitro; 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 full genome DNA synthesis, or full 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, silica column-based purification, or similar RNA purification methods.

[0107] Also provided herein are methods of making any of the above composition claims for delivery of an antigen expression system, comprising: (a) providing components for 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 combine to form a composition for delivery of the antigen expression system. In some embodiments, the conditions are provided by microfluidic mixing.

[0108] Also provided herein is a method for treating a subject having a disease, optionally wherein the disease is cancer or an infectious disease, comprising administering to the subject an antigen-based vaccine, wherein the antigen-based vaccine comprises an antigen-encoding cassette, or a polypeptide sequence encoded by a cassette, wherein the antigen-encoding cassette has, from 5' to 3', the formula: (E x -(EN n ) y ) z wherein E represents a nucleotide sequence comprising at least one of the at least one distinct epitope-encoding nucleic acid sequences, n represents the number of separate distinct epitope-encoding nucleic acid sequences and is any integer including 0, and E N represents a nucleotide sequence constituting a separate and distinct epitope-encoding nucleic acid sequence for each corresponding n, where for every z repeats, x=0 or 1, y=0 or 1 for each n, and at least one of x or y=1, and z=2 or more, where the antigen-encoding nucleic acid sequence is E, a given E N or a combination thereof, wherein at least one of the at least one distinct epitope-encoding nucleic acid sequence comprising at least two repeats encodes an EGFR-associated MHC class I neoepitope.

[0109] Also provided herein is a method for treating a subject having a disease, optionally wherein the disease is cancer, comprising administering to the subject an antigen-based vaccine, wherein the antigen-based vaccine comprises 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 epitope-encoding nucleic acid sequence encoding an EGFR-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. (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, the second poly(A) sequence being a native poly(A) sequence or an exogenous poly(A) sequence 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 an epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope.

[0110] In some embodiments, at least one epitope-encoding nucleic acid sequence is derived from a tumor of a subject with cancer. In some embodiments, at least one epitope-encoding nucleic acid sequence is not derived from a tumor of a subject with cancer.

[0111] Also provided herein is a method for stimulating an immune response in a subject, comprising administering to the subject an antigen-based vaccine, wherein the antigen-based vaccine comprises an antigen-encoding cassette, or a polypeptide sequence encoded by a cassette, wherein the antigen-encoding cassette has, from 5' to 3', the formula: (E x -(E N n ) y ) z wherein E represents a nucleotide sequence comprising at least one of the at least one distinct epitope-encoding nucleic acid sequences, n represents the number of separate distinct epitope-encoding nucleic acid sequences and is any integer including 0, and E N represents a nucleotide sequence constituting a separate and distinct epitope-encoding nucleic acid sequence for each corresponding n, where for every z repeats, x=0 or 1, y=0 or 1 for each n, and at least one of x or y=1, and z=2 or more, where the antigen-encoding nucleic acid sequence is E, a given E N or a combination thereof, wherein at least one of the at least one distinct epitope-encoding nucleic acid sequence comprising at least two repeats encodes an EGFR-associated MHC class I neoepitope.

[0112] Also provided herein is a method for stimulating an immune response in a subject, comprising administering to the subject an antigen-based vaccine, wherein the antigen-based vaccine comprises 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 backbone comprising: (I) at least one epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope, wherein each epitope-encoding nucleic acid sequence 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, the at least one second poly(A) sequence being a native poly(A) sequence or an exogenous poly(A) sequence 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 an epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope.

[0113] In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present an EGFR-associated MHC class I neoepitope. In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present an EGFR-associated MHC class I neoepitope, wherein the at least one HLA allele is HLA A * 03:01, HLA C * 15:02, and / or HLA A * In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present an EGFR-associated MHC class I neoepitope, and the at least one HLA allele is HLA A * In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present an EGFR-associated MHC class I neoepitope, and the at least one HLA allele is HLA C * In some embodiments, the subject expresses at least one HLA allele that is predicted or known to present an EGFR-associated MHC class I neoepitope, and the at least one HLA allele is HLA A * It's 11:01.

[0114] In some embodiments, the subject expresses at least one HLA allele predicted or known to present at least one epitope sequence, wherein the at least one epitope sequence comprises an epitope known or suspected to be presented by MHC class I on the surface of the cell, wherein the at least one epitope sequence predicted or known to be presented comprises an EGFR-associated MHC class I neoepitope. In some embodiments, the surface of the 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 tumor is non-small cell lung cancer (NSCLC).

[0115] Also provided herein is a method for inducing an immune response in a subject, comprising administering to the subject an antigen-based vaccine, wherein the antigen-based vaccine comprises an antigen-encoding cassette, or a polypeptide sequence encoded by a cassette, wherein the antigen-encoding cassette has, from 5' to 3', the formula: (E x -(E N n ) y ) z wherein E represents a nucleotide sequence comprising at least one of the at least one distinct epitope-encoding nucleic acid sequences, n represents the number of separate distinct epitope-encoding nucleic acid sequences and is any integer including 0, and E N represents a nucleotide sequence constituting a separate and distinct epitope-encoding nucleic acid sequence for each corresponding n, where for every z repeats, x=0 or 1, y=0 or 1 for each n, and at least one of x or y=1, and z=2 or more, where the antigen-encoding nucleic acid sequence is E, a given E Nor a combination thereof, wherein at least one of the at least one distinct epitope-encoding nucleic acid sequences comprising at least two repeats encodes an EGFR-associated MHC class I neoepitope, and the subject expresses at least one HLA allele predicted or known to present at least one EGFR-associated MHC class I neoepitope.

[0116] Also provided herein is a method for inducing an immune response in a subject, comprising administering to the subject an antigen-based vaccine, wherein the antigen-based vaccine comprises 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 vector backbone comprising: (I) at least one epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neo-epitope, wherein each epitope-encoding nucleic acid sequence comprises: (A) optionally, a 5' linker sequence, and (B) optionally, a 3' linker sequence; and (ii) optionally, a nucleic acid sequence functionally 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, which is a native poly(A) sequence or an exogenous poly(A) sequence 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 an epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope, wherein the subject expresses at least one HLA allele predicted or known to present at least one EGFR-associated MHC class I neoepitope.

[0117] In some embodiments, the antigen-encoding nucleic acid sequence encodes a peptide comprising the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the antigen-encoding nucleic acid sequence encodes a peptide comprising the amino acid sequence set forth in SEQ ID NO: 61.

[0118] In some embodiments, at least one promoter sequence is a CMV, SV40, EF-1, RSV, PGK, HSA, MCK, or EBV promoter sequence. In some embodiments, at least one promoter sequence is a regulatable promoter, optionally, the regulatable promoter is a tetracycline (TET) repressor protein (TETr)-controlled promoter, optionally, the regulatable promoter comprises multiple TET operator (TETo) sequences 5' or 3' to the RNA polymerase binding sequence of the promoter. In some embodiments, P comprises a promoter sequence derived from CMV, optionally, the CMV-derived promoter sequence comprises a TETr-controlled CMV-derived promoter.

[0119] 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.

[0120] 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 boost doses. In some embodiments, the boost dose is different from the priming dose. In some embodiments, a) the priming dose comprises a chimpanzee adenovirus vector and the boost dose comprises an alphavirus vector; or b) the priming dose comprises an alphavirus vector and the boost dose comprises a chimpanzee adenovirus vector. In some embodiments, the boost dose is the same as the priming dose. In some embodiments, the injection site of one or more boost doses is as close as possible to the injection site of the priming dose.

[0121] In some embodiments, the methods further comprise determining or having determined the subject's HLA haplotype.

[0122] 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 IM administration is administered 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 gluteal or rectus femoris muscle sites.

[0123] 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 immunomodulator. In some embodiments, the immunomodulator 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.

[0124] Also disclosed herein are vectors that comprise the isolated nucleotide sequences disclosed herein.

[0125] Also disclosed herein are kits that include a vector or composition disclosed herein and instructions for use.

[0126] Also disclosed herein are methods of treating a subject, comprising administering to the subject a vector disclosed herein or a pharmaceutical composition disclosed herein. Also disclosed herein are methods for inducing an immune response in a subject, comprising administering to the subject any of the compositions, vectors, or pharmaceutical compositions described herein. In some embodiments, the subject expresses at least one HLA allele predicted or known to present an MHC class I epitope. In some embodiments, the vector or composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV).

[0127] Also disclosed herein are methods for producing one or more vectors of any of the above compositions, 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 further comprising adding an m7g cap to the resulting RNA in vitro; 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 either bacterial recombination or full genome DNA synthesis, or full 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, silica column-based purification, or similar RNA purification methods.

[0128] Also disclosed herein are methods of making any of the compositions disclosed herein, comprising: providing components for a nanoparticulate delivery vehicle; providing an antigen expression system; and providing conditions sufficient for the nanoparticulate delivery vehicle and the antigen expression system to produce a composition for delivery of the antigen expression system. In some aspects, the conditions are provided by microfluidic mixing.

[0129] Also disclosed herein is a method for 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.

[0130] 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.

[0131] In some embodiments, the plasmid sequences are generated using one of bacterial recombination or full genome DNA synthesis, or full genome 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 comprises one or more of chromatographic separation, centrifugation, virus precipitation, and filtration.

[0132] Also provided herein is a method for treating a subject having a disease, optionally wherein the disease is cancer or an infectious disease, comprising administering to the subject an antigen-based vaccine, wherein the antigen-based vaccine comprises an antigen-encoding cassette, or a polypeptide sequence encoded by a cassette, wherein the antigen-encoding cassette is (i) nucleic acid sequence A(E A ); and (ii) nucleic acid sequence B(E B ), including E A and E B each encodes one MHC epitope, E A MHC epitopes encoded by and E B the MHC epitopes encoded by are distinct and non-identical, The cassette is E A and at least two repetitions of E B at least two repetitions of E A and E B each repeat of each comprises an identical nucleic acid sequence, E A encodes an EGFR-associated MHC class I neoepitope.

[0133] In some embodiments, the antigen-encoding cassette comprises any one of the antigen-encoding cassettes described herein. In some embodiments, the antigen-based vaccine comprises any one of the pharmaceutical compositions described herein. 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 boosting doses. In some embodiments, the boosting dose is different from the priming dose. In some embodiments, (a) the priming dose comprises a chimpanzee adenovirus vector and the boosting dose comprises an alphavirus vector; or (b) the priming dose comprises an alphavirus vector and the boosting dose comprises a chimpanzee adenovirus vector. In some embodiments, the boosting dose is the same as the priming dose. In some embodiments, the injection site of one or more boosting doses is as close as possible to the injection site of the priming dose.

[0134] In some embodiments, any one of the methods further comprises determining or having determined the HLA haplotype of the subject. In some embodiments, the determined HLA haplotype of the subject comprises an HLA allele predicted or validated to present at least one EGFR-associated MHC class I neoepitope encoded by the antigen-encoding cassette. In some embodiments, the determined HLA haplotype of the subject comprises an HLA allele predicted or validated to present at least one EGFR-associated MHC class I neoepitope encoded by the antigen-encoding cassette. In some embodiments, the determined HLA haplotype of the subject comprises an HLA allele validated to present at least one EGFR-associated MHC class I neoepitope encoded by the antigen-encoding cassette. In some embodiments, the determined HLA haplotype of the subject comprises an HLA allele predicted or validated to present at least one EGFR-associated MHC class I neoepitope encoded by the antigen-encoding cassette, wherein the HLA allele is HLA A * 03:01, HLA C * 15:02, and / or HLA A* In some embodiments, the subject's determined HLA haplotype comprises an HLA allele predicted or validated to present at least one EGFR-associated MHC class I neoepitope encoded by the antigen-encoding cassette, the HLA allele being HLA A * In some embodiments, the subject's determined HLA haplotype comprises an HLA allele predicted or validated to present at least one EGFR-associated MHC class I neoepitope encoded by the antigen-encoding cassette, the HLA allele being HLA C * In some embodiments, the subject's determined HLA haplotype comprises an HLA allele predicted or validated to present at least one EGFR-associated MHC class I neoepitope encoded by the antigen-encoding cassette, and the HLA allele is HLA A*11:01. In some embodiments, the subject's determined HLA haplotype comprises at least HLA A*11:01. * 11:01 and / or HLA A * The amino acid sequence KITDFGRAK, which has been verified by mass spectrometry to be presented by 03:01, and / or at least HLA A * In some embodiments, the subject's determined HLA haplotype comprises at least an HLA allele that is verified to present KITDFGRAKL, which is verified by mass spectrometry to be presented by 03:01. * The HLA alleles are verified to present the amino acid sequence STVQLIMQL or LTSTVQLIM, which have been verified by mass spectrometry to be presented by 15:02. [Brief explanation of the drawings]

[0135] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings. [Figure 1A]The analysis results of EGFR neoepitopes and HLA prevalence are presented. HLA frequencies were provided by the CWD China and CIWD databases. Mutation frequencies were estimated from Zhang et al. Oncotarget 7(48) 2016 and multiplied by HLA frequencies. Figure 1A (top panel) also shows an overview of the prediction and mass spectrometry validation of HLA presentation of EGFR neoantigens. [Figure 1B] The analysis results of EGFR neoepitopes and HLA prevalence are presented. HLA frequencies were provided by the CWD China and CIWD databases. Mutation frequencies were estimated from Zhang et al. Oncotarget 7(48) 2016 and multiplied by HLA frequencies. [Figure 2A] Mass spectrometry validation of EGFR L858R neoepitope KITDFGRAK presentation by HLA A*03:01. While there was no positive signal in uninduced A*03:01 K562 cells (left panel), a significant peak was detected in the same strain expressing the neoantigen cassette carrying EGFR_L858R (center panel). Right panel - An isotope-labeled synthetic standard peptide injected in the same MS run showed multiple matching transitions at the correct retention times (*adjusted based on a typical process recovery estimate of 9% [median of 100 process recovery analyses across 11 different pHLA types]). [Figure 2B] Mass spectrometry validation of EGFR L858R neoepitope KITDFGRAK presentation by HLA A*11:01. While there was no positive signal in uninduced A*11:01 K562 cells (left panel), a significant peak was detected in the same strain expressing the neoantigen cassette carrying EGFR_L858R (center panel). Right panel - An isotope-labeled synthetic standard peptide injected in the same MS run showed multiple matching transitions at the correct retention times (*adjusted based on a typical process recovery estimate of 9% [median of 100 process recovery analyses across 11 different pHLA types]). [Figure 2C]Mass spectrometry validation of EGFR T790M neoepitope STVQLIMQL presentation by HLA C*15:02. No positive signal was detected in untransduced K562 cells with HLA C*15:02 and the EGFR T790M 25mer (left panel), whereas a significant peak was detected in the same parental line expressing both the HLA C*15:02 and EGFR_T790M 25mer cassettes (center panel). Right panel - An isotope-labeled synthetic standard peptide injected in the same MS run showed multiple matching transitions at the correct retention times. [Figure 3] The designs of the 4× and 8× EGFR L858R cassettes are shown. [Figure 4] Figure 1 shows T cell responses in mice immunized with SAM or ChAd vectors encoding 4x and 8x EGFR L858R cassettes. IFNγ ELISpot after ex vivo stimulation with EFGR L858 mutant peptide (KITDFGRAK). Background subtraction. [Figure 5] Figure 1 shows T cell responses in mice immunized with SAM or ChAd vectors encoding 4x and 8x EGFR L858R cassettes. Figure 2 shows IFNγ ELISpot after ex vivo stimulation with a peptide pool of 38 peptides spanning 25mers encoding EGFR L858. Background subtraction. [Figure 6] Figure 1 shows T cell responses to the universal MHC class II PADRE antigen in mice immunized with SAM or ChAd vectors encoding the 4x and 8x EGFRL858R cassettes. IFNγ ELISpot after ex vivo stimulation with the PADRE epitope is shown. [Figure 7]Figure 1 shows analysis of target density of the EGFR L858R neoepitope in monoallelic cell lines expressing HLA A*03:01 and A*11:01. Expression of expression-matched cell lines transduced with the indicated "1x4" and "1x8" EGFR L858R epitope expression cassettes as assessed by qPCR (left panel), and HLA peptide copy number per cell (target density) of the EGFR L858R peptide as assessed by mass spectrometry (right panel). [Figure 8] HLA peptide copy number per cell (target density) of the EGFR L858R peptide as assessed by mass spectrometry is shown. Three independent immunoprecipitation replicates from the same respective cell line are shown, processed separately for monoallelic cell lines expressing HLA A*11:01 (upper panel) and A*03:01 (lower panel). Paired T-test. [Figure 9] 1 shows the design of various EGFR cassettes containing different repeats of the EGFR neoepitope with EGFR mutations L858R, T790M, and / or E746_A750del "E19del." [Figure 10] 1 shows the design and size characteristics of redesigned 3x8 EGFR cassettes with mutations L858R, T790M, and / or E746_A750del "E19del." [Figure 11A] Immunogenicity of mice engineered to express human HLA-A*11:01 immunized with 5x10 VP using ChAdV68 delivery vectors encoding 1x8 and 2x8 cassettes. Efficacy was assessed by IFNg ELISpot stimulation with a peptide pool containing 38 minimal epitopes. [Figure 11B] Immunogenicity of mice engineered to express human HLA-A*11:01 immunized with 5x10 VP using a ChAdV68 delivery vector encoding a 3x8 cassette is shown. Efficacy was assessed by IFNg ELISpot stimulation with a peptide pool containing 38 minimal epitopes. DETAILED DESCRIPTION OF THE INVENTION

[0136] Detailed Description I. Definition In general, the terms used in the claims and specification are intended to be interpreted as having their plain meaning as understood by a person skilled in the art. For further clarity, certain terms are defined below. If there is a conflict between the plain meaning and a provided definition, the provided definition should be used.

[0137] As used herein, the term "antigen" refers to a substance that induces 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.

[0138] 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, through a mutation in a tumor cell or a tumor cell-specific post-translational modification. Neoantigens can 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 can be identified for administration through the use of various diagnostic methods, such as the patient selection methods detailed below.

[0139] 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.

[0140] 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.

[0141] As used herein, the term "candidate antigen" is a mutation or other abnormality that gives rise to a sequence that may represent an antigen.

[0142] As used herein, the term "coding region" is the portion or portions of a gene that encode a protein.

[0143] As used herein, the term "coding mutation" is a mutation that occurs in a coding region.

[0144] As used herein, the term "ORF" means open reading frame.

[0145] As used herein, the term "NEO-ORF" refers to a tumor-specific ORF that results from mutation or other abnormalities such as splicing.

[0146] As used herein, the term "missense mutation" is a mutation that results in the substitution of one amino acid for another.

[0147] 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 standard start codon.

[0148] As used herein, the term "frameshift mutation" is a mutation that causes an alteration in the frame of a protein.

[0149] As used herein, the term "indel" is an insertion or deletion of one or more nucleic acids.

[0150] 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 the specified percentage of nucleotides or amino acid residues are the same when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the percent "identity" may exist over a region of the sequences being compared, e.g., over a functional domain, or in other cases, over the entire length of the two sequences being compared.

[0151] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered 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(s) relative to the reference sequence based on the designated program parameters. Alternatively, sequence similarity or dissimilarity can be established by the presence or absence of particular nucleotides, or, in the case of translated sequences, amino acids at selected sequence positions (e.g., sequence motifs).

[0152] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), 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, for an overview, Ausubel et al., infra).

[0153] One example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is 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.

[0154] As used herein, the term "nonstop or readthrough" refers to a mutation that results in the removal of the natural stop codon.

[0155] As used herein, the term "epitope" is the specific portion of an antigen that is typically bound by an antibody or T-cell receptor.

[0156] As used herein, the term "immunogenic" is the ability to elicit an immune response, for example, via T cells, B cells, or both.

[0157] As used herein, the terms "HLA binding affinity," "MHC binding affinity," refer to the affinity of binding between a particular antigen and a particular MHC allele.

[0158] As used herein, the term "bait" is a nucleic acid probe used to enrich for a specific sequence of DNA or RNA from a sample.

[0159] As used herein, the term "variant" refers to a difference between a nucleic acid of interest and a reference human genome used as a control.

[0160] As used herein, the term "variant calling" is the algorithmic determination, typically from sequencing, of the presence of a variant.

[0161] As used herein, the term "polymorphism" refers to a germline variant, i.e., a variant that is found in all DNA-bearing cells of an individual.

[0162] As used herein, the term "somatic variant" is a variant that occurs in the non-germline cells of an individual.

[0163] As used herein, the term "allele" is a version of a gene or a version of a gene sequence or a version of a protein.

[0164] As used herein, the term "HLA type" is complementary to HLA gene alleles.

[0165] As used herein, the term "nonsense-mediated decay" or "NMD" is the degradation of mRNA by a cell due to a premature stop codon.

[0166] As used herein, the term "truncal mutation" is a mutation that occurs early in tumor development and is present in the majority of cells of a tumor.

[0167] As used herein, the term "subclonal mutation" is a mutation that arises late in tumor development and is present in only a subset of tumor cells.

[0168] As used herein, the term "exome" is the subset of the genome that encodes proteins. The exome can be a collection of exons within the genome.

[0169] As used herein, the term "logistic regression" is a regression model for binary data from statistics that models the logit of the probability that the dependent variable is equal to 1 as a linear function of the dependent variable.

[0170] As used herein, the term "neural network" is a machine learning model for classification or regression that consists of multiple layers of linear transformations followed by element-wise nonlinearities that are typically trained by stochastic gradient descent and backpropagation.

[0171] As used herein, the term "proteome" is the set of all proteins expressed and / or translated by a cell, a group of cells, or an individual.

[0172] 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 refer to the properties of a cell or a collection of cells (e.g., a tumor peptidome, which refers to the combination of the peptidomes of all cells that make up a tumor).

[0173] As used herein, the term "ELISPOT" means enzyme-linked immunosorbent spot assay, which is a common method for monitoring immune responses in humans and animals.

[0174] As used herein, the term "dextramer" refers to a dextran-based peptide-MHC multimer used for antigen-specific T cell staining in flow cytometry.

[0175] As used herein, the term "tolerance or immune tolerance" is a state of immune non-responsiveness to one or more antigens, eg, self-antigens.

[0176] As used herein, the term "central tolerance" refers to tolerance that is affected in the thymus by either eliminating autoreactive T cell clones or promoting their differentiation into immunosuppressive regulatory T cells (Tregs).

[0177] As used herein, the term "peripheral tolerance" is tolerance that is affected in the periphery by downregulating or anergizing autoreactive T cells that survive central tolerance, or by promoting these T cells to differentiate into Tregs.

[0178] The term "sample" can include a single cell or multiple cells or cell fragments 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 other intervention or means known in the art.

[0179] The term "subject" encompasses a cell, tissue, or organism, human or non-human, whether male or female, in vivo, ex vivo, or in vitro. The term subject includes mammals, such as humans.

[0180] The term "mammal" encompasses both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0181] The term "clinical factor" refers to a measure of a subject's condition, for example, disease activity or severity. "Clinical factor" includes all markers of a subject's health status, including non-sample markers, and / or other characteristics of the subject, such as, without limitation, age and sex. A clinical factor can be a score, value, or set of values ​​that can be obtained from a sample (or a group of samples) from a subject or from the evaluation of a subject under determined conditions. Clinical factors 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.

[0182] The term "antigen-encoding nucleic acid sequence derived from a tumor" refers to nucleic acid sequences extracted directly from a tumor, e.g., via RT-PCR; or sequence data obtained by sequencing the tumor and then synthesizing nucleic acid sequences using the sequencing data, e.g., via various synthetic or PCR-based methods known in the art.

[0183] The term "antigen-encoding nucleic acid sequence derived from an infectious disease" refers to nucleic acid sequences extracted directly from infected cells or infectious disease organisms, e.g., via RT-PCR; or sequence data obtained by sequencing infected cells or infectious disease organisms and then synthesizing nucleic acid sequences using the sequencing data, e.g., via various synthetic or PCR-based methods known in the art.

[0184] The term "alphavirus" refers to members of the Togaviridae family and is a single-stranded, positive-sense RNA virus. Alphaviruses are typically classified as either Old World, such as Sindbis virus, Ross River virus, Mayaro virus, Chikungunya virus, and Semliki Forest virus, or New World, such as Eastern equine encephalitis, Aura virus, Fort Morgan virus, or Venezuelan equine encephalitis virus and its derivative TC-83. Alphaviruses are typically self-replicating RNA viruses.

[0185] The term "alphavirus backbone" refers to the minimal sequence(s) of an alphavirus that allows for autonomous replication of the viral genome. The minimal sequence may 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 viral subgenomic RNA, such as the 26S promoter element.

[0186] The term "sequence for nonstructural protein-mediated amplification" includes alphavirus conserved sequence elements (CSEs) well known to those of skill in the art, including, but not limited to, the alphavirus 5'UTR, a 51 nt CSE, a 24 nt CSE, or other 26S subgenomic promoter sequence, a 19 nt CSE, and the alphavirus 3'UTR.

[0187] The term "RNA polymerase" includes polymerases that catalyze the production of an RNA polynucleotide from a DNA template, including, but not limited to, polymerases from bacteriophages such as T3, T7, and SP6.

[0188] The term "lipid" includes hydrophobic and / or amphipathic molecules. Lipids can be cationic, anionic, or neutral. Lipids can be synthetic or naturally derived, and in some cases, can be biodegradable. Lipids can 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 can also include dilinoleylmethyl-4-dimethylaminobutyrate (MC3) and MC3-like molecules.

[0189] The term "lipid nanoparticle" or "LNP" includes vesicle-like structures formed using a lipid-containing membrane surrounding an aqueous interior, also called liposomes. Lipid nanoparticles include lipid-based compositions with a solid lipid core stabilized by surfactants. The core lipid can be fatty acids, acylglycerols, waxes, and mixtures of these surfactants. Biological membrane lipids, such as phospholipids, sphingomyelin, bile salts (sodium taurocholate), and sterols (cholesterol), can be used as stabilizers. Lipid nanoparticles can be formed using defined ratios of different lipid molecules, such as, 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 contact target cells to deliver the encapsulated molecules to the host cell cytosol. Lipid nanoparticles, including their surface, can be modified or functionalized with non-lipid molecules. Lipid nanoparticles can be unilamellar or multilamellar. Lipid nanoparticles can form complexes with nucleic acids. Unilamellar lipid nanoparticles can form complexes with nucleic acids, where the nucleic acid is in the aqueous interior. Multilamellar lipid nanoparticles can form complexes with nucleic acids, where the nucleic acid is in, forms, or is sandwiched between the aqueous interior.

[0190] Abbreviations: MHC: major histocompatibility complex; HLA: human leukocyte antigen or human MHC gene locus; NGS: next-generation sequencing; PPV: positive predictive value; TSNA: tumor-specific neoantigen; FFPE: formalin-fixed, paraffin-embedded; NMD: nonsense-mediated decay; NSCLC: non-small-cell lung cancer; DC: dendritic cell.

[0191] It should be noted 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.

[0192] Unless specifically stated or clear from the context, the term "about" as used herein is understood to be within the normal tolerances in the art, for example, within 2 standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0193] Any terms not directly defined herein should be understood to have the meaning commonly associated with those terms as understood within the technical field of the present invention. Certain terms are discussed herein to provide additional guidance to practitioners in describing the compositions, devices, methods, etc. of embodiments of the present invention and how to make or use them. It will be recognized that the same thing may be said in multiple ways. Thus, alternative language and synonyms may be used for any one or more of the terms discussed herein. Whether or not a term is detailed or discussed herein is immaterial. Several synonyms or substitute methods, materials, etc. are provided. Unless explicitly stated, the recitation of one or a few synonyms or equivalents does not exclude the use of other synonyms or equivalents. The use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the embodiments of the present invention herein.

[0194] 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.

[0195] II. Antigen Identification Research methods for NGS analysis of tumor and normal exomes and transcriptomes have been reported and applied to antigen identification areas. 6、14、15Specific optimizations can be considered to increase 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 research methods can also be applied to the identification of antigens in other situations, such as identifying infectious disease organisms, infections in subjects, or identifying antigens from infected cells in subjects. Examples of optimizations are known to those skilled in the art, and are described in detail in, for example, U.S. Patent No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1, and International Patent Application Publication Nos. WO / 2018 / 195357 and WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.

[0196] Methods for identifying antigens (e.g., antigens derived from tumors or infectious disease organisms) include identifying antigens that are likely to be cell surface-presented (e.g., presented by MHC on tumor cells, infected cells, or immune cells, including professional antigen-presenting cells such as dendritic cells) and / or that are likely to be immunogenic. As an example, one such method may include obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing and / or expression data from a tumor, an infected cell, or an infectious disease organism, where the nucleotide sequencing data and / or expression data is used to obtain data representing the peptide sequence of each of a set of antigens (e.g., antigens derived from the tumor or infectious disease organism); inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical possibilities that each of the antigens will be presented on a cell surface, e.g., on the surface of a tumor cell or infected cell of a subject, by one or more MHC alleles, where the set of numerical possibilities has been identified based at least on the received mass spectrometry data; and selecting a subset of the set of antigens based on the set of numerical possibilities to generate a set of selected antigens.

[0197] III. Identification of tumor-specific mutations in neoantigens Also disclosed herein are methods for identifying certain mutations (e.g., variants or alleles present in cancer cells). In particular, these mutations may be present in the genome, transcriptome, proteome, or exome of cancer cells in a subject with cancer, but not present in the subject's normal tissues. Specific methods for identifying tumor-specific neoantigens, including common neoantigens, are known to those skilled in the art, and are described in detail in, for example, U.S. Patent No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1, and International Patent Application Publication Nos. WO / 2018 / 195357 and WO / 2018 / 208856, each of which is incorporated by reference in its entirety for all purposes. Examples of common tumor-specific neoantigens are described in detail in International Patent Application Publication No. WO2019226941A1, which is incorporated by reference in its entirety for all purposes. Common neoantigens include, but are not limited to, EGFR-associated mutations (e.g., EGFR L858R, EGFR T790M, and / or EGFR E19 deletion mutations such as E746-A750 deletions). For example, EGFR-associated mutations can include mutations in wild-type (WT) human EGFR, e.g., mutations in the isoform A precursor exemplified by the following amino acid sequence: TIFF2025534986000005.tif169152

[0198] EGFR-associated neoantigens may refer only to EGFR-associated neoepitope sequences.

[0199] The EGFR-associated neoantigen can be an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, an EGFR E746-A750 deletion MHC class I epitope, or a combination thereof.

[0200] The EGFR-associated neoantigen may be an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, or an EGFR E746-A750 deletion MHC class I epitope.

[0201] The antigen-encoding cassettes described herein may encode two or more EGFR-associated neoantigens. The antigen-encoding cassettes described herein may encode an EGFR_L858R MHC class I epitope and an EGFR T790M MHC class I epitope. The antigen-encoding cassettes described herein may encode an EGFR_L858R MHC class I epitope and an EGFR E746-A750 deletion MHC class I epitope. The antigen-encoding cassettes described herein may encode an EGFR E746-A750 deletion MHC class I epitope and an EGFR T790M MHC class I epitope.

[0202] The antigen-encoding cassettes described herein may encode two or more repeats of two or more EGFR-associated neoantigens. The antigen-encoding cassettes described herein may encode two or more repeats of the EGFR_L858R MHC class I epitope and the EGFR T790M MHC class I epitope. The antigen-encoding cassettes described herein may encode two or more repeats of the EGFR_L858R MHC class I epitope and the EGFR E746-A750 deletion MHC class I epitope. The antigen-encoding cassettes described herein may encode two or more repeats of the EGFR E746-A750 deletion MHC class I epitope and the EGFR T790M MHC class I epitope.

[0203] The antigen-encoding cassettes described herein may encode eight repeats of two or more EGFR-associated neoantigens. The antigen-encoding cassettes described herein may encode eight repeats of the EGFR_L858R MHC class I epitope and the EGFR T790M MHC class I epitope. The antigen-encoding cassettes described herein may encode eight repeats of the EGFR_L858R MHC class I epitope and the EGFR E746-A750 deletion MHC class I epitope. The antigen-encoding cassettes described herein may encode eight repeats of the EGFR E746-A750 deletion MHC class I epitope and the EGFR T790M MHC class I epitope.

[0204] The antigen-encoding cassettes described herein may encode three or more EGFR-associated neoantigens. The antigen-encoding cassettes described herein may encode the EGFR_L858R MHC class I epitope, the EGFR T790M MHC class I epitope, and the EGFR E746-A750 deletion MHC class I epitope. The antigen-encoding cassettes described herein may encode two or more repeats of three or more EGFR-associated neoantigens. The antigen-encoding cassettes described herein may encode eight repeats of three or more EGFR-associated neoantigens.

[0205] The antigen-encoding cassettes described herein can encode two or more repeats of three or more EGFR-associated neoantigens, and can encode three or more repeats of the EGFR_L858R MHC class I epitope, the EGFR T790M MHC class I epitope, and the EGFR E746-A750 deletion MHC class I epitope.

[0206] The antigen-encoding cassettes described herein can encode eight or more repeats of three or more EGFR-associated neoantigens, such as the EGFR_L858R MHC class I epitope, the EGFR T790M MHC class I epitope, and the EGFR E746-A750 deletion MHC class I epitope.

[0207] The EGFR-associated epitope may comprise the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, ITDFGRAKL, STVQLIMQL, TVQLIMQL, LTSTVQLIM, PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK.The EGFR-associated epitope may comprise the amino acid sequence KITDFGRAK, KITDFGRAKL, STVQLIMQL, or LSTVQLIM.

[0208] The EGFR-associated epitope may comprise the amino acid sequence KITDFGRAK. The EGFR-associated epitope may comprise the amino acid sequence ITDFGRAK. The EGFR-associated epitope may comprise the amino acid sequence KITDFGRAKL. The EGFR-associated epitope may comprise the amino acid sequence ITDFGRAKL. The EGFR-associated epitope may comprise the amino acid sequence STVQLIMQL. The EGFR-associated epitope may comprise the amino acid sequence TVQLIMQL. The EGFR-associated epitope may comprise the amino acid sequence LTSTVQLIM. The EGFR-associated epitope may comprise the amino acid sequence PVAIKTSPK. The EGFR-associated epitope may comprise the amino acid sequence AIKTSPKANK. The EGFR-associated epitope may comprise the amino acid sequence VAIKTSPK. The EGFR-associated epitope may comprise the amino acid sequence KIPVAIKTSPK.

[0209] The EGFR-associated neoepitope may be an EGFR_L858R MHC class I epitope having the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, or ITDFGRAKL. The EGFR-associated neoepitope may be an EGFR_L858R MHC class I epitope having the amino acid sequence KITDFGRAK. The EGFR-associated neoepitope may be an EGFR_L858R MHC class I epitope having the amino acid sequence ITDFGRAK. The EGFR-associated neoepitope may be an EGFR_L858R MHC class I epitope having the amino acid sequence KITDFGRAKL. The EGFR-associated neoepitope may be an EGFR_L858R MHC class I epitope having the amino acid sequence ITDFGRAKL. The EGFR-associated neoepitope may be, for example, an EGFR-associated neoepitope that is specifically targeted to at least HLA A. * 11:01 and HLA A * The amino acid sequence KITDFGRAK, which has been verified by mass spectrometry to be presented by 03:01, and / or at least HLA A * The EGFR_L858R MHC class I epitope was verified to be presented by HLA with KITDFGRAKL verified by mass spectrometry to be presented by 03:01.

[0210] The EGFR-associated epitope may be an EGFR T790M MHC class I epitope having the amino acid sequence STVQLIMQL, TVQLIMQL, or LTSTVQLIM. The EGFR-associated epitope may be an EGFR T790M MHC class I epitope having the amino acid sequence STVQLIMQL. The EGFR-associated neoepitope may be an EGFR T790M MHC class I epitope having the amino acid sequence LTSTVQLIM. The EGFR-associated neoepitope may be an EGFR T790M MHC class I epitope having the amino acid sequence TVQLIMQL. The EGFR-associated neoepitope may, for example, be an EGFR T790M MHC class I epitope that is associated with at least HLA C *The epitope may be an EGFR T790M MHC class I epitope verified to be presented by HLA having the amino acid sequence STVQLIMQL or LTSTVQLIM, verified by mass spectrometry to be presented by 15:02.

[0211] The EGFR-associated neoepitope can be an EGFR E746-A750 deletion (also referred to as "E19Del") MHC class I epitope having the amino acid sequence PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK. The EGFR-associated neoepitope can be an EGFR E746-A750 deletion MHC class I epitope having the amino acid sequence PVAIKTSPK. The EGFR-associated neoepitope can be an EGFR E746-A750 deletion MHC class I epitope having the amino acid sequence VAIKTSPK. The EGFR-associated neoepitope can be an EGFR E746-A750 deletion MHC class I epitope having the amino acid sequence KIPVAIKTSPK. The EGFR-associated neoepitope can be an EGFR E746-A750 deletion MHC class I epitope having the amino acid sequence AIKTSPKANK.

[0212] An EGFR-associated neoantigen can refer to an antigenic peptide comprising an EGFR-associated neoepitope, for example, the EGFR_L858R MHC class I epitope comprising the amino acid sequence KITDFGRAK, the T790M MHC class I epitope comprising the amino acid sequence STVQLIMQL or LTSTVQLIM, and / or the EGFR E746-A750 deletion MHC class I epitope comprising the amino acid sequence PVAIKTSPK, VAIKTSPK, or KIPVAIKTSPK. The EGFR-associated neoepitope can be an EGFR E746-A750 deletion MHC class I epitope that has been validated as being presented by HLA.

[0213] An EGFR-associated neoantigen may refer to an antigenic peptide comprising an EGFR-associated neoepitope and an additional peptide sequence. For example, an EGFR-associated neoantigen comprising an EGFR-associated neoepitope may embed the epitope within its native surrounding peptide sequence. In one illustrative, non-limiting example, the EGFR_L858R MHC class I epitope KITDFGRAK can be embedded within an antigenic peptide having the sequence KTPQHVKITDFGRAKLLGAEEKEYH, KTPQHVKITDFGRAKLLGAEEKEYHA, and / or KTPQHVKITDFGRAKLLGAEEKEYHAEGGKVP. In another series of illustrative, non-limiting examples, the T790M MHC class I epitope STVQLIMQL or LTSTVQLIM can be embedded within an antigenic peptide having the sequence LGICLTSTVQLIMQLMPFGCLLDYV, GICLTSTVQLIMQLMPFGCLLDYVR, LGICLTSTVQLIMQLMPFGCLLDYVR, and / or CRLLGICLTSTVQLIMQLMPFGCLLDYVR. In another series of illustrative, non-limiting examples, the EGFR E746-A750 deletion MHC class I epitope PVAIKTSPK, VAIKTSPK, or KIPVAIKTSPK can be embedded within an antigenic peptide having the sequence EKVKIPVAIKTSPKANKEILDEAY, GEKVKIPVAIKTSPKANKEILDEAY, and / or EKVKIPVAIKTSPKANKEILDEAYVMASVDNPHVCR.

[0214] An antigenic peptide comprising an EGFR-associated neoepitope may comprise multiple distinct EGFR-associated neoepitopes. An antigenic peptide comprising an EGFR-associated neoepitope may comprise multiple repeats of one or more EGFR-associated neoepitopes. An antigenic peptide comprising an EGFR-associated neoepitope may comprise multiple repeats of multiple distinct EGFR-associated neoepitopes. An antigenic peptide comprising an EGFR-associated neoepitope may comprise multiple repeats of at least two different EGFR-associated neoepitopes. An antigenic peptide comprising an EGFR-associated neoepitope may comprise multiple repeats of at least three different EGFR-associated neoepitopes.

[0215] The antigenic peptide comprising an EGFR-associated neoepitope may comprise at least each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. The antigenic peptide comprising an EGFR-associated neoepitope may comprise multiple repeats of at least each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. The antigenic peptide comprising an EGFR-associated neoepitope may comprise at least four repeats of each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope. The antigenic peptide comprising an EGFR-associated neoepitope may comprise at least eight repeats of each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope.

[0216] The antigenic peptide containing an EGFR-associated neoepitope may include at least each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope. The antigenic peptide containing an EGFR-associated neoepitope may include multiple repeats of at least each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope. The antigenic peptide containing an EGFR-associated neoepitope may include at least four repeats of each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope. The antigenic peptide containing an EGFR-associated neoepitope may include at least eight repeats of each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope.

[0217] Genetic mutations in tumors can be considered useful for immunological tumor targeting if they result in changes in the amino acid sequence of a protein only in tumors. Useful mutations include (1) nonsynonymous mutations that result in different amino acids in the protein, (2) read-through mutations that alter or delete a stop codon, resulting in translation of a longer protein with a new tumor-specific sequence at the C-terminus, (3) splice site mutations that result in the inclusion of an intron in the mature mRNA, thereby resulting in a unique tumor-specific protein sequence, (4) chromosomal rearrangements (i.e., gene fusions) that result in a chimeric protein with a tumor-specific sequence at the junction of two proteins, and (5) frameshift mutations or deletions that result in a new open reading frame with a new tumor-specific protein sequence. Mutations can also include one or more of the following genomic or expression changes: non-frameshift indels, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic or expression changes that result in neo-ORFs.

[0218] In tumor cells, peptides or mutant polypeptides with mutations resulting from, for example, splice site, frameshift, readthrough, or gene fusion mutations can be identified by sequencing DNA, RNA, or protein in tumor versus normal cells.

[0219] Mutations can also include previously identified tumor-specific mutations. Known tumor mutations can be found in the Catalogue of Somatic Mutations in Cancer (COSMIC) database.

[0220] 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 led to accurate, easy, and inexpensive large-scale SNP genotyping. For example, several techniques have been reported, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and various DNA "chip" technologies such as the Affymetrix SNP chip. These methods utilize amplification of target gene regions, typically by PCR. Still other methods rely on invasive cleavage followed by mass spectrometry or the generation of small signal molecules by immobilized padlock probes and rolling circle amplification. Some of the methods known in the art for detecting specific mutations are summarized below.

[0221] PCR-based detection means can include 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, differentially labeled primers can be used to amplify different markers, so that each can be differentially detected.Of course, hybridization-based detection means allow differential detection of multiple PCR products in a sample.Other techniques that allow multiplex analysis of multiple markers are known in the art.

[0222] 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 specialized exonuclease-resistant nucleotides, as disclosed in Mundy, CR (U.S. Patent No. 4,656,127). According to this method, a primer complementary to the allele sequence adjacent to the 3' end of a 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 will be incorporated into the end of the hybridized primer. This incorporation makes the primer resistant to exonucleases, thereby enabling its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, the finding that the primer has become resistant to exonucleases reveals that the nucleotide(s) present at the polymorphic site of the target molecule are complementary to the nucleotide of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of exogenous sequence data.

[0223] 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. WO91 / 02087). As in the method of Mundy in U.S. Patent No. 4,656,127, a primer complementary to the allele sequence 3' adjacent to the polymorphic site is used. This method uses a labeled dideoxynucleotide derivative incorporated at 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.

[0224] An alternative method known as Genetic Bit Analysis (GBA) has been reported 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' to 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 Cohen et al. method (French Patent No. 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.

[0225] Several other primer-directed nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Komher, J.S. 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, unlike GBA, utilize the incorporation of labeled deoxynucleotides to discriminate between bases at polymorphic sites. In such formats, signal is proportional to the number of incorporated deoxynucleotides, so polymorphisms occurring in 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)).

[0226] Numerous initiatives are obtaining sequence information directly from millions of individual molecules of DNA or RNA in parallel. Real-time single-molecule sequencing during synthesis relies on the detection of fluorescent nucleotides, which are incorporated into nascent strands of DNA complementary to the template to be sequenced. In one method, 30-50 base-long oligonucleotides are covalently tethered at their 5' ends to a coverslip. These tethered strands serve two functions. First, they act as capture sites for target template strands when the template is configured with a capture tail complementary to the surface-bound oligonucleotide. They also act as primers for template-directed primer extension, which forms the basis for sequence readout. The capture primers serve as fixed sites for multiple cycles of synthesis, detection, and sequencing using chemical cleavage of the dye-linker to remove the dye. Each cycle involves the addition of a polymerase / labeled nucleotide mixture, rinsing, imaging, and dye cleavage. In an alternative method, the polymerase is modified with a fluorescent donor molecule and immobilized on a glass slide, and each nucleotide is color-coded with an acceptor fluorescent moiety attached to the gamma-phosphate. The system detects the interaction of the fluorescently tagged polymerase with the fluorescently modified nucleotide as it is incorporated into the new strand. Other decoding-by-synthesis techniques also exist.

[0227] Any suitable sequencing-by-synthesis platform can be used to identify mutations. As mentioned above, four major sequencing-by-synthesis platforms are currently available: Roche / 454 Life Sciences' Genome Sequencers, Illumina / Solexa's 1G Analyzer, Applied BioSystems' SOLiD system, and Helicos Biosciences' Heliscope system. Sequencing-by-synthesis platforms have also been reported by Pacific BioSciences and VisiGen Biotechnologies. In some embodiments, multiple nucleic acid molecules to be sequenced are attached to a support (e.g., a solid support). To immobilize the nucleic acid on the support, a capture sequence / universal priming site can be added to the 3' and / or 5' end of the template. The nucleic acid can be attached to the support by hybridizing the capture sequence to a complementary sequence covalently attached to the support. The capture sequence (also called a universal capture sequence) is a nucleic acid sequence complementary to the support-attached sequence, which can double as a universal primer.

[0228] As an alternative to capture sequences, a member of a binding pair (e.g., antibody / antigen, receptor / ligand, or avidin-biotin pairs, such as those described in U.S. Patent Application Publication No. 2006 / 0252077) can be linked to each fragment to be captured on a surface coated with the second member of the respective binding pair.

[0229] After capture, the sequence can be analyzed by single-molecule detection / sequencing, including, for example, template-dependent decoding-by-synthesis, as described, for example, in the Examples and U.S. Patent No. 7,283,337. In decoding-by-synthesis, the surface-bound molecules are exposed to multiple labeled nucleotide triphosphates in the presence of a polymerase. The sequence of the template is determined by the order of labeled nucleotides incorporated onto the 3' end of the growing strand. This can be performed in real time or in a step-and-repeat fashion. For real-time analysis, each nucleotide can incorporate a different optical label, and multiple lasers can be used to stimulate the incorporated nucleotides.

[0230] Sequencing can also include other massively parallel sequencing or next-generation sequencing (NGS) technologies and platforms. Further examples of massively parallel sequencing technologies and platforms are Illumina HiSeq or MiSeq, Thermo PGM or Proton, Pac Bio RS II or Sequel, Qiagen Gene Reader, and Oxford Nanopore MinION. Additional similar current massively parallel sequencing technologies, and future generations of these technologies, can be used.

[0231] 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 techniques (e.g., venipuncture).Alternatively, nucleic acid testing can be performed on dried samples (e.g., hair or skin).In addition, one sample can be obtained from tumors for sequencing, and another sample can be obtained from normal tissues with the same tissue type as tumors for sequencing.One sample can be obtained from tumors for sequencing, and another sample can be obtained from normal tissues with different tissue types than tumors for sequencing.

[0232] 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.

[0233] 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.

[0234] IV. Antigen Antigens can include nucleotides or polypeptides. For example, antigens can be RNA sequences that encode polypeptide sequences. Thus, antigens useful for vaccines can include nucleotide sequences or polypeptide sequences.

[0235] 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. Neoantigen peptides can be described in relation to their coding sequences, where neoantigens include nucleotide sequences (e.g., DNA or RNA) that encode the relevant polypeptide sequence.

[0236] Also disclosed herein are peptides derived from any polypeptide known or found to be altered in expression in tumor cells or cancerous tissues compared to normal cells or tissues, for example, any polypeptide known or found to be abnormally expressed in tumor cells or cancerous tissues compared to normal cells or tissues.Suitable polypeptides from which antigen peptides can be derived can be found, for example, in the COSMIC database.COSMIC maintains comprehensive information on somatic mutations in human cancers.Peptides contain tumor-specific mutations.

[0237] Antigens can be selected that are predicted to be presented on the cell surface of cells such as tumor cells, infected cells, or immune cells, including professional antigen-presenting cells such as dendritic cells. Antigens can be selected that are predicted to be immunogenic.

[0238] 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; for MHC class I peptides, a length of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids; 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; and for MHC class II peptides, a length of 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids; and the presence of a sequence motif within or near the peptide that promotes extracellular or lysosomal protease (e.g., cathepsin) cleavage or HLA-DM catalyzed HLA binding.

[0239] One or more antigens may be presented on the surface of the tumor.

[0240] The one or more antigens may be immunogenic in a tumor-bearing subject, for example, capable of stimulating a T cell response and / or a B cell response in the subject.

[0241] 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 tumors). Antibodies can recognize linear polypeptide sequences or can recognize secondary and tertiary structures. Thus, B cell antigens can include linear polypeptide sequences or polypeptides with secondary and tertiary structures, including, but not limited to, full-length proteins, protein subunits, protein domains, or any polypeptide sequence known or predicted to have secondary and tertiary structures. Antigens capable of stimulating a B cell response against tumors can be antigens found on the surface of tumor cells or infectious disease organisms, respectively. Antigens capable of stimulating a B cell response against tumors can be intracellular neoantigens expressed in tumors.

[0242] The one or more antigens may include a combination of antigens capable of stimulating a T cell response (e.g., peptides such as predicted T cell epitope sequences) and separate antigens capable of stimulating a B cell response (e.g., full-length proteins, protein subunits, protein domains).

[0243] 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 the subject.

[0244] The size of the at least one antigenic peptide molecule (e.g., epitope sequence) can be, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 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 amino acids or less.

[0245] Antigen peptides and polypeptides may be 15 residues or less in length, usually about 8 to about 11 residues, particularly 9 or 10 residues, for MHC class I, and 6 to 30 residues for MHC class II.

[0246] If necessary, longer peptides can be designed in several ways. In some cases, when the potential for peptide presentation by HLA alleles is predicted or known, longer peptides can consist of either (1) individual presented peptides with extensions of 2 to 5 amino acids toward the N- and C-termini of each corresponding gene product, or (2) a concatenation of part or all of the presented peptide with the respective extended sequence. In other cases, when sequencing reveals long (more than 10 residues) neoepitope sequences present in tumors (e.g., due to frameshifts, readthrough, or intron inclusion resulting in novel peptide sequences), longer peptides can (3) consist of the entire region of novel tumor- or infection-specific amino acids, thus obviating the need for computational or in vitro testing-based selection of the most potent HLA-presented short peptides. In either case, the use of longer peptides can allow for endogenous processing by patient cells, resulting in more effective antigen presentation and stimulation of T cell responses. Longer peptides can also include full-length proteins, protein subunits, protein domains, and combinations thereof, such as those expressed in tumors or infectious disease organisms, respectively. Longer peptides (e.g., full-length proteins, protein subunits, or protein domains) and combinations thereof can be included to stimulate a B cell response.

[0247] Antigenic peptides and polypeptides can be presented on HLA proteins. In some embodiments, the antigenic peptides and polypeptides are presented on HLA proteins and have higher affinity than wild-type peptides. In some embodiments, the antigenic peptide or polypeptide can have an IC50 of at least 5000 nM, at least 1000 nM, at least 500 nM, at least 250 nM, at least 200 nM, at least 150 nM, at least 100 nM, at least 50 nM, or less.

[0248] 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.

[0249] Also provided are compositions comprising at least two or more antigenic peptides. In some embodiments, the composition contains at least two distinct peptides. The at least two distinct peptides may be derived from the same polypeptide. Distinct polypeptides mean that the peptides differ in length, amino acid sequence, or both. The peptide may contain a tumor-specific mutation. The tumor-specific peptide may be derived from any polypeptide known or found to contain a tumor-specific mutation, or from any polypeptide known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues, for example, any polypeptide known or found to be abnormally expressed in tumor cells or cancerous tissues compared to normal cells or tissues. The peptide may be derived from any polypeptide known or suspected to be associated with an infectious disease organism, or 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 Genomics Evidence Neoplasia Information Exchange (GENIE) database. COSMIC maintains comprehensive information on somatic mutations in human cancers. AACR GENIE aggregates clinical-grade cancer genomic data and correlates them with clinical outcomes from tens of thousands of cancer patients. In some embodiments, tumor-specific mutations are driver mutations for specific cancer types.

[0250] Antigenic peptides and polypeptides with desired activities or properties can be modified to confer certain desired attributes, e.g., improved pharmacological properties, while increasing or at least substantially retaining the biological activity of the unmodified peptide, i.e., binding to desired MHC molecules and activating appropriate T cells. For example, antigenic peptides and polypeptides can undergo 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 one amino acid residue with another that is biologically and / or chemically similar, e.g., the replacement of one hydrophobic residue with another, or one polar residue with another. Substitutions include Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and combinations such as Phe and Tyr. The effects of single amino acid substitutions can also be probed using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, for example, as described in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979), and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2nd Ed. (1984).

[0251] Modification of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful in 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, for example, Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Peptide half-life can be conveniently determined using a 25% human serum (v / v) assay. The protocol is generally as follows: Pooled human serum (type AB, non-heat-inactivated) is 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, a small amount of the reaction solution is removed and added to an aqueous solution of 6% trichloroacetic acid or ethanol. The turbid reaction sample is cooled (4°C) for 15 minutes, after which precipitated serum proteins are pelleted by centrifugation. The presence of the peptide is then determined by reverse phase HPLC using stability specific chromatographic conditions.

[0252] Peptides and polypeptides can be modified to achieve desired attributes other than improved serum half-life. For example, the ability of a peptide to stimulate CTL activity can be enhanced by conjugation 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 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 is generally at least one or two residues, more typically three to six residues. Alternatively, the peptide can be linked to the T helper peptide without a spacer.

[0253] The antigenic peptide can be linked directly to the T helper peptide, or via a spacer at either the amino or carboxy terminus of the peptide. The amino terminus of either the antigenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398, and 378-389.

[0254] Proteins or peptides can be produced by any technique known to those of skill in the art, such as expressing the protein, polypeptide, or peptide by standard molecular biology techniques, isolating the protein or peptide from a natural source, or chemically synthesizing the protein or peptide. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed and can be found in computerized databases known to those of skill in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information at the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using techniques disclosed herein or that would be known to those of skill in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those of skill in the art.

[0255] In a further aspect, the antigen includes a nucleic acid (e.g., a polynucleotide) encoding an antigenic peptide or a portion thereof. The polynucleotide can be, for example, DNA, cDNA, PNA, CNA, or RNA (e.g., mRNA), either single-stranded and / or double-stranded, or a naturally occurring polynucleotide or a stabilized form of a polynucleotide, such as a polynucleotide having a phosphorothioate backbone, or a combination thereof, and may or may not contain introns. The polynucleotide sequence encoding the antigen can be sequence-optimized, such as by improving transcription, translation, post-transcriptional processing, and / or RNA stability to improve expression. For example, the polynucleotide sequence encoding the antigen can be codon-optimized. As used herein, "codon optimization" refers to replacing less frequently used codons with more frequently used synonymous codons relative 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 reduce unintended splicing, and / or by introducing exogenous splicing motifs (e.g., splice donor, branch, and / or acceptor sequences) to bias preferred splicing events. Exogenous intron sequences include, but are not limited to, those derived from SV40 (e.g., SV40 mini-introns) and immunoglobulins (e.g., human β-globin genes). 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), incorporated herein by reference for all purposes. Polynucleotide sequences can be optimized to improve transcript stability, for example, through removal of RNA instability motifs (eg, AU-rich elements and 3'UTR motifs) and / or repetitive nucleotide sequences.Polynucleotide sequences can be optimized to improve accurate transcription, for example, by removing potential transcription initiators and / or terminators. Polynucleotide sequences can be optimized to improve translation and translation accuracy, for example, by removing potential 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 constitutive transport elements (CTEs), RNA export factors (RTEs), or woodchuck posttranscriptional regulatory elements (WPREs). Nuclear export signals for use in expression vectors are described in detail in Callendret et al. (Virology. 2007 Jul 5;363(2):288-302), incorporated herein by reference 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, posttranscriptional processing, and / or RNA stability. Sequence optimization can generate an optimal sequence that balances transcription, translation, post-transcriptional processing, and RNA stability. Sequence optimization algorithms such as GeneArt (Thermo Fisher), Codon Optimization Tool (IDT), CoolTool (University of Singapore), and SGI-DNA (La Jolla California) are known to those skilled in the art. One or more regions of the protein encoding the antigen can be sequence-optimized separately.

[0256] Yet another embodiment 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 appropriate orientation and correct reading frame for expression. If necessary, the DNA can be linked to appropriate transcriptional and translational regulatory nucleotide sequences recognized by the desired host; such controls are generally available in the expression vector. The vector is then introduced into the host using standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0257] V. Vaccine Compositions Also disclosed herein are immunogenic compositions, e.g., vaccine compositions, capable of generating a specific immune response, e.g., a tumor-specific immune response or an infectious disease organism-specific immune response. Vaccine compositions typically include multiple antigens, selected, e.g., using the methods described herein. Vaccine compositions may also be referred to as vaccines.

[0258] The vaccine can contain 1 to 30 peptides, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different peptides, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different peptides, or 12, 13, or 14 different peptides. The peptides can include post-translational modifications. The vaccines may contain 1 to 100 or more nucleotide sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 It may contain 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different nucleotide sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different nucleotide sequences, or 12, 13, or 14 different nucleotide sequences.The vaccine contains antigen sequences 1-30, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, , 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.

[0259] The vaccines contain antigen-encoding nucleic acid sequences 1-30, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, The nucleic acid sequence may contain 6, 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 refer to an antigen encoding part of an "antigen cassette." Characteristics of antigen cassettes are described in detail herein. An antigen-encoding nucleic acid sequence may contain one or more epitope-encoding nucleic acid sequences (e.g., an antigen-encoding nucleic acid sequence encoding linked T cell epitopes).

[0260] The vaccines comprise 1 to 30 distinct epitope-encoding nucleic acid sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more distinct epitope-encoding nucleic acid sequences, 6, 7, 8, 9, 10, 11, 12, 13, or 14 distinct epitope-encoding nucleic acid sequences, or 12, 13, or 14 distinct epitope-encoding nucleic acid sequences. An epitope-encoding nucleic acid sequence can refer to a sequence for an individual epitope sequence, for example, each of the T cell epitopes in an antigen-encoding nucleic acid sequence that encodes linked T cell epitopes.

[0261] The vaccine can contain at least two repeats of an epitope-encoding nucleic acid sequence. As used herein, "repeat" (or interchangeably, "repeat") refers to two or more identical nucleic acid epitope-encoding nucleic acid sequences (including optional 5' linker sequences and / or optional 3' linker sequences 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 distinct epitopes, at least one of the distinct epitopes being encoded by at least two repeats of a nucleic acid sequence encoding a distinct epitope (i.e., at least two distinct epitope-encoding nucleic acid sequences). In an illustrative non-limiting example, the antigen-encoding nucleic acid sequence encodes epitopes A, B, and C encoded by epitope-encoding nucleic acid sequences, with epitope-encoding sequence A(EA ), epitope coding sequence B (E B ), and epitope coding sequence C(E C ), as well as exemplary antigen-encoding nucleic acid sequences having at least one repeat of a distinct epitope are exemplified, but not limited to, by the following formula: - Repeat of one distinct 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 distinct 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 several distinct 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。

[0262] The above examples are not limiting, and an antigen-encoding nucleic acid sequence having at least one repeat of a distinct epitope can encode each of the distinct epitopes in any order or frequency. For example, the order and frequency can be a random arrangement of the distinct epitopes, e.g., in the example using epitopes A, B, and C, the sequence of the formula E A -E B -E C -E C -E A -E B -E A -E C -E A -E C -E C -E B is possible.

[0263] An exemplary antigen-encoding cassette design having two or more repeats of two or more distinct and non-identical MHC epitopes is described by the following, wherein the antigen-encoding cassette comprises: (i) nucleic acid sequence A(E A ); and (ii) nucleic acid sequence B(E B ) Including, E A and E B each encodes one MHC epitope, E A MHC epitopes encoded by and E B the MHC epitopes encoded by are distinct and non-identical, The cassette is E A and at least two repetitions of E B at least two repetitions of E A and E B Each repeat of is an identical nucleic acid sequence.

[0264] The above cassette is E A and E B The cassette may encode at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 repeats of one or both of E Aand E B The above cassettes, which can encode at least eight repeats of one or both of the following, are A and E B The above cassettes, which may encode two repeats of one or both of the following, are A and E B The above cassettes, which may encode three repeats of one or both of the following, are A and E B The above cassettes, which may encode four repeats of one or both of the following, are A and E B The above cassettes, which may encode five repeats of one or both of the following, are A and E B The above cassettes, which may encode six repeats of one or both of the following, are A and E B The above cassettes, which may encode one or both heptad repeats of E A and E B It may encode eight repeats of one or both of the following:

[0265] The above cassette is E A and E B The cassette encodes at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 repeats of each of the following: A and E B The cassette may encode at least eight repeats of each of the following: A and E B The above cassette can encode two repeats of each of E A and E B The above cassette can encode three repeats of each of E A and E B The above cassette can encode four repeats of each of E A and E B The above cassette can encode five repeats of each of E A and E B The above cassette can encode six repeats of each of E A and E BThe above cassette can encode seven repeats of each of E A and E B It can encode eight repeats of each of the following:

[0266] E A and / or E B The linker-encoding nucleic acid can be used to link other MHC epitopes (e.g., other E A and / or E B ) can be linked to a nucleic acid encoding the

[0267] The above cassette is E A and E B Each of the repeating units E A -E B For example, the four repeated units E A -E B is shown by: E A -E B -E A -E B -E A -E B -E A -E B .

[0268] The cassette comprises the nucleic acid sequence C(E C ), and E C encodes one MHC epitope, and E C The MHC epitopes encoded by E A MHC epitopes encoded by and E B The MHC epitopes encoded by the cassette are distinct and non-identical to those encoded by E C and E C Each repeat of E is an identical nucleic acid sequence. A -E B -E C Each of the four repeated units E A -E B -E C is shown by: E A -EB -E C -E A -E B -E C -E A -E B -E C -E A -E B -E C .

[0269] The above cassette is E A , E B , and E C The cassette encodes at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 repeats of each of the following: A , E B , and E C The cassette may encode at least eight repeats of each of the following: A , E B , and E C The above cassette can encode two repeats of each of E A , E B , and E C The above cassette can encode three repeats of each of E A , E B , and E C The above cassette can encode four repeats of each of E A , E B , and E C The above cassette can encode five repeats of each of E A , E B , and E C The above cassette can encode six repeats of each of E A , E B , and E C The above cassette can encode seven repeats of each of E A , E B , and E C It can encode eight repeats of each of the following:

[0270] E A , E B , and / or E C The repetition of the EAA -E B -E C etc., but in alternative orders, including orders designed to minimize junctional epitope formation, e.g., E A -E B -E C -E C -E B -E A -E A -E B -E C -E B -E A -E C or E A -E A -E A -E A -E B -E B -E B -E B -E C -E C -E C -E C It may be repeated, etc.

[0271] E A , E B , and / or E C The linker-encoding nucleic acid can be used to link other MHC epitopes (e.g., other E A , E B , and / or E C ) can be linked to a nucleic acid encoding the

[0272] [ZN for incorporating claim 1 style formula format]

[0273] Also provided herein is an antigen-encoding cassette, which comprises, from 5' to 3', the following formula: (E x -(E N n ) y ) z wherein E represents a nucleotide sequence, such as a distinct 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 constituting a separate, distinct epitope-encoding nucleic acid sequence for each corresponding n, For every z iterations, x=0 or 1, y=0 or 1 for each n, and at least one of x or y=1, and z=2 or more, where the antigen-encoding nucleic acid sequence is E, given E N or a combination thereof. In some embodiments, at least one of the distinct epitope-encoding nucleic acid sequences comprising at least two repeats encodes an EGFR-associated MHC class I neoepitope.

[0274] Each E or E N can independently comprise any epitope-encoding nucleic acid sequence (e.g., a peptide encoding a neoantigen epitope) described herein. For example, each E or E N are independently converted from 5' to 3' into the formula (L5 b -N c -L3 d ), in which N can be any of E or E N and a distinct epitope-encoding nucleic acid sequence associated with L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L25, L26, L27, L28, L29, L30, L31, L32, L33, L34, L35, L40, L41, L42, L43, L44, L45, L46, L47, L48, L49, L50, L51, L52, L53, L54, L55, L56, L57, L58, L59, L60, L61, L62, L63, L64, L65, L66, L67, L68, L69, L7

[0275] Repeats of epitope-encoding nucleic acid sequences (including optional 5' linker sequences and / or optional 3' linker sequences) can be directly linked together in a linear fashion (e.g., E A -E A-...). The repeats of the epitope-encoding nucleic acid sequence can be separated by one or more additional nucleotide sequences. Generally, the repeats of the epitope-encoding nucleic acid sequence can be separated by a nucleotide sequence of any size applicable to the compositions described herein. In one example, the repeats of the epitope-encoding nucleic acid sequence are separated by separate, distinct epitope-encoding nucleic acid sequences (e.g., E A -E B -E C -E A In an example where the repeats are separated by a single, separate, and distinct epitope-encoding nucleic acid sequence, and each epitope-encoding nucleic acid sequence (including an optional 5' linker sequence and / or an optional 3' linker sequence) encodes a peptide 25 amino acids in length, the repeats may be separated by 75 nucleotides, e.g., E A -E B -E A In the case of an antigen-encoding nucleic acid represented by E A are separated by 75 nucleotides. By way of example, in an antigen-encoding nucleic acid having the sequence VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT encoding repeats of the 25-mer antigen Trp1 (VTNTEMFVTAPDNLGYMYEVQWPGQ) and Trp2 (TQPQIANCSVYDFFVWLHYYSVRDT), the Trp1 repeats are separated by the 25-mer Trp2, and therefore 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, distinct epitope-encoding nucleic acid sequences, and each epitope-encoding nucleic acid sequence (including an optional 5' linker sequence and / or an optional 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.

[0276] In one embodiment, the different peptides and / or polypeptides, or the nucleotide sequences encoding them, are selected such that the peptides and / or polypeptides have the ability to associate with different MHC molecules, such as different MHC class I molecules and / or different MHC class II molecules. In some aspects, a vaccine composition comprises coding sequences for peptides and / or polypeptides capable of associating with 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 associating with at least two preferred, at least three preferred, or at least four preferred MHC class I molecules and / or different MHC class II molecules.

[0277] The composition may be capable of stimulating a specific cytotoxic T cell response and / or a specific helper T cell response. The vaccine composition may be capable of stimulating a specific cytotoxic T cell response and a specific helper T cell response.

[0278] The vaccine composition may be capable of stimulating a specific B cell response (eg, an antibody response).

[0279] The vaccine composition may be capable of stimulating a specific cytotoxic T cell response, a specific helper T cell response, and / or a specific B cell response. The vaccine composition may be capable of stimulating a specific cytotoxic T cell response and a specific B cell response. The vaccine composition may be capable of stimulating a specific helper T cell response and a specific B cell response. The vaccine composition may be capable of stimulating a specific cytotoxic T cell response, a specific helper T cell response, and a specific B cell response.

[0280] The vaccine composition may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are described herein below. The composition may be associated with a carrier, such as a protein or an antigen-presenting cell, such as a dendritic cell (DC), capable of presenting peptides to T cells.

[0281] An adjuvant is any substance that, when incorporated into a vaccine composition, enhances or otherwise modifies the immune response to an antigen. A carrier can be a scaffolding structure, such as a polypeptide or polysaccharide, to which an antigen can be associated. Optionally, the adjuvant is conjugated by covalent or non-covalent bonding.

[0282] The ability of adjuvants to enhance the immune response to antigens is typically manifested by a significant or substantial increase in immune-mediated responses or the alleviation of disease symptoms.For example, the increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies produced against antigens, and the increase in T cell activity is typically manifested by an increase in cell proliferation, cytotoxicity, or cytokine secretion.Adjuvants can also change immune response, for example, by changing a predominantly humoral or Th response to a predominantly cellular or Th response.

[0283] Suitable adjuvants include 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 ISA 206, Montanide ISA 50V, Montanide Adjuvants include, but are not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector systems, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF traps, R848, beta-glucans, Pam3Cys, Aquila's QS21 Stimulon (Aquila Biotech, Worcester, Mass., USA) derived from saponins, mycobacterial extracts and synthetic bacterial cell wall 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 immune adjuvants specific for dendritic cells (e.g., MF59) and their preparations have been previously reported (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison AC; Dev Biol Stand. 1998;92:3-11). Cytokines can also be used. Several cytokines have been directly implicated in influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-alpha), promoting the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. 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).

[0284] CpG immunostimulatory oligonucleotides have also been reported to enhance the effect of adjuvants in the context of vaccines. Other TLR-binding molecules, such as RNAs that bind to TLR7, TLR8, and / or TLR9, can also be used.

[0285] 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 such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act as therapeutic agents and / or adjuvants. The amounts and concentrations of adjuvants and additives can be readily determined by those skilled in the art without undue experimentation. Additional adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0286] A vaccine composition can include multiple different adjuvants. Additionally, a therapeutic composition can include any adjuvant material, including any of the above or combinations thereof. It is also contemplated that the vaccine and adjuvant can be administered together or separately in any suitable order.

[0287] The carrier (or excipient) can exist independently of the adjuvant. The function of the carrier can be, for example, to increase the molecular weight of the variant, particularly to enhance activity or immunogenicity, to confer stability, to increase biological activity, or to extend serum half-life. Furthermore, the carrier can aid in presenting the peptide to T cells. The carrier can be any suitable carrier known to those skilled in the art, such as a protein or an antigen-presenting cell. The carrier protein can be, but is not limited to, keyhole limpet hemocyanin, a serum protein such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin, or ovalbumin, an immunoglobulin, or a hormone such as insulin or palmitic acid. For human immunization, the carrier is generally a physiologically acceptable carrier that is tolerated and safe for humans. However, tetanus toxoid and / or diphtheria toxoid are preferred carriers. Alternatively, the carrier can be a dextran, such as Sepharose.

[0288] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules, rather than the intact foreign antigen itself. MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, CTL activation is possible when a trimeric complex of peptide antigen, MHC molecule, and APC is present. Consistent with this, immune responses can be enhanced when not only peptides are used to activate CTLs, but additional APCs are added with their respective MHC molecules. Thus, in some embodiments, the vaccine composition further contains at least one antigen-presenting cell.

[0289] Antigens can also be used in viral vector-based vaccine platforms, such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including, but not limited to, second-generation, third-generation, or second / third-generation hybrid lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors). (See, e.g., 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), etc. Depending on the packaging capacity of the viral vector-based vaccine platform, this approach can deliver one or more nucleotide sequences encoding one or more antigenic peptides.The sequence may be adjacent to non-mutated sequence, separated by a linker, or preceded by one or more sequences that target a subcellular compartment (see, e.g., Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22(4):433-8; Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science. (2016) 352(6291):1337-41; Lu et al., Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20(13):3401-10). Upon introduction into the host, infected cells express the antigen, thereby eliciting a host immune (e.g., CTL) response against the peptide(s). Vaccinia vectors and methods useful for immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette-Guerin). 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.

[0290] VA antigen cassette The selection of one or more antigens, the cloning and construction of an "antigen cassette," and the methods used to insert it into a viral vector are within the skill of one of ordinary skill in the art, given 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 necessary to transcribe the antigen(s) and express the transcribed product. The selected antigen or antigens may refer to separate epitope sequences; for example, an antigen-encoding nucleic acid sequence within a cassette may 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 may 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 may also contain a selected promoter linked to the antigen(s) and located, along with other optional regulatory elements, within the selected viral sequence of the recombinant vector. A cassette can have one or more antigen-encoding nucleic acid sequences; for example, cassettes containing multiple antigen-encoding nucleic acid sequences are each independently operably linked to separate promoters 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. Linkers can also have cleavage sites, such as TEV or furin cleavage sites. Linkers with cleavage sites can be used in combination with other elements, such as 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, such that the furin protease cleavage site is configured to facilitate post-translational removal of the 2A sequence.In a cassette containing multiple antigen-encoding nucleic acid sequences, each antigen-encoding nucleic acid sequence can contain one or more epitope-encoding nucleic acid sequences (e.g., antigen-encoding nucleic acid sequences encoding linked T cell epitopes).

[0291] Useful promoters can be constitutive promoters or regulated (inducible) promoters, allowing for control of the amount of antigen(s) expressed. For example, a desirable promoter is the cytomegalovirus immediate early promoter / enhancer promoter [see, e.g., Boshart et al., Cell, 41:521-530 (1985)]. Another desirable promoter includes the Rous sarcoma virus LTR promoter / enhancer. Yet another promoter / enhancer sequence is the chicken cytoplasmic beta-actin promoter [TAKost et al., Nucl. Acids Res., 11(23):8287 (1983)]. Other suitable or desirable promoters can be selected by those skilled in the art.

[0292] 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 TET promoter system can be used to minimize transcription of a payload nucleic acid encoded in the cassette, such as an antigen encoded in a vaccine cassette, during virus production. The TET promoter system is described in detail in International Patent Application Publication No. WO 2020 / 243719, which is incorporated herein by reference for all purposes.

[0293] The TET promoter system may include a tetracycline (TET) repressor protein (TETr)-regulated promoter. Accordingly, a viral vector is also disclosed herein, comprising a cassette having at least one payload sequence operably linked to a tetracycline (TET) repressor protein (TETr)-regulated promoter. The TETr-regulated promoter may include the 19-bp TET operator (TETo) sequence TCCCTATCAGTGATAGAGA (SEQ ID NO: 66). The TETr-regulated promoter may include 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more TETo nucleic acid sequences. The TETr-regulated promoter may have two or more TETo nucleic acid sequences, and the TETo sequences may be linked together. The TETr-regulated promoter may have two or more TETo nucleic acid sequences, and the TETo sequences may be directly linked together. When a TETr-regulated promoter has two or more TETo nucleic acid sequences, the TETo sequences can be linked together 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-regulated promoter can use any desired promoter sequence, such as an SV40, EF-1, RSV, PGK, HSA, MCK, or EBV promoter sequence. The TETr-regulated promoter can use a CMV promoter sequence. The TETr-regulated 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 an illustrative example, seven TETo sequences are located upstream (5') of the promoter sequence. The TETr-regulated promoter has a TETo sequence upstream of the promoter sequence region and is operably linked to at least one payload nucleic acid sequence, and has the formula in the 5' to 3' direction: (TL Y ) X -PN where 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, where Y=0 or 1 for each X, and where X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In an illustrative example, X=7 and Y=1 for each X describes when seven TETo sequences are upstream (5') of the promoter sequence, with each TETo sequence separated by a linker.

[0294] 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. The TETr-regulated promoter, with the TETo sequence downstream of the promoter sequence region, is operably linked to at least one payload nucleic acid sequence, and has the formula: P-(TL Y ) X -N where 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, where Y=0 or 1 for each X, and where X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In an illustrative example, X=2 and Y=1 for each X describes the case where two TETo sequences are downstream (3') of the promoter sequence, with each TETo sequence separated by a linker.

[0295] For viral production of vectors having a TETr-regulated promoter, any viral-producing cell line engineered to express the TETr sequence (tTS), such as the 293 cell line or its derivatives (e.g., the 293F cell line), 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-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to production in non-tTS-expressing cells. Viral production of a vector having a TETr-regulated promoter in tTS-expressing cells can improve viral production and / or viral infectivity by at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to production in non-tTS-expressing cells. Viral production of a vector having a TETr-regulated promoter in tTS-expressing cells can improve viral production and / or viral infectivity by at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to production of a vector without a TETr-regulated promoter.Viral production of a vector having a TETr-regulated promoter in tTS-expressing cells can improve viral production and / or viral infectivity by at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to production of a vector without a TETr-regulated promoter.

[0296] 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 with functional splice donor and splice acceptor sites. A common polyA sequence used in exemplary vectors of the present invention is derived from the papovavirus SV-40. The polyA sequence is generally 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-40 T intron sequence. The antigen cassette may also contain an intron, such as one 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 and Genbank.

[0297] An antigen cassette can have one or more antigens. For example, a given cassette can include 1-10, 1-20, 1-30, 10-20, 15-25, 15-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens. Antigens can be linked directly to each other. Antigens can also be linked to each other with a linker. Antigens can be in any orientation relative to each other, such as N-to-C or C-to-N.

[0298] As mentioned above, the antigen cassette can be located at any selected deletion site within the viral vector, for example, at the site of the E1 gene region deletion or the E3 gene region deletion, among other sites that may be selected.

[0299] The antigen cassette has the following formula from 5' to 3' to represent the ordered sequence of each element: (P a -(L5 b -N c -L3 d ) X ) Z -(P2 h -(G5 e -U f ) Y ) W -G3 g The universal sequence can be represented using the formula: where P and P2 comprise promoter nucleotide sequences, N comprises a separate 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 the at least one nucleic acid sequences encoding an amino acid linker, U comprises an MHC class II antigen-encoding nucleic acid sequence, and 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 composition and ordered array can be further defined by selecting the number of elements present, e.g., 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.

[0300] In one example, the elements present include a=0, b=1, d=1, e=1, g=1, h=0, X=10, Y=2, Z=1, and W=1, indicating that there are no additional promoters (e.g., only promoter nucleotide sequences provided in the vector backbone, such as an RNA alphavirus backbone), there are 10 epitopes, a 5' linker is present at each N, a 3' linker is present at each N, two MHC class II epitopes are present, a linker connecting the two MHC class II epitopes is present, a linker connecting the 5' ends of the two MHC class II epitopes to the 3' linker of the final MHC class I epitope is present, and 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 linking directly to a 3' UTR element provided by the vector backbone, such as the 3' 19 nt CSE. Examples of linking the 5' end of the antigen cassette to a vector backbone (e.g., an RNA alphavirus backbone) include linking directly to a promoter or 5' UTR element of the vector backbone, such as a subgenomic promoter sequence (e.g., a 26S subgenomic promoter sequence), an alphavirus 5' UTR, a 51 nt CSE, or a 24 nt CSE.

[0301] Other examples include: a=1, indicating that a promoter other than the promoter nucleotide sequence provided by the vector backbone (e.g., an RNA alphavirus backbone) is present; when a=1 and Z is greater than 1, multiple promoters other than the promoter nucleotide sequence provided by the vector backbone are present, each directing expression of one or more distinct MHC Class I epitope encoding nucleic acid sequences; h=1, indicating that a separate promoter is present for directing expression of an MHC Class II epitope-encoding nucleic acid sequence; and g=0, indicating that the MHC Class II epitope-encoding nucleic acid sequence, if present, is directly linked to the vector backbone (e.g., an RNA alphavirus backbone).

[0302] Other examples include cases where each MHC class I epitope present can have 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 can have both a 5' linker and a 3' linker, while other MHC class I epitopes can 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 can have either a 5' linker or a 3' linker, while other MHC class I epitopes can have a 5' linker, a 3' linker, or neither.

[0303] 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.

[0304] Other examples include cases where each antigen present may have 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.

[0305] 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.

[0306] 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 be non-natural sequences, or one can be natural and the other non-natural. For each X, the amino acid linkers L5 and / or L3 are each independently 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, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, It can be 0, 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 amino acids in length. For each X, the amino acid linkers L5 and / or L3 can each independently 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. For each X, the amino acid linkers L5 and / or L3 can each independently be at least 5 amino acids in length. For each X, the amino acid linkers L5 and / or L3 can each independently be at least 6 amino acids in length. For each X, the amino acid linkers L5 and / or L3 can each independently be at least 7 amino acids in length. For each X, the amino acid linkers L5 and / or L3 can each independently be at least 8 amino acids in length.For each X, the amino acid linkers L5 and / or L3 can each independently be at least 9 amino acids in length. For each X, the amino acid linkers L5 and / or L3 can each independently be 2 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, or 10 to 20 amino acids in length. For each X, the amino acid linkers L5 and / or L3 can each independently be 2 to 15, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, or 10 to 20 amino acids in length.

[0307] 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, It can be 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids in length. For each Y, the amino acid linker can also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0308] 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, It can be 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids in length. G3 can also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0309] 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 7-15 amino acids in length. For each X, each N can 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 can encode an MHC class I epitope 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.

[0310] 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 distinct epitope-encoding nucleic acid sequences. A cassette encoding one or more antigens may be 700 nucleotides or less and may encode at least two distinct epitope-encoding nucleic acid sequences. A cassette encoding one or more antigens may be 700 nucleotides or less and may encode three distinct epitope-encoding nucleic acid sequences. A cassette encoding one or more antigens may be 700 nucleotides or less and may encode at least three distinct 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.

[0311] 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 distinct epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-700 nucleotides in length and may encode at least two distinct epitope-encoding nucleic acid sequences (e.g., may encode nucleic acid sequences derived from two distinct tumors that encode immunogenic polypeptides). The cassette encoding one or more antigens may be 375-700 nucleotides in length and may encode three distinct 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 distinct 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.

[0312] The cassette encoding one or more antigens may be 600, 500, 400, 300, 200, or 100 nucleotides in length or less. The cassette encoding one or more antigens may be 600, 500, 400, 300, 200, or 100 nucleotides in length or less and may encode two distinct epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 600, 500, 400, 300, 200, or 100 nucleotides in length or less and may encode at least two distinct epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 600, 500, 400, 300, 200, or 100 nucleotides in length or less and may encode three distinct epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens can be 600, 500, 400, 300, 200, or 100 nucleotides or less in length and can encode at least three distinct epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens can be 600, 500, 400, 300, 200, or 100 nucleotides or less in length and can include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0313] 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 distinct 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 distinct 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 distinct 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 encode at least three distinct 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 include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0314] In some cases, the antigen or epitope within the cassette encoding the additional antigen and / or epitope may be an epitope that is immunodominant relative to the others encoded. Immunodominance generally refers to the bias of the immune response to only one or a few specific immunogenic peptides. Immunodominance can be assessed as part of an immune monitoring protocol. For example, immunodominance can be assessed by assessing T cell and / or B cell responses to the encoded antigen.

[0315] Immunodominance can be assessed as the effect that the presence of an immunodominant antigen has on the immune response to one or more other antigens. For example, an immunodominant antigen and its respective immune response (e.g., an immunodominant MHC class I epitope) can reduce the immune response to another antigen compared to the immune response in the absence of the immunodominant antigen. This reduction can be such that the immune response in the presence of the immunodominant antigen is no longer considered a therapeutically effective response. For example, an MHC class I epitope is generally considered immunodominant if the T cell response to the other antigen is no longer considered a therapeutically effective response compared to the response elicited in the absence of the immunodominant MHC class I epitope. The immune response can also be reduced to below or near the limit of detection compared to the response in the absence of the immunodominant antigen. For example, an MHC class I epitope is generally considered immunodominant if the T cell response to the other antigen is below the limit of detection compared to the response elicited in the absence of the immunodominant MHC class I epitope. Generally, immunodominance is assessed between two antigens capable of stimulating an immune response, e.g., between two T cell epitopes in a vaccine composition administered to a subject with cognate MHC alleles known or predicted to present each epitope, respectively. Immunodominance can be assessed by assessing the relative immune response to other antigens in the presence and absence of the suspected immunodominant antigen.

[0316] Immunodominance can be evaluated as the relative difference in immune response between two or more antigens. Immunodominance can refer to an immune response of a specific antigen being 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than that of another antigen encoded in the same cassette. Immunodominance can refer to an immune response of a specific antigen being 100-fold, 200-fold, 300-fold, 400-fold, or 500-fold greater than that of another antigen encoded in the same cassette. Immunodominance can refer to an immune response of a specific antigen being 1000-fold, 2000-fold, 3000-fold, 4000-fold, or 5000-fold greater than that of another antigen encoded in the same cassette. Immunodominance can refer to an immune response of a specific antigen being 10,000-fold greater than that of another antigen encoded in the same cassette.

[0317] 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-administering and / or encoding an immunodominant epitope with an additional epitope may reduce the immune response to the additional epitope, including potentially ultimately reducing the efficacy of the vaccine against the additional epitope. As an illustrative, non-limiting example, a vaccine composition such as a TP53-associated neoepitope may bias an immune response, e.g., a T cell response, toward the TP53-associated neoepitope, negatively impacting immune responses to other antigens or epitopes in the vaccine composition (e.g., one or more EGFR-associated neoepitopes in the vaccine composition) (e.g., reducing the immune response to the point where it is not a therapeutically effective response and / or below the limit of detection). Thus, a vaccine composition can be designed to not contain immunodominant epitopes, e.g., a vaccine cassette (e.g., a (neo)antigen-encoding cassette) can be designed to 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 an 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, and the immune response is not a therapeutically effective response.In another example, the cassette does not encode an epitope that stimulates an immune response 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold or more than another epitope encoded within the same cassette in a vaccine composition administered to a subject, where each antigen is capable of stimulating an immune response in the subject. In another example, the cassette does not encode an epitope that stimulates an immune response 100-fold, 200-fold, 300-fold, 400-fold, or 500-fold or more than another epitope encoded within the same cassette in a vaccine composition administered to a subject, where each antigen is capable of stimulating an immune response in the subject. In another example, the cassette does not encode an epitope that stimulates an immune response 1000-fold, 2000-fold, 3000-fold, 4000-fold, or 5000-fold or more relative to another epitope encoded within the same cassette in a vaccine composition administered to a subject, where each antigen is capable of stimulating an immune response in the subject. In another example, the cassette does not encode an epitope that produces an immune response 10,000-fold or more relative to another epitope encoded within the same cassette in a vaccine composition administered to a subject, where each antigen is capable of stimulating an immune response in the subject.

[0318] VB immunomodulator A vector described herein, such as the C68 vector described herein or the 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. Immunomodulators can include binding molecules (e.g., antibodies such as scFv) that bind to and block the activity of immune checkpoint molecules. Immunomodulators can include cytokines such as IL-2, IL-7, IL-12 (including p35, p40, p70, and / or p70 fusion constructs of IL-12), IL-15, or IL-21. Immunomodulators can include modified cytokines (e.g., pegylated IL-2). A vector can include an antigen cassette and one or more nucleic acid molecules encoding an immunomodulator.

[0319] Exemplary immune checkpoint molecules that can be targeted for blockade or inhibition include, but are not limited to, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (a 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), CT-011 (an anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (an anti-PDL1 antibody), BMS-936559 (an anti-PDL1 antibody), MPLDL3280A (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 such as C68 using ordinary skill in the art. An 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.

[0320] Additional considerations for the design and manufacture of VC vaccines VC1. Determining a set of peptides targeting all tumor subclones Truncated peptides, meaning peptides presented by all or most tumor subclones, can be prioritized for inclusion in the vaccine. 53 Optionally, if there are no stem peptides that are presented and predicted to be highly immunogenic, or if the number of stem peptides that are presented and predicted to be highly immunogenic is small enough that additional non-stem peptides can be included in the vaccine, further peptides can be prioritized by estimating the number and identity of tumor subclones and selecting peptides that maximize the number of tumor subclones covered by the vaccine. 54

[0321] VC2. Antigen prioritization Even after all of the above antigen filters have been applied, there may still be more candidate antigens available for inclusion in a vaccine than vaccine technology can support. Furthermore, various aspects of the antigen analysis may still be uncertain, and there may be trade-offs between different properties of the vaccine antigen candidates. Therefore, instead of predefined filters at each step of the selection process, one can consider an integrative multidimensional model, i.e., a model that places candidate antigens in a space with at least the following axes and uses an integrative approach to optimize selection: 1. Risk of autoimmunity or tolerance (germline risk) (low autoimmunity risk is typically preferred) 2. Probability of sequencing artifacts (typically, a lower probability of artifacts is preferable) 3. Probability of immunogenicity (typically, a higher probability of immunogenicity is preferred) 4. Probability of presentation (typically, a higher probability of presentation is preferred) 5. Gene expression (higher expression is typically better) 6. HLA gene coverage (the greater the number of HLA molecules involved in presenting a set of antigens, the lower the chance that a tumor may escape immune attack through downregulation or mutation of HLA molecules) 7. HLA class coverage (targeting both HLA-I and HLA-II may increase the chance of therapeutic efficacy and decrease the chance of tumor escape)

[0322] Furthermore, optionally, 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 the patient's tumor or infected cells. Loss of HLA alleles occurs 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 LOH (loss of heterozygosity) and homozygous deletion (including HLA loci) have also been well reported (Carter et al., 2012; McGranahan et al., 2017; Van Loo et al., 2010). Antigens can also be deprioritized if mass spectrometry data indicates that the predicted antigen is not presented by the predicted HLA allele.

[0323] VD alphavirus VD1. Alphavirus Biology Alphaviruses are members of the Togaviridae family and are single-stranded, positive-sense RNA viruses. They are typically classified as either Old World viruses, such as Sindbis virus, Ross River virus, Mayaro virus, Chikungunya virus, and Semliki Forest virus, or New World viruses, such as Eastern equine encephalitis virus, Aura virus, Fort Morgan virus, or Venezuelan equine encephalitis virus and its derivative TC-83 (Strauss Microbial Review 1994). Native 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 third containing subgenomic expression cassettes encoding structural proteins for virion production (Frolov RNA 2001).

[0324] The model life cycle of alphaviruses involves several distinct stages (Strauss Microbial Review 1994, Jose Future Microbiol 2009). Following viral adsorption to the host cell, the virion fuses with membranes within the intracellular compartment, ultimately resulting in the release of genomic RNA into the cytosol. The genomic RNA, which is in a positive-strand orientation and contains a 5' methylguanylate cap and a 3' poly(A) tail, is translated to generate the nonstructural proteins nsP1-4, which form a replication complex. Early in infection, the positive-strand is then replicated by the complex onto a negative-strand template. In the current model, the replication complex undergoes further processing as infection progresses, and the resulting processed complex switches to transcribing negative strands into both full-length positive-strand genomic RNA and the positive-strand RNA of the 26S subgenomic genome, which contains the structural genes. Several conserved sequence elements (CSEs) in alphaviruses have been identified to play roles in various RNA replication steps, including the complementary strand 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 at the junction region between nsP and 26S RNA in transcription of subgenomic RNA from the minus strand, and a 3' 19-nt CSE in minus-strand synthesis from a plus-strand template.

[0325] Following replication of various RNA species, viral particles are typically assembled in the natural life cycle of viruses. The 26S RNA is translated, and the resulting proteins undergo further processing to generate structural proteins, including capsid proteins, glycoproteins E1 and E2, and two small polypeptides, E3 and 6K (Strauss 1994). Encapsidation of the viral RNA occurs, usually with capsid proteins specific only to the genomic RNA being packaged, followed by assembly of the virion and budding onto the membrane surface.

[0326] VD2. Alphaviruses as Delivery Vectors Alphaviruses (including alphavirus sequences, characteristics, and other elements) can be used to generate alphavirus-based delivery vectors (also called alphavirus vectors, alphavirus viral vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying RNA (samRNA) vectors). Alphaviruses have previously been engineered for use as expression vector systems (Pushko 1997; Rheme 2004). Alphaviruses offer several advantages, particularly in the context of vaccines where heterologous antigen expression may be desirable. Due to their ability to replicate autonomously in the host cytosol, alphavirus vectors can generally generate high copy numbers of expression cassettes within cells, resulting in high levels of heterologous antigen production. Furthermore, the vectors are generally transient, resulting in improved biosafety and reduced induction of immune tolerance to the vector. The public also generally lacks pre-existing immunity to alphavirus vectors compared to other standard viral vectors, such as human adenoviruses. Alphavirus-based vectors also generally result in a cytotoxic response to infected cells. Cytotoxicity can be somewhat important in the context of a vaccine in order to adequately stimulate an immune response to the expressed heterologous antigen. However, because the desired degree of cytotoxicity can be a tightrope walk, several attenuated alphaviruses have been developed, including the TC-83 strain of VEE. Thus, an exemplary antigen expression vector described herein can utilize an alphavirus backbone that allows for high-level antigen expression, stimulates a strong immune response to the antigen, does not stimulate an immune response to the vector itself, and can be used in a safe manner. Furthermore, antigen expression cassettes can be designed to stimulate different levels of immune response through optimization of the alphavirus sequences used by the vector, including, but not limited to, sequences derived from VEE or its attenuated derivative, TC-83.

[0327] Several expression vector design strategies have been developed using alphavirus sequences (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). Thus, in addition to the native nonstructural and structural proteins, an additional subgenomic RNA expressing the heterologous protein is generated. In this system, repeated infection of the expression vector in uninfected cells can occur because all elements for the production of infectious virions are present.

[0328] Another expression vector design utilizes a helper virus system (Pushko 1997). In this strategy, structural proteins are replaced with heterologous genes. Thus, following autonomous replication of viral RNA mediated by intact nonstructural genes, expression of the heterologous protein is provided by the 26S subgenomic RNA. Traditionally, an additional vector expressing the structural proteins is then supplied in trans, such as by cotransfection of a cell line, to generate infectious virus. This system is described in detail in US Pat. No. 8,093,021, which is incorporated herein by reference in its entirety for all purposes. Helper vector systems offer the benefit of limiting the likelihood of infectious particle formation, thus improving biosafety. Furthermore, helper vector systems reduce the overall vector length, improving replication and expression efficiency. Thus, one example of an antigen expression vector described herein can utilize an alphavirus backbone, in which structural proteins are replaced with an antigen cassette, and the resulting vector promotes efficient expression by reducing the overall size of the expression vector, while reducing biosafety concerns.

[0329] VD3. In vitro generation of alphavirus Alphavirus delivery vectors are generally positive-strand RNA polynucleotides. A convenient technique for RNA production well known in the art is in vitro transcription (IVT). In this technique, a DNA template of the desired vector is first generated by standard molecular biology techniques well known to those skilled in the art, such as cloning, restriction enzyme digestion, ligation, gene synthesis (e.g., chemical and / or enzymatic synthesis), and polymerase chain reaction (PCR). The DNA template contains an RNA polymerase promoter at the 5' end of the sequence desired to be transcribed into RNA. Promoters include, but are not limited to, bacteriophage polymerase promoters such as T3, T7, or SP6. The DNA template is then incubated with an appropriate RNA polymerase enzyme, buffer, and nucleotides (NTPs). The resulting RNA polynucleotide can optionally be further modified, 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 techniques well known in the art, such as phenol-chloroform extraction or column purification (eg, purification using chromatography).

[0330] VD4. Delivery via lipid nanoparticles An important aspect to consider in vaccine vector design is immunity to the vector itself (Riley 2017). This can be in the form of pre-existing immunity to the vector itself, as is the case with certain human adenovirus systems, or it can be in the form of immunity to the vector developed after vaccination. The latter is an important consideration when multiple doses of the same vaccine are administered, such as with separate priming and boosting doses, or when the same vaccine vector system is used to deliver different antigen cassettes.

[0331] For alphavirus vectors, the standard delivery method is the previously discussed helper virus system, which provides the capsid, E1, and E2 proteins in trans to generate infectious viral particles. However, it is important to note that the E1 and E2 proteins are often the primary targets of neutralizing antibodies (Strauss 1994). Therefore, 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.

[0332] An alternative to viral particle-mediated gene delivery is the use of nanomaterials to deliver expression vectors (Riley 2017). Importantly, nanomaterial vehicles can be made of non-immunogenic materials, generally avoiding immune stimulation 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. Materials can be synthetic or naturally derived and, in some cases, 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.

[0333] Lipid nanoparticles (LNPs) are attractive delivery systems because the amphiphilic nature of lipids allows them to form membranes and vesicle-like 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 generally contain defined mixtures of cationic, neutral, anionic, and amphiphilic lipids. In some cases, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups to facilitate attachment of additional moieties. The lipid composition can affect the overall size and stability of the LNP. In one example, the lipid composition contains dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as PEG or PEG-conjugated lipids, sterols, or neutral lipids.

[0334] Nucleic acid vectors, such as expression vectors, that are directly exposed to serum can result in several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target immune system stimulation by released nucleic acids. Therefore, encapsulation of alphavirus vectors can be used to avoid degradation while also avoiding potential off-target effects. In certain examples, the alphavirus vector is fully encapsulated within the delivery vehicle, e.g., within the aqueous interior of an LNP. Encapsulation of alphavirus vectors within LNPs can be achieved by techniques well known to those skilled in the art, such as microfluidic mixing and droplet generation in a microfluidic droplet generating device. Such devices include, but are not limited to, standard T-shaped devices or flow-focusing devices. In one example, a desired lipid formulation, such as a composition containing MC3 or MC3-like, is provided to a droplet generating device in parallel with the alphavirus delivery vector and other desired substances, and the delivery vector and desired substances are fully encapsulated within the interior of the LNP using MC3 or MC3-like. In one example, the droplet generating device can control the size range and particle size distribution of the LNPs produced. For example, LNPs can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. After droplet generation, the delivery vehicles encapsulating the expression vectors can be further processed or modified to prepare them for administration.

[0335] VE chimpanzee adenovirus (ChAd) VE1. Viral delivery with chimpanzee adenovirus Vaccine compositions for delivery of one or more antigens (e.g., via antigen cassettes) can be produced by providing chimpanzee-derived adenovirus nucleotide sequences, various novel vectors, and cell lines expressing chimpanzee adenovirus genes. The nucleotide sequence (see SEQ ID NO: 1) of chimpanzee C68 adenovirus (also referred to herein as ChAdV68) can be used in vaccine compositions for antigen delivery. C68 adenovirus-derived vectors are described in detail in USPN 6,083,716, which is incorporated herein by reference in its entirety for all purposes. Vectors and delivery systems using ChAdV68 are described in detail in US Patent Publication No. US20200197500A1 and International Patent Publication No. WO2020243719A1, each of which is incorporated herein by reference for all purposes.

[0336] 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 directing its expression. The recombinant virus is capable of infecting mammalian cells, preferably human cells, and expressing the antigen cassette product 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 of interest against which an antigen-stimulated immune response is desired.

[0337] In another aspect, provided herein are mammalian cells infected with a chimpanzee adenovirus, such as C68.

[0338] In yet another aspect, novel mammalian cell lines are provided that express chimpanzee adenovirus genes (eg, from C68) or functional fragments thereof.

[0339] In yet another aspect, provided herein is a method for delivering an antigen cassette to a mammalian cell, comprising the step of introducing into the cell an effective amount of a chimpanzee adenovirus, such as C68, that has been engineered to express the antigen cassette.

[0340] In yet another aspect, a method is provided for 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 from a tumor to which the immune response is targeted.

[0341] In yet another aspect, a method is provided for stimulating an immune response in a mammalian host to treat or prevent a disease in a subject, such as an infectious disease, 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 from the infectious disease or the like that the immune response is targeted against.

[0342] Also disclosed are non-simian mammalian cells expressing chimpanzee adenovirus genes obtained from the sequence of SEQ ID NO: 1. The genes may 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.

[0343] Also disclosed are nucleic acid molecules comprising a chimpanzee adenovirus DNA sequence 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 and lacks at least one gene selected from the group consisting of the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of SEQ ID NO: 1.

[0344] Also disclosed are vectors comprising chimpanzee adenoviral DNA sequences derived from SEQ ID NO:1 and an antigen cassette operably linked to one or more regulatory sequences directing expression of such cassette in a heterologous host cell, optionally wherein the chimpanzee adenoviral DNA sequences comprise at least cis elements necessary for replication and virion encapsidation, the cis elements flanking the antigen cassette and regulatory sequences. In some embodiments, the chimpanzee adenoviral DNA sequences comprise genes selected from the group consisting of 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 can lack the E1A and / or E1B genes.

[0345] Also disclosed herein is an adenoviral vector comprising a partially deleted E4 gene, the adenoviral vector comprising a deleted or partially deleted E4orf2 region, a deleted or partially deleted E4orf3 region, and optionally a deleted or partially deleted E4orf4 region. The partially deleted E4 can comprise an E4 deletion of at least nucleotides 34,916 to 35,642 of the sequence set forth in SEQ ID NO:1, wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1. The partially deleted E4 can include an E4 deletion of at least nucleotides 34,916 to 34,942 of the sequence set forth in SEQ ID NO: 1, at least a partial deletion of nucleotides 34,952 to 35,305 of the sequence set forth in SEQ ID NO: 1, and at least a partial deletion of nucleotides 35,302 to 35,642 of the sequence set forth in SEQ ID NO: 1, wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 can include an E4 deletion of at least nucleotides 34,980 to 36,516 of the sequence set forth in SEQ ID NO: 1, wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 can include an E4 deletion of at least nucleotides 34,979 to 35,642 of the sequence set forth in SEQ ID NO: 1, wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO: 1. A partially deleted E4 can include an E4 deletion that is at least a partial deletion of E4Orf2, a complete deletion of E4Orf3, and at least a partial deletion of E4Orf4. A partially deleted E4 can include an E4 deletion that is at least a partial deletion of E4Orf2, at least a partial deletion of E4Orf3, and at least a partial deletion of E4Orf4. A partially deleted E4 can include an E4 deletion that is at least a partial deletion of E4Orf1, a complete deletion of E4Orf2, and at least a partial deletion of E4Orf3. A partially deleted E4 can include an E4 deletion that is at least a partial deletion of E4Orf2 and at least a partial deletion of E4Orf3.The partially deleted E4 can include an E4 deletion from the start site of E4Orf1 to the start site of E4Orf5. The partially deleted E4 can be an E4 deletion adjacent to the start site of E4Orf1. The partially deleted E4 can be an E4 deletion adjacent to the start site of E4Orf2. The partially deleted E4 can be an E4 deletion adjacent to the start site of E4Orf3. The partially deleted E4 can be an E4 deletion adjacent to the start site of E4Orf4. The E4 deletion can be at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, at least 1100 nucleotides, at least 1200 nucleotides, at least 1300 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1600 nucleotides, at least 1700 nucleotides, at least 1800 nucleotides, at least 1900 nucleotides, or at least 2000 nucleotides. The E4 deletion can be at least 700 nucleotides. The E4 deletion can be at least 1500 nucleotides. The E4 deletion can be 50 nucleotides or less, 100 nucleotides or less, 200 nucleotides or less, 300 nucleotides or less, 400 nucleotides or less, 500 nucleotides or less, 600 nucleotides or less, 700 nucleotides or less, 800 nucleotides or less, 900 nucleotides or less, 1000 nucleotides or less, 1100 nucleotides or less, 1200 nucleotides or less, 1300 nucleotides or less, 1400 nucleotides or less, 1500 nucleotides or less, 1600 nucleotides or less, 1700 nucleotides or less, 1800 nucleotides or less, 1900 nucleotides or less, or 2000 nucleotides or less. The E4 deletion can be 750 nucleotides or less. The E4 deletion can be at least 1550 nucleotides or less.

[0346] 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 at least nucleotides 34,916 to 34,942, nucleotides 34,952 to 35,305, and nucleotides 35,302 to 35,642 of the sequence shown in SEQ ID NO: 1. The partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, lacking at least nucleotides 34,980 to 36,516 of the sequence shown in SEQ ID NO: 1. The partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, lacking at least nucleotides 34,979 to 35,642 of the sequence shown in SEQ ID NO: 1. The adenoviral vector having a partially deleted E4 gene can have a cassette, wherein the cassette includes at least one payload nucleic acid sequence, and the cassette includes at least one promoter sequence operably linked to the at least one payload nucleic acid sequence. The adenoviral vector having a partially deleted E4 gene can have one or more genes or regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO: 1, and optionally, the one or more genes or regulatory sequences include at least one of the chimpanzee adenovirus inverted terminal repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence shown in SEQ ID NO: 1. An adenoviral vector having a partially deleted E4 gene can have nucleotides 2 to 34,916 of the sequence set forth in SEQ ID NO:1, wherein the partially deleted E4 gene is 3' of nucleotides 2 to 34,916, and optionally, nucleotides 2 to 34,916 further lack nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, corresponding to an E1 deletion, and / or nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO:1, corresponding to an E3 deletion.An adenoviral vector having a partially deleted E4 gene can have nucleotides 35,643 to 36,518 of the sequence set forth in SEQ ID NO: 1, where the partially deleted E4 gene is 5' of nucleotides 35,643 to 36,518. An adenoviral vector having a partially deleted E4 gene can have nucleotides 2 to 34,916 of the sequence set forth in SEQ ID NO: 1, where the partially deleted E4 gene is 3' of nucleotides 2 to 34,916, where nucleotides 2 to 34,916 further lack nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO: 1, corresponding to an E1 deletion, and lack nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO: 1, corresponding to an E3 deletion. An adenoviral vector having a partially deleted E4 gene can have nucleotides 2 to 34,916 of the sequence set forth in SEQ ID NO:1, where the partially deleted E4 gene is 3' of nucleotides 2 to 34,916, which further lacks nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, corresponding to an E1 deletion, and nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO:1, corresponding to an E3 deletion, and has nucleotides 35,643 to 36,518 of the sequence set forth in SEQ ID NO:1, where the partially deleted E4 gene is 5' of nucleotides 35,643 to 36,518.

[0347] The partially deleted E4 gene can be the E4 gene sequence set forth in SEQ ID NO:1 lacking at least nucleotides 34,916-35,642 of the sequence set forth in SEQ ID NO:1 and being nucleotides 2-34,916 of the sequence set forth in SEQ ID NO:1, wherein the partially deleted E4 gene is 3' of nucleotides 2-34,916, which nucleotides 2-34,916 further lack nucleotides 577-3403 of the sequence set forth in SEQ ID NO:1, corresponding to an E1 deletion, and lack nucleotides 27,125-31,825 of the sequence set forth in SEQ ID NO:1, corresponding to an E3 deletion, and has nucleotides 35,643-36,518 of the sequence set forth in SEQ ID NO:1, wherein the partially deleted E4 gene is 5' of nucleotides 35,643-36,518.

[0348] Also disclosed herein are host cells transfected with the vectors disclosed herein, such as the C68 vector engineered to express an antigen cassette. Also disclosed herein are human cells that express a selected gene introduced via the introduction of the vectors disclosed herein into the human cells.

[0349] Also disclosed herein is a method for delivering an antigen cassette to a mammalian cell, comprising introducing into said cell an effective amount of a vector disclosed herein, such as a C68 vector engineered to express the antigen cassette.

[0350] Also disclosed herein is a method for producing an antigen, comprising introducing a vector disclosed herein into a mammalian cell, culturing the cell under suitable conditions, and producing the antigen.

[0351] VE2.E1-expressing complementing cell line To generate recombinant chimpanzee adenoviruses (Ad) lacking any of the genes described herein, the function of the deleted gene region, if essential for viral replication and infectivity, can be supplied to the recombinant virus by a helper virus or cell line, i.e., a complementing or packaging cell line. For example, to generate replication-deficient chimpanzee adenovirus vectors, cell lines expressing the E1 gene product of human or chimpanzee adenovirus can be used, including HEK293 or variants thereof. Cell lines expressing any selected chimpanzee adenovirus gene can be generated according to the protocol for generating cell lines expressing chimpanzee E1 gene products (see Examples 3 and 4 of US Pat. No. 6,083,716).

[0352] To identify chimpanzee adenovirus E1-expressing cell lines, AAV enhancement assay can be used. This assay is useful for confirming E1 function in cell lines created using uncharacterized adenovirus E1 genes (e.g., from other species). The assay is described in Example 4B of USPN 6,083,716.

[0353] The selected chimpanzee adenovirus gene, e.g., E1, can be under the transcriptional control of a promoter for expression in the selected parent cell line. For this purpose, an inducible or constitutive promoter can be used. Inducible promoters include the sheep metallothionine promoter, which is inducible by zinc, or the mouse mammary tumor virus (MMTV) promoter, which is inducible by glucocorticoids, particularly dexamethasone. Other inducible promoters, such as those identified in International Patent Application Publication No. WO 95 / 13392, incorporated herein by reference, can also be used to produce packaging cell lines. Constitutive promoters can also be used to control the expression of chimpanzee adenovirus genes.

[0354] Parental cells can be selected to generate novel cell lines expressing any desired C68 gene. Without limitation, such parental cell lines can be HeLa [ATCC Accession No. CCL2], A549 [ATCC Accession No. CCL185], KB [CCL17], Detroit [e.g., Detroit 510, CCL72], and WI-38 [CCL75] cells. Other suitable parental cell lines are available from other sources. Parental cell lines can include CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a.

[0355] E1-expressing cell lines can be useful for producing recombinant chimpanzee adenovirus E1-deleted vectors. Cell lines that express one or more other chimpanzee adenovirus gene products, constructed using essentially the same procedures, are useful for producing recombinant chimpanzee adenovirus vectors in which the genes encoding those products have been deleted. In addition, cell lines that express other human Ad E1 gene products are also useful for producing chimpanzee recombinant Ad.

[0356] VE3. Recombinant viral particles as vectors The compositions disclosed herein can include a viral vector that delivers at least one antigen to a cell. Such a vector includes a chimpanzee adenovirus DNA sequence, such as C68, and an antigen cassette operably linked to a regulatory sequence that directs expression of the cassette. The C68 vector is capable of expressing the cassette in infected mammalian cells. The C68 vector can be functionally deleted for one or more viral genes. The antigen cassette includes at least one antigen under the control of one or more regulatory sequences, such as a promoter. An optional helper virus and / or packaging cell line can provide any necessary components of the deleted adenovirus gene to the chimpanzee viral vector.

[0357] The term "functionally deleted" means that a sufficient amount of a gene region is removed or otherwise altered, for example, by mutation or modification, so that the gene region is no longer capable of producing one or more functional products of gene expression. Mutations or modifications that can result in functional deletion include, but are not limited to, nonsense mutations such as the introduction of premature stop codons and the removal of standard and non-standard start codons, mutations that alter mRNA splicing or other transcription processing, or combinations thereof. If desired, the entire gene region can be removed.

[0358] Modifications of the nucleic acid sequences forming the vectors disclosed herein, such as sequence deletions, insertions, and other mutations, can be produced using standard molecular biology techniques and are within the scope of the present invention.

[0359] VE4. Viral Plasmid Vector Construction Chimpanzee adenovirus C68 vectors useful in the present invention include recombinant defective adenoviruses, i.e., chimpanzee adenovirus sequences functionally deleted in the E1a or E1b genes, and optionally with other mutations, such as temperature-sensitive mutations or deletions in other genes. These chimpanzee sequences are also expected to be useful in forming hybrid vectors from other adenovirus and / or adeno-associated virus sequences. Homologous adenovirus vectors prepared from human adenoviruses have been described in the published literature [see, e.g., Kozarsky I and II, supra, and references cited therein, U.S. Pat. No. 5,240,846].

[0360] In constructing chimpanzee adenovirus C68 vectors useful for delivering antigen cassettes to human (or other mammalian) cells, a range of adenovirus nucleic acid sequences can be used in the vector. Vectors containing minimal chimpanzee C68 adenovirus sequences can be used in conjunction with a helper virus to generate infectious recombinant viral particles. The helper virus provides essential gene products required for viral infectivity and propagation of the minimal chimpanzee adenovirus vector. When only one or more selected deletions of chimpanzee adenovirus genes are made in an otherwise functional viral vector, the deleted gene products can be supplied during the viral vector production process by propagating the virus in a selected packaging cell line that provides the deleted gene functions in trans.

[0361] VE5. Recombinant Minimal Adenovirus The minimal chimpanzee Ad C68 virus is a viral particle that contains only the adenoviral cis elements necessary for replication and virion encapsidation. That is, the vector contains the adenoviral cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (which function as origins of replication) and the native 5' packaging / enhancer domain (which contains sequences necessary for packaging of the linear Ad genome and the enhancer element of the E1 promoter). See, for example, the techniques described for the preparation of "minimal" human Ad vectors in International Patent Application No. WO 96 / 13597, incorporated herein by reference.

[0362] VE6. Other defective adenoviruses Recombinant replication-defective adenoviruses can also contain more than the minimal chimpanzee adenovirus sequences. These other Ad vectors can feature deletions of various portions of the viral gene region and the optional use of helper viruses and / or packaging cell lines to form infectious viral particles.

[0363] As an example, a suitable vector can be formed by deleting all or a sufficient portion of the C68 adenovirus immediate-early gene E1a and delayed-early gene E1b so that their normal biological function is eliminated. Replication-deficient E1-deleted viruses are capable of replicating and generating infectious virus when grown in complementation cell lines transformed with chimpanzee adenoviruses containing functional adenovirus E1a and E1b genes, providing the corresponding gene products in trans. Based on homology with known adenovirus sequences, as is also true for human recombinant E1-deleted adenoviruses in the art, the resulting recombinant chimpanzee adenoviruses are expected to be capable of infecting many cell types and expressing antigen(s), but will be unable to replicate in most cells lacking chimpanzee E1 region DNA unless the cells are infected at a very high multiplicity of infection.

[0364] As another example, all or part of the C68 adenovirus delayed early gene E3 can be excluded from the chimpanzee adenovirus sequences that form part of the recombinant virus.

[0365] Chimpanzee adenovirus C68 vectors can also be constructed with a deletion of the E4 gene. Yet another vector can contain a deletion in the delayed early gene E2a.

[0366] Deletions can also be made in any of the late genes L1 through L5 of the chimpanzee C68 adenovirus genome. Similarly, deletions in intermediate genes IX and IVa2 may also be useful for some purposes. Other deletions can be made in other structural or nonstructural adenovirus genes.

[0367] The deletions discussed above can be used individually, i.e., the adenoviral sequence can contain only an E1 deletion. Alternatively, deletion of entire genes or portions thereof effective to disrupt or reduce biological activity can be used in any combination. For example, in one exemplary vector, the adenoviral C68 sequence can have deletions of the E1 and E4 genes, or deletions of the E1, E2a, and E3 genes, or deletions of the E1 and E3 genes, or deletions of the E1, E2a, and E4 genes, with or without an E3 deletion. As mentioned above, such deletions can be used in combination with other mutations, such as temperature-sensitive mutations, to achieve the desired results.

[0368] The cassette containing the antigen(s) can optionally be inserted into any deleted region of the chimpanzee C68 Ad virus. Alternatively, if desired, the cassette can be inserted into an existing gene region to disrupt the function of that region.

[0369] VE7.Helper virus Depending on the chimpanzee adenovirus gene content of the viral vector used to carry the antigen cassette, helper adenovirus or non-replicating viral fragments can be used to provide sufficient chimpanzee adenovirus gene sequences to generate infectious recombinant viral particles containing the cassette.

[0370] Useful helper viruses contain selected adenoviral gene sequences that are not present in the adenoviral vector construct and / or are not expressed by the packaging cell line into which the vector is transfected. The helper virus may be replication-defective and may contain various adenoviral genes in addition to the sequences described above. Helper viruses can be used in combination with the E1-expressing cell lines described herein.

[0371] In the case of C68, the "helper" virus can be a fragment formed by clipping the C-terminus of the C68 genome to SspI, removing approximately 1300 bp from the left end of the virus. This clipped virus is then co-transfected with plasmid DNA into an E1-expressing cell line, thereby forming recombinant virus by homologous recombination with the C68 sequences in the plasmid.

[0372] The helper virus can also be formed into a polycation conjugate as described in Wu et al., J. Biol. Chem., 264:16985-16987 (1989); KJ Fisher and JM Wilson, Biochem. J., 299:49 (Apr. 1, 1994). The helper virus can optionally contain a reporter gene. Many such reporter genes are known in the art. The presence of a reporter gene on the helper virus that is different from the antigen cassette on the adenovirus vector allows for independent monitoring of both the Ad vector and the helper virus. This second reporter is used to allow for separation of the resulting recombinant virus from the helper virus during purification.

[0373] VE8. Assembly of Viral Particles and Infection of Cell Lines The assembly of selected adenoviral DNA sequences, antigen cassettes, and other vector elements into various intermediate plasmids and shuttle vectors, as well as the use of the plasmids and vectors to generate recombinant viral particles, can all be accomplished using conventional techniques. These include conventional cDNA cloning techniques, in vitro recombination techniques (e.g., Gibson assembly), the use of overlapping oligonucleotide sequences from the adenoviral genome, polymerase chain reaction, and any suitable method for generating the desired nucleotide sequence. Standard transfection and cotransfection techniques, such as CaPO precipitation or liposome-mediated transfection methods such as lipofectamine, are used. Other conventional methods that can be used include homologous recombination of the viral genome, viral plaque formation on an agar overlay, and measuring signal generation.

[0374] For example, after construction and assembly of a viral vector containing a desired antigen cassette, the vector can be transfected in vitro into a packaging cell line in the presence of a helper virus, allowing homologous recombination between the helper and vector sequences to occur, allowing the adenoviral antigen sequences in the vector to be replicated and packaged into virion capsids, resulting in recombinant viral vector particles.

[0375] The resulting recombinant chimpanzee C68 adenovirus is useful for introducing antigen cassettes into selected cells. In vivo studies using recombinant viruses propagated in packaging cell lines have demonstrated the utility of the E1-deleted recombinant chimpanzee adenovirus for introducing cassettes into non-chimpanzee cells, preferably human cells.

[0376] VE9. Use of Recombinant Viral Vectors Thus, the resulting recombinant chimpanzee C68 adenovirus containing the antigen cassette (generated by the cooperation of an adenoviral vector and a helper virus or an adenoviral vector and a packaging cell line as described above) provides an efficient gene transfer vehicle capable of delivering antigen(s) to a subject in vivo or ex vivo.

[0377] The recombinant vectors described above are administered to humans according to published gene therapy methods. The chimpanzee viral vector carrying the antigen cassette can be administered to patients preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle. Suitable vehicles include sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and familiar to those skilled in the art can be used for this purpose.

[0378] The chimpanzee adenoviral vector is administered in an amount sufficient to transduce human cells and to result in a sufficient level of antigen transduction and expression to provide a therapeutic benefit without undue adverse effects or with a medically acceptable physiological effect, as can be determined by one skilled in the medical field. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the liver, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parenteral routes of administration. Routes of administration may be combined as desired.

[0379] The dosage of a viral vector may vary from patient to patient, depending primarily on factors such as the condition being treated and the patient's age, weight, and health. Dosages are adjusted to balance the therapeutic benefit against any side effects, and such dosages may vary depending on the therapeutic application for which the recombinant vector is used. Expression levels of the antigen(s) can be monitored to determine the frequency of administration of the dosage.

[0380] The recombinant replication-deficient adenovirus can be administered in a "pharmaceutically effective amount," i.e., an amount of recombinant adenovirus effective in the route of administration to transfect the desired cells and provide a sufficient level of expression of the selected gene to achieve a vaccine effect, i.e., some measurable level of protective immunity. The C68 vector containing the antigen cassette can be co-administered with an adjuvant. The adjuvant can be separate from the vector (e.g., alum) or encoded within the vector, particularly if the adjuvant is a protein. Adjuvants are well known in the art.

[0381] Conventional pharmaceutically acceptable routes of administration include, but are not limited to, intranasal, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral, and other parenteral routes of administration. Routes of administration may be combined as needed or adjusted depending on the immunogen or disease. For example, for rabies prophylaxis, subcutaneous, intratracheal, and intranasal routes are preferred. The route of administration will primarily depend on the nature of the disease being treated.

[0382] The level of immunity to the antigen(s) can be monitored to determine the need for boosters, if any. For example, after assessment of antibody titers in the serum, optional booster immunizations may be desirable.

[0383] VI. Methods of Treatment and Manufacturing Also provided are methods of inducing a tumor-specific immune response in a subject, vaccinating against a tumor, and treating and / or alleviating symptoms of cancer in a subject by administering to the subject one or more antigens, e.g., multiple antigens identified using the methods disclosed herein.

[0384] In some embodiments, the subject has been diagnosed with cancer or is at risk of developing cancer. The subject may be a human, dog, cat, horse, or any animal in which a tumor-specific immune response is desired. The tumor may be any solid tumor, such as breast, ovarian, prostate, lung, kidney, stomach, colon, testicular, head and neck, pancreas, brain, melanoma, and other tumors of tissue organs, as well as hematological tumors, such as lymphomas and leukemias, for example, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma. The cancer may be non-small cell lung cancer (NSCLC).

[0385] The antigen may be administered in an amount sufficient to stimulate a CTL response. The antigen may be administered in an amount sufficient to stimulate a T cell response. The antigen may be administered in an amount sufficient to stimulate a B cell response. The antigen may be administered in an amount sufficient to stimulate both a T cell response and a B cell response.

[0386] The antigen may be administered alone or in combination with other therapeutic agents, which may include those that target the infectious disease organism, such as antivirals or antibiotics.

[0387] Furthermore, an anti-immunosuppressant / immunostimulatory agent such as a checkpoint inhibitor can be further administered to the subject. For example, an anti-CTLA antibody or anti-PD-1 or anti-PD-L1 can be further administered to the subject. Blockade of CTLA-4 or PD-L1 with an antibody can enhance the immune response against cancerous cells in the patient. In particular, blockade of CTLA-4 has been shown to be effective when used in a vaccination protocol.

[0388] The optimal amount of each antigen to be included in the vaccine composition and the optimal administration regimen can be determined. For example, the antigen or its variant can be prepared for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Methods of injection include sc, id, ip, im, and iv. Methods of DNA or RNA injection include id, im, sc, ip, and iv. Other methods of administering the vaccine composition are known to those skilled in the art.

[0389] Vaccines can be edited so that the selection, number, and / or amount of antigens present in the composition are tissue-, cancer-, infection-, and / or patient-specific. For example, the precise selection of peptides can be guided by the expression pattern of the parent protein in a given tissue, as well as by the patient's mutation or disease state. Selection can vary depending on the specific type of cancer, the specific infection, the disease state, the purpose of vaccination (e.g., preventative or targeting ongoing disease), prior treatment regimens, the patient's immune status, and, of course, the patient's HLA haplotype. Furthermore, vaccines can contain components that are personalized according to the individual needs of a particular patient. Examples include altering antigen selection according to the expression of antigens in a particular patient, or adjusting secondary treatments following a primary treatment or treatment scheme.

[0390] Patients can be identified for administration of antigen vaccines through the use of various diagnostic methods, such as the patient selection methods described in detail below. Patient selection can involve identifying mutations in one or more genes or expression patterns of one or more genes. Patient selection can involve identifying an ongoing infection. Patient selection can involve identifying the risk of infection from an infectious disease. In some cases, patient selection involves identifying the patient's haplotype. Various patient selection methods can be performed in parallel; for example, a sequencing diagnostic can identify both the patient's mutation and haplotype. Various patient selection methods can be performed sequentially; for example, one diagnostic test can identify a mutation and another diagnostic test can identify the patient's haplotype, where each test can be the same diagnostic method (e.g., both high-throughput sequencing) or different diagnostic methods (e.g., one high-throughput sequencing and the other Sanger sequencing).

[0391] For compositions used as vaccines for cancer or infectious diseases, antigens with normal self-peptides similar to those highly expressed in normal tissues may be avoided or present in low amounts in the compositions described herein, whereas if a patient's tumor or infected cells are known to express high amounts of a particular antigen, a pharmaceutical composition for treating this cancer or infectious disease, respectively, may be present in high amounts and / or may include multiple antigens specific to this particular antigen or the pathway of this antigen.

[0392] Compositions containing antigens can be administered to individuals already suffering from cancer or infectious diseases. In therapeutic applications, the compositions are administered to subjects in an amount sufficient to stimulate an effective CTL response against tumor antigens and cure or at least partially halt symptoms and / or complications. An amount appropriate to achieve this is defined as a "therapeutically effective dose." Effective amounts for this use will vary depending, for example, on the composition, the mode of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician. It should be noted that compositions are generally used in serious conditions, i.e., life-threatening or potentially life-threatening situations, particularly when cancer has metastasized. In such cases, the physician may find it possible and desirable to administer substantial excesses of these compositions, taking into account the minimization of exogenous substances and the relative non-toxicity of the antigen.

[0393] For therapeutic use, administration can begin at the time of tumor detection or surgical removal, or at the time of detection or treatment of infection, followed by boosting doses until at least symptoms are substantially alleviated and for a period thereafter, or until immunity is deemed to be provided (e.g., memory B or T cell populations, or antigen-specific B cells or antibodies are produced).

[0394] Pharmaceutical compositions for therapeutic treatment (e.g., vaccine compositions) are intended for parenteral, topical, nasal, oral, or local administration. Pharmaceutical compositions can be administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. Compositions can be administered at a surgical excision site to stimulate a local immune response against a tumor. Compositions can be administered to target specific infected tissues and / or cells in a subject. Disclosed herein are compositions for parenteral administration, which comprise a solution of an antigen, or vaccine composition, dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. Various aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional, well-known sterilization techniques or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is or lyophilized, with the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, etc., e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0395] Antigens can also be administered via liposomes, which target them to specific cellular tissues, such as lymphoid tissues. Liposomes are also useful for extending half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these preparations, the antigen to be delivered is incorporated as part of the liposome, either alone or in combination with a molecule that binds to a receptor frequently found on lymphoid cells, such as a monoclonal antibody that binds to the CD45 antigen, or in combination with other therapeutic or immunogenic compositions. Liposomes loaded with the desired antigen can thus be directed to the site of lymphoid cells, where they deliver the selected therapeutic / immunogenic composition. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid is generally guided by considerations, for example, of liposome size, acid lability, and liposome stability in the bloodstream. Various methods are available for preparing liposomes, as described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

[0396] When directed to immune cells, the ligand incorporated within the liposome can include, for example, an antibody or fragment thereof specific for a cell surface determinant of the desired immune system cell. The liposome suspension can be administered intravenously, locally, topically, etc., at doses that vary depending, inter alia, on the mode of administration, the peptide being delivered, and the stage of disease being treated.

[0397] For therapeutic or immunization purposes, nucleic acids encoding peptides, optionally encoding one or more of the peptides described herein, can also be administered to patients. Numerous methods are conveniently used to deliver nucleic acids to patients. For example, nucleic acids can be delivered directly as "naked DNA." This technique is described, for example, in Wolff et al., Science 247:1465-1468 (1990) and U.S. Patent Nos. 5,580,859 and 5,589,466. Nucleic acids can also be administered using ballistic delivery, as described, for example, in U.S. Patent No. 5,204,253. Particles composed solely of DNA can be administered. Alternatively, DNA can be attached to particles such as gold particles. Techniques for delivering nucleic acid sequences can include viral vectors, mRNA vectors, and DNA vectors, with or without electroporation.

[0398] Nucleic acids can also be delivered in complexes with cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in 9618372 WOAWO96 / 18372, 9324640 WOAWO93 / 24640, Mannino & Gould-Fogerite, BioTechniques 6(7):682-691 (1988), U.S. Patent No. 5,279,833 (Rose), U.S. Patent No. 5,279,833, 9106309 WOAWO91 / 06309, and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987).

[0399] Antigens can also be used in viral vector-based vaccine platforms, such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including, but not limited to, second-generation, third-generation, or second / third-generation hybrid lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors). (See, e.g., 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), etc. Depending on the packaging capacity of the viral vector-based vaccine platform, this approach can deliver one or more nucleotide sequences encoding one or more antigenic peptides.The sequence may be adjacent to non-mutated sequence, separated by a linker, or preceded by one or more sequences that target a subcellular compartment (see, e.g., Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22(4):433-8; Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science. (2016) 352(6291):1337-41; Lu et al., Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20(13):3401-10). Upon introduction into the host, infected cells express the antigen, thereby stimulating a host immune (e.g., CTL) response against the peptide(s). Vaccinia vectors and methods useful for immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette-Guerin). 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.

[0400] A means of administering nucleic acids uses minigene constructs encoding one or more epitopes. To generate DNA sequences encoding selected CTL epitopes (minigenes) for expression in human cells, the amino acid sequences of the epitopes are reverse-translated. A human codon usage table is used to guide codon selection for each amino acid. These epitope-encoding DNA sequences are directly adjacent to each other to create a contiguous polypeptide sequence. Additional elements can be incorporated into the minigene design to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be reverse-translated and included in the minigene sequence include helper T lymphocytes, epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. Additionally, synthetic (e.g., polyalanine) or naturally occurring flanking sequences can be included adjacent to the CTL epitopes to improve CTL epitope presentation to the MHC. The minigene sequence is converted to DNA by assembling oligonucleotides encoding the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified, and annealed under appropriate conditions using well-known techniques. The ends of the oligonucleotides are ligated using T4 DNA ligase. This synthetic minigene encoding the CTL epitope polypeptide can then be cloned into a desired expression vector.

[0401] Purified plasmid DNA can be prepared for injection using a variety of formulations. The most convenient of these is reconstitution of lyophilized DNA with sterile phosphate-buffered saline (PBS). Various methods have been reported, and new techniques may become available. As noted above, nucleic acids are conveniently formulated with cationic lipids. Additionally, glycolipids, fusogenic liposomes, peptides, and compounds collectively known as protective interacting non-condensing (PINC) compounds, can also be complexed with purified plasmid DNA to affect variables such as stability, intramuscular distribution, or transport to specific organs or cell types.

[0402] Also disclosed are methods of producing a vaccine comprising carrying out the steps of the methods disclosed herein and producing a vaccine comprising a plurality of antigens or a subset of a plurality of antigens.

[0403] The antigens disclosed herein can be produced using methods known in the art. For example, methods for producing the antigens or vectors disclosed herein (e.g., vectors containing at least one sequence encoding one or more antigens) can include culturing host cells containing at least one polynucleotide encoding the antigen or vector under conditions suitable for expression of the antigen or vector, and purifying the antigen or vector. Standard purification methods include chromatography, electrophoresis, immunological techniques, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques.

[0404] The host cell may include Chinese hamster ovary (CHO) cells, NS0 cells, yeast, or HEK293 cells. The host cell can be transformed with one or more polynucleotides comprising at least one nucleic acid sequence encoding an antigen or vector disclosed herein, wherein optionally, the isolated polynucleotide further comprises a promoter sequence operably linked to the at least one nucleic acid sequence encoding the antigen or vector. In certain embodiments, the isolated polynucleotide may be a cDNA.

[0405] VII. Use and Administration of Antigens Vaccination protocols can be used to administer one or more antigens to subjects. Priming vaccines and boosting vaccines can be used to administer to subjects. Vaccination methods, protocols, and schedules that can be used include, but are not limited to, those described in International Application Publication No. WO2021092095, which is incorporated herein by reference for all purposes.

[0406] The priming vaccine can be based on C68 (e.g., the sequence shown in SEQ ID NO: 1 or 2) or SAM (e.g., the sequence shown in SEQ ID NO: 3 or 4). The boosting vaccine can also be based on C68 (e.g., the sequence shown in SEQ ID NO: 1 or 2) or SAM (e.g., the sequence shown in SEQ ID NO: 3 or 4).

[0407] Each vector in a prime / boost strategy typically contains a cassette containing an antigen. The cassette can contain approximately 1 to 50 antigens, usually separated by spacers, such as natural sequences surrounding each antigen, or other non-natural spacer sequences, such as AAY. The cassette can also contain MHC II antigens, such as tetanus toxoid and PADRE antigens, which can be considered universal class II antigens. The cassette can also contain a targeting sequence, such as a ubiquitin-targeting sequence. Furthermore, each vaccine dose can be administered to a subject in conjunction with (e.g., simultaneously with, before, or after) an immunomodulator. Each vaccine dose can be administered to a subject in conjunction with (e.g., simultaneously with, before, or after) a checkpoint inhibitor (CPI). The CPI c...

Claims

1. An antigen-encoding cassette, or a polypeptide sequence encoded by said cassette, comprising: the antigen-encoding cassette (i) nucleic acid sequence A(E Ａ ); and (ii) nucleic acid sequence B(E Ｂ ) Including, E Ａ and E Ｂ each encoding one MHC epitope, E Ａ The MHC epitope and E Ｂ are distinct and non-identical; The cassette is Ａ and at least two repeats of E Ｂ at least two repeats of E Ａ and E Ｂ each repeat of each comprises an identical nucleic acid sequence, E Ａ encodes an EGFR-associated MHC class I neoepitope, the antigen-encoding cassette, or the polypeptide sequence encoded by the cassette.

2. E Ａ 2. The cassette of claim 1, wherein the EGFR-associated MHC class I neoepitope encoded by is selected from the group consisting of an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, an EGFR E746-A750 deletion MHC class I epitope, and combinations thereof.

3. E Ａ 3. The cassette of claim 1 or 2, wherein the EGFR-associated MHC class I neoepitope encoded by comprises the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, ITDFGRAKL, STVQLIMQL, TVQLIMQL, LTSTVQLIM, PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK.

4. E Ａ 3. The cassette of claim 1 or 2, wherein the EGFR-associated MHC class I neoepitope encoded by comprises the amino acid sequence KITDFGRAK, KITDFGRAKL, STVQLIMQL, or LSTVQLIM.

5. E Ｂ But, E Ａ The cassette of any one of claims 1 to 4, encoding a non-identical EGFR-associated neoepitope with respect to

6. E Ｂ 6. The cassette of claim 1, wherein the EGFR-associated neoepitope encoded by comprises an MHC class I neoepitope.

7. E Ｂ 7. The cassette of claim 1, wherein the EGFR-associated MHC class I neoepitope encoded by is selected from the group consisting of an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, an EGFR E746-A750 deletion MHC class I epitope, and combinations thereof.

8. E Ｂ 8. The cassette of claim 1, wherein the EGFR-associated MHC class I neoepitope encoded by comprises the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, ITDFGRAKL, STVQLIMQL, TVQLIMQL, LTSTVQLIM, PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK.

9. E Ｂ 8. The cassette of claim 1, wherein the EGFR-associated MHC class I neoepitope encoded by comprises the amino acid sequence KITDFGRAK, KITDFGRAKL, STVQLIMQL, or LSTVQLIM.

10. E Ａ and E Ｂ 10. The cassette of claim 1, wherein said EGFR_L858R and EGFR T790M MHC class I epitopes are combined together to form a cassette.

11. E Ａ and E Ｂ 11. The cassette of any one of claims 1 to 10, encoding at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 repeats of one or both of:

12. E Ａ and E Ｂ 11. The cassette of any one of claims 1 to 10, encoding at least eight repeats of each of:

13. The cassette comprises units E Ａ -E Ｂ and optionally encoding two or more repeats of E Ａ and E Ｂ are linked by a linker-encoding nucleic acid.

14. The unit E Ａ -E Ｂ 14. The cassette of claim 13, which encodes eight repeats of:

15. E Ａ The epitope encoded by the Ｂ 15. The cassette of claim 14, wherein the epitope encoded by comprises an EGFR T790M MHC class I epitope.

16. 16. The cassette of claim 15, encoding the amino acid sequence of SEQ ID NO:

71.

17. The antigen-encoding cassette comprises the nucleic acid sequence C(E Ｃ ) further comprising E Ｃ encodes one MHC epitope, E Ｃ The MHC epitope encoded by E Ａ The MHC epitope and E Ｂ and the MHC epitope encoded by The cassette is Ｃ at least two repeats of E Ｃ each repeat of The cassette according to any one of claims 1 to 15.

18. E Ｃ The cassette of claim 13, wherein said cassette encodes an EGFR-associated MHC class I neoepitope.

19. E Ｃ But EA Ａ and E Ｂ The cassette of claim 18, encoding an EGFR-associated neoepitope that is non-identical to both of

20. E Ｃ 20. The cassette of any one of claims 13 to 19, wherein the EGFR-associated neoepitope encoded by comprises an MHC class I neoepitope.

21. E Ｃ 21. The cassette of any one of claims 13 to 20, wherein the EGFR-associated MHC class I neoepitope encoded by is selected from the group consisting of an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, an EGFR E746-A750 deletion MHC class I epitope, and combinations thereof.

22. E Ｃ 22. The cassette of any one of claims 13 to 21, wherein the EGFR-associated MHC class I neoepitope encoded by comprises the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, ITDFGRAKL, STVQLIMQL, TVQLIMQL, LTSTVQLIM, PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK.

23. E Ｃ 22. The cassette of any one of claims 13 to 21, wherein the EGFR-associated MHC class I neoepitope encoded by comprises the amino acid sequence KITDFGRAK, KITDFGRAKL, STVQLIMQL, or LSTVQLIM.

24. E Ａ , E Ｂ , and E Ｃ 24. The cassette of claim 13, wherein said cassette comprises, in combination, each of an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, and an EGFR E746-A750 deletion MHC class I epitope.

25. E Ａ , E Ｂ , and E Ｃ 24. The cassette of any one of claims 13 to 23, encoding at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 repeats of one or each of:

26. E Ａ , E Ｂ , and E Ｃ 26. The cassette of any one of claims 13 to 25, encoding at least eight repeats of each of:

27. The cassette comprises units E Ａ -E Ｂ -E Ｃ and optionally encoding two or more repeats of E Ａ , E Ｂ , and E Ｃ are linked by a linker-encoding nucleic acid.

28. The unit E Ａ -E Ｂ -E Ｃ 28. The cassette of claim 27, encoding eight repeats of:

29. E Ａ The epitope encoded by the Ｂ 29. The cassette of claim 28, wherein the epitope encoded by comprises an EGFR T790M MHC class I epitope.

30. 30. The cassette of claim 29, encoding the amino acid sequence of SEQ ID NO:

94.

31. 31. The composition of any one of claims 1 to 30, wherein the EGFR-associated MHC class I neoepitope comprises the EGFR_L858R MHC class I epitope.

32. 32. The composition of claim 31 , wherein the EGFR_L858R MHC class I epitope comprises the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, or ITDFGRAKL.

33. 32. The composition of claim 31, wherein the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, or ITDFGRAKL.

34. 32. The composition of claim 31, wherein the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAK or KITDFGRAKL.

35. 35. The composition of any one of claims 1 to 34, wherein the EGFR-associated MHC class I neoepitope comprises the T790M MHC class I epitope.

36. 36. The composition of claim 35, wherein the T790M MHC class I epitope comprises the amino acid sequence STVQLIMQL, TVQLIMQL, or LTSTVQLIM.

37. 36. The composition of claim 35, wherein the T790M MHC class I epitope is the amino acid sequence STVQLIMQL, TVQLIMQL, or LTSTVQLIM.

38. 36. The composition of claim 35, wherein the T790M MHC class I epitope is the amino acid sequence STVQLIMQL or LTSTVQLIM.

39. 39. The composition of any one of claims 1 to 38, wherein the EGFR-associated MHC class I neoepitope comprises an E746-A750 deleted MHC class I epitope.

40. 40. The composition of claim 39, wherein the E746-A750 deleted MHC class I epitope comprises the amino acid sequence PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK.

41. 40. The composition of claim 39, wherein the E746-A750 deleted MHC class I epitope is the amino acid sequence PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK.

42. 42. The composition of any one of claims 1 to 41, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope.

43. 43. The composition of claim 42, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least four repeats of each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope.

44. 43. The composition of claim 42, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least eight repeats of each of the EGFR_L858R MHC class I epitope and the T790M MHC class I epitope.

45. 42. The composition of any one of claims 1 to 41, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope.

46. 43. The composition of claim 42, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least four repeats of each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope.

47. 43. The composition of claim 42, wherein the antigen-encoding cassette comprises at least one antigen-encoding nucleic acid sequence encoding at least eight repeats of each of the EGFR_L858R MHC class I epitope, the T790M MHC class I epitope, and the E746-A750 deletion MHC class I epitope.

48. E Ａ and E Ｂ and optionally E Ｃ From 5' to 3', the formula (L5 ｂ -N ｃ -L3 ｄ ), and is represented by In the formula, N is E Ａ , E Ｂ , and / or E Ｃ wherein c=1; and L5 comprises a 5' linker sequence, where b=0 or 1; L3 comprises a 3' linker sequence, where d=0 or 1; The composition according to any one of claims 1 to 47.

49. each N encodes an epitope 7 to 15 amino acids in length; L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of the epitope, wherein the 5' linker sequence encodes a peptide that is at least 2 amino acids in length, and optionally 2 to 20 amino acids in length; L3 is a natural 3' linker sequence encoding the natural C-terminal amino acid sequence of the epitope, wherein the 3' linker sequence encodes a peptide that is at least 2 amino acids in length, and optionally 2 to 20 amino acids in length; Optionally, E Ａ , E Ｂ , and / or E Ｃ each of which encodes a polypeptide that is 12 to 35 amino acids in length, 49. The composition of claim 48.

50. E Ａ and E Ｂ , and optionally E Ｃ 50. The composition of any one of claims 1 to 49, wherein each of the sequences encodes an epitope at least 7 amino acids in length.

51. E Ａ and E Ｂ , and optionally E Ｃ 50. The composition of any one of claims 1 to 49, wherein each of said sequences encodes an epitope of 7 to 15 amino acids in length.

52. E Ａ and E Ｂ , and optionally E Ｃ 52. The composition of any one of claims 1 to 51, wherein each of is a nucleotide sequence at least 21 nucleotides in length.

53. E Ａ and E Ｂ , and optionally E Ｃ 52. The composition of any one of claims 1 to 51, wherein each of is a nucleotide sequence 75 nucleotides in length.

54. 1. A composition for delivering an antigen expression system, comprising: the antigen expression system, the antigen expression system comprises one or more vectors; The one or more vectors are: (a) a vector backbone comprising: (i) at least one promoter nucleotide sequence, and (ii) at least one polyadenylation (poly(A)) sequence; the backbone comprising: (b) a cassette comprising: (i) at least one antigen-encoding nucleic acid sequence, comprising: (I) an epitope-encoding nucleic acid sequence encoding an EGFR-associated MHC class I neoepitope, wherein the EGFR-associated MHC class I neoepitope is selected from the group consisting of an EGFR_L858R MHC class I epitope, an EGFR T790M MHC class I epitope, an EGFR E746-A750 deletion MHC class I epitope, and a combination thereof; wherein each of said epitope-encoding nucleic acid sequences comprises: (A) optionally, a 5' linker sequence, and (B) Optionally, a 3' linker sequence. The epitope-encoding nucleic acid sequence comprises 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; (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 an exogenous poly(A) sequence relative to the vector backbone; 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 at least one antigen-encoding nucleic acid sequence comprises at least two repeats of the epitope-encoding nucleic acid sequence encoding the EGFR-associated MHC class I neoepitope; The cassette The composition comprising:

55. 55. The composition of claim 54, wherein the EGFR_L858R MHC class I epitope comprises the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, or ITDFGRAKL.

56. 55. The composition of claim 54, wherein the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAK, ITDFGRAK, KITDFGRAKL, or ITDFGRAKL.

57. 55. The composition of claim 54, wherein the EGFR_L858R MHC class I epitope is the amino acid sequence KITDFGRAK or KITDFGRAKL.

58. 58. The composition of any one of claims 54-57, wherein the T790M MHC class I epitope comprises the amino acid sequence STVQLIMQL, TVQLIMQL, or LTSTVQLIM.

59. 58. The composition of any one of claims 54 to 57, wherein the T790M MHC class I epitope is the amino acid sequence STVQLIMQL, TVQLIMQL, or LTSTVQLIM.

60. 58. The composition of any one of claims 54 to 57, wherein the T790M MHC class I epitope is the amino acid sequence STVQLIMQL or LTSTVQLIM.

61. 61. The composition of any one of claims 54 to 60, wherein the E746-A750 deleted MHC class I epitope comprises the amino acid sequence PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK.

62. 61. The composition of any one of claims 54 to 60, wherein the E746-A750 deleted MHC class I epitope is the amino acid sequence PVAIKTSPK, AIKTSPKANK, VAIKTSPK, or KIPVAIKTSPK.

63. 63. The composition of any one of claims 54 to 62, wherein the at least two repeats are separated by at least one separate and distinct epitope-encoding nucleic acid sequence.

64. 63. The composition of any one of claims 54 to 62, wherein the at least two repeats are separated by at least two separate and distinct epitope-encoding nucleic acid sequences.

65. 63. The composition of any one of claims 54 to 62, wherein the at least two repeats comprising the optional 5' linker sequence and / or the optional 3' linker sequence are separated by at least 75 nucleotides.

66. 10. The composition of any one of the preceding claims, wherein the at least two repeats comprise a number of repeats sufficient to stimulate a greater immune response compared to an antigen-encoding nucleic acid sequence comprising a single repeat of the at least one epitope-encoding nucleic acid sequence.

67. 10. The composition of any one of the preceding claims, wherein the at least two repeats comprise a number of repeats sufficient to stimulate an immune response, and wherein a single repeat of the at least one epitope-encoding nucleic acid sequence is insufficient to stimulate the immune response or is insufficient to stimulate a detectable immune response.

68. 68. The composition of claim 66 or 67, wherein the immune response is the proliferation of epitope-specific T cells after in vivo immunization with the composition for delivery of the antigen expression system.

69. 68. The composition of claim 66 or 67, wherein the immune response is increased activation of epitope-specific T cells and / or increased epitope-specific killing by epitope-specific T cells after in vivo immunization with the composition for delivery of the antigen expression system.

70. 10. The composition of any one of the preceding claims, further comprising a nanoparticulate delivery vehicle.

71. 10. The composition of claim 1, wherein the cassette is integrated between the at least one promoter nucleotide sequence and the at least one poly(A) sequence.

72. 10. The composition of any one of the preceding claims, wherein the second promoter is absent and the at least one promoter nucleotide sequence is operably linked to the antigen-encoding nucleic acid sequence.

73. the one or more vectors (i) one or more positive-strand RNA vectors; (ii) a 5′ 7-methylguanosine (m7g) cap; (iii) an RNA vector produced by in vitro transcription; and / or (iv) a vector that is self-replicating in mammalian cells 10. The composition of any one of the preceding claims, comprising:

74. 10. The composition of any one of the preceding claims, wherein the backbone comprises at least one nucleotide sequence of Venezuelan Equine Encephalitis virus.

75. 75. The composition of claim 74, wherein the vector backbone comprises at least a sequence for nonstructural protein-mediated amplification, a 26S promoter sequence, and a poly(A) sequence encoded by a nucleotide sequence of Venezuelan Equine Encephalitis virus, wherein the sequence for nonstructural protein-mediated amplification is selected from the group consisting of an alphavirus 5'UTR, a 51nt CSE, a 24nt CSE, a 26S subgenomic promoter sequence, a 19nt CSE, an alphavirus 3'UTR, or a combination thereof, and / or the backbone does not encode the structural virion protein capsids E2 and E1.

76. 76. The composition of claim 74 or 75, wherein the Venezuelan equine encephalitis virus comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 5, further comprising a deletion between base pairs 7544 and 11175, and the antigen 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.

77. 10. The composition of any one of the preceding claims, wherein the backbone comprises at least one nucleotide sequence of a chimpanzee adenoviral vector.

78. The chimpanzee adenoviral vector is a ChAdV68 vector, and optionally, the ChAdV68 vector comprises: - the sequence set out in SEQ ID NO: 1; the sequence set forth in SEQ ID NO: 1, with the proviso that said sequence is completely 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 set forth in SEQ ID NO: 1, and optionally said sequence is completely or functionally deleted in: (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; - a gene or regulatory sequence derived from the sequence SEQ ID NO: 1, optionally said gene being selected from the group consisting of the chimpanzee adenovirus inverted terminal repeats (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genes of the sequence set out in 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, and optionally a deleted or partially deleted E4orf4 region; - at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1, further comprising: (1) an E1 deletion of at least nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, (2) an E3 deletion of at least nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO:1, and (3) an E4 deletion of at least nucleotides 34,916 to 35,642 of the sequence set forth in SEQ ID NO:1; optionally, wherein the antigen cassette is inserted within the E1 deletion; - the sequence set forth in SEQ ID NO: 68, optionally wherein said antigen cassette is inserted within said E1 deletion; one or more deletions between base pairs 577 and 3403 or between base pairs 456 and 3014, optionally wherein the vector further comprises one or more deletions between base pairs 27,125 and 31,825 or between base pairs 27,816 and 31,333 of the sequence set forth in SEQ ID NO: 1; or - one or more deletions between base pair numbers 3957 and 10346, between base pair numbers 21787 and 23370, and between base pair numbers 33486 and 36193 of the sequence set forth in SEQ ID NO: 1 ChAdV68 vector backbone comprising and Optionally, the cassette is inserted into the E1 region, E3 region, and / or any deleted AdV region of the ChAdV vector backbone that allows integration of the cassette; 78. The composition of claim 77.

79. 10. The composition of any one of the preceding claims, wherein the at least one promoter nucleotide sequence is the native 26S promoter nucleotide sequence encoded by the backbone.

80. 10. The composition of any one of the preceding claims, wherein the second promoter nucleotide sequence comprises multiple 26S promoter nucleotide sequences, each 26S promoter nucleotide sequence providing transcription of one or more of the separate open reading frames.

81. 10. The composition of any one of the preceding claims, wherein the at least one promoter sequence is a regulatable promoter, optionally wherein the regulatable promoter is a tetracycline (TET) repressor protein (TETr)-controlled promoter, and optionally wherein the regulatable promoter comprises multiple TET operator (TETo) sequences 5' or 3' to an RNA polymerase binding sequence of the promoter.

82. 10. The composition of any one of the preceding claims, wherein the antigen-encoding cassette encodes a peptide comprising the amino acid sequence of SEQ ID NO:

61.

83. 10. The composition of any one of the preceding claims, wherein the antigen-encoding cassette encodes a peptide comprising the amino acid sequence of SEQ ID NO:

62.

84. 10. The composition of any one of the preceding claims, wherein the antigen-encoding cassette encodes a peptide comprising the amino acid sequence of SEQ ID NO:

71.

85. 10. The composition of any one of the preceding claims, wherein the antigen-encoding cassette encodes a peptide comprising the amino acid sequence of SEQ ID NO:

94.

86. 10. The composition of any one of the preceding claims, wherein the EGFR-associated MHC class I neoepitope has been validated for presentation by at least one HLA allele, and each antigen / HLA pairing has an antigen / HLA prevalence in the population of at least 1%.

87. 10. The composition of any one of the preceding claims, wherein the EGFR-associated MHC class I neoepitope has been validated for presentation by at least one HLA allele, and each antigen / HLA pairing has an antigen / HLA prevalence in the population of at least 10%.

88. 10. The composition of any one of the preceding claims, wherein the EGFR-associated MHC class I neoepitope has been validated for presentation by at least one HLA allele, and each antigen / HLA pairing has an antigen / HLA prevalence in the population of at least 14%.

89. The at least one HLA allele is HLA A ＊ 03:01, HLA C ＊ 15:02, and / or HLA A ＊ 11:

01. The composition of any one of claims 86 to 88.

90. 90. The composition of any one of claims 86 to 89, wherein the population is the Chinese Hubei Han population.

91. A pharmaceutical composition comprising a composition according to any one of the preceding claims and a pharmaceutically acceptable carrier.

92. 1. An isolated nucleotide sequence or set of isolated nucleotide sequences comprising:

1. A composition comprising a cassette according to any of the preceding claims and one or more elements taken from the sequence of SEQ ID NO: 3 or SEQ ID NO: 5, 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 nsP1-4 genes of the sequences set forth in SEQ ID NO:3 or SEQ ID NO:5; Optionally, the nucleotide sequence is cDNA. The isolated nucleotide sequence or set of isolated nucleotide sequences.

93. 93. A vector or set of vectors comprising the nucleotide sequence of claim 92.

94. 94. An isolated cell comprising the nucleotide sequence or isolated nucleotide sequence of claim 92 or 93, Optionally, the cells are BHK-21, CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a cells; The cells.

95. A kit comprising a composition according to any of the preceding composition claims and instructions for use.

96. 1. A method for treating a subject having cancer, comprising: administering to said subject a composition according to any of the preceding composition claims or a composition according to any of the preceding pharmaceutical composition claims. The method.

97. 97. The method of claim 96, wherein the cancer is non-small cell lung cancer (NSCLC).

98. 1. A method for stimulating an immune response in a subject, comprising: administering to said subject a composition according to any of the preceding composition claims, or any of the preceding pharmaceutical compositions. The method.

99. The subject expresses at least one HLA allele that is predicted or known to present the EGFR-associated MHC class I neoepitope, and the at least one HLA allele is HLA A ＊ 03:01, HLA C ＊ 15:02, and / or HLA A ＊ 11:

01.

100. 100. The method of any one of claims 96 to 99, further comprising administering to the subject a second vaccine composition.

101. The method of claim 100, wherein the second vaccine composition is administered prior to the administration of the composition or pharmaceutical composition of any one of claims 96 to 99.

102. The method of claim 100, wherein the second vaccine composition is administered after administration of the composition or pharmaceutical composition of any one of claims 96 to 99.

103. The method of claim 101 or 102, wherein said second vaccine composition is the same as the composition or pharmaceutical composition of any one of claims 96 to 99.

104. The method of claim 101 or 102, wherein said second vaccine composition is different from the composition or pharmaceutical composition of any one of claims 96 to 99.

105. 105. The method of claim 104, wherein the second vaccine composition comprises a chimpanzee adenoviral vector encoding at least one antigen-encoding nucleic acid sequence.

106. 106. The method of claim 105, wherein the at least one antigen-encoding nucleic acid sequence encoded by the chimpanzee adenoviral vector is an antigen-encoding cassette described in any of the preceding composition claims.

107. 1. A method for producing one or more vectors according to any of the preceding composition claims, comprising: (a) obtaining a linearized DNA sequence comprising the backbone and the 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 to transcribe the linearized DNA sequence into RNA, optionally further comprising adding an m7g cap to the resulting RNA in vitro; and (c) isolating the one or more vectors from the in vitro transcription reaction. The method comprising:

108. 1. A method for producing a composition according to any of the preceding composition claims for delivery of an antigen expression system, comprising: (a) providing components for said nanoparticulate delivery vehicle; (b) providing the antigen expression system; and (c) providing conditions sufficient for the nanoparticulate delivery vehicle and the antigen expression system to produce the composition for delivery of the antigen expression system. The method comprising:

109. 1. A method for treating a subject having a disease, comprising: Optionally, the disease is cancer or an infectious disease; administering to said subject an antigen-based vaccine to said subject; wherein said antigen-based vaccine comprises an antigen-encoding cassette, or a polypeptide sequence encoded by said cassette, wherein said antigen-encoding cassette comprises: (i) nucleic acid sequence A(E Ａ ); and (ii) nucleic acid sequence B(E Ｂ ) Including, E Ａ and E Ｂ each encoding one MHC epitope, E Ａ The MHC epitope and E Ｂ are distinct and non-identical; The cassette is Ａ and at least two repeats of E Ｂ at least two repeats of E Ａ and E Ｂ each repeat of each comprises an identical nucleic acid sequence, E Ａ encodes an EGFR-associated MHC class I neoepitope, The method.

110. 110. The method of claim 109, wherein the antigen-encoding cassette comprises any one of the antigen-encoding cassettes of any one of claims 1 to 90.

111. The method of any of claims 109 to 110, wherein the antigen-based vaccine comprises the pharmaceutical composition of claim 91.

112. 112. The method of any of claims 109 to 111, wherein the antigen-based vaccine is administered as a priming dose.

113. 113. The method of any of claims 109-112, wherein the antigen-based vaccine is administered as one or more booster doses.

114. 114. The method of claim 113, wherein the boost dose is different from the priming dose.

115. a) the priming dose comprises a chimpanzee adenoviral vector and the boosting dose comprises an alphavirus vector; or b) the priming dose comprises an alphavirus vector and the boosting dose comprises a chimpanzee adenovirus vector; The method of claim 114.

116. 114. The method of claim 113, wherein the boost dose is the same as the priming dose.

117. 117. The method of any one of claims 113 to 116, wherein the injection site of the one or more boost doses is as close as possible to the injection site of the priming dose.

118. Determining or having determined the subject's HLA haplotype. further comprising Optionally, the determined HLA haplotype of the subject comprises an HLA allele predicted or verified to present at least one of the EGFR-associated MHC class I neoepitopes encoded by the antigen-encoding cassette, wherein the HLA allele is selected from the group consisting of HLA A ＊ 03:01, HLA C ＊ 15:02, and / or HLA A ＊ It is 11:

01.

10. A method according to any one of the preceding method claims.

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