WO2018102585A1 - Personalized immunotherapy in combination with immunotherapy targeting recurrent cancer mutations - Google Patents

Abstract

Provided herein are systems comprising recurrent cancer mutation immunotherapy compositions and personalized neoepitope immunotherapy compositions. Also provided are recurrent cancer mutation immunotherapy compositions comprising a recombinant Listeria strain comprising a nucleic acid comprising a nucleic acid comprising an open reading frame encoding a recombinant fusion polypeptide comprising one or more antigenic peptides (e.g., fused to a PEST-containing peptide) from cancer-associated proteins. The antigenic peptides can comprise one or more or all of an antigenic peptide comprising a recurrent cancer mutation, an antigenic peptide comprising a heteroclitic mutation, or an antigenic peptide fused to a ubiquitin protein. Also provided are recurrent cancer mutation immunotherapy compositions comprising a recombinant Listeria strain comprising a nucleic acid comprising an open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to two or more antigenic peptides, wherein each of the antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein. Also provided are personalized immunotherapy compositions comprising a recombinant Listeria strain comprising a nucleic acid comprising an open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to one or more antigenic peptides, wherein each of the antigenic peptides comprises a cancer-specific neoepitope comprising a cancer-specific mutation found in a cancer sample from a subject but not in a healthy biological sample from the subject. Also provided are recombinant fusion polypeptides, nucleic acids encoding fusion polypeptides, and methods of generating such compositions. Also provided are methods of inducing an anti-tumor-associated-antigen immune response in a subject, methods of inducing an anti-tumor or anti-cancer immune response in a subject, methods of treating a tumor or cancer in a subject, methods of preventing a tumor or cancer in a subject, and methods of protecting a subject against a tumor or cancer using such immunotherapy compositions, recombinant fusion polypeptides, nucleic acids, or recombinant bacteria or Listeria strains.

Description

PERSONALIZED IMMUNOTHERAPY IN COMBINATION WITH IMMUNOTHERAPY TARGETING RECURRENT CANCER MUTATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims the benefit of US Application No. 62/428,524, filed November 30, 2016, US Application No. 62/443,490, filed January 6, 2017, and US

Application No. 62/583,288, filed November 8, 2017, each of which is herein incorporated by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

[0002] The Sequence Listing written in file 507181SEQLIST.txt is 2.29 megabytes, was created on November 30, 2017, and is hereby incorporated by reference.

BACKGROUND

[0003] Many cancer patients share common mutations in the functional domains of critical tumor driver genes that are the most frequently mutated or that are at least partially responsible for the creating a malignant phenotype. These "hotspot" mutations are commonly shared by cancer patients across multiple tumor types. The acquisition of somatic driver mutations is one of the major mechanisms responsible for the dysregulation of proliferation, invasion, and apoptosis, which are required for oncogenesis. Many of these mutations frequently occur in the functional regions of biologically active proteins (for example, kinase domains or binding domains) or interrupt active sites (for example, phosphorylation sites) resulting in loss-of- function or gain-of-function mutations, or they can occur in such a way that the three-dimensional structure and/or charge balance of the protein is perturbed sufficiently to interfere with normal function.

[0004] Pre-clinical evidence and early clinical trial data suggests that the anti-tumor capabilities of the immune system can be harnessed to treat patients with established cancers. The vaccine strategy takes advantage of tumor antigens associated with various types of cancers. Immunizing with live vaccines such as viral or bacterial vectors expressing a tumor- associated antigen is one strategy for eliciting strong CTL responses against tumors.

[0005] Before personalized medicine, most patients with a specific type and stage of cancer received the same treatment. However, it has become clear that some treatments work well for certain patients and not as well for others. Thus, there is a need to develop effective, personalized immunotherapies effective for a particular tumor. Personalized treatment strategies may be more effective for an individual and cause fewer side effects than would be expected with standard treatments.

[0006] Tumors develop due to mutations in a person's DNA, which can cause the production of mutated or abnormal proteins, comprising potential neoepitopes not present within the corresponding normal protein produced by the host. Some of these neoepitopes may stimulate T cell responses and mediate the destruction of early- stage cancerous cells by the immune system so that clinical evidence of a cancer does not develop. In cases of established cancer, however, the immune response has been insufficient. A large body of data has been generated regarding the development of therapeutic immunotherapies that target natural sequence tumor-associated, overexpressed or inappropriately expressed biomarkers in cancer. However, demonstration of clear clinical benefit associated with these treatments has proven quite difficult. A major reason for this is that as part of central tolerance that develops in all individuals, any T cells that have high binding affinity toward natural sequence peptides are identified as self-antigens and these self-reactive clones are eliminated by the thymus early in life, or otherwise inactivated through mechanisms of tolerance to prevent auto-immunity.

[0007] Neoepitopes are potentially immunogenic epitopes present within a protein associated with a disease that result from a change in the DNA that occurs later in life, such as an acquired mutation or genomic change caused by changes in the DNA of certain cells. For example, in cancer, a specific neoepitope may be present in a cancer cell but not present within the corresponding normal protein associated with cells (in the same individual) that do not harbor the acquired DNA abnormality. The specific acquired DNA abnormalities are very individual to both the specific patient's diseased cells as well as the particular epitope that their immune system might recognize. Because these factors vary from person to person, a personalized approach must be employed to target the multiple neoepitopes, which may number in the thousands, that occur in a person with cancer.

SUMMARY

[0008] Methods and compositions are provided for cancer immunotherapy. In one aspect, provided herein are methods for inducing an immune response against a tumor or cancer in a subject or treating a tumor or cancer in a subject, comprising: (a) administering a recurrent cancer mutation immunotherapy composition to the subject, wherein the recurrent cancer mutation immunotherapy composition comprises a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST-containing peptide fused to two or more first antigenic peptides, wherein each of the first antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the first antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer- associated protein; and (b) administering a personalized immunotherapy composition to the subject, wherein the personalized immunotherapy composition comprises a second recombinant Listeria strain comprising a second nucleic acid comprising a second open reading frame encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises a second PEST-containing peptide fused to one or more second antigenic peptides, wherein each of the second antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject. Alternatively, each of the antigenic peptides in (a) comprises a different recurrent cancer mutation from a different cancer-associated protein. Optionally, each of the antigenic peptides in (a) comprises a different recurrent cancer mutation from a single type of cancer.

[0009] In another aspect, provided herein are methods for inducing an immune response against a tumor or cancer in a subject or treating a tumor or cancer in a subject, comprising: (a) administering a recurrent cancer mutation immunotherapy composition to the subject, wherein the recurrent cancer mutation immunotherapy composition comprises a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST-containing peptide fused to two or more first antigenic peptides, wherein at least one antigenic peptide is from a cancer-associated protein and comprises a recurrent cancer mutation, and at least one antigenic peptide is from a cancer-associated protein and comprises a heteroclitic mutation; and (b) administering a personalized immunotherapy composition to the subject, wherein the personalized immunotherapy composition comprises a second recombinant Listeria strain comprising a second nucleic acid comprising a second open reading frame encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises a second PEST-containing peptide fused to one or more second antigenic peptides, wherein each of the second antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject. Optionally, the first PEST-containing peptide comprises a bacterial secretion signal sequence, and the first fusion polypeptide further comprises a ubiquitin protein fused to a carboxy-terminal antigenic peptide, wherein the first PEST-containing peptide, the first two or more antigenic peptides, the ubiquitin, and the carboxy-terminal antigenic peptide are arranged in tandem from the amino-terminal end to the carboxy-terminal end of the first fusion polypeptide.

[0010] In another aspect, provided herein are systems for use cancer immunotherapy in a subject, comprising: (a) a recurrent cancer mutation immunotherapy composition, wherein the recurrent cancer mutation immunotherapy composition comprises a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST- containing peptide fused to two or more first antigenic peptides, wherein each of the first antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the first antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein; and (b) a personalized immunotherapy composition, wherein the personalized immunotherapy composition comprises a second recombinant Listeria strain comprising a second nucleic acid comprising a second open reading frame encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises a second PEST- containing peptide fused to one or more second antigenic peptides, wherein each of the second antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject. Alternatively, each of the antigenic peptides in (a) comprises a different recurrent cancer mutation from a different cancer-associated protein. Optionally, each of the antigenic peptides in (a) comprises a different recurrent cancer mutation from a single type of cancer.

[0011 ] In another aspect, provided herein are systems for use cancer immunotherapy in a subject, comprising: (a) a recurrent cancer mutation immunotherapy composition, wherein the recurrent cancer mutation immunotherapy composition comprises a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST- containing peptide fused to two or more first antigenic peptides, wherein at least one antigenic peptide is from a cancer-associated protein and comprises a recurrent cancer mutation, and at least one antigenic peptide is from a cancer-associated protein and comprises a heteroclitic mutation; and (b) a personalized immunotherapy composition, wherein the personalized immunotherapy composition comprises a second recombinant Listeria strain comprising a second nucleic acid comprising a second open reading frame encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises a second PEST- containing peptide fused to one or more second antigenic peptides, wherein each of the second antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject. Optionally, the first PEST-containing peptide comprises a bacterial secretion signal sequence, and the first fusion polypeptide further comprises a ubiquitin protein fused to a carboxy-terminal antigenic peptide, wherein the first PEST-containing peptide, the first two or more antigenic peptides, the ubiquitin, and the carboxy-terminal antigenic peptide are arranged in tandem from the amino-terminal end to the carboxy- terminal end of the first fusion polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows CT26 tumor volume in mice treated with PBS control, LmddA- 21 A control, Lm KRAS_G12D_Kd_minigene, Lm KRAS_G12D_Dd_minigene, and Lm KRAS-G12D_21mer.

[0013] Figures 2A and 2B show schematics of WT1 minigene constructs. Figure 2A shows a WT1 minigene construct designed to express a single WT1 chimeric polypeptide antigen. Figure 2B shows a WT1 minigene construct designed to express three separate WT1 chimeric polypeptide antigens.

[0014] Figures 3A and 3B show Western blots of the Lmdda-ΨΊ 1 -tLLO-FLAG-Ub- heteroclitic phenylalanine minigene construct (Figure 3A) and the Lmdda-WTl- tLLO-Pl- P2-P3-FLAG-Ub-heteroclitic tyrosine minigene construct (Figure 3B). In Figure 3A, lane 1 is the ladder, lane 2 is the Lmdda-WTl- tLLO-Pl-P2-P3-FLAG-Ub-heteroclitic tyrosine minigene construct (68 kDa), and lane 3 is a negative control. In Figure 3B, lane 1 is the ladder, lane 2 is the negative control, and lane 3 is the WT1- tLLO-FLAG-Ub-heteroclitic phenylalanine minigene construct (construct #1).

[0015] Figure 4 shows colony PCR results for several Lm-minigene constructs expressing heteroclitic mutant WT1 peptides. Mutated residues are bolded and underlined.

[0016] Figure 5 shows an ELISPOT assay in splenocytes stimulated ex vivo with WT1 peptides RMFPNAPYL (SEQ ID NO: 749) and FMFPNAPYL (SEQ ID NO: 732). The splenocytes are from HLA2 transgenic mice immunized with the WT1-F minigene construct. PBS and LmddA274 were used as negative controls. [0017] Figure 6 shows an ELISPOT assay in splenocytes stimulated ex vivo with WT1 peptides RMFPNAPYL (SEQ ID NO: 749) and YMFPNAPYL (SEQ ID NO: 741). The splenocytes are from HLA2 transgenic mice immunized with the WTl-AHl-Tyr minigene construct. PBS and LmddA274 were used as negative controls.

[0018] Figures 7A and 7B show IFN-γ spot- forming cells (SFC) per million splenocytes stimulated ex vivo with WT1 peptides RMFPNAPYL (SEQ ID NO: 749; Figure 7A) and FMFPNAPYL (SEQ ID NO: 732; Figure 7B). The splenocytes are from HLA2 transgenic mice immunized with the WT1-F minigene construct. PBS and LmddA274 were used as negative controls.

[0019] Figures 8A and 8B show IFN-γ spot- forming cells (SFC) per million splenocytes stimulated ex vivo with WT1 peptides RMFPNAPYL (SEQ ID NO: 749; Figure 8A) and YMFPNAPYL (SEQ ID NO: 741; Figure 8B). The splenocytes are from HLA2 transgenic mice immunized with the WTl-AHl-Tyr minigene construct. PBS and LmddA274 were used as negative controls.

[0020] Figure 9 shows MC38 tumor volume in mice treated with LmddA-214 control, Lm

Dpagtl+Adpgk non-minigene, Lm Adpgk minigene, and Lm Dpagtl minigene.

[0021 ] Figures 10A and 10B show CT26 tumor volume in mice treated with PBS control, LmddA-214 control, Lm AHl_21mer, and Lm AHl_minigene after intraperitoneal

(IP) dosing (Figure 10A) or intravenous (IV) dosing (Figure 10B).

[0022] Figure 11 shows CT26 tumor volume in mice treated with PBS control or Lm

AH1_HC.

[0023] Figure 12 shows Western blot data for different NSCLC constructs. The upper left panel shows detection, using an anti-Flag antibody, of NSCLC constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0024] Figure 13 shows Western blot data for different prostate cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of prostate cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots. [0025] Figure 14 shows Western blot data for different bladder cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of bladder cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0026] Figure 15 shows Western blot data for different bladder cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of bladder cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0027] Figure 16 shows Western blot data for different breast cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of breast cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0028] Figure 17 shows Western blot data for different pancreatic cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of pancreatic cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0029] Figure 18 shows Western blot data for different NSCLC constructs. The upper left panel shows detection, using an anti-Flag antibody, of NSCLC constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0030] Figure 19 shows Western blot data for different prostate cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of prostate cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0031 ] Figure 20 shows IFN-γ spot-forming cells (SFC) per 2xl0⁵ splenocytes stimulated ex vivo with the minimal SIINFEKL peptide (SEQ ID NO: 1007). The splenocytes were from mice immunized with various low-expressing Lm constructs.

[0032] Figure 21 shows a construct design schematic. The top panel shows the tLLO fusion protein design with the C-terminal 3XFLAG and SIINFEKL tag moieties but no linker sequences. The middle panel shows the tLLO fusion protein with C-terminal tags and flanking linker sequences. The bottom panel defines each component of the tLLO fusion protein, with 21mer flanking linkers (^A), long spacers (*), and immunoproteasome spacers (#).

[0033] Figure 22 shows expression and secretion of a Lm construct targeting 15 non- synonymous mutations from the murine MC38 colorectal cancer cell line with or without various linker combinations. The left panel shows a representative anti-FLAG antibody Western blot of culture supernatant from ten unique constructs targeting the same 15 mutations. The right panel shows the construct design strategy and expected size (kDa) of each construct. The same base MT15 amino acid sequence was used in all constructs; the constructs differed by the absence or inclusion of various permutations of flanking linkers and long spacers that have either flexible, rigid, or preferential proteasomal cleavage enhancing properties.

[0034] Figure 23A. General overview of the tumor sequencing and DNA generation work stream.

[0035] Figure 23B. General overview of DNA cloning and immunotherapy

manufacturing work stream.

[0036] Figure 24. Diagram of a cluster of fully enclosed single use cell growth systems arranged for parallel manufacturing of personalized immunotherapy compositions.

[0037] Figure 25. Detailed diagram of the inoculation and fermentation segments of fully enclosed single use cell growth system.

[0038] Figure 26. Detailed diagram of the concentration segment of fully enclosed single use cell growth system.

[0039] Figure 27. Detailed diagram of the diafiltration segment of fully enclosed single use cell growth system.

[0040] Figure 28. Detailed diagram of the product dispensation segment of fully enclosed single use cell growth system. [0041 ] Figure 29 A. Diagram of the process of using a serial selection of neo-epitopes in order to improve efficiency of immunotherapy.

[0042] Figure 29B. Diagram of the process of using a parallel selection multiple neo- epitopes.

[0043] Figure 30. Flow chart of a process (manual or automated) that generates the DNA sequence of a personalized plasmid vector comprising one or more neo-epitopes for use in a delivery vector, e.g., Listeria monocytogenes using output data containing all neo-antigens and patient HLA types.

[0044] Figure 31 shows the effects of moving the SIINFEKL tag on 25D detection. The SIINFEKL tag identifies a secreted neo-epitope whether the tag is located at the C-terminus, the N-terminus, or in between.

[0045] Figure 32A shows the timeline for B 16F10 tumor experiments, including treatments with Lm Neo constructs.

[0046] Figure 32B shows tumor regression with Lmdd ΑΠ A, Lm-Neo-12, and Lm-Neo- 20, with PBS used as a negative control.

[0047] Figure 32C compares survival of mice with B 16F10 tumors following treatment with LmddAlTA, Lm-Neo-12, or Lm-Neo-20, with PBS used as a negative control.

[0048] Figures 33A-33C show expression and secretion levels for PSA-Survivin- SIINFEKL (Figure 33A), PSA-Survivin without SIINFEKL (Figure 33B), and Neo 20- SIINFEKL (Figure 33C).

[0049] Figure 34 shows CD8 T-cell response to the Neo 20 antigen (with C-terminal SIINFEKL tag) or a negative control. The graph indicates the percent SIINFEKL-specific CD8 T-cell response for each condition.

[0050] Figure 35A shows tumor regression with LmddAllA, Lm-Neo-12, Lm-Neo-20, and Lm-Neo 30, with PBS used as a negative control.

[0051 ] Figure 35B compares survival of mice with B 16F10 tumors following treatment with LmddAHA, Lm-Neo-12, Lm-Neo-20, and Lm-Neo 30, with PBS used as a negative control.

[0052] Figure 36 shows the effects of randomizing the order of neo-epitopes within a construct or breaking down the combination of neo-epitopes into subcombinations of neo- epitopes and randomizing those subcombinations to modify secretion.

[0053] Figure 37 shows the relative CD8 cell response in mice immunized with lung neo- epitope constructs. [0054] Figure 38 shows Western blot data for different breast cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of breast cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0055] Figures 39A and 39B show a Lm-HOT (KRAS_G12D) construct induced KRAS- induced specific IFNg immune responses in the periphery of non-tumor-bearing mice.

Figure 39A shows BALB/c mice (n=4/group) were immunized at days 0 and 7 with the Lm- HOT KRAS_G12D construct, and spleens were harvested one week post final immunization (day 14) to assess the cellular immune responses. In Figure 39B, induction of a TH1 response is shown by the number of KRAS_G12D-specific IFNg spot-forming colonies (SFC) per million splenocytes determined by IFNg ELISpot assay. Splenocytes were stimulated for 18 hours using KRAS_G12D pooled peptides (15-mers overlapping by 9 amino acids; 2.5 μg/mL final concentration) spanning the entire KRAS G12D 21mer antigen target. ***P<0.001. Errors bars indicate SEM; n = 4/group.

[0056] Figures 40A-40D show Lm-HOT construct therapy altered the cellular

composition of the tumor immune microenvironment in the CT26 colorectal tumor model and induced KRAS tumor- specific T cells. Naive BALB/c mice were implanted with 300,000 CT26 colorectal tumor cells in the flank. Four days after tumor implantation, mice were immunized with the HOT-Lm KRAS_G12D construct, followed with a boost one week after initial immunization. TILs from tumors of treated CT26 mice were harvested 14 days after tumor implantation. In Figures 40A and 40B, CD45⁺ leukocyte infiltrate and CD8⁺ TILs as percentage of total CD45⁺ cells are shown in treated versus control groups. In Figure 40C, the induction of a TH1 response is shown by the number of KRAS_G12D- specific IFNg spot-forming colonies (SFC) per million TILs determined by IFNg ELISpot assay. In Figure 40D, summary plot data show the percentages of FOXP3+CD4+ and FOXP3+CD25+CD4+ Tregs, respectively, of CD45+ TILs and CD4+FOXP3- TILs as percentage of total CD45+ cells. TILs populations were identified by flow cytometry. *P<0.05; **P<0.01; ***P<0.001; ns not significant. Error bars indicate SEM of n = 4/group.

[0057] Figure 41 shows Western blot data for different bladder cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of bladder cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0058] Figure 42 shows Western blot data for different non-small cell lung cancer (NSCLC) constructs. The upper left panel shows detection, using an anti-Flag antibody, of NSCLC constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0059] Figure 43 shows Western blot data for different prostate cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of prostate cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0060] Figure 44 shows Western blot data for different colorectal cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of colorectal cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0061 ] Figure 45 shows Western blot data for different pancreatic cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of pancreatic cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0062] Figure 46 shows Western blot data for different bladder cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of bladder cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0063] Figure 47 shows Western blot data for a non-small cell lung cancer (NSCLC) construct. The upper left panel shows detection, using an anti-Flag antibody, of NSCLC constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0064] Figure 48 shows Western blot data for different prostate cancer constructs. The upper left panel shows detection, using an anti-Flag antibody, of prostate cancer constructs expressed and secreted into supernatant by LmddA (Western blot). The lower left panel shows detection, using an anti-p60 antibody, of the loading control p60 protein expressed and secreted into supernatant by LmddA (Western blot). The table on the right shows the lane orders for the Western blots.

[0065] Figure 49 shows CT26 tumor volume in naive mice and mice treated with LmddA-llA control, Lm KR AS - G 12D_21 mer , and Lm NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) (HOT-Lung). **** indicates P<0.001; error bars indicate SEM of n = 10/group.

DEFINITIONS

[0066] The terms "protein," "polypeptide," and "peptide," used interchangeably herein, refer to polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms include polymers that have been modified, such as polypeptides having modified peptide backbones.

[0067] Proteins are said to have an "N-terminus" and a "C-terminus." The term "N- terminus" relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (-NH2). The term "C-terminus" relates to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).

[0068] The term "fusion protein" refers to a protein comprising two or more peptides linked together by peptide bonds or other chemical bonds. The peptides can be linked together directly by a peptide or other chemical bond. For example, a chimeric molecule can be recombinantly expressed as a single-chain fusion protein. Alternatively, the peptides can be linked together by a "linker" such as one or more amino acids or another suitable linker between the two or more peptides.

[0069] The terms "nucleic acid" and "polynucleotide," used interchangeably herein, refer to polymeric forms of nucleotides of any length, including ribonucleotides,

deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double- , and multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0070] Nucleic acids are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one

mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements.

[0071 ] "Codon optimization" refers to a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a polynucleotide encoding a fusion polypeptide can be modified to substitute codons having a higher frequency of usage in a given Listeria cell or any other host cell as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at the "Codon Usage Database." The optimal codons utilized by L. monocytogenes for each amino acid are shown US 2007/0207170, herein incorporated by reference in its entirety for all purposes. These tables can be adapted in a number of ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available {see, e.g., Gene Forge).

[0072] The term "plasmid" or "vector" includes any known delivery vector including a bacterial delivery vector, a viral vector delivery vector, a peptide immunotherapy delivery vector, a DNA immunotherapy delivery vector, an episomal plasmid, an integrative plasmid, or a phage vector. The term "vector" refers to a construct which is capable of delivering, and, optionally, expressing, one or more fusion polypeptides in a host cell.

[0073] The term "episomal plasmid" or "extrachromosomal plasmid" refers to a nucleic acid vector that is physically separate from chromosomal DNA (i.e., episomal or

extrachromosomal and does not integrated into a host cell's genome) and replicates independently of chromosomal DNA. A plasmid may be linear or circular, and it may be single- stranded or double-stranded. Episomal plasmids may optionally persist in multiple copies in a host cell's cytoplasm (e.g., Listeria), resulting in amplification of any genes of interest within the episomal plasmid.

[0074] The term "genomically integrated" refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence integrates into the genome of the cell and is capable of being inherited by progeny thereof. Any protocol may be used for the stable incorporation of a nucleic acid into the genome of a cell.

[0075] The term "stably maintained" refers to maintenance of a nucleic acid molecule or plasmid in the absence of selection (e.g., antibiotic selection) for at least 10 generations without detectable loss. For example, the period can be at least 15 generations, 20

generations, at least 25 generations, at least 30 generations, at least 40 generations, at least 50 generations, at least 60 generations, at least 80 generations, at least 100 generations, at least 150 generations, at least 200 generations, at least 300 generations, or at least 500 generations. Stably maintained can refer to a nucleic acid molecule or plasmid being maintained stably in cells in vitro (e.g., in culture), being maintained stably in vivo, or both.

[0076] An "open reading frame" or "ORF" is a portion of a DNA which contains a sequence of bases that could potentially encode a protein. As an example, an ORF can be located between the start-code sequence (initiation codon) and the stop-codon sequence (termination codon) of a gene.

[0077] A "promoter" is a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked

polynucleotide. A promoter can be active in one or more of the cell types disclosed herein (e.g., a eukaryotic cell, a non-human mammalian cell, a human cell, a rodent cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue- specific promoter). Examples of promoters can be found, for example, in WO 2013/176772, herein incorporated by reference in its entirety.

[0078] "Operable linkage" or being "operably linked" refers to the juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence).

[0079] "Sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).

[0080] "Percentage of sequence identity" refers to the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0081 ] Unless otherwise stated, sequence identity/similarity values refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. "Equivalent program" includes any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0082] The term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue. Typical amino acid categorizations are summarized below.

Alanine Ala A Nonpolar Neutral 1.8

Arginine Arg R Polar Positive -4.5

Asparagine Asn N Polar Neutral -3.5

Aspartic acid Asp D Polar Negative -3.5

Cysteine Cys C Nonpolar Neutral 2.5

Glutamic acid Glu E Polar Negative -3.5

Glutamine Gin Q Polar Neutral -3.5

Glycine Gly G Nonpolar Neutral -0.4

Histidine His H Polar Positive -3.2

Isoleucine lie I Nonpolar Neutral 4.5

Leucine Leu L Nonpolar Neutral 3.8

Lysine Lys K Polar Positive -3.9

Methionine Met M Nonpolar Neutral 1.9

Phenylalanine Phe F Nonpolar Neutral 2.8

Proline Pro P Nonpolar Neutral -1.6

Serine Ser S Polar Neutral -0.8

Threonine Thr T Polar Neutral -0.7

Tryptophan Trp w Nonpolar Neutral -0.9

Tyrosine Tyr Y Polar Neutral -1.3

Valine Val V Nonpolar Neutral 4.2

[0083] A "homologous" sequence (e.g., nucleic acid sequence) refers to a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence.

[0084] The term "wild type" refers to entities having a structure and/or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type gene and polypeptides often exist in multiple different forms (e.g., alleles).

[0085] The term "isolated" with respect to proteins and nucleic acid refers to proteins and nucleic acids that are relatively purified with respect to other bacterial, viral or cellular components that may normally be present in situ, up to and including a substantially pure preparation of the protein and the polynucleotide. The term "isolated" also includes proteins and nucleic acids that have no naturally occurring counterpart, have been chemically synthesized and are thus substantially uncontaminated by other proteins or nucleic acids, or has been separated or purified from most other cellular components with which they are naturally accompanied (e.g., other cellular proteins, polynucleotides, or cellular components). [0086] "Exogenous" or "heterologous" molecules or sequences are molecules or sequences that are not normally expressed in a cell or are not normally present in a cell in that form. Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. An exogenous or heterologous molecule or sequence, for example, can include a mutated version of a corresponding endogenous sequence within the cell or can include a sequence corresponding to an endogenous sequence within the cell but in a different form (i.e., not within a chromosome). An exogenous or heterologous molecule or sequence in a particular cell can also be a molecule or sequence derived from a different species than a reference species of the cell or from a different organism within the same species. For example, in the case of a Listeria strain expressing a heterologous polypeptide, the heterologous polypeptide could be a polypeptide that is not native or endogenous to the Listeria strain, that is not normally expressed by the Listeria strain, from a source other than the Listeria strain, derived from a different organism within the same species.

[0087] In contrast, "endogenous" molecules or sequences or "native" molecules or sequences are molecules or sequences that are normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0088] The term "variant" refers to an amino acid or nucleic acid sequence (or an organism or tissue) that is different from the majority of the population but is still sufficiently similar to the common mode to be considered to be one of them (e.g., splice variants).

[0089] The term "isoform" refers to a version of a molecule (e.g., a protein) with only slight differences compared to another isoform, or version (e.g., of the same protein). For example, protein isoforms may be produced from different but related genes, they may arise from the same gene by alternative splicing, or they may arise from single nucleotide polymorphisms.

[0090] The term "fragment" when referring to a protein means a protein that is shorter or has fewer amino acids than the full length protein. The term "fragment" when referring to a nucleic acid means a nucleic acid that is shorter or has fewer nucleotides than the full length nucleic acid. A fragment can be, for example, an N-terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment. A fragment can also be, for example, a functional fragment or an immunogenic fragment. [0091 ] The term "analog" when referring to a protein means a protein that differs from a naturally occurring protein by conservative amino acid differences, by modifications which do not affect amino acid sequence, or by both.

[0092] The term "functional" refers to the innate ability of a protein or nucleic acid (or a fragment, isoform, or variant thereof) to exhibit a biological activity or function. Such biological activities or functions can include, for example, the ability to elicit an immune response when administered to a subject. Such biological activities or functions can also include, for example, binding to an interaction partner. In the case of functional fragments, isoforms, or variants, these biological functions may in fact be changed (e.g., with respect to their specificity or selectivity), but with retention of the basic biological function.

[0093] The terms "immunogenicity" or "immunogenic" refer to the innate ability of a molecule (e.g., a protein, a nucleic acid, an antigen, or an organism) to elicit an immune response in a subject when administered to the subject. Immunogenicity can be measured, for example, by a greater number of antibodies to the molecule, a greater diversity of antibodies to the molecule, a greater number of T-cells specific for the molecule, a greater cytotoxic or helper T-cell response to the molecule, and the like.

[0094] The term "antigen" is used herein to refer to a substance that, when placed in contact with a subject or organism (e.g., when present in or when detected by the subject or organism), results in a detectable immune response from the subject or organism. An antigen may be, for example, a lipid, a protein, a carbohydrate, a nucleic acid, or combinations and variations thereof. For example, an "antigenic peptide" refers to a peptide that leads to the mounting of an immune response in a subject or organism when present in or detected by the subject or organism. For example, such an "antigenic peptide" may encompass proteins that are loaded onto and presented on MHC class I and/or class II molecules on a host cell's surface and can be recognized or detected by an immune cell of the host, thereby leading to the mounting of an immune response against the protein. Such an immune response may also extend to other cells within the host, such as diseased cells (e.g., tumor or cancer cells) that express the same protein.

[0095] The term "epitope" refers to a site on an antigen that is recognized by the immune system (e.g., to which an antibody binds). An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996), herein incorporated by reference in its entirety for all purposes.

[0096] The term "mutation" refers to the any change of the structure of a gene or a protein. For example, a mutation can result from a deletion, an insertion, a substitution, or a rearrangement of chromosome or a protein. An "insertion" changes the number of nucleotides in a gene or the number of amino acids in a protein by adding one or more additional nucleotides or amino acids. A "deletion" changes the number of nucleotides in a gene or the number of amino acids in a protein by reducing one or more additional nucleotides or amino acids.

[0097] A "frameshift" mutation in DNA occurs when the addition or loss of nucleotides changes a gene's reading frame. A reading frame consists of groups of 3 bases that each code for one amino acid. A frameshift mutation shifts the grouping of these bases and changes the code for amino acids. The resulting protein is usually nonfunctional. Insertions and deletions can each be frameshift mutations.

[0098] A "missense" mutation or substitution refers to a change in one amino acid of a protein or a point mutation in a single nucleotide resulting in a change in an encoded amino acid. A point mutation in a single nucleotide that results in a change in one amino acid is a "nonsynonymous" substitution in the DNA sequence. Nonsynonymous substitutions can also result in a "nonsense" mutation in which a codon is changed to a premature stop codon that results in truncation of the resulting protein. In contrast, a "synonymous" mutation in a DNA is one that does not alter the amino acid sequence of a protein (due to codon degeneracy).

[0099] The term "somatic mutation" includes genetic alterations acquired by a cell other than a germ cell (e.g., sperm or egg). Such mutations can be passed on to progeny of the mutated cell in the course of cell division but are not inheritable. In contrast, a germinal mutation occurs in the germ line and can be passed on to the next generation of offspring.

[00100] A "recurrent cancer mutation" is a change in the amino acid sequence of a protein that occurs in multiple types of cancer and/or in multiple subjects having a particular types of cancer. Such mutations associated with a cancer can result in tumor-associated antigens that are not normally present in corresponding healthy tissue.

[00101 ] The term "in vitro" refers to artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube). [00102] The term "in vivo" refers to natural environments (e.g., a cell or organism or body) and to processes or reactions that occur within a natural environment.

[00103] Compositions or methods "comprising" or "including" one or more recited elements may include other elements not specifically recited. For example, a composition that "comprises" or "includes" a protein may contain the protein alone or in combination with other ingredients.

[00104] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.

[00105] Unless otherwise apparent from the context, the term "about" encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations + 0.5%, 1%, 5%, or 10% from a specified value.

[00106] The singular forms of the articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an antigen" or "at least one antigen" can include a plurality of antigens, including mixtures thereof.

[00107] Statistically significant means p <0.05.

DETAILED DESCRIPTION

J. Overview

[00108] Provided herein are systems comprising recurrent cancer mutation immunotherapy compositions and personalized neoepitope immunotherapy compositions. Also provided are recurrent cancer mutation immunotherapy compositions comprising one or more antigenic peptides (e.g., fused to a PEST-containing peptide) from cancer-associated proteins. The antigenic peptides can comprise one or more or all of an antigenic peptide comprising a recurrent cancer mutation, an antigenic peptide comprising a heteroclitic mutation, or an antigenic peptide fused to a ubiquitin protein. Also provided are recurrent cancer mutation immunotherapy compositions comprising a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST-containing peptide fused to two or more first antigenic peptides, wherein each of the first antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the first antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein. Also provided are personalized immunotherapy compositions comprising a second recombinant Listeria strain comprising a second nucleic acid comprising a second open reading frame encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises a second PEST-containing peptide fused to one or more second antigenic peptides, wherein each of the second antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject. Also provided are recombinant fusion polypeptides from such compositions, nucleic acids encoding such fusion proteins. Also provided herein are methods of generating such fusion polypeptides, such nucleic acids, and such recombinant bacteria or Listeria strains. Also provided are immunogenic compositions, pharmaceutical compositions, and vaccines comprising such immunotherapy compositions, such fusion polypeptides, such nucleic acids, or such recombinant bacteria or Listeria strains. Also provided are methods of inducing an anti-tumor-associated-antigen immune response in a subject, methods of inducing an anti-tumor or anti-cancer immune response in a subject, methods of treating a tumor or cancer in a subject, methods of preventing a tumor or cancer in a subject, and methods of protecting a subject against a tumor or cancer using such immunotherapy compositions, recombinant fusion polypeptides, nucleic acids, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines.

[00109] Some therapeutic targets in human cancers are proteins encoded by tumor-driver genes with tumor- specific mutational "hotspots," such as TP53, PIK3CA, PIK3R1, PTEN, KRAS, NRAS, BRAF, and EGFR. Hotspots are areas within the DNA molecule which are most likely to mutate. The acquisition of somatic driver mutations is one of the major mechanisms responsible for the dysregulation of proliferation, invasion, and apoptosis, which are required for oncogenesis. Many of these mutations frequently occur in the functional regions of biologically active proteins (for example, kinase domains or binding domains) or interrupt active sites (for example, phosphorylation sites) resulting in loss-of- function or gain-of-function mutations. Many patients share common mutations in the functional domains of critical tumor driver genes that are the most frequently mutated or that are at least partially responsible for the creating a malignant phenotype. For example, one study evaluated over 11,000 tumors in 41 different tumor types and reported 470 somatic mutational hotspots that affected 275 genes. It was also reported that approximately 55% of all solid tumors have one or more hotspots (Chang et al. (2016) Nat Biotechnol 34(2): 155- 163, herein incorporated by reference in its entirety for all purposes). Evaluating the specific missense amino acid substitutions resulting from these hotspots reveals that many mutations are commonly shared by cancer patients across multiple tumor types. For example, it has been hypothesized that p53 function is compromised in most human tumors while at least half of all tumors exhibit mutation of p53 (Po lager and Ginsberg (2009) Nat Rev. Cancer 9(10):738-748, herein incorporated by reference in its entirety for all purposes). This mutational "sharing" across patients and tumor types creates an opportunity for the "off the shelf development of treatment constructs that target these common hotspots. Targeting of acquired tumor- specific or cancer- specific mutations is not prevented by central tolerance and minimizes off-target effects in normal cells. Disclosed herein are such "off the shelf constructs using Listeria monocytogenes {Lm) technology (ADXS-HOT) and their use in therapeutic methods.

[00110] The Lm technology has a mechanism of action that incorporates potent innate immune stimulation, delivery of a target peptide directly into the cytosol of dendritic cells and antigen presenting cells, generation of a targeted T cell response, and reduced immune suppression by regulatory T cells and myeloid-derived suppressor cells in the tumor microenvironment. Multiple treatments can be given and/or combined without neutralizing antibodies. The Lm technology can use, for example, live, attenuated, bioengineered Lm bacteria to stimulate the immune system to view tumor cells as potentially bacterial- infected cells and target them for elimination. The technology process can start with a live, attenuated strain of Listeria and can add, for example, multiple copies of a plasmid that encodes a fusion protein sequence including a fragment of, for example, the LLO (listeriolysin O) molecule joined to the antigen of interest. This fusion protein is secreted by the Listeria inside antigen- presenting cells. This results in a stimulation of both the innate and adaptive arms of the immune system that reduces tumor defense mechanisms and makes it easier for the immune system to attack and destroy the cancer cells.

[00111 ] Immunologically, Lm-based vectors are a far superior platform for the generation of CD8+ dominant T cell responses compared to peptide vaccines. First, there is no need to add adjuvants of filgrastim injections. This is because the live attenuated bacteria vectors inherently trigger numerous innate immune activation triggers which include several TLRs, PAMP, and DAMP receptors and have a potent ability to agonize the STING receptor within the cytosol of the antigen-presenting cells. This is a much broader alteration of the immunologic microenvironment that primes the patients' immune system for an adaptive immune response. Second, the Lm vector is infused intravenously. This allows it to reach significantly more antigen-presenting cells than may reside in a finite area of subcutaneous tissue. It also eliminates the requirement for subcutaneous injections, the use of filgrastim, and the risk of delayed type hypersensitivity. It is also likely to generate high T cell titers faster as optimum CD8+ T cell numbers typically peak after 3 treatments, not greater than 10. Third, Lm promotes a predominant CD8+ T cell response with CD4+ cross-reactivity for T cell help. CD8+ T cells are the most effective at killing cancer cells and because Lm vectors present their antigen in the cytoplasm of the APC, those peptides are rapidly shunted to the proteasome for processing, complexed with MHC Class 1 and transported to the APC surface for presentation to predominantly CD8+ T cells. This should bring the advantage of generating more CD8+ T cells that a subcutaneous Montanide presentation of antigen peptides. Fourth, Lm vectors increase the expression of chemokine and chemokine receptors on tumors and surrounding lymph nodes. This facilitates the attraction of activated T cells to the vicinity of solid tumors. Fifth, Lm vectors decrease the relative number and suppressive function of immunosuppressive cells that may protect a tumor from T cell attack, better enabling T cell killing of cancer cells. This reduction of the immunosuppressive ability of regulatory T cells and myeloid derived suppressor cells will better enable T cells generated against these peptides to have better activity in solid tumors. Sixth, Lm vectors do not generate neutralizing antibodies. Because of this, these vectors can be administered repeatedly for extended periods of time without the loss of efficacy from neutralizing antibodies and the development of delayed-type hypersensitivity or acute hypersensitivity which may include anaphylaxis.

[00112] Lm vectors act via multiple immunotherapy mechanisms: potent innate immune stimulation via toll- like receptors (TLRs) and pathogen-associated molecular patterns (PAMPs) including the stimulator of interferon genes (STING) receptor, strong CD8⁺ and CD4⁺ T cell responses, epitope spreading, and immune suppression by disabling Tregs and myeloid derived suppressor cells (MDSCs) in the tumor microenvironment. In addition, the unique intracellular life cycle of Listeria avoids neutralizing antibodies, allowing for repeat dosing. Lm is also advantageous because it has synergies with checkpoint inhibitors, costimulatory agonists, and others agents. It also has a large capacity and can be adapted to target many different tumor types. As an example, live, attenuated strains of Lm can be bioengineered to secrete an antigen-adjuvant fusion protein comprising, consisting essentially of, or consisting of a truncated fragment of listeriolysin O (tLLO), which has adjuvant properties, and one or more tumor-associated antigens. Upon infusion into a patient, bioengineered Lm can be phagocytosed by antigen-presenting cells, where the fusion protein is secreted by the Lm, processed, and presented onto major histocompatibility complex (MHC) class I and II molecules. Target peptides presented on the surface of the antigen- presenting cells stimulate tumor-associated-antigen-specific CD4⁺ and CD8⁺ T cells. Activated CD8⁺ T cells can then seek out and kill tumor-associated-antigen-expressing cancer cells and modulate the tumor microenvironment to overcome immune suppression.

[00113] Lm vectors have some clinical advantages. Any side effects associated with treatment appear in the hours immediately post-infusion while the patient is still in the clinic, are almost exclusively mild-moderate and respond readily to treatment, and resolve the day of dosing without evidence of delayed onset, cumulative toxicity, or lasting sequalae. Practical advantages include the fact that there is no need to administer multiple agents and switch to alternate dosing sites for subsequent administrations.

[00114] From a manufacturing standpoint, there are several advantages. First, there is no need to manufacture the individual peptides to high concentrations and high degrees of purity. The Lm bacteria transcribe the DNA simultaneously on multiple copies of DNA plasmids inside the bacteria and secrete these peptides directly into the cytoplasm of the APC, where they are almost immediately transported to the proteasome for processing.

Essentially, the peptides are manufactured by the bacteria right at the point of use for antigen processing. Second, Lm vectors are highly scalable. Once the genetic engineering is complete, the bacteria replicate themselves in broth cultures. The cultures can be scaled up to vastly reduce cost of goods. Third, there is no need to formulate in a complex carrier like Montanide or create an emulsion. Fourth, the bacteria are very stable, some more than 5 years, without worry of peptide degradation or breakdown product contamination that can lead to loss of potency of a peptide formulation.

[00115] The ADXS-HOT constructs disclosed herein utilize the Lm vector technology to target the specific epitopes (e.g., T cell epitopes) represented by multiple recurrent cancer mutations (e.g., shared tumor driver hotspot mutations) occurring in cancer-associated genes (e.g., key tumor driver genes). As an example, one Lm vector can be prepared that can cover the specific hotspot mis sense mutations that are found in the majority of patients who share a mutation in a specific tumor driver gene. This approach would allow a single product to represent the potential mutated epitopes that would be found in, for example, 90% or more (e.g., 98% or more) of patients who have an acquired mutation in a particular gene such as TP53, PIK3CA, or NRAS or KRAS. For example, mutated epitopes at 17 positions could cover > 90% of the recurrent missense cancer mutations in TP53. Combining the majority of the potential mutations in a tumor driver gene into one product is possible because many of these mutations are shared by a significant proportion of cancer patients. As a result, the total spectrum of potential tumor driver gene missense mutations for solid tumors can be covered within the capacity of one Lm construct. This makes the Lm vector technology a highly efficient and adaptable technology for engineering "off the shelf hotspot constructs to target common mutations.

[00116] As another example, one Lm vector can be prepared that can cover the specific hotspot missense mutations that are found in the majority of patients (or in a certain percentage of patients, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) who have a specific type of cancer. This approach would allow a single product to represent the potential mutated epitopes that would be found in, for example, 50% or more of patients who have a particular type of cancer. Combining the majority of or a significant percentage of the potential mutations in a particular type of cancer into one product is possible because many of these mutations are shared by a significant proportion of cancer patients. As a result, the total spectrum of potential tumor driver gene missense mutations for solid tumors can be covered within the capacity of one Lm construct. This makes the Lm vector technology a highly efficient and adaptable technology for engineering "off the shelf hotspot constructs to target common mutations.

[00117] ADXS-HOT constructs can be bioengineered to target the most common tumor driver hotspot mutations. These products can be manufactured and available immediately for a patient who is found through biomarker testing to carry a mutation included in the ADXS- HOT product's mutational coverage for a specific tumor driver gene. Likewise, these products can be manufactured and available immediately for a patient who is found through biomarker testing to carry a mutation included in the ADXS-HOT product's mutational coverage for two or more specific tumor driver genes. The presence of this mutation can be determined or confirmed for each patient by rapid PCR testing, Nanostring, DNA

sequencing, RNA sequencing, or another diagnostic biomarker procedure, on a biopsy or archived tumor tissue or DNA or RNA sequencing information that may already exist. The ability to use biomarker test results to rapidly confirm eligibility facilitates a rapid delivery of the ADXS-HOT product directly to the patient and eliminates any waiting period needed to develop a customized treatment. Presence of hotspot mutations can be rapidly determined through biomarker testing, and "off the shelf treatments can be initiated immediately. DNA sequencing is not required, and manufacture of a patient- specific product is not necessary. This "off the shelf delivery of hotspot-targeted immunotherapies to qualified patients represents a significant therapeutic option in cancer treatment.

[00118] Design and use of heteroclitic sequences (i.e., sequence-optimized peptides) derived from tumor-associated antigen genes (e.g., from cancer testis antigens or oncofetal antigens) can increase presentation by MHC Class I alleles. Heteroclitic sequences have been shown to be sufficient to prime a T cell response, to overcome central tolerance, and to elicit a successful cross-reactive immune response to the wild-type peptide. Addition of heteroclitic epitopes to hotspot-targeted immunotherapies can complement the original hotspot mutation peptides in that total patient coverage within a cancer type can approach 100%. We therefore do not need to sequence a patient prior to treatment as we assume that they will express a tumor-associated antigen that we have designed heteroclitic peptides for to cover the most prevalent HLAs (HLA-A0201, HLA-A0301, HLA-A2402, and HLA- B0702).

[00119] Use of the minigene construct approach disclosed herein for the expression of specific MHC class I binding antigenic determinants allows for the highly efficient delivery of short peptide sequences to the antigen presentation pathway of professional antigen presenting cells (pAPC). A specific advantage of the minigene technology is that it bypasses the requirement for proteasome mediated degradation of larger proteins in order to liberate short peptide sequences that can be bound and presented on MHC class I molecules. This results in a much higher efficiency of peptide-MHC class I antigen presentation on the surface of the pAPC and, therefore, a much higher level of antigen expression for the priming of antigen specific T cell responses.

[00120] In some approaches disclosed herein, up to or more than four distinct attributes can be combined into a single, disease-specific, off-the-shelf product that maximizes target coverage and minimizes off-target toxicities. These attributes can include: attenuated Listeria monocytogenes {Lm) vectors, tLLO fusion proteins, hotspot mutations, and optimized peptides derived from cancer testis antigens (CTAs) or oncofetal antigens (OFAs). Lm in the body is actively taken up by antigen-presenting cells and moves into the cytoplasm; therefore, it is an ideal vector for the delivery of antigens to be presented through both the MHC I and II pathways. Lm also produces virulence factors which allow survival in the host cytosol and potently stimulate the immune system. These virulence factors can enhance the

immunogenicity of tumor-associated antigens. Multiple plasmids within Lm can encode for expression of tumor-associated antigen fusion proteins (e.g., tLLO fusion proteins) inside antigen-presenting cells, which triggers a powerful CD8+ T cell response along the MHC I pathway. The Lm and tLLO fusion protein can also neutralize the regulatory T cells and MDSCs protecting the tumor, increasing CD8+ T cell efficacy. Having multiple copies of plasmids within the Lm increases antigen presentation and tumor microenvironment effects. The fusion protein can include hotspot peptides and/or sequence-optimized peptides (i.e., peptides with heteroclitic mutations) derived, for example, from CTAs or OFAs. Hotspot mutations are high- value targets against tumor drivers, and targeting them can generate a strong immune response and inhibit tumor proliferation. Incorporating multiple hotspot mutation peptides broadens the patient coverage in the targeted diseases. Hotspots are somatic mutations frequently observed in multiple patients, often in tumor driver genes contributing to oncogenesis. These hotspot mutations represent a source of "shared" or "public" antigens. Hotspots targets in the constructs described herein can be designed to generate epitopes to virtually any of the 12,500+ identified HLA Class I alleles and can be prioritized agnostic to in silico algorithms. OFAs and CTAs are expressed in up to 100% of patients within a cancer indication, but are not expressed in healthy tissue of adults (e.g., normally expressed only in embryonic tissues). Many OFAs/CTAs have primary roles in oncogenesis. Because of OFA/CTAs highly restricted tissue expression in cancer, they are attractive targets for immunotherapy. Adding multiple sequence-optimized, proprietary immunogenic OFA/CTA peptides or tumor-associated antigen peptides (i.e., sequence- optimized to improve immunogenicity) provides additional targets capable of generating strong T cell responses. In combination, these components take advantage of somatic mutations, cancer testis antigens, and oncofetal antigens more capable of generating potent, tumor specific, high strength (avidity) T cells to kill tumor cells than more traditional, over- expressed, native-sequence tumor-associated antigens. Most hotspot mutations and

OFA/CTA proteins play critical roles in oncogenesis. Targeting both at once could significantly impair cancer proliferation. Combining hotspot mutations with multiple OFA/CTAs peptides presents multiple high avidity targets in one treatment that are expressed in all patients with, the target disease.

[00121 ] Patients with multiple mutations in cancer-associated genes (e.g., tumor driver genes) can be treated with a combination (e.g., a single dosing regimen consisting of two or more immunotherapies) targeting their particular mutated genes identified in biomarker testing, or, alternatively, a combination kit or panel (e.g., a single dosing regimen consisting of two or more immunotherapies) for their type of cancer can be used that covers mutated genes commonly found in patients with that disease (e.g., a lung adenocarcinoma panel, a colorectal cancer panel, and so forth). Patients with a particular type of cancer can then be treated with a fixed combination or panel of ADXS-HOT constructs targeting commonly observed mutated genes in that particular type of cancer. Alternatively, such patients can be treated with a single immunotherapy targeting their particular mutated genes identified in biomarker testing or a single immunotherapy specific for their type of cancer that covers mutated genes found in multiple different cancer-associated proteins found in patients with that disease. All patients with a given tumor type can be treated in the same way. For example, in certain diseases there are relatively few genes that carry mutations in a large percentage of patients. In these instances, for example, it may be more expeditious to give all patients with the same disease type the same combination of ADXS-HOT constructs. For example, 93% of ovarian cancer patients have a mutation in TP53, so there may be no need for a diagnostic test. In colorectal cancer (CRC), four tumor driver genes are mutated most frequently, and most patients will harbor more than one mutation in these four genes. A "standard" combination for CRC could include ADXS-HOT constructs for APC, TP53, PIK3CA, and RAS because tumor driver mutations in CRC include APC in 76% of patients, TP-53 in 52% of patients, RAS (KRAS/NRAS) in 52 % of patients, and PIK3CA in 19% of patients. Alternatively, a "standard" for CRC could include a single ADXS-HOT construct including a set of the most common CRC mutations in APC, TP53, PIK3CA, and RAS. There is a great likelihood that most patients would express anywhere from 2-4 of these, so multiple recurrent cancer mutations would be targeted.

[00122] The ADXS-HOT immunotherapies disclosed herein have the potential to revolutionize the treatment of cancer by providing highly efficacious, targeted attacks on hotspots with little to no impact on healthy cells. Tumor immunotherapies take advantage of the most effective cancer- fighting agents that nature has devised: the host's own immune cells.

[00123] Tumor- specific antigens that arise as a consequence of tumor- specific mutations are important targets for effective cancer immunotherapy. The most effective and longest lasting responses to immunotherapy of cancer can be attributed to amplification of T cell responses against tumor- specific antigens or tumor- specific epitopes associated with mutations in the tumors. Furthermore, mutations in tumor driver genes are most often associated with loss of function or gain of function phenotypes that drive persistence or growth of cancer cells. Targeting these driver mutations specifically may offer the best chance for immunotherapy to inhibit disease progression and eliminate cancer cells without compromising normal cells. Although recurrent cancer mutations may or may not be included in a personalized treatment, the ADXS-HOT approach has inherent advantages over personalized, neoepitope-targeted, patient- specific products for the treatment of cancer. First, it targets what may be the most critical mutations associated with cancer growth. Second, targeting shared, recurrent cancer mutations allows the same product to be used for multiple patients. The capacity of Lm-LLO vectors allows coverage of nearly all of the mutations that may occur in a single gene-targeted product such that the product can treat nearly all patients who have any acquired mutation in a particular cancer-associated gene (e.g., tumor driver gene). ADXS-HOT constructs can be manufactured in bulk, and Lm-LLO products have shown good stability for 5 years or more. In addition, the ability to combine multiple constructs increases coverage. Finally, the ADXS-HOT are ready, on the shelf, and are available for patients to start treatment immediately but still target tumor- specific epitopes. Cost of goods can be kept low by making larger batches as opposed to a one-off per patient product. Product stability for previous LM-LLO constructs, for example, can exceed five years. Patients with advanced cancer may not be able to wait months to begin treatment with a personal neoepitope product, but by leveraging ADXS-HOT panels, treatment against tumor- specific epitopes can start almost immediately. In some cases, ADXS-HOT constructs can be used immediately targeting recurrent cancer mutations found in a patient's cancer while a personalized neoepitope construct is being prepared. When the personalized product is ready, it can replace the ADXS-HOT regimen and/or add targeting of the personalized neoepitopes to the recurrent cancer mutations being targeted.

[00124] Multiple Lm-LLO constructs as disclosed herein that will have broad utility across multiple tumor types and multiple patients who share common mutations in tumor driver genes. The products target acquired recurrent cancer mutations that are shared by multiple patients and should have greater immunogenicity than the natural sequence peptide in normal cells, which is protected by tolerance. Mutations in P-53 and PI3 Kinase alone occur in over 50% of all cancer patients, and panels can be formed for major cancers as disclosed herein where hot-spot mutations in tumor driver genes are common.

[00125] Multiple ADXS-HOT constructs can be made to provide a "spice rack" approach, driven by biomarker testing determinations. Readily available rapid biomarker testing and/or RNA or DNA sequencing can determine the presence of a target for creation of a

personalized medicine "kit" for individual patients. Disease- specific panels can target the majority of patients with a specific disease that share common mutations. Alternatively, a set combination can be given for certain disease types and will include mutations found in a majority of patients with a certain disease without the need for a diagnostic test.

[00126] Constructs can be used as a monotherapy, but the potential also exists to use ADXS-HOT constructs as part of a combination treatment regimen either as several individual hotspot products together or in combination with other therapeutic cancer treatments. As an example, where more than one gene is mutated in the same patient, the representative constructs for each gene can be mixed just before infusion. For example, if a patient is found to have missense mutations in hotspots for TP53, RAS, and BRAF, then these three ADXS-HOT products could be given in combination (ADXS-htTP53, ADXS-htRAS, and ADXS-htBRAF) as a treatment regimen. In addition, similar to other Lm constructs, hotspot treatments can be given in combination or sequentially with other cancer treatments like checkpoint inhibitors, costimulatory agonists, radiation therapy, or personalized neoepitope immunization. The reason for this is that animal models and early data from clinical trials have shown that Lm-LLO immunotherapies have the potential for significant synergy with active immunotherapy agents, particularly PD-1 and/or PD-L1 blocking antibodies.

[00127] For example, the combination of an Lm-LLO-based vaccine with anti-PD-1 antibody leads to increased antigen- specific immune responses and tumor- infiltrating CD8+ T cells, along with a decrease in immune suppressor cells (Tregs and MDSCs). The combination regimen led to synergistic activity, with significant inhibition of tumor growth and prolonged survival/complete regression of tumors in treated animals. The combination of an Lm-LLO-based vaccine with blocking of PD-l/PD-Ll can lead to overall enhancement of the efficacy of anti-tumor immunotherapy over either agent alone. It was also shown that in vitro infection with Lm results in significant upregulation of surface PD-L1 expression on human monocyte-derived dendritic cells, which suggests the translational capacity of this finding.

[00128] Preclinical data also suggests synergy with immune costimulatory agonists like Ox-40 and GITR (Mkrtichyan et al. (2013) J Immunother Cancer 1: 15, doi: 10.1186/2051- 1426-1-15, herein incorporated by reference in its entirety for all purposes). Synergy of Lm- LLO vectors with radiation therapy has been demonstrated in preclinical models (Hannan et. al. (2012) Cancer Immunol Immunother 61(12):2227-2238, herein incorporated by reference in its entirety for all purposes) and has also been observed in ongoing veterinary trials in non- resected canine osteosarcoma. Lm treatments can also be given sequentially with

chemotherapies provided there has been sufficient hematopoietic recovery. In addition, research to date shows there is no development of neutralizing antibodies with Lm vectors, so repeated treatments with a single Lm vector or simultaneous or sequential treatment with multiple vectors is possible.

//. Recombinant Fusion Polypeptides Comprising Recurrent Cancer Mutations

[00129] Disclosed herein are recombinant fusion polypeptides comprising a PEST- containing peptide fused to two or more antigenic peptides (i.e., in tandem, such as PEST- peptide l-peptide2), wherein each antigenic peptide comprises a single, recurrent cancer mutation (i.e., a single, recurrent change in the amino acid sequence of a protein, or a sequence encoded by a single, different, nonsynonymous, recurrent cancer mutation in a gene). Also disclosed herein are recombinant fusion polypeptides comprising a PEST- containing peptide fused to two or more antigenic peptides (i.e., in tandem, such as PEST- peptidel-peptide2), wherein each antigenic peptide comprises a single, recurrent cancer mutation (i.e., a single, recurrent change in the amino acid sequence of a protein, or a sequence encoded by a single, different, nonsynonymous, recurrent cancer mutation in a gene), and wherein at least two of the antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein. Alternatively, each of the antigenic peptides comprises a different recurrent cancer mutation from a different cancer- associated protein. Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own PEST-containing peptide (e.g., PEST1- peptidel ; PEST2-peptide2). Optionally, some or all of the fragments are non-contiguous fragments of the same cancer-associated protein. Non-contiguous fragments are fragments that do not occur sequentially in a protein sequence (e.g., the first fragment consists of residues 10-30, and the second fragment consists of residues 100-120; or the first fragment consists of residues 10-30, and the second fragment consists of residues 20-40). Optionally, each of the antigenic peptides comprises a different recurrent cancer mutation from a single type of cancer. For example, the single type of cancer can be non-small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer (e.g., ER+ breast cancer), uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer (e.g., MSS colorectal cancer), or head and neck cancer.

[00130] Also disclosed herein are recombinant fusion polypeptides comprising two or more antigenic peptides, wherein each antigenic peptide comprises a single, recurrent cancer mutation (i.e., a single, recurrent change in the amino acid sequence of a protein, or a sequence encoded by a single, different, nonsynonymous, recurrent cancer mutation in a gene), and wherein the fusion polypeptide does not comprise a PEST-containing peptide. Also disclosed herein are recombinant fusion polypeptides comprising two or more antigenic peptides, wherein each antigenic peptide comprises a single, recurrent cancer mutation (i.e., a single, recurrent change in the amino acid sequence of a protein, or a sequence encoded by a single, different, nonsynonymous, recurrent cancer mutation in a gene), wherein at least two of the antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein, and wherein the fusion polypeptide does not comprise a PEST-containing peptide. Alternatively, each of the antigenic peptides comprises a different recurrent cancer mutation from a different cancer-associated protein. Optionally, some or all of the fragments are non-contiguous fragments of the same cancer-associated protein.

Optionally, each of the antigenic peptides comprises a different recurrent cancer mutation from a single type of cancer. For example, the single type of cancer can be non-small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer (e.g., ER+ breast cancer), uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer (e.g., MSS colorectal cancer), or head and neck cancer.

[00131 ] Also provided herein are recombinant fusion polypeptides comprising from N- terminal end to C-terminal end a bacterial secretion sequence, a ubiquitin (Ub) protein, and two or more antigenic peptides (i.e., in tandem, such as Ub-peptidel-peptide2), wherein each antigenic peptide comprises a single, recurrent cancer mutation (i.e., a single, recurrent change in the amino acid sequence of a protein, or a sequence encoded by a single, different, nonsynonymous, recurrent cancer mutation in a gene), and wherein at least two of the antigenic peptides are fragments of the same cancer-associated protein. Alternatively, each of the antigenic peptides comprises a different recurrent cancer mutation from a different cancer-associated protein. Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own secretion sequence and Ub protein (e.g., Ubl-peptidel ; Ub2-peptide2). Optionally, some or all of the fragments are noncontiguous fragments of the same cancer-associated protein. Optionally, each of the antigenic peptides comprises a different recurrent cancer mutation from a single type of cancer. For example, the single type of cancer can be no n- small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer (e.g., ER+ breast cancer), uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer (e.g., MSS colorectal cancer), or head and neck cancer.

[00132] Nucleic acids (termed minigene constructs) encoding such recombinant fusion polypeptides are also disclosed. Such minigene nucleic acid constructs can further comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a minigene nucleic acid construct can further comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acid constructs, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis. [00133] The bacterial signal sequence can be a Listerial signal sequence, such as an Hly or an ActA signal sequence, or any other known signal sequence. In other cases, the signal sequence can be an LLO signal sequence. The signal sequence can be bacterial, can be native to a host bacterium (e.g., Listeria monocytogenes, such as a secAl signal peptide), or can be foreign to a host bacterium. Specific examples of signal peptides include an Usp45 signal peptide from Lactococcus lactis, a Protective Antigen signal peptide from Bacillus anthracis, a secA2 signal peptide such the p60 signal peptide from Listeria monocytogenes, and a Tat signal peptide such as a B. subtilis Tat signal peptide (e.g., PhoD). In specific examples, the secretion signal sequence is from a Listeria protein, such as an ActA₃oo secretion signal or an ActAioo secretion signal.

[00134] The ubiquitin can be, for example, a full-length protein. The ubiquitin expressed from the nucleic acid construct provided herein can be cleaved at the carboxy terminus from the rest of the recombinant fusion polypeptide expressed from the nucleic acid construct through the action of hydrolases upon entry to the host cell cytosol. This liberates the amino terminus of the fusion polypeptide, producing a peptide in the host cell cytosol.

[00135] Selection of, variations of, and arrangement of antigenic peptides within a fusion polypeptide are discussed in detail elsewhere herein, and cancer-associated proteins are discussed in more detail elsewhere herein.

[00136] The recombinant fusion polypeptides can comprise one or more tags. For example, the recombinant fusion polypeptides can comprise one or more peptide tags N- terminal and/or C-terminal to the combination of the two or more antigenic peptides. A tag can be fused directly to an antigenic peptide or linked to an antigenic peptide via a linker (examples of which are disclosed elsewhere herein). Examples of tags include the following: FLAG tag, 2xFLAG tag, 3xFLAG tag; His tag, 6xHis tag; and SIINFEKL tag. An exemplary SIINFEKL tag is set forth in SEQ ID NO: 293 (encoded by any one of the nucleic acids set forth in SEQ ID NOS: 278-292). Another exemplary SIINFEKL tag is set forth in SEQ ID NO: 922. An exemplary 3xFLAG tag is set forth in SEQ ID NO: 309 (encoded by any one of the nucleic acids set forth in SEQ ID NOS: 294-308). Another exemplary FLAG tag is set forth in SEQ ID NO: 762. Two or more flags can be used together, such as a 2xFLAG tag and a SIINFEKL tag, a 3xFLAG tag and a SIINFEKL tag, or a 6xHis tag and a SIINFEKL tag. If two or more tags are used, they can be located anywhere within the recombinant fusion polypeptide and in any order. For example, the two tags can be at the C-terminus of the recombinant fusion polypeptide, the two tags can be at the N-terminus of the recombinant fusion polypeptide, the two tags can be located internally within the recombinant fusion polypeptide, one tag can be at the C-terminus and one tag at the N-terminus of the

recombinant fusion polypeptide, one tag can be at the C-terminus and one internally within the recombinant fusion polypeptide, or one tag can be at the N-terminus and one internally within the recombinant fusion polypeptide. Other tags include chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX), and poly(NANP). Particular recombinant fusion polypeptides comprise a C-terminal SIINFEKL tag. Such tags can allow for easy detection of the recombinant fusion protein, confirmation of secretion of the recombinant fusion protein, or for following the immunogenicity of the secreted fusion polypeptide by following immune responses to these "tag" sequence peptides. Such immune response can be monitored using a number of reagents including, for example, monoclonal antibodies and DNA or RNA probes specific for these tags.

[00137] The recombinant fusion polypeptides disclosed herein can be expressed by recombinant Listeria strains or can be expressed and isolated from other vectors and cell systems used for protein expression and isolation. Recombinant Listeria strains comprising expressing such antigenic peptides can be used, for example in immunogenic compositions comprising such recombinant Listeria and in vaccines comprising the recombinant Listeria strain and an adjuvant. Expression of one or more antigenic peptides as a fusion polypeptides with a nonhemolytic truncated form of LLO, ActA, or a PEST-like sequence in host cell systems in Listeria strains and host cell systems other than Listeria can result in enhanced immunogenicity of the antigenic peptides.

[00138] The recombinant fusion polypeptide can be any molecular weight. For example, the recombinant fusion polypeptide can be less than or no more than about 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, or 125 kilodaltons (kDa). In a specific example, the recombinant fusion polypeptide is less than or no more than about 150 kDa or less than or no more about 130 kDa. As another example the recombinant fusion polypeptide can be between about 50-200, 50-195, 50-190, 50-185, 50-180, 50-175, 50-170, 50-165, 50-160, 50-155, 50-150, 50-145, 50-140, 50-135, 50-130, 50-125, 100-200, 100-195, 100-190, 100-185, 100-180, 100-175, 100-170, 100-165, 100-160, 100-155, 100-150, 100- 145, 100-140, 100-135, 100-130, or 100-125 kDa. In a specific example, the recombinant fusion polypeptide is between about 50-150, 100-150, 50-125, or 100-125 kDa. As another example, the recombinant fusion polypeptide can be at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 kDa. As a specific example, the recombinant fusion polypeptide can be at least about 100 kDa. [00139] Nucleic acids encoding such recombinant fusion polypeptides are also disclosed. The nucleic acid can be in any form. The nucleic acid can comprise or consist of DNA or RNA, and can be single- stranded or double-stranded. The nucleic acid can be in the form of a plasmid, such as an episomal plasmid, a multicopy episomal plasmid, or an integrative plasmid. Alternatively, the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome. Such nucleic acids can have one open reading frame or can have two or more open reading frames (e.g., an open reading frame encoding the recombinant fusion polypeptide and a second open reading frame encoding a metabolic enzyme). In one example, such nucleic acids can comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a nucleic acid can comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acids, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.

A. Antigenic Peptides Comprising Recurrent Cancer Mutations

[00140] Each antigenic peptide can be a fragment of a cancer-associated protein (i.e., a contiguous sequence of amino acids from a cancer-associated protein). Each antigenic peptide can be of any length sufficient to induce an immune response, and each antigenic peptide can be the same length or the antigenic peptides can have different lengths. For example, an antigenic peptide disclosed herein can be 5-200, 5-100, 7-200, 7-100, 15-50, or 21-27 amino acids in length, or 15-100, 15-95, 15-90, 15-85, 15-80, 15-75, 15-70, 15-65, 15- 60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, 20-100, 20-95, 20-90, 20-85, 20-80, 20-75, 20- 70, 20-65, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 11-21, 15-21, 21-31, 31-41, 41- 51, 51-61, 61-71, 71-81, 81-91, 91-101, 101-121, 121-141, 141-161, 161-181, 181-201, 8-27, 10-30, 10-40, 15-30, 15-40, 15-25, 1-10, 10-20, 20-30, 30-40, 1-100, 5-75, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 1-75, 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 8-11, or 11-16 amino acids in length. For example, an antigenic peptide can be 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, or 60 amino acids in length. Some specific examples of antigenic peptides are 21 or 27 amino acids in length. [00141 ] Each antigenic peptide can also be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria

monocytogenes or another bacteria of interest. For example, antigenic peptides can be scored by a Kyte and Doolittle hydropathy index 21 amino acid window, and all scoring above a cutoff (around 1.6) can be excluded as they are unlikely to be secretable by Listeria monocytogenes.

[00142] Each antigenic peptide can comprise a single recurrent cancer mutation or can comprise two or more recurrent cancer mutations (e.g., two recurrent cancer mutations). For example, an antigenic peptide can comprise more than one recurrent cancer mutation (e.g., 2 or 3 recurrent cancer mutations) because of the close proximity of the mutated residues to each other in the cancer-associated protein. The recurrent cancer mutations can be any type of mutation (e.g., somatic missense mutation or frameshift mutation). The recurrent cancer mutation in each antigenic peptide can be flanked on each side by an equal number of amino acids, or can be flanked on each side by a different number of amino acids (e.g., with 9 amino acids flanking N-terminal and 10 amino acids flanking C-terminal, or with 10 amino acids flanking N-terminal and 13 amino acids flanking C-terminal). The flanking sequence on each side of the recurrent cancer mutation can be the sequence that naturally flanks the mutation in the cancer-associated protein. For example, the recurrent cancer mutation in an antigenic peptide can be flanked on each side by an equal number of amino acids, wherein the flanking sequence is identical to the sequences that naturally flanks the recurrent cancer mutation in the cancer-associated protein. The number of flanking amino acids on each side of the recurrent cancer mutation can be any length, such as 5-30 amino acids flanking each side. As one example, the recurrent cancer mutation can be flanked on each side by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acids (e.g., by at least 10 amino acids or by at least 13 amino acids). Preferably, at least about 10 flanking amino acids on each side of the detected recurrent cancer mutation are incorporated to accommodate class 1 MHC-1 presentation, in order to provide at least some of the different HLA T-cell receptor (TCR) reading frames, or at least about 13 flanking amino acids on each side of the detected recurrent cancer mutation are incorporated to accommodate class 2 MHC-2 presentation, in order to provide at least some of the different HLA T-cell receptor (TCR) reading frames for CD4+ T cell antigen presentation. However, this does not necessarily need to be the case, and in some cases may not be possible (e.g., if a recurrent cancer mutation occurs in the first 10 amino acids of a protein or the last 10 amino acids of a protein). In some cases, the location of the recurrent cancer mutation in the cancer- associated protein may dictate how many amino acids are flanking on one particular side (e.g., if the mutation is in the first 10 amino acids of the protein or the last 10 amino acids of the protein). In the case of a frameshift mutation, any number of predicted amino acids downstream of the frameshift mutation can be included. For example, all of the predicted amino acids downstream of the frameshift mutation can be included.

[00143] The antigenic peptides can be linked together in any manner. For example, the antigenic peptides can be fused directly to each other with no intervening sequence.

Alternatively, the antigenic peptides can be linked to each other indirectly via one or more linkers, such as peptide linkers. In some cases, some pairs of adjacent antigenic peptides can be fused directly to each other, and other pairs of antigenic peptides can be linked to each other indirectly via one or more linkers. The same linker can be used between each pair of adjacent antigenic peptides, or any number of different linkers can be used between different pairs of adjacent antigenic peptides. In addition, one linker can be used between a pair of adjacent antigenic peptides, or multiple linkers can be used between a pair of adjacent antigenic peptides.

[00144] Any suitable sequence can be used for a peptide linker. As an example, a linker sequence may be, for example, from 1 to about 50 amino acids in length. Some linkers may be hydrophilic. The linkers can serve varying purposes. For example, the linkers can serve to increase bacterial secretion, to facilitate antigen processing, to increase flexibility of the fusion polypeptide, to increase rigidity of the fusion polypeptide, or any other purpose. As a specific example, one or more or all of a flexibility linker, a rigidity linker, and an immunoproteasome processing linker can be used. Examples of such linkers are provided below. In some cases, different amino acid linker sequences are distributed between the antigenic peptides or different nucleic acids encoding the same amino acid linker sequence are distributed between the antigenic peptides (e.g., SEQ ID NOS: 572-582) in order to minimize repeats. This can also serve to reduce secondary structures, thereby allowing efficient transcription, translation, secretion, maintenance, or stabilization of the nucleic acid (e.g., plasmid) encoding the fusion polypeptide within a Lm recombinant vector strain population. Other suitable peptide linker sequences may be chosen, for example, based on one or more of the following factors: (1) their ability to adopt a flexible extended

conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the antigenic peptides; and (3) the lack of hydrophobic or charged residues that might react with the functional epitopes. For example, peptide linker sequences may contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al. (1985) Gene 40:39-46; Murphy et al. (1986) Proc Natl Acad Sci USA 83:8258-8262; US Pat. No. 4,935,233; and US

4,751,180, each of which is herein incorporated by reference in its entirety for all purposes. Specific examples of linkers include those in the following table (each of which can be used by itself as a linker, in a linker comprising repeats of the sequence, or in a linker further comprising one or more of the other sequences in the table), although others can also be envisioned {see, e.g., Reddy Chichili et al. (2013) Protein Science 22: 153-167, herein incorporated by reference in its entirety for all purposes). Unless specified, "n" represents an undetermined number of repeats in the listed linker.

[00145] The VGKGGSGG linker (SEQ ID NO: 314) can be used, for example, as a longer linker after the tLLO and also before the tag sequences to provide additional space between the tLLO and the antigenic portion of the fusion peptide and before the tag sequences. It also can provide flexibility and to charge balance the fusion protein. The EAAAK linker (SEQ ID NO: 316) is a rigid/stiff linker that can be used to facilitate expression and secretion, for example, if the fusion protein would otherwise fold on itself. The GGGGS linker (SEQ ID NO: 313) is a flexible linker that can be used, for example, to add increased flexibility to the fusion protein to help facilitate expression and secretion. The "i20" linkers (e.g., SEQ ID NOS: 821-829) are immunoproteasome linkers that are designed, for example, to help facilitate cleavage of the fusion protein by the immunoproteasome and increase the frequency of obtaining the exact minimal binding fragment that is desired. Combinations of GGGGS and EAAAK linkers (SEQ ID NOS: 313 and 316, respectively) can be used, for example, to alternate flexibility and rigidity to help balance the construct for improved expression and secretion and to help facilitate DNA synthesis by providing more unique codons to choose from.

[00146] The fusion polypeptide can comprise any number of antigenic peptides. In some cases, the fusion polypeptide comprises any number of antigenic peptides such that the fusion polypeptide is able to be produced and secreted from a recombinant Listeria strain. For example, the fusion polypeptide can comprise at least 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 antigenic peptides, or 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 antigenic polypeptides. In another example, the fusion polypeptide can include a single antigenic peptide. In another example, the fusion polypeptide can include a number of antigenic peptides ranging from about 1-100, 1-5, 5-10, 10-15, 15-20, 10-20, 20- 30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 5-15, 5-20, 5-25, 15-20, 15-25, 15-30, 15-35, 20-25, 20-35, 20-45, 30-45, 30-55 ,40-55, 40-65, 50-65, 50-75, 60-75, 60-85, 70-85, 70-95, 80-95, 80-105, 95-105, 50-100, 1-100, 5-100, 5-75, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 1-100, 1-75, 1-50, 1-40, 1-30, 1-20, 1-15, or 1-10 antigenic peptides. In another example, the fusion polypeptide can include up to about 100, 10, 20, 30, 40, or 50 antigenic peptides. In another example, the fusion polypeptide can comprise about 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, or 100 antigenic peptides. In another example, the fusion polypeptide can comprise at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 antigenic peptides or between about 5-50, 10-40, or 20-30 antigenic peptides.

[00147] In addition, the fusion polypeptide can comprise any number of antigenic peptides from the same cancer-associated protein (i.e., any number of non-contiguous fragments from the same cancer-associated protein). Alternatively, the fusion polypeptide can comprise any number of antigenic peptides from two or more different cancer-associated proteins, such as from 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer-associated proteins. For example, the two or more cancer-associated proteins can be about 2-30, about 2-25, about 2-20, about 2-15, or about 2- 10 cancer-associated proteins. For example, the fusion polypeptide can comprise at least 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 antigenic peptides from the same cancer-associated protein, or 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 antigenic polypeptides from the same cancer-associated protein. Likewise, the fusion polypeptide can comprise at least 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 antigenic peptides from the same cancer-associated protein, or 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-10, 10-15, 15-20, 20- 25, 25-30, 30-35, 35-40, 40-45, or 45-50 antigenic polypeptides from two or more different cancer-associated proteins. In addition, the fusion polypeptide can comprise any number of non-contiguous antigenic peptides from the same cancer-associated protein (i.e., any number of non-contiguous fragments from the same cancer-associated protein). For example, the fusion polypeptide can comprise at least 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 non-contiguous antigenic peptides from the same cancer-associated protein, or 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 non-contiguous antigenic polypeptides from the same cancer-associated protein. In some cases, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the antigenic peptides are non-contiguous antigenic peptides from the same cancer-associated protein, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the antigenic peptides that are from a single cancer-associated protein are non-contiguous antigenic peptides from that cancer-associated protein.

[00148] Each antigenic peptide can comprise a different (i.e., unique) recurrent cancer mutation. Alternatively, two or more of the antigenic peptides in the fusion polypeptide can comprise the same recurrent cancer mutation. For example, two or more copies of the same antigenic peptide can be included in the fusion polypeptide (i.e., the fusion polypeptide comprises two or more copies of the same antigenic peptide). In some fusion polypeptides, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the antigenic peptides comprise a different (i.e., unique) recurrent cancer mutation that is not present in any of the other antigenic peptides.

[00149] In some cases, at least two of the antigenic peptides can comprise overlapping fragments of the same cancer-associated protein. Likewise, the recurrent cancer mutations in at least two of the antigenic peptides can be recurrent cancer mutations that do not occur naturally together in the same subject. For example, two or more of the antigenic peptides can comprise different recurrent cancer mutations at the same amino acid residue of the cancer-associated protein (e.g., R248L, R248Q, and R248W in the protein encoded by TP53).

[00150] Some antigenic peptides can comprise at least two different recurrent cancer mutations, at least three different recurrent cancer mutations, or at least four different recurrent cancer mutations.

[00151 ] Any combination of recurrent cancer mutations can be included in the fusion polypeptide. Each of the recurrent cancer mutations can be a somatic missense mutation, or the recurrent cancer mutations can comprise other mutations as well. For example, in some fusion polypeptides, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the recurrent cancer mutations are somatic missense mutations. As one example, the antigenic peptides can comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the 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, or 40 most common recurrent cancer mutations in the cancer-associated protein. For example, the antigenic peptides can comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the 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, or 40 most common recurrent somatic missense cancer mutations in the cancer-associated protein. As another example, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a mutation in the cancer-associated protein have a recurrent cancer mutation in the cancer-associated protein that is included in the combination of antigenic peptides in the fusion polypeptide. For example, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a somatic missense mutation in the cancer-associated protein have a recurrent cancer mutation in the cancer-associated protein that is included in the combination of antigenic peptides in the fusion polypeptide. As another example, the antigenic peptides can comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the 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, or 40 most common recurrent cancer mutations or most common recurrent somatic missense cancer mutations in a particular type of cancer. As another example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a particular type of cancer have a recurrent cancer mutation that is included in the combination of antigenic peptides in the fusion polypeptide (or in a combination of two or more fusion polypeptides). For example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with particular type of cancer have a recurrent cancer mutation that is included in the combination of antigenic peptides in the fusion polypeptide (or in a combination of two or more fusion polypeptides). In a particular example, the antigenic peptides comprise at least 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, or 60 different recurrent cancer mutations or different recurrent somatic missense mutations from the same type of cancer, or the antigenic peptides comprise 2-80, 10-60, 10- 50, 10-40, or 10-30 different recurrent cancer mutations or different recurrent somatic missense mutations from a single type of cancer. For example, the single type of cancer can be no n- small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer (e.g., ER+ breast cancer), uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer (e.g., MSS colorectal cancer), or head and neck cancer.

[00152] Each of the antigenic peptides in the fusion polypeptide can comprise a recurrent cancer mutation from the same cancer-associated protein, or the combination of antigenic peptides in the fusion polypeptide can comprise recurrent cancer mutations from two or more cancer-associated proteins. For example, the fusion polypeptide can comprise recurrent cancer mutations from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cancer-associated proteins, or 2-5, 5-10, 10-15, or 15-20 cancer-associated proteins. For example, the two or more cancer-associated proteins can be about 2-30, about 2-25, about 2- 20, about 2-15, or about 2-10 cancer-associated proteins. In one example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the antigenic peptides comprise a recurrent cancer mutation from the same cancer-associated protein. In another example, none of the antigenic peptides comprise a recurrent cancer mutation from the same cancer-associated protein.

[00153] Exemplary sequences of antigenic peptides are disclosed elsewhere herein. As an example, an antigenic peptide can comprise, consist essentially of, or consist of a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the antigenic peptide sequences disclosed herein. B. Cancer- Associated Proteins and Recurrent Cancer Mutations

[00154] The fusion polypeptides disclosed herein comprise antigenic peptides comprising recurrent cancer mutations from cancer-associated proteins. Any combination of recurrent cancer mutations disclosed herein can be included in a fusion polypeptide. The term "cancer- associated protein" includes proteins having mutations that occur in multiple types of cancer, that occur in multiple subjects having a particular type of cancer, or that are correlated with the occurrence or progression of one or more types of cancer. For example, a cancer- associated protein can be an oncogenic protein (i.e., a protein with activity that can contribute to cancer progression, such as proteins that regulate cell growth), or it can be a tumor- suppressor protein (i.e., a protein that typically acts to alleviate the potential for cancer formation, such as through negative regulation of the cell cycle or by promoting apoptosis). Preferably, a cancer-associated protein has a "mutational hotspot." A mutational hotspot is an amino acid position in a protein-coding gene that is mutated (preferably by somatic substitutions rather than other somatic abnormalities, such as translocations, amplifications, and deletions) more frequently than would be expected in the absence of selection. Such hotspot mutations can occur across multiple types of cancer and/or can be shared among multiple cancer patients. Mutational hotspots indicate selective pressure across a population of tumor samples. Tumor genomes contain recurrent cancer mutations that "drive" tumorigenesis by affecting genes (i.e., tumor driver genes) that confer selective growth advantages to the tumor cells upon alteration. Such tumor driver genes can be identified, for example, by identifying genes that are mutated more frequently than expected from the background mutation rate (i.e., recurrence); by identifying genes that exhibit other signals of positive selection across tumor samples (e.g., a high rate of non-silent mutations compared to silent mutations, or a bias towards the accumulation of functional mutations); by exploiting the tendency to sustain mutations in certain regions of the protein sequence based on the knowledge that whereas inactivating mutations are distributed along the sequence of the protein, gain-of- function mutations tend to occur specifically in particular residues or domains; or by exploiting the overrepresentation of mutations in specific functional residues, such as phosphorylation sites. Many of these mutations frequently occur in the functional regions of biologically active proteins (for example, kinase domains or binding domains) or interrupt active sites (for example, phosphorylation sites) resulting in loss-of- function or gain-of- function mutations, or they can occur in such a way that the three-dimensional structure and/or charge balance of the protein is perturbed sufficiently to interfere with normal function. Genomic analysis of large numbers of tumors reveals that mutations often occur at a limited number of amino acid positions. Therefore, a majority of the common mutations can be represented by a relatively small number of potential tumor-associated antigens or T cell epitopes.

[00155] For example, the cancer-associated protein can be any one of the following:

[00156] Other tumor-driver genes and cancer-associated proteins having common mutations that occur across multiple cancers or among multiple cancer patients are also known, and sequencing data across multiple tumor samples and multiple tumor types exists. See, e.g., Chang et al. (2016) Nat Biotechnol 34(2): 155-163; Tamborero et al. (2013) Sci Rep 3:2650, each of which is herein incorporated by reference in its entirety.

[00157] As a set of specific examples, the cancer-associated protein can be encoded by one of the following genes: BRAF, EGFR, PIK3CA, PIK3R1, PTEN, RAS (e.g., KRAS), TP53, APC, FBXW7, KEAP1, STK11, NF1, KMT2D, CDKN2A, NFE2L2, SPOP, GAT A3, AKT1, MAP3K1, and MAP2K4. As a set of specific examples, the cancer-associated protein can be encoded by one of the following genes: BRAF, EGFR, PIK3CA, PIK3R1, PTEN, RAS (e.g., KRAS), TP53, APC, FBXW7, KEAP1, STK11, NF1, KMT2D, CDKN2A, NFE2L2, SPOP, GATA3, AKT1, MAP3K1, MAP2K4, AHNAK2, ANKRD36C, CHEK2, KRTAP4-11, RGPD8, FAM47C, and TAN. As another set of specific examples, the cancer-associated protein can be encoded by one of the following genes: ACVR2A, ADAM28, AKT1, ANKRD36C, AR, ARID1A, BMPR2, BRAF, CHEK2, C12orf4, CTNNB1, DOCK3, EGFR, ESR1, FBXW7, FGFR3, FHOD3, GNAS, HRAS, IDH1, IDH2, KIAA2026, KRAS, KRTAP1-5, KRTAP4-11, LARP4B, MBOAT2, NFE2L2, PGM5, PIK3CA, PLEKHA6, POLE, PTEN, RGPD8, RNF43, RXRA, SMAD4, SPOP, SVIL, TGFBR2, TP53, TRIM48, UBR5, U2AF1, WNT16, XYLT2, ZBTB20, and ZNF814.

[00158] The fusion polypeptides disclosed herein can comprise antigenic peptides comprising any combination of recurrent cancer mutation from any combination of cancer- associated proteins (i.e., one or more cancer-associated proteins) and in any order. The combination of antigenic peptides or the fusion polypeptide can be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria monocytogenes or another bacteria of interest.

[00159] As one example, the cancer-associated protein can be encoded by BRAF, and the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following recurrent cancer mutations: G466E; G466V; G469A; G469R; G469S; G469V; V600E; and V600K. The wild type BRAF reference sequence is set forth in SEQ ID NO: 361. The mutations can be in any order. For example, the fusion polypeptide can comprise antigenic peptides comprising the following BRAF mutations, from N-terminal to C-terminal: G469V; G469R; V600E; G469S; G466V; V600K; G469A; and G466E. See, e.g., SEQ ID NOS: 1-6. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following BRAF mutations, from N-terminal to C-terminal: V600K; G469R; G469V; G466V; G466E; V600E; G469A; and G469S. See, e.g., SEQ ID NOS: 7- 12. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following BRAF mutations, from N-terminal to C-terminal: G469V; V600K; G469S; G466V; G469A; V600E; G466E; and G469R. See, e.g., SEQ ID NOS: 13-18. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following BRAF mutations, from N-terminal to C-terminal: V600E; V600K; G469A; G469S; G469R; G469V; G466V; and G466E. See, e.g., SEQ ID NOS: 19-24. In a specific example, the BRAF antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. [00160] As another example, the cancer-associated protein can be encoded by EGFR, and the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the following recurrent cancer mutations: R108K; A289V; G598V; E709A; E709K; G719A; G719C; G719S; L747P; L747S; S768I; T790M; L833V/H835L; T833V; L858R; and L861Q. The wild type EGFR reference sequence is set forth in SEQ ID NO: 362. For example, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the following recurrent cancer mutations: R108K; A289V; G598V; E709A; E709K; G719A; G719C; G719S; L747P; L747S; S768I; T790M; L833V/H835L; T833V; L858R; and L861Q. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the following recurrent cancer mutations: A289V; G598V; E709K; G719A; G719C; G719S; S768I; T790M; L833V/H835L; L858R; and L861Q. The mutations can be in any order. For example, the fusion polypeptide can comprise antigenic peptides comprising the following EGFR mutations, from N-terminal to C-terminal: G719S; L747P; G719C; R108K; S768I; L833V/H835L; T833V; E709A; G598V; T790M; E709K; A289V; L861Q; G719A; L747S; and L858R. See, e.g., SEQ ID NOS: 25-30. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following EGFR mutations, from N-terminal to C-terminal: T790M; S768I; G719C; R108K; L747P; G719A; L747S; E709K; T833V; L861Q; E709A; L858R; G598V; A289V; L833V/H835L; and G719S. See, e.g., SEQ ID NOS: 31-36. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following EGFR mutations, from N-terminal to C-terminal: R108K; T833V; L747S; T790M; G719C; A289V; L858R; E709A; G719S; E709K; G719A; L747P; G598V; L861Q; S768I; and L833V/H835L. See, e.g., SEQ ID NOS: 37-42.

Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following EGFR mutations, from N-terminal to C-terminal: G719A; L858R; G719C; A289V; T790M; S768I; T833V; G598V; G719S; L747S; L747P; L833V/H835L; E709A; R108K; L861Q; and E709K. See, e.g., SEQ ID NOS: 43-48. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following EGFR mutations, from N-terminal to C-terminal: A289V; G598V; E709K; G719A; S768I; G719S; L861Q; T790M; G719C; L833V/H835L; and L858R. See, e.g., SEQ ID NOS: 229-235. In a specific example, the EGFR antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation.

[00161 ] As another example, the cancer-associated protein can be encoded by PIK3CA, and the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or all of the following recurrent cancer mutations: R38C; R38H; E81K; R88Q; R93Q; R93W; R108H; G118D; L334G; N345K; C420R; E453K;

E542K; E545A; E545G; E545K; E545Q; Q546K; Q546R; E726K; M1043I; M1043V;

H1047L; H1047R; and G1049R. The wild type PIK3CA reference sequence is set forth in SEQ ID NO: 363. For example, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or all of the following recurrent cancer mutations: R38H; E81K; R88Q; R108H; G118D; N345K; C420R; E542K; E545A; E545G; E545K; Q546K; Q546R; M1043I; H1047L; H1047R; and G1049R. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following recurrent cancer mutations: R88Q; E542K; E545A; E545G; E545K; Q546K; H1047L; and H1047. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all of the following recurrent cancer mutations: R38H; E81K; R108H; G118D; N345K; C420R; Q546R; M1043I; and G1049R. The mutations can be in any order. For example, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA mutations, from N-terminal to C-terminal: M1043V; E545G; E726K; Q546R; L334G; G1049R; M1043I; Q546K; E542K; R93Q; H1047R; R108H; R93W; E81K; R38H; N345K; R88Q; G118D; E545Q; H1047L; E545A; E453K; E545K; R38C; and C420R. See, e.g., SEQ ID NOS: 49-54. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA mutations, from N-terminal to C-terminal: E726K; E81K; M1043V; E545A; E545K; R38C; G118D; R93W; E545G; E542K; G1049R; N345K; Q546K; E453K; C420R; H1047L;

L334G; E545Q; R88Q; H1047R; M1043I; R93Q; R108H; Q546R; and R38H. See, e.g., SEQ ID NOS: 55-60. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA mutations, from N-terminal to C-terminal: R108H;

M1043V; R88Q; R93W; R38H; H1047R; E545K; M1043I; Q546R; E542K; N345K; R38C; E545G; E81K; Q546K; R93Q; E453K; G1049R; E545A; C420R; H1047L; L334G; G118D; E726K; and E545Q. See, e.g., SEQ ID NOS: 61-66. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA mutations, from N- terminal to C-terminal: N345K; R38H; E545K; G1049R; H1047L; E726K; R88Q; E81K; R93Q; E545Q; L334G; R38C; H1047R; C420R; R93W; Q546K; M1043V; M1043I; E545G; E545A; G118D; E453K; Q546R; R108H; and E542K. See, e.g., SEQ ID NOS: 67-72.

Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA mutations, from N-terminal to C-terminal: E542K; E545K; R88Q;

E545A; H1047R; E545G; H1047L; Q546K; R38H; E81K; R108H; N345K; C420R; Q546R; M1043I; G118D; and G1049R. See, e.g., SEQ ID NOS: 236-242. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA mutations, from N-terminal to C-terminal: E542K; E545K; R88Q; E545A; H1047R; E545G; H1047L; and Q546K. See, e.g., SEQ ID NOS: 243-249. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA mutations, from N-terminal to C-terminal: R38H; E81K; R108H; N345K; C420R; Q546R; M1043I; G118D; and G1049R. See, e.g., SEQ ID NOS: 250-256. In a specific example, the PIK3CA antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation.

[00162] As another example, the cancer-associated protein can be encoded by PIK3R1, and the antigenic peptides comprise 2 or more or all of the following recurrent cancer mutations: G376R; N564D; and K567E. The wild type PIK3R1 reference sequence is set forth in SEQ ID NO: 364. The mutations can be in any order. For example, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3R1 mutations, from N-terminal to C-terminal: G376R; N564D; and K567E. See, e.g., SEQ ID NOS: 73-78. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3R1 mutations, from N-terminal to C-terminal: N564D; K567E; and G376R. See, e.g., SEQ ID NOS: 79-84. In a specific example, the PIK3R1 antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation.

[00163] As another example, the cancer-associated protein can be encoded by PIK3CA and PIK3R1, and the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, or all of the following recurrent cancer mutations: PIK3CAIR38C; PIK3CAIR38H; PIK3CAIE81K;

PIK3CAIR88Q; PIK3CAIR93Q; PIK3CAIR93W; PIK3CAIR108H; PIK3CAIG118D; PIK3CAIL334G; PIK3CAIN345K; PIK3CAIC420R; PIK3CAIE453K; PIK3CAIE542K; PIK3CAIE545A; PIK3CAIE545G; PIK3CAIE545K; PIK3CAIE545Q; PIK3CAIQ546K; PIK3CAIQ546R; PIK3CAIE726K; PIK3CAIM1043I; PIK3CAIM1043V; PIK3CAIH1047L; PIK3CAIH1047R; PIK3CAIG1049R; PIK3R1IG376R; PIK3R1IN564D; and PIK3R1IK567E. The mutations can be in any order. For example, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA and PIK3R1 mutations, from N- terminal to C-terminal: PIK3CAIR38C; PIK3CAIN345K; PIK3CAIE726K; PIK3CAIE453K; PIK3CAIR93Q; PIK3CAIH1047R; PIK3CAIE545A; PIK3CAIM1043V; PIK3R1IN564D; PIK3R1IK567E; PIK3CAIE81K; PIK3CAIR108H; PIK3CAIQ546R; PIK3CAIQ546K;

PIK3CAIE545Q; PIK3CAIG1049R; PIK3CAIC420R; PIK3CAIH1047L; PIK3CAIR93W; PIK3CAIR88Q; PIK3CAIM1043I; PIK3CAIE545G; PIK3CAIG118D; PIK3CAIR38H;

PIK3R1IG376R; PIK3CAIE542K; PIK3CAIE545K; and PIK3CAIL334G. See, e.g., SEQ ID NOS: 85-90. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA and PIK3R1 mutations, from N-terminal to C-terminal: PIK3CAIR38C; PIK3CAIR108H; PIK3CAIC420R; PIK3CAIR93Q; PIK3CAIE453K;

PIK3CAIM1043V; PIK3CAIH1047L; PIK3R1IN564D; PIK3CAIE726K; PIK3CAIG118D; PIK3CAIQ546K; PIK3CAIQ546R; PIK3CAIE542K; PIK3CAIE545K; PIK3CAIG1049R; PIK3CAIM1043I; PIK3CAIL334G; PIK3R1IK567E; PIK3CAIR38H; PIK3R1IG376R;

PIK3CAIR93W; PIK3CAIH1047R; PIK3CAIE545G; PIK3CAIE81K; PIK3CAIR88Q;

PIK3CAIN345K; PIK3CAIE545A; and PIK3CAIE545Q. See, e.g., SEQ ID NOS: 91-96. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA and PIK3R1 mutations, from N-terminal to C-terminal: PIK3CAIR108H; PIK3CAIM1043V; PIK3CAIR88Q; PIK3CAIR93W; PIK3CAIR38H; PIK3CAIH1047R; PIK3CAIE545K; PIK3CAIM1043I; PIK3CAIQ546R; PIK3CAIE542K; PIK3CAIN345K; PIK3CAIR38C; PIK3CAIE545G; PIK3CAIE81K; PIK3CAIQ546K; PIK3CAIR93Q;

PIK3CAIE453K; PIK3CAIG1049R; PIK3CAIE545A; PIK3CAIC420R; PIK3CAIH1047L; PIK3CAIL334G; PIK3CAIG118D; PIK3CAIE726K; and PIK3CAIE545Q. See, e.g., SEQ ID NOS: 97-102. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA and PIK3R1 mutations, from N-terminal to C-terminal: PIK3CAIE545Q; PIK3CAIR93W; PIK3CAIH1047R; PIK3CAIG1049R; PIK3CAIN345K; PIK3CAIQ546R; PIK3CAIE545K; PIK3CAIE453K; PIK3CAIL334G; PIK3CAIH1047L; PIK3R1IG376R; PIK3CAIM1043V; PIK3CAIR88Q; PIK3CAIR38H; PIK3CAIG118D;

PIK3R1IK567E; PIK3CAIR38C; PIK3CAIE542K; PIK3CAIQ546K; PIK3CAIE726K;

PIK3CAIC420R; PIK3CAIE545A; PIK3CAIR93Q; PIK3R1IN564D; PIK3CAIR108H; PIK3CAIM1043I; PIK3CAIE545G; and PIK3CAIE81K. See, e.g., SEQ ID NOS: 103-108. In a specific example, the PIK3CA and PIK3R1 antigenic peptides can be 21-mers (e.g., 21- mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation.

[00164] As another example, the cancer-associated protein can be encoded by PTEN, and the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or all of the following recurrent cancer mutations: Y68H; Y88C; D92E; dell21-131 ; R130G; R130L; R130P;

R130Q; C136Y; R142W; Y155C; R173H; and P246L. The wild type PTEN reference sequence is set forth in SEQ ID NO: 365. The mutations can be in any order. For example, the fusion polypeptide can comprise antigenic peptides comprising the following PTEN mutations, from N-terminal to C-terminal: dell21- 131; Y88C; R130G; Y155C; D92E;

C136Y; R130Q; Y68H; R142W; R173H; R130L; R130P; and P246L. See, e.g., SEQ ID NOS: 109-114. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PTEN mutations, from N-terminal to C-terminal: R130P; R130G; Y155C; R130L; C136Y; dell21-131; P246L; D92E; R173H; Y68H; R130Q; Y88C; and R142W. See, e.g., SEQ ID NOS: 115-120. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PTEN mutations, from N-terminal to C-terminal: R130Q; R130G; dell21-131; C136Y; R130L; P246L; Y155C; D92E; R142W; R130P; Y88C; Y68H; and R173H. See, e.g., SEQ ID NOS: 121-126. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following PTEN mutations, from N-terminal to C-terminal: dell21- 131; C136Y; Y68H; R142W; R173H; IR130L; P246L; R130G; R130P; Y88C; D92E; R130Q; and Y155C. See, e.g., SEQ ID NOS: 127-132. In a specific example, the PTEN antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation.

[00165] As another example, the cancer-associated protein can be encoded by KRAS, and the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or all of the following recurrent cancer mutations: G12A; G12C; G12D; G12R; G12S; G12V; G13C; G13D; G13R; G13S; G13V; L19F; Q61K; Q61H; Q61L; Q61R; K117N; A146T; A146V; and A164G. The wild type KRAS reference sequence is set forth in SEQ ID NO: 366. The mutations can be in any order. For example, the fusion polypeptide can comprise antigenic peptides comprising the following KRAS mutations, from N-terminal to C-terminal: Q61R; Q61K; Q61L; Q61H; L19F; K117N; G12A; A164G; G12D; G13D; G13S; G12S; A146V; G13R; G13C; G12C; G12R; G13V; G12V; and A146T. See, e.g., SEQ ID NOS: 133-138. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following KRAS mutations, from N-terminal to C-terminal: Q61H; K117N; G13C; G13R; G12D; G12S;

G12V; G12A; Q61K; G13V; G12C; L19F; Q61R; Q61L; A146V; A164G; G12R; G13S; A146T; and G13D. See, e.g., SEQ ID NOS: 139-144. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following KRAS mutations, from N-terminal to C-terminal: G12D; L19F; A146V; Q61H; G12V; A164G; G12C; Q61L; A146T; G13S; G12A; G13V; G13C; G13D; G12R; G12S; Q61R; Q61K; G13R; and K117N. See, e.g., SEQ ID NOS: 145-150. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following KRAS mutations, from N-terminal to C-terminal: G13V; G13S; G12V; G12R; A146V; G13D; G12D; K117N; Q61H; G12C; G13C; A146T; G12A; Q61L; Q61K; A164G; G12S; L19F; G13R; and Q61R. See, e.g., SEQ ID NOS: 151-156. In a specific example, the KRAS antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation.

[00166] As another example, the cancer-associated protein can be encoded by TP53, and the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, or all of the following recurrent cancer mutations: Y107D; K132N; C141Y; V143A; V157F; Y163C; R175H; C176F; C176Y; H179R; H179W; H193R; I195T; V216M; Y220C; Y234C; Y234H; S241F; S242F; G245D; G245S; R248L; R248Q; R248W; R249S; R273C; R273H; R273L; P278L; P278S; R282G; R282W; and R337H. The wild type TP53 reference sequence is set forth in SEQ ID NO: 367. For example, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, or all of the following recurrent cancer mutations: Y107D; C141Y; V143A; V157F; Y163C; R175H; C176F; H193R; I195T; V216M; Y220C; Y234C; Y234H; G245D; G245S; R248Q; R248W; R249S; R273C; R273H; R273L; R282G; and R282W. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the following recurrent cancer mutations: V143A; R175H; H193R; Y220C; G245D; R248Q; R248W; R249S; R273C; R273H; and R282W. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all of the following recurrent cancer mutations: Y107D; C141Y; V157F; Y163C; C176F; I195T; V216M;

Y234C; Y234H; G245S; R273L; and R282G. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or all of the following recurrent cancer mutations: Y107D; C141Y; V143A; Y163C; C176Y; H179R; H179W; H193R; V216M; Y234H; S241F; G245D; R248Q; R248W; R273C; R273L; and P278S. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or all of the following recurrent cancer mutations: C141Y; R175H; H179R; H193R; V216M; Y234H; G245D; G245S; R248L; R248W; R273C; R273H; P278L; P278S; R282G; R282W;and R337H. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or all of the following recurrent cancer mutations: Y107D; C141Y; V143A; C176F; H179R; V216M; Y220C; S241F; S242F; G245S; R248L; R248W; R273L; P278L; P278S; R282G; and R282W. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or all of the following recurrent cancer mutations: Y107D; K132N; V143A; V157F; Y163C; R175H; C176Y; Y234C; Y234H;

S241F; S242F; G245D; G245S; R273C; P278S; R282W; and R337H. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the following recurrent cancer mutations: K132N; V157F; R175H; C176F; I195T; Y220C; Y234C; S242F; G245S; R248L; R249S; R273H; P278L; R282G; R282W; and R337H. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the following recurrent cancer mutations: Y107D; K132N; V143A; V157F; Y163C; C176F; C176Y; H179W; I195T; Y220C; Y234C; S241F; S242F; R248Q; R249S; and R273L. Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the following recurrent cancer mutations: K132N; V157F; Y163C; R175H; C176Y; H179W; H193R;

I195T; Y234C; Y234H; G245D; R248Q; R249S; R273C; R273H; and R337H.

Alternatively, the antigenic peptides comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the following recurrent cancer mutations: C141Y; C176F;

H179R; H179W; H193R; I195T; V216M; Y220C; R248L; R248Q; R248W; R249S; R273H; R273L; P278L; and R282G. The mutations can be in any order. For example, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: H179W; R273L; R249S; R248Q; Y234H; G245D; Y220C; R248L; H193R; K132N; S242F; Y234C; G245S; C176F; R282W; R273H; R282G; C141Y; R273C; V216M; R337H; R248W; V143A; I195T; P278S; S241F; C176Y; Y107D; R175H; H179R; V157F; P278L; and Y163C. See, e.g., SEQ ID NOS: 157-162. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: R248W; R248L; Y220C; Y163C; G245D; Y107D; H179R;

V216M; P278S; S241F; R273L; P278L; C176F; C141Y; S242F; R249S; V143A; I195T; R273H; R273C; R282G; H179W; R175H; R248Q; G245S; H193R; R337H; R282W;

Y234C; V157F; Y234H; C176Y; and K132N. See, e.g., SEQ ID NOS: 163-166.

Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: R248W; H179R; R273H; Y107D; R337H; R282G; V157F; V143A; Y234H; Y220C; R282W; R248L; S241F; H179W; R273C; C141Y; R249S; P278L; G245S; I195T; R175H; G245D; R273L; K132N; V216M; Y163C; C176F; S242F; Y234C; H193R; R248Q; P278S; and C176Y. See, e.g., SEQ ID NOS: 167- 174. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: V143A; R282W; V157F; H179W; K132N; Y163C; C176Y; G245D; Y220C; S242F; Y234C; R249S; H179R; R273H; C141Y; R273L; P278S; C176F; R337H; H193R; R273C; R282G; R175H; R248W; P278L; I195T; S241F; R248L; Y234H; V216M; G245S; Y107D; and R248Q. See, e.g., SEQ ID NOS: 175- 180. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: S241F; G245D; V143A; P278S; R273C; C176Y; Y234H; R248W; V216M; R248Q; C141Y; Y163C; H193R; H179R;

H179W; Y107D; and R273L. See, e.g., SEQ ID NOS: 181-186. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: K132N; R282W; G245S; Y234C; S242F; R175H; Y220C; V157F; R282G; C176F; R337H; I195T; R249S; P278L; R273H; and R248L. See, e.g., SEQ ID NOS: 187-192. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: H193R; P278L; R273C; R248W; H179R; P278S; R248L; V216M; R282G; R337H; R175H; Y234H; G245D; R273H; G245S; R282W; and C141Y. See, e.g., SEQ ID NOS: 193-198. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: Y107D; K132N; C176F; C176Y; R273L; Y220C; R248Q; V143A; I195T; R249S; S242F; Y234C; H179W; V157F; Y163C; and S241F. See, e.g., SEQ ID NOS: 199-204. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: P278S; C176F; H179R; R282G; S241F; R273L; P278L; C141Y; Y107D; R248W; V216M; R282W; S242F; Y220C; V143A; G245S; and R248L. See, e.g., SEQ ID NOS: 205-210.

Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: R175H; H179W; R249S; Y234H; I195T; R248Q; R273H; C176Y; V157F; H193R; Y234C; K132N; R273C; Y163C; G245D; and R337H. See, e.g., SEQ ID NOS: 211-216. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C- terminal: C176Y; R175H; G245D; R337H; S241F; K132N; V143A; P278S; R282W; Y163C; Y107D; R273C; S242F; G245S; V157F; Y234C; and Y234H. See, e.g., SEQ ID NOS: 217- 222. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: C176F; R273L; H179R; R282G; Y220C; I195T; C141Y; R248L; R273H; H179W; H193R; R249S; V216M; P278L; R248W; and R248Q. See, e.g., SEQ ID NOS: 223-228. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C- terminal: R248W; R273H; V143A; R249S; R175H; H193R; Y220C; G245D; R248Q;

R273C; R282W; Y107D; C141Y; V157F; Y163C; C176F; I195T; V216M; Y234H; G245S; R273L; Y234C; and R282G. See, e.g., SEQ ID NOS: 257-263. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: R248W; R273H; V143A; R249S; R175H; H193R; Y220C;

G245D; R248Q; R273C; and R282W. See, e.g., SEQ ID NOS: 264-270. Alternatively, the fusion polypeptide can comprise antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: Y107D; C141Y; V157F; Y163C; C176F; I195T; V216M; Y234H; G245S; R273L; Y234C; and R282G. See, e.g., SEQ ID NOS: 271-277. In a specific example, the TP53 antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation.

[00167] In some cases, the recurrent cancer mutations can be from multiple cancer- associated proteins. For example, each of the recurrent cancer mutations in a particular fusion polypeptide (or in a set of fusion polypeptides to be used, for example, in a single dosing regimen) can be a recurrent cancer mutation that occurs in the same type of cancer. As an example, the two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: PI3KCA, AKT1, AHNAK2, ERBB2, and TP53. The antigenic peptides can comprise, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more of the following recurrent cancer mutations: PIK3CAIH1047R; PIK3CAIE545K; PIK3CAIE542K; PIK3CAIH1047L;

PIK3CAIQ546K; PIK3CAIE545A; PIK3CAIE545G; AKT1IE17K; AHNAK2IV2016L, ERBB2IL755S, and TP53IR175H. The mutations can be in any order. In a specific example, the antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such 21-mers are set forth in Example 9 and in SEQ ID NOS: 584-594.

[00168] As another example, the two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: BRAF, KRAS/NRAS, TP53, PIK3CA, and SMAD4. The antigenic peptides can comprise, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more. 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, or 18 or more of the following recurrent cancer mutations: BRAFIV600E; KRASIG12D; KRASIG13D;

KRASIG12V; KRASIG12C; KRASIQ61K; KRASIG12A; KRASIG12S; TP53IR175H;

TP53IR248W; TP53IR273C; TP53IR282W; TP53IR273H; TP53IR248Q; TP53IG245S;

PIK3CAIE545K; PIK3CAIH1047R; PIK3CAIR88Q; and SMAD4IR361H. The mutations can be in any order. In a specific example, the antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such 21-mers are set forth in Example 9 and in SEQ ID NOS: 595-613.

[00169] As another example, two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: KRAS, TP53, EGFR, U2AF1, BRAF, and PIK3CA. The antigenic peptides can comprise, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more. 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, or 28 or more of the following recurrent cancer mutations: KRASIG12C; KRASIG12V; KRASIG12D; KRASIG12F; KRASIG12R; KRASIQ61L; KRASIG12Y; TP53IR158L;

TP53IR273L; TP53IG245V; TP53IR175H; TP53IA159P; TP53IR249M; TP53IR273H;

TP53IR280I; TP53IQ144L; TP53IR273C; TP53IR280G; TP53IR280T; EGFRIL858R;

EGFRIL861Q; EGFRIG719A; U2AF1IS34F; BRAF1IV600E; BRAF1IG466V;

BRAF1IN581S; PIK3CAIE545K; PIK3CAIE726K; and PIK3CAIH1047R. The mutations can be in any order. In a specific example, the antigenic peptides can be 21-mers (e.g., 21- mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such 21-mers are set forth in

Example 9 and in SEQ ID NOS: 614-643.

[00170] In another example, two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: TP53, PIK3CA, NFE2L2, CDKN2A, and PTEN. The antigenic peptides can comprise, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more. 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, or 59 or more of the following recurrent cancer mutations: TP53IY163C; TP53IR175G; TP53IC242F; TP53IR273L; TP53IH179L; TP53IH193L; TP53IH214R;

TP53IY220C; TP53IY234C; TP53IG245V; TP53IL111Q; TP53IT125P; TP53IK132R;

TP53IC135W; TP53IC141W; TP53IC176F; TP53IC176Y; TP53IH179R; TP53IH179Y;

TP53IH193R; TP53II195S; TP53IY205C; TP53IR213G; TP53IV216E; TP53IY234S;

TP53IY236C; TP53IM237I; TP53IG244C; TP53IG245S; TP53IR248L; TP53IR248P;

TP53IR248Q; TP53IR248W; TP53IR249G; TP53IR249S; TP53IR249W; TP53IG266V;

TP53IF270I; TP53IR273C; TP53IR273H; TP53IR273P; TP53IR280I; TP53ID281Y;

TP53IR282Q; TP53IR282W; PIK3CAIE545K; PIK3CAIE542K; PIK3CAIH1047R;

PIK3CAIE726K; PIK3CAIC420R; NFE2L2IE79Q; NFE2L2IR34Q; NFE2L2IL30F;

NFE2L2IG81S; NFE2L2IG31A; NFE2L2ID29G; NFE2L2IG81V; CDKN2AID108Y;

CDKN2AID18N; and PTENIR130Q. The mutations can be in any order. In a specific example, the antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such 21-mers are set forth in Example 9 and in SEQ ID NOS: 644- 703.

[00171 ] As another example, two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: ANKRD36C, SPOP, CHEK2,

KRTAP4-11, RGPD8, TP53, FAM47C, ZAN, and PIK3CA. The antigenic peptides can comprise, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more. 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more of the following recurrent cancer mutations: ANKRD36CII645T; ANKRD36CID629Y; ANKRD36CID629N; SPOPIW131G; SPOPIF133L; SPOPIF133V; SPOPIF133C; SPOPIW131R; SPOPIW131L; CHEK2IK373E; KRTAP4-11IM93V; KRTAP4-11IR51K; KRTAP4-11IL161V; RGPD8IP1760A;

TP53IR248Q; TP53IG245S; TP53IG245D; FAM47CIN648D; ZANIL878P; PIK3CAIE542K; and PIK3CAIH1047R. The mutations can be in any order. In a specific example, the antigenic peptides can be 21-mers (e.g., 21-mers fused directly together), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation.

Examples of such 21-mers are set forth in Example 9 and in SEQ ID NOS: 704-724.

[00172] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more or all of the following genes: KRAS, EGFR, U2AF1, BRAF, PIK3CA, and TP53. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the following recurrent cancer mutations:

KRAS_G12C, EGFR_L858R, KRAS_G12D, U2AF1_S34F, BRAF_V600E, KRAS_G12V, PIK3CA_E545K, TP53_R158L, KRAS_G12A, EGFR_L861Q, and TP53_R273L. Such mutations are associated with, for example, non-small cell lung cancer (NSCLC). The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the antigenic peptides in Table 35. [00173] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, or all of the following genes: SPOP, CHEK2, RGPD8, ANKRD36C, and AT?. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all of the following recurrent cancer mutations: SPOP_F133V, CHEK2_K373E, RGPD8_P1760A, ANKRD36C_I634T,

ANKRD36C_D629Y, SPOP_W131G, ANKRD36C_D626N, SPOP_F133L, AR_T878A, AR_L702H, AR_W742C, AR_H875Y, and AR_F877L. Such mutations are associated with, for example, prostate cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all of the antigenic peptides in Table 52.

[00174] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, or all of the following genes: KRAS, U2AF1, TP53, SMAD4, and GNAS. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the following recurrent cancer mutations: KRAS_G12C, KRAS_G12D, U2AF1_S34F, KRAS_G12V, TP53_R248Q, TP53_R248W, TP53_R175H, TP53_R273C, KRAS_G12R, KRAS_Q61H, TP53_R282W, TP53_R273H, TP53_G245S, SMAD4_R361C, GNAS_R201C, and

GNAS_R201H. Such mutations are associated with, for example, pancreatic cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the antigenic peptides in Table 68. [00175] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes:

PIK3CA, FGFR3, TP53, RXRA, FBXW7, and NFE2L2. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or all of the following recurrent cancer mutations: PIK3CA_E545K, FGFR3_S249C, TP53_R248Q, PIK3CA_E542K,

PvXRA_S427F, FBXW7_R505G, TP53_R280T, NFE2L2_E79K, FGFR3_R248C,

TP53_K132N, TP53_R248W, TP53_R175H, and TP53_R273C. Such mutations are associated with, for example, bladder cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or all of the antigenic peptides in Table 76.

[00176] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, or all of the following genes: PIK3CA, AKT1, and ESR1. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all of the following recurrent cancer mutations: PIK3CA_E545K,

PIK3CA_E542K, PIK3CA_H1047R, AKT1_E17K, PIK3CA_H1047L, PIK3CA_Q546K, PIK3CA_E545A, PIK3CA_E545G, ESR1_K303R, ESR1_D538G, ESR1_Y537S,

ESR1_Y537N, ESR1_Y537C, and ESR1_E380Q. Such mutations are associated with, for example, breast cancer (e.g., ER+ breast cancer). The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all of the antigenic peptides in Table 87. [00177] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes:

PTEN, KRAS, PIK3CA, CTNNBl, FBXW7, and TP53. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the following recurrent cancer mutations: PTEN_R130G, PTEN_R130Q,

KRAS_G12D, KRAS_G12V, PIK3CA_H1047R; PIK3CA_R88Q, PIK3CA_E545K, PIK3CA_E542K, CTNNB 1_S37F, KRAS_G13D, CTNNB 1_S37C, PIK3CA_H1047L, PIK3CA_G118D, KRAS_G12A, FBXW7_R505C, and TP53_R248W. Such mutations are associated with, for example, uterine cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the antigenic peptides in Table 95.

[00178] As another example, the cancer-associated protein can comprise the protein encoded by TP53. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all of the following recurrent cancer mutations: TP53_R248Q, TP53_R248W, TP53_R175H, TP53_R273C, TP53_R282W, TP53_R273H, TP53_Y220C, TP53_I195T, TP53_C176Y, TP53_H179R, TP53_S241F, and TP53_H193R. Such mutations are associated with, for example, ovarian cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all of the antigenic peptides in Table 100. [00179] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, or all of the following genes: TP53, PIK3CA, IDH1, IDH2, and EGFR. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the following recurrent cancer mutations: TP53_R273L, TP53_R273C, TP53_R273H, PIK3CA_G118D, IDH1_R132C, IDH1_R132G, IDH1_R132H,

IDH1_R132S, IDH2_R172K, PIK3CA_E453K, and EGFR_G598V. Such mutations are associated with, for example, low-grade glioma. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the antigenic peptides in Table 104.

[00180] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, or all of the following genes: KRAS, BRAF, PIK3CA, and TP53. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all of the following recurrent cancer mutations: KRAS_G12C, KRAS_G12D, BRAF_V600E, KRAS_G12V, PIK3CA_E545K, TP53_R248W, TP53_R175H, TP53_R273C,

PIK3CA_H1047R, TP53_R282W, TP53_R273H, and KRAS_G13D. Such mutations are associated with, for example, colorectal cancer (e.g., MSS colorectal cancer). The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all of the antigenic peptides in Table 108.

[00181 ] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all of the following genes: PIK3CA, CHEK2, RGPD8, ANKRD36C, TP53, ZNF814, KRTAP1-5, KRTAP4-11, and HRAS. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or all of the following recurrent cancer mutations: PIK3CA_E545K, CHEK2_K373E,

RGPD8_P1760A, ANKRD36C_I634T, TP53_R248Q, PIK3CA_E542K, TP53_R248W, TP53_R175H, PIK3CA_H1047R, TP53_R282W, TP53_R273H, TP53_G245S,

TP53_Y220C, ZNF814_D404E, KRTAP1-5_I88T, KRTAP4-11_L161V, and HRAS_G13V. Such mutations are associated with, for example, head and neck cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or all of the antigenic peptides in Table 112.

[00182] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more,

17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, or all of the following genes: KRAS, BRAF, PIK3CA, TRIM48, PTEN, POLE, PGM5, MBOAT2, KIAA2026, FBXW7, C12orf4, ZBTB20, XYLT2, WNT16, UBR5, TGFBR2, SVIL, RNF43, PLEKHA6, LARP4B, FHOD3, DOCK3, BMPR2, ARID1A,

ADAM28, and ACVR2A. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more,

18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, or all of the following recurrent cancer mutations:

KRAS_G12D, BRAF_V600E, PIK3CA_H1047R, TRIM48_Y192H, PTEN_R130N, POLE_V411L, POLE_P286R, PIK3CA_R88N, PGM5_I98V, MBOAT2_R43N,

KIAA2026_R574C, FBXW7_R465C, C12orf4_R335N, ZBTB20_p.Pro692LeufsTer43, XYLT2_p.Gly529AlafsTer78, WNT16_p.Glyl67AlafsTerl7, UBR5_p.Glu2121LysfsTer28, TGFBR2_p.Glu 150GlyfsTer35, S VIL_p.Met 1863TrpfsTer44, RNF43_p.Gly659ValfsTer41 , PLEKHA6_p.Val328TyrfsTerl72, LARP4B_p.Thrl63HisfsTer47, FHOD3_p.Ser336ValfsTerl38, DOCK3_p.Prol852GlnfsTer45,

BMPR2_p.Asn583ThrfsTer44, ARIDlA_p.Aspl850ThrfsTer33,

ADAM28_p.Asn75LysfsTerl5, and ACVR2A_p.Lys435GlufsTerl9. Such mutations are associated with, for example, DNA mismatch repair deficient cancers or tumors. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, or all of the antigenic peptides in Table 116. An exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprises, consists essentially of, or consists of a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 917. A breakdown of the amino acids positions of the individual components in each construct is provided in Table 117.

C. PEST- Containing Peptides

[00183] The recombinant fusion proteins disclosed herein comprise a PEST-containing peptide. The PEST-containing peptide may at the amino terminal (N-terminal) end of the fusion polypeptide (i.e., N-terminal to the antigenic peptides), may be at the carboxy terminal (C-terminal) end of the fusion polypeptide (i.e., C-terminal to the antigenic peptides), or may be embedded within the antigenic peptides. In some recombinant Listeria strains and methods, a PEST containing peptide is not part of and is separate from the fusion

polypeptide. Fusion of an antigenic peptides to a PEST-like sequence, such as an LLO peptide, can enhance the immunogenicity of the antigenic peptides and can increase cell- mediated and antitumor immune responses (i.e., increase cell- mediated and anti-tumor immunity). See, e.g., Singh et al. (2005) J Immunol 175(6):3663-3673, herein incorporated by reference in its entirety for all purposes.

[00184] A PEST-containing peptide is one that comprises a PEST sequence or a PEST-like sequence. PEST sequences in eukaryotic proteins have long been identified. For example, proteins containing amino acid sequences that are rich in prolines (P), glutamic acids (E), serines (S) and threonines (T) (PEST), generally, but not always, flanked by clusters containing several positively charged amino acids, have rapid intracellular half-lives (Rogers et al. (1986) Science 234:364-369, herein incorporated by reference in its entirety for all purposes). Further, it has been reported that these sequences target the protein to the ubiquitin-proteosome pathway for degradation (Rechsteiner and Rogers (1996) Trends Biochem. Sci. 21:267-271, herein incorporated by reference in its entirety for all purposes). This pathway is also used by eukaryotic cells to generate immunogenic peptides that bind to MHC class I and it has been hypothesized that PEST sequences are abundant among eukaryotic proteins that give rise to immunogenic peptides (Realini et al. (1994) FEBS Lett. 348: 109- 113, herein incorporated by reference in its entirety for all purposes). Prokaryotic proteins do not normally contain PEST sequences because they do not have this enzymatic pathway. However, a PEST-like sequence rich in the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T) has been reported at the amino terminus of LLO and has been reported to be essential for L. monocytogenes pathogenicity (Decatur and Portnoy (2000) Science 290:992-995, herein incorporated by reference in its entirety for all purposes). The presence of this PEST-like sequence in LLO targets the protein for destruction by proteolytic machinery of the host cell so that once the LLO has served its function and facilitated the escape of L. monocytogenes from the phagosomal or phagolysosomal vacuole, it is destroyed before it can damage the cells.

[00185] Identification of PEST and PEST-like sequences is well known in the art and is described, for example, in Rogers et al. (1986) Science 234(4774):364-378 and in

Rechsteiner and Rogers (1996) Trends Biochem. Sci. 21:26 '-271, each of which is herein incorporated by reference in its entirety for all purposes. A PEST or PEST-like sequence can be identified using the PEST-find program. For example, a PEST-like sequence can be a region rich in proline (P), glutamic acid (E), serine (S), and threonine (T) residues.

Optionally, the PEST-like sequence can be flanked by one or more clusters containing several positively charged amino acids. For example, a PEST-like sequence can be defined as a hydrophilic stretch of at least 12 amino acids in length with a high local concentration of proline (P), aspartate (D), glutamate (E), serine (S), and/or threonine (T) residues. In some cases, a PEST-like sequence contains no positively charged amino acids, namely arginine (R), histidine (H), and lysine (K). Some PEST-like sequences can contain one or more internal phosphorylation sites, and phosphorylation at these sites precedes protein

degradation. [00186] In one example, the PEST-like sequence fits an algorithm disclosed in Rogers et al. In another example, the PEST-like sequence fits an algorithm disclosed in Rechsteiner and Rogers. PEST-like sequences can also be identified by an initial scan for positively charged amino acids R, H, and K within the specified protein sequence. All amino acids between the positively charged flanks are counted, and only those motifs containing a number of amino acids equal to or higher than the window- size parameter are considered further. Optionally, a PEST-like sequence must contain at least one P, at least one D or E, and at least one S or T.

[00187] The quality of a PEST motif can be refined by means of a scoring parameter based on the local enrichment of critical amino acids as well as the motifs hydrophobicity.

Enrichment of D, E, P, S, and T is expressed in mass percent (w/w) and corrected for one equivalent of D or E, onel of P, and one of S or T. Calculation of hydrophobicity can also follow in principle the method of Kyte and Doolittle (1982) J. Mol. Biol. 157: 105, herein incorporated by reference in its entirety for all purposes. For simplified calculations, Kyte- Doolittle hydropathy indices, which originally ranged from -4.5 for arginine to +4.5 for isoleucine, are converted to positive integers, using the following linear transformation, which yielded values from 0 for arginine to 90 for isoleucine: Hydropathy index = 10 * Kyte- Doolittle hydropathy index + 45.

[00188] A potential PEST motif's hydrophobicity can also be calculated as the sum over the products of mole percent and hydrophobicity index for each amino acid species. The desired PEST score is obtained as combination of local enrichment term and hydrophobicity term as expressed by the following equation: PEST score = 0.55 * DEPST - 0.5 *

hydrophobicity index.

[00189] Thus, a PEST-containing peptide can refer to a peptide having a score of at least +5 using the above algorithm. Alternatively, it can refer to a peptide having a score of 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, at least 30, at least 32, at least 35, at least 38, at least 40, or at least 45.

[00190] Any other available methods or algorithms known in the art can also be used to identify PEST-like sequences. See, e.g., the CaSPredictor (Garay-Malpartida et al. (2005) Bioinformatics 21 Suppl l:il69-76, herein incorporated by reference in its entirety for all purposes). Another method that can be used is the following: a PEST index is calculated for each stretch of appropriate length (e.g. a 30-35 amino acid stretch) by assigning a value of one to the amino acids Ser, Thr, Pro, Glu, Asp, Asn, or Gin. The coefficient value (CV) for each of the PEST residues is one and the CV for each of the other AA (non-PEST) is zero.

[00191 ] Examples of PEST-like amino acid sequences are those set forth in SEQ ID NOS: 320-328. One example of a PEST-like sequence is

KENS IS S M APP AS PP AS PKTPIEKKH ADEID K (SEQ ID NO: 320). Another example of a PEST-like sequence is KENSISSMAPPASPPASPK (SEQ ID NO: 321). However, any PEST or PEST-like amino acid sequence can be used. PEST sequence peptides are known and are described, for example, in US 7,635,479; US 7,665,238; and US 2014/0186387, each of which is herein incorporated by reference in its entirety for all purposes.

[00192] The PEST-like sequence can be from a Listeria species, such as from Listeria monocytogenes. For example, the Listeria monocytogenes ActA protein contains at least four such sequences (SEQ ID NOS: 322-325), any of which are suitable for use in the

compositions and methods disclosed herein. Other similar PEST-like sequences include SEQ ID NOS: 329-331. Streptolysin O proteins from Streptococcus sp. also contain a PEST sequence. For example, Streptococcus pyogenes streptolysin O comprises the PEST sequence KQNTASTETTTTNEQPK (SEQ ID NO: 326) at amino acids 35-51 and

Streptococcus equisimilis streptolysin O comprises the PEST-like sequence

KQNTANTETTTTNEQPK (SEQ ID NO: 327) at amino acids 38-54. Another example of a PEST-like sequence is from Listeria seeligeri cytolysin, encoded by the Iso gene:

RSEVTISPAETPESPPATP (e.g., SEQ ID NO: 328).

[00193] Alternatively, the PEST-like sequence can be derived from other prokaryotic organisms. Other prokaryotic organisms wherein PEST-like amino acid sequences would be expected include, for example, other Listeria species.

(1) Listeriolysin O (LLO)

[00194] One example of a PEST-containing peptide that can be utilized in the

compositions and methods disclosed herein is a listeriolysin O (LLO) peptide. An example of an LLO protein is the protein assigned GenBank Accession No. P13128 (SEQ ID NO: 332; nucleic acid sequence is set forth in GenBank Accession No. X15127). SEQ ID NO: 332 is a proprotein including a signal sequence. The first 25 amino acids of the proprotein is the signal sequence and is cleaved from LLO when it is secreted by the bacterium, thereby resulting in the full-length active LLO protein of 504 amino acids without the signal sequence. An LLO peptide disclosed herein can comprise the signal sequence or can comprise a peptide that does not include the signal sequence. Exemplary LLO proteins that can be used comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 332 or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of SEQ ID NO: 332. Any sequence that encodes a fragment of an LLO protein or a homologue, variant, isoform, analog, fragment of a homologue, fragment of a variant, or fragment of an analog of an LLO protein can be used. A homologous LLO protein can have a sequence identity with a reference LLO protein, for example, of greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%.

[00195] Another example of an LLO protein is set forth in SEQ ID NO: 333. LLO proteins that can be used can comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 333 or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of SEQ ID NO: 333.

[00196] Another example of an LLO protein is an LLO protein from the Listeria monocytogenes 10403S strain, as set forth in GenBank Accession No.: ZP_01942330 or EBA21833, or as encoded by the nucleic acid sequence as set forth in GenBank Accession No.: NZ_AARZ01000015 or AARZ01000015.1. Another example of an LLO protein is an LLO protein from the Listeria monocytogenes 4b F2365 strain {see, e.g., GenBank Accession No.: YP_012823), EGD-e strain {see, e.g., GenBank Accession No.: NP_463733), or any other strain of Listeria monocytogenes. Yet another example of an LLO protein is an LLO protein from Flavobacteriales bacterium HTCC2170 {see, e.g., GenBank Accession No.: ZP_01106747 or EAR01433, or encoded by GenBank Accession No.: NZ_AAOC01000003). LLO proteins that can be used can comprise, consist essentially of, or consist of any of the above LLO proteins or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of the above LLO proteins.

[00197] Proteins that are homologous to LLO, or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms thereof, can also be used. One such example is alveolysin, which can be found, for example, in Paenibacillus alvei {see, e.g., GenBank Accession No.: P23564 or AAA22224, or encoded by GenBank Accession No.: M62709). Other such homologous proteins are known. [00198] The LLO peptide can be a full-length LLO protein or a truncated LLO protein or LLO fragment. Likewise, the LLO peptide can be one that retains one or more functionalities of a native LLO protein or lacks one or more functionalities of a native LLO protein. For example, the retained LLO functionality can be allowing a bacteria (e.g., Listeria) to escape from a phagosome or phagolysosome, or enhancing the immunogenicity of a peptide to which it is fused. The retained functionality can also be hemolytic function or antigenic function. Alternatively, the LLO peptide can be a non-hemolytic LLO. Other functions of LLO are known, as are methods and assays for evaluating LLO functionality.

[00199] An LLO fragment can be a PEST-like sequence or can comprise a PEST-like sequence. LLO fragments can comprise one or more of an internal deletion, a truncation from the C-terminal end, and a truncation from the N-terminal end. In some cases, an LLO fragment can comprise more than one internal deletion. Other LLO peptides can be full- length LLO proteins with one or more mutations.

[00200] Some LLO proteins or fragments have reduced hemolytic activity relative to wild type LLO or are non-hemolytic fragments. For example, an LLO protein can be rendered non-hemolytic by deletion or mutation of the activation domain at the carboxy terminus, by deletion or mutation of cysteine 484, or by deletion or mutation at another location.

[00201 ] Other LLO proteins are rendered non-hemolytic by a deletion or mutation of the cholesterol binding domain (CBD) as detailed in US 8,771,702, herein incorporated by reference in its entirety for all purposes. The mutations can comprise, for example, a substitution or a deletion. The entire CBD can be mutated, portions of the CBD can be mutated, or specific residues within the CBD can be mutated. For example, the LLO protein can comprise a mutation of one or more of residues C484, W491, and W492 (e.g., C484, W491, W492, C484 and W491, C484 and W492, W491 and W492, or all three residues) of SEQ ID NO: 332 or corresponding residues when optimally aligned with SEQ ID NO: 332 (e.g., a corresponding cysteine or tryptophan residue). As an example, a mutant LLO protein can be created wherein residues C484, W491, and W492 of LLO are substituted with alanine residues, which will substantially reduce hemolytic activity relative to wild type LLO. The mutant LLO protein with C484A, W491A, and W492A mutations is termed "mutLLO."

[00202] As another example, a mutant LLO protein can be created with an internal deletion comprising the cholesterol-binding domain. The sequence of the cholesterol-binding domain of SEQ ID NO: 332 set forth in SEQ ID NO: 351. For example, the internal deletion can be a 1-11 amino acid deletion, an 11-50 amino acid deletion, or longer. Likewise, the mutated region can be 1-11 amino acids, 11-50 amino acids, or longer (e.g., 1-50, 1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2- 3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-20, 11- 25, 11-30, 11-35, 11-40, 11-50, 11-60, 11-70, 11-80, 11-90, 11-100, 11-150, 15-20, 15-25, 15-30, 15-35, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90, 15-100, 15-150, 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100, or 30-150 amino acids). For example, a mutated region consisting of residues 470-500, 470-510, or 480-500 of SEQ ID NO: 332 will result in a deleted sequence comprising the CBD (residues 483-493 of SEQ ID NO: 332). However, the mutated region can also be a fragment of the CBD or can overlap with a portion of the CBD. For example, the mutated region can consist of residues 470-490, 480-488, 485-490, 486-488, 490-500, or 486-510 of SEQ ID NO: 332. For example, a fragment of the CBD (residues 484-492) can be replaced with a heterologous sequence, which will substantially reduce hemolytic activity relative to wild type LLO. For example, the CBD (ECTGLAWEWWR; SEQ ID NO: 351) can be replaced with a CTL epitope from the antigen NY-ESO-1 (ESLLMWITQCR; SEQ ID NO: 352), which contains the HLA-A2 restricted epitope 157-165 from NY-ESO-1. The resulting LLO is termed "ctLLO."

[00203] In some mutated LLO proteins, the mutated region can be replaced by a heterologous sequence. For example, the mutated region can be replaced by an equal number of heterologous amino acids, a smaller number of heterologous amino acids, or a larger number of amino acids (e.g., 1-50, 1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10- 11, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-20, 11-25, 11-30, 11-35, 11-40, 11-50, 11-60, 11- 70, 11-80, 11-90, 11-100, 11-150, 15-20, 15-25, 15-30, 15-35, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90, 15-100, 15-150, 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100, or 30-150 amino acids). Other mutated LLO proteins have one or more point mutations (e.g., a point mutation of 1 residue, 2 residues, 3 residues, or more). The mutated residues can be contiguous or not contiguous.

[00204] In one example embodiment, an LLO peptide may have a deletion in the signal sequence and a mutation or substitution in the CBD.

[00205] Some LLO peptides are N-terminal LLO fragments (i.e., LLO proteins with a C- terminal deletion). Some LLO peptides are at least 494, 489, 492, 493, 500, 505, 510, 515, 520, or 525 amino acids in length or 492-528 amino acids in length. For example, the LLO fragment can consist of about the first 440 or 441 amino acids of an LLO protein (e.g., the first 441 amino acids of SEQ ID NO: 332 or 333, or a corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO: 332 or 333). Other N-terminal LLO fragments can consist of the first 420 amino acids of an LLO protein (e.g., the first 420 amino acids of SEQ ID NO: 332 or 333, or a corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO: 332 or 333). Other N-terminal fragments can consist of about amino acids 20-442 of an LLO protein (e.g., amino acids 20-442 of SEQ ID NO: 332 or 333, or a corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO: 332 or 333). Other N-terminal LLO fragments comprise any ALLO without the activation domain comprising cysteine 484, and in particular without cysteine 484. For example, the N-terminal LLO fragment can correspond to the first 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, or 25 amino acids of an LLO protein (e.g., the first 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, or 25 amino acids of SEQ ID NO: 332 or 333, or a corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO: 332 or 333). Preferably, the fragment comprises one or more PEST-like sequences. LLO fragments and truncated LLO proteins can contain residues of a homologous LLO protein that correspond to any one of the above specific amino acid ranges. The residue numbers need not correspond exactly with the residue numbers enumerated above (e.g., if the homologous LLO protein has an insertion or deletion relative to a specific LLO protein disclosed herein). Examples of N-terminal LLO fragments include SEQ ID NOS: 334, 335, and 336. LLO proteins that can be used comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 334, 335, or 336 or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of SEQ ID NO: 334, 335, or 336. In some compositions and methods, the N-terminal LLO fragment set forth in SEQ ID NO: 336 is used. An example of a nucleic acid encoding the N-terminal LLO fragment set forth in SEQ ID NO: 336 is SEQ ID NO: 337.

(2) ActA

[00206] Another example of a PEST-containing peptide that can be utilized in the compositions and methods disclosed herein is an ActA peptide. ActA is a surface-associated protein and acts as a scaffold in infected host cells to facilitate the polymerization, assembly, and activation of host actin polymers in order to propel a Listeria monocytogenes through the cytoplasm. Shortly after entry into the mammalian cell cytosol, L. monocytogenes induces the polymerization of host actin filaments and uses the force generated by actin polymerization to move, first intracellularly and then from cell to cell. ActA is responsible for mediating actin nucleation and actin-based motility. The ActA protein provides multiple binding sites for host cytoskeletal components, thereby acting as a scaffold to assemble the cellular actin polymerization machinery. The N-terminus of ActA binds to monomeric actin and acts as a constitutively active nucleation promoting factor by stimulating the intrinsic actin nucleation activity. The actA and hly genes are both members of the 10-kb gene cluster regulated by the transcriptional activator PrfA, and actA is upregulated approximately 226- fold in the mammalian cytosol. Any sequence that encodes an ActA protein or a homologue, variant, isoform, analog, fragment of a homologue, fragment of a variant, or fragment of an analog of an ActA protein can be used. A homologous ActA protein can have a sequence identity with a reference ActA protein, for example, of greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%.

[00207] One example of an ActA protein comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 338. Another example of an ActA protein comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 339. The first 29 amino acid of the proprotein corresponding to either of these sequences are the signal sequence and are cleaved from ActA protein when it is secreted by the bacterium. An ActA peptide can comprise the signal sequence (e.g., amino acids 1-29 of SEQ ID NO: 338 or 339), or can comprise a peptide that does not include the signal sequence. Other examples of ActA proteins comprise, consist essentially of, or consist of homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of isoforms, or fragments of analogs of SEQ ID NO: 338 or 339.

[00208] Another example of an ActA protein is an ActA protein from the Listeria monocytogenes 10403S strain (GenBank Accession No.: DQ054585) the NICPBP 54002 strain (GenBank Accession No.: EU394959), the S3 strain (GenBank Accession No.:

EU394960), NCTC 5348 strain (GenBank Accession No.: EU394961), NICPBP 54006 strain (GenBank Accession No.: EU394962), M7 strain (GenBank Accession No.: EU394963), S 19 strain (GenBank Accession No.: EU394964), or any other strain of Listeria monocytogenes. LLO proteins that can be used can comprise, consist essentially of, or consist of any of the above LLO proteins or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of the above LLO proteins. [00209] ActA peptides can be full-length ActA proteins or truncated ActA proteins or ActA fragments (e.g., N-terminal ActA fragments in which a C-terminal portion is removed). Preferably, truncated ActA proteins comprise at least one PEST sequence (e.g., more than one PEST sequence). In addition, truncated ActA proteins can optionally comprise an ActA signal peptide. Examples of PEST-like sequences contained in truncated ActA proteins include SEQ ID NOS: 322-325. Some such truncated ActA proteins comprise at least two of the PEST-like sequences set forth in SEQ ID NOS: 322-325 or homo logs thereof, at least three of the PEST-like sequences set forth in SEQ ID NOS: 322-325 or homo logs thereof, or all four of the PEST-like sequences set forth in SEQ ID NOS: 322-325 or homologs thereof. Examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of about residues 30-122, about residues 30-229, about residues 30-332, about residues 30-200, or about residues 30-399 of a full length ActA protein sequence (e.g., SEQ ID NO: 339). Other examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of about the first 50, 100, 150, 200, 233, 250, 300, 390, 400, or 418 residues of a full length ActA protein sequence (e.g., SEQ ID NO: 339). Other examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of about residues 200-300 or residues 300-400 of a full length ActA protein sequence (e.g., SEQ ID NO: 339). For example, the truncated ActA consists of the first 390 amino acids of the wild type ActA protein as described in US 7,655,238, herein incorporated by reference in its entirety for all purposes. As another example, the truncated ActA can be an ActA-NlOO or a modified version thereof (referred to as ActA-NlOO*) in which a PEST motif has been deleted and containing the nonconservative QDNKR (SEQ ID NO: 350) substitution as described in US 2014/0186387, herein incorporated by references in its entirety for all purposes. Alternatively, truncated ActA proteins can contain residues of a homologous ActA protein that corresponds to one of the above amino acid ranges or the amino acid ranges of any of the ActA peptides disclosed herein. The residue numbers need not correspond exactly with the residue numbers enumerated herein (e.g., if the homologous ActA protein has an insertion or deletion, relative to an ActA protein utilized herein, then the residue numbers can be adjusted accordingly).

[00210] Examples of truncated ActA proteins include, for example, proteins comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 340, 341, 342, or 343or homologues, variants, isoforms, analogs, fragments of variants, fragments of isoforms, or fragments of analogs of SEQ ID NO: 340, 341,342, or 343. SEQ ID NO: 340 referred to as ActA/PESTl and consists of amino acids 30-122 of the full length ActA sequence set forth in SEQ ID NO: 339. SEQ ID NO: 341 is referred to as ActA/PEST2 or LA229 and consists of amino acids 30-229 of the full length ActA sequence set forth in the full-length ActA sequence set forth in SEQ ID NO: 339. SEQ ID NO: 342 is referred to as ActA/PEST3 and consists of amino acids 30-332 of the full-length ActA sequence set forth in SEQ ID NO: 339. SEQ ID NO: 343 is referred to as ActA/PEST4 and consists of amino acids 30-399 of the full-length ActA sequence set forth in SEQ ID NO: 339. As a specific example, the truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 341 can be used.

[00211 ] Examples of truncated ActA proteins include, for example, proteins comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 344, 346, 347, or 349 or homologues, variants, isoforms, analogs, fragments of variants, fragments of isoforms, or fragments of analogs of SEQ ID NO: 344, 346, 347, or 349. As a specific example, the truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 344 (encoded by the nucleic acid set forth in SEQ ID NO: 345) can be used. As another specific example, the truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 347 (encoded by the nucleic acid set forth in SEQ ID NO: 348) can be used. SEQ ID NO: 348 is the first 1170 nucleotides encoding ActA in the Listeria monocytogenes 10403S strain. In some cases, the ActA fragment can be fused to a heterologous signal peptide. For example, SEQ ID NO: 349 sets forth an ActA fragment fused to an Hly signal peptide.

D. Generating Immunotherapy Constructs Encoding Recombinant Fusion Polypeptides Comprising Recurrent Cancer Mutations

[00212] Also provided herein are methods for generating immunotherapy constructs encoding or compositions comprising the recombinant fusion polypeptides disclosed herein. For example, such methods can comprise selecting a set of recurrent cancer mutations to include in the immunotherapy construct, designing antigenic peptides comprising each of the recurrent cancer mutations (and, for example, testing the hydropathy of the each antigenic peptide, and modifying or deselecting an antigenic peptide if it scores above a selected hydropathy index threshold value), selecting one or more sets of antigenic peptides, designing one or more fusion polypeptides comprising each of the selected antigenic peptides, and generating a nucleic acid construct encoding the fusion polypeptide.

[00213] Individual recurrent cancer mutations can be selected based on any criteria. For example, individual selected recurrent cancer mutations can be selected based on frequency of occurrence across multiple types of cancer (e.g., occurrence in at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all cancer patients), frequency of occurrence in a particular type of cancer (e.g., occurrence in at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all patients having a particular type of cancer), location within a functional domain of a cancer-associated protein, status as a known cancer driver mutation, status as a known chemotherapy resistance mutation, or identification as a somatic missense mutation. A particular cancer-associated protein can be selected, for example, if mutations in a particular cancer-associated protein may occur in at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all instances of cancer or a particular type of cancer. After selection of one or more cancer-associated proteins, the highest frequency shared somatic mutations can be identified. This can be done, for example, using databases such as COSMIC (Catalogue of Somatic Mutations in Cancer; cancer.Sanger.ac.uk) or Cancer Genome Analysis or other similar cancer-associated gene database. Examples of other such databases include TCGA, IGGC, and cBioportal. The mutations can be ranked, for example, according to one of more of the following: frequency of occurrence in a particular type of cancer or across all cancers;

locations within mutational hotspots as disclosed elsewhere herein; and effect of the mutation on function of the protein (e.g., loss of function of a tumor suppressor protein; known cancer "driver" mutations; known chemotherapy resistance mutations). Optionally, one or more of nonsense mutations, deletion mutations, insertion mutations, frameshift mutations, or translocation mutations can be excluded. In some cases, only somatic missense mutations are considered. In some cases, only frameshift (e.g., somatic frameshift mutations) are considered. In some cases, both somatic missense and frameshift mutations are considered.

[00214] A set of recurrent cancer mutations can be selected based on one or more additional criteria. For example, the set of recurrent cancer mutations can be selected based on the set including the potential mutated epitopes that would be found in at least 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients who have a mutation in a single cancer-associated protein, or at least 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients who have a somatic missense mutation in a single cancer-associated protein. Likewise, the set of recurrent cancer mutations can be selected based on the set including the potential mutated epitopes that would be found in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients who have a particular type of cancer. The set can also be selected based on the set comprising at least 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, or 40 different recurrent cancer mutations from a single cancer-associated protein, or at least 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, or 40 different recurrent somatic missense cancer mutations from a single cancer-associated protein. Likewise, the set can also be selected based on the set comprising at least 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, or 40 different recurrent cancer mutations from a single type of cancer, or at least 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, or 40 different recurrent somatic missense cancer mutations from a single type of cancer. For example, the single type of cancer can be no n- small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer (e.g., ER+ breast cancer), uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer (e.g., MSS colorectal cancer), or head and neck cancer. The set can also be selected based on the set comprising no more than 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49 or 50 recurrent cancer mutations, or any other threshold based on the capacity for a particular delivery system (e.g., bacterial delivery system). In addition, the set can be selected such that at least 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the selected recurrent cancer mutations in step (a) are from a single cancer-associated protein, or that no more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or none of the recurrent cancer mutations in step (a) are from the same cancer-associated protein.

[00215] In a specific example, mutation data can be sub- stratified by disease indication type. Particular types of mutations can be selected for consideration. For example, recurrent somatic mutations can include missense substitutions and insertions/deletions (INDELs) resulting in in-frame and frameshift mutations. The somatic mutations can be rank-ordered within a specific-indication cohort based on frequency of the total number of mutation events observed across all samples. Mutations occurring with frequencies below a certain frequency (e.g., 1%, 2%, 3%, 4%, 5%, or 10%) can be excluded. Recurrent mutations with disease- indication frequencies equal to and above, e.g., 1%, 2%, 3%, 4%, 5%, or 10% can be selected for panel.

[00216] After identification of a set of possible recurrent cancer mutations to include in a fusion polypeptide, sequences for antigenic peptides comprising each recurrent cancer mutation can be selected. Each antigenic peptide can be designed, for example, to comprise a fragment of the cancer-associated protein comprising a recurrent cancer mutation and flanking sequence on each side. Different size antigenic peptides can be used, as disclosed elsewhere herein. Preferably, however, at least about 10 flanking amino acids on each side of the recurrent cancer mutation are incorporated to accommodate class 1 MHC-1 presentation, in order to provide at least some of the different HLA T-cell receptor (TCR) reading frames. For example, an antigenic peptide can be selected to include a recurrent cancer mutation and 10 flanking amino acids from the cancer-associated protein on each side (i.e., a 21-mer). Alternatively, for example, an antigenic peptide can be selected to include a recurrent cancer mutation and 13 flanking amino acids from the cancer-associated protein on each side (i.e., a 27-mer).

[00217] The antigenic peptides can then be screened for hydrophobicity or hydrophilicity. Antigenic peptides can be selected, for example, if they are hydrophilic or if they score up to or below a certain hydropathy threshold, which can be predictive of secretability in a particular bacteria of interest (e.g., Listeria monocytogenes). For example, antigenic peptides can be scored by Kyte and Doolittle hydropathy index with a 21 amino acid window, all scoring above cutoff (around 1.6) are excluded as they are unlikely to be secretable by Listeria monocytogenes. See, e.g., Kyte-Doolittle (1982) J Mol Biol 157(1): 105—132; herein incorporated by reference in its entirety for all purposes. Alternatively, an antigenic peptide scoring about a selected cutoff can be altered (e.g., changing the length of the antigenic peptide or shifting the region of the cancer-associated protein included in the antigenic peptide (so long as the antigenic peptide still contains the recurrent cancer mutation and sufficient flanking sequence on each side). Other sliding window sizes that can be used include, for example, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or more amino acids. For example, the sliding window size can be 9-11 amino acids, 11-13 amino acids, 13-15 amino acids, 15- 17 amino acids, 17-19 amino acids, 19-21 amino acids, 21-23 amino acids, 23-25 amino acids, or 25-27 amino acids. Other cutoffs that can be used include, for example, the following ranges 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2.0-2.2 2.2-2.5, 2.5-3.0, 3.0-3.5, 3.5-4.0, or 4.0-4.5, or the cutoff can be 1.4, 1.5, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.3, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5. The cutoff can vary, for example, depending on the genus or species of the bacteria being used to deliver the fusion polypeptide.

[00218] Other suitable hydropathy plots or other appropriate scales include, for example, those reported in Rose et al. (1993) Annu Rev Biomol Struct 22:381-415; Biswas et al. (2003) Journal of Chromatography A 1000:637-655; Eisenberg (1984) Ann Rev Biochem 53:595- 623; Abraham and Leo (1987) Proteins: Structure, Function and Genetics 2: 130-152; Sweet and Eisenberg (1983) Mol Biol 171:479-488; Bull and Breese (1974) Arch Biochem Biophys 161:665-670; Guy (1985) Biophys J 47:61-70; Miyazawa et al. (1985) Macromolecules 18:534-552; Roseman (1988) J Mol Biol 200:513-522; Wolfenden et al. (1981) Biochemistry 20:849-855; Wilson (1981) Biochem J 199:31-41; Cowan and Whittaker (1990) Peptide Research 3:75-80; Aboderin (1971) Int J Biochem 2:537-544; Eisenberg et al. (1984) J Mol Biol 179: 125-142; Hopp and Woods (1981) Proc Natl Acad Sci USA 78:3824-3828;

Manavalan and Ponnuswamy (1978) Nature 275:673-674; Black and Mould (1991) Anal Biochem 193:72-82; Fauchere and Pliska (1983) Eur J Med Chem 18:369-375; Janin (1979) Nature 277:491-492; Rao and Argos (1986) Biochim Biophys Acta 869: 197-214; Tanford (1962) Am Chem Soc 84:4240-4274; Welling et al. (1985) FEBS Lett 188:215-218; Parker et al. (1986) Biochemistry 25:5425-5431; and Cowan and Whittaker (1990) Peptide Research 3:75-80, each of which is herein incorporated by reference in its entirety for all purposes.

[00219] Optionally, the remaining antigenic peptides can then be scored for their ability to bind to the subject human leukocyte antigen (HLA) type (for example by using the Immune Epitope Database (IED) available at www.iedb.org, which includes netMHCpan, ANN, SMMPMBEC. SMM, CombLib_Sidney2008, PickPocket, and netMHCcons) and ranked by best MHC binding score from each antigenic peptide. Other sources include TEpredict (tepredict.sourceforge.net/help.html) or other available MHC binding measurement scales. Cutoffs may be different for different expression vectors such as Salmonella.

[00220] Optionally, the antigenic peptides can be further screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL-10-inducing T helper epitopes, and so forth) to deselect antigenic peptides or to avoid immunosuppressive influences.

[00221 ] Optionally, a predicative algorithm for immunogenicity of the epitopes can be used to screen the antigenic peptides. However, these algorithms are at best 20% accurate in predicting which peptide will generate a T cell response. Alternatively, no

screening/predictive algorithms are used. Alternatively, the antigenic peptides can be screened for immunogenicity. For example, this can comprise contacting one or more T cells with an antigenic peptide, and analyzing for an immunogenic T cell response, wherein an immunogenic T cell response identifies the peptide as an immunogenic peptide. This can also comprise using an immunogenic assay to measure secretion of at least one of CD25, CD44, or CD69 or to measure secretion of a cytokine selected from the group comprising IFN-γ, TNF-a, IL-1, and IL-2 upon contacting the one or more T cells with the peptide, wherein increased secretion identifies the peptide as comprising one or more T cell epitopes. [00222] In a specific example in which target peptides are generated for recurrent mutations, for missense substitutions, the mutant amino acid can be flanked by, e.g., up to 10 wild-type amino acids immediately before and after missense mutation position. For frameshift substitutions, the predicted peptide sequence arising from out-of- frame INDEL substitution can be generated from the annotation transcript and up to, e.g., 10 wild-type amino acids can be added upstream of frameshift mutation position. For in-frame INDEL substitutions, up to, e.g., 10 wild-type amino acid sequences before and after INDEL position can be joined together. Specific identifiers can be generated for each hotspot target peptide that consist of the gene symbol (HGNC format) and mutation substitution information (HGVS format) separated by an underscore. For example, the substitution of glycine for aspartic acid at position 12 in KRAS would create a specific identifier of KRAS_G12D.

Target peptides can then subjected to BLAST analysis against the non-redundant protein sequences (nr) database for human. This step can ensure that target peptide sequences generated from frameshift mutations do not represent known, wild-type sequences. For missense substations, this step can ensure that flanking wild-type amino acids match the known human reference proteome.

[00223] The selected antigenic peptides can then be arranged into one or more candidate orders for a potential fusion polypeptide. If there are more usable antigenic peptides than can fit into a single plasmid, different antigenic peptides can be assigned priority ranks as needed/desired and/or split up into different fusion polypeptides (e.g., for inclusion in different recombinant Listeria strains). Priority rank can be determined by factors such as relative size, priority of transcription, and/or overall hydrophobicity of the translated polypeptide. The antigenic peptides can be arranged so that they are joined directly together without linkers, or any combination of linkers between any number of pairs of antigenic peptides, as disclosed in more detail elsewhere herein. The number of linear antigenic peptides to be included can be determined based on consideration of the number of constructs needed versus the mutational burden, the efficiency of translation and secretion of multiple epitopes from a single plasmid, the MOI needed for each bacteria or Lm comprising a plasmid, the number of recurrent cancer mutations or hotspot mutations in a particular cancer-associated protein, or how many recurrent cancer mutations need to be included to cover at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a mutation or somatic mutation in that cancer-associated protein. Likewise, the number of linear antigenic peptides to be included can be determined based in part on consideration of the number of recurrent cancer mutations or hotspot mutations in a particular type of cancer, or how many recurrent cancer mutations need to be included to cover at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a particular type of cancer. For example, ranges of linear antigenic peptides can be starting, for example, with about 50, 40, 30, 20, or 10 antigenic peptides per plasmid.

[00224] Different possible arrangements of the same antigenic peptides in a fusion polypeptide can be generated through one or more iterations of randomizing the order of the antigenic peptides. Such randomizing can include, for example, randomizing the order of the entire set of antigenic peptides, or can comprise randomizing the order of a subset of the antigenic peptides. For example, if there are 20 antigenic peptides (ordered 1-20), the randomizing can comprise randomizing the order of all 20 peptides or can comprise randomizing the order of only a subset of the peptides (e.g., peptides 1-5 or 6-10). Such randomization of the order can facilitate secretion and presentation of the fusion polypeptide and of each individual antigenic peptide. Alternatively, the order of the antigenic peptides can be generated using selected parameters, such as a predefined ranking of the antigenic peptides.

[00225] The combination of antigenic peptides or the entire fusion polypeptide (i.e., comprising the antigenic peptides and the PEST-containing peptide and any tags) can also be scored for hydrophobicity. For example, the entirety of the fused antigenic peptides or the entire fusion polypeptide can be scored for hydropathy by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window. If any region scores above a cutoff (e.g., around 1.6), the antigenic peptides can be reordered or shuffled within the fusion polypeptide using selected parameters or using randomization until an acceptable order of antigenic peptides is found (i.e., one in which no region scores above the cutoff). Alternatively, any problematic antigenic peptides can be removed or redesigned to be of a different size, or to shift the sequence of the cancer-associated protein included in the antigenic peptide (so long as the antigenic peptide still comprises the recurrent cancer mutation and sufficiently sized flanking sequences). Alternatively or additionally, one or more linkers between antigenic peptides as disclosed elsewhere herein can be added or modified to change the hydrophobicity. As with hydropathy testing for the individual antigenic peptides, other window sizes can be used, or other cutoffs can be used (e.g., depending on the genus or species of the bacteria being used to deliver the fusion polypeptide). In addition, other suitable hydropathy plots or other appropriate scales could be used. [00226] Optionally, the combination of antigenic peptides or the entire fusion polypeptide can be further screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL- 10- inducing T helper epitopes, and so forth) to deselect antigenic peptides or to avoid immunosuppressive influences.

[00227] A nucleic acid encoding a candidate combination of antigenic peptides or fusion polypeptide can then be designed and optimized. For example, the sequence can be optimized for increased levels of translation, duration of expression, levels of secretion, levels of transcription, and any combination thereof. For example, the increase can be 2-fold to 1000-fold, 2-fold to 500-fold, 2-fold to 100-fold, 2-fold to 50-fold, 2-fold to 20-fold, 2-fold to 10-fold, or 3-fold to 5-fold relative to a control, non-optimized sequence.

[00228] For example, the fusion polypeptide or nucleic acid encoding the fusion polypeptide can be optimized for decreased levels of secondary structures possibly formed in the oligonucleotide sequence, or alternatively optimized to prevent attachment of any enzyme that may modify the sequence. Expression in bacterial cells can be hampered, for example, by transcriptional silencing, low mRNA half-life, secondary structure formation, attachment sites of oligonucleotide binding molecules such as repressors and inhibitors, and availability of rare tRNAs pools. The source of many problems in bacterial expressions is found within the original sequence. The optimization of RNAs may include modification of cis acting elements, adaptation of its GC-content, modifying codon bias with respect to non-limiting tRNAs pools of the bacterial cell, and avoiding internal homologous regions. Thus, optimizing a sequence can entail, for example, adjusting regions of very high (> 80%) or very low (< 30%) GC content. Optimizing a sequence can also entail, for example, avoiding one or more of the following cis-acting sequence motifs: internal TATA-boxes, chi-sites, and ribosomal entry sites; AT-rich or GC-rich sequence stretches; repeat sequences and RNA secondary structures; (cryptic) splice donor and acceptor sites; branch points; or a combination thereof. Optimizing expression can also entail adding sequence elements to flanking regions of a gene and/or elsewhere in the plasmid.

[00229] Optimizing a sequence can also entail, for example, adapting the codon usage to the codon bias of host genes (e.g., Listeria monocytogenes genes). For example, the codons below can be used for Listeria monocytogenes. A = GCA G = GGT L = TTA Q = CAA V = GTT

C = TGT H = CAT M = ATG R = CGT W = TGG

D = GAT 1 = ATT N = AAC S = TCT Y = TAT

E = GAA K = AAA P = CCA T = ACA STOP = TAA

F = TTC

[00230] A nucleic acid encoding a fusion polypeptide can be generated and introduced into a delivery vehicle such as a bacteria strain or Listeria strain. Other delivery vehicles may be suitable for DNA immunotherapy or peptide immunotherapy, such as a vaccinia virus or virus-like particle. Once a plasmid encoding a fusion polypeptide is generated and introduced into a bacteria strain or Listeria strain, the bacteria or Listeria strain can be cultured and characterized to confirm expression and secretion of the fusion polypeptide comprising the antigenic peptides.

///. Recombinant Fusion Polypeptides Comprising Heteroclitic Antigenic Peptides

[00231 ] Disclosed herein are recombinant fusion polypeptides comprising a PEST- containing peptide fused to two or more antigenic peptides (i.e., in tandem, such as PEST- peptidel-peptide2), wherein each antigenic peptide (e.g., from a cancer-associated protein) comprises a heteroclitic mutation. Also disclosed herein are recombinant fusion polypeptides comprising a PEST-containing peptide fused to two or more antigenic peptides (i.e., in tandem, such as PEST-peptidel-peptide2), wherein each antigenic peptide (e.g., from a cancer-associated protein) comprises a heteroclitic mutation, and wherein at least two of the antigenic peptides comprise different heteroclitic mutations and are fragments of the same cancer-associated protein. Alternatively, each of the antigenic peptides comprises a different heteroclitic mutation from a different cancer-associated protein. Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own PEST-containing peptide (e.g., PESTl-peptidel; PEST2-peptide2). Optionally, some or all of the fragments are non-contiguous fragments of the same cancer-associated protein. Non-contiguous fragments are fragments that do not occur sequentially in a protein sequence (e.g., the first fragment consists of residues 10-30, and the second fragment consists of residues 100-120; or the first fragment consists of residues 10-30, and the second fragment consists of residues 20-40).

[00232] Also disclosed herein are recombinant fusion polypeptides comprising two or more antigenic peptides, wherein each antigenic peptide (e.g., from a cancer-associated protein) comprises a heteroclitic mutation, wherein at least two of the antigenic peptides comprise different heteroclitic mutations and are fragments of the same cancer-associated protein, and wherein the fusion polypeptide does not comprise a PEST-containing peptide. Also disclosed herein are recombinant fusion polypeptides comprising two or more antigenic peptides, wherein each antigenic peptide (e.g., from a cancer-associated protein) comprises a heteroclitic mutation, wherein at least two of the antigenic peptides comprise different heteroclitic mutations and are fragments of the same cancer-associated protein, and wherein the fusion polypeptide does not comprise a PEST-containing peptide. Alternatively, each of the antigenic peptides comprises a different heteroclitic mutation from a different cancer- associated protein. Optionally, some or all of the fragments are non-contiguous fragments of the same cancer-associated protein.

[00233] Also provided herein are recombinant fusion polypeptides comprising from N- terminal end to C-terminal end a bacterial secretion sequence, a ubiquitin (Ub) protein, and two or more antigenic peptides (i.e., in tandem, such as Ub-peptidel-peptide2), wherein each antigenic peptide (e.g., from a cancer-associated protein) comprises a heteroclitic mutation. Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own secretion sequence and Ub protein (e.g., Ubl-peptidel ; Ub2-peptide2). Optionally, some or all of the fragments are non-contiguous fragments of the same cancer-associated protein.

[00234] Nucleic acids (termed minigene constructs) encoding such recombinant fusion polypeptides are also disclosed. Such minigene nucleic acid constructs can further comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a minigene nucleic acid construct can further comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acid constructs, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.

[00235] The bacterial signal sequence can be a Listerial signal sequence, such as an Hly or an ActA signal sequence, or any other known signal sequence. In other cases, the signal sequence can be an LLO signal sequence. The signal sequence can be bacterial, can be native to a host bacterium (e.g., Listeria monocytogenes, such as a secAl signal peptide), or can be foreign to a host bacterium. Specific examples of signal peptides include an Usp45 signal peptide from Lactococcus lactis, a Protective Antigen signal peptide from Bacillus anthracis, a secA2 signal peptide such the p60 signal peptide from Listeria monocytogenes, and a Tat signal peptide such as a B. subtilis Tat signal peptide (e.g., PhoD). In specific examples, the secretion signal sequence is from a Listeria protein, such as an ActA₃oo secretion signal or an ActAioo secretion signal.

[00236] The ubiquitin can be, for example, a full-length protein. The ubiquitin expressed from the nucleic acid construct provided herein can be cleaved at the carboxy terminus from the rest of the recombinant fusion polypeptide expressed from the nucleic acid construct through the action of hydrolases upon entry to the host cell cytosol. This liberates the amino terminus of the fusion polypeptide, producing a peptide in the host cell cytosol.

[00237] Selection of, variations of, and arrangement of antigenic peptides within a fusion polypeptide are discussed in detail elsewhere herein, and cancer-associated proteins are discussed in more detail elsewhere herein. The recombinant fusion polypeptides can comprise one or more tags as disclosed in more detail elsewhere herein.

[00238] The recombinant fusion polypeptides disclosed herein can be expressed by recombinant Listeria strains or can be expressed and isolated from other vectors and cell systems used for protein expression and isolation. Recombinant Listeria strains comprising expressing such antigenic peptides can be used, for example in immunogenic compositions comprising such recombinant Listeria and in vaccines comprising the recombinant Listeria strain and an adjuvant. Expression of one or more antigenic peptides as a fusion polypeptides with a nonhemolytic truncated form of LLO, ActA, or a PEST-like sequence in host cell systems in Listeria strains and host cell systems other than Listeria can result in enhanced immunogenicity of the antigenic peptides.

[00239] The recombinant fusion polypeptide can be any molecular weight. For example, the recombinant fusion polypeptide can be less than or no more than about 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, or 125 kilodaltons (kDa). In a specific example, the recombinant fusion polypeptide is less than or no more than about 150 kDa or less than or no more than about 130 kDa. As another example the recombinant fusion polypeptide can be between about 50-200, 50-195, 50-190, 50-185, 50-180, 50-175, 50-170, 50-165, 50-160, 50-155, 50-150, 50-145, 50-140, 50-135, 50-130, 50-125, 100-200, 100-195, 100-190, 100-185, 100-180, 100-175, 100-170, 100-165, 100-160, 100-155, 100-150, 100- 145, 100-140, 100-135, 100-130, or 100-125 kDa. In a specific example, the recombinant fusion polypeptide is between about 50-150, 100-150, 50-125, or 100-125 kDa. As another example, the recombinant fusion polypeptide can be at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 kDa. As a specific example, the recombinant fusion polypeptide can be at least about 100 kDa.

[00240] Nucleic acids encoding such recombinant fusion polypeptides are also disclosed. The nucleic acid can be in any form. The nucleic acid can comprise or consist of DNA or RNA, and can be single- stranded or double-stranded. The nucleic acid can be in the form of a plasmid, such as an episomal plasmid, a multicopy episomal plasmid, or an integrative plasmid. Alternatively, the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome. Such nucleic acids can have one open reading frame or can have two or more open reading frames (e.g., an open reading frame encoding the recombinant fusion polypeptide and a second open reading frame encoding a metabolic enzyme). In one example, such nucleic acids can comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a nucleic acid can comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acids, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.

A. Heteroclitic Antigenic Peptides

[00241 ] Each heteroclitic antigenic peptide can be a fragment of a cancer-associated protein (i.e., a contiguous sequence of amino acids from a cancer-associated protein) comprising a heteroclitic mutation. Each heteroclitic antigenic peptide can be of any length sufficient to induce an immune response, and each heteroclitic antigenic peptide can be the same length or the heteroclitic antigenic peptides can have different lengths. For example, a heteroclitic antigenic peptide disclosed herein can be 5-100, 15-50, or 21-27 amino acids in length, or 15-100, 15-95, 15-90, 15-85, 15-80, 15-75, 15-70, 15-65, 15-60, 15-55, 15-50, 15- 45, 15-40, 15-35, 15-30, 20-100, 20-95, 20-90, 20-85, 20-80, 20-75, 20-70, 20-65, 20-60, 20- 55, 20-50, 20-45, 20-40, 20-35, 20-30, 11-21, 15-21, 21-31, 31-41, 41-51, 51-61, 61-71, 71- 81, 81-91, 91-101, 101-121, 121-141, 141-161, 161-181, 181-201, 8-27, 10-30, 10-40, 15-30, 15-40, 15-25, 1-10, 10-20, 20-30, 30-40, 1-100, 5-75, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 1- 75, 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 8-11, or 11-16 amino acids in length. For example, a heteroclitic antigenic peptide can be 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, or 60 amino acids in length. For example, a heteroclitic antigenic peptide can be 8-100, 8-50, 8-30, 8-25, 8-22, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 7-11, or 8-10 amino acids in length. In one example, a heteroclitic antigenic peptide can be 9 amino acids in length.

[00242] Each heteroclitic antigenic peptide can also be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria monocytogenes or another bacteria of interest. For example, heteroclitic antigenic peptides can be scored by a Kyte and Doolittle hydropathy index 21 amino acid window, and all scoring above a cutoff (around 1.6) can be excluded as they are unlikely to be secretable by Listeria monocytogenes.

[00243] Each heteroclitic antigenic peptide can comprise a single heteroclitic mutation or can comprise two or more heteroclitic mutations (e.g., two heteroclitic mutations).

Exemplary heteroclitic mutant peptides are provided in the following table along with the corresponding wild type (native) peptides. The residues in the wild type peptides that are modified in the corresponding heteroclitic peptides are bolded and underlined.

[00244] Table 140. Heteroclitic Antigenic Peptides and Corresponding Native

Peptides.

Peptide

Heteroclitic Peptide Native Peptide

(GENE_HLA Type)

STEAP1_A0201 LLLGTIHAV (SEQ ID NO: 799) LLLGTIHAL (SEQ ID NO: 311)

STEAP1_A2402 KYKKFPWWL (SEQ ID NO: 800) KYKKFPHWL (SEQ ID NO: 312)

SURVIVIN_A0201 KMSSGCAFL (SEQ ID NO: 818) KHSSGCAFL (SEQ ID NO: 317)

SURVIVIN_A2402 SWFKNWPFF (SEQ ID NO: 819) STFKNWPFL (SEQ ID NO: 318)

[00245] The heteroclitic antigenic peptides can be linked together in any manner. For example, the heteroclitic antigenic peptides can be fused directly to each other with no intervening sequence. Alternatively, the heteroclitic antigenic peptides can be linked to each other indirectly via one or more linkers, such as peptide linkers. In some cases, some pairs of adjacent heteroclitic antigenic peptides can be fused directly to each other, and other pairs of heteroclitic antigenic peptides can be linked to each other indirectly via one or more linkers. The same linker can be used between each pair of adjacent heteroclitic antigenic peptides, or any number of different linkers can be used between different pairs of adjacent heteroclitic antigenic peptides. In addition, one linker can be used between a pair of adjacent heteroclitic antigenic peptides, or multiple linkers can be used between a pair of adjacent heteroclitic antigenic peptides.

[00246] Any suitable sequence can be used for a peptide linker. As an example, a linker sequence may be, for example, from 1 to about 50 amino acids in length. Some linkers may be hydrophilic. The linkers can serve varying purposes. For example, the linkers can serve to increase bacterial secretion, to facilitate antigen processing, to increase flexibility of the fusion polypeptide, to increase rigidity of the fusion polypeptide, or any other purpose. In some cases, different amino acid linker sequences are distributed between the heteroclitic antigenic peptides or different nucleic acids encoding the same amino acid linker sequence are distributed between the heteroclitic antigenic peptides (e.g., SEQ ID NOS: 572-582) in order to minimize repeats. This can also serve to reduce secondary structures, thereby allowing efficient transcription, translation, secretion, maintenance, or stabilization of the nucleic acid (e.g., plasmid) encoding the fusion polypeptide within a Lm recombinant vector strain population. Other suitable peptide linker sequences may be chosen, for example, based on one or more of the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the heteroclitic antigenic peptides; and (3) the lack of hydrophobic or charged residues that might react with the functional epitopes. For example, peptide linker sequences may contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al. (1985) Gene 40:39-46; Murphy et al. (1986) Proc Natl Acad Sci USA 83:8258-8262; US Pat. No. 4,935,233; and US 4,751,180, each of which is herein incorporated by reference in its entirety for all purposes. Specific examples of linkers are disclosed elsewhere herein.

[00247] The fusion polypeptide can comprise any number of heteroclitic antigenic peptides. In some cases, the fusion polypeptide comprises any number of heteroclitic antigenic peptides such that the fusion polypeptide is able to be produced and secreted from a recombinant Listeria strain. For example, the fusion polypeptide can comprise at least 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 heteroclitic antigenic peptides, or 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5- 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 heteroclitic antigenic polypeptides. In another example, the fusion polypeptide can include a single heteroclitic antigenic peptide. In another example, the fusion polypeptide can include a number of heteroclitic antigenic peptides ranging from about 1-100, 1-5, 5-10, 10-15, 15-20, 10-20, 20- 30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 5-15, 5-20, 5-25, 15-20, 15-25, 15-30, 15-35, 20-25, 20-35, 20-45, 30-45, 30-55 ,40-55, 40-65, 50-65, 50-75, 60-75, 60-85, 70-85, 70-95, 80-95, 80-105, 95-105, 50-100, 1-100, 5-100, 5-75, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 1-100, 1-75, 1-50, 1-40, 1-30, 1-20, 1-15, or 1-10 heteroclitic antigenic peptides. In another example, the fusion polypeptide can include up to about 100, 10, 20, 30, 40, or 50 heteroclitic antigenic peptides. In another example, the fusion polypeptide can comprise about 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, or 100 heteroclitic antigenic peptides.

[00248] In addition, the fusion polypeptide can comprise any number of heteroclitic antigenic peptides from the same cancer-associated protein (i.e., any number of noncontiguous fragments from the same cancer-associated protein). Alternatively, the fusion polypeptide can comprise any number of heteroclitic antigenic peptides from two or more different cancer-associated proteins, such as from 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer-associated proteins. For example, the fusion polypeptide can comprise heteroclitic mutations from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cancer-associated proteins, or 2-5, 5-10, 10-15, or 15-20 cancer-associated proteins. For example, the two or more cancer-associated proteins can be about 2-30, about 2-25, about 2-20, about 2-15, or about 2-10 cancer-associated proteins. For example, the fusion polypeptide can comprise at least 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 heteroclitic antigenic peptides from the same cancer-associated protein, or 2-50, 2- 45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35- 40, 40-45, or 45-50 heteroclitic antigenic polypeptides from the same cancer-associated protein. Likewise, the fusion polypeptide can comprise at least 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 heteroclitic antigenic peptides from the same cancer-associated protein, or 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 heteroclitic antigenic polypeptides from two or more different cancer-associated proteins. In addition, the fusion polypeptide can comprise any number of non-contiguous heteroclitic antigenic peptides from the same cancer-associated protein (i.e., any number of non-contiguous fragments from the same cancer-associated protein). For example, the fusion polypeptide can comprise at least 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 non-contiguous heteroclitic antigenic peptides from the same cancer- associated protein, or 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 non-contiguous heteroclitic antigenic polypeptides from the same cancer-associated protein. In some cases, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the heteroclitic antigenic peptides are non-contiguous heteroclitic antigenic peptides from the same cancer- associated protein, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the heteroclitic antigenic peptides that are from a single cancer- associated protein are non-contiguous heteroclitic antigenic peptides from that cancer- associated protein.

[00249] Each heteroclitic antigenic peptide can comprise a different (i.e., unique) heteroclitic mutation. Alternatively, two or more of the heteroclitic antigenic peptides in the fusion polypeptide can comprise the same heteroclitic mutation. For example, two or more copies of the same heteroclitic antigenic polypeptide can be included in the fusion

polypeptide (i.e., the fusion polypeptide comprises two or more copies of the same heteroclitic antigenic peptide). In some fusion polypeptides, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the heteroclitic antigenic peptides comprise a different (i.e., unique) heteroclitic mutation that is not present in any of the other heteroclitic antigenic peptides. [00250] In some cases, at least two of the heteroclitic antigenic peptides can comprise overlapping fragments of the same cancer-associated protein. For example, two or more of the heteroclitic antigenic peptides can comprise different heteroclitic mutations at the same amino acid residue of the cancer-associated protein.

[00251 ] Some heteroclitic antigenic peptides can comprise at least two different heteroclitic mutations, at least three different heteroclitic mutations, or at least four different heteroclitic mutations.

[00252] Any combination of heteroclitic mutations can be included in the fusion polypeptide. For example, heteroclitic antigenic peptides can be included that bind to one or more different HLA types. For example, heteroclitic antigenic peptides can be identified that bind to one or more or all of the following HLA types: HLA-A*02:01, HLA-A*03:01, HLA- A*24:02, and HLA-B*07:02.

[00253] Each of the heteroclitic antigenic peptides in the fusion polypeptide can comprise a heteroclitic mutation from the same cancer-associated protein, or the combination of heteroclitic antigenic peptides in the fusion polypeptide can comprise heteroclitic mutations from two or more cancer-associated proteins. For example, the fusion polypeptide can comprise heteroclitic mutations from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cancer-associated proteins, or 2-5, 5-10, 10-15, or 15-20 cancer-associated proteins. For example, the two or more cancer-associated proteins can be about 2-30, about 2-25, about 2-20, about 2-15, or about 2-10 cancer-associated proteins. In one example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the heteroclitic antigenic peptides comprise a heteroclitic mutation from the same cancer- associated protein. In another example, none of the heteroclitic antigenic peptides comprise a heteroclitic mutation from the same cancer-associated protein.

[00254] Exemplary sequences of heteroclitic antigenic peptides are disclosed elsewhere herein. As an example, a heteroclitic antigenic peptide can comprise, consist essentially of, or consist of a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the antigenic peptide sequences disclosed herein.

B. Cancer- Associated Proteins and Heteroclitic Mutations

[00255] The fusion polypeptides disclosed herein comprise antigenic peptides comprising heteroclitic mutations from cancer-associated proteins. Any combination of heteroclitic mutations disclosed herein can be included in a fusion polypeptide. The term "cancer- associated protein" in the context of heteroclitic peptides refers to proteins whose expression is correlated with the occurrence or progression of one or more types of cancer. Optionally, such proteins includes proteins having mutations that occur in multiple types of cancer, that occur in multiple subjects having a particular type of cancer, or that are correlated with the occurrence or progression of one or more types of cancer. For example, a cancer-associated protein can be an oncogenic protein (i.e., a protein with activity that can contribute to cancer progression, such as proteins that regulate cell growth), or it can be a tumor-suppressor protein (i.e., a protein that typically acts to alleviate the potential for cancer formation, such as through negative regulation of the cell cycle or by promoting apoptosis). Preferably, a cancer-associated protein from which a heteroclitic peptide is derived is a protein that is expressed in a particular type of cancer but is not normally expressed in healthy adult tissue (i.e., a protein with cancer- specific expression, cancer-restricted expression, tumor- specific expression, or tumor-restricted expression). However, a cancer-associated protein does not have to have cancer-specific, cancer-restricted, tumor-specific, or tumor-restricted expression. Examples of proteins that are considered cancer- specific or cancer-restricted are cancer testis antigens or oncofetal antigens. Cancer testis antigens (CTAs) are a large family of tumor-associated antigens expressed in human tumors of different histological origin but not in normal tissue, except for male germ cells. In cancer, these developmental antigens can be re-expressed and can serve as a locus of immune activation. Oncofetal antigens (OFAs) are proteins that are typically present only during fetal development but are found in adults with certain kinds of cancer. The tumor-restricted pattern of expression of CTAs and OFAs make them ideal targets for tumor- specific immunotherapy. Most OFA/CTA proteins play critical roles in oncogenesis.

[00256] The term "heteroclitic" refers to a peptide that generates an immune response that recognizes the native peptide from which the heteroclitic peptide was derived (e.g., the peptide not containing the anchor residue mutations). For example, YLMPVNSEV (SEQ ID NO: 726) was generated from YMMPVNSEV (SEQ ID NO: 725) by mutation of residue 2 to methionine. A heteroclitic peptide can generate an immune response that recognizes the native peptide from which the heteroclitic peptide was derived. For example, the immune response against the native peptide generated by vaccination with the heteroclitic peptide can be equal or greater in magnitude than the immune response generated by vaccination with the native peptide. The immune response can be increased, for example, by 2-fold, 3-fold, 5- fold, 7-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 100-fold, 150-fold, 200-fold, 300- fold, 500-fold, 1000-fold, or more. [00257] A heteroclitic peptide disclosed herein can bind to one or more human leukocyte antigens (HLA) molecules. HLA molecules, also known as major histocompatibility complex (MHC) molecules, bind peptides and present them to immune cells. The immunogenicity of a peptide can be partially determined by its affinity for HLA molecules. HLA class I molecules interact with CD8 molecules, which are generally present on cytotoxic T lymphocytes (CTL). HLA class II molecules interact with CD4 molecules, which are generally present on helper T lymphocytes. For example, a heteroclitic peptide disclosed herein can bind to an HLA molecule with sufficient affinity to activate a T cell precursor or with sufficient affinity to mediate recognition by a T cell.

[00258] A heteroclitic peptide disclosed herein can bind to one or more HLA class II molecules. For example, a heteroclitic peptide can bind to an HLA-DRB molecule, an HLA- DRA molecule, an HLA-DQA1 molecule, an HLA-DQB 1 molecule, an HLA-DPA1 molecule, an HLA-DPB 1 molecule, an HLA-DMA molecule, an HLA-DMB molecule, an HLA-DOA molecule, or an HLA-DOB molecule.

[00259] A native or heteroclitic peptide disclosed herein can bind to one or more HLA class I molecules. For example, a heteroclitic peptide can bind to an HLA-A molecule, an HLA-B molecule, an HLA-C molecule, an HLA-A0201 molecule, HLA Al, HLA A2, HLA A2.1, HLA A3, HLA A3.2, HLA Al l, HLA A24, HLA B7, HLA B27, or HLA B8.

Similarly, a heteroclitic peptide can bind to a superfamily of HLA class I molecules, such as the A2 superfamily, the A3 superfamily, the A24 superfamily, the B7 superfamily, the B27 superfamily, the B44 superfamily, the CI superfamily, or the C4 superfamily.

[00260] Heteroclitic peptides can comprise a mutation that enhances binding of the peptide to an HLA class II molecule relative to the corresponding native peptide. Alternatively, or additionally, heteroclitic peptides can comprise a mutation that enhances binding of the peptide to an HLA class I molecule relative to the corresponding native peptide. For example, the mutated residue can be an HLA class II motif anchor residue. "Anchor motifs" or "anchor residues" refers, in another embodiment, to one or a set of preferred residues at particular positions in an HLA-binding sequence (e.g., an HLA class II binding sequence or an HLA class I binding sequence).

[00261 ] Various methods are well-known for generating predicted heteroclitic epitopes with the potential to elicit cross-reactive immunogenic responses to a wild-type epitope. For example, to design heteroclitic epitopes with the potential to elicit cross-reactive

immunogenic responses to a wild-type epitope, baseline predicted peptide-MHC binding affinity of the wild-type epitopes can be determined using NetMHCpan 3.0 Server (www.cbs.dtu.dk/services/NetMHCpan/). A peptide-MHC binding affinity percent rank of less than or equal to 1.0 is considered a strong binder that is likely to elicit an immune response. Potential heteroclitic epitopes are generated by random substitution of 1 or more amino acids at, but not limited to, positions 1, 2, 3, or the C-terminal position of the wild-type epitope that is predicted to be a strong binder. The peptide-MHC binding affinity of the potential heteroclitic epitopes is then estimated using NetMHCpan 3.0 Server. Heteroclitic epitopes with percentage ranking binding affinities similar to wild-type epitopes and less than or equal to 1.0 percentage rank can be considered potential antigens for future validation.

[00262] Other methods for identifying HLA class I and class II residues, and for improving HLA binding by mutating the residues, are well-known. See, e.g., US 8,765,687, US 7,488,718, US 9,233,149, and US 7,598,221, each of which is herein incorporated by reference in its entirety for all purposes. For example, methods for predicting MHC class II epitopes are well-known. As one example, the MHC class II epitope can be predicted using TEPITOPE (Meister et al. (1995) Vaccine 13:581-591, herein incorporated by reference in its entirety for all purposes). As another example, the MHC class II epitope can be predicted using EpiMatrix (De Groot et al. (1997) AIDS Res. Hum. Retroviruses 13:529-531, herein incorporated by reference in its entirety for all purposes). As yet another example, the MHC class II epitope can be predicted using the Predict Method (Yu K et al. (2002) Mol. Med. 8: 137-148, herein incorporated by reference in its entirety for all purposes). As yet another example, the MHC class II epitope can be predicted using the SYFPEITHI epitope prediction algorithm. SYFPEITHI is a database comprising more than 4500 peptide sequences known to bind class I and class II MHC molecules. SYFPEITHI provides a score based on the presence of certain amino acids in certain positions along the MHC-binding groove. Ideal amino acid anchors are valued at 10 points, unusual anchors are worth 6-8 points, auxiliary anchors are worth 4-6 points, preferred residues are worth 1-4 points; negative amino acid effect on the binding score between -1 and -3. The maximum score for HLA-A*0201 is 36. As yet another example, the MHC class II epitope can be predicted using Rankpep. Rankpep uses position specific scoring matrices (PSSMs) or profiles from sets of aligned peptides known to bind to a given MHC molecule as the predictor of MHC-peptide binding. Rankpep includes information on the score of the peptide and the % optimum or percentile score of the predicted peptide relative to that of a consensus sequence that yields the maximum score, with the selected profile. Rankpep includes a selection of 102 and 80 PSSMs for the prediction of peptide binding to MHC I and MHC II molecules, respectively. Several PSSMs for the prediction of peptide binders of different sizes are usually available for each MHC I molecule. As another example, the MHC class II epitope can be identified using SVMHC (Donnes and Elofsson (2002) BMC Bio informatics 11; 3:25, herein incorporated by reference in its entirety for all purposes).

[00263] Methods for identifying MHC class I epitopes are also well-known. As one example, the MHC class I epitope can be predicted using BIMAS software. A BIMAS score is based on the calculation of the theoretical half-life of the MHC-I/p2-microglobulin/peptide complex, which is a measure of peptide-binding affinity. The program uses information about HLA-I peptides of 8-10 amino acids in length. The higher the binding affinity of a peptide to the MHC, the higher the likelihood that this peptide represents an epitope. The BIMAS algorithm assumes that each amino acid in the peptide contributes independently to binding to the class I molecule. Dominant anchor residues, which are critical for binding, have coefficients in the tables that are significantly higher than 1. Unfavorable amino acids have positive coefficients that are less than 1. If an amino acid is not known to make either a favorable or unfavorable contribution to binding, then it is assigned the value 1. All the values assigned to the amino acids are multiplied and the resulting running score is multiplied by a constant to yield an estimate of half-time of dissociation. As another example, the MHC class I epitope can be identified using SYFPEITHI. As yet another example, the MHC class I epitope can be identified using SVMHC. As yet another example, the MHC class I epitope can be identified using NetMHC-2.0 (Buus et al. (2003) Tissue Antigens 62:378-384, herein incorporated by reference in its entirety for all purposes).

[00264] Different residues in HLA binding motifs can be mutated to enhance MHC binding. In one example, a mutation that enhances MHC binding is in the residue at position 1 of the HLA class I binding motif (e.g., a mutation to tyrosine, glycine, threonine, or phenylalanine). As another example, the mutation can be in position 2 of the HLA class I binding motif (e.g., a mutation to leucine, valine, isoleucine, or methionine). As another example, the mutation can be in position 6 of the HLA class I binding motif (e.g., to valine, cysteine, glutamine, or histidine). As another example, the mutation can be in position 9 of the HLA class I binding motif or in the C-terminal position (e.g., to valine, threonine, isoleucine, leucine, alanine, or cysteine). The mutation can be in a primary anchor residue or in a secondary anchor residue. For example, the HLA class I primary anchor residues can be positions 2 and 9, and the secondary anchor residues can be positions 1 and 8 or positions 1, 3, 6, 7, and 8. In another example, a point mutation can be in a position selected from positions 4, 5, and 8. [00265] Similarly, different residues in HLA class II binding sites can be mutated. For example, an HLA class II motif anchor residue can be modified. For example, the PI position, the P2 position, the P6 position, or the P9 position can be mutated. Alternatively, theP4 position, the P5 position, the P10 position, the PI 1 position, the P12 position, or the P13 position can be mutated.

[00266] The term "cancer-associated protein" includes proteins having mutations that occur in multiple types of cancer, that occur in multiple subjects having a particular type of cancer, or that are correlated with the occurrence or progression of one or more types of cancer. For example, a cancer-associated protein can be an oncogenic protein (i.e., a protein with activity that can contribute to cancer progression, such as proteins that regulate cell growth), or it can be a tumor-suppressor protein (i.e., a protein that typically acts to alleviate the potential for cancer formation, such as through negative regulation of the cell cycle or by promoting apoptosis.

[00267] For example, the cancer-associated protein can be any one of the cancer- associated proteins listed elsewhere herein. For example, the cancer-associated protein can be encoded by one of the following genes: CEACAM5, GAGE1, hTERT, KLHL7, MAGEA3, MAGEA4, MAGEA6, NUF2, NYESOl, PAGE4, PRAME, PSA, PSMA, RNF43, SART3, SSX2, STEAP1, and SURVIVIN.

[00268] The fusion polypeptides disclosed herein can comprise heteroclitic antigenic peptides comprising any combination of heteroclitic mutations from any combination of cancer-associated proteins (i.e., one or more cancer-associated proteins) and in any order. The combination of heteroclitic antigenic peptides or the fusion polypeptide can be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria monocytogenes or another bacteria of interest. In some cases, the heteroclitic antigenic peptides can be from multiple cancer-associated proteins (e.g., two or more cancer-associated proteins).

[00269] As one example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESOl, STEAP1, and RNF43. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, non-small cell lung cancer (NSCLC). The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the heteroclitic antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the heteroclitic antigenic peptides in Table 36.

[00270] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all of the following genes: CEACAM5, MAGEA4, STEAPl, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, prostate cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroclitic antigenic peptides in Table 53.

[00271 ] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes:

CEACAM5, STEAPl, MAGEA3, PRAME, hTERT, and SURVIVIN. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, pancreatic cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12 of the heteroclitic antigenic peptides in Table 69.

[00272] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, GAGE1, NYESOl, RNF43, NUF2, KLHL7, MAGEA3, and PRAME. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, bladder cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroclitic antigenic peptides in Table 77.

[00273] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes:

CEACAM5, STEAP1, RNF43, MAGEA3, PRAME, and hTERT. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, breast cancer (e.g., ER+ breast cancer). The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the heteroclitic antigenic peptides in Table 88.

[00274] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, ore or all of the following genes: CEACAM5, PRAME, hTERT, STEAP1, RNF43, NUF2, KLHL7, and SART3. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, uterine cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroclitic antigenic peptides in Table 96.

[00275] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, STEAP1, RNF43, SART3, NUF2, KLHL7, PRAME, and hTERT. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, ovarian cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroclitic antigenic peptides in Table 101.

[00276] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, MAGEA6, STEAP1, RNF43, SART3, NUF2, KLHL7, and hTERT. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, low-grade glioma. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroclitic antigenic peptides in Table 105.

[00277] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESOl, STEAP1, RNF43, and MAGEA3. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, colorectal cancer (e.g., MSS colorectal cancer). The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroclitic antigenic peptides in Table 109.

[00278] As another example, the cancer-associated proteins can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes:

CEACAM5, MAGEA4, STEAP1, NYESOl, PRAME, and hTERT. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, head and neck cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroclitic antigenic peptides in Table 113.

C. PEST- Containing Peptides

[00279] The recombinant fusion proteins disclosed herein comprise a PEST-containing peptide. The PEST-containing peptide may at the amino terminal (N-terminal) end of the fusion polypeptide (i.e., N-terminal to the antigenic peptides), may be at the carboxy terminal (C-terminal) end of the fusion polypeptide (i.e., C-terminal to the antigenic peptides), or may be embedded within the antigenic peptides. In some recombinant Listeria strains and methods, a PEST containing peptide is not part of and is separate from the fusion

polypeptide. Fusion of antigenic peptides to a PEST-like sequence, such as an LLO peptide, can enhance the immunogenicity of the antigenic peptides and can increase cell-mediated and antitumor immune responses (i.e., increase cell- mediated and anti-tumor immunity). See, e.g., Singh et al. (2005) J Immunol 175(6):3663-3673, herein incorporated by reference in its entirety for all purposes. PEST-containing peptides are disclosed in more detail elsewhere herein.

D. Generating Immunotherapy Constructs Encoding Recombinant Fusion Polypeptides

[00280] Also provided herein are methods for generating immunotherapy constructs encoding or compositions comprising the recombinant fusion polypeptides disclosed herein. For example, such methods can comprise selecting a set of heteroclitic mutations to include in the immunotherapy construct, designing a heteroclitic antigenic peptides comprising each of the heteroclitic mutations (and, for example, testing the hydropathy of the each heteroclitic antigenic peptide, and modifying or deselecting a heteroclitic antigenic peptide if it scores above a selected hydropathy index threshold value), selecting one or more sets of heteroclitic antigenic peptides, designing one or more fusion polypeptides comprising each of the selected heteroclitic antigenic peptides, and generating a nucleic acid construct encoding the fusion polypeptide.

[00281 ] Individual heteroclitic mutations can be selected based on any criteria as discussed in further detail elsewhere herein. For example, individual heteroclitic mutations or heteroclitic peptides can be selected if they are known to generate CD8+ T lymphocyte responses.

[00282] After identification of a set of possible heteroclitic mutations to include in a fusion polypeptide, sequences for heteroclitic antigenic peptides comprising each heteroclitic mutation can be selected. Different size antigenic peptides can be used, as disclosed elsewhere herein. For example, heteroclitic mutations or heteroclitic antigenic peptides can be focused, for example, on MHC Class I epitopes consisting of 9 amino acids.

[00283] The sequence of the heteroclitic antigenic peptide can then be optimized to enhance binding to MHC Class I molecules. To optimize binding to each HLA, the Peptide MHC Binding Motif and Amino Acid Binding Chart can be assessed from the Immune Epitope Database and Analysis Resource (for example: iedb.org/MHCalleleid/143). The preferred amino acids at the anchor positions can be inserted into the heteroclitic antigenic peptide sequence (e.g., NUF2 - wild type: YMMPVNSEV (SEQ ID NO: 725); and NUF2 - heteroclitic: YLMPVNSEV (SEQ ID NO: 726)).

[00284] The binding affinities of sequence-optimized heteroclitic antigenic peptides can then be assessed, for example, using one of the following algorithms: NetMHC4.0 Server; NetMHCpan4.0 Server; and mhcflurry vO.2.0. The heteroclitic antigenic peptides can be considered, for example, if predicting binding affinity to a specific HLA is equivalent or stronger than the corresponding native sequence. Selected sequence-optimized heteroclitic antigenic peptides can then be screened for in vitro binding to specific HLAs using

Pro Immune' s REVEAL assay. For example, heteroclitic antigenic peptides with binding affinity >= 45% of the REVEAL assay's positive control peptide were considered binders.

[00285] The RNA expression level of heteroclitic antigenic peptides can also be measured in a specific-indication in TCGA RNAseq V2 dataset. The percentage of TCGA samples with normalized RNA expression reads greater than 0 can be calculated. Heteroclitic antigenic peptides with TCGA expression in a majority of samples can be prioritized.

[00286] Such methods can also comprise, for example, testing the hydropathy of each heteroclitic antigenic peptide, and modifying or deselecting a heteroclitic antigenic peptide if it scores above a selected hydropathy index threshold value), designing one or more fusion polypeptides comprising each of the selected heteroclitic antigenic peptides, and generating a nucleic acid construct encoding the fusion polypeptide. Such methods are disclosed in more detail elsewhere herein.

[00287] In a specific example, a literature review can be done to survey the genomic landscape of indication- specific tumor-associated antigens to generate a short-list of potential TAAs. A second literature review can be done to determine if short-list TAAs contain known immunogenic peptides that generate CD8+ T lymphocyte response. This approach can focus, for example, primarily on MHC Class I epitopes consisting of 9 amino acids (9mer) from TAAs. This step can, for example, identify potential target peptides in 9mer format that bind to one of four HLAs types (HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, and HLA- B*07:02).

[00288] Target peptides can then be sequence optimized to enhance binding to MHC Class I molecules (aka heteroclitic peptide). To optimize binding to each HLA, the Peptide MHC Binding Motif and Amino Acid Binding Chart can be assessed from the Immune Epitope Database and Analysis Resource (for example: iedb.org/MHCalleleid/143). The preferred amino acids at the anchor positions can be inserted into the target peptide sequence (e.g., NUF2 - wild type: YMMPVNSEV (SEQ ID NO: 725); and NUF2 - heteroclitic:

YLMPVNSEV (SEQ ID NO: 726)). The binding affinities of sequence-optimized target peptides and wild-type target peptides can then be assessed, e.g., using one of the following algorithms: NetMHC4.0 Server; NetMHCpan4.0 Server; and mhcflurry vO.2.0. Sequence- optimized target peptides can be considered, for example, if predicting binding affinity to a specific HLA is equivalent or stronger than the wild-type target peptide sequence. Selected sequence-optimized target peptides can then be screened for in vitro binding to specific HLAs using Pro Immune' s REVEAL assay. For example, target peptides with binding affinity >= 45% of the REVEAL assay's positive control peptide can be considered binders. Finally, the RNA expression level of target peptides can be measured in a specific-indication in TCGA RNAseqV2 dataset. For example, the percentage of TCGA samples with normalized RNA expression reads greater than 0 can be calculated. For example, target peptides with TCGA expression in a majority of samples can be prioritized.

IV. Recombinant Fusion Polypeptides Encoded by Minigene Constructs

[00289] Disclosed herein are recombinant fusion polypeptides comprising from N-terminal end to C-terminal end a bacterial secretion signal sequence, a ubiquitin (Ub) protein, and an antigenic peptide (or one or more antigenic peptides) from a cancer-associated protein. If two or more antigenic peptides are included, the antigenic peptides can be in tandem (e.g., Ub-peptidel-peptide2). Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own secretion sequence and Ub protein (e.g., Ubl-peptidel ; Ub2-peptide2). Examples of suitable antigenic peptides are disclosed elsewhere herein. The antigenic peptides can comprise recurrent cancer mutations as disclosed elsewhere herein. Alternatively, the antigenic peptides can comprise heteroclitic mutations as disclosed elsewhere herein.

[00290] Nucleic acids (termed minigene constructs) encoding such recombinant fusion polypeptides are also disclosed. Such minigene nucleic acid constructs can further comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a minigene nucleic acid construct can further comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different fusion polypeptide comprising from N-terminal end to C-terminal end a bacterial secretion sequence, a ubiquitin (Ub) protein, and one or more antigenic peptides. The codon encoding the carboxy terminus of the fusion polypeptide can be followed by two stop codons to ensure termination of protein synthesis.

[00291 ] In some fusion polypeptides encoded by minigene constructs, there are one or more additional antigenic peptides from cancer-associated proteins (e.g., comprising a recurrent cancer mutation and/or a heteroclitic mutation) between the bacterial secretion sequence and the ubiquitin protein. For example, there can be 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additional antigenic peptides between the bacterial secretion sequence and the ubiquitin protein. If there are two or more additional antigenic peptides, they can be fused directly to each other or linked via a peptide linker. Exemplary linkers are disclosed elsewhere herein. The additional antigenic peptides can comprise one or more antigenic peptides comprising recurrent cancer mutations and/or one or more heteroclitic antigenic peptides. Examples of such peptides are disclosed elsewhere herein.

[00292] Examples of bacterial secretion signal sequences are disclosed in more detail elsewhere herein. The ubiquitin can be, for example, a full-length protein. An exemplary ubiquitin peptide encoded by a minigene construct comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 747. The ubiquitin expressed from the nucleic acid construct provided herein can be cleaved at the carboxy terminus of the ubiquitin from the rest of the recombinant fusion polypeptide expressed from the nucleic acid construct through the action of hydrolases upon entry to the host cell cytosol. This liberates the rest of the fusion polypeptide, producing a peptide in the host cell cytosol.

[00293] Selection of, variations of, and arrangement of antigenic peptides within a fusion polypeptide are discussed in detail elsewhere herein, and methods of generating heteroclitic mutant antigenic peptides are discussed in more detail elsewhere herein. The recombinant fusion polypeptides can comprise one or more tags as disclosed elsewhere herein. For example, the recombinant fusion polypeptides can comprise one or more peptide tags N- terminal and/or C-terminal to the one or more antigenic peptides or to the ubiquitin (e.g., N- terminal to the ubiquitin). A tag can be fused directly to an antigenic peptide or ubiquitin or linked to an antigenic peptide or ubiquitin via a linker (examples of which are disclosed elsewhere herein).

[00294] The recombinant fusion polypeptides disclosed herein can be expressed by recombinant Listeria strains or can be expressed and isolated from other vectors and cell systems used for protein expression and isolation. Recombinant Listeria strains comprising expressing such antigenic peptides can be used, for example in immunogenic compositions comprising such recombinant Listeria and in vaccines comprising the recombinant Listeria strain and an adjuvant.

[00295] Nucleic acids (minigene constructs) encoding such recombinant fusion

polypeptides are also disclosed. The nucleic acid can be in any form. The nucleic acid can comprise or consist of DNA or RNA, and can be single- stranded or double-stranded. The nucleic acid can be in the form of a plasmid, such as an episomal plasmid, a multicopy episomal plasmid, or an integrative plasmid. Alternatively, the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome. Such nucleic acids can have one open reading frame or can have two or more open reading frames (e.g., an open reading frame encoding the recombinant fusion polypeptide and a second open reading frame encoding a metabolic enzyme). In one example, such nucleic acids can comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a nucleic acid can comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. The codon encoding the carboxy terminus of the fusion polypeptide can be followed by two stop codons to ensure termination of protein synthesis.

[00296] Some exemplary antigenic peptides for inclusion in minigene constructs include those in the table below.

A. Antigenic Peptides Encoded by Minigene Constructs

[00297] Antigenic peptides encoded by the minigene constructs disclosed herein can be recurrent cancer mutation antigenic peptides and/or heteroclitic antigenic peptides (e.g., HLA class I and class II heteroclitic peptides). Examples of such peptides are disclosed elsewhere herein. For example, the antigenic peptide encoded by a minigene construct can be a heteroclitic antigenic peptide that binds to one or more of the following HLA types: HLA- A*02:01, HLA-A*03:01, HLA-A*24:02, and HLA-B*07:02. As a specific example, the antigenic peptide encoded by the minigene construct can be from a protein encoded by one of the following genes: STEAP1, CEACAM5, NYESOl, and NUF2.

[00298] The fusion polypeptide encoded by the minigene construct can include a single antigenic peptide or can include two or more antigenic peptides. Each antigenic peptide can be of any length sufficient to induce an immune response, and each antigenic peptide can be the same length or the antigenic peptides can have different lengths. For example, an antigenic peptide encoded by a minigene construct can be 8-100, 8-50, 8-30, 8-25, 8-22, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 7-11, or 8-10 amino acids in length. In one example, an antigenic peptide can be 9 amino acids in length.

[00299] Each antigenic peptide can also be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria

monocytogenes or another bacteria of interest. For example, antigenic peptides can be scored by a Kyte and Doolittle hydropathy index 21 amino acid window, and all scoring above a cutoff (around 1.6) can be excluded as they are unlikely to be secretable by Listeria monocytogenes. Likewise, the combination of antigenic peptides or the fusion polypeptide can be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria monocytogenes or another bacteria of interest.

[00300] If the fusion polypeptide includes more than one antigenic peptide, the antigenic peptides can be linked together in any manner. For example, the antigenic peptides can be fused directly to each other with no intervening sequence. Alternatively, the antigenic peptides can be linked to each other indirectly via one or more linkers, such as peptide linkers. In some cases, some pairs of adjacent antigenic peptides can be fused directly to each other, and other pairs of antigenic peptides can be linked to each other indirectly via one or more linkers. The same linker can be used between each pair of adjacent antigenic peptides, or any number of different linkers can be used between different pairs of adjacent antigenic peptides. In addition, one linker can be used between a pair of adjacent antigenic peptides, or multiple linkers can be used between a pair of adjacent antigenic peptides. Any suitable sequence can be used for a peptide linker. Examples of suitable linkers are disclosed elsewhere herein.

[00301 ] Exemplary sequences of antigenic peptides for use in minigene constructs are disclosed elsewhere herein. As an example, an antigenic peptide can comprise, consist essentially of, or consist of a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the antigenic peptide sequences disclosed herein.

B. Bacterial Secretion Signal Sequences

[00302] The bacterial secretion signal sequence can be a Listerial signal sequence, such as an Hly or an ActA signal sequence, or any other known signal sequence. In other cases, the signal sequence can be an LLO signal sequence. An exemplary LLO signal sequence is set forth in SEQ ID NO: 920. For example, a bacterial secretion signal sequence encoded by a minigene construct herein can be an N-terminal fragment of LLO such as that set forth in SEQ ID NO: 336. The signal sequence can be bacterial, can be native to a host bacterium (e.g., Listeria monocytogenes, such as a secAl signal peptide), or can be foreign to a host bacterium. Specific examples of signal peptides include an Usp45 signal peptide from Lactococcus lactis, a Protective Antigen signal peptide from Bacillus anthracis, a secA2 signal peptide such the p60 signal peptide from Listeria monocytogenes, and a Tat signal peptide such as a B. subtilis Tat signal peptide (e.g., PhoD). In specific examples, the secretion signal sequence is from a Listeria protein, such as an ActA₃oo secretion signal or an ActAioo secretion signal (comprising the first 100 amino acids of the ActA secretion signal sequence). An exemplary ActA signal sequence is set forth in SEQ ID NO: 921.

C. Generating Immunotherapy Constructs Encoding Recombinant Fusion Polypeptides Encoded by Minigene Constructs

[00303] Also provided herein are methods for generating immunotherapy constructs encoding or compositions comprising the recombinant fusion polypeptides disclosed herein. For example, such methods can comprise selecting and designing antigenic or immunogenic peptides to include in the immunotherapy construct (and, for example, testing the hydropathy of each antigenic peptide, and modifying or deselecting an antigenic peptide if it scores above a selected hydropathy index threshold value), designing one or more fusion polypeptides comprising each of the selected antigenic peptides, and generating a nucleic acid construct encoding the fusion polypeptide. Such methods are disclosed in more detail elsewhere herein. In addition, methods for generating predicted heteroclitic epitopes with the potential to elicit cross-reactive immunogenic responses to a wild-type epitope are described in more detail elsewhere herein.

V. Recombinant Fusion Polypeptides Comprising Combinations of Recurrent Cancer Mutation Antigenic Peptides, Heteroclitic Antigenic Peptides, and Minigene- Construct- Encoded Peptides

[00304] The recombinant fusion polypeptides disclosed herein can comprise any combination of antigenic peptides comprising any of the recurrent cancer mutations disclosed herein, antigenic peptides (e.g., from cancer-associated proteins) comprising any of the heteroclitic mutations disclosed herein, and antigenic peptides (e.g., from cancer-associated proteins) expressed from any of the minigene constructs disclosed herein (i.e., antigenic peptides fused to ubiquitin). Any of the antigenic peptides disclosed herein can be included in a recombinant fusion polypeptide. For example, the recombinant fusion polypeptides can comprise recurrent cancer mutation antigenic peptides only, heteroclitic antigenic peptides only, or minigene construct antigenic peptides only. Similarly, the recombinant fusion polypeptides can comprise both recurrent cancer mutation antigenic peptides and heteroclitic antigenic peptides but no minigene construct antigenic peptides. Similarly, the recombinant fusion polypeptides can comprise both recurrent cancer mutation antigenic peptides and minigene construct antigenic peptides but no heteroclitic antigenic peptides. Similarly, the recombinant fusion polypeptides can comprise both heteroclitic antigenic peptides and minigene construct antigenic peptides but no recurrent cancer mutation antigenic peptides.

[00305] For example, disclosed herein are recombinant fusion polypeptides comprising a PEST-containing peptide fused to two or more antigenic peptides (i.e., in tandem, such as PEST-peptidel-peptide2), wherein at least one antigenic peptide comprises a recurrent cancer mutation, and at least one antigenic peptide (e.g., from a cancer-associated protein) comprises a heteroclitic mutation. Also herein are recombinant fusion polypeptides comprising a PEST- containing peptide fused to two or more antigenic peptides (i.e., in tandem, such as PEST- peptidel-peptide2), wherein at least one antigenic peptide comprises a recurrent cancer mutation, and at least one antigenic peptide comprises a heteroclitic mutation, and wherein the fusion polypeptide does not comprise a PEST-containing peptide. Examples of recurrent cancer mutations and heteroclitic mutations are disclosed elsewhere herein.

[00306] Also disclosed herein are recombinant fusion polypeptides comprising from N- terminal end to C-terminal end a PEST-containing peptide comprising a bacterial secretion signal sequence, one or more antigenic peptides comprising a recurrent cancer mutation, a ubiquitin (Ub) protein, and an antigenic peptide (or one or more antigenic peptides) from a cancer-associated protein. If two or more antigenic peptides are included at the C-terminal end, the antigenic peptides can be in tandem (e.g., Ub-peptidel-peptide2). Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own secretion sequence and Ub protein (e.g., Ubl -peptide 1 ; Ub2-peptide2).

Examples of suitable antigenic peptides are disclosed elsewhere herein. Examples of antigenic peptides comprising recurrent cancer mutations are disclosed elsewhere herein.

[00307] Also disclosed herein are recombinant fusion polypeptides comprising from N- terminal end to C-terminal end a PEST-containing peptide comprising a bacterial secretion signal sequence, one or more antigenic peptides (e.g., from a cancer-associated protein) comprising a heteroclitic mutation, a ubiquitin (Ub) protein, and an antigenic peptide (or one or more antigenic peptides) from a cancer-associated protein. If two or more antigenic peptides are included at the C-terminal end, the antigenic peptides can be in tandem (e.g., Ub- peptidel-peptide2). Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own secretion sequence and Ub protein (e.g., Ubl-peptidel ; Ub2-peptide2). Examples of suitable antigenic peptides are disclosed elsewhere herein. Examples of antigenic peptides comprising heteroclitic mutations are disclosed elsewhere herein.

[00308] Also disclosed herein are recombinant fusion polypeptides comprising from N- terminal end to C-terminal end a PEST-containing peptide comprising a bacterial secretion signal sequence, two or more antigenic peptides (wherein at least one antigenic peptide comprises a recurrent cancer mutation, and at least one antigenic peptide (e.g., from a cancer- associated protein) comprises a heteroclitic mutation), a ubiquitin (Ub) protein, and an antigenic peptide (or one or more antigenic peptides) from a cancer-associated protein. If two or more antigenic peptides are included at the C-terminal end, the antigenic peptides can be in tandem (e.g., Ub-peptidel-peptide2). Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own secretion sequence and Ub protein (e.g., Ubl-peptidel ; Ub2-peptide2). Examples of suitable antigenic peptides are disclosed elsewhere herein. Examples of antigenic peptides comprising recurrent cancer mutations are disclosed elsewhere herein. Examples of antigenic peptides comprising heteroclitic mutations are disclosed elsewhere herein.

[00309] The recombinant fusion polypeptides can comprise one or more tags as disclosed in more detail elsewhere herein. Selection of and examples of recurrent cancer mutation antigenic peptides, heteroclitic antigenic peptides, and minigene construct antigenic peptides are disclosed elsewhere herein. Selection of, variations of, and arrangement of antigenic peptides within a fusion polypeptide are discussed in detail elsewhere herein, and cancer- associated proteins are discussed in more detail elsewhere herein. Examples of PEST- containing peptides and bacterial secretion signal sequences are disclosed elsewhere herein. Generation of immunotherapy constructs encoding such recombinant fusion polypeptides is disclosed elsewhere herein.

[00310] The recombinant fusion polypeptides disclosed herein can be expressed by recombinant Listeria strains or can be expressed and isolated from other vectors and cell systems used for protein expression and isolation. Recombinant Listeria strains comprising expressing such antigenic peptides can be used, for example in immunogenic compositions comprising such recombinant Listeria and in vaccines comprising the recombinant Listeria strain and an adjuvant. Expression of one or more antigenic peptides as a fusion polypeptides with a nonhemolytic truncated form of LLO, ActA, or a PEST-like sequence in host cell systems in Listeria strains and host cell systems other than Listeria can result in enhanced immunogenicity of the antigenic peptides.

[00311 ] The fusion polypeptide can comprise any number of antigenic peptides. In some cases, the fusion polypeptide comprises any number of antigenic peptides such that the fusion polypeptide is able to be produced and secreted from a recombinant Listeria strain. For example, the fusion polypeptide can comprise at least 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 antigenic peptides, or 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 antigenic polypeptides. In another example, the fusion polypeptide can include a single antigenic peptide. In another example, the fusion polypeptide can include a number of antigenic peptides ranging from about 1-100, 1-5, 5-10, 10-15, 15-20, 10-20, 20- 30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 5-15, 5-20, 5-25, 15-20, 15-25, 15-30, 15-35, 20-25, 20-35, 20-45, 30-45, 30-55 ,40-55, 40-65, 50-65, 50-75, 60-75, 60-85, 70-85, 70-95, 80-95, 80-105, 95-105, 50-100, 1-100, 5-100, 5-75, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 1-100, 1-75, 1-50, 1-40, 1-30, 1-20, 1-15, or 1-10 antigenic peptides. In another example, the fusion polypeptide can include up to about 100, 10, 20, 30, 40, or 50 antigenic peptides. In another example, the fusion polypeptide can comprise about 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, or 100 antigenic peptides.

[00312] In another example, the fusion polypeptide can comprise at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 antigenic peptides or between about 5-50, 10-40, or 20-30 antigenic peptides. For example, the fusion polypeptide can comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic peptides comprising a recurrent cancer mutation or between about 5 to about 30 or about 10 to about 20 antigenic peptides comprising a recurrent cancer mutation and/or can comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic peptides comprising a heteroclitic mutation or between about 5 to about 30 or about 10 to about 20 antigenic peptides comprising a heteroclitic mutation.

[00313] The antigenic peptides can be from any number of cancer-associated proteins. For example, the fusion polypeptide can comprise antigenic peptides from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cancer-associated proteins, or 2-5, 5-10, 10- 15, or 15-20 cancer-associated proteins. For example, the cancer-associated proteins can be about 2-30, about 2-25, about 2-20, about 2-15, or about 2-10 cancer-associated proteins.

[00314] In fusion polypeptides comprising two or more antigenic peptides comprising a recurrent cancer mutation and/or two or more antigenic peptides comprising a heteroclitic mutation, the antigenic peptides comprising a recurrent cancer mutation can be in tandem, and the antigenic peptides comprising a heteroclitic mutation can be in tandem.

Alternatively, the antigenic peptides comprising a recurrent cancer mutation and the antigenic peptides comprising a heteroclitic mutation can be intermixed within the fusion polypeptide.

[00315] Components within a fusion polypeptide may be fused directly to each other or linked via linkers (e.g., peptide linkers) as disclosed in more detail elsewhere herein. For example, the peptide linkers used can comprise flexibility linkers and/or rigidity linkers and/or immunoproteasome linkers or can comprise one or more of the linkers set forth in SEQ ID NOS: 310-319 and 821-829 (e.g., to link two or more antigenic peptides). In one examples, the peptide linker upstream of each antigenic peptide comprising a heteroclitic mutation is an immunoproteasome linker or is selected from the linkers set forth in SEQ ID NOS: 821-829.

[00316] The VGKGGSGG linker (SEQ ID NO: 314) can be used, for example, as a longer linker after the tLLO and also before the tag sequences to provide additional space between the tLLO and the antigenic portion of the fusion peptide and before the tag sequences. It also can provide flexibility and to charge balance the fusion protein. The EAAAK linker (SEQ ID NO: 316) is a rigid/stiff linker that can be used to facilitate expression and secretion, for example, if the fusion protein would otherwise fold on itself. The GGGGS linker (SEQ ID NO: 313) is a flexible linker that can be used, for example, to add increased flexibility to the fusion protein to help facilitate expression and secretion. The "i20" linkers (e.g., SEQ ID NOS: 821-829) are immunoproteasome linkers that are designed, for example, to help facilitate cleavage of the fusion protein by the immunoproteasome and increase the frequency of obtaining the exact minimal binding fragment that is desired. Combinations of GGGGS and EAAAK linkers (SEQ ID NOS: 313 and 316, respectively) can be used, for example, to alternate flexibility and rigidity to help balance the construct for improved expression and secretion and to help facilitate DNA synthesis by providing more unique codons to choose from.

[00317] The recombinant fusion polypeptide can be any molecular weight. For example, the recombinant fusion polypeptide can be less than or no more than about 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, or 125 kilodaltons (kDa). In a specific example, the recombinant fusion polypeptide is less than or no more than about 150 kDa or less than or no more than about 130 kDa. As another example the recombinant fusion polypeptide can be between about 50-200, 50-195, 50-190, 50-185, 50-180, 50-175, 50-170, 50-165, 50-160, 50-155, 50-150, 50-145, 50-140, 50-135, 50-130, 50-125, 100-200, 100-195, 100-190, 100-185, 100-180, 100-175, 100-170, 100-165, 100-160, 100-155, 100-150, 100- 145, 100-140, 100-135, 100-130, or 100-125 kDa. In a specific example, the recombinant fusion polypeptide is between about 50-150, 100-150, 50-125, or 100-125 kDa. As another example, the recombinant fusion polypeptide can be at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 kDa. As a specific example, the recombinant fusion polypeptide can be at least about 100 kDa.

[00318] Nucleic acids encoding such recombinant fusion polypeptides are also disclosed. The nucleic acid can be in any form. The nucleic acid can comprise or consist of DNA or RNA, and can be single- stranded or double-stranded. The nucleic acid can be in the form of a plasmid, such as an episomal plasmid, a multicopy episomal plasmid, or an integrative plasmid. Alternatively, the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome. Such nucleic acids can have one open reading frame or can have two or more open reading frames (e.g., an open reading frame encoding the recombinant fusion polypeptide and a second open reading frame encoding a metabolic enzyme). In one example, such nucleic acids can comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a nucleic acid can comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acids, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.

[00319] The fusion polypeptides disclosed herein can comprise antigenic peptides from any combination of cancer-associated proteins (i.e., one or more cancer-associated proteins) and in any order. The combination of antigenic peptides or the fusion polypeptide can be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria monocytogenes or another bacteria of interest. In some cases, the antigenic peptides can be from multiple cancer-associated proteins (e.g., two or more cancer-associated proteins).

[00320] As one example, the cancer-associated proteins from which recurrent cancer mutation peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: KRAS, EGFR, U2AF1, BRAF, PIK3CA, and TP53. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the following recurrent cancer mutations: KRAS_G12C, EGFR_L858R, KRAS_G12D, U2AF1_S34F, BRAF_V600E, KRAS_G12V, PIK3CA_E545K, TP53_R158L,

KRAS_G12A, EGFR_L861Q, and TP53_R273L. Such mutations are associated with, for example, non-small cell lung cancer (NSCLC). The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the antigenic peptides in Table 35. The cancer-associated proteins from which heteroclitic antigenic peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESOl, STEAP1, and RNF43. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, non-small cell lung cancer (NSCLC). The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the heteroclitic antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the heteroclitic antigenic peptides in Table 36. In a specific example, the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by CEACAM5. For example, the minigene antigenic peptide can comprise SEQ ID NO: 798 or SEQ ID NO: 796. In one example, the antigenic peptides in the fusion polypeptide can comprise one or more or all of the peptides set forth in Table 35 and Table 36. Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprise, consist essentially of, or consist of sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequence set forth in SEQ ID NO: 859; SEQ ID NO: 860; SEQ ID NO: 861; SEQ ID NO: 862; SEQ ID NO: 863; SEQ ID NO: 864; SEQ ID NO: 865; SEQ ID NO: 894; SEQ ID NO: 895; SEQ ID NO: 905, SEQ ID NO: 909, SEQ ID NO: 910, SEQ ID NO: 911, or SEQ ID NO: 912. A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 38-51.

[00321 ] As another example, the cancer-associated proteins from which recurrent cancer mutation peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, or all of the following genes: SPOP, CHEK2, RGPD8, ANKRD36C, and AR. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all of the following recurrent cancer mutations: SPOP_F133V,

CHEK2_K373E, RGPD8_P1760A, ANKRD36C_I634T, ANKRD36C_D629Y,

SPOP_W131G, ANKRD36C_D626N, SPOP_F133L, AR_T878A, AR_L702H, AR_W742C, AR_H875Y, and AR_F877L. Such mutations are associated with, for example, prostate cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all of the antigenic peptides in Table 52. The cancer- associated proteins from which heteroclitic antigenic peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer- associated proteins are associated with, for example, prostate cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. . The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroclitic antigenic peptides in Table 53. In a specific example, the cancer- associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAPl. For example, the minigene antigenic peptide can comprise SEQ ID NO: 799 or SEQ ID NO: 800. In one example, the antigenic peptides in the fusion polypeptide can comprise one or more or all of the peptides set forth in Table 52 and Table 54. Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprise, consist essentially of, or consist of sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequence set forth in SEQ ID NO: 871; SEQ ID NO: 872; SEQ ID NO: 873; SEQ ID NO: 874; SEQ ID NO: 875; SEQ ID NO: 876; SEQ ID NO: 877; SEQ ID NO: 892; SEQ ID NO: 893; SEQ ID NO: 906, SEQ ID NO: 913, SEQ ID NO: 914, SEQ ID NO: 915, or SEQ ID NO: 916. A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 54-67.

[00322] As another example, the cancer-associated proteins from which recurrent cancer mutation peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, or all of the following genes: KRAS, U2AF1, TP53, SMAD4, and GNAS. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the following recurrent cancer mutations: KRAS_G12C, KRAS_G12D, U2AF1_S34F, KRAS_G12V, TP53_R248Q, TP53_R248W, TP53_R175H, TP53_R273C, KRAS_G12R, KRAS_Q61H, TP53_R282W, TP53_R273H, TP53_G245S, SMAD4_R361C, GNAS_R201C, and GNAS_R201H. Such mutations are associated with, for example, pancreatic cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the antigenic peptides in Table 68. The cancer-associated proteins from which heteroclitic antigenic peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, STEAPl, MAGEA3, PRAME, hTERT, and SURVIVIN. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, pancreatic cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12 of the heteroclitic antigenic peptides in Table 69. In a specific example, the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by CEACAM5. For example, the minigene antigenic peptide can comprise SEQ ID NO: 798 or SEQ ID NO: 796. In one example, the antigenic peptides in the fusion polypeptide can comprise one or more or all of the peptides set forth in Table 68 and Table 69. Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprise, consist essentially of, or consist of sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequence set forth in SEQ ID NO: 866; SEQ ID NO: 867; SEQ ID NO: 868; SEQ ID NO: 869; SEQ ID NO: 870; or SEQ ID NO: 908. A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 70-75.

[00323] As another example, the cancer-associated proteins from which recurrent cancer mutation peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: PIK3CA, FGFR3, TP53, RXRA, FBXW7, and NFE2L2. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or all of the following recurrent cancer mutations:

PIK3CA_E545K, FGFR3_S249C, TP53_R248Q, PIK3CA_E542K, RXRA_S427F, FBXW7_R505G, TP53_R280T, NFE2L2_E79K, FGFR3_R248C, TP53_K132N,

TP53_R248W, TP53_R175H, and TP53_R273C. Such mutations are associated with, for example, bladder cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or all of the antigenic peptides in Table 76. The cancer- associated proteins from which heteroclitic antigenic peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, GAGE1, NYESOl, RNF43, NUF2, KLHL7, MAGEA3, and PRAME. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, bladder cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroclitic antigenic peptides in Table 77. In a specific example, the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by NYESOl or NUF2. For example, the minigene antigenic peptide can comprise SEQ ID NO: 797 or SEQ ID NO: 800. In one example, the antigenic peptides in the fusion polypeptide can comprise one or more or all of the peptides set forth in Table 76 and Table 77. Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprise, consist essentially of, or consist of sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequence set forth in SEQ ID NO: 878; SEQ ID NO: 879; SEQ ID NO: 880; SEQ ID NO: 881; SEQ ID NO: 882; SEQ ID NO: 888; SEQ ID NO: 889; SEQ ID NO: 890; or SEQ ID NO: 891. A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 78-86.

[00324] As another example, the cancer-associated proteins from which recurrent cancer mutation peptides are generated can comprise proteins encoded by 1 or more, 2 or more, or all of the following genes: PIK3CA, AKT1, and ESR1. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all of the following recurrent cancer mutations: PIK3CA_E545K, PIK3CA_E542K, PIK3CA_H1047R,

AKT1_E17K, PIK3CA_H1047L, PIK3CA_Q546K, PIK3CA_E545A, PIK3CA_E545G, ESR1_K303R, ESR1_D538G, ESR1_Y537S, ESR1_Y537N, ESR1_Y537C, and ESR1_E380Q. Such mutations are associated with, for example, breast cancer (e.g., ER+ breast cancer). The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all of the antigenic peptides in Table 87. The cancer-associated proteins from which heteroclitic antigenic peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, STEAP1, RNF43, MAGEA3, PRAME, and hTERT. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, breast cancer (e.g., ER+ breast cancer). The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the heteroclitic antigenic peptides in Table 88. In a specific example, the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAP1. For example, the minigene antigenic peptide can comprise SEQ ID NO: 799 or SEQ ID NO: 800. In one example, the antigenic peptides in the fusion polypeptide can comprise one or more or all of the peptides set forth in Table 87 and Table 88. Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprise, consist essentially of, or consist of sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequence set forth in SEQ ID NO: 883; SEQ ID NO: 884; SEQ ID NO: 885; SEQ ID NO: 886; SEQ ID NO: 887; or SEQ ID NO: 907. A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 89-94. [00325] As another example, the cancer-associated proteins from which recurrent cancer mutation peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: PTEN, KRAS, PIK3CA, CTNNB1, FBXW7, and TP53. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the following recurrent cancer mutations: PTEN_R130G, PTEN_R130Q, KRAS_G12D, KRAS_G12V,

PIK3CA_H1047R; PIK3CA_R88Q, PIK3CA_E545K, PIK3CA_E542K, CTNNB 1_S37F, KRAS_G13D, CTNNB 1_S37C, PIK3CA_H1047L, PIK3CA_G118D, KRAS_G12A, FBXW7_R505C, and TP53_R248W. Such mutations are associated with, for example, uterine cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all of the antigenic peptides in Table 95. The cancer-associated proteins from which heteroclitic antigenic peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, PRAME, hTERT, STEAP1, RNF43, NUF2, KLHL7, and SART3. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, uterine cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroclitic antigenic peptides in Table 96. In a specific example, the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAP1. For example, the minigene antigenic peptide can comprise SEQ ID NO: 799 or SEQ ID NO: 800. In one example, the antigenic peptides in the fusion polypeptide can comprise one or more or all of the peptides set forth in Table 95 and Table 96. Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprise, consist essentially of, or consist of sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequence set forth in SEQ ID NO: 896; SEQ ID NO: 897; or SEQ ID NO: 904. A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 97-99.

[00326] As another example, the cancer-associated proteins from which recurrent cancer mutation peptides are generated can comprise TP53. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all of the following recurrent cancer mutations: TP53_R248Q, TP53_R248W, TP53_R175H, TP53_R273C, TP53_R282W, TP53_R273H, TP53_Y220C, TP53_I195T, TP53_C176Y, TP53_H179R, TP53_S241F, and TP53_H193R. Such mutations are associated with, for example, ovarian cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all of the antigenic peptides in Table 100. The cancer-associated proteins from which heteroclitic antigenic peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, STEAP1, RNF43, SART3, NUF2, KLHL7, PRAME, and hTERT. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, ovarian cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroclitic antigenic peptides in Table 101. In a specific example, the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAP1. For example, the minigene antigenic peptide can comprise SEQ ID NO: 799. In one example, the antigenic peptides in the fusion polypeptide can comprise one or more or all of the peptides set forth in Table 100 and Table 101. Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprise, consist essentially of, or consist of sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequence set forth in SEQ ID NO: 898 or 899. A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 102-103.

[00327] As another example, the cancer-associated proteins from which recurrent cancer mutation peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, or all of the following genes: TP53, PIK3CA, IDHl, IDH2, and EGFR. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the following recurrent cancer mutations: TP53_R273L, TP53_R273C, TP53_R273H, PIK3CA_G118D, IDH1_R132C, IDH1_R132G, IDH1_R132H, IDH1_R132S, IDH2_R172K,

PIK3CA_E453K, and EGFR_G598V. Such mutations are associated with, for example, low- grade glioma. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the antigenic peptides in Table 104. The cancer-associated proteins from which heteroclitic antigenic peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, MAGEA6, STEAP1, RNF43, SART3, NUF2, KLHL7, and hTERT. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, low-grade glioma. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroclitic antigenic peptides in Table 105. In a specific example, the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by NUF2. For example, the minigene antigenic peptide can comprise SEQ ID NO: 807. In one example, the antigenic peptides in the fusion polypeptide can comprise one or more or all of the peptides set forth in Table 104 and Table 105. Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprise, consist essentially of, or consist of sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequence set forth in SEQ ID NO: 900 or SEQ ID NO: 901. A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 106-107.

[00328] As another example, the cancer-associated proteins from which recurrent cancer mutation peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, or all of the following genes: KRAS, BRAF, PIK3CA, and TP53. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all of the following recurrent cancer mutations: KRAS_G12C, KRAS_G12D, BRAF_V600E, KRAS_G12V, PIK3CA_E545K, TP53_R248W, TP53_R175H, TP53_R273C, PIK3CA_H1047R,

TP53_R282W, TP53_R273H, and KRAS_G13D. Such mutations are associated with, for example, colorectal cancer (e.g., MSS colorectal cancer). The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all of the antigenic peptides in Table 108. The cancer-associated proteins from which heteroclitic antigenic peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESOl, STEAP1, RNF43, and MAGEA3. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer- associated proteins are associated with, for example, colorectal cancer (e.g., MSS colorectal cancer). The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroclitic antigenic peptides in Table 109. In a specific example, the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAP1. For example, the minigene antigenic peptide can comprise SEQ ID NO: 799. In one example, the antigenic peptides in the fusion polypeptide can comprise one or more or all of the peptides set forth in Table 108 and Table 109. Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprise, consist essentially of, or consist of sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequence set forth in SEQ ID NO: 902 or SEQ ID NO: 903. A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 110-111.

[00329] As another example, the cancer-associated proteins from which recurrent cancer mutation peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all of the following genes: PIK3CA, CHEK2, RGPD8, ANKRD36C, TP53, ZNF814, KRTAP1-5, KRTAP4-11, and HRAS. The antigenic peptides can comprise, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or all of the following recurrent cancer mutations: PIK3CA_E545K, CHEK2_K373E, RGPD8_P1760A, ANKRD36C_I634T, TP53_R248Q, PIK3CA_E542K, TP53_R248W, TP53_R175H, PIK3CA_H1047R,

TP53_R282W, TP53_R273H, TP53_G245S, TP53_Y220C, ZNF814_D404E, KRTAP1- 5_I88T, KRTAP4-11_L161V, and HRAS_G13V. Such mutations are associated with, for example, head and neck cancer. The mutations can be in any order. The antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 21-mers (e.g., 21-mers linked together by linkers), each including the naturally occurring 10 amino acids flanking each side of the recurrent cancer mutation. Examples of such antigenic peptides are provided in Example 11. The antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or all of the antigenic peptides in Table 112. The cancer-associated proteins from which heteroclitic antigenic peptides are generated can comprise proteins encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, MAGEA4, STEAPl, NYESOl, PRAME, and hTERT. The heteroclitic antigenic peptides can bind, for example, one or more or all of HLA types A0201, A0301, A2402, and B0702. Such cancer-associated proteins are associated with, for example, head and neck cancer. The heteroclitic antigenic peptides can be in any order. The heteroclitic antigenic peptides can be fused directly together or linked together by linkers, examples of which are disclosed elsewhere herein. In a specific example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by linkers). Examples of such heteroclitic antigenic peptides are provided in Example 11. The heteroclitic antigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroclitic antigenic peptides in Table 113. In a specific example, the cancer- associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAPl. For example, the minigene antigenic peptide can comprise SEQ ID NO: 799. In one example, the antigenic peptides in the fusion polypeptide can comprise one or more or all of the peptides set forth in Table 112 and Table 113. Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) comprise, consist essentially of, or consist of sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of the sequence set forth in SEQ ID NO: 918 or SEQ ID NO: 919. A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 114-115.

[00330] Also provided herein are methods for generating immunotherapy constructs encoding or compositions comprising the recombinant fusion polypeptides disclosed herein. For example, such methods can comprise selecting and designing antigenic or immunogenic peptides to include in the immunotherapy construct (and, for example, testing the hydropathy of each antigenic peptide, and modifying or deselecting an antigenic peptide if it scores above a selected hydropathy index threshold value), designing one or more fusion polypeptides comprising each of the selected antigenic peptides, and generating a nucleic acid construct encoding the fusion polypeptide. Such methods are disclosed in more detail elsewhere herein. As a specific example, such a method can comprise: (a) selecting a set of recurrent cancer mutations and a set of heteroclitic mutations in cancer-associated proteins to include in the immunotherapy construct; (b) designing antigenic peptides comprising each of the recurrent cancer mutations and each of the heteroclitic mutations; (c) selecting a set of antigenic peptides, comprising testing the hydropathy of the each antigenic peptide, and modifying or deselecting an antigenic peptide if it scores above a selected hydropathy index threshold value; (d) designing a fusion polypeptide comprising each of the selected antigenic peptides; and (e) generating a nucleic acid construct encoding the fusion polypeptide.

[00331 ] The individual selected recurrent cancer mutations can be selected in step (a), for example, based on one or more of the following criteria: (i) frequency of occurrence across multiple types of cancers or a particular type of cancer; (ii) location within a functional domain of a cancer-associated protein; (iii) status as a known cancer driver mutation or chemotherapy resistance mutation; and (iv) identification as a somatic missense mutation or a somatic frameshift mutation. Likewise, the set of recurrent cancer mutations selected in step (a) can be selected based on one or more of the following criteria: (i) the set includes no more than about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 recurrent cancer mutations and/or no more than about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 heteroclitic mutations; (ii) the set includes recurrent cancer mutations that would be found in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients who have a single type of cancer; and (iii) the set comprises at least 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, or 40 different recurrent cancer mutations or recurrent somatic missense mutations from a single type of cancer.

[00332] The individual selected heteroclitic mutations can be selected in step (a), for example, based on one or more of the following criteria: (i) ability to bind to one or more of the following HLA types: HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, and HLA-B*07:02; (ii) ability to generate a CD8+ T lymphocyte response; and (iii) binding affinity to a specific HLA that is equivalent or stronger than the corresponding wild type sequence. Likewise, the set of heteroclitic mutations selected in step (a) can be selected based on collective ability to bind to one or more or all of the following HLA types: HLA-A*02:01, HLA-A*03:01, HLA- A*24:02, and HLA-B*07:02.

[00333] One or more or all of the antigenic peptides designed in step (b) to comprise a recurrent cancer mutation can be designed, for example, to comprise a fragment of the cancer-associated protein comprising the recurrent cancer mutation and flanking sequence on each side. For example, one or more or all of the antigenic peptides comprising a recurrent cancer mutation can include at least about 10 flanking amino acids on each side of the recurrent cancer mutation.

[00334] One or more or all of the antigenic peptides designed in step (b) to comprise a heteroclitic mutation can be designed, for example, to have a preferred amino acid at an anchor position.

[00335] The antigenic peptides can be selected in step (c), for example, if they are below a hydropathy threshold predictive of secretability in Listeria monocytogenes. For example, the antigenic peptides can be scored by a Kyte and Doolittle hydropathy index 21 amino acid window, and any peptides scoring above a cutoff of about 1.6 can be excluded or are modified to score below the cutoff. Likewise, the hydropathy of the fusion polypeptide can be tested, followed by either reordering the antigenic peptides or removing problematic antigenic peptides if any region of the fusion polypeptide scores above a selected hydropathy index threshold value (e.g., a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, wherein the threshold value is about 1.6). In addition, the fusion polypeptide can be designed to have a molecular weight of, for example, no more than about 150 kDa, or no more than about 120 kDa. For example, the recombinant fusion polypeptide can be less than or no more than about 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, or 125 kilodaltons (kDa). In a specific example, the recombinant fusion polypeptide is less than or no more than about 150 kDa or less than or no more than about 130 kDa. As another example the recombinant fusion polypeptide can be between about 50- 200, 50-195, 50-190, 50-185, 50-180, 50-175, 50-170, 50-165, 50-160, 50-155, 50-150, 50- 145, 50-140, 50-135, 50-130, 50-125, 100-200, 100-195, 100-190, 100-185, 100-180, 100- 175, 100-170, 100-165, 100-160, 100-155, 100-150, 100-145, 100-140, 100-135, 100-130, or 100-125 kDa. In a specific example, the recombinant fusion polypeptide is between about 50-150, 100-150, 50-125, or 100-125 kDa. As another example, the recombinant fusion polypeptide can be at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 kDa. As a specific example, the recombinant fusion polypeptide can be at least about 100 kDa. Other parameters for design and selection of antigenic peptides and fusion polypeptides are disclosed in more detail elsewhere herein and can also be used. VI. Recombinant Fusion Polypeptides Comprising Personalized Neoepitopes

[00336] Disclosed herein are recombinant fusion polypeptides comprising a PEST- containing peptide fused to one or more antigenic peptides (i.e., in tandem, such as PEST- peptidel-peptide2), wherein each antigenic peptide comprises a neoepitope present in a cancer sample or tumor sample from a subject (e.g., an altered amino acid sequence encoded by a nonsynonymous mutation in a gene) that is not present in a healthy biological sample (e.g., a healthy biological sample from the subject). PEST-containing peptides suitable for inclusion in the fusion polypeptides are disclosed elsewhere herein. Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own PEST-containing peptide (e.g., PES Tl -peptide 1 ; PEST2-peptide2).

[00337] Also disclosed herein are recombinant fusion polypeptides comprising one or more antigenic peptides, wherein each antigenic peptide comprises a neoepitope present in a cancer cell or tumor cell from a subject that is not present in a healthy cell from the subject, and wherein the fusion polypeptide does not comprise a PEST-containing peptide.

[00338] Also provided herein are recombinant fusion polypeptides comprising from N- terminal end to C-terminal end a bacterial secretion sequence, a ubiquitin (Ub) protein, and two or more antigenic peptides (i.e., in tandem, such as Ub-peptidel-peptide2), wherein each antigenic peptide comprises a neoepitope present in a cancer sample or tumor sample from a subject (e.g., an altered amino acid sequence encoded by a nonsynonymous mutation in a gene) that is not present in a healthy biological sample (e.g., a healthy biological sample from the subject). Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own secretion sequence and Ub protein (e.g., Ub 1 -peptide 1 ; Ub2-peptide2) .

[00339] Nucleic acids (termed minigene constructs) encoding such recombinant fusion polypeptides are also disclosed. Such minigene nucleic acid constructs can further comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a minigene nucleic acid construct can further comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acid constructs, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis. [00340] The bacterial signal sequence can be a Listerial signal sequence, such as an Hly or an ActA signal sequence, or any other known signal sequence. In other cases, the signal sequence can be an LLO signal sequence. The signal sequence can be bacterial, can be native to a host bacterium (e.g., Listeria monocytogenes, such as a secAl signal peptide), or can be foreign to a host bacterium. Specific examples of signal peptides include an Usp45 signal peptide from Lactococcus lactis, a Protective Antigen signal peptide from Bacillus anthracis, a secA2 signal peptide such the p60 signal peptide from Listeria monocytogenes, and a Tat signal peptide such as a B. subtilis Tat signal peptide (e.g., PhoD). In specific examples, the secretion signal sequence is from a Listeria protein, such as an ActA₃oo secretion signal or an ActAioo secretion signal.

[00341 ] The ubiquitin can be, for example, a full-length protein. The ubiquitin expressed from the nucleic acid construct provided herein can be cleaved at the carboxy terminus from the rest of the recombinant fusion polypeptide expressed from the nucleic acid construct through the action of hydrolases upon entry to the host cell cytosol. This liberates the amino terminus of the fusion polypeptide, producing a peptide in the host cell cytosol.

[00342] Selection of, variations of, and arrangement of antigenic peptides within a fusion polypeptide are discussed in detail elsewhere herein.

[00343] The recombinant fusion polypeptides can comprise one or more tags. For example, the recombinant fusion polypeptides can comprise one or more peptide tags N- terminal and/or C-terminal to the combination of the two or more antigenic peptides. A tag can be fused directly to an antigenic peptide or linked to an antigenic peptide via a linker (examples of which are disclosed elsewhere herein). Examples of tags include the following: FLAG tag, 3xFLAG tag; His tag, 6xHis tag; and SIINFEKL tag. An exemplary SIINFEKL tag is set forth in SEQ ID NO: 293 (encoded by any one of the nucleic acids set forth in SEQ ID NOS: 278-292). An exemplary 3xFLAG tag is set forth in SEQ ID NO: 309 (encoded by any one of the nucleic acids set forth in SEQ ID NOS: 294-309). Other tags include chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX), and poly(NANP). Particular recombinant fusion polypeptides comprise a C-terminal SIINFEKL tag. Such tags can allow for easy detection of the recombinant fusion protein, confirmation of secretion of the recombinant fusion protein, or for following the immunogenicity of the secreted fusion polypeptide by following immune responses to these "tag" sequence peptides. Such immune response can be monitored using a number of reagents including, for example, monoclonal antibodies and DNA or RNA probes specific for these tags. [00344] The recombinant fusion polypeptides disclosed herein can be expressed by recombinant Listeria strains or can be expressed and isolated from other vectors and cell systems used for protein expression and isolation. Recombinant Listeria strains comprising expressing such antigenic peptides can be used, for example in immunogenic compositions comprising such recombinant Listeria strains and in vaccines comprising the recombinant Listeria strains and an adjuvant. Expression of one or more antigenic peptides as a fusion polypeptides with a nonhemolytic truncated form of LLO, ActA, or a PEST-like sequence in host cell systems in Listeria strains and host cell systems other than Listeria strains can result in enhanced immunogenicity of the antigenic peptides.

[00345] Nucleic acids encoding such recombinant fusion polypeptides are also disclosed. The nucleic acid can be in any form. The nucleic acid can comprise or consist of DNA or RNA, and can be single- stranded or double-stranded. The nucleic acid can be in the form of a plasmid, such as an episomal plasmid, a multicopy episomal plasmid, or an integrative plasmid. Alternatively, the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome. Such nucleic acids can have one open reading frame or can have two or more open reading frames (e.g., an open reading frame encoding the recombinant fusion polypeptide and a second open reading frame encoding a metabolic enzyme). In one example, such nucleic acids can comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a nucleic acid can comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acids, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.

A. Antigenic Peptides Comprising Personalized Neoepitopes

[00346] Each antigenic peptide can be of any length sufficient to induce an immune response, and each antigenic peptide can be the same length or the antigenic peptides can have different lengths. For example, an antigenic peptide disclosed herein can be 5-100, 15- 50, or 21-27 amino acids in length, or 15-100, 15-95, 15-90, 15-85, 15-80, 15-75, 15-70, 15- 65, 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, 20-100, 20-95, 20-90, 20-85, 20-80, 20- 75, 20-70, 20-65, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 11-21, 15-21, 21-31, 31- 41, 41-51, 51-61, 61-71, 71-81, 81-91, 91-101, 101-121, 121-141, 141-161, 161-181, 181- 201, 8-27, 10-30, 10-40, 15-30, 15-40, 15-25, 1-10, 10-20, 20-30, 30-40, 1-100, 5-75, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 1-75, 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 8-11, or 11-16 amino acids in length. For example, an antigenic peptide can be 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, or 60 amino acids in length. Some specific examples of antigenic peptides are 21 or 27 amino acids in length.

[00347] Each antigenic peptide can also be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria

monocytogenes or another bacteria of interest. For example, antigenic peptides can be scored by a Kyte and Doolittle hydropathy index 21 amino acid window, and all scoring above a cutoff (around 1.6) can be excluded as they are unlikely to be secretable by Listeria monocytogenes.

[00348] Each antigenic peptide can comprise, for example, a single neoepitope comprising a single cancer- specific mutation. Alternatively, an antigenic peptide can comprise two or more neoepitopes or two or more cancer- specific mutations. For example, an antigenic peptide can comprise more than one cancer- specific mutation (e.g., 2 or 3 cancer- specific mutations) because of the close proximity of the mutated residues to each other in a protein.

[00349] Each antigenic peptide can comprise cancer- specific mutation (i.e., a mutation present in a cancer sample from a subject but not a healthy biological sample), such as a cancer- specific mutation caused by a single nonsynonymous mutation. Alternatively, an antigenic peptide can comprise two or more (e.g., at least 2 or at least 3) cancer- specific mutations (e.g., caused by two or more nonsynonymous mutations). The cancer- specific mutation in each antigenic peptide can be flanked on each side by an equal number of amino acids, or can be flanked on each side by a different number of amino acids (e.g., with 9 amino acids flanking N-terminal and 10 amino acids flanking C-terminal, or with 10 amino acids flanking N-terminal and 13 amino acids flanking C-terminal). The flanking sequence on each side of the cancer- specific mutation can be the sequence that naturally flanks the cancer- specific mutation. For example, the cancer- specific mutation in an antigenic peptide can be flanked on each side by an equal number of amino acids, wherein the flanking sequence is identical to the sequences that naturally flanks the cancer- specific mutation in the mutated protein. The number of flanking amino acids on each side of the cancer- specific mutation can be any length, such as 5-30 amino acids flanking each side. As one example, the cancer- specific mutation can be flanked on each side by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acids (e.g., by at least 10 amino acids or by at least 13 amino acids). Preferably, at least about 10 flanking amino acids on each side of the detected cancer- specific mutation are incorporated to accommodate class 1 MHC- 1 presentation, in order to provide at least some of the different HLA T-cell receptor (TCR) reading frames, or at least about 13 flanking amino acids on each side of the detected cancer- specific mutation are incorporated to accommodate class 2 MHC-2 presentation, in order to provide at least some of the different HLA T-cell receptor (TCR) reading frames for CD4+ T cell antigen presentation. However, this does not necessarily need to be the case. In some cases, the location of the cancer- specific mutation in the protein in which it naturally occurs may dictate how many amino acids are flanking on one particular side (e.g., if the mutation is in the first 10 amino acids of the protein or the last 10 amino acids of the protein).

[00350] The antigenic peptides can be linked together in any manner. For example, the antigenic peptides can be fused directly to each other with no intervening sequence.

Alternatively, the antigenic peptides can be linked to each other indirectly via one or more linkers, such as peptide linkers. In some cases, some pairs of adjacent antigenic peptides can be fused directly to each other, and other pairs of antigenic peptides can be linked to each other indirectly via one or more linkers. The same linker can be used between each pair of adjacent antigenic peptides, or any number of different linkers can be used between different pairs of adjacent antigenic peptides. In addition, one linker can be used between a pair of adjacent antigenic peptides, or multiple linkers can be used between a pair of adjacent antigenic peptides.

[00351 ] Any suitable sequence can be used for a peptide linker. As an example, a linker sequence may be, for example, from 1 to about 50 amino acids in length. Some linkers may be hydrophilic. The linkers can serve varying purposes. For example, the linkers can serve to increase bacterial secretion, to facilitate antigen processing, to increase flexibility of the fusion polypeptide, to increase rigidity of the fusion polypeptide, or any other purpose. In some cases, different amino acid linker sequences are distributed between the antigenic peptides in order to minimize repeats, or different nucleic acids encoding the same amino acid linker sequence are distributed between the antigenic peptides (e.g., SEQ ID NOS: 572- 582) in order to minimize repeats. This can also serve to reduce secondary structures, thereby allowing efficient transcription, translation, secretion, maintenance, or stabilization of the nucleic acid (e.g., plasmid) encoding the fusion polypeptide within a Lm recombinant vector strain population. Other suitable peptide linker sequences may be chosen, for example, based on one or more of the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the antigenic peptides; and (3) the lack of hydrophobic or charged residues that might react with the functional epitopes. For example, peptide linker sequences may contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al. (1985) Gene 40:39-46; Murphy et al. (1986) Proc Natl Acad Sci USA 83:8258-8262; US Pat. No. 4,935,233; and US

4,751,180, each of which is herein incorporated by reference in its entirety for all purposes. Specific examples of linkers include those in the following table (each of which can be used by itself as a linker, in a linker comprising repeats of the sequence, or in a linker further comprising one or more of the other sequences in the table), although others can also be envisioned {see, e.g., Reddy Chichili et al. (2013) Protein Science 22: 153-167, herein incorporated by reference in its entirety for all purposes). Unless specified, "n" represents an undetermined number of repeats in the listed linker. Any other linker disclosed elsewhere herein (e.g., SEQ ID NOS: 313-316, 319, and 821-829) can also be used.

[00352] The fusion polypeptide can comprise any number of antigenic peptides. In some cases, the fusion polypeptide comprises any number of antigenic peptides such that the fusion polypeptide is able to be produced and secreted from a recombinant Listeria strain. For example, the fusion polypeptide can comprise at least 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 antigenic peptides, or 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 antigenic polypeptides. In another example, the fusion polypeptide can include a single antigenic peptide or neoepitope. In another example, the fusion polypeptide can include a number of antigenic peptides or neoepitopes ranging from about 1-100, 1-5, 5- 10, 10-15, 15-20, 10-20, 20-30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 5-15, 5-20, 5-25, 15-20, 15-25, 15-30, 15-35, 20-25, 20-35, 20-45, 30-45, 30-55 ,40-55, 40-65, 50-65, 50-75, 60-75, 60-85, 70-85, 70-95, 80-95, 80-105, 95-105, 50-100, 1-100, 5-100, 5-75, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 1-100, 1-75, 1-50, 1-40, 1-30, 1-20, 1-15, or 1-10 antigenic peptides or neoepitopes. In another example, the fusion polypeptide can include up to about 100, 10, 20, 30, 40, or 50 antigenic peptides or neoepitopes. In another example, the fusion polypeptide can comprise about 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, or 100 antigenic peptides or neoepitopes.

[00353] Each antigenic peptide can comprise a different (i.e., unique) neoepitope.

Alternatively, two or more of the antigenic peptides in the fusion polypeptide can comprise the same neoepitope. For example, two or more copies of the same antigenic polypeptide can be included in the fusion polypeptide (i.e., the fusion polypeptide comprises two or more copies of the same antigenic peptide). In some fusion polypeptides, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the antigenic

polypeptides comprise a different (i.e., unique) neoepitope that is not present in any of the other antigenic peptides. In some cases, at least two of the antigenic peptides can comprise overlapping fragments of the same protein.

[00354] An antigenic peptide can comprise at least two different neoepitopes or cancer- specific mutations, at least three different neoepitopes or cancer- specific mutations, or at least four different neoepitopes or cancer- specific mutations.

[00355] Any combination of cancer- specific mutations or neoepitopes can be included in the fusion polypeptide. Each of the cancer- specific mutations can be a somatic missense mutation, or the cancer- specific mutations can comprise other mutations as well. For example, in some fusion polypeptides, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cancer- specific mutations are somatic missense mutations.

[00356] In some cases, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the neoepitopes in a subject formed by nonsynonymous, somatic, cancer- specific mutations are included in the fusion polypeptide. In some cases, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the neoepitopes in the subject formed by nonsynonymous, somatic, missense cancer- specific mutations are included in the fusion polypeptide. [00357] The fusion polypeptide can comprise neoepitopes from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins, or 2-5, 5-10, 10-15, or 15-20 proteins.

B. Neoepitopes

[00358] The fusion polypeptides disclosed herein comprise antigenic peptides comprising neoepitopes. These neoepitopes can be patient- specific (i.e., subject- specific) cancer mutations. A process of for creating a personalized immunotherapy may comprise use of extracted nucleic acid a cancer sample from a subject and extracted nucleic acid from a normal or healthy reference sample in order to identify somatic mutations or sequence differences present in the cancer sample as compared with the normal or healthy sample, wherein these sequence having somatic mutations or differences encode an expressed amino acid sequence. A peptide expressing such somatic mutations or sequence differences can be referred to as a "neoepitope." A cancer- specific neoepitope may refer to an epitope that is not present in a reference sample (such as a normal non-cancerous or germline cell or tissue) but is found in a cancer sample. This includes, for example, situations wherein in a normal non-cancerous or germline cell a corresponding epitope is found; however, due to one or more mutations in a cancer cell, the sequence of the epitope is changed so as to result in the neoepitope. A neoepitope can comprise a mutated epitope, and can comprise non-mutated sequence on either or both sides of the mutation.

[00359] A neoepitope can be a linear epitope, a solvent-exposed epitope, a conformational epitope, or a T-cell epitope. A neoepitope can be tumor-specific, for example, or metastasis- specific. A neoepitope can be a linear epitope. A neoepitope can be considered solvent- exposed and therefore accessible to T-cell antigen receptors. Neoepitopes can comprise immunogenic epitopes, T cell epitopes, or adaptive immune response epitopes. Neoepitopes can be recognized as "non-self antigens by the adaptive immune system.

[00360] Neoepitopes can be epitopes that do not comprise immunosuppressive epitopes or immunosuppressive T-regulatory epitopes. In some cases, a neoepitope does not activate T- regulatory (T-reg) cells.

[00361 ] Neoepitopes can comprise a single mutation or two or more mutations. For example, a neoepitope can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations, or can comprise 1-10, 11-20, 20-30, or 30-40 mutations.

[00362] The cancer- specific neoepitopes disclosed herein are present within a cancer sample but not in a reference healthy biological sample. The neoepitope can be causative of the cancer or turn or in some cases, or can be present in the cancer sample without being causative. A neoepitope can also be associated with a cancer (e.g., correlate with occurrence of a type of cancer) or may not be associated with the cancer.

[00363] Neoepitopes can be identified by whole genome sequencing, exome sequencing transcriptome sequencing, T-cell receptor sequencing, or any other means. The term genome refers to the total amount of genetic information in the chromosomes of an organism, the term exome refers to the coding regions of the genome, and the term transcriptome refers to the set of all mRNA molecules. Any suitable sequencing method can be used. For example, next generation sequencing (NGS) technologies can be used. The term NGS refers to all novel high throughput sequencing technologies which, in contrast to the conventional sequencing methodology known as Sanger sequencing, read nucleic acid templates randomly in parallel along the entire genome by breaking the entire genome into small pieces. Such NGS technologies (also known as massively parallel sequencing technologies) are able to deliver nucleic acid sequence information of a whole genome, exome, transcriptome (all transcribed sequences of a genome) or methylome (all methylated sequences of a genome) in very short time periods (e.g., within 1-2 weeks, preferably within 1-7 days or most preferably within less than 24 hours) and allow, in principle, single cell sequencing approaches. See, e.g., Zhang et al. (2011) Genet Genomics 38(3):95-109 and Voelkerding et al. (2009) Clinical Chemistry 55:641-658, each of which is herein incorporated by reference in its entirety for all purposes.

[00364] The fusion polypeptides disclosed herein can comprise antigenic peptides comprising any combination of neoepitopes from any combination of proteins (i.e., one or more proteins) and in any order. The combination of antigenic peptides or the fusion polypeptide can be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria monocytogenes or another bacteria of interest.

C. Generating Personalized Immunotherapy Constructs

[00365] Also provided herein are methods for generating personalized immunotherapy constructs encoding or compositions comprising the recombinant fusion polypeptides disclosed herein. Such a personalized immunotherapy uses neoepitopes within mutated and variant antigens (neoantigens) that are specific to a particular subject's cancer or tumor. [00366] For example, such methods can comprise selecting a set of neoepitopes to include in the immunotherapy construct, designing antigenic peptides comprising each of the neoepitopes (and, for example, testing the hydropathy of the each antigenic peptide, and modifying or deselecting an antigenic peptide if it scores above a selected hydropathy index threshold value), selecting one or more sets of antigenic peptides, designing one or more fusion polypeptides comprising each of the selected antigenic peptides, and generating a nucleic acid construct encoding the fusion polypeptide.

[00367] In one example, generating a personalized immunotherapy composition can comprise comparing one or more open reading frame sequences or mRNA sequences from a cancer sample from a subject having a cancer or tumor with one or more open reading frame sequences or mRNA sequences from a healthy biological sample, wherein the comparing identifies one or more cancer- specific neoepitopes, each comprising a different cancer- specific mutation. Such method can further comprise selecting a set of cancer- specific neoepitopes to include in the second nucleic acid and designing the second nucleic acid, and then transforming a Listeria strain with the second nucleic acid.

[00368] Optionally, such methods can further comprise obtaining the cancer sample from the subject and/or obtaining the healthy biological sample. The cancer sample and/or the healthy biological sample can comprise, for example, a tissue, cells isolated from blood, cells isolated from sputum, cells isolated from saliva, or cells isolated from cerebrospinal fluid. A cancer sample can be from a primary tumor sample, from a metastasis, or from circulating tumor cells. A cancer sample can be from any type of cancer, specific examples of which are disclosed elsewhere herein. Samples may be obtained, for example, using routine biopsy procedures. Biopsies may comprise the removal of cells or tissues from a subject by skilled medical personnel, for example a pathologist. There are many different types of biopsy procedures. The most common types include: (1) incisional biopsy, in which only a sample of tissue is removed; (2) excisional biopsy, in which an entire lump or suspicious area is removed; and (3) needle biopsy, in which a sample of tissue or fluid is removed with a needle. When a wide needle is used, the procedure is called a core biopsy. When a thin needle is used, the procedure is called a fine-needle aspiration biopsy.

[00369] The healthy biological sample can be from the same subject (i.e., normal or healthy cells from the same subject) as the cancer sample from another individual of the same species. If the sample is from another individual, it can be, for example, a relative of the subject. A cancer sample and a healthy biological sample can both be obtained from the same tissue (e.g., a tissue section containing both tumor tissue and surrounding normal tissue). Preferably, healthy biological samples consist essentially or entirely of normal, healthy cells and can be used in comparison to a cancer sample. Preferably, the samples are of the same type (e.g., both blood or both sera). For example, if the cancer sample comprises cells, preferably the cells in the healthy biological sample have the same tissue origin as the cancer cells (e.g., lung or brain) and arise from the same cell type (e.g., neuronal, epithelial, mesenchymal, hematopoietic). Optionally, the normal or healthy biological sample can be obtained at the same time. Alternatively, the normal or healthy biological sample can be obtained at a different time, wherein the time may be such that the normal of healthy sample is obtained prior to obtaining the cancer sample or afterwards.

[00370] Nucleic acids can be extracted in triplicates and can be from a primary tumor sample, from a metastasis, or from circulating tumor cells. Additional mutations not resident in the initial biopsy may be present in a metastasis or circulating turn or cell and could be included to specifically target cytotoxic T cells (CTC) or metastases that have mutated differently than a primary biopsy that was sequenced.

[00371 ] Neoepitopes can be selected from a subject by comparing one or more open reading frames (ORFs) or mRNAs in nucleic acid sequences extracted from a cancer sample from the subject with one or more ORFs or mRNAs in nucleic acid sequences extracted from a healthy biological sample, wherein one or more neoepitopes are identified encoded within the one or more ORFs from the disease-bearing sample that are not present in the healthy biological sample. The neoepitopes can be determined, for example, using exome sequencing (to determine open reading frame sequences) or transcriptome sequencing (to determine mRNA sequences) to determine the sequences in the cancer sample and the healthy biological sample. Alternatively, the entire genome can be sequenced. Neoepitopes can also be identified using T-cell receptor sequencing. The comparing can comprise use of a screening assay or screening tool and associated digital software for comparing one or more ORFs in nucleic acid sequences extracted from the tumor or cancer sample with one or more ORFs in nucleic acid sequences extracted from the healthy biological sample, optionally wherein the associated digital software comprises access to a sequence database that allows screening of mutations within the ORFs in the nucleic acid sequences extracted from the tumor or cancer sample for identification of immunogenic potential of the neoepitopes.

[00372] The methods can further comprise designing an antigenic peptides for some (e.g., one or more) or each of the one or more cancer- specific neoepitopes. Neoepitopes can be selected based on any criteria. The neoepitopes can be ranked, for example, according to one of more of the following: locations within mutational hotspots as disclosed elsewhere herein; and effect of the cancer- specific mutation on function of the protein (e.g., loss of function of a tumor suppressor protein; known cancer "driver" mutations; known chemotherapy resistance mutations). Optionally, one or more of nonsense mutations, deletion mutations, insertion mutations, frameshift mutations, or translocation mutations can be excluded. In some cases, only somatic missense mutations are considered. Alternatively, every cancer- specific neoepitope can be selected, every cancer- specific neoepitope comprising a cancer- specific somatic missense mutation can be selected (i.e., amino acid change created by a somatic, nonsynonymous, missense mutation in a gene), every cancer- specific neoepitope that scores below a hydropathy threshold predictive of secretability in Listeria

monocytogenes can be selected, or every cancer- specific neoepitope that comprises a cancer- specific somatic missense mutation and scores below a hydropathy threshold predictive of secretability in Listeria monocytogenes can be selected.

[00373] After identification of a set of cancer- specific neoepitopes comprising cancer- specific mutations to include in a fusion polypeptide, sequences for antigenic peptides comprising each cancer- specific mutation can be selected. Each antigenic peptide can be designed, for example, to comprise a fragment of the protein comprising a cancer- specific neoepitope having a cancer- specific mutation and flanking sequence on each side. Different size antigenic peptides can be used, as disclosed elsewhere herein. Preferably, however, at least about 10 flanking amino acids on each side of the cancer- specific mutation are incorporated to accommodate class 1 MHC-1 presentation, in order to provide at least some of the different HLA T-cell receptor (TCR) reading frames. For example, an antigenic peptide can be selected to include a cancer- specific mutation and 10 flanking amino acids from the protein on each side (i.e., a 21-mer). Alternatively, for example, an antigenic peptide can be selected to include a cancer- specific mutation and 13 flanking amino acids from the protein on each side (i.e., a 27-mer).

[00374] The antigenic peptides or cancer- specific neoepitopes can then be screened for hydrophobicity or hydrophilicity. Antigenic peptides or cancer- specific neoepitopes can be selected, for example, if they are hydrophilic or if they score up to or below a certain hydropathy threshold, which can be predictive of secretability in a particular bacteria of interest (e.g., Listeria monocytogenes). For example, antigenic peptides or cancer- specific neoepitopes can be scored by Kyte and Doolittle hydropathy index with a 21 amino acid window, all scoring above cutoff (around 1.6) are excluded as they are unlikely to be secretable by Listeria monocytogenes. See, e.g., Kyte-Doolittle (1982) J Mol Biol

157(1): 105-132; herein incorporated by reference in its entirety for all purposes. Alternatively, an antigenic peptide or cancer- specific neoepitope scoring about a selected cutoff can be altered (e.g., changing the length of the antigenic peptide or shifting the region of the protein included in the antigenic peptide (so long as the antigenic peptide still contains the cancer- specific mutation and sufficient flanking sequence on each side). Other sliding window sizes that can be used include, for example, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or more amino acids. For example, the sliding window size can be 9-11 amino acids, 11-13 amino acids, 13-15 amino acids, 15-17 amino acids, 17-19 amino acids, 19-21 amino acids, 21-23 amino acids, 23-25 amino acids, or 25-27 amino acids. Other cutoffs that can be used include, for example, the following ranges 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2.0-2.2 2.2-2.5, 2.5-3.0, 3.0-3.5, 3.5-4.0, or 4.0-4.5, or the cutoff can be 1.4, 1.5, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,

2.3, 2.3, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,

4.4, or 4.5. The cutoff can vary, for example, depending on the genus or species of the bacteria being used to deliver the fusion polypeptide.

[00375] Other suitable hydropathy plots or other appropriate scales include, for example, those reported in Rose et al. (1993) Annu Rev Biomol Struct 22:381-415; Biswas et al. (2003) Journal of Chromatography A 1000:637-655; Eisenberg (1984) Ann Rev Biochem 53:595- 623; Abraham and Leo (1987) Proteins: Structure, Function and Genetics 2: 130-152; Sweet and Eisenberg (1983) Mol Biol 171:479-488; Bull and Breese (1974) Arch Biochem Biophys 161:665-670; Guy (1985) Biophys J 47:61-70; Miyazawa et al. (1985) Macromolecules 18:534-552; Roseman (1988) J Mol Biol 200:513-522; Wolfenden et al. (1981) Biochemistry 20:849-855; Wilson (1981) Biochem J 199:31-41; Cowan and Whittaker (1990) Peptide Research 3:75-80; Aboderin (1971) Int J Biochem 2:537-544; Eisenberg et al. (1984) J Mol Biol 179: 125-142; Hopp and Woods (1981) Proc Natl Acad Sci USA 78:3824-3828;

Manavalan and Ponnuswamy (1978) Nature 275:673-674; Black and Mould (1991) Anal Biochem 193:72-82; Fauchere and Pliska (1983) Eur J Med Chem 18:369-375; Janin (1979) Nature 277:491-492; Rao and Argos (1986) Biochim Biophys Acta 869: 197-214; Tanford (1962) Am Chem Soc 84:4240-4274; Welling et al. (1985) FEBS Lett 188:215-218; Parker et al. (1986) Biochemistry 25:5425-5431; and Cowan and Whittaker (1990) Peptide Research 3:75-80, each of which is herein incorporated by reference in its entirety for all purposes.

[00376] Optionally, the remaining antigenic peptides or cancer- specific neoepitopes can then be scored for their ability to bind subject (patient) HLA (for example by using the Immune Epitope Database (IED) available at www.iedb.org, which includes netMHCpan, ANN, SMMPMBEC. SMM, CombLib_Sidney2008, PickPocket, and netMHCcons) and ranked by best MHC binding score from each antigenic peptide. Other sources include TEpredict (tepredict.sourceforge.net/help.html) or other available MHC binding measurement scales. Cutoffs may be different for different expression vectors such as Salmonella.

[00377] Optionally, the antigenic peptides or cancer- specific neoepitopes can be further screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL- 10- inducing T helper epitopes, and so forth) to deselect antigenic peptides or cancer- specific neoepitopes or to avoid immunosuppressive influences.

[00378] Optionally, a predicative algorithm for immunogenicity of the epitopes can be used to screen the antigenic peptides or cancer- specific neoepitopes. However, these algorithms are at best 20% accurate in predicting which peptide will generate a T cell response. Alternatively, no screening/predictive algorithms are used. Alternatively, the antigenic peptides or cancer- specific neoepitopes can be screened for immunogenicity. For example, this can comprise contacting one or more T cells with an antigenic peptide or cancer- specific neoepitope, and analyzing for an immunogenic T cell response, wherein an immunogenic T cell response identifies the peptide as an immunogenic peptide. This can also comprise using an immunogenic assay to measure secretion of at least one of CD25, CD44, or CD69 or to measure secretion of a cytokine selected from the group comprising IFN-γ, TNF-a, IL-1, and IL-2 upon contacting the one or more T cells with the peptide, wherein increased secretion identifies the peptide as comprising one or more T cell neoepitopes.

[00379] Optionally, antigenic peptides comprising the one or more neoepitopes can be screened for an immunogenic response. For example, this can comprise transforming a Listeria strain with a nucleic acid encoding the antigenic peptide to create recombinant Listeria strain as disclosed elsewhere herein and administering the recombinant Listeria strain to the subject. A biological sample can then be obtained from the subject comprising a T-cell clone or T-infiltrating cell from the T-cell immune response, and the method can further comprise characterizing specific peptides comprising one or more neoepitopes bound by MHC Class I or MHC Class II molecules on the T cells to identify immunogenic neoepitopes. The characterizing can comprise, for example, identifying, isolating, and expanding T cell clones or T-infiltrating cells that respond against the cancer, and screening for and identifying one or more peptides comprising one or more immunogenic neoepitopes loaded on specific MHC Class I or MHC Class II molecules to which a T-cell receptor on the T cells binds. The screening for and identifying can comprise, for example, T-cell receptor sequencing, multiplex based flow cytometry, or high-performance liquid chromatography. Optionally, the sequencing can comprise the use of associated digital software and a database. Such methods can further comprise screening for and selecting a nucleic acid construct encoding one or more peptides comprising one or more identified immunogenic neoepitopes, and then transforming a second Listeria strain with a nucleic acid encoding one or more of the identified immunogenic neoepitopes to create a recombinant Listeria strain as described elsewhere herein. This second recombinant Listeria strain can then, for example, be administered to the subject.

[00380] Other immune response assays include, for example, T-cell proliferation assays, in vitro tumor regression assays using T cells activated with a neoepitope and co-incubated with tumor cells using a ⁵¹Cr-release assay or a ³H-thymidine assay, an ELISA assay, an ELIspot assay, and FACS analysis {see, e.g., US 8,771,702, herein incorporated by reference in its entirety for all purposes). Alternatively, a step for screening for an immunogenic response examines a non-T-cell response. Such assays can be similar to those above for T-cells, except that examining cytokine production focuses on a different subset of cytokines, namely, IL-10 and IL-Ιβ {see, e.g., US 8,962,319 and EP 177432, each of which is herein

incorporated by reference in its entirety for all purposes). For example, a T-cell immune response may be assayed by a ⁵¹Cr release assay, comprising the steps of immunizing mice with a immunotherapy comprising one or more neo-epitopes, followed by harvesting spleens about ten days post- immunization, wherein splenocytes may then be established in culture with irradiated TC-1 cells (100: 1, splenocytes:TC-l) as feeder cells; stimulated in vitro for 5 days, then used in a standard ⁵¹Cr release assay, using a peptide/polypeptide comprising one or more neoepitopes as the target. In one example, a step for screening for an immune response comprises use of an HLA-A2 transgenic mouse {see, e.g., US 2011/0129499, herein incorporated by reference in its entirety for all purposes).

[00381 ] The selected antigenic peptides can then be arranged into one or more candidate orders for a potential fusion polypeptide. If there are more usable antigenic peptides than can fit into a single plasmid, different antigenic peptides can be assigned priority ranks as needed/desired and/or split up into different fusion polypeptides (e.g., for inclusion in different recombinant Listeria strains). Priority rank can be determined by factors such as relative size, priority of transcription, and/or overall hydrophobicity of the translated polypeptide. The antigenic peptides can be arranged so that they are joined directly together without linkers, or any combination of linkers between any number of pairs of antigenic peptides, as disclosed in more detail elsewhere herein. The number of linear antigenic peptides to be included can be determined based on consideration of the number of constructs needed versus the mutational burden, the efficiency of translation and secretion of multiple epitopes from a single plasmid, or the MOI needed for each bacteria or Lm comprising a plasmid. For example, ranges of linear antigenic peptides can be starting, for example, with about 50, 40, 30, 20, or 10 antigenic peptides per plasmid.

[00382] Different possible arrangements of the same antigenic peptides in a fusion polypeptide can be generated through one or more iterations of randomizing the order of the antigenic peptides. Such randomizing can include, for example, randomizing the order of the entire set of antigenic peptides, or can comprise randomizing the order of a subset of the antigenic peptides. For example, if there are 20 antigenic peptides (ordered 1-20), the randomizing can comprise randomizing the order of all 20 peptides or can comprise randomizing the order of only a subset of the peptides (e.g., peptides 1-5 or 6-10). Such randomization of the order can facilitate secretion and presentation of the fusion polypeptide and of each individual antigenic peptide. Alternatively, the order of the antigenic peptides can be generated using selected parameters, such as a predefined ranking of the antigenic peptides.

[00383] The combination of antigenic peptides or the entire fusion polypeptide (i.e., comprising the antigenic peptides and the PEST-containing peptide and any tags) can also be scored for hydrophobicity. For example, the entirety of the fused antigenic peptides or the entire fusion polypeptide can be scored for hydropathy by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window. If any region scores above a cutoff (e.g., around 1.6), the antigenic peptides can be reordered or shuffled within the fusion polypeptide using selected parameters or using randomization until an acceptable order of antigenic peptides is found (i.e., one in which no region scores above the cutoff). Alternatively, any problematic antigenic peptides can be removed or redesigned to be of a different size, or to shift the sequence of the protein included in the antigenic peptide (so long as the antigenic peptide still comprises the cancer- specific neoepitope or cancer- specific mutation and sufficiently sized flanking sequences). Alternatively or additionally, one or more linkers between antigenic peptides as disclosed elsewhere herein can be added or modified to change the

hydrophobicity. As with hydropathy testing for the individual antigenic peptides, other window sizes can be used, or other cutoffs can be used (e.g., depending on the genus or species of the bacteria being used to deliver the fusion polypeptide). In addition, other suitable hydropathy plots or other appropriate scales could be used.

[00384] Optionally, the combination of antigenic peptides or the entire fusion polypeptide can be further screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL- 10- inducing T helper epitopes, and so forth) to deselect antigenic peptides or to avoid immunosuppressive influences.

[00385] A nucleic acid encoding a candidate combination of antigenic peptides or fusion polypeptide can then be designed and optimized. For example, the sequence can be optimized for increased levels of translation, duration of expression, levels of secretion, levels of transcription, and any combination thereof. For example, the increase can be 2-fold to 1000-fold, 2-fold to 500-fold, 2-fold to 100-fold, 2-fold to 50-fold, 2-fold to 20-fold, 2-fold to 10-fold, or 3-fold to 5-fold relative to a control, non-optimized sequence.

[00386] For example, the fusion polypeptide or nucleic acid encoding the fusion polypeptide can be optimized for decreased levels of secondary structures possibly formed in the oligonucleotide sequence, or alternatively optimized to prevent attachment of any enzyme that may modify the sequence. Expression in bacterial cells can be hampered, for example, by transcriptional silencing, low mRNA half-life, secondary structure formation, attachment sites of oligonucleotide binding molecules such as repressors and inhibitors, and availability of rare tRNAs pools. The source of many problems in bacterial expressions is found within the original sequence. The optimization of RNAs may include modification of cis acting elements, adaptation of its GC-content, modifying codon bias with respect to non-limiting tRNAs pools of the bacterial cell, and avoiding internal homologous regions. Thus, optimizing a sequence can entail, for example, adjusting regions of very high (> 80%) or very low (< 30%) GC content. Optimizing a sequence can also entail, for example, avoiding one or more of the following cis-acting sequence motifs: internal TATA-boxes, chi-sites, and ribosomal entry sites; AT-rich or GC-rich sequence stretches; repeat sequences and RNA secondary structures; (cryptic) splice donor and acceptor sites; branch points; or a combination thereof. Optimizing expression can also entail adding sequence elements to flanking regions of a gene and/or elsewhere in the plasmid.

[00387] Optimizing a sequence can also entail, for example, adapting the codon usage to the codon bias of host genes (e.g., Listeria monocytogenes genes). For example, the codons below can be used for Listeria monocytogenes.

[00388] A nucleic acid encoding a fusion polypeptide can be generated and introduced into a delivery vehicle such as a bacteria strain or Listeria strain. Other delivery vehicles may be suitable for DNA immunotherapy or peptide immunotherapy, such as a vaccinia virus or virus-like particle. Once a nucleic acid encoding a fusion polypeptide is generated and introduced into a bacteria strain or Listeria strain, the bacteria or Listeria strain can be cultured and characterized to confirm expression and secretion of the fusion polypeptide comprising the antigenic peptides.

[00389] In one specific example, a process for creating a personalized immunotherapy can comprise: (a) obtaining a cancer sample from a subject having the cancer; (b) extracting nucleic acids from the cancer sample; (c) obtaining a healthy biological sample from the subject or from a different individual of the same species; (d) extracting nucleic acids from the healthy sample; (e) sequencing the extracted nucleic acids from steps (b) and (d); (f) comparing one or more open reading frames (ORFs) in nucleic acid sequences extracted from the cancer sample with open reading frames in nucleic acid sequences extracted from the healthy biological sample; (g) identifying mutated nucleic acid sequences within the ORFs of the cancer sample, wherein the ORFs encodes a peptide comprising one or more neoepitopes (wherein the neoepitopes are identified, for example, using well-known methods such as T- cell receptor (TCR) sequencing or whole exome sequencing); (h) expressing the one or more peptides comprising the identified mutated nucleic acid sequences; (i) screening each peptide for an immunogenic T-cell response, wherein the presence of an immunogenic T-cell response correlates with presence of one or more neoepitopes comprising a T-cell epitope; (j) identifying and selecting a nucleic acid sequence that encodes a one or more immunogenic peptides comprising one or more immunogenic neoepitopes that are T-cell epitopes, and transforming an attenuated Listeria strain with a plasmid comprising the sequence; (k) culturing and characterizing the transformed attenuated Listeria strain to confirm expression and secretion of the one or more immunogenic peptides; and (1) storing the transformed attenuated Listeria for administering to the subject at a pre-determined period or

administering the attenuated Listeria strain to the subject, wherein the transformed attenuated Listeria strain is administered as part of an immunogenic composition.

[00390] Also provided herein is a system for providing a personalized immunotherapy for a subject having a tumor or cancer, comprising the following components: (1) a tumor or cancer sample from the subject; (2) a healthy biological sample from the subject with the cancer or tumor or from another healthy subject; (3) a screening assay or screening tool and associated digital software for comparing one or more open reading frames (ORFs) in nucleic acid sequences extracted from the tumor or cancer sample with open reading frames in nucleic acid sequences extracted from the healthy biological sample, and for identifying mutations in the ORFs tumor or cancer sample that are not in the healthy biological sample; (4) a nucleic acid cloning and expression kit for cloning and expressing a nucleic acid encoding one or more peptides comprising one or more neoepitopes comprising the tumor- specific or cancer- specific mutations from the tumor or cancer sample; (5) optionally an immunogenic assay for testing the T-cell immunogenicity of candidate peptides comprising one or more neoepitopes; and (6) a recombinant Listeria strain for transforming with a nucleic acid (e.g., plasmid) comprising one or more open reading frames encoding said identified immunogenic peptides comprising one or more immunogenic neoepitopes.

[00391 ] Also provided herein is a system for creating personalized immunotherapy for a subject, comprising: at least one processor and at least one storage medium containing program instructions for execution by the processor, the program instructions causing the processor to execute steps comprising: (a) receiving output data containing all neoepitopes and the human leukocyte antigen (HLA) type of the subject; (b) scoring the hydrophobicity of each neoepitope and removing epitopes that score above a certain threshold; (c) numerically rating the remaining neoepitopes based on their ability to bind to subject HLA and on their predictive MHC binding scores; (d) inserting an amino acid sequence of each neoepitope into a plasmid; (e) scoring the hydrophobicity of each construct and removing any constructs that score above a certain threshold; (f) reverse translating the amino acid sequence of each construct into the corresponding DNA sequence, starting with the highest scored construct; (g) inserting additional neoepitopes into the plasmid construct in order of ranking until a predetermined upper limit is reached; (h) adding a DNA sequence tag to the end of the construct in order to facilitate measuring the immunotherapeutic response in the subject; and (i) optimizing the DNA sequence encoding the neoepitopes and the DNA sequence tag for expression and secretion in Listeria monocytogenes. In some such systems, the preferred output data can be in FASTA format.

[00392] Another specific example of a system for providing a personalized

immunotherapy for a subject having a cancer comprises the following components: (a) a cancer sample obtained from the subject; (b) a healthy biological sample, wherein the healthy biological sample is obtained from the human subject having the cancer or another healthy human subject; (c) a screening assay or screening tool and associated digital software for comparing one or more open reading frames (ORFs) in nucleic acid sequences extracted from the cancer sample with open reading frames in nucleic acid sequences extracted from the healthy biological sample, and for identifying mutations in the ORFs encoded by the nucleic acid sequences of the cancer sample, wherein the mutations comprise one or more neoepitopes (e.g., the said associated digital software comprises access to a sequence database that allows screening of the mutations within the ORFs for identification of T-cell epitope(s) or

immunogenic potential, or any combination thereof); (d) a nucleic acid cloning and expression kit for cloning and expressing a nucleic acid encoding one or more peptides comprising the one or more neoepitopes from the cancer sample; (e) an immunogenic assay for testing the T-cell immunogenicity and/or binding of candidate peptides comprising one or more neo-epitopes; (f) analytic equipment, and associated software for sequencing and analyzing nucleic acid sequences, peptide amino acid sequences and T-cell receptor amino acid sequences; (g) an attenuated Listeria delivery vector for transforming with a plasmid comprising a nucleic acid construct comprising one or more open reading frames encoding the identified immunogenic peptides comprising one or more immunogenic neoepitopes of step (e) (e.g., wherein once transformed, said Listeria is stored or is administered to said human subject in (a) as part of an immunogenic composition); or a delivery vector; and optionally a vector for transforming the delivery vector, the vector comprising a nucleic acid construct comprising one or more open reading frames encoding one or more peptides comprising one or more neoepitopes, wherein the neoepitopes comprise immunogenic epitopes present the cancer sample.

VII. Recombinant Bacteria or Listeria Strains

[00393] Also provided herein are recombinant bacterial strains, such as a Listeria strain, comprising a recombinant fusion polypeptide disclosed herein or a nucleic acid encoding the recombinant fusion polypeptide as disclosed elsewhere herein. Preferably, the bacterial strain is a Listeria strain, such as a Listeria monocytogenes (Lm) strain. Lm has a number of inherent advantages as a vaccine vector. The bacterium grows very efficiently in vitro without special requirements, and it lacks LPS, which is a major toxicity factor in gram- negative bacteria, such as Salmonella. Genetically attenuated Lm vectors also offer additional safety as they can be readily eliminated with antibiotics, in case of serious adverse effects, and unlike some viral vectors, no integration of genetic material into the host genome occurs.

[00394] The recombinant Listeria strain can be any Listeria strain. Examples of suitable Listeria strains include Listeria seeligeri, Listeria grayi, Listeria ivanovii, Listeria murrayi, Listeria welshimeri, Listeria monocytogenes (Lm), or any other Listeria species known in the art. Preferably, the recombinant listeria strain is a strain of the species Listeria monocytogenes. Examples of Listeria monocytogenes strains include the following: L.

monocytogenes 10403S wild type {see, e.g., Bishop and Hinrichs (1987) J Immunol

139:2005-2009; Lauer et al. (2002) J Bact 184:4177-4186); L. monocytogenes DP-L4056, which is phage cured {see, e.g., Lauer et al. (2002) J Bact 184:4177-4186); L. monocytogenes DP-L4027, which is phage cured and has an hly gene deletion {see, e.g., Lauer et al. (2002) Bact 184:4177- 4186; Jones and Portnoy (1994) Infect Immunity 65:5608-5613); L.

monocytogenes DP-L4029, which is phage cured and has an actA gene deletion {see, e.g., Lauer et al. (2002) J Bact 184:4177-4186; Skoble et al. (2000) J Cell Biol 150:527- 538); L. monocytogenes DP-L4042 (delta PEST) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci. USA 101: 13832-13837 and supporting information); L. monocytogenes DP-L4097 (LLO- S44A) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101: 13832-13837 and supporting information); L. monocytogenes DP- L4364 (delta IplA; lipoate protein ligase) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101: 13832-13837 and supporting information); L. monocytogenes DP-L4405 (delta inlA) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101: 13832-13837 and supporting information); L. monocytogenes DP-L4406 (delta MB) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA

101: 13832-13837 and supporting information); L. monocytogenes CS-LOOOl (delta actA; delta MB) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101: 13832-13837 and supporting information); L. monocytogenes CS-L0002 (delta actA; delta IplA) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101: 13832-13837 and supporting

information); L. monocytogenes CS-L0003 (LLO L461T; delta IplA) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101: 13832-13837 and supporting information); L.

monocytogenes DP-L4038 (delta actA; LLO L461T) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101: 13832-13837 and supporting information); L. monocytogenes DP- L4384 (LLO S44A; LLO L461T) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101: 13832-13837 and supporting information); a L. monocytogenes strain with an IplAl deletion (encoding lipoate protein ligase LplAl) {see, e.g., O'Riordan et al. (2003) Science 302:462-464); L. monocytogenes DP-L4017 (10403S with LLO L461T) {see, e.g., US 7,691,393); L. monocytogenes EGD {see, e.g., GenBank Accession No. AL591824). In another embodiment, the Listeria strain is L. monocytogenes EGD-e (see GenBank

Accession No. NC_003210; ATCC Accession No. BAA-679); L. monocytogenes DP-L4029 {actA deletion, optionally in combination with uvrAB deletion (DP-L4029uvrAB) {see, e.g., US 7,691,393); L. monocytogenes actA-IMB - double mutant {see, e.g., ATCC Accession No. PTA-5562); L. monocytogenes IplA mutant or hly mutant {see, e.g., US 2004/0013690); L. monocytogenes dalldat double mutant {see, e.g., US 2005/0048081). Other L.

monocytogenes strains includes those that are modified (e.g., by a plasmid and/or by genomic integration) to contain a nucleic acid encoding one of, or any combination of, the following genes: hly (LLO; listeriolysin); iap (p60); inlA; inlB; inlC; dal (alanine racemase); dat (D- amino acid aminotransferase); plcA; plcB; actA; or any nucleic acid that mediates growth, spread, breakdown of a single walled vesicle, breakdown of a double walled vesicle, binding to a host cell, or uptake by a host cell. Each of the above references is herein incorporated by reference in its entirety for all purposes.

[00395] The recombinant bacteria or Listeria can have wild-type virulence, can have attenuated virulence, or can be avirulent. For example, a recombinant Listeria of can be sufficiently virulent to escape the phagosome or phagolysosome and enter the cytosol. Such Listeria strains can also be live-attenuated Listeria strains, which comprise at least one attenuating mutation, deletion, or inactivation as disclosed elsewhere herein. Preferably, the recombinant Listeria is an attenuated auxotrophic strain. An auxotrophic strain is one that is unable to synthesize a particular organic compound required for its growth. Examples of such strains are described in US 8,114,414, herein incorporated by reference in its entirety for all purposes.

[00396] Preferably, the recombinant Listeria strain lacks antibiotic resistance genes. For example, such recombinant Listeria strains can comprise a plasmid that does not encode an antibiotic resistance gene. However, some recombinant Listeria strains provided herein comprise a plasmid comprising a nucleic acid encoding an antibiotic resistance gene.

Antibiotic resistance genes may be used in the conventional selection and cloning processes commonly employed in molecular biology and vaccine preparation. Exemplary antibiotic resistance genes include gene products that confer resistance to ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, chloramphenicol (CAT), neomycin, hygromycin, and gentamicin.

A. Bacteria or Listeria Strains Comprising Recombinant Fusion Polypeptides or Nucleic Acids Encoding Recombinant Fusion Polypeptides

[00397] The recombinant bacterial strains (e.g., Listeria strains) disclosed herein comprise a recombinant fusion polypeptide disclosed herein or a nucleic acid encoding the recombinant fusion polypeptide as disclosed elsewhere herein. [00398] In bacteria or Listeria strains comprising a nucleic acid encoding a recombinant fusion protein, the nucleic acid can be codon optimized. The optimal codons utilized by L. monocytogenes for each amino acid are shown US 2007/0207170, herein incorporated by reference in its entirety for all purposes. A nucleic acid is codon-optimized if at least one codon in the nucleic acid is replaced with a codon that is more frequently used by L.

monocytogenes for that amino acid than the codon in the original sequence.

[00399] The nucleic acid can be present in an episomal plasmid within the bacteria or Listeria strain and/or the nucleic acid can be genomically integrated in the bacteria or Listeria strain. Some recombinant bacteria or Listeria strains comprise two separate nucleic acids encoding two recombinant fusion polypeptides as disclosed herein: one nucleic acid in an episomal plasmid, and one genomically integrated in the bacteria or Listeria strain.

[00400] The episomal plasmid can be one that is stably maintained in vitro (in cell culture), in vivo (in a host), or both in vitro and in vivo. If in an episomal plasmid, the open reading frame encoding the recombinant fusion polypeptide can be operably linked to a promoter/regulatory sequence in the plasmid. If genomically integrated in the bacteria or Listeria strain, the open reading frame encoding the recombinant fusion polypeptide can be operably linked to an exogenous promoter/regulatory sequence or to an endogenous promoter/regulatory sequence. Examples of promoters/regulatory sequences useful for driving constitutive expression of a gene are well known and include, for example, an hly, hlyA, actA, prfA, and p60 promoters of Listeria, the Streptococcus bac promoter, the

Streptomyces griseus sgiA promoter, and the B. thuringiensis phaZ promoter. In some cases, an inserted gene of interest is not interrupted or subjected to regulatory constraints which often occur from integration into genomic DNA, and in some cases, the presence of the inserted heterologous gene does not lead to rearrangement or interruption of the cell's own important regions.

[00401 ] Such recombinant bacteria or Listeria strains can be made by transforming a bacteria or Listeria strain or an attenuated bacteria or Listeria strain described elsewhere herein with a plasmid or vector comprising a nucleic acid encoding the recombinant fusion polypeptide. The plasmid can be an episomal plasmid that does not integrate into a host chromosome. Alternatively, the plasmid can be an integrative plasmid that integrates into a chromosome of the bacteria or Listeria strain. The plasmids used herein can also be multicopy plasmids. Methods for transforming bacteria are well known, and include calcium-chloride competent cell-based methods, electroporation methods, bacteriophage- mediated transduction, chemical transformation techniques, and physical transformation techniques. See, e.g., de Boer et al. (1989) Cell 56:641-649; Miller et al. (1995) FASEB J. 9: 190-199; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al. (1997) Current Protocols in Molecular

Biology, John Wiley & Sons, New York; Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, D.C.; and Miller, 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., each of which is herein incorporated by reference in its entirety for all purposes.

[00402] Bacteria or Listeria strains with genomically integrated heterologous nucleic acids can be made, for example, by using a site- specific integration vector, whereby the bacteria or Listeria comprising the integrated gene is created using homologous recombination. The integration vector can be any site- specific integration vector that is capable of infecting a bacteria or Listeria strain. Such an integration vector can comprise, for example, a PSA attPP' site, a gene encoding a PSA integrase, a U153 attPP' site, a gene encoding a U153 integrase, an Al 18 attPP' site, a gene encoding an Al 18 integrase, or any other known attPP' site or any other phage integrase.

[00403] Such bacteria or Listeria strains comprising an integrated gene can also be created using any other known method for integrating a heterologous nucleic acid into a bacteria or Listeria chromosome. Techniques for homologous recombination are well known, and are described, for example, in Baloglu et al. (2005) Vet Microbiol 109(1-2): 11-17); Jiang et al. 2005) Acta Biochim Biophys Sin (Shanghai) 37(l):19-24), and US 6,855,320, each of which is herein incorporated by reference in its entirety for all purposes.

[00404] Integration into a bacteria or Listerial chromosome can also be achieved using transposon insertion. Techniques for transposon insertion are well known, and are described, for example, for the construction of DP-L967 by Sun et al. (1990) Infection and Immunity 58: 3770-3778, herein incorporated by reference in its entirety for all purposes. Transposon mutagenesis can achieve stable genomic insertion, but the position in the genome where the heterologous nucleic acids has been inserted is unknown.

[00405] Integration into a bacterial or Listerial chromosome can also be achieved using phage integration sites (see, e.g., Lauer et al. (2002) J Bacteriol 184(15):4177-4186, herein incorporated by reference in its entirety for all purposes). For example, an integrase gene and attachment site of a bacteriophage (e.g., U153 or PSA listeriophage) can be used to insert a heterologous gene into the corresponding attachment site, which may be any appropriate site in the genome (e.g. comK or the 3' end of the arg tRNA gene). Endogenous prophages can be cured from the utilized attachment site prior to integration of the heterologous nucleic acid. Such methods can result, for example, in single-copy integrants. In order to avoid a "phage curing step," a phage integration system based on PSA phage can be used (see, e.g., Lauer et al. (2002) J Bacteriol 184:4177-4186, herein incorporated by reference in its entirety for all purposes). Maintaining the integrated gene can require, for example, continuous selection by antibiotics. Alternatively, a phage-based chromosomal integration system can be established that does not require selection with antibiotics. Instead, an auxotrophic host strain can be complemented. For example, a phage-based chromosomal integration system for clinical applications can be used, where a host strain that is auxotrophic for essential enzymes, including, for example, D-alanine racemase is used (e.g., Lm dal(-)dat(-)).

[00406] Conjugation can also be used to introduce genetic material and/or plasmids into bacteria. Methods for conjugation are well known, and are described, for example, in Nikodinovic et al. (2006) Plasmid 56(3):223-227 and Auchtung et al. (2005) Proc Natl Acad Sci USA 102(35): 12554-12559, each of which is herein incorporated by reference in its entirety for all purposes.

[00407] In a specific example, a recombinant bacteria or Listeria strain can comprise a nucleic acid encoding a recombinant fusion polypeptide genomically integrated into the bacteria or Listeria genome as an open reading frame with an endogenous actA sequence (encoding an ActA protein) or an endogenous hly sequence (encoding an LLO protein). For example, the expression and secretion of the fusion polypeptide can be under the control of the endogenous actA promoter and ActA signal sequence or can be under the control of the endogenous hly promoter and LLO signal sequence. As another example, the nucleic acid encoding a recombinant fusion polypeptide can replace an actA sequence encoding an ActA protein or an hly sequence encoding an LLO protein.

[00408] Selection of recombinant bacteria or Listeria strains can be achieved by any means. For example, antibiotic selection can be used. Antibiotic resistance genes may be used in the conventional selection and cloning processes commonly employed in molecular biology and vaccine preparation. Exemplary antibiotic resistance genes include gene products that confer resistance to ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, chloramphenicol (CAT), neomycin, hygromycin, and gentamicin. Alternatively, auxotrophic strains can be used, and an exogenous metabolic gene can be used for selection instead of or in addition to an antibiotic resistance gene. As an example, in order to select for auxotrophic bacteria comprising a plasmid encoding a metabolic enzyme or a complementing gene provided herein, transformed auxotrophic bacteria can be grown in a medium that will select for expression of the gene encoding the metabolic enzyme (e.g., amino acid metabolism gene) or the complementing gene.

Alternatively, a temperature- sensitive plasmid can be used to select recombinants or any other known means for selecting recombinants.

B. Attenuation of Bacteria or Listeria Strains

[00409] The recombinant bacteria strains (e.g., recombinant Listeria strains) disclosed herein can be attenuated. The term "attenuation" encompasses a diminution in the ability of the bacterium to cause disease in a host animal. For example, the pathogenic characteristics of an attenuated Listeria strain may be lessened compared with wild-type Listeria, although the attenuated Listeria is capable of growth and maintenance in culture. Using as an example the intravenous inoculation of BALB/c mice with an attenuated Listeria, the lethal dose at which 50% of inoculated animals survive (LD50) is preferably increased above the LD50 of wild-type Listeria by at least about 10-fold, more preferably by at least about 100-fold, more preferably at least about 1,000 fold, even more preferably at least about 10,000 fold, and most preferably at least about 100,000-fold. An attenuated strain of Listeria is thus one that does not kill an animal to which it is administered, or is one that kills the animal only when the number of bacteria administered is vastly greater than the number of wild-type non- attenuated bacteria which would be required to kill the same animal. An attenuated bacterium should also be construed to mean one which is incapable of replication in the general environment because the nutrient required for its growth is not present therein. Thus, the bacterium is limited to replication in a controlled environment wherein the required nutrient is provided. Attenuated strains are environmentally safe in that they are incapable of uncontrolled replication

(1) Methods of Attenuating Bacteria and Listeria Strains

[00410] Attenuation can be accomplished by any known means. For example, such attenuated strains can be deficient in one or more endogenous virulence genes or one or more endogenous metabolic genes. Examples of such genes are disclosed herein, and attenuation can be achieved by inactivation of any one of or any combination of the genes disclosed herein. Inactivation can be achieved, for example, through deletion or through mutation (e.g., an inactivating mutation). The term "mutation" includes any type of mutation or

modification to the sequence (nucleic acid or amino acid sequence) and may encompass a deletion, a truncation, an insertion, a substitution, a disruption, or a translocation. For example, a mutation can include a frameshift mutation, a mutation which causes premature termination of a protein, or a mutation of regulatory sequences which affect gene expression. Mutagenesis can be accomplished using recombinant DNA techniques or using traditional mutagenesis technology using mutagenic chemicals or radiation and subsequent selection of mutants. Deletion mutants may be preferred because of the accompanying low probability of reversion. The term "metabolic gene" refers to a gene encoding an enzyme involved in or required for synthesis of a nutrient utilized or required by a host bacteria. For example, the enzyme can be involved in or required for the synthesis of a nutrient required for sustained growth of the host bacteria. The term "virulence" gene includes a gene whose presence or activity in an organism's genome that contributes to the pathogenicity of the organism (e.g., enabling the organism to achieve colonization of a niche in the host (including attachment to cells), immunoevasion (evasion of host's immune response), immunosuppression (inhibition of host's immune response), entry into and exit out of cells, or obtaining nutrition from the host).

[00411 ] A specific example of such an attenuated strain is Listeria monocytogenes (Lm) dal(-)dat(-) (Lmdd). Another example of such an attenuated strain is Lm dal(-)dat(-) actA (LmddA). See, e.g., US 2011/0142791, herein incorporated by references in its entirety for all purposes. LmddA is based on a Listeria strain which is attenuated due to the deletion of the endogenous virulence gene actA. Such strains can retain a plasmid for antigen expression in vivo and in vitro by complementation of the dal gene. Alternatively, the LmddA can be a dal/dat/actA Listeria having mutations in the endogenous dal, dat, and actA genes. Such mutations can be, for example, a deletion or other inactivating mutation.

[00412] Another specific example of an attenuated strain is Lm prfA(-) or a strain having a partial deletion or inactivating mutation in the prfA gene. The PrfA protein controls the expression of a regulon comprising essential virulence genes required by Lm to colonize its vertebrate hosts; hence the prfA mutation strongly impairs PrfA ability to activate expression of Prf A-dependent virulence genes.

[00413] Yet another specific example of an attenuated strain is Lm inlB(-)actA(-) in which two genes critical to the bacterium's natural virulence— internalin B and act A— are deleted.

[00414] Other examples of attenuated bacteria or Listeria strains include bacteria or Listeria strains deficient in one or more endogenous virulence genes. Examples of such genes include actA, prfA, plcB, plcA, inlA, inlB, inlC, inlJ, and bsh in Listeria. Attenuated Listeria strains can also be the double mutant or triple mutant of any of the above-mentioned strains. Attenuated Listeria strains can comprise a mutation or deletion of each one of the genes, or comprise a mutation or deletion of, for example, up to ten of any of the genes provided herein (e.g., including the actA, prfA, and dal/dat genes). For example, an attenuated Listeria strain can comprise a mutation or deletion of an endogenous internalin C (inlC) gene and/or a mutation or deletion of an endogenous actA gene. Alternatively, an attenuated Listeria strain can comprise a mutation or deletion of an endogenous internalin B (MB) gene and/or a mutation or deletion of an endogenous actA gene. Alternatively, an attenuated Listeria strain can comprise a mutation or deletion of endogenous MB, inlC, and actA genes. Translocation of Listeria to adjacent cells is inhibited by the deletion of the endogenous actA gene and/or the endogenous inlC gene or endogenous inlB gene, which are involved in the process, thereby resulting in high levels of attenuation with increased immunogenicity and utility as a strain backbone. An attenuated Listeria strain can also be a double mutant comprising mutations or deletions of both plcA and plcB. In some cases, the strain can be constructed from the EGD Listeria backbone.

[00415] A bacteria or Listeria strain can also be an auxotrophic strain having a mutation in a metabolic gene. As one example, the strain can be deficient in one or more endogenous amino acid metabolism genes. For example, the generation of auxotrophic strains of Listeria deficient in D-alanine, for example, may be accomplished in a number of ways that are well known, including deletion mutations, insertion mutations, frameshift mutations, mutations which cause premature termination of a protein, or mutation of regulatory sequences which affect gene expression. Deletion mutants may be preferred because of the accompanying low probability of reversion of the auxotrophic phenotype. As an example, mutants of D-alanine which are generated according to the protocols presented herein may be tested for the ability to grow in the absence of D-alanine in a simple laboratory culture assay. Those mutants which are unable to grow in the absence of this compound can be selected.

[00416] Examples of endogenous amino acid metabolism genes include a vitamin synthesis gene, a gene encoding pantothenic acid synthase, a D-glutamic acid synthase gene, a D-alanine amino transferase {dat) gene, a D-alanine racemase {dal) gene, dga, a gene involved in the synthesis of diaminopimelic acid (DAP), a gene involved in the synthesis of Cysteine synthase A {cysK), a vitamin-B 12 independent methionine synthase, trpA, trpB, trpE, asnB, gltD, gltB, leuA, argG, and thrC. The Listeria strain can be deficient in two or more such genes (e.g., dat and dal). D-glutamic acid synthesis is controlled in part by the dal gene, which is involved in the conversion of D-glu + pyr to alpha-ketoglutarate + D-ala, and the reverse reaction. [00417] As another example, an attenuated Listeria strain can be deficient in an

endogenous synthase gene, such as an amino acid synthesis gene. Examples of such genes include folP, a gene encoding a dihydro uridine synthase family protein, ispD, ispF, a gene encoding a phosphoenolpyruvate synthase, hisF, hisH,fliI, a gene encoding a ribosomal large subunit pseudouridine synthase, ispD, a gene encoding a bifunctional GMP

synthase/glutamine amidotransferase protein, cobS, cobB, cbiD, a gene encoding a uroporphyrin-III C-methyltransferase/uroporphyrinogen-III synthase, cobQ, uppS, truB, dxs, mvaS, dapA, ispG,folC, a gene encoding a citrate synthase, argj, a gene encoding a 3-deoxy- 7-phosphoheptulonate synthase, a gene encoding an indole-3-glycerol-phosphate synthase, a gene encoding an anthranilate synthase/glutamine amidotransferase component, inenB, a gene encoding a menaquinone-specific isochorismate synthase, a gene encoding a

phosphoribosylformylglycinamidine synthase I or II, a gene encoding a

phosphoribosylaminoimidazole-succinocarboxamide synthase, carB, carA, thyA, mgsA, aroB, hepB, rluB, ilvB, ilvN, cilsS,fabF,fabH, a gene encoding a pseudouridine synthase, pyrG, truA, pabB, and an atp synthase gene (e.g., atpC, atpD-2, aptG, atpA-2, and so forth).

[00418] Attenuated Listeria strains can be deficient in endogenous phoP, aroA, aroC, aroD, or plcB. As yet another example, an attenuated Listeria strain can be deficient in an endogenous peptide transporter. Examples include genes encoding an ABC transporter/ ATP- binding/permease protein, an oligopeptide ABC transporter/oligopeptide-binding protein, an oligopeptide ABC transporter/permease protein, a zinc ABC transporter/zinc-binding protein, a sugar ABC transporter, a phosphate transporter, a ZIP zinc transporter, a drug resistance transporter of the EmrBIQacA family, a sulfate transporter, a proton-dependent oligopeptide transporter, a magnesium transporter, a formate/nitrite transporter, a spermidine/putrescine ABC transporter, a Na/Pi-cotransporter, a sugar phosphate transporter, a glutamine ABC transporter, a major facilitator family transporter, a glycine betaine/L-proline ABC

transporter, a molybdenum ABC transporter, a techoic acid ABC transporter, a cobalt ABC transporter, an ammonium transporter, an amino acid ABC transporter, a cell division ABC transporter, a manganese ABC transporter, an iron compound ABC transporter, a

maltose/maltodextrin ABC transporter, a drug resistance transporter of the BcrlCflA family, and a subunit of one of the above proteins.

[00419] Other attenuated bacteria and Listeria strains can be deficient in an endogenous metabolic enzyme that metabolizes an amino acid that is used for a bacterial growth process, a replication process, cell wall synthesis, protein synthesis, metabolism of a fatty acid, or for any other growth or replication process. Likewise, an attenuated strain can be deficient in an endogenous metabolic enzyme that can catalyze the formation of an amino acid used in cell wall synthesis, can catalyze the synthesis of an amino acid used in cell wall synthesis, or can be involved in synthesis of an amino acid used in cell wall synthesis. Alternatively, the amino acid can be used in cell wall biogenesis. Alternatively, the metabolic enzyme is a synthetic enzyme for D-glutamic acid, a cell wall component.

[00420] Other attenuated Listeria strains can be deficient in metabolic enzymes encoded by a D-glutamic acid synthesis gene, dga, an air (alanine racemase) gene, or any other enzymes that are involved in alanine synthesis. Yet other examples of metabolic enzymes for which the Listeria strain can be deficient include enzymes encoded by serC (a phosphoserine aminotransferase), asd (aspartate betasemialdehyde dehydrogenase; involved in synthesis of the cell wall constituent diaminopimelic acid), the gene encoding gsaB- glutamate-1- semialdehyde aminotransferase (catalyzes the formation of 5-aminolevulinate from (S)-4- amino-5-oxopentanoate), hemL (catalyzes the formation of 5-aminolevulinate from (S)-4- amino-5-oxopentanoate), aspB (an aspartate aminotransferase that catalyzes the formation of oxalozcetate and L-glutamate from L-aspartate and 2-oxoglutarate), argF-1 (involved in arginine biosynthesis), aroE (involved in amino acid biosynthesis), aroB (involved in 3- dehydroquinate biosynthesis), aroD (involved in amino acid biosynthesis), aroC (involved in amino acid biosynthesis), hisB (involved in histidine biosynthesis), hisD (involved in histidine biosynthesis), hisG (involved in histidine biosynthesis), metX (involved in methionine biosynthesis), proB (involved in proline biosynthesis), argR (involved in arginine biosynthesis), argj (involved in arginine biosynthesis), thil (involved in thiamine

biosynthesis), LMOf2365_1652 (involved in tryptophan biosynthesis), aroA (involved in tryptophan biosynthesis), ilvD (involved in valine and isoleucine biosynthesis), ilvC

(involved in valine and isoleucine biosynthesis), leuA (involved in leucine biosynthesis), dapF (involved in lysine biosynthesis), and thrB (involved in threonine biosynthesis) (all GenBank Accession No. NC_002973).

[00421 ] An attenuated Listeria strain can be generated by mutation of other metabolic enzymes, such as a tRNA synthetase. For example, the metabolic enzyme can be encoded by the trpS gene, encoding tryptophanyltRNA synthetase. For example, the host strain bacteria can be A(trpS aroA), and both markers can be contained in an integration vector.

[00422] Other examples of metabolic enzymes that can be mutated to generate an attenuated Listeria strain include an enzyme encoded by murE (involved in synthesis of diaminopimelic acid; GenBank Accession No: NC_003485), LMOf2365_2494 (involved in teichoic acid biosynthesis), WecE (Lipopolysaccharide biosynthesis protein rffA; GenBank Accession No: AE014075.1), or amiA (an N-acetylmuramoyl-L- alanine amidase). Yet other examples of metabolic enzymes include aspartate aminotransferase, histidinol-phosphate aminotransferase (GenBank Accession No. NP_466347), or the cell wall teichoic acid glycosylation protein GtcA.

[00423] Other examples of metabolic enzymes that can be mutated to generate an attenuated Listeria strain include a synthetic enzyme for a peptidoglycan component or precursor. The component can be, for example, UDP-N-acetylmuramylpentapeptide, UDP- N-acetylglucosamine, MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol, GlcNAc-p- (l,4)-MurNAc-(pentapeptide)-pyrophosphorylundecaprenol, or any other peptidoglycan component or precursor.

[00424] Yet other examples of metabolic enzymes that can be mutated to generate an attenuated Listeria strain include metabolic enzymes encoded by murG, murD, inurA-1, or murA-2 (all set forth in GenBank Accession No. NC_002973). Alternatively, the metabolic enzyme can be any other synthetic enzyme for a peptidoglycan component or precursor. The metabolic enzyme can also be a trans-glycosylase, a trans-peptidase, a carboxy-peptidase, any other class of metabolic enzyme, or any other metabolic enzyme. For example, the metabolic enzyme can be any other Listeria metabolic enzyme or any other Listeria monocytogenes metabolic enzyme.

[00425] Other bacterial strains can be attenuated as described above for Listeria by mutating the corresponding orthologous genes in the other bacterial strains.

(2) Methods of Complementing Attenuated Bacteria and Listeria Strains

[00426] The attenuated bacteria or Listeria strains disclosed herein can further comprise a nucleic acid comprising a complementing gene or encoding a metabolic enzyme that complements an attenuating mutation (e.g., complements the auxotrophy of the auxotrophic Listeria strain). For example, a nucleic acid having a first open reading frame encoding a fusion polypeptide as disclosed herein can further comprise a second open reading frame comprising the complementing gene or encoding the complementing metabolic enzyme. Alternatively, a first nucleic acid can encode the fusion polypeptide and a separate second nucleic acid can comprise the complementing gene or encode the complementing metabolic enzyme.

[00427] The complementing gene can be extrachromosomal or can be integrated into the bacteria or Listeria genome. For example, the auxotrophic Listeria strain can comprise an episomal plasmid comprising a nucleic acid encoding a metabolic enzyme. Such plasmids will be contained in the Listeria in an episomal or extrachromosomal fashion. Alternatively, the auxotrophic Listeria strain can comprise an integrative plasmid (i.e., integration vector) comprising a nucleic acid encoding a metabolic enzyme. Such integrative plasmids can be used for integration into a Listeria chromosome. Preferably, the episomal plasmid or the integrative plasmid lacks an antibiotic resistance marker.

[00428] The metabolic gene can be used for selection instead of or in addition to an antibiotic resistance gene. As an example, in order to select for auxotrophic bacteria comprising a plasmid encoding a metabolic enzyme or a complementing gene provided herein, transformed auxotrophic bacteria can be grown in a medium that will select for expression of the gene encoding the metabolic enzyme (e.g., amino acid metabolism gene) or the complementing gene. For example, a bacteria auxotrophic for D-glutamic acid synthesis can be transformed with a plasmid comprising a gene for D-glutamic acid synthesis, and the auxotrophic bacteria will grow in the absence of D-glutamic acid, whereas auxotrophic bacteria that have not been transformed with the plasmid, or are not expressing the plasmid encoding a protein for D-glutamic acid synthesis, will not grow. Similarly, a bacterium auxotrophic for D-alanine synthesis will grow in the absence of D-alanine when transformed and expressing a plasmid comprising a nucleic acid encoding an amino acid metabolism enzyme for D-alanine synthesis. Such methods for making appropriate media comprising or lacking necessary growth factors, supplements, amino acids, vitamins, antibiotics, and the like are well-known and are available commercially.

[00429] Once the auxotrophic bacteria comprising the plasmid encoding a metabolic enzyme or a complementing gene provided herein have been selected in appropriate medium, the bacteria can be propagated in the presence of a selective pressure. Such propagation can comprise growing the bacteria in media without the auxotrophic factor. The presence of the plasmid expressing the metabolic enzyme or the complementing gene in the auxotrophic bacteria ensures that the plasmid will replicate along with the bacteria, thus continually selecting for bacteria harboring the plasmid. Production of the bacteria or Listeria strain can be readily scaled up by adjusting the volume of the medium in which the auxotrophic bacteria comprising the plasmid are growing.

[00430] In one specific example, the attenuated strain is a strain having a deletion of or an inactivating mutation in dal and dat (e.g., Listeria monocytogenes {Lm) dal{-)dat{-) (Lmdd) or Lm dal(-)dat(-) actA (LmddA)), and the complementing gene encodes an alanine racemase enzyme (e.g., encoded by dal gene) or a D-amino acid aminotransferase enzyme (e.g., encoded by dat gene). An exemplary alanine racemase protein can have the sequence set forth in SEQ ID NO: 353 (encoded by SEQ ID NO: 355; GenBank Accession No:

AF038438) or can be a homologue, variant, isoform, analog, fragment, fragment of a homologue, fragment of a variant, fragment of an analog, or fragment of an isoform of SEQ ID NO: 353 . The alanine racemase protein can also be any other Listeria alanine racemase protein. Alternatively, the alanine racemase protein can be any other gram-positive alanine racemase protein or any other alanine racemase protein. An exemplary D-amino acid aminotransferase protein can have the sequence set forth in SEQ ID NO: 354 (encoded by SEQ ID NO: 356; GenBank Accession No: AF038439) or can be a homologue, variant, isoform, analog, fragment, fragment of a homologue, fragment of a variant, fragment of an analog, or fragment of an isoform of SEQ ID NO: 354. The D-amino acid aminotransferase protein can also be any other Listeria D-amino acid aminotransferase protein. Alternatively, the D-amino acid aminotransferase protein can be any other gram-positive D-amino acid aminotransferase protein or any other D-amino acid aminotransferase protein.

[00431 ] In another specific example, the attenuated strain is a strain having a deletion of or an inactivating mutation in prfA (e.g., Lm prfA(-)), and the complementing gene encodes a PrfA protein. For example, the complementing gene can encode a mutant PrfA (D133V) protein that restores partial PrfA function. An example of a wild type PrfA protein is set forth in SEQ ID NO: 357 (encoded by nucleic acid set forth in SEQ ID NO: 358), and an example of a D133V mutant PrfA protein is set forth in SEQ ID NO: 359 (encoded by nucleic acid set forth in SEQ ID NO: 360). The complementing PrfA protein can be a homologue, variant, isoform, analog, fragment, fragment of a homologue, fragment of a variant, fragment of an analog, or fragment of an isoform of SEQ ID NO: 357 or 359. The PrfA protein can also be any other Listeria PrfA protein. Alternatively, the PrfA protein can be any other gram-positive PrfA protein or any other PrfA protein.

[00432] In another example, the bacteria strain or Listeria strain can comprise a deletion of or an inactivating mutation in an actA gene, and the complementing gene can comprise an actA gene to complement the mutation and restore function to the Listeria strain.

[00433] Other auxotroph strains and complementation systems can also be adopted for the use with the methods and compositions provided herein. C. Preparation and Storage of Bacteria or Listeria Strains

[00434] The recombinant bacteria strain (e.g., Listeria strain) optionally has been passaged through an animal host. Such passaging can maximize efficacy of the Listeria strain as a vaccine vector, can stabilize the immunogenicity of the Listeria strain, can stabilize the virulence of the Listeria strain, can increase the immunogenicity of the Listeria strain, can increase the virulence of the Listeria strain, can remove unstable sub-strains of the Listeria strain, or can reduce the prevalence of unstable sub- strains of the Listeria strain. Methods for passaging a recombinant Listeria strain through an animal host are well known in the art and are described, for example, in US 2006/0233835, herein incorporated by reference in its entirety for all purposes.

[00435] The recombinant bacteria strain (e.g., Listeria strain) can be stored in a frozen cell bank or stored in a lyophilized cell bank. Such a cell bank can be, for example, a master cell bank, a working cell bank, or a Good Manufacturing Practice (GMP) cell bank. Examples of "Good Manufacturing Practices" include those defined by 21 CFR 210-211 of the United States Code of Federal Regulations. However, "Good Manufacturing Practices" can also be defined by other standards for production of clinical-grade material or for human

consumption, such as standards of a country other than the United States. Such cell banks can be intended for production of clinical-grade material or can conform to regulatory practices for human use.

[00436] Such a cell bank can comprise, for example, 1-5, 5-10, 10-15, 15-20, 20-25, 25- 30, 30-35, 35-40, 40-45, or 45-50 or more recombinant Listeria strains disclosed herein. Such recombinant Listeria strains can comprise recurrent cancer mutations in, for example, 1- 5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 cancer-associated proteins. For example, the recombinant Listeria strains can comprise the 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 most common recurrent cancer mutations in each cancer-associated protein. Likewise, for each cancer- associated protein, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a mutation in the cancer-associated protein have a recurrent cancer mutation in the cancer-associated protein that is included in the combination of antigenic peptides in the recombinant Listeria strains in the cell bank.

[00437] Recombinant bacteria strains (e.g., Listeria strains) can also be from a batch of vaccine doses, from a frozen stock, or from a lyophilized stock. [00438] Such cell banks, frozen stocks, or batches of vaccine doses can, for example, exhibit viability upon thawing of greater than 90%. The thawing, for example, can follow storage for cryopreservation or frozen storage for 24 hours. Alternatively, the storage can last, for example, for 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 5 months, 6 months, 9 months, or 1 year.

[00439] The cell bank, frozen stock, or batch of vaccine doses can be cryopreserved, for example, by a method that comprises growing a culture of the bacteria strain (e.g., Listeria strain) in a nutrient media, freezing the culture in a solution comprising glycerol, and storing the Listeria strain at below -20°C. The temperature can be, for example, about -70°C or between about -70 to about -80°C. Alternatively, the cell bank, frozen stock, or batch of vaccine doses can be cryopreserved by a method that comprises growing a culture of the Listeria strain in a defined medium, freezing the culture in a solution comprising glycerol, and storing the Listeria strain at below -20°C. The temperature can be, for example, about - 70°C or between about -70 to about -80°C. Any defined microbiological medium may be used in this method.

[00440] The culture (e.g., the culture of a Listeria vaccine strain that is used to produce a batch of Listeria vaccine doses) can be inoculated, for example, from a cell bank, from a frozen stock, from a starter culture, or from a colony. The culture can be inoculated, for example, at mid-log growth phase, at approximately mid-log growth phase, or at another growth phase.

[00441 ] The solution used for freezing optionally contain another colligative additive or additive with anti- freeze properties in place of glycerol or in addition to glycerol. Examples of such additives include, for example, mannitol, DMSO, sucrose, or any other colligative additive or additive with anti-freeze properties.

[00442] The nutrient medium utilized for growing a culture of a bacteria strain (e.g., a Listeria strain) can be any suitable nutrient medium. Examples of suitable media include, for example, LB; TB; a modified, animal-product-free Terrific Broth; or a defined medium.

[00443] The step of growing can be performed by any known means of growing bacteria. For example, the step of growing can be performed with a shake flask (such as a baffled shake flask), a batch fermenter, a stirred tank or flask, an airlift fermenter, a fed batch, a continuous cell reactor, an immobilized cell reactor, or any other means of growing bacteria.

[00444] Optionally, a constant pH is maintained during growth of the culture (e.g. in a batch fermenter). For example, the pH can be maintained at about 6.0, at about 6.5, at about 7.0, at about 7.5, or about 8.0. Likewise, the pH can be, for example, from about 6.5 to about 7.5, from about 6.0 to about 8.0, from about 6.0 to about 7.0, from about 6.0 to about 7.0, or from about 6.5 to about 7.5.

[00445] Optionally, a constant temperature can be maintained during growth of the culture. For example, the temperature can be maintained at about 37°C or at 37°C.

Alternatively, the temperature can be maintained at 25°C, 27°C, 28°C, 30°C, 32°C, 34°C, 35°C, 36°C, 38°C, or 39°C.

[00446] Optionally, a constant dissolved oxygen concentration can be maintained during growth of the culture. For example, the dissolved oxygen concentration can be maintained at 20% of saturation, 15% of saturation, 16% of saturation, 18% of saturation, 22% of saturation, 25% of saturation, 30% of saturation, 35% of saturation, 40% of saturation, 45% of saturation, 50% of saturation, 55% of saturation, 60% of saturation, 65% of saturation, 70% of saturation, 75% of saturation, 80% of saturation, 85% of saturation, 90% of saturation, 95% of saturation, 100% of saturation, or near 100% of saturation.

[00447] Methods for lyophilization and cryopreservation of recombinant bacteria strains (e.g., Listeria strains are known. For example, a Listeria culture can be flash-frozen in liquid nitrogen, followed by storage at the final freezing temperature. Alternatively, the culture can be frozen in a more gradual manner (e.g., by placing in a vial of the culture in the final storage temperature). The culture can also be frozen by any other known method for freezing a bacterial culture.

[00448] The storage temperature of the culture can be, for example, between -20 and - 80°C. For example, the temperature can be significantly below -20°C or not warmer than - 70°C. Alternatively, the temperature can be about -70°C, -20°C, -30°C, -40°C, -50°C, -60°C, -80°C, -30 to -70°C, -40 to -70°C, -50 to -70°C, -60 to -70°C, -30 to -80°C, -40 to -80°C, -50 to -80°C, -60 to -80°C, or -70 to -80°C. Alternatively, the temperature can be colder than 70°C or colder than -80°C.

VIII. Immunogenic Compositions, Pharmaceutical Compositions, and Vaccines

[00449] Also provided are immunogenic compositions, pharmaceutical compositions, or vaccines comprising a recombinant fusion polypeptide as disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide as disclosed herein, or a recombinant bacteria or Listeria strain as disclosed herein. An immunogenic composition comprising a Listeria strain can be inherently immunogenic by virtue of its comprising a Listeria strain and/or the composition can also further comprise an adjuvant. Other immunogenic compositions comprise DNA immunotherapy or peptide immunotherapy compositions.

[00450] The term "immunogenic composition" refers to any composition containing an antigen that elicits an immune response against the antigen in a subject upon exposure to the composition. The immune response elicited by an immunogenic composition can be to a particular antigen or to a particular epitope on the antigen.

[00451 ] An immunogenic composition can comprise a single recombinant fusion polypeptide as disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide as disclosed herein, or a recombinant bacteria or Listeria strain as disclosed herein, or it can comprise multiple different recombinant fusion polypeptides as disclosed herein, nucleic acids encoding recombinant fusion polypeptides as disclosed herein, or recombinant bacteria or Listeria strains as disclosed herein. A first recombinant fusion polypeptide is different from a second recombinant fusion polypeptide, for example, if it includes one antigenic peptide that the second recombinant fusion polypeptide does not. The two recombinant fusion polypeptides can include many of the same antigenic peptides and still be considered different. As one example, an immunogenic composition can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, or recombinant bacteria or Listeria strains.

Alternatively, an immunogenic composition can comprise a mixture of 1-2, 1-5, 1-10, 1-20 or 1-40, or a mixture of 1-5, 5-10, 10-15, 15-20, 10-20, 20-30, 30-40, or 40-50 recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, or recombinant bacteria or Listeria strains. Such different recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, or recombinant bacteria or Listeria strains can be administered concomitantly to a subject or sequentially to a subject. Sequential

administration can be particularly useful when a drug substance comprising a recombinant Listeria strain (or recombinant fusion polypeptide or nucleic acid) disclosed herein is in different dosage forms (e.g., one agent is a tablet or capsule and another agent is a sterile liquid) and/or is administered on different dosing schedules (e.g., one composition from the mixture is administered at least daily and another is administered less frequently, such as once weekly, once every two weeks, or once every three weeks). The multiple recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, or recombinant bacteria or Listeria strains can each comprise a different set of antigenic peptides.

Alternatively, two or more of the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, or recombinant bacteria or Listeria strains can comprise the same set of antigenic peptides (e.g., the same set of antigenic peptides in a different order).

[00452] An immunogenic composition can also comprise one or more recurrent cancer mutation immunotherapy compositions as disclosed herein (e.g., recombinant fusion polypeptides as disclosed herein, nucleic acids encoding recombinant fusion polypeptides as disclosed herein, or recombinant bacteria or Listeria strains as disclosed herein) in

combination with one or more personalized neoepitope immunotherapy compositions as disclosed herein (e.g., ., recombinant fusion polypeptides as disclosed herein, nucleic acids encoding recombinant fusion polypeptides as disclosed herein, or recombinant bacteria or Listeria strains as disclosed herein).

[00453] For the recurrent cancer mutation compositions, the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, or recombinant bacteria or Listeria strains can comprise antigenic peptides from a single cancer-associated protein or from multiple cancer-associated proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cancer-associated proteins).

[00454] In addition, the combination of recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, or recombinant bacteria or Listeria strains can comprise any number of different antigenic peptides, such as about 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-120, 120- 140, 140-160, 160-180, 180-200, 200-220, 220-240, 240-260, 260-280, 280-300, 300-320, 320-340, 340-360, 360-380, or 380-400 different antigenic peptides. The number of different antigenic peptides can be up to about 100, above about 100, up to about 10, up to about 20, up to about 50 antigenic peptides. Alternatively, it can be about 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, or 50 antigenic peptides or about 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, or 100 antigenic peptides or about 5-15, 5-20, 5-25, 15-20, 15-25, 15-30, 15-35, 20-25, 20-35, 20- 45, 30-45, 30-55 ,40-55, 40-65, 50-65, 50-75, 60-75, 60-85, 70-85, 70-95, 80-95, 80-105 or 95-105 antigenic peptides or about 1-5, 1-10 , 1-20, 1-30, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-150, 1-200, 1-250, 1-300, or 1-500 antigenic peptides.

[00455] Any combination of recurrent cancer mutations can be included in the

immunogenic composition. Each of the recurrent cancer mutations can be a somatic missense mutation, or the recurrent cancer mutations can comprise other mutations as well. For example, in some immunogenic compositions, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the recurrent cancer mutations are somatic missense mutations. As one example, the antigenic peptides can comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the 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, or 40 most common recurrent cancer mutations in the cancer-associated protein. For example, the antigenic peptides can comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the 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, or 40 most common recurrent somatic missense cancer mutations in the cancer-associated protein. As another example, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a mutation in the cancer- associated protein have a recurrent cancer mutation in the cancer-associated protein that is included in the combination of antigenic peptides in the immunogenic composition. For example, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a somatic missense mutation in the cancer-associated protein have a recurrent cancer mutation in the cancer-associated protein that is included in the combination of antigenic peptides in the immunogenic composition. As another example, the antigenic peptides can comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the 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, or 40 most common recurrent cancer mutations or most common recurrent somatic missense cancer mutations in a particular type of cancer. As another example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a particular type of cancer have a recurrent cancer mutation that is included in the combination of antigenic peptides in the immunogenic composition. For example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with particular type of cancer have a recurrent cancer mutation that is included in the combination of antigenic peptides in the immunogenic composition. In a particular example, the antigenic peptides comprise at least 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, or 60 different recurrent cancer mutations or different recurrent somatic missense mutations from the same type of cancer, or the antigenic peptides comprise 2-80, 10-60, 10-50, 10-40, or 10-30 different recurrent cancer mutations or different recurrent somatic mis sense mutations from a single type of cancer. For example, the single type of cancer can be no n- small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer (e.g., ER+ breast cancer), uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer (e.g., MSS colorectal cancer), or head and neck cancer.

[00456] An immunogenic composition can additionally comprise an adjuvant (e.g., two or more adjuvants), a cytokine, a chemokine, or combination thereof. Optionally, an immunogenic composition can additionally comprises antigen presenting cells (APCs), which can be autologous or can be allogeneic to the subject.

[00457] The term adjuvant includes compounds or mixtures that enhance the immune response to an antigen. For example, an adjuvant can be a non-specific stimulator of an immune response or substances that allow generation of a depot in a subject which when combined with an immunogenic composition disclosed herein provides for an even more enhanced and/or prolonged immune response. An adjuvant can favor, for example, a predominantly Thl-mediated immune response, a Thl-type immune response, or a Thl- mediated immune response. Likewise, an adjuvant can favor a cell-mediated immune response over an antibody-mediated response. Alternatively, an adjuvant can favor an antibody-mediated response. Some adjuvants can enhance the immune response by slowly releasing the antigen, while other adjuvants can mediate their effects by any of the following mechanisms: increasing cellular infiltration, inflammation, and trafficking to the injection site, particularly for antigen-presenting cells (APC); promoting the activation state of APCs by upregulating costimulatory signals or major histocompatibility complex (MHC) expression; enhancing antigen presentation; or inducing cytokine release for indirect effect.

[00458] Examples of adjuvants include saponin QS21, CpG oligonucleotides,

unmethylated CpG-containing oligonucleotides, MPL, TLR agonists, TLR4 agonists, TLR9 agonists, Resiquimod^®, imiquimod, cytokines or nucleic acids encoding the same, chemokines or nucleic acids encoding same, IL- 12 or a nucleic acid encoding the same, IL-6 or a nucleic acid encoding the same, and lipopolysaccharides. Another example of a suitable adjuvant is Montanide ISA 51. Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier. Other examples of a suitable adjuvant include granulocyte/macrophage colony- stimulating factor (GM-CSF) or a nucleic acid encoding the same and keyhole limpet hemocyanin (KLH) proteins or nucleic acids encoding the same. The GM-CSF can be, for example, a human protein grown in a yeast (S. cerevisiae) vector. GM-CSF promotes clonal expansion and differentiation of hematopoietic progenitor cells, antigen presenting cells (APCs), dendritic cells, and T cells. Yet another example of a suitable adjuvant is detoxified listeriolysin O (dtLLO) protein. One example of a dtLLO suitable for use as an adjuvant is encoded by SEQ ID NO: 583. A dtLLO encoded by a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 583 is also suitable for use as an adjuvant. Other examples of adjuvants include growth factors or nucleic acids encoding the same, cell populations, Freund' s incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG (bacille Calmette-Guerin), alum, interleukins or nucleic acids encoding the same, quill glycosides, monophosphoryl lipid A, liposomes, bacterial mitogens, bacterial toxins, or any other type of known adjuvant (see, e.g., Fundamental Immunology, 5th ed. (August 2003): William E. Paul (Editor); Lippincott Williams & Wilkins Publishers; Chapter 43: Vaccines, GJV Nossal, which is herein incorporated by reference in its entirety for all purposes).

[00459] An immunogenic composition can further comprise one or more

immunomodulatory molecules. Examples include interferon gamma, a cytokine, a chemokine, and a T cell stimulant.

[00460] An immunogenic composition can be in the form of a vaccine or pharmaceutical composition. The terms "vaccine" and "pharmaceutical composition" are interchangeable and refer to an immunogenic composition in a pharmaceutically acceptable carrier for in vivo administration to a subject. A vaccine may be, for example, a peptide vaccine (e.g., comprising a recombinant fusion polypeptide as disclosed herein), a DNA vaccine (e.g., comprising a nucleic acid encoding a recombinant fusion polypeptide as disclosed herein), or a vaccine contained within and delivered by a cell (e.g., a recombinant Listeria as disclosed herein). A vaccine may prevent a subject from contracting or developing a disease or condition and/or a vaccine may be therapeutic to a subject having a disease or condition. Methods for preparing peptide vaccines are well known and are described, for example, in EP 1408048, US 2007/0154953, and Ogasawara et al. (1992) Proc. Natl Acad Sci USA 89:8995- 8999, each of which is herein incorporated by reference in its entirety for all purposes.

Optionally, peptide evolution techniques can be used to create an antigen with higher immunogenicity. Techniques for peptide evolution are well known and are described, for example, in US 6,773,900, herein incorporated by reference in its entirety for all purposes.

[00461 ] A "pharmaceutically acceptable carrier" refers to a vehicle for containing an immunogenic composition that can be introduced into a subject without significant adverse effects and without having deleterious effects on the immunogenic composition. That is, "pharmaceutically acceptable" refers to any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of at least one immunogenic composition for use in the methods disclosed herein. Pharmaceutically acceptable carriers or vehicles or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers, and factors involved in their selection, are found in a variety of readily available sources such as, for example, Remington 's Pharmaceutical Sciences, 18th ed., 1990, herein incorporated by reference in its entirety for all purposes. Such carriers can be suitable for any route of administration (e.g., parenteral, enteral (e.g., oral), or topical application). Such pharmaceutical compositions can be buffered, for example, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the immunogenic compositions and route of administration.

[00462] Suitable pharmaceutically acceptable carriers include, for example, sterile water, salt solutions such as saline, glucose, buffered solutions such as phosphate buffered solutions or bicarbonate buffered solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates (e.g., lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, and the like. Pharmaceutical compositions or vaccines may also include auxiliary agents including, for example, diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffering agents, viscosity enhancing additives, lubricants, salts for influencing osmotic pressure, buffers, vitamins, coloring, flavoring, aromatic substances, and the like which do not deleteriously react with the immunogenic composition.

[00463] For liquid formulations, for example, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils include those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish- liver oil. Solid carriers/diluents include, for example, a gum, a starch (e.g., corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, or dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. [00464] Optionally, sustained or directed release pharmaceutical compositions or vaccines can be formulated. This can be accomplished, for example, through use of liposomes or compositions wherein the active compound is protected with differentially degradable coatings (e.g., by microencapsulation, multiple coatings, and so forth). Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the compositions and use the lyophilisates obtained (e.g., for the preparation of products for injection).

[00465] An immunogenic composition, pharmaceutical composition, or vaccine disclosed herein may also comprise one or more additional compounds effective in preventing or treating cancer. For example, the additional compound may comprise a compound useful in chemotherapy, such as amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine, gliadelimplants, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomaldoxorubicin, liposomaldaunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel (Taxol), pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. The additional compound can also comprise other biologies, including Herceptin^® (trastuzumab) against the HER2 antigen, Avastin^® (bevacizumab) against VEGF, or antibodies to the EGF receptor, such as Erbitux^® (cetuximab), and Vectibix^® (panitumumab). The additional compound can also comprise, for example, an additional immunotherapy.

[00466] An additional compound can also comprise an immune checkpoint inhibitor antagonist, such as a PD-1 signaling pathway inhibitor, a CD-80/86 and CTLA-4 signaling pathway inhibitor, a T cell membrane protein 3 (TIM3) signaling pathway inhibitor, an adenosine A2a receptor (A2aR) signaling pathway inhibitor, a lymphocyte activation gene 3 (LAG3) signaling pathway inhibitor, a killer immunoglobulin receptor (KIR) signaling pathway inhibitor, a CD40 signaling pathway inhibitor, or any other antigen-presenting cell/T cell signaling pathway inhibitor. Examples of immune checkpoint inhibitor antagonists include an anti-PD-Ll/PD-L2 antibody or fragment thereof, an anti-PD-1 antibody or fragment thereof, an anti-CTLA-4 antibody or fragment thereof, or an anti-B7-H4 antibody or fragment thereof. An additional compound can also comprise a T cell stimulator, such as an antibody or functional fragment thereof binding to a T-cell receptor co-stimulatory molecule, an antigen presenting cell receptor binding co-stimulatory molecule, or a member of the TNF receptor superfamily. The T-cell receptor co-stimulatory molecule can comprise, for example, CD28 or ICOS. The antigen presenting cell receptor binding co-stimulatory molecule can comprise, for example, a CD80 receptor, a CD86 receptor, or a CD46 receptor. The TNF receptor superfamily member can comprise, for example, glucocorticoid-induced TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor), or TNFR25. See, e.g., WO2016100929, WO2016011362, and WO2016011357, each of which is incorporated by reference in its entirety for all purposes.

IX. Therapeutic Methods and Systems

[00467] The recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, and vaccines disclosed herein can be used in various methods. In addition, systems for use in cancer immunotherapy in a subject with cancer are provided. Such systems comprise a recurrent cancer mutation immunotherapy composition as described elsewhere herein (i.e., antigenic peptide comprising recurrent cancer mutations), and a personalized neoepitope immunotherapy composition as described elsewhere herein (i.e., antigenic peptides comprising personalized, cancer- specific neoepitopes). For example, they can be used in methods of inducing an anti-tumor-associated-antigen immune response in a subject, in methods of inducing an anti-tumor or anti-cancer immune response in a subject, in methods of treating a tumor or cancer in a subject, in methods of preventing a tumor or cancer in a subject, or in methods of protecting a subject against a tumor or cancer. They can also be used in methods of increasing the ratio of T effector cells to regulatory T cells (Tregs) in the spleen and tumor of a subject, wherein the T effector cells are targeted to a tumor- associated antigen. They can also be used in methods for increasing tumor-associated- antigen T cells in a subject, increasing survival time of a subject having a tumor or cancer, delaying the onset of cancer in a subject, or reducing tumor or metastasis size in a subject.

[00468] A method of inducing an anti-tumor-associated-antigen immune response in a subject can comprise, for example, administering to the subject a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein (e.g., that comprises a recombinant fusion polypeptide comprising the tumor-associated antigen or a nucleic acid encoding the recombinant fusion polypeptide). An anti-tumor-associated-antigen immune response can thereby be induced in the subject. For example, in the case of a recombinant Listeria strain, the Listeria strain can express the fusion polypeptide, thereby eliciting an immune response in the subject. The immune response can comprise, for example, a T-cell response, such as a CD4+FoxP3- T cell response, a CD8+ T cell response, or a CD4+FoxP3- and CD8+ T cell response. Such methods can also increase the ratio of T effector cells to regulatory T cells (Tregs) in the spleen and tumor microenvironments of the subject, allowing for a more profound anti-tumor response in the subject.

[00469] A method of inducing an anti-tumor or anti-cancer immune response in a subject can comprise, for example, administering to the subject a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein. An anti-tumor or anti-cancer immune response can thereby be induced in the subject. For example, in the case of a recombinant Listeria strain, the Listeria strain can express the fusion polypeptide, thereby eliciting an anti-tumor or anti-cancer response in the subject.

[00470] A method of treating a tumor or cancer in a subject (e.g., wherein the tumor or cancer expresses one or more tumor-associated antigens or has one or more recurrent cancer mutations), .can comprise, for example, administering to the subject a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein. The subject can then mount an immune response against the tumor or cancer expressing the one or more tumor-associated antigens or the one or more recurrent cancer mutations, thereby treating the tumor or cancer in the subject.

[00471 ] A method of preventing a tumor or cancer in a subject or protecting a subject against developing a tumor or cancer (e.g., wherein the tumor or cancer is associated with expression of one or more tumor-associated antigens or one or more recurrent cancer mutations), can comprise, for example, administering to the subject a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein. The subject can then mount an immune response against the one or more tumor-associated antigens or the one or more recurrent cancer mutations, thereby preventing a tumor or cancer or protecting the subject against developing a tumor or cancer.

[00472] The recombinant fusion polypeptide, nucleic acid encoding a recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine can comprise a recurrent cancer mutation

immunotherapy composition (i.e., antigenic peptide comprising recurrent cancer mutations), a personalized neoepitope immunotherapy composition (i.e., antigenic peptides comprising personalized, cancer- specific neoepitopes), or a combination of both. The recurrent cancer mutation immunotherapy composition can be administered prior to, concurrent with, or subsequent to the personalized immunotherapy composition.

[00473] Some methods can further comprise generating the personalized immunotherapy composition for the subject. The generating can be done concurrent with, prior to, or subsequent to administering the recurrent cancer mutation immunotherapy composition to the subject. For example, a first treatment regime can be started with the recurrent cancer mutation immunotherapy. During the first treatment regime, the personalized neoepitope immunotherapy composition can be generated. A second treatment regime can then be started with the personalized neoepitope immunotherapy composition. Methods of generating personalized neoepitope immunotherapy compositions are disclosed elsewhere herein.

[00474] In some of the above methods, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines are administered. For example, a first Listeria strain comprising antigenic peptides comprising recurrent cancer mutations from a first cancer-associated protein can be administered, and a second Listeria strain comprising antigenic peptides comprising recurrent cancer mutations from a second cancer-associated protein can be administered. Likewise, a first Listeria strain comprising antigenic peptides comprising a first set of personalized cancer- specific neoepitopes can be administered, and a second Listeria strain comprising a second set of personalized cancer- specific neoepitopes can be administered. The multiple recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can be administered sequentially in any order or combination, or can be administered simultaneously in any combination. As an example, if four different Listeria strains are being administered, they can be administered sequentially, they can be administered simultaneously, or they can be administered in any combination (e.g., administering the first and second strains simultaneously and subsequently administering the third and fourth strains simultaneously). Optionally, in the case of sequential administration, the compositions can be administered during the same immune response, preferably within 0-10 or 3-7 days of each other. The multiple recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can each comprise a different set of antigenic peptides. Alternatively, two or more can comprise the same set of antigenic peptides (e.g., the same set of antigenic peptides in a different order). The multiple recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can comprise antigenic peptides from two or more cancer-associated proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cancer-associated proteins). In addition, the combination of multiple recombinant fusion polypeptides, nucleic acids encoding

recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can comprise about 5-10, 10-15, 15- 20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-120, 120-140, 140-160, 160-180, 180-200, 200-220, 220-240, 240-260, 260-280, 280-300, 300- 320, 320-340, 340-360, 360-380, or 380-400 different antigenic peptides.

[00475] In any of the above methods, any combination of recurrent cancer mutations can be included in the administered recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines. Each of the recurrent cancer mutations can be a somatic mis sense mutation, or the recurrent cancer mutations can comprise other mutations as well. For example, in some methods, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the recurrent cancer mutations are somatic missense mutations. As one example, the antigenic peptides can comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the 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, or 40 most common recurrent cancer mutations in the cancer-associated protein. For example, the antigenic peptides can comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the 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, or 40 most common recurrent somatic missense cancer mutations in the cancer-associated protein. As another example, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a mutation in the cancer-associated protein have a recurrent cancer mutation in the cancer- associated protein that is included in the combination of antigenic peptides administered. For example, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a somatic missense mutation in the cancer-associated protein have a recurrent cancer mutation in the cancer-associated protein that is included in the combination of antigenic peptides administered. As another example, the antigenic peptides can comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the 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, or 40 most common recurrent cancer mutations or most common recurrent somatic missense cancer mutations in a particular type of cancer. As another example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a particular type of cancer have a recurrent cancer mutation that is included in the combination of antigenic peptides administered. For example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with particular type of cancer have a recurrent cancer mutation that is included in the combination of antigenic peptides administered. In a particular example, the antigenic peptides comprise at least 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, or 60 different recurrent cancer mutations or different recurrent somatic missense mutations from the same type of cancer, or the antigenic peptides comprise 2-80, 10-60, 10-50, 10-40, or 10-30 different recurrent cancer mutations or different recurrent somatic missense mutations from a single type of cancer. For example, the single type of cancer can be no n- small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer (e.g., ER+ breast cancer), uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer (e.g., MSS colorectal cancer), or head and neck cancer.

[00476] Any of the above methods can further comprise screening the subject for and identifying one or more recurrent cancer mutations prior to the administering step, and then administering to the subject a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine comprising antigenic peptides comprising the one or more recurrent cancer mutations identified in the subject.

Alternatively, in cases in which the subject has a cancer associated with recurrent cancer mutations in one or more cancer-associated proteins, the method can comprise administering to the subject a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine comprising the recurrent cancer mutations associated with the cancer. For example mutations in a particular cancer-associated protein may occur in at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all instances of a particular type of cancer, or mutations at a particular residue (i.e., hotspot) or set of residues (i.e., hotspots) in a cancer- associated protein may occur in at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all instances of a particular type of cancer. Likewise, a particular recurrent cancer mutation may occur in at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all instances of a particular type of cancer (e.g., all subjects having a particular type of cancer). Similarly, a particular set of recurrent cancer mutations may occur in at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all instances of a particular type of cancer (e.g., all subjects having a particular type of cancer).

[00477] Cancer is a physiological condition in mammals that is typically characterized by unregulated cell growth and proliferation. Cancers can be hematopoietic malignancies or solid tumors (i.e., masses of cells that result from excessive cell growth or proliferation, including pre-cancerous legions). Metastatic cancer refers to a cancer that has spread from the place where it first started to another place in the body. Tumors formed by metastatic cancer cells are called a metastatic tumor or a metastasis, which is a term also used to refer to the process by which cancer cells spread to other parts of the body. In general, metastatic cancer has the same name and same type of cancer cells as the original, or primary, cancer. Examples of solid tumors include melanoma, carcinoma, blastoma, and sarcoma.

Hematologic malignancies include, for example, leukemia or lymphoid malignancies, such as lymphoma. Exemplary categories of cancers include brain, breast, gastrointestinal, genitourinary, gynecologic, head and neck, heme, skin and thoracic. Brain malignancies include, for example, glioblastoma, high-grade pontine glioma, low-grade glioma, medulloblastoma, neuroblastoma, and pilocytic astrocytoma. Gastrointestinal cancers include, for example, colorectal, gallbladder, hepatocellular, pancreas, PNET, gastric, and esophageal. Genitourinary cancers include, for example, adrenocortical, bladder, kidney chromophobe, renal (clear cell), renal (papillary), rhabdoid cancers, and prostate.

Gynecologic cancers include, for example, uterine carcinosarcoma, uterine endometrial, serous ovarian, and cervical. Head and neck cancers include, for example, thyroid, nasopharyngeal, head and neck, and adenoid cystic. Heme cancers include, for example, multiple myeloma, myelodysplasia, mantle-cell lymphoma, acute lymphoblastic leukemia (ALL), non-lymphoma, chronic lymphocytic leukemia (CLL), and acute myeloid leukemia (AML). Skin cancers includes, for example, cutaneous melanoma and squamous cell carcinoma. Thoracic cancers include, for example, squamous lung, small-cell lung, and lung adenocarcinoma.

[00478] More particular examples of such cancers include squamous cell cancer or carcinoma (e.g., oral squamous cell carcinoma), myeloma, oral cancer, juvenile

nasopharyngeal angiofibroma, neuroendocrine tumors, lung cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, glial tumors, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, breast cancer, triple-negative breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer or carcinoma, salivary gland carcinoma, kidney or renal cancer (e.g., renal cell carcinoma), prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, fibrosarcoma, gallbladder cancer, osteosarcoma, mesothelioma, as well as head and neck cancer. A cancer can also be a brain cancer or another type of CNS or intracranial tumor. For example, a subject can have an astrocytic tumor (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, pilocytic astrocytoma, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma), oligodendroglial tumor (e.g., oligodendroglioma, anaplastic oligodendroglioma), ependymal cell tumor (e.g., ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma), mixed glioma (e.g., mixed oligoastrocytoma, anaplastic oligoastrocytoma), neuroepithelial tumor of uncertain origin (e.g., polar spongioblastoma, astroblastoma, gliomatosis cerebri), tumor of the choroid plexus (e.g., choroid plexus papilloma, choroid plexus carcinoma), neuronal or mixed neuronal-glial tumor (e.g., gangliocytoma, dyplastic gangliocytoma of cerebellum, ganglioglioma, anaplastic ganglioglioma, desmoplastic infantile ganglioma, central neurocytoma, dysembryoplastic neuroepthelial tumor, olfactory neuroblastoma), pineal parenchyma tumor (e.g., pineocytoma, pineoblastoma, mixed pineocytoma/pineoblastoma), or tumor with mixed neuroblastic or glioblastic elements (e.g., medulloepithelioma, medulloblastoma,

neuroblastoma, retinoblastoma, ependymoblastoma). Other examples of cancer include low- grade glioma, non-small cell lung cancer (NSCLC), estrogen-receptor-positive (ER+) breast cancer, and DNA mismatch repair deficient cancers or tumors. A cancer is called estrogen- receptor-positive if it has receptors for estrogen. Another example of a cancer is a micro satellite stable (MSS) colorectal cancer.

[00479] The term "treat" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted tumor or cancer. Treating may include one or more of directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, slowing the progression of, stabilizing the progression of, inducing remission of, preventing or delaying the metastasis of, reducing/ameliorating symptoms associated with the tumor or cancer, or a combination thereof. For example, treating may include increasing expected survival time or decreasing tumor or metastasis size. The effect (e.g., suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, slowing the progression of, stabilizing the progression of, inducing remission of, preventing or delaying the metastasis of, reducing/ameliorating symptoms of, and so forth, can be relative to a control subject not receiving a treatment or receiving a placebo treatment. The term "treat" or "treating" can also refer to increasing percent chance of survival or increasing expected time of survival for a subject with the tumor or cancer (e.g., relative to a control subject not receiving a treatment or receiving a placebo treatment). In one example, "treating" refers to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of alternative therapeutics, decreasing resistance to alternative therapeutics, or a combination thereof (e.g., relative to a control subject not receiving a treatment or receiving a placebo treatment). The terms "preventing" or "impeding" can refer, for example to delaying the onset of symptoms, preventing relapse of a tumor or cancer, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, preventing metastasis of a tumor or cancer, or a combination thereof. The terms "suppressing" or "inhibiting" can refer, for example, to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

[00480] The term "subject" refers to a mammal (e.g., a human) in need of therapy for, or susceptible to developing, a tumor or a cancer. The term subject also refers to a mammal (e.g., a human) that receives either prophylactic or therapeutic treatment. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, non-human mammals, and humans. The term "subject" does not necessarily exclude an individual that is healthy in all respects and does not have or show signs of cancer or a tumor.

[00481 ] An individual is at increased risk of developing a tumor or a cancer if the subject has at least one known risk-factor (e.g., genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the tumor or cancer than individuals without the risk factor.

[00482] A "symptom" or "sign" refers to objective evidence of a disease as observed by a physician or subjective evidence of a disease, such as altered gait, as perceived by the subject. A symptom or sign may be any manifestation of a disease. Symptoms can be primary or secondary. The term "primary" refers to a symptom that is a direct result of a particular disease or disorder (e.g., a tumor or cancer), while the term "secondary" refers to a symptom that is derived from or consequent to a primary cause. The recombinant fusion polypeptides, nucleic acids encoding the recombinant fusion polypeptides, the immunogenic compositions, the pharmaceutical compositions, and the vaccines disclosed herein can treat primary or secondary symptoms or secondary complications.

[00483] The recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines are administered in an effective regime, meaning a dosage, route of administration, and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of the tumor or cancer. Alternatively, the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines are administered in an effective regime, meaning a dosage, route of administration, and frequency of administration that induces an immune response to a heterologous antigen in the recombinant fusion polypeptide (or encoded by the nucleic acid), the recombinant bacteria or Listeria strain, the immunogenic composition, the pharmaceutical composition, or the vaccine, or in the case of recombinant bacteria or Listeria strains, that induces an immune response to the bacteria or Listeria strain itself. If a subject is already suffering from the tumor or cancer, the regime can be referred to as a therapeutically effective regime. If the subject is at elevated risk of developing the tumor or cancer relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients. For example, a regime can be considered therapeutically or prophylactic ally effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods described herein, or if a more favorable outcome is demonstrated in treated patients versus control patients in a controlled clinical trial (e.g., a phase II, phase II/III or phase III trial) at the p < 0.05 or 0.01 or even 0.001 level.

[00484] Exemplary dosages for a recombinant Listeria strain are, for example, 1 x 10⁶ - 1 x 10⁷ CFU, 1 x 10⁷ - 1 x 10^s CFU, 1 x 10^s - 3.31 x 10¹⁰ CFU, 1 x 10⁹ - 3.31 x 10¹⁰ CFU, 5- 500 x 10⁸ CFU, 7-500 x 10⁸ CFU, 10-500 x 10⁸ CFU, 20-500 x 10⁸ CFU, 30-500 x 10⁸ CFU, 50-500 x 10⁸ CFU, 70-500 x 10⁸ CFU, 100-500 x 10⁸ CFU, 150-500 x 10⁸ CFU, 5-300 x 10⁸ CFU, 5-200 x 10⁸ CFU, 5-15 x 10⁸ CFU, 5-100 x 10⁸ CFU, 5-70 x 10⁸ CFU, 5-50 x 10⁸ CFU, 5-30 x 10⁸ CFU, 5-20 x 10⁸ CFU, 1-30 x 10⁹ CFU, 1-20 x 10⁹CFU, 2-30 x 10⁹ CFU, 1-10 x 10⁹ CFU, 2-10 x 10⁹ CFU, 3-10 x 10⁹ CFU, 2-7 x 10⁹ CFU, 2-5 x 10⁹ CFU, and 3-5 x 10⁹ CFU. Other exemplary dosages for a recombinant Listeria strain are, for example, 1 x 10⁷ organisms, 1.5 x 10⁷ organisms, 2 x 10⁸ organisms, 3 x 10⁷ organisms, 4 x 10⁷ organisms, 5 x 10⁷ organisms, 6 x 10⁷ organisms, 7 x 10⁷ organisms, 8 x 10⁷ organisms, 10 x 10⁷ organisms, 1.5 x 10⁸ organisms, 2 x 10⁸ organisms, 2.5 x 10⁸ organisms, 3 x 10⁸ organisms, 3.3 x 10⁸ organisms, 4 x 10⁸ organisms, 5 x 10⁸ organisms, 1 x 10⁹ organisms, 1.5 x 10⁹ organisms, 2 x 10⁹ organisms, 3 x 10⁹ organisms, 4 x 10⁹ organisms, 5 x 10⁹ organisms, 6 x 10⁹ organisms, 7 x 10⁹ organisms, 8 x 10⁹ organisms, 10 x 10⁹ organisms, 1.5 x 10¹⁰ organisms, 2 x 10¹⁰ organisms, 2.5 x 10¹⁰ organisms, 3 x 10¹⁰ organisms, 3.3 x 10¹⁰ organisms, 4 x 10¹⁰ organisms, and 5 x 10¹⁰ organisms. The dosage can depend on the condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic, and other factors.

[00485] Administration can be by any suitable means. For example, administration can be parenteral, intravenous, oral, subcutaneous, intra- arterial, intracranial, intrathecal,

intracerebroventricular, intraperitoneal, topical, intranasal, intramuscular, intra-ocular, intrarectal, conjunctival, transdermal, intradermal, vaginal, rectal, intratumoral, parcanceral, transmucosal, intravascular, intraventricular, inhalation (aerosol), nasal aspiration (spray), sublingual, aerosol, suppository, or a combination thereof. For intranasal administration or application by inhalation, solutions or suspensions of the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines mixed and aerosolized or nebulized in the presence of the appropriate carrier are suitable. Such an aerosol may comprise any recombinant fusion polypeptide, nucleic acids encoding a recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine described herein. Administration may also be in the form of a suppository (e.g., rectal suppository or urethral suppository), in the form of a pellet for subcutaneous implantation (e.g., providing for controlled release over a period of time), or in the form of a capsule. Administration may also be via injection into a tumor site or into a tumor. Regimens of administration can be readily determined based on factors such as exact nature and type of the tumor or cancer being treated, the severity of the tumor or cancer, the age and general physical condition of the subject, body weight of the subject, response of the individual subject, and the like.

[00486] The frequency of administration can depend on the half-life of the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines in the subject, the condition of the subject, and the route of administration, among other factors. The frequency can be, for example, daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the subject's condition or progression of the tumor or cancer being treated. The course of treatment can depend on the condition of the subject and other factors. For example, the course of treatment can be several weeks, several months, or several years (e.g., up to 2 years). For example, repeat administrations (doses) may be undertaken immediately following the first course of treatment or after an interval of days, weeks or months to achieve tumor regression or suppression of tumor growth.

Assessment may be determined by any known technique, including diagnostic methods such as imaging techniques, analysis of serum tumor markers, biopsy, or the presence, absence, or amelioration of tumor-associated symptoms. As a specific example, the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can be administered every 3 weeks for up to 2 years. In one example, a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein is administered in increasing doses in order to increase the T- effector cell to regulatory T cell ratio and generate a more potent anti-tumor immune response. Anti-tumor immune responses can be further strengthened by providing the subject with cytokines including, for example, IFN-γ, TNF-a, and other cytokines known to enhance cellular immune response. See, e.g., US 6,991,785, herein incorporated by reference in its entirety for all purposes.

[00487] Some methods may further comprise "boosting" the subject with additional recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines or administering the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines multiple times.

"Boosting" refers to administering an additional dose to a subject. For example, in some methods, 2 boosts (or a total of 3 inoculations) are administered, 3 boosts are administered, 4 boosts are administered, 5 boosts are administered, or 6 or more boosts are administered. The number of dosages administered can depend on, for example, the response of the tumor or cancer to the treatment.

[00488] Optionally, the recombinant fusion polypeptide, nucleic acids encoding a recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine used in the booster inoculation is the same as the recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine used in the initial "priming" inoculation. Alternatively, the booster recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine is different from the priming recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine. Optionally, the same dosages are used in the priming and boosting inoculations. Alternatively, a larger dosage is used in the booster, or a smaller dosage is used in the booster. The period between priming and boosting inoculations can be experimentally determined. For example, the period between priming and boosting inoculations can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6-8 weeks, or 8-10 weeks.

[00489] Heterologous prime boost strategies have been effective for enhancing immune responses and protection against numerous pathogens. See, e.g., Schneider et al. (1999) Immunol. Rev. 170:29-38; Robinson (2002) Nat. Rev. Immunol. 2:239-250; Gonzalo et al. (2002) Vaccine 20: 1226-1231; and Tanghe (2001) Infect. Immun. 69:3041-3047, each of which is herein incorporated by reference in its entirety for all purposes. Providing antigen in different forms in the prime and the boost injections can maximize the immune response to the antigen. DNA vaccine priming followed by boosting with protein in adjuvant or by viral vector delivery of DNA encoding antigen is one effective way of improving antigen- specific antibody and CD4⁺ T-cell responses or CD8⁺ T-cell responses. See, e.g., Shiver et al. (2002) Nature 415: 331-335; Gilbert et al. (2002) Vaccine 20: 1039-1045; BiUaut-Mulot et al. (2000) Vaccine 19:95-102; and Sin et al. (1999) DNA Cell Biol. 18:771-779, each of which is herein incorporated by reference in its entirety for all purposes. As one example, adding CRL1005 poloxamer (12 kDa, 5% POE) to DNA encoding an antigen can enhance T-cell responses when subjects are vaccinated with a DNA prime followed by a boost with an adenoviral vector expressing the antigen. See, e.g., Shiver et al. (2002) Nature 415:331-335, herein incorporated by reference in its entirety for all purposes. As another example, a vector construct encoding an immunogenic portion of an antigen and a protein comprising the immunogenic portion of the antigen can be administered. See, e.g., US 2002/0165172, herein incorporated by reference in its entirety for all purposes. Similarly, an immune response of nucleic acid vaccination can be enhanced by simultaneous administration of (e.g., during the same immune response, preferably within 0-10 or 3-7 days of each other) a polynucleotide and polypeptide of interest. See, e.g., US 6,500,432, herein incorporated by reference in its entirety for all purposes.

[00490] The therapeutic methods disclosed herein can also comprise administering one or more additional compounds effective in preventing or treating cancer. For example, an additional compound may comprise a compound useful in chemotherapy, such as amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine, gliadelimplants, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomaldoxorubicin, liposomaldaunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel (Taxol), pemetrexed, pento statin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur- uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. Alternatively, an additional compound can also comprise other biologies, including Herceptin^® (trastuzumab) against the HER2 antigen, Avastin^® (bevacizumab) against VEGF, or antibodies to the EGF receptor, such as Erbitux^® (cetuximab), and Vectibix^® (panitumumab). Alternatively, an additional compound can comprise other immunotherapies. Alternatively, the additional compound can be an indoleamine 2,3-dioxygenase (IDO) pathway inhibitor, such as 1-methyltryptophan (1MT), 1-methyltryptophan (1MT), Necro statin- 1, Pyridoxal Isonicotinoyl Hydrazone, Ebselen, 5-Methylindole-3-carboxaldehyde, CAY 10581, an anti-IDO antibody, or a small molecule IDO inhibitor. IDO inhibition can enhance the efficacy of chemotherapeutic agents. The therapeutic methods disclosed herein can also be combined with radiation, stem cell treatment, surgery, or any other treatment.

[00491 ] Such additional compounds or treatments can precede the administration of a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein, follow the administration of a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein, or be simultaneous to the administration of a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein.

[00492] Targeted immunomodulatory therapy is focused primarily on the activation of costimulatory receptors, for example by using agonist antibodies that target members of the tumor necrosis factor receptor superfamily, including 4- IBB, OX40, and GITR

(glucocorticoid-induced TNF receptor-related). The modulation of GITR has demonstrated potential in both antitumor and vaccine settings. Another target for agonist antibodies are co- stimulatory signal molecules for T cell activation. Targeting costimulatory signal molecules may lead to enhanced activation of T cells and facilitation of a more potent immune response. Co-stimulation may also help prevent inhibitory influences from checkpoint inhibition and increase antigen- specific T cell proliferation.

[00493] Listeria-based immunotherapy acts by inducing the de novo generation of tumor antigen- specific T cells that infiltrate and destroy the tumor and by reducing the numbers and activities of immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Antibodies (or functional fragments thereof) for T cell co-inhibitory or co-stimulatory receptors (e.g., checkpoint inhibitors CTLA-4, PD- 1, TIM-3, LAG3 and co-stimulators CD137, OX40, GITR, and CD40) can have synergy with Listeria-based immunotherapy.

[00494] Thus, some methods can comprise further administering a composition

comprising an immune checkpoint inhibitor antagonist, such as a PD-1 signaling pathway inhibitor, a CD-80/86 and CTLA-4 signaling pathway inhibitor, a T cell membrane protein 3 (TIM3) signaling pathway inhibitor, an adenosine A2a receptor (A2aR) signaling pathway inhibitor, a lymphocyte activation gene 3 (LAG3) signaling pathway inhibitor, a killer immunoglobulin receptor (KIR) signaling pathway inhibitor, a CD40 signaling pathway inhibitor, or any other antigen-presenting cell/T cell signaling pathway inhibitor. Examples of immune checkpoint inhibitor antagonists include an anti-PD-Ll/PD-L2 antibody or fragment thereof, an anti-PD- 1 antibody or fragment thereof, an anti-CTLA-4 antibody or fragment thereof, or an anti-B7-H4 antibody or fragment thereof. For example, an anti PD-1 antibody can be administered to a subject at 5-10 mg/kg every 2 weeks, 5-10 mg/kg every 3 weeks, 1-2 mg/kg every 3 weeks, 1-10 mg/kg every week, 1-10 mg/kg every 2 weeks, 1-10 mg/kg every 3 weeks, or 1-10 mg/kg every 4 weeks.

[00495] Likewise, some methods can further comprise administering a T cell stimulator, such as an antibody or functional fragment thereof binding to a T-cell receptor co- stimulatory molecule, an antigen presenting cell receptor binding co-stimulatory molecule, or a member of the TNF receptor superfamily. The T-cell receptor co-stimulatory molecule can comprise, for example, CD28 or ICOS. The antigen presenting cell receptor binding co-stimulatory molecule can comprise, for example, a CD80 receptor, a CD86 receptor, or a CD46 receptor. The TNF receptor superfamily member can comprise, for example, glucocorticoid-induced TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor), or TNFR25.

[00496] For example, some methods can further comprise administering an effective amount of a composition comprising an antibody or functional fragment thereof binding to a T-cell receptor co-stimulatory molecule or an antibody or functional fragment thereof binding to an antigen presenting cell receptor binding a co-stimulatory molecule. The antibody can be, for example, an anti-TNF receptor antibody or antigen-binding fragment thereof (e.g., TNF receptor superfamily member glucocorticoid-induced TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor), or TNFR25), an anti-OX40 antibody or antigen- binding fragment thereof, or an anti-GITR antibody or antigen binding fragment thereof. Alternatively, other agonistic molecules can be administered (e.g., GITRL, an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containing an active fragment of GITRL, an antigen presenting cell (APC)/T cell agonist, CD 134 or a ligand or fragment thereof, CD137 or a ligand or fragment thereof, or an inducible T cell costimulatory (ICOS) or a ligand or fragment thereof, or an agonistic small molecule).

[00497] In a specific example, some methods can further comprise administering an anti- CTLA-4 antibody or a functional fragment thereof and/or an anti-CD 137 antibody or functional fragment thereof. For example, the anti-CTLA-4 antibody or a functional fragment thereof or the anti-CD 137 antibody or functional fragment thereof can be administered about 72 hours after the first dose of recombinant fusion polypeptide, nucleic acids encoding a recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine, or about 48 hours after the first dose of recombinant fusion polypeptide, nucleic acids encoding a recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition,

pharmaceutical composition, or vaccine. The anti-CTLA-4 antibody or a functional fragment thereof or anti-CD137 antibody or functional fragment thereof can be administered at a dose, for example, of about 0.05 mg/kg and about 5 mg/kg. A recombinant Listeria strain or immunogenic composition comprising a recombinant Listeria strain can be administered at a dose, for example, of about 1 x 10⁹ CFU. Some such methods can further comprise administering an effective amount of an anti-PD- 1 antibody or functional fragment thereof.

[00498] Methods for assessing efficacy of cancer immunotherapies are well known and are described, for example, in Dzojic et al. (2006) Prostate 66(8):831-838; Naruishi et al. (2006) Cancer Gene Ther. 13(7):658-663, Sehgal et al. (2006) Cancer Cell Int. 6:21), and Heinrich et al. (2007) Cancer Immunol Immunother 56(5):725-730, each of which is herein

incorporated by reference in its entirety for all purposes. As one example, for prostate cancer, a prostate cancer model can be to test methods and compositions disclosed herein, such as a TRAMP-C2 mouse model, a 178-2 BMA cell model, a PAIII adenocarcinoma cells model, a PC-3M model, or any other prostate cancer model.

[00499] Alternatively or additionally, the immunotherapy can be tested in human subjects, and efficacy can be monitored using known. Such methods can include, for example, directly measuring CD4+ and CD8+ T cell responses, or measuring disease progression (e.g., by determining the number or size of tumor metastases, or monitoring disease symptoms such as cough, chest pain, weight loss, and so forth). Methods for assessing the efficacy of a cancer immunotherapy in human subjects are well known and are described, for example, in Uenaka et al. (2007) Cancer Immun. 7:9 and Thomas-Kaskel et al. (2006) Int J Cancer 119(10):2428- 2434, each of which is herein incorporated by reference in its entirety for all purposes.

X. Kits

[00500] Also provided are kits comprising a reagent utilized in performing a method disclosed herein or kits comprising a composition, tool, or instrument disclosed herein. [00501 ] For example, such kits can comprise a recombinant fusion polypeptide disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide disclosed herein, a recombinant bacteria or Listeria strain disclosed herein, an immunogenic composition disclosed herein, a pharmaceutical composition disclosed herein, or a vaccine disclosed herein. Such kits can additionally comprise an instructional material which describes use of the recombinant fusion polypeptide, the nucleic acid encoding the recombinant fusion polypeptide, the recombinant Listeria strain, the immunogenic composition, the

pharmaceutical composition, or the vaccine to perform the methods disclosed herein. Such kits can optionally further comprise an applicator. Although model kits are described below, the contents of other useful kits will be apparent in light of the present disclosure.

[00502] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

LISTING OF EMBODIMENTS

[00503] The subject matter disclosed herein includes, but is not limited to, the following embodiments.

[00504] 1. A method for inducing an immune response against a tumor or cancer in a subject or treating a tumor or cancer in a subject, comprising: (a) administering a recurrent cancer mutation immunotherapy composition to the subject, wherein the recurrent cancer mutation immunotherapy composition comprises a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST-containing peptide fused to two or more first antigenic peptides, wherein each of the first antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the first antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer- associated protein; and (b) administering a personalized immunotherapy composition to the subject, wherein the personalized immunotherapy composition comprises a second recombinant Listeria strain comprising a second nucleic acid comprising a second open reading frame encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises a second PEST-containing peptide fused to one or more second antigenic peptides, wherein each of the second antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject.

[00505] 2. The method of embodiment 1, wherein the recurrent cancer mutation immunotherapy composition is administered prior to the personalized immunotherapy composition.

[00506] 3. The method of embodiment 1, wherein the recurrent cancer mutation immunotherapy composition is administered subsequent to the personalized immunotherapy composition.

[00507] 4. The method of embodiment 1, wherein the recurrent cancer mutation immunotherapy composition and the personalized immunotherapy composition are administered concurrently.

[00508] 5. The method of any preceding embodiment, wherein the recurrent cancer mutation immunotherapy composition is administered with an adjuvant and/or the personalized immunotherapy composition is administered with an adjuvant.

[00509] 6. The method of embodiment 4, wherein the adjuvant comprises a

granulocyte/macrophage colony- stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide, or wherein the adjuvant comprises a

granulocyte/macrophage colony- stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, an unmethylated CpG- containing oligonucleotide, or a detoxified listeriolysin O protein.

[00510] 7. The method of any preceding embodiment, wherein the method further comprises administering an immune checkpoint inhibitor antagonist. [00511 ] 8. The method of embodiment 7, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody or an antigen-binding fragment thereof and/or an anti- CTLA-4 antibody or an antigen-binding fragment thereof.

[00512] 9. The method of any preceding embodiment, wherein the method further comprises administering a T cell stimulator.

[00513] 10. The method of embodiment 9, wherein the T cell stimulator comprises an anti- OX40 antibody or an antigen-binding fragment thereof or an anti-GITR antibody or an antigen-binding fragment thereof.

[00514] 11. The method of any preceding embodiment, wherein the subject has a cancer associated with recurrent cancer mutations in one or more cancer-associated proteins, and the first recombinant Listeria strain comprises antigenic peptides comprising the recurrent cancer mutations associated with the cancer.

[00515] 12. The method of any preceding embodiment, wherein the method comprises screening the subject for and identifying one or more recurrent cancer mutations prior to the administering the recurrent cancer mutation immunotherapy composition, wherein the first recombinant Listeria strain comprises antigenic peptides comprising the one or more recurrent cancer mutations identified in the subject.

[00516] 13. The method of any preceding embodiment, further comprising generating the personalized immunotherapy composition for the subject.

[00517] 14. The method of embodiment 13, wherein the generating the personalized immunotherapy composition is concurrent with administering the recurrent cancer mutation immunotherapy composition to the subject.

[00518] 15. The method of embodiment 13, wherein the generating the personalized immunotherapy composition is subsequent to administering the recurrent cancer mutation immunotherapy composition to the subject.

[00519] 16. The method of embodiment 13, wherein the generating the personalized immunotherapy composition is prior to administering the recurrent cancer mutation immunotherapy composition to the subject.

[00520] 17. The method of any one of embodiments 13-16, wherein generating the personalized immunotherapy composition comprises: (a) comparing one or more open reading frame sequences or mRNA sequences from the cancer sample with one or more open reading frame sequences or mRNA sequences from the healthy biological sample, wherein the comparing identifies one or more cancer- specific neoepitopes, each comprising a different cancer- specific mutation; (b) selecting a set of cancer- specific neoepitopes to include in the second nucleic acid and designing the second nucleic acid; and (c) transforming a Listeria strain with the second nucleic acid.

[00521 ] 18. The method of embodiment 17, further comprising obtaining the cancer sample from the subject and/or obtaining the healthy biological sample from the subject.

[00522] 19. The method of embodiment 17 or 18, wherein the cancer sample and/or the healthy biological sample comprise a tissue, cells isolated from blood, cells isolated from sputum, cells isolated from saliva, or cells isolated from cerebrospinal fluid.

[00523] 20. The method of any one of embodiments 17-19, wherein the open reading frame sequences are compared, and the open reading frame sequences are determined using exome sequencing.

[00524] 21. The method of any one of embodiments 17-19, wherein the mRNA sequences are compared, and the mRNA sequences are determined using transcriptome sequencing.

[00525] 22. The method of any one of embodiments 17-19, wherein the comparing comprises use of a screening assay or screening tool and associated digital software for comparing one or more open reading frames in nucleic acid sequences, wherein the associated digital software comprises access to a sequence database that allows screening of mutations within open reading frames for identification of the immunogenic potential of the one or more cancer- specific neoepitopes.

[00526] 23. The method of any one of embodiments 17-22, wherein step (b) comprises designing an antigenic peptide for each of the one or more cancer- specific neoepitopes.

[00527] 24. The method of embodiment 23, wherein each antigenic peptide comprises a different cancer- specific mutation and flanking sequence on each side.

[00528] 25. The method of embodiment 24, wherein each antigenic peptide includes at least about 10 flanking amino acids on each side

[00529] 26. The method of any one of embodiments 23-25, wherein step (b) comprises scoring the each antigenic peptide and selecting antigenic peptide if it scores below a hydropathy threshold predictive of secretability in Listeria monocytogenes.

[00530] 27. The method of embodiment 26, wherein the scoring is by a Kyte and Doolittle hydropathy index 21 amino acid window, and any antigenic peptides scoring above a cutoff of about 1.6 are excluded or are modified to score below the cutoff.

[00531 ] 28. The method of any embodiment 26 or 27, wherein every identified cancer- specific neoepitope for which an antigenic peptide can be designed that scores below the threshold is selected for inclusion in the second nucleic acid in the second recombinant Listeria strain or in the second nucleic acid and one or more additional nucleic acids for transforming one or more additional recombinant Listeria strains.

[00532] 29. The method of any one of embodiments 17-28, wherein step (b) further comprises rating the ability of the one or more cancer- specific neoepitopes to bind subject

HLA.

[00533] 30. The method of any one of embodiments 17-29, wherein step (b) further comprises screening the one or more cancer- specific neoepitopes for immunosuppressive epitopes and deselecting or modifying cancer- specific neoepitopes that have

immunosuppressive epitopes.

[00534] 31. The method of any one of embodiments 17-30, wherein step (b) further comprises screening one or more peptides comprising the one or more cancer- specific neoepitopes for immunogenicity.

[00535] 32. The method of embodiment 31, wherein for each of the one or more peptides the screening comprises: (a) contacting one or more T cells with the peptide, and (b) analyzing for an immunogenic T cell response, wherein an immunogenic T cell response identifies the peptide as an immunogenic peptide.

[00536] 33. The method of embodiment 31, wherein the screening comprises using an immunogenic assay to measure secretion of at least one of CD25, CD44, or CD69 or to measure secretion of a cytokine selected from the group comprising IFN-γ, TNF-a, IL-1, and IL-2 upon contacting the one or more T cells with the peptide, wherein increased secretion identifies the peptide as comprising one or more T cell neoepitopes.

[00537] 34. The method of any one of embodiments 17-33, wherein designing the second nucleic acid in step (b) comprises determining an order for the cancer- specific neoepitopes in the second fusion polypeptide.

[00538] 35. The method of embodiment 34, wherein the order is selected using randomization.

[00539] 36. The method of any one of embodiments 17-35, wherein designing the second nucleic acid in step (b) comprises scoring the hydropathy of the second fusion polypeptide, and either reordering the cancer- specific neoepitopes or removing problematic cancer- specific neoepitopes if any region of the second fusion polypeptide scores above a selected hydropathy index threshold value.

[00540] 37. The method of embodiment 36, wherein the second fusion polypeptide is scored by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, and wherein the threshold value is about 1.6. [00541 ] 38. The method of any one of embodiments 17-37, wherein designing the second nucleic acid in step (b) comprises codon optimizing the second nucleic acid for expression and secretion in Listeria monocytogenes.

[00542] 39. The method of any one of embodiments 17-38, wherein the transforming is accomplished using a plasmid or a phage vector.

[00543] 40. The method of any one of embodiments 17-39, further comprising culturing and characterizing the transformed recombinant Listeria strain to confirm expression and/or secretion of the second fusion polypeptide.

[00544] 41. The method of any preceding embodiment, wherein each of the first antigenic peptides is a fragment of a cancer-associated protein and is about 5-100, 15-50, or 21-27 amino acids in length.

[00545] 42. The method of any preceding embodiment, wherein each of the first antigenic peptides comprises a recurrent cancer mutation flanked on each side by an equal number of amino acids.

[00546] 43. The method of any preceding embodiment, wherein each of the first antigenic peptides comprises a recurrent cancer mutation flanked on each side by at least 10 or at least 13 amino acids.

[00547] 44. The method of any preceding embodiment, wherein the two or more of the first antigenic peptides are fused directly to each other without intervening sequence.

[00548] 45. The method of any one of embodiments 1-43, wherein the first antigenic peptides are linked to each other via peptide linkers.

[00549] 46. The method of embodiment 45, wherein one or more of the linkers set forth in SEQ ID NOS: 310-319 are used to link the two or more antigenic peptides.

[00550] 47. The method of any preceding embodiment, wherein the first fusion polypeptide comprises at least 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 antigenic peptides from the same cancer-associated protein or 3-40 antigenic peptides from the same cancer-associated protein, or wherein the first fusion polypeptide comprises at least 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 non-contiguous antigenic peptides from the same cancer-associated protein or 2-40 non-contiguous antigenic peptides from the same cancer-associated protein.

[00551 ] 48. The method of embodiment 47, wherein the first antigenic peptides comprise the 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 most common recurrent cancer mutations in the cancer-associated protein. [00552] 49. The method of embodiment 48, wherein the first antigenic peptides comprise the 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 most common recurrent somatic missense cancer mutations in the cancer- associated protein.

[00553] 50. The method of any preceding embodiment, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a mutation in the cancer- associated protein have a recurrent cancer mutation in the cancer-associated protein that is included in the combination of first antigenic peptides in the first recombinant Listeria strain.

[00554] 51. The method of any preceding embodiment, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cancer patients with a somatic missense mutation in the cancer-associated protein have a recurrent cancer mutation in the cancer- associated protein that is included in the combination of first antigenic peptides in the first recombinant Listeria strain.

[00555] 52. The method of any preceding embodiment, wherein the recurrent cancer mutations in at least two of the first antigenic peptides are from the same cancer-associated protein and do not occur naturally together.

[00556] 53. The method of any preceding embodiment, wherein at least two of the first antigenic peptides are overlapping fragments of the same cancer-associated protein.

[00557] 54. The method of embodiment 53, wherein the recurrent cancer mutations in at least two of the first antigenic peptides are from the same cancer-associated protein and occur at the same amino acid residue of the cancer-associated protein.

[00558] 55. The method of embodiment 54, wherein the first fusion polypeptide comprises two or more copies of a single antigenic peptide, or wherein two of the first antigenic peptides comprise the same recurrent cancer mutation.

[00559] 56. The method of any one of embodiments 1-54, wherein each of the first antigenic peptides comprises a different recurrent cancer mutation.

[00560] 57. The method of any preceding embodiment, wherein each recurrent cancer mutation in the first fusion polypeptide is a somatic missense mutation.

[00561 ] 58. The method of any preceding embodiment, wherein the first antigenic peptides are from the cancer associated protein and one or more additional proteins.

[00562] 59. The method of any preceding embodiment, wherein the cancer-associated protein is an oncogenic protein.

[00563] 60. The method of any one of embodiments 1-58, wherein the cancer-associated protein is a tumor suppressor protein. [00564] 61. The method of any one of embodiments 1-58, wherein the cancer-associated protein is encoded by one of the following human genes: TP53, PIK3CA, APC, CTNNB1, CDKN2A, NFE2L2, BRAF, KRAS, EGFR, ERBB2, SF3B1, FBXW7, PIK3R1, SMAD4, SPOP, PTPN11, NRAS, PTEN, HRAS, U2AF1, ERBB3, FGFR3, ARID 1 A, MAP2K1, FGFR2, RHOA, MTOR, BCL2L12, RAC1, IDH2, H3F3A, PPP2R1A, POLE, ATM, EP300, ALK, RQCDl, GPRIN2, THSD7B, CDK4, NUP93, CCNDl, FGFRl, MAX, VHL, ACVR1, MEF2A, MYC, FRMD6, SRC, KIT, KEAP1, STK11, NF1, KMT2D, GATA3, AKT1, MAP3K1,

MAP2K4, KMT2C, FAT1, PBRM1, SETD2, CREBBP, RBI, SMARCA4, CHD4, FLT3, ARID2, CDH1, DNMT3A, ARHGAP35, BCOR, CTCF, KDM5C, KDM6A, CASP8, ASXL1, RASA1, RUNX1, NPM1, CDKN1B, HLA-A, B2M, RPL5, MYD88, CBFB, and GPS2, or wherein the cancer-associated protein is encoded by one of the following human genes:

TP53, PIK3CA, APC, CTNNB1, CDKN2A, NFE2L2, BRAF, KRAS, EGFR, ERBB2, SF3B1, FBXW7, PIK3R1, SMAD4, SPOP, PTPN11, NRAS, PTEN, HRAS, U2AF1, ERBB3, FGFR3, ARID 1 A, MAP2K1, FGFR2, RHOA, MTOR, BCL2L12, RAC1, IDH2, H3F3A, PPP2R1A, POLE, ATM, EP300, ALK, RQCDl, GPRIN2, THSD7B, CDK4, NUP93, CCNDl, FGFRl, MAX, VHL, ACVR1, MEF2A, MYC, FRMD6, SRC, KIT, KEAP1, STK11, NF1, KMT2D, GATA3, AKT1, MAP3K1, MAP2K4, KMT2C, FAT1, PBRM1, SETD2, CREBBP, RBI, SMARCA4, CHD4, FLT3, ARID2, CDH1, DNMT3A, ARHGAP35, BCOR, CTCF, KDM5C, KDM6A, CASP8, ASXL1, RASA1, RUNX1, NPM1, CDKN1B, HLA-A, B2M, RPL5, MYD88, CBFB, GPS2, AHNAK2, ANKRD36C, CHEK2, KRTAP4-11, RGPD8, FAM47C, and TAN.

[00565] 62. The method of embodiment 61, wherein the cancer-associated protein is encoded by one of the following genes: BRAF, EGFR, PIK3CA, PIK3R1, PTEN, KRAS, TP53, APC, FBXW7, KEAP1, STK11, NF1, KMT2D, CDKN2A, NFE2L2, SPOP, GATA3, AKT1, MAP3K1, and MAP2K4, or wherein the cancer-associated protein is encoded by one of the following genes: BRAF, EGFR, PIK3CA, PIK3R1, PTEN, KRAS, TP53, APC, FBXW7, KEAP1, STK11, NF1, KMT2D, CDKN2A, NFE2L2, SPOP, GATA3, AKT1, MAP3K1, MAP2K4, AHNAK2, ANKRD36C, CHEK2, KRTAP4-11, RGPD8, FAM47C, and TAN.

[00566] 63. The method of embodiment 62, wherein the cancer-associated protein is encoded by BRAF, and the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: G466E; G466V; G469A; G469R; G469S; G469V; V600E; and V600K.

[00567] 64. The method of embodiment 63, wherein the first fusion polypeptide comprises antigenic peptides comprising the following recurrent cancer mutations in one of the following N-terminal to C-terminal orders: (a) G469V; G469R; V600E; G469S; G466V; V600K; G469A; and G466E; (b) V600K; G469R; G469V; G466V; G466E; V600E; G469A; and G469S; (c) G469V; V600K; G469S; G466V; G469A; V600E; G466E; and G469R; and (d) V600E; V600K; G469A; G469S; G469R; G469V; G466V; and G466E.

[00568] 65. The method of embodiment 64, wherein the combination of the first antigenic peptides in the first fusion polypeptide comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 2, 8, 14, and 20.

[00569] 66. The method of embodiment 65, wherein the portion of the first open reading frame encoding the combination of the first antigenic peptides comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 1, 7, 13, and 19.

[00570] 67. The method of embodiment 62, wherein the cancer-associated protein is encoded by EGFR, and the first antigenic peptides comprise two or more of the following recurrent cancer mutations: R108K; A289V; G598V; E709A; E709K; G719A; G719C;

G719S; L747P; L747S; S768I; T790M; L833V/H835L; T833V; L858R; and L861Q.

[00571 ] 68. The method of embodiment 67, wherein the first fusion polypeptide comprises antigenic peptides comprising the following recurrent cancer mutations in one of the following N-terminal to C-terminal orders: (a) G719S; L747P; G719C; R108K; S768I;

L833V/H835L; T833V; E709A; G598V; T790M; E709K; A289V; L861Q; G719A; L747S; and L858R; (b) T790M; S768I; G719C; R108K; L747P; G719A; L747S; E709K; T833V; L861Q; E709A; L858R; G598V; A289V; L833V/H835L; and G719S; (c) R108K; T833V; L747S; T790M; G719C; A289V; L858R; E709A; G719S; E709K; G719A; L747P; G598V; L861Q; S768I; and L833V/H835L; (d) G719A; L858R; G719C; A289V; T790M; S768I; T833V; G598V; G719S; L747S; L747P; L833V/H835L; E709A; R108K; L861Q; and E709K; and (e) A289V; G598V; E709K; G719A; S768I; G719S; L861Q; T790M; G719C; L833V/H835L; and L858R.

[00572] 69. The method of embodiment 68, wherein the combination of the first antigenic peptides in the first fusion polypeptide comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 26, 32, 38, 44, and 231.

[00573] 70. The method of embodiment 69, wherein the portion of the first open reading frame encoding the combination of the first antigenic peptides comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 25, 31, 37, 43, 229, and 230.

[00574] 71. The method of embodiment 62, wherein the cancer-associated protein is encoded by PIK3CA, and the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: R38C; R38H; E81K; R88Q; R93Q; R93W; R108H; G118D; L334G; N345K; C420R; E453K; E542K; E545A; E545G; E545K; E545Q; Q546K; Q546R; E726K; M1043I; M1043V; H1047L; H1047R; and G1049R.

[00575] 72. The method of embodiment 71, wherein the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: R88Q; E542K; E545A;

E545G; E545K; Q546K; H1047L; and H1047R.

[00576] 73. The method of embodiment 71, wherein the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: R38H; E81K; R108H;

G118D; N345K; C420R; Q546R; M1043I; and G1049R.

[00577] 74. The method of embodiment 71, wherein the first fusion polypeptide comprises antigenic peptides comprising the following recurrent cancer mutations in one of the following N-terminal to C-terminal orders: (a) M1043V; E545G; E726K; Q546R; L334G; G1049R; M1043I; Q546K; E542K; R93Q; H1047R; R108H; R93W; E81K; R38H; N345K; R88Q; G118D; E545Q; H1047L; E545A; E453K; E545K; R38C; and C420R; (b) E726K; E81K; M1043V; E545A; E545K; R38C; G118D; R93W; E545G; E542K; G1049R; N345K; Q546K; E453K; C420R; H1047L; L334G; E545Q; R88Q; H1047R; M1043I; R93Q; R108H; Q546R; and R38H; (c) R108H; M1043V; R88Q; R93W; R38H; H1047R; E545K; M1043I; Q546R; E542K; N345K; R38C; E545G; E81K; Q546K; R93Q; E453K; G1049R; E545A; C420R; H1047L; L334G; G118D; E726K; and E545Q; (d) N345K; R38H; E545K; G1049R; H1047L; E726K; R88Q; E81K; R93Q; E545Q; L334G; R38C; H1047R; C420R; R93W; Q546K; M1043V; M1043I; E545G; E545A; G118D; E453K; Q546R; R108H; and E542K; (e) E542K; E545K; R88Q; E545A; H1047R; E545G; H1047L; Q546K; R38H; E81K;

R108H; N345K; C420R; Q546R; M1043I; G118D; and G1049R; (f) E542K; E545K; R88Q; E545A; H1047R; E545G; H1047L; and Q546K; and (g) R38H; E81K; R108H; N345K; C420R; Q546R; M1043I; G118D; and G1049R.

[00578] 75. The method of embodiment 74, wherein the combination of the first antigenic peptides in the first fusion polypeptide comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 50, 56, 62, 68, 238, 245, and 252.

[00579] 76. The method of embodiment 75, wherein the portion of the first open reading frame encoding the combination of the first antigenic peptides comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 49, 55, 61, 67, 236, 237, 243, 244, 250, and 251.

[00580] 77. The method of embodiment 62, wherein the cancer-associated protein is encoded by PIK3R1, and the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: G376R; N564D; and K567E. [00581 ] 78. The method of embodiment 77, wherein the first fusion polypeptide comprises antigenic peptides comprising the following recurrent cancer mutations in one of the following N-terminal to C-terminal orders: (a) G376R; N564D; and K567E; and (b) N564D; K567E; and G376R.

[00582] 79. The method of embodiment 78, wherein the combination of the first antigenic peptides in the first fusion polypeptide comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 74 and 80.

[00583] 80. The method of embodiment 79, wherein the portion of the first open reading frame encoding the combination of the first antigenic peptides comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 73 and 79.

[00584] 81. The method of embodiment 62, wherein the cancer-associated protein is encoded by PIK3CA, and the first antigenic peptides from PIK3CA comprise two or more or all of the following recurrent PIK3CA mutations: R38C; R38H; E81K; R88Q; R93Q; R93W; R108H; G118D; L334G; N345K; C420R; E453K; E542K; E545A; E545G; E545K; E545Q; Q546K; Q546R; E726K; M1043I; M1043V; H1047L; H1047R; and G1049R; and wherein the first antigenic peptides further comprise antigenic peptides from the protein encoded by PIK3R1, and the antigenic peptides from PIK3R1 comprise two or more or all of the following recurrent PIK3R1 mutations: G376R; N564D; and K567E.

[00585] 82. The method of embodiment 81, wherein the first fusion polypeptide comprises antigenic peptides comprising the following recurrent cancer mutations in one of the following N-terminal to C-terminal orders: (a) PIK3CAIR38C; PIK3CAIN345K;

PIK3CAIE726K; PIK3CAIE453K; PIK3CAIR93Q; PIK3CAIH1047R; PIK3CAIE545A; PIK3CAIM1043V; PIK3R1IN564D; PIK3R1IK567E; PIK3CAIE81K; PIK3CAIR108H; PIK3CAIQ546R; PIK3CAIQ546K; PIK3CAIE545Q; PIK3CAIG1049R; PIK3CAIC420R; PIK3CAIH1047L; PIK3CAIR93W; PIK3CAIR88Q; PIK3CAIM1043I; PIK3CAIE545G; PIK3CAIG118D; PIK3CAIR38H; PIK3R1IG376R; PIK3CAIE542K; PIK3CAIE545K; and PIK3CAIL334G; (b) PIK3CAIR38C; PIK3CAIR108H; PIK3CAIC420R; PIK3CAIR93Q; PIK3CAIE453K; PIK3CAIM1043V; PIK3CAIH1047L; PIK3R1IN564D; PIK3CAIE726K; PIK3CAIG118D; PIK3CAIQ546K; PIK3CAIQ546R; PIK3CAIE542K; PIK3CAIE545K; PIK3CAIG1049R; PIK3CAIM1043I; PIK3CAIL334G; PIK3R1IK567E; PIK3CAIR38H; PIK3R1IG376R; PIK3CAIR93W; PIK3CAIH1047R; PIK3CAIE545G; PIK3CAIE81K;

PIK3CAIR88Q; PIK3CAIN345K; PIK3CAIE545A; and PIK3CAIE545Q; (c)

PIK3CAIR108H; PIK3CAIM1043V; PIK3CAIR88Q; PIK3CAIR93W; PIK3CAIR38H;

PIK3CAIH1047R; PIK3CAIE545K; PIK3CAIM1043I; PIK3CAIQ546R; PIK3CAIE542K; PIK3CAIN345K; PIK3CAIR38C; PIK3CAIE545G; PIK3CAIE81K; PIK3CAIQ546K;

PIK3CAIR93Q; PIK3CAIE453K; PIK3CAIG1049R; PIK3CAIE545A; PIK3CAIC420R; PIK3CAIH1047L; PIK3CAIL334G; PIK3CAIG118D; PIK3CAIE726K; and PIK3CAIE545Q; and (d) PIK3CAIE545Q; PIK3CAIR93W; PIK3CAIH1047R; PIK3CAIG1049R;

PIK3CAIN345K; PIK3CAIQ546R; PIK3CAIE545K; PIK3CAIE453K; PIK3CAIL334G; PIK3CAIH1047L; PIK3R1IG376R; PIK3CAIM1043V; PIK3CAIR88Q; PIK3CAIR38H; PIK3CAIG118D; PIK3R1IK567E; PIK3CAIR38C; PIK3CAIE542K; PIK3CAIQ546K;

PIK3CAIE726K; PIK3CAIC420R; PIK3CAIE545A; PIK3CAIR93Q; PIK3R1IN564D;

PIK3CAIR108H; PIK3CAIM1043I; PIK3CAIE545G; and PIK3CAIE81K.

[00586] 83. The method of embodiment 82, wherein the combination of the first antigenic peptides in the first fusion polypeptide comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 86, 92, 98, and 104.

[00587] 84. The method of embodiment 83, wherein the portion of the first open reading frame encoding the combination of the first antigenic peptides comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 85, 91, 97, and 103.

[00588] 85. The method of embodiment 62, wherein the cancer-associated protein is encoded by PTEN, and the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: Y68H; Y88C; D92E; dell21-131; R130G; R130L; R130P; R130Q; C136Y; R142W; Y155C; R173H; and P246L.

[00589] 86. The method of embodiment 85, wherein the first fusion polypeptide comprises antigenic peptides comprising the following recurrent cancer mutations in one of the following N-terminal to C-terminal orders: (a) dell21-131; Y88C; R130G; Y155C; D92E; C136Y; R130Q; Y68H; R142W; R173H; R130L; R130P; and P246L; (b) R130P; R130G; Y155C; R130L; C136Y; dell21-131; P246L; D92E; R173H; Y68H; R130Q; Y88C; and R142W; (c) R130Q; R130G; dell21-131; C136Y; R130L; P246L; Y155C; D92E; R142W; R130P; Y88C; Y68H; and R173H; and (d) dell21- 131; C136Y; Y68H; R142W; R173H; IR130L; P246L; R130G; R130P; Y88C; D92E; R130Q; and Y155C.

[00590] 87. The method of embodiment 86, wherein the combination of the first antigenic peptides in the first fusion polypeptide comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 110, 116, 122, and 128.

[00591 ] 88. The method of embodiment 87, wherein the portion of the first open reading frame encoding the combination of the first antigenic peptides comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 109, 115, 121, and 127.

[00592] 89. The method of embodiment 62, wherein the cancer-associated protein is encoded by KRAS, and the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: G12A; G12C; G12D; G12R; G12S; G12V; G13C; G13D; G13R; G13S; G13V; L19F; Q61K; Q61H; Q61L; Q61R; K117N; A146T; A146V; and A164G.

[00593] 90. The method of embodiment 89, wherein the first fusion polypeptide comprises antigenic peptides comprising the following recurrent cancer mutations in one of the following N-terminal to C-terminal orders: (a) Q61R; Q61K; Q61L; Q61H; L19F; K117N; G12A; A164G; G12D; G13D; G13S; G12S; A146V; G13R; G13C; G12C; G12R; G13V; G12V; and A146T; (b) Q61H; K117N; G13C; G13R; G12D; G12S; G12V; G12A; Q61K; G13V; G12C; L19F; Q61R; Q61L; A146V; A164G; G12R; G13S; A146T; and G13D; (c) G12D; L19F; A146V; Q61H; G12V; A164G; G12C; Q61L; A146T; G13S; G12A; G13V; G13C; G13D; G12R; G12S; Q61R; Q61K; G13R; and K117N; and (d) G13V; G13S; G12V; G12R; A146V; G13D; G12D; K117N; Q61H; G12C; G13C; A146T; G12A; Q61L; Q61K; A164G; G12S; L19F; G13R; and Q61R.

[00594] 91. The method of embodiment 90, wherein the combination of the first antigenic peptides in the first fusion polypeptide comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 134, 140, 146, and 152.

[00595] 92. The method of embodiment 91, wherein the portion of the first open reading frame encoding the combination of the first antigenic peptides comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 133, 139, 145, and 151.

[00596] 93. The method of embodiment 62, wherein the cancer-associated protein is encoded by TP53, and the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: Y107D; K132N; C141Y; V143A; V157F; Y163C; R175H; C176F; C176Y; H179R; H179W; H193R; I195T; V216M; Y220C; Y234C; Y234H; S241F; S242F; G245D; G245S; R248L; R248Q; R248W; R249S; R273C; R273H; R273L; P278L; P278S; R282G; R282W; and R337H.

[00597] 94. The method of embodiment 93, wherein the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: Y107D; C141Y; V143A; V157F; Y163C; R175H; C176F; H193R; I195T; V216M; Y220C; Y234C; Y234H; G245D; G245S; R248Q; R248W; R249S; R273C; R273H; R273L; R282G; and R282W. [00598] 95. The method of embodiment 93, wherein the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: V143A; R175H; H193R; Y220C; G245D; R248Q; R248W; R249S; R273C; R273H; and R282W.

[00599] 96. The method of embodiment 93, wherein the first antigenic peptides comprise two or more or all of the following recurrent cancer mutations: Y107D; C141Y; V157F; Y163C; C176F; I195T; V216M; Y234C; Y234H; G245S; R273L; and R282G.

[00600] 97. The method of embodiment 93, wherein the first antigenic peptides comprise: (a) two or more or all of the following recurrent cancer mutations: Y107D; C141Y; V143A; Y163C; C176Y; H179R; H179W; H193R; V216M; Y234H; S241F; G245D; R248Q;

R248W; R273C; R273L; and P278S; (b) two or more or all of the following recurrent cancer mutations: C141Y; R175H; H179R; H193R; V216M; Y234H; G245D; G245S; R248L;

R248W; R273C; R273H; P278L; P278S; R282G; R282W;and R337H; (c) two or more or all of the following recurrent cancer mutations: Y107D; C141Y; V143A; C176F; H179R;

V216M; Y220C; S241F; S242F; G245S; R248L; R248W; R273L; P278L; P278S; R282G; and R282W; or (d) two or more or all of the following recurrent cancer mutations: Y107D; K132N; V143A; V157F; Y163C; R175H; C176Y; Y234C; Y234H; S241F; S242F; G245D; G245S; R273C; P278S; R282W; and R337H.

[00601 ] 98. The method of embodiment 93, wherein the first antigenic peptides comprise: (a) two or more or all of the following recurrent cancer mutations: K132N; V157F; R175H; C176F; I195T; Y220C; Y234C; S242F; G245S; R248L; R249S; R273H; P278L; R282G; R282W; and R337H; (b) two or more or all of the following recurrent cancer mutations: Y107D; K132N; V143A; V157F; Y163C; C176F; C176Y; H179W; I195T; Y220C; Y234C; S241F; S242F; R248Q; R249S; and R273L; (c) two or more or all of the following recurrent cancer mutations: K132N; V157F; Y163C; R175H; C176Y; H179W; H193R; I195T; Y234C; Y234H; G245D; R248Q; R249S; R273C; R273H; and R337H; or (d) two or more or all of the following recurrent cancer mutations: C141Y; C176F; H179R; H179W; H193R; I195T; V216M; Y220C; R248L; R248Q; R248W; R249S; R273H; R273L; P278L; and R282G.

[00602] 99. The method of embodiment 93, wherein the first fusion polypeptide comprises antigenic peptides comprising the following recurrent cancer mutations in one of the following N-terminal to C-terminal orders: (a) H179W; R273L; R249S; R248Q; Y234H; G245D; Y220C; R248L; H193R; K132N; S242F; Y234C; G245S; C176F; R282W; R273H; R282G; C141Y; R273C; V216M; R337H; R248W; V143A; I195T; P278S; S241F; C176Y; Y107D; R175H; H179R; V157F; P278L; and Y163C; (b) R248W; R248L; Y220C; Y163C; G245D; Y107D; H179R; V216M; P278S; S241F; R273L; P278L; C176F; C141Y; S242F; R249S; V143A; I195T; R273H; R273C; R282G; H179W; R175H; R248Q; G245S; H193R; R337H; R282W; Y234C; V157F; Y234H; C176Y; and K132N; (c) R248W; H179R; R273H; Y107D; R337H; R282G; V157F; V143A; Y234H; Y220C; R282W; R248L; S241F; H179W; R273C; C141Y; R249S; P278L; G245S; I195T; R175H; G245D; R273L; K132N; V216M; Y163C; C176F; S242F; Y234C; H193R; R248Q; P278S; and C176Y; (d) V143A; R282W; V157F; H179W; K132N; Y163C; C176Y; G245D; Y220C; S242F; Y234C; R249S; H179R; R273H; C141Y; R273L; P278S; C176F; R337H; H193R; R273C; R282G; R175H; R248W; P278L; I195T; S241F; R248L; Y234H; V216M; G245S; Y107D; and R248Q; (e) S241F; G245D; V143A; P278S; R273C; C176Y; Y234H; R248W; V216M; R248Q; C141Y; Y163C; H193R; H179R; H179W; Y107D; and R273L; (f) K132N; R282W; G245S; Y234C; S242F; R175H; Y220C; V157F; R282G; C176F; R337H; I195T; R249S; P278L; R273H; and R248L; (g) H193R; P278L; R273C; R248W; H179R; P278S; R248L; V216M; R282G;

R337H; R175H; Y234H; G245D; R273H; G245S; R282W; and C141Y; (h) Y107D; K132N; C176F; C176Y; R273L; Y220C; R248Q; V143A; I195T; R249S; S242F; Y234C; H179W; V157F; Y163C; and S241F; (i) P278S; C176F; H179R; R282G; S241F; R273L; P278L; C141Y; Y107D; R248W; V216M; R282W; S242F; Y220C; V143A; G245S; and R248L; (j) R175H; H179W; R249S; Y234H; I195T; R248Q; R273H; C176Y; V157F; H193R; Y234C; K132N; R273C; Y163C; G245D; and R337H; (k) C176Y; R175H; G245D; R337H; S241F; K132N; V143A; P278S; R282W; Y163C; Y107D; R273C; S242F; G245S; V157F; Y234C; and Y234H; (1) C176F; R273L; H179R; R282G; Y220C; I195T; C141Y; R248L; R273H; H179W; H193R; R249S; V216M; P278L; R248W; and R248Q; (m) R248W; R273H;

V143A; R249S; R175H; H193R; Y220C; G245D; R248Q; R273C; R282W; Y107D; C141Y; V157F; Y163C; C176F; I195T; V216M; Y234H; G245S; R273L; Y234C; and R282G; (n) R248W; R273H; V143A; R249S; R175H; H193R; Y220C; G245D; R248Q; R273C; and R282W; and (o) Y107D; C141Y; V157F; Y163C; C176F; I195T; V216M; Y234H; G245S; R273L; Y234C; and R282G.

[00603] 100. The method of embodiment 99, wherein the combination of the first antigenic peptides in the first fusion polypeptide comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 259, 266, and 273.

[00604] 101. The method of embodiment 100, wherein the portion of the first open reading frame encoding the combination of the first antigenic peptides comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOS: 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 257, 258, 264, 265, 271, and 272. [00605] 101b. The method of any one of embodiments 58-61, wherein the antigenic peptides are from two or more cancer associated proteins.

[00606] 101c. The method of embodiment 101b, wherein the two or more cancer associated proteins are 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer-associated proteins.

[00607] lOld. The method of embodiment 101b or 101c, wherein the antigenic peptides comprise at least 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, or 60 different recurrent cancer mutations from the same type of cancer, or wherein the antigenic peptides comprise 2-80, 10-60, 10-50, 10-40, or 10-30 different recurrent cancer mutations from a single type of cancer, or wherein the antigenic peptides comprise at least 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, or 60 different recurrent somatic missense cancer mutations from a single type of cancer, or wherein the antigenic peptides comprise 2-80, 10-60, 10-50, 10-40, or 10-30 different recurrent somatic missense cancer mutations from a single type of cancer

[00608] lOle. The method of any one of embodiments 101b - lOld, wherein: (a) the two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: PI3KCA, AKT1, AHNAK2, ERBB2, and TP53; (b) the two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: BRAF, KRASINRAS, TP53, PIK3CA, and SMAD4; (c) the two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: KRAS, TP53, EGFR, U2AF1, BRAF, and PIK3CA; (d) the two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: TP53, PIK3CA, NFE2L2, CDKN2A, and PTEN; or (e) the two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: ANKRD36C, SPOP, CHEK2, KRTAP4-11, RGPD8, TP53, FAM47C, ZAN, and PIK3CA.

[00609] lOlf. The method of embodiment lOle, wherein the antigenic peptides comprise two or more or all of the following recurrent cancer mutations: PIK3CAIH1047R;

PIK3CAIE545K; PIK3CAIE542K; PIK3CAIH1047L; PIK3CAIQ546K; PIK3CAIE545A; PIK3CAIE545G; AKT1IE17K; AHNAK2IV2016L, ERBB2IL755S, and TP53IR175H.

[00610] 101g. The method of embodiment lOle, wherein the antigenic peptides comprise two or more or all of the following recurrent cancer mutations: BRAFIV600E; KRASIG12D; KRASIG13D; KRASIG12V; KRASIG12C; KRASIQ61K; KRASIG12A; KRASIG12S; TP53IR175H; TP53IR248W; TP53IR273C; TP53IR282W; TP53IR273H; TP53IR248Q;

TP53IG245S; PIK3CAIE545K; PIK3CAIH1047R; PIK3CAIR88Q; and SMAD4IR361H.

[00611 ] lOlh. The method of embodiment 105, wherein the antigenic peptides comprise two or more or all of the following recurrent cancer mutations: KRASIG12C; KRASIG12V; KRASIG12D; KRASIG12F; KRASIG12R; KRASIQ61L; KRASIG12Y; TP53IR158L;

TP53IR273L; TP53IG245V; TP53IR175H; TP53IA159P; TP53IR249M; TP53IR273H;

TP53IR280I; TP53IQ144L; TP53IR273C; TP53IR280G; TP53IR280T; EGFRIL858R;

EGFRIL861Q; EGFRIG719A; U2AF1IS34F; BRAF1IV600E; BRAF1IG466V;

BRAF1IN581S; PIK3CAIE545K; PIK3CAIE726K; and PIK3CAIH1047R.

[00612] lOli. The method of embodiment lOle, wherein the antigenic peptides comprise two or more or all of the following recurrent cancer mutations: TP53IY163C; TP53IR175G; TP53IC242F; TP53IR273L; TP53IH179L; TP53IH193L; TP53IH214R; TP53IY220C;

TP53IY234C; TP53IG245V; TP53IL111Q; TP53IT125P; TP53IK132R; TP53IC135W;

TP53IC141W; TP53IC176F; TP53IC176Y; TP53IH179R; TP53IH179Y; TP53IH193R;

TP53II195S; TP53IY205C; TP53IR213G; TP53IV216E; TP53IY234S; TP53IY236C;

TP53IM237I; TP53IG244C; TP53IG245S; TP53IR248L; TP53IR248P; TP53IR248Q;

TP53IR248W; TP53IR249G; TP53IR249S; TP53IR249W; TP53IG266V; TP53IF270I;

TP53IR273C; TP53IR273H; TP53IR273P; TP53IR280I; TP53ID281Y; TP53IR282Q;

TP53IR282W; PIK3CAIE545K; PIK3CAIE542K; PIK3CAIH1047R; PIK3CAIE726K;

PIK3CAIC420R; NFE2L2IE79Q; NFE2L2IR34Q; NFE2L2IL30F; NFE2L2IG81S;

NFE2L2IG31A; NFE2L2ID29G; NFE2L2IG81V; CDKN2AID108Y; CDKN2AID18N; and PTENIR130Q.

[00613] lOlj. The method of embodiment lOle, wherein the antigenic peptides comprise two or more or all of the following recurrent cancer mutations: ANKRD36CII645T;

ANKRD36CID629Y; ANKRD36CID629N; SPOPIW131G; SPOPIF133L; SPOPIF133V; SPOPIF133C; SPOPIW131R; SPOPIW131L; CHEK2IK373E; KRTAP4-11IM93V; KRTAP4- 11IR51K; KRTAP4-11IL161V; RGPD8IP1760A; TP53IR248Q; TP53IG245S; TP53IG245D; FAM47CIN648D; ZANIL878P; PIK3CAIE542K; and PIK3CAIH1047R.

[00614] 102. The method of any preceding embodiment, wherein the recurrent cancer mutation immunotherapy composition comprises two or more recurrent cancer mutation recombinant Listeria strains including the first recombinant Listeria strain, wherein each of the two or more recurrent cancer mutation recombinant Listeria strains comprises two or more antigenic peptides, wherein each of the antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein, and wherein each of the two or more recurrent cancer mutation recombinant Listeria strains comprises a different set of antigenic peptides or the same set of antigenic peptides in a different order.

[00615] 103. The method of embodiment 102, wherein the two or more recurrent cancer mutation recombinant Listeria strains are administered to the subject sequentially.

[00616] 104. The method of embodiment 102, wherein the two or more recurrent cancer mutation recombinant Listeria strains are administered to the subject simultaneously.

[00617] 105. The method of embodiment 102, wherein each of the two or more recurrent cancer mutation recombinant Listeria strains comprises a different set of antigenic peptides.

[00618] 106. The method of any one of embodiments 102-105, wherein the two or more recurrent cancer mutation recombinant Listeria strains comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 recombinant Listeria strains.

[00619] 107. The method of any one of embodiments 102-106, wherein the two or more recurrent cancer mutation recombinant Listeria strains comprise antigenic peptides from two or more cancer-associated proteins.

[00620] 108. The method of embodiment 107, wherein the two or more cancer-associated proteins are 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer-associated proteins.

[00621 ] 109. The method of embodiment 107 or 108, wherein the two or more cancer- associated proteins are encoded by two or more of the following human genes: TP53, PIK3CA, APC, CTNNBl, CDKN2A, NFE2L2, BRAF, KRAS, EGFR, ERBB2, SF3B1, FBXW7, PIK3R1, SMAD4, SPOP, PTPN11, NRAS, PTEN, HRAS, U2AF1, ERBB3, FGFR3, ARID 1 A, MAP2K1, FGFR2, RHOA, MTOR, BCL2L12, RAC1, IDH2, H3F3A, PPP2R1A, POLE, ATM, EP300, ALK, RQCD1, GPRIN2, THSD7B, CDK4, NUP93, CCND1, FGFR1, MAX, VHL, ACVR1, MEF2A, MYC, FRMD6, SRC, KIT, KEAP1, STK11, NF1, KMT2D, GATA3, AKT1, MAP3K1, MAP2K4, KMT2C, FAT1, PBRM1, SETD2, CREBBP, RBI, SMARCA4, CHD4, FLT3, ARID2, CDH1, DNMT3A, ARHGAP35, BCOR, CTCF, KDM5C, KDM6A, CASP8, ASXL1, RASA1, RUNX1, NPM1, CDKN1B, HLA-A, B2M, RPL5, MYD88, CBFB, and GPS2, or wherein the two or more cancer-associated proteins are encoded by two or more of the following human genes: TP53, PIK3CA, APC, CTNNBl, CDKN2A, NFE2L2, BRAF, KRAS, EGFR, ERBB2, SF3B1, FBXW7, PIK3R1, SMAD4, SPOP, PTPN11, NRAS, PTEN, HRAS, U2AF1, ERBB3, FGFR3, ARID 1 A, MAP2K1, FGFR2, RHOA, MTOR, BCL2L12, RAC1, IDH2, H3F3A, PPP2R1A, POLE, ATM, EP300, ALK, RQCD1, GPRIN2, THSD7B, CDK4, NUP93, CCND1, FGFR1, MAX, VHL, ACVR1, MEF2A, MYC, FRMD6, SRC, KIT, KEAP1, STK11, NF1, KMT2D, GATA3, AKT1, MAP3K1, MAP2K4, KMT2C, FAT1, PBRMl, SETD2, CREBBP, RBI, SMARCA4, CHD4, FLT3, ARID2, CDHl, DNMT3A, ARHGAP35, BCOR, CTCF, KDM5C, KDM6A, CASP8, ASXL1, RASA1, RUNX1, NPM1, CDKN1B, HLA-A, B2M, RPL5, MYD88, CBFB, GPS2, AHNAK2, ANKRD36C, CHEK2, KRTAP4-11, RGPD8, FAM47C, and ZAN.

[00622] 110. The method of embodiment 109, wherein the two or more cancer-associated proteins are encoded by two or more of the following human genes: BRAF, EGFR, PIK3CA, PIK3R1, PTEN, KRAS, TP53, APC, FBXW7, KEAP1, STK11, NF1, KMT2D, CDKN2A, NFE2L2, SPOP, GATA3, AKT1, MAP3K1, and MAP2K4, or wherein the two or more cancer- associated proteins are encoded by two or more of the following human genes: BRAF, EGFR, PIK3CA, PIK3R1, PTEN, KRAS, TP53, APC, FBXW7, KEAP1, STK11, NF1, KMT2D, CDKN2A, NFE2L2, SPOP, GATA3, AKT1, MAP3K1, MAP2K4, AHNAK2, ANKRD36C, CHEK2, KRTAP4-11, RGPD8, FAM47C, and ZAN.

[00623] 111. The method of any one of embodiments 102-110, wherein the combination of the two or more recurrent cancer mutation recombinant Listeria strains comprises about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-120, 120-140, 140-160, 160-180, 180-200, 200-220, 220-240, 240-260, 260-280, or 280- 300 different antigenic peptides.

[00624] 112. The method of any preceding embodiment, wherein the neoepitope in each of or some of the one or more second antigenic peptides comprises a linear neoepitope, a conformational neoepitope, a solvent-exposed neoepitope, or a combination thereof.

[00625] 113. The method of any preceding embodiment, wherein the neoepitope in each of or some of the one or more second antigenic peptides comprises a T-cell epitope.

[00626] 114. The method of any preceding embodiment, wherein each of the second antigenic peptides is about 5-100, 15-50, or 21-27 amino acids in length.

[00627] 115. The method of any preceding embodiment, wherein each of the second antigenic peptides comprises a cancer- specific mutation flanked on each side by an equal number of amino acids.

[00628] 116. The method of any preceding embodiment, wherein each of the second antigenic peptides comprises a cancer- specific mutation flanked on each side by at least 10 or at least 13 amino acids.

[00629] 117. The method of any preceding embodiment, wherein the one or more second antigenic peptides comprise at least 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 antigenic peptides or 2-40 antigenic peptides. [00630] 118. The method of embodiment 117, wherein the second antigenic peptides are fused directly to each other without intervening sequence.

[00631 ] 119. The method of embodiment 117, wherein the second antigenic peptides are linked to each other via peptide linkers.

[00632] 120. The method of embodiment 119, wherein one or more of the linkers set forth in SEQ ID NOS: 310-319 are used to link the second antigenic peptides.

[00633] 121. The method of any preceding embodiment, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the neoepitopes in the subject formed by nonsynonymous, somatic, cancer- specific mutations.

[00634] 122. The method of any preceding embodiment, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the neoepitopes in the subject formed by nonsynonymous, somatic, missense, cancer- specific mutations.

[00635] 123. The method of any preceding embodiment, wherein the second fusion polypeptide comprises two or more copies of a single antigenic peptide, or wherein two of the second antigenic peptides comprise the same cancer- specific mutation.

[00636] 124. The method of any one of embodiments 1-122, wherein each of the second antigenic peptides comprises a different neoepitope or a different cancer- specific mutation.

[00637] 125. The method of any preceding embodiment, wherein each cancer- specific mutation in the second fusion polypeptide is a somatic missense mutation.

[00638] 126. The method of any preceding embodiment, wherein the personalized immunotherapy composition comprises two or more personalized neoepitope recombinant Listeria strains including the second recombinant Listeria strain, wherein each of the two or more personalized neoepitope recombinant Listeria strains comprises an open reading frame encoding a fusion polypeptide, wherein each fusion polypeptide comprises a PEST- containing peptide fused to one or more antigenic peptides, wherein each of the antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject, and wherein each of the two or more personalized neoepitope recombinant Listeria strains comprises a different set of antigenic peptides or the same set of antigenic peptides in a different order.

[00639] 127. The method of embodiment 126, wherein the two or more personalized neoepitope recombinant Listeria strains are administered to the subject sequentially.

[00640] 128. The method of embodiment 126, wherein the two or more personalized neoepitope recombinant Listeria strains are administered to the subject simultaneously. [00641 ] 129. The method of any one of embodiments 126-128, wherein each of the two or more personalized neoepitope recombinant Listeria strains comprises a different set of antigenic peptides.

[00642] 130. The method of any one of embodiments 126-129, wherein the two or more personalized neoepitope recombinant Listeria strains comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 recombinant Listeria strains.

[00643] 131. The method of any one of embodiments 126-130, wherein the combination of the two or more personalized neoepitope recombinant Listeria strains comprises about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-120, 120-140, 140-160, 160-180, 180-200, 200-220, 220-240, 240-260, 260-280, or 280- 300 different antigenic peptides.

[00644] 132. The method of any preceding embodiment, wherein the first fusion polypeptide further comprises one or more peptide tags N-terminal and/or C-terminal to the combination of the first antigenic peptides and/or the second fusion polypeptide further comprises one or second peptide tags N-terminal, and/or C-terminal to the combination of the second antigenic peptides.

[00645] 133. The method of embodiment 132, wherein the one or more first peptide tags comprise one or more of the following: 3xFLAG tag; 6xHis tag; and SIINFEKL tag.

[00646] 134. The method of any preceding embodiment, wherein the first PEST- containing peptide is on the N-terminal end of the first fusion polypeptide and/or the second PEST-containing peptide is on the N-terminal end of the second fusion polypeptide.

[00647] 135. The method of any preceding embodiment, wherein the first PEST- containing peptide is a listeriolysin O (LLO) protein or a fragment thereof or an ActA protein or a fragment thereof and/or the second PEST-containing peptide is a listeriolysin O (LLO) protein or a fragment thereof or an ActA protein or a fragment thereof.

[00648] 136. The method of embodiment 135, wherein the first PEST-containing peptide is an N-terminal fragment of LLO and/or the second PEST-containing peptide is an N- terminal fragment of LLO.

[00649] 137. The method of embodiment 136, wherein the N-terminal fragment of LLO has the sequence set forth in SEQ ID NO: 336.

[00650] 138. The method of embodiment 135, wherein the first PEST-containing peptide is the LLO protein or the fragment thereof and comprises a mutation in a cholesterol-binding domain, and/or the second PEST-containing peptide is the LLO protein or the fragment thereof and comprises a mutation in a cholesterol-binding domain. [00651 ] 139. The method of embodiment 138, wherein the LLO mutation comprises one of the following: (1) a substitution of residues C484, W491, or W492 of SEQ ID NO: 332 or corresponding substitutions when the LLO protein is optimally aligned with SEQ ID NO: 332; or (2) a deletion of 1-11 amino acids within the residues 483-493 of SEQ ID NO: 332 or a corresponding deletion when the LLO protein is optimally aligned with SEQ ID NO: 332.

[00652] 140. The method of any preceding embodiment, wherein the first nucleic acid is operably integrated into the Listeria genome and/or the second nucleic acid is operably integrated into the Listeria genome.

[00653] 141. The method of any one of embodiments 1-139, wherein the first nucleic acid is in an episomal plasmid and/or the second nucleic acid is in an episomal plasmid.

[00654] 142. The method of any preceding embodiment, wherein the first nucleic acid does not confer antibiotic resistance upon the first recombinant Listeria strain and/or the second nucleic acid does not confer antibiotic resistance upon the second recombinant Listeria strain.

[00655] 143. The method of any preceding embodiment, wherein the first recombinant Listeria strain is attenuated and/or the second recombinant Listeria strain is attenuated.

[00656] 144. The method of any preceding embodiment, wherein the first recombinant Listeria strain is an auxotrophic Listeria strain and/or the second recombinant Listeria strain is an auxotrophic Listeria strain.

[00657] 145. The method of embodiment 143 or 144, wherein the attenuated Listeria strain comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes.

[00658] 146. The method of embodiment 145, wherein the one or more endogenous genes comprise prfA.

[00659] 147. The method of embodiment 145, wherein the one or more endogenous genes comprise actA.

[00660] 148. The method of embodiment 145, wherein the one or more endogenous genes comprise actA and inlB.

[00661 ] 149. The method of embodiment 145, wherein the one or more endogenous genes comprise actA, dal, and dat.

[00662] 150. The method of any preceding embodiment, wherein the first nucleic acid comprises a third open reading frame encoding a metabolic enzyme and/or the second nucleic acid comprises a fourth open reading frame encoding a metabolic enzyme.

[00663] 151. The method of embodiment 150, wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid aminotransferase enzyme.

[00664] 152. The method of any preceding embodiment, wherein the first fusion polypeptide is expressed from an hly promoter, a prfA promoter, an actA promoter, or a p60 promoter, and/or the second fusion polypeptide is expressed from an hly promoter, a prfA promoter, an actA promoter, or a p60 promoter.

[00665] 153. The method of embodiment 152, wherein the first fusion polypeptide is expressed from an hly promoter and/or the second fusion polypeptide is expressed from an hly promoter.

[00666] 154. The method of any preceding embodiment, wherein the first recombinant Listeria strain is a recombinant Listeria monocytogenes strain and/or the second recombinant Listeria strain is a recombinant Listeria monocytogenes strain.

[00667] 155. The method of any one of embodiments 1-134, wherein the first recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in prfA, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein, and/or the second recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in prfA, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein.

[00668] 156. The method of any one of embodiments 1-134, wherein the first recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D- amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N- terminal fragment of LLO, and/or the second recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

[00669] 157. The method of any one of embodiments 1-134, wherein the first recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA and inlB, wherein the nucleic acid is genomically integrated, and wherein the PEST-containing peptide is an ActA protein or a fragment thereof, and/or the second recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA and inlB, wherein the nucleic acid is genomically integrated, and wherein the PEST-containing peptide is an ActA protein or a fragment thereof.

[00670] 158. The method of any preceding embodiment, wherein the first recombinant Listeria strain has been passaged through an animal host and/or the second recombinant Listeria strain has been passaged through an animal host.

[00671 ] 159. The method of any preceding embodiment, wherein the first recombinant Listeria strain is capable of escaping a phagolysosome and/or the second recombinant Listeria strain is capable of escaping a phagolysosome.

[00672] 160. The method of any preceding embodiment, wherein no region of the first fusion polypeptide scores above a cutoff of around 1.6 when scored for hydropathy by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, and/or wherein no region of the first fusion polypeptide scores above a cutoff of around 1.6 when scored for hydropathy by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window.

[00673] 161. A system for use in cancer immunotherapy in a subject, comprising: (a) the recurrent cancer mutation immunotherapy composition of any preceding embodiment; and (b) the personalized immunotherapy composition of any preceding embodiment.

[00674] The subject matter disclosed herein also includes, but is not limited to, the following embodiments.

[00675] 1. A method for inducing an immune response against a tumor or cancer in a subject or treating a tumor or cancer in a subject, comprising: (a) administering a recurrent cancer mutation immunotherapy composition to the subject, wherein the recurrent cancer mutation immunotherapy composition comprises a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST-containing peptide fused to two or more first antigenic peptides, wherein at least one antigenic peptide is from a cancer-associated protein and comprises a recurrent cancer mutation, and at least one antigenic peptide is from a cancer-associated protein and comprises a heteroclitic mutation; and (b) administering a personalized immunotherapy composition to the subject, wherein the personalized immunotherapy composition comprises a second recombinant Listeria strain comprising a second nucleic acid comprising a second open reading frame encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises a second PEST- containing peptide fused to one or more second antigenic peptides, wherein each of the second antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject.

[00676] 2. The method of embodiment 1, wherein the recurrent cancer mutation immunotherapy composition is administered prior to the personalized immunotherapy composition.

[00677] 3. The method of embodiment 1, wherein the recurrent cancer mutation immunotherapy composition is administered subsequent to the personalized immunotherapy composition.

[00678] 4. The method of embodiment 1, wherein the recurrent cancer mutation immunotherapy composition and the personalized immunotherapy composition are administered concurrently.

[00679] 5. The method of any preceding embodiment, wherein the recurrent cancer mutation immunotherapy composition is administered with an adjuvant and/or the personalized immunotherapy composition is administered with an adjuvant.

[00680] 6. The method of any preceding embodiment, wherein the subject has a cancer associated with one or more recurrent cancer mutations in one or more cancer-associated proteins, and the first recombinant Listeria strain comprises antigenic peptides comprising one or more recurrent cancer mutations associated with the cancer.

[00681 ] 7. The method of any preceding embodiment, wherein the method comprises screening the subject for and identifying at least one of the one or more recurrent cancer mutations prior to the administering the recurrent cancer mutation immunotherapy composition, wherein the first recombinant Listeria strain comprises antigenic peptides comprising the at least one of the one or more recurrent cancer mutations identified in the subject.

[00682] 8. The method of any one of embodiments 1-6, wherein the method does not comprise screening the subject for and identifying recurrent cancer mutations prior to administering the recurrent cancer mutation immunotherapy composition.

[00683] 9. The method of any preceding embodiment, further comprising generating the personalized immunotherapy composition for the subject.

[00684] 10. The method of embodiment 9, wherein the personalized immunotherapy composition is generated concurrently with administering the recurrent cancer mutation immunotherapy composition to the subject. [00685] 11. The method of embodiment 9, wherein the personalized immunotherapy composition is generated subsequent to administering the recurrent cancer mutation immunotherapy composition to the subject.

[00686] 12. The method of embodiment 9, wherein the personalized immunotherapy composition is generated prior to administering the recurrent cancer mutation immunotherapy composition to the subject.

[00687] 13. The method of any one of embodiments 9-12, wherein generating the personalized immunotherapy composition comprises: (a) comparing one or more open reading frame sequences or mRNA sequences from the cancer sample with one or more open reading frame sequences or mRNA sequences from the healthy biological sample, wherein the comparing identifies one or more cancer- specific neoepitopes, each comprising a different cancer- specific mutation; (b) selecting a set of cancer- specific neoepitopes to include in the second nucleic acid and designing the second nucleic acid; and (c) transforming a Listeria strain with the second nucleic acid.

[00688] 14. The method of embodiment 13, further comprising obtaining the cancer sample from the subject and/or obtaining the healthy biological sample from the subject.

[00689] 15. The method of embodiment 13 or 14, wherein the cancer sample and/or the healthy biological sample comprise a tissue, cells isolated from blood, cells isolated from sputum, cells isolated from saliva, or cells isolated from cerebrospinal fluid.

[00690] 16. The method of any one of embodiments 13-15, wherein the open reading frame sequences are compared, and the open reading frame sequences are determined using exome sequencing.

[00691 ] 17. The method of any one of embodiments 13-15, wherein the mRNA sequences are compared, and the mRNA sequences are determined using transcriptome sequencing.

[00692] 18. The method of any one of embodiments 13-15, wherein the comparing comprises use of a screening assay or screening tool and associated digital software for comparing one or more open reading frames in nucleic acid sequences, wherein the associated digital software comprises access to a sequence database that allows screening of mutations within open reading frames for identification of the immunogenic potential of the one or more cancer- specific neoepitopes.

[00693] 19. The method of any one of embodiments 13-18, wherein step (b) comprises designing an antigenic peptide for each of the one or more cancer- specific neoepitopes.

[00694] 20. The method of embodiment 19, wherein each antigenic peptide comprises a different cancer- specific mutation and flanking sequence on each side. [00695] 21. The method of embodiment 20, wherein each antigenic peptide includes at least about 10 flanking amino acids on each side

[00696] 22. The method of any one of embodiments 19-21, wherein step (b) comprises scoring the each antigenic peptide and selecting an antigenic peptide if it scores below a hydropathy threshold predictive of secretability in Listeria monocytogenes.

[00697] 23. The method of embodiment 22, wherein the scoring is by a Kyte and Doolittle hydropathy index 21 amino acid window, and any antigenic peptides scoring above a cutoff of about 1.6 are excluded or are modified to score below the cutoff.

[00698] 24. The method of any embodiment 22 or 23, wherein every identified cancer- specific neoepitope for which an antigenic peptide can be designed that scores below the threshold is selected for inclusion in the second nucleic acid in the second recombinant Listeria strain or in the second nucleic acid and one or more additional nucleic acids for transforming one or more additional recombinant Listeria strains.

[00699] 25. The method of any one of embodiments 13-24, wherein designing the second nucleic acid in step (b) comprises determining an order for the cancer- specific neoepitopes in the second fusion polypeptide.

[00700] 26. The method of embodiment 25, wherein the order is selected using randomization.

[00701 ] 27. The method of any one of embodiments 13-26, wherein designing the second nucleic acid in step (b) comprises scoring the hydropathy of the second fusion polypeptide, and either reordering the cancer- specific neoepitopes or removing problematic cancer- specific neoepitopes if any region of the second fusion polypeptide scores above a selected hydropathy index threshold value.

[00702] 28. The method of embodiment 27, wherein the second fusion polypeptide is scored by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, and wherein the threshold value is about 1.6.

[00703] 29. The method of any one of embodiments 13-28, wherein designing the second nucleic acid in step (b) comprises codon optimizing the second nucleic acid for expression and secretion in Listeria monocytogenes.

[00704] 30. The method of any one of embodiments 13-29, wherein the transforming is accomplished using a plasmid or a phage vector.

[00705] 31. The method of any one of embodiments 13-30, further comprising culturing and characterizing the transformed recombinant Listeria strain to confirm expression and/or secretion of the second fusion polypeptide. [00706] 32. The method of any preceding embodiment, wherein the first PEST-containing peptide comprises a bacterial secretion signal sequence, and the first fusion polypeptide further comprises a ubiquitin protein fused to a carboxy-terminal antigenic peptide, wherein the first PEST-containing peptide, the two or more first antigenic peptides, the ubiquitin, and the carboxy-terminal antigenic peptide are arranged in tandem from the amino-terminal end to the carboxy-terminal end of the first fusion polypeptide.

[00707] 33. The method of embodiment 32, wherein the carboxy-terminal antigenic peptide is from a cancer-associated protein and comprises a heteroclitic mutation.

[00708] 34. The method of embodiment 32 or 33, wherein the carboxy-terminal antigenic peptide is about 7-11, 8-10, or 9 amino acids in length.

[00709] 35. The method of any one of embodiments 32-34, wherein the carboxy-terminal antigenic peptide binds to one or more of the following HLA types: HLA-A*02:01, HLA- A*03:01, HLA-A*24:02, and HLA-B*07:02.

[00710] 36. The method of any one of embodiments 32-35, wherein the carboxy-terminal antigenic peptide is from a protein encoded by one of the following genes: STEAP1,

CEACAM5, NYESOl, and NUF2.

[00711 ] 37. The method of embodiment 36, wherein the carboxy-terminal antigenic peptide is selected from the peptides set forth in SEQ ID NOS: 796, 797, 798, 799, 800, and 807.

[00712] 38. The method of any preceding embodiment, wherein each antigenic peptide in the first fusion polypeptide is a fragment of a cancer-associated protein and is about 7-200 amino acids in length.

[00713] 39. The method of any preceding embodiment, wherein the first fusion

polypeptide comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 antigenic peptides or comprises between about 5-50, 10-40, or 20-30 antigenic peptides.

[00714] 40. The method of any preceding embodiment, wherein the first fusion

polypeptide comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic peptides comprising a recurrent cancer mutation or between about 5-30 or 10-20 antigenic peptides comprising a recurrent cancer mutation, and/or wherein the first fusion polypeptide comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic peptides comprising a heteroclitic mutation or between about 5-30 or 10-20 antigenic peptides comprising a heteroclitic mutation. [00715] 41. The method of embodiment 40, wherein the antigenic peptides comprising a recurrent cancer mutation in the first fusion polypeptide are in tandem, and the antigenic peptides comprising a heteroclitic mutation in the first fusion polypeptide are in tandem.

[00716] 42. The method of embodiment 40, wherein the antigenic peptides comprising a recurrent cancer mutation and the antigenic peptides comprising a heteroclitic mutation are intermixed within the first fusion polypeptide.

[00717] 43. The method of any preceding embodiment, wherein the two or more antigenic peptides in the first fusion polypeptide are linked to each other via peptide linkers.

[00718] 44. The method of embodiment 43, wherein the peptide linkers comprise flexibility linkers and/or rigidity linkers and/or immunoproteasome processing linkers, or wherein one or more of the linkers set forth in SEQ ID NOS: 310-319 and 821-829 are used to link the two or more antigenic peptides.

[00719] 45. The method of embodiment 44, wherein the peptide linker upstream of one or more of the antigenic peptides comprising a heteroclitic mutation is an immunoproteasome processing linker or is selected from the linkers set forth in SEQ ID NOS: 821-829.

[00720] 46. The method of any preceding embodiment, wherein no region of the first fusion polypeptide scores above a cutoff of around 1.6 when scored for hydropathy by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window.

[00721 ] 47. The method of any preceding embodiment, wherein at least two of the antigenic peptides in the first fusion polypeptide comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein.

[00722] 48. The method of any preceding embodiment, wherein the recurrent cancer mutations in at least two of the antigenic peptides in the first fusion polypeptide are from the same cancer-associated protein and do not occur naturally together.

[00723] 49. The method of any preceding embodiment, wherein at least two of the antigenic peptides in the first fusion polypeptide are overlapping fragments of the same cancer-associated protein.

[00724] 50. The method of embodiment 49, wherein the recurrent cancer mutations in at least two of the antigenic peptides in the first fusion polypeptide are from the same cancer- associated protein and occur at the same amino acid residue of the cancer-associated protein.

[00725] 51. The method of embodiment 50, wherein two of the antigenic peptides in the first fusion polypeptide comprise the same recurrent cancer mutation. [00726] 52. The method of any one of embodiments 1-50, wherein each antigenic peptide comprising a recurrent cancer mutation in the first fusion polypeptide comprises a different recurrent cancer mutation.

[00727] 53. The method of any preceding embodiment, wherein each recurrent cancer mutation in the first fusion polypeptide is a somatic frameshift mutation or a somatic missense mutation.

[00728] 54. The method of embodiment 53, wherein each recurrent cancer mutation in the first fusion polypeptide is a somatic missense mutation.

[00729] 55. The method of any preceding embodiment, wherein one or more or all of the antigenic peptides comprising a recurrent cancer mutation in the first fusion polypeptide have an equal number of amino acids flanking each side of the recurrent cancer mutation.

[00730] 56. The method of embodiment 55, wherein the number of flanking amino acids on each side of the recurrent cancer mutation is at least 10 amino acids.

[00731 ] 57. The method of any preceding embodiment, wherein the antigenic peptides in the first fusion polypeptide comprise the 2, 3, 4, 5, 6, 7, 8, 9, or 10 most common recurrent cancer mutations or recurrent somatic missense cancer mutations from a particular type of cancer.

[00732] 58. The method of any preceding embodiment, wherein at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 35%, 50%, 60%, 70%, 80%, or 90% of patients with a particular type of cancer have a recurrent cancer mutation that is included in the combination of antigenic peptides in the first fusion polypeptide.

[00733] 59. The method of any preceding embodiment, wherein the antigenic peptides in the first fusion polypeptide comprise at least about 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 recurrent cancer mutations or recurrent somatic missense cancer mutations from a particular type of cancer, or wherein the antigenic peptides in the first fusion polypeptide comprise about 2-80, 10-60, 10- 50, 10-40, or 10-30 different recurrent cancer mutations or recurrent somatic missense cancer mutations from a particular type of cancer.

[00734] 60. The method of any one of embodiments 57-59, wherein the particular type of cancer is no n- small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer, or head and neck cancer.

[00735] 61. The method of any preceding embodiment, wherein the antigenic peptides in the first fusion polypeptide are from two or more cancer-associated proteins. [00736] 62. The method of embodiment 61, wherein the two or more cancer-associated proteins are at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer-associated proteins, or wherein the two or more cancer-associated proteins are about 2-30, 2-25, 2-20, 2-15, or 2-10 cancer- associated proteins.

[00737] 63. The method of any preceding embodiment, wherein the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more of the following genes: ACVR2A, ADAM28, AKT1, ANKRD36C, AR, ARID1A, BMPR2, BRAF, CHEK2, C12orf4, CTNNB1, DOCK3, EGFR, ESR1, FBXW7, FGFR3, FHOD3, GNAS, HRAS, IDH1, IDH2, KIAA2026, KRAS, KRTAP1-5, KRTAP4-11, LARP4B, MBOAT2, NFE2L2, PGM5, PIK3CA, PLEKHA6, POLE, PTEN, RGPD8, RNF43, RXRA, SMAD4, SPOP, SVIL, TGFBR2, TP53, TRIM48, UBR5, U2AF1, WNT16, XYLT2, ZBTB20, and ZNF814.

[00738] 64. The method of embodiment 63, wherein: (a) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS, EGFR, U2AF1, BRAF, PIK3CA, and TP53; (b) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: SPOP, CHEK2, RGPD8, ANKRD36C, and AT?; (c) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS, U2AF1, TP53, SMAD4, and GNAS; (d) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: PIK3CA, FGFR3, TP53, RXRA, FBXW7, and NFE2L2; (e) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: PIK3CA, AKT1, and ESR1; (f) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: PTEN, KRAS, PIK3CA, CTNNB1, FBXW7, and TP53; (g) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: TP53; (h) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: TP53, PIK3CA, IDH1, IDH2, and EGFR; (i) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS, BRAF, PIK3CA, and TP53; or j) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: PIK3CA, CHEK2, RGPD8, ANKRD36C, TP53, ZNF814, KRTAP1-5, KRTAP4-11, and HRAS.

[00739] 65. The method of embodiment 64, wherein: (a) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: KRAS_G12C, EGFR_L858R, KRAS_G12D, U2AF1_S34F, BRAF_V600E, KRAS_G12V, PIK3CA_E545K, TP53_R158L, KRAS_G12A, EGFR_L861Q, and TP53_R273L; (b) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: SPOP_F133V, CHEK2_K373E, RGPD8_P1760A,

ANKRD36C_I634T, ANKRD36C_D629Y, SPOP_W131G, ANKRD36C_D626N,

SPOP_F133L, AR_T878A, AR_L702H, AR_W742C, AR_H875Y, and AR_F877L; (c) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: KRAS_G12C, KRAS_G12D, U2AF1_S34F, KRAS_G12V, TP53_R248Q, TP53_R248W, TP53_R175H, TP53_R273C, KRAS_G12R, KRAS_Q61H, TP53_R282W, TP53_R273H, TP53_G245S, SMAD4_R361C, GNAS_R201C, and

GNAS_R201H; (d) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: PIK3CA_E545K, FGFR3_S249C, TP53_R248Q, PIK3CA_E542K, RXRA_S427F, FBXW7_R505G, TP53_R280T,

NFE2L2_E79K, FGFR3_R248C, TP53_K132N, TP53_R248W, TP53_R175H, and

TP53_R273C; (e) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: PIK3CA_E545K, PIK3CA_E542K, PIK3CA_H1047R, AKT1_E17K, PIK3CA_H1047L, PIK3CA_Q546K, PIK3CA_E545A, PIK3CA_E545G, ESR1_K303R, ESR1_D538G, ESR1_Y537S, ESR1_Y537N,

ESR1_Y537C, and ESR1_E380Q; (f) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: PTEN_R130G, PTEN_R130Q, KRAS_G12D, KRAS_G12V, PIK3CA_H1047R; PIK3CA_R88Q,

PIK3CA_E545K, PIK3CA_E542K, CTNNB 1_S37F, KRAS_G13D, CTNNB 1_S37C, PIK3CA_H1047L, PIK3CA_G118D, KRAS_G12A, FBXW7_R505C, and TP53_R248W; (g) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: TP53_R248Q, TP53_R248W, TP53_R175H,

TP53_R273C, TP53_R282W, TP53_R273H, TP53_Y220C, TP53_I195T, TP53_C176Y, TP53_H179R, TP53_S241F, and TP53_H193R; (h) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations:

TP53_R273L, TP53_R273C, TP53_R273H, PIK3CA_G118D, IDH1_R132C, IDH1_R132G, IDH1_R132H, IDH1_R132S, IDH2_R172K, PIK3CA_E453K, and EGFR_G598V; (i) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: KRAS_G12C, KRAS_G12D, BRAF_V600E, KRAS_G12V, PIK3CA_E545K, TP53_R248W, TP53_R175H, TP53_R273C, PIK3CA_H1047R,

TP53_R282W, TP53_R273H, and KRAS_G13D; or (j) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: PIK3CA_E545K, CHEK2_K373E, RGPD8_P1760A, ANKRD36C_I634T, TP53_R248Q, PIK3CA_E542K, TP53_R248W, TP53_R175H, PIK3CA_H1047R, TP53_R282W,

TP53_R273H, TP53_G245S, TP53_Y220C, ZNF814_D404E, KRTAP1-5_I88T, KRTAP4- 11_L161V, and HRAS_G13V.

[00740] 66. The method of embodiment 65, wherein: (a) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 35; (b) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 52; (c) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 68; (d) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 76; (e) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 87; (f) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 95; (g) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 100; (h) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 104; (i) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 108; or (j) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 112.

[00741 ] 67. The method of any preceding embodiment, wherein each antigenic peptide comprising a heteroclitic mutation in the first fusion polypeptide is about 7-11, 8-10, or 9 amino acids in length.

[00742] 68. The method of any preceding embodiment, wherein the antigenic peptides comprising a heteroclitic mutation in the first fusion polypeptide bind to one or more or all of the following HLA types: HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, and HLA-B*07:02.

[00743] 69. The method of any preceding embodiment, wherein the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more of the following genes: CEACAM5, GAGE1, hTERT, KLHL7, MAGEA3, MAGEA4, MAGEA6, NUF2, NYESOl, PAGE4, PRAME, PSA, PSMA, RNF43, SART3, SSX2, STEAP1, and SURVIVIN. [00744] 70. The method of embodiment 69, wherein: (a) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESOl, STEAP1, and RNF43; (b) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA; (c) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, STEAP1, MAGEA3, PRAME, hTERT, and SURVIVIN; (d) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, GAGE1, NYESOl, RNF43, NUF2, KLHL7, MAGEA3, and PRAME; (e) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, STEAP1, RNF43, MAGEA3, PRAME, and hTERT; (f) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, PRAME, hTERT, STEAP1, RNF43, NUF2, KLHL7, and SART3; (g) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, STEAP1, RNF43, SART3, NUF2, KLHL7, PRAME, and hTERT; (h) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes:

CEACAM5, MAGEA6, STEAP1, RNF43, SART3, NUF2, KLHL7, and hTERT; (i) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESOl, STEAP1, RNF43, and MAGEA3; or (j) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, MAGEA4, STEAP1, NYESOl, PRAME, and hTERT.

[00745] 71. The method of embodiment 70, wherein: (a) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 36; (b) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 53; (c) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 69; (d) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 77; (e) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 88; (f) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 96; (g) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 101; (h) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 105; (i) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 109; or (j) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 113.

[00746] 72. The method of any preceding embodiment, wherein: (a) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 35 and 36; (b) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 52 and 53; (c) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 68 and 69; (d) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 76 and 77; (e) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 87 and 88; (f) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 95 and 96; (g) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 100 and 101; (h) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 104 and 105; (i) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 108 and 109; or (j) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 112 and 113.

[00747] 73. The method of embodiment 42, wherein: (a) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 859, 860, 861, 862, 863, 864, 865, 894, 895, and 905; (b) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 871, 872, 873, 874, 875, 876, 877, 892, 893, and 906; (c) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 866, 867, 868, 869, 870, and 908; (d) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 878, 879, 880, 881, 882, 888, 889, 890, and 891; (e) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 883, 884, 885, 886, 887, and 907; (f) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 896, 897, and 904; (g) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 898 and 899; (h) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 900 and 901; (i) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 902 and 903; or (j) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 918 and 919.

[00748] 74. The method of any preceding embodiment, wherein the first fusion polypeptide has a molecular weight of no more than about 150 kDa or no more than about 125 kDa.

[00749] 75. The method of embodiment 1, wherein the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by all of the following genes: KRAS, EGFR, U2AF1, BRAF, PIK3CA, and TP53, or the method of any preceding embodiment, wherein the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by all of the following genes: KRAS, EGFR, U2AF1, BRAF, PIK3CA, and TP53.

[00750] 76. The method of embodiment 75, wherein the antigenic peptides in the first fusion polypeptide comprise all of the following recurrent cancer mutations: KRAS_G12C, EGFR_L858R, KRAS_G12D, U2AF1_S34F, BRAF_V600E, KRAS_G12V,

PIK3CA_E545K, TP53_R158L, KRAS_G12A, EGFR_L861Q, and TP53_R273L.

[00751 ] 77. The method of embodiment 76, wherein the antigenic peptides in the first fusion polypeptide comprise all of the peptides set forth in Table 35.

[00752] 78. The method of embodiment 1, wherein the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESOl, STEAP1, and RNF43, or the method of any preceding embodiment, wherein the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESOl, STEAP1, and RNF43.

[00753] 79. The method of embodiment 78, wherein the antigenic peptides comprise all of the peptides set forth in Table 36.

[00754] 80. The method of embodiment 77 or 79, wherein the antigenic peptides in the first fusion polypeptide comprise all of the peptides set forth in Tables 35 and 36.

[00755] 81. The method of embodiment 80, wherein one or more of the antigenic peptides in the first fusion polypeptide comprising a recurrent cancer mutation are preceded by the linker set forth in SEQ ID NO: 316, and wherein one or more of the antigenic peptides in the first fusion polypeptide comprising a heteroclitic mutation are preceded by the linker set forth in any one of SEQ ID NOS: 821-829.

[00756] 82. The method of embodiment 81, wherein the first fusion polypeptide comprises the sequence set forth in SEQ ID NO: 895. [00757] 83. The method of embodiment 1, wherein the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by all of the following genes: SPOP, CHEK2, RGPD8, ANKRD36C, and AR, or the method of any preceding embodiment, wherein the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by all of the following genes: SPOP, CHEK2, RGPD8, ANKRD36C, and AR.

[00758] 84. The method of embodiment 83, wherein the antigenic peptides in the first fusion polypeptide comprise all of the following recurrent cancer mutations: SPOP_F133V, CHEK2_K373E, RGPD8_P1760A, ANKRD36C_I634T, ANKRD36C_D629Y,

SPOP_W131G, ANKRD36C_D626N, SPOP_F133L, AR_T878A, AR_L702H, AR_W742C, AR_H875Y, and AR_F877L.

[00759] 85. The method of embodiment 84, wherein the antigenic peptides in the first fusion polypeptide comprise all of the peptides set forth in Table 52.

[00760] 86. The method of embodiment 1, wherein the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA, or the method of any preceding embodiment, wherein the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA.

[00761 ] 87. The method of embodiment 86, wherein the antigenic peptides in the first fusion polypeptide comprise all of the peptides set forth in Table 53.

[00762] 88. The method of embodiment 85 or 87, wherein the antigenic peptides in the first fusion polypeptide comprise all of the peptides set forth in Tables 52 and 53.

[00763] 89. The method of embodiment 88, wherein one or more of the antigenic peptides in the first fusion polypeptide comprising a recurrent cancer mutation are preceded by the linker set forth in SEQ ID NO: 316, and wherein one or more of the antigenic peptides in the first fusion polypeptide comprising a heteroclitic mutation are preceded by the linker set forth in any one of SEQ ID NOS: 821-829.

[00764] 90. The method of embodiment 89, wherein the first fusion polypeptide comprises the sequence set forth in SEQ ID NO: 893.

[00765] 91. A method for inducing an immune response against a tumor or cancer in a subject or treating a tumor or cancer in a subject, comprising: (a) administering a recurrent cancer mutation immunotherapy composition to the subject, wherein the recurrent cancer mutation immunotherapy composition comprises a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST-containing peptide fused to two or more first antigenic peptides, wherein each of the first antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the first antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer- associated protein; or administering a recurrent cancer mutation immunotherapy composition to the subject, wherein the recurrent cancer mutation immunotherapy composition comprises a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST-containing peptide fused to two or more first antigenic peptides, wherein at least two of the first antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein; and (b) administering a personalized immunotherapy composition to the subject, wherein the personalized immunotherapy composition comprises a second recombinant Listeria strain comprising a second nucleic acid comprising a second open reading frame encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises a second PEST-containing peptide fused to one or more second antigenic peptides, wherein each of the second antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject.

[00766] 92. The method of embodiment 91, wherein the recurrent cancer mutations in at least two of the antigenic peptides are from the same cancer-associated protein and do not occur naturally together.

[00767] 93. The method of embodiment 91 or 92, wherein at least two of the antigenic peptides are overlapping fragments of the same cancer-associated protein.

[00768] 94. The method of embodiment 93, wherein the recurrent cancer mutations in at least two of the antigenic peptides are from the same cancer-associated protein and occur at the same amino acid residue of the cancer-associated protein.

[00769] 95. The method of any one of embodiments 91-94, wherein one or more of the recurrent cancer mutations in the fusion polypeptide is a somatic missense mutation.

[00770] 96. The method of any one of embodiments 91-95, wherein one or more of the recurrent cancer mutations in the fusion polypeptide is a somatic frameshift mutation.

[00771 ] 97. The method of any one of embodiments 91-96, wherein the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS, BRAF, PIK3CA, TRIM48, PTEN, POLE, PGM5, MBOAT2, KIAA2026, FBXW7, C12orf4, ZBTB20, XYLT2, WNT16, UBR5, TGFBR2, SVIL, RNF43, PLEKHA6, LARP4B, FHOD3, DOCK3, BMPR2, ARID1A, ADAM28, and ACVR2A.

[00772] 98. The method of embodiment 97, wherein the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: TRIM48_Y192H, PTEN_R130N, POLE_V411L, POLE_P286R, PIK3CA_H1047R, PIK3CA_R88N, PGM5_I98V,

MBOAT2_R43N, KRAS_G12D, KIAA2026_R574C, FBXW7_R465C, C12orf4_R335N, BRAF_V600E, ZBTB20_p.Pro692LeufsTer43, XYLT2_p.Gly529AlafsTer78,

WNT16_p.Glyl67AlafsTerl7, UBR5_p.Glu2121LysfsTer28,

TGFBR2_p.Glu 150GlyfsTer35, S VIL_p.Met 1863TrpfsTer44, RNF43_p.Gly659ValfsTer41 , PLEKHA6_p.Val328TyrfsTerl72, LARP4B_p.Thrl63HisfsTer47,

FHOD3_p.Ser336ValfsTerl38, DOCK3_p.Prol852GlnfsTer45,

BMPR2_p.Asn583ThrfsTer44, ARIDlA_p.Aspl850ThrfsTer33,

ADAM28_p.Asn75LysfsTerl5, and ACVR2A_p.Lys435GlufsTerl9.

[00773] 99. The method of embodiment 98, wherein the antigenic peptides comprise one or more or all of the peptides set forth in Table 116.

[00774] 100. The method of embodiment 99, wherein the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NO: 917.

[00775] 101. The method of any preceding embodiment, wherein the neoepitope in each of or some of the one or more second antigenic peptides comprises a linear neoepitope, a conformational neoepitope, a solvent-exposed neoepitope, or a combination thereof.

[00776] 102. The method of any preceding embodiment, wherein the neoepitope in each of or some of the one or more second antigenic peptides comprises a T-cell epitope.

[00777] 103. The method of any preceding embodiment, wherein each of the second antigenic peptides is about 5-100, 15-50, or 21-27 amino acids in length.

[00778] 104. The method of any preceding embodiment, wherein each of the second antigenic peptides comprises a cancer- specific mutation flanked on each side by an equal number of amino acids.

[00779] 105. The method of any preceding embodiment, wherein each of the second antigenic peptides comprises a cancer- specific mutation flanked on each side by at least 10 or at least 13 amino acids.

[00780] 106. The method of any preceding embodiment, wherein the one or more second antigenic peptides comprise at least 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 antigenic peptides or 2-40 antigenic peptides. [00781 ] 107. The method of embodiment 106, wherein the second antigenic peptides are linked to each other via peptide linkers.

[00782] 108. The method of embodiment 107, wherein one or more of the linkers set forth in SEQ ID NOS: 313-316, 319, and 821-829 are used to link the second antigenic peptides.

[00783] 109. The method of any preceding embodiment, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the neoepitopes in the subject formed by nonsynonymous, somatic, cancer- specific mutations or formed by nonsynonymous, somatic, missense, cancer- specific mutations are included in the second fusion polypeptide.

[00784] 110. The method of any preceding embodiment, wherein each of the second antigenic peptides comprises a different neoepitope or a different cancer- specific mutation.

[00785] 111. The method of any preceding embodiment, wherein each cancer- specific mutation in the second fusion polypeptide is a somatic missense mutation.

[00786] 112. The method of any preceding embodiment, wherein the first and/or second fusion polypeptide further comprises one or more peptide tags N-terminal and/or C-terminal to the combination of the two or more antigenic peptides, wherein the one or more peptide tags comprise one or both of the following: FLAG tag and SIINFEKL tag.

[00787] 113. The method of any preceding embodiment, wherein the first and/or second PEST-containing peptide is on the N-terminal end of the first and/or second fusion polypeptide.

[00788] 114. The method of embodiment 113, wherein the first and/or second PEST- containing peptide is an N-terminal fragment of LLO.

[00789] 115. The method of embodiment 114, wherein the N-terminal fragment of LLO has the sequence set forth in SEQ ID NO: 336.

[00790] 116. The method of any preceding embodiment, wherein the first and/or second nucleic acid is in an episomal plasmid.

[00791 ] 117. The method of any preceding embodiment, wherein the first and/or second nucleic acid does not confer antibiotic resistance upon the first and/or second recombinant Listeria strain.

[00792] 118. The method of any preceding embodiment, wherein the first and/or second recombinant Listeria strain is an attenuated, auxotrophic Listeria strain.

[00793] 119. The method of embodiment 118, wherein the attenuated, auxotrophic Listeria strain comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes. [00794] 120. The method of embodiment 119, wherein the one or more endogenous genes comprise actA, dal, and dat.

[00795] 121. The method of any preceding embodiment, wherein the first and/or second nucleic acid comprises an open reading frame encoding a metabolic enzyme.

[00796] 122. The method of embodiment 121, wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid aminotransferase enzyme.

[00797] 123. The method of any preceding embodiment, wherein the first and/or second fusion polypeptide is expressed from an hly promoter.

[00798] 124. The method of any preceding embodiment, wherein the first and/or second recombinant Listeria strain is a recombinant Listeria monocytogenes strain.

[00799] 125. The method of any preceding embodiment, wherein the first and/or second recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the first and/or second nucleic acid is in an episomal plasmid and comprises an open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

[00800] 126. A system for use in cancer immunotherapy in a subject, comprising: (a) the recurrent cancer mutation immunotherapy composition of any preceding embodiment; and (b) the personalized immunotherapy composition of any preceding embodiment.

BRIEF DESCRIPTION OF THE SEQUENCES

[00801 ] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5' end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. When a nucleotide sequence encoding an amino acid sequence is provided, it is understood that codon degenerate variants thereof that encode the same amino acid sequence are also provided. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus. SEQ ID Type Description NO

1 DNA BRAF1 Insert (no Tags)

2 Protein BRAF1 Insert (no Tags)

3 DNA (1) 3xFLAG-BRAFl-SIINFEKL

4 Protein (1) 3xFLAG-BRAFl-SIINFEKL

5 DNA (2) BRAFl-3xFLAG-SIINFEKL

6 Protein (2) BRAFl-3xFLAG-SIINFEKL

7 DNA BRAF2 Insert (no Tags)

8 Protein BRAF2 Insert (no Tags)

9 DNA (3) 3xFLAG-BRAF2-SIINFEKL

10 Protein (3) 3xFLAG-BRAF2-SIINFEKL

11 DNA (4) BRAF2-3xFLAG-SIINFEKL

12 Protein (4) BRAF2-3xFLAG-SIINFEKL

13 DNA BRAF3 Insert (no Tags)

14 Protein BRAF3 Insert (no Tags)

15 DNA (5) 3xFLAG-BRAF3-SIINFEKL

16 Protein (5) 3xFLAG-BRAF3-SIINFEKL

17 DNA (6) BRAF3-3xFLAG-SIINFEKL

18 Protein (6) BRAF3-3xFLAG-SIINFEKL

19 DNA BRAF4 Insert (no Tags)

20 Protein BRAF4 Insert (no Tags)

21 DNA (7) 3xFLAG-BRAF4-SIINFEKL

22 Protein (7) 3xFLAG-BRAF4-SIINFEKL

23 DNA (8) BRAF4-3xFLAG-SIINFEKL

24 Protein (8) BRAF4-3xFLAG-SIINFEKL

25 DNA EGFR1 Insert (no Tags)

26 Protein EGFR1 Insert (no Tags)

27 DNA (9) 3xFLAG-EGFRl -SIINFEKL

28 Protein (9) 3xFLAG-EGFRl -SIINFEKL

29 DNA (10) EGFRl-3xFLAG-SIINFEKL

30 Protein (10) EGFRl-3xFLAG-SIINFEKL

31 DNA EGFR2 Insert (no Tags)

32 Protein EGFR2 Insert (no Tags)

33 DNA (11) 3xFLAG-EGFR2-SIINFEKL

34 Protein (11) 3xFLAG-EGFR2-SIINFEKL

35 DNA (12) EGFR2-3xFLAG-SIINFEKL

36 Protein (12) EGFR2-3xFLAG-SIINFEKL

37 DNA EGFR3 Insert (no Tags)

38 Protein EGFR3 Insert (no Tags)

39 DNA (13) 3xFLAG-EGFR3-SIINFEKL

40 Protein (13) 3xFLAG-EGFR3-SIINFEKL

41 DNA (14) EGFR3-3xFLAG-SIINFEKL

42 Protein (14) EGFR3-3xFLAG-SIINFEKL

43 DNA EGFR4 Insert (no Tags)

44 Protein EGFR4 Insert (no Tags)

45 DNA (15) 3xFLAG-EGFR4-SIINFEKL

46 Protein (15) 3xFLAG-EGFR4-SIINFEKL

47 DNA (16) EGFR4-3xFLAG-SIINFEKL

48 Protein (16) EGFR4-3xFLAG-SIINFEKL

49 DNA PIK3CA1 Insert (no Tags)

50 Protein PIK3CA1 Insert (no Tags)

51 DNA (17) 3xFLAG-PIK3CAl-SIINFEKL

52 Protein (17) 3xFLAG-PIK3CAl-SIINFEKL

53 DNA (18) PIK3CAl-3xFLAG-SIINFEKL

54 Protein (18) PIK3CAl-3xFLAG-SIINFEKL

55 DNA PIK3CA2 Insert (no Tags)

56 Protein PIK3CA2 Insert (no Tags) SEQ ID Type Description NO

57 DNA (19) 3xFLAG-PIK3CA2-SIINFEKL

58 Protein (19) 3xFLAG-PIK3CA2-SIINFEKL

59 DNA (20) PIK3CA2-3xFLAG-SIINFEKL

60 Protein (20) PIK3CA2-3xFLAG-SIINFEKL

61 DNA PIK3CA3 Insert (no Tags)

62 Protein PIK3CA3 Insert (no Tags)

63 DNA (21) 3xFLAG-PIK3CA3-SIINFEKL

64 Protein (21) 3xFLAG-PIK3CA3-SIINFEKL

65 DNA (22) PIK3CA3-3xFLAG-SIINFEKL

66 Protein (22) PIK3CA3-3xFLAG-SIINFEKL

67 DNA PIK3CA4 Insert (no Tags)

68 Protein PIK3CA4 Insert (no Tags)

69 DNA (23) 3xFLAG-PIK3CA4-SIINFEKL

70 Protein (23) 3xFLAG-PIK3CA4-SIINFEKL

71 DNA (24) PIK3CA4-3xFLAG-SIINFEKL

72 Protein (24) PIK3CA4-3xFLAG-SIINFEKL

73 DNA PIK3R1-1 Insert (no Tags)

74 Protein PIK3R1-1 Insert (no Tags)

75 DNA (25) 3xFLAG-PIK3Rl-l-SIINFEKL

76 Protein (25) 3xFLAG-PIK3Rl-l-SIINFEKL

77 DNA (26) PIK3Rl-l-3xFLAG-SIINFEKL

78 Protein (26) PIK3Rl-l-3xFLAG-SIINFEKL

79 DNA PIK3R1-2 Insert (no Tags)

80 Protein PIK3R1-2 Insert (no Tags)

81 DNA (27) 3xFLAG-PIK3Rl-2-SIINFEKL

82 Protein (27) 3xFLAG-PIK3Rl-2-SIINFEKL

83 DNA (28) PIK3Rl-2-3xFLAG-SIINFEKL

84 Protein (28) PIK3Rl-2-3xFLAG-SIINFEKL

85 DNA PIK3combol Insert (no Tags)

86 Protein PIK3combol Insert (no Tags)

87 DNA (29) 3xFLAG-PIK3combol-SIINFEKL

88 Protein (29) 3xFLAG-PIK3combol-SIINFEKL

89 DNA (30) PIK3combol-3xFLAG-SIINFEKL

90 Protein (30) PIK3combol-3xFLAG-SIINFEKL

91 DNA PIK3combo2 Insert (no Tags)

92 Protein PIK3combo2 Insert (no Tags)

93 DNA (31) 3xFLAG-PIK3combo2-SIINFEKL

94 Protein (31) 3xFLAG-PIK3combo2-SIINFEKL

95 DNA (32) PIK3combo2-3xFLAG-SIINFEKL

96 Protein (32) PIK3combo2-3xFLAG-SIINFEKL

97 DNA PIK3combo3 Insert (no Tags)

98 Protein PIK3combo3 Insert (no Tags)

99 DNA (33) 3xFLAG-PIK3combo3-SIINFEKL

100 Protein (33) 3xFLAG-PIK3combo3-SIINFEKL

101 DNA (34) PIK3combo3-3xFLAG-SIINFEKL

102 Protein (34) PIK3combo3-3xFLAG-SIINFEKL

103 DNA PIK3combo4 Insert (no Tags)

104 Protein PIK3combo4 Insert (no Tags)

105 DNA (35) 3xFLAG-PIK3combo4-SIINFEKL

106 Protein (35) 3xFLAG-PIK3combo4-SIINFEKL

107 DNA (36) PIK3combo4-3xFLAG-SIINFEKL

108 Protein (36) PIK3combo4-3xFLAG-SIINFEKL

109 DNA PTEN1 Insert (no Tags)

110 Protein PTEN1 Insert (no Tags)

111 DNA (37) 3xFLAG-PTENl-SIINFEKL

112 Protein (37) 3xFLAG-PTENl-SIINFEKL SEQ ID Type Description NO

113 DNA (38) PTENl-3xFLAG-SIINFEKL

114 Protein (38) PTENl-3xFLAG-SIINFEKL

115 DNA PTEN2 Insert (no Tags)

116 Protein PTEN2 Insert (no Tags)

117 DNA (39) 3xFLAG-PTEN2-SIINFEKL

118 Protein (39) 3xFLAG-PTEN2-SIINFEKL

119 DNA (40) PTEN2-3xFLAG-SIINFEKL

120 Protein (40) PTEN2-3xFLAG-SIINFEKL

121 DNA PTEN3 Insert (no Tags)

122 Protein PTEN3 Insert (no Tags)

123 DNA (41) 3xFLAG-PTEN3-SIINFEKL

124 Protein (41) 3xFLAG-PTEN3-SIINFEKL

125 DNA (42) PTEN3-3xFLAG-SIINFEKL

126 Protein (42) PTEN3-3xFLAG-SIINFEKL

127 DNA PTEN4 Insert (no Tags)

128 Protein PTEN4 Insert (no Tags)

129 DNA (43) 3xFLAG-PTEN4-SIINFEKL

130 Protein (43) 3xFLAG-PTEN4-SIINFEKL

131 DNA (44) PTEN4-3xFLAG-SIINFEKL

132 Protein (44) PTEN4-3xFLAG-SIINFEKL

133 DNA KRAS1 Insert (no Tags)

134 Protein KRAS1 Insert (no Tags)

135 DNA (45) 3xFLAG-KRAS 1 -SIINFEKL

136 Protein (45) 3xFLAG-KRASl -SIINFEKL

137 DNA (46) KRAS 1 -3xFLAG-SIINFEKL

138 Protein (46) KRAS 1 -3xFLAG-SIINFEKL

139 DNA KRAS2 Insert (no Tags)

140 Protein KRAS2 Insert (no Tags)

141 DNA (47) 3xFLAG-KRAS2-SIINFEKL

142 Protein (47) 3xFLAG-KRAS2-SIINFEKL

143 DNA (48) KRAS2-3xFLAG-SIINFEKL

144 Protein (48) KRAS2-3xFLAG-SIINFEKL

145 DNA KRAS3 Insert (no Tags)

146 Protein KRAS3 Insert (no Tags)

147 DNA (49) 3xFLAG-KRAS3-SIINFEKL

148 Protein (49) 3xFLAG-KRAS3-SIINFEKL

149 DNA (50) KRAS3-3xFLAG-SIINFEKL

150 Protein (50) KRAS3-3xFLAG-SIINFEKL

151 DNA KRAS4 Insert (no Tags)

152 Protein KRAS4 Insert (no Tags)

153 DNA (51) 3xFLAG-KRAS4-SIINFEKL

154 Protein (51) 3xFLAG-KRAS4-SIINFEKL

155 DNA (52) KRAS4-3xFLAG-SIINFEKL

156 Protein (52) KRAS4-3xFLAG-SIINFEKL

157 DNA TP53 33merl Insert (no Tags)

158 Protein TP53 33merl Insert (no Tags)

159 DNA (53) 3xFLAG-TP53 33merl -SIINFEKL

160 Protein (53) 3xFLAG-TP53 33merl -SIINFEKL

161 DNA (54) TP53 33mer 1 -3xFLAG-SIINFEKL

162 Protein (54) TP53 33mer 1 -3xFLAG-SIINFEKL

163 DNA TP53 33mer2 Insert (no Tags)

164 Protein TP53 33mer2 Insert (no Tags)

165 DNA (55) 3xFLAG-TP53 33mer2-SIINFEKL

166 Protein (55) 3xFLAG-TP53 33mer2-SIINFEKL

167 DNA (56) TP53 33mer2-3xFLAG-SIINFEKL

168 Protein (56) TP53 33mer2-3xFLAG-SIINFEKL SEQ ID Type Description NO

169 DNA TP53 33mer3 Insert (no Tags)

170 Protein TP53 33mer3 Insert (no Tags)

171 DNA (57) 3xFLAG-TP53 33mer3-SIINFEKL

172 Protein (57) 3xFLAG-TP53 33mer3-SIINFEKL

173 DNA (58) TP53 33mer3-3xFLAG-SIINFEKL

174 Protein (58) TP53 33mer3-3xFLAG-SIINFEKL

175 DNA TP53 33mer4 Insert (no Tags)

176 Protein TP53 33mer4 Insert (no Tags)

177 DNA (59) 3xFLAG-TP53 33mer4-SIINFEKL

178 Protein (59) 3xFLAG-TP53 33mer4-SIINFEKL

179 DNA (60) TP53 33mer4-3xFLAG-SIINFEKL

180 Protein (60) TP53 33mer4-3xFLAG-SIINFEKL

181 DNA TP53 17merA Insert (no Tags)

182 Protein TP53 17merA Insert (no Tags)

183 DNA (61) 3xFLAG-TP53 17merA-SIINFEKL

184 Protein (61) 3xFLAG-TP53 17merA-SIINFEKL

185 DNA (62) TP53 17merA-3xFLAG-SIINFEKL

186 Protein (62) TP53 17merA-3xFLAG-SIINFEKL

187 DNA TP53 16merA Insert (no Tags)

188 Protein TP53 16merA Insert (no Tags)

189 DNA (63) 3xFLAG-TP53 16merA-SIINFEKL

190 Protein (63) 3xFLAG-TP53 16merA-SIINFEKL

191 DNA (64) TP53 16merA-3xFLAG-SIINFEKL

192 Protein (64) TP53 16merA-3xFLAG-SIINFEKL

193 DNA TP53 17merB Insert (no Tags)

194 Protein TP53 17merB Insert (no Tags)

195 DNA (65) 3xFLAG-TP53 17merB-SIINFEKL

196 Protein (65) 3xFLAG-TP53 17merB-SIINFEKL

197 DNA (66) TP53 17merB-3xFLAG-SIINFEKL

198 Protein (66) TP53 17merB-3xFLAG-SIINFEKL

199 DNA TP53 16merB Insert (no Tags)

200 Protein TP53 16merB Insert (no Tags)

201 DNA (67) 3xFLAG-TP53 16merB-SIINFEKL

202 Protein (67) 3xFLAG-TP53 16merB-SIINFEKL

203 DNA (68) TP53 16merB-3xFLAG-SIINFEKL

204 Protein (68) TP53 16merB-3xFLAG-SIINFEKL

205 DNA TP53 17merC Insert (no Tags)

206 Protein TP53 17merC Insert (no Tags)

207 DNA (69) 3xFLAG-TP53 17merC-SIINFEKL

208 Protein (69) 3xFLAG-TP53 17merC-SIINFEKL

209 DNA (70) TP53 17merC-3xFLAG-SIINFEKL

210 Protein (70) TP53 17merC-3xFLAG-SIINFEKL

211 DNA TP53 16merC Insert (no Tags)

212 Protein TP53 16merC Insert (no Tags)

213 DNA (71) 3xFLAG-TP53 16merC-SIINFEKL

214 Protein (71) 3xFLAG-TP53 16merC-SIINFEKL

215 DNA (72) TP53 16merC -3xFLAG-SIINFEKL

216 Protein (72) TP53 16merC -3xFLAG-SIINFEKL

217 DNA TP53 17merD Insert (no Tags)

218 Protein TP53 17merD Insert (no Tags)

219 DNA (73) 3xFLAG-TP53 17merD-SIINFEKL

220 Protein (73) 3xFLAG-TP53 17merD-SIINFEKL

221 DNA (74) TP53 17merD-3xFLAG-SIINFEKL

222 Protein (74) TP53 17merD-3xFLAG-SIINFEKL

223 DNA TP53 16merD Insert (no Tags)

224 Protein TP53 16merD Insert (no Tags) SEQ ID Type Description NO

225 DNA (75) 3xFLAG-TP53 16merD-SIINFEKL

226 Protein (75) 3xFLAG-TP53 16merD-SIINFEKL

227 DNA (76) TP53 16merD-3xFLAG-SIINFEKL

228 Protein (76) TP53 16merD-3xFLAG-SIINFEKL

229 DNA EGFR Insert MP vl (no Tags)

230 DNA EGFR Insert MP v2 (no Tags)

231 Protein EGFR Insert MP (no Tags)

232 DNA (77) 3xFLAG-EGFR-SIINFEKL MP

233 Protein (77) 3xFLAG-EGFR-SIINFEKL MP

234 DNA (78) EGFR-3xFLAG-SIINFEKL MP

235 Protein (78) EGFR-3xFLAG-SIINFEKL MP

236 DNA PIK3CAall Insert MP vl (no Tags)

237 DNA PIK3CAall Insert MP v2 (no Tags)

238 Protein PIK3CAall Insert MP (no Tags)

239 DNA (79) 3xFLAG-PIK3CAall-SIINFEKL MP

240 Protein (79) 3xFLAG-PIK3CAall-SIINFEKL MP

241 DNA (80) PIK3CAall-3xFLAG-SIINFEKL MP

242 Protein (80) PIK3CAall-3xFLAG-SIINFEKL MP

243 DNA PIK3CAmajor Insert MP vl (no Tags)

244 DNA PIK3CAmajor Insert MP v2 (no Tags)

245 Protein PIK3CAmajor Insert MP (no Tags)

246 DNA (81) 3xFLAG-PIK3CAmajor-SIINFEKL MP

247 Protein (81) 3xFLAG-PIK3CAmajor-SIINFEKL MP

248 DNA (82) PIK3CAmajor-3xFLAG-SIINFEKL MP

249 Protein (82) PIK3CAmajor-3xFLAG-SIINFEKL MP

250 DNA PIK3CAminor Insert MP vl (no Tags)

251 DNA PIK3CAminor Insert MP v2 (no Tags)

252 Protein PIK3CAminor Insert MP (no Tags)

253 DNA (83) 3xFLAG-PIK3CAminor-SIINFEKL MP

254 Protein (83) 3xFLAG-PIK3CAminor-SIINFEKL MP

255 DNA (84) PIK3CAminor-3xFLAG-SIINFEKL MP

256 Protein (84) PIK3CAminor-3xFLAG-SIINFEKL MP

257 DNA TP53all Insert MP vl (no Tags)

258 DNA TP53all Insert MP v2 (no Tags)

259 Protein TP53all Insert MP (no Tags)

260 DNA (85) 3xFLAG-TP53all-SIINFEKL MP

261 Protein (85) 3xFLAG-TP53all-SIINFEKL MP

262 DNA (86) TP53all-3xFLAG-SIINFEKL MP

263 Protein (86) TP53all-3xFLAG-SIINFEKL MP

264 DNA TP53major Insert MP vl (no Tags)

265 DNA TP53major Insert MP v2 (no Tags)

266 Protein TP53major Insert MP (no Tags)

267 DNA (87) 3xFLAG-TP53major-SIINFEKL MP

268 Protein (87) 3xFLAG-TP53major-SIINFEKL MP

269 DNA (88) TP53major-3xFLAG-SIINFEKL MP

270 Protein (88) TP53major-3xFLAG-SIINFEKL MP

271 DNA TP53minor Insert MP vl (no Tags)

272 DNA TP53minor Insert MP v2 (no Tags)

273 Protein TP53minor Insert MP (no Tags)

274 DNA (89) 3xFLAG-TP53minor-SIINFEKL MP

275 Protein (89) 3xFLAG-TP53minor-SIINFEKL MP

276 DNA (90) TP53minor-3xFLAG-SIINFEKL MP

277 Protein (90) TP53minor-3xFLAG-SIINFEKL MP

278 DNA SIINFEKL Tag vl

279 DNA SIINFEKL Tag v2

280 DNA SIINFEKL Tag v3 SEQ ID Type Description NO

281 DNA SIINFEKL Tag v4

282 DNA SIINFEKL Tag v5

283 DNA SIINFEKL Tag v6

284 DNA SIINFEKL Tag v7

285 DNA SIINFEKL Tag v8

286 DNA SIINFEKL Tag v9

287 DNA SIINFEKL Tag vlO

288 DNA SIINFEKL Tag vl l

289 DNA SIINFEKL Tag vl2

290 DNA SIINFEKL Tag vl3

291 DNA SIINFEKL Tag vl4

292 DNA SIINFEKL Tag vl5

293 Protein SIINFEKL Tag

294 DNA 3xFLAG Tag vl

295 DNA 3xFLAG Tag v2

296 DNA 3xFLAG Tag v3

297 DNA 3xFLAG Tag v4

298 DNA 3xFLAG Tag v5

299 DNA 3xFLAG Tag v6

300 DNA 3xFLAG Tag v7

301 DNA 3xFLAG Tag v8

302 DNA 3xFLAG Tag v9

303 DNA 3xFLAG Tag vlO

304 DNA 3xFLAG Tag vl l

305 DNA 3xFLAG Tag vl2

306 DNA 3xFLAG Tag vl3

307 DNA 3xFLAG Tag vl4

308 DNA 3xFLAG Tag vl5

309 Protein 3xFLAG Tag

310 Protein SSX2_A0201 Native Peptide

311 Protein STEAP1_A0201 Native Peptide

312 Protein STEAP1_A2402 Native Peptide

313 Protein Peptide Linker v4

314 Protein Peptide Linker v5

315 Protein Peptide Linker v6

316 Protein Peptide Linker v7

317 Protein SURVIVIN_A0201 Native Peptide

318 Protein SURVIVIN_A2402 Native Peptide

319 Protein Peptide Linker vlO

320 Protein PEST-Like Sequence vl

321 Protein PEST-Like Sequence v2

322 Protein PEST-Like Sequence v3

323 Protein PEST-Like Sequence v4

324 Protein PEST-Like Sequence v5

325 Protein PEST-Like Sequence v6

326 Protein PEST-Like Sequence v7

327 Protein PEST-Like Sequence v8

328 Protein PEST-Like Sequence v9

329 Protein PEST-Like Sequence vlO

330 Protein PEST-Like Sequence vl l

331 Protein PEST-Like Sequence vl2

332 Protein LLO Protein vl

333 Protein LLO Protein v2

334 Protein N-Terminal Truncated LLO vl

335 Protein N-Terminal Truncated LLO v2

336 Protein N-Terminal Truncated LLO v3 SEQ ID Type Description

NO

337 DNA Nucleic Acid Encoding N-Terminal Truncated LLO v3

338 Protein ActA Protein vl

339 Protein ActA Protein v2

340 Protein ActA Fragment vl

341 Protein ActA Fragment v2

342 Protein ActA Fragment v3

343 Protein ActA Fragment v4

344 Protein ActA Fragment v5

345 DNA Nucleic Acid Encoding ActA Fragment v5

346 Protein ActA Fragment v6

347 Protein ActA Fragment v7

348 DNA Nucleic Acid Encoding ActA Fragment v7

349 Protein ActA Fragment Fused to Hly Signal Peptide

350 Protein ActA Substitution

351 Protein Cholesterol-Binding Domain of LLO

352 Protein HLA-A2 restricted Epitope from NY-ESO-1

353 Protein Lm Alanine Racemase

354 Protein Lm D- Amino Acid Aminotransferase

355 DNA Nucleic Acid Encoding Lm Alanine Racemase

356 DNA Nucleic Acid Encoding Lm D- Amino Acid Aminotransferase

357 Protein Wild Type PrfA

358 DNA Nucleic Acid Encoding Wild Type PrfA

359 Protein D133V PrfA

360 DNA Nucleic Acid Encoding D133V PrfA

361 Protein WT BRAF

362 Protein WT EGFR

363 Protein WT PIK3CA

364 Protein WT PIK3R1

365 Protein WT PTEN

366 Protein WT KRAS

367 Protein WT TP53

368-571 Protein See Example 1

572 DNA 4X Glycine Linker Gl

573 DNA 4X Glycine Linker G2

574 DNA 4X Glycine Linker G3

575 DNA 4X Glycine Linker G4

576 DNA 4X Glycine Linker G5

577 DNA 4X Glycine Linker G6

578 DNA 4X Glycine Linker G7

579 DNA 4X Glycine Linker G8

580 DNA 4X Glycine Linker G9

581 DNA 4X Glycine Linker G10

582 DNA 4X Glycine Linker Gi l

583 Protein dtLLO

584-594 Protein See Example 3

595-613 Protein See Example 3

614-643 Protein See Example 3

644-703 Protein See Example 3

704-724 Protein See Example 3

725 Protein NUF2 Wild Type

726 Protein NUF2 Heteroclitic

727 DNA Advl6 f

728 DNA Adv295 r

729 Protein KRAS_G12D_21-Mer Insert

730 Protein KRAS_G 12D_Kd Minigene Insert

731 Protein KRAS_G 12D_Dd Minigene Insert SEQ ID Type Description NO

732 Protein Heteroclitic WTl Peptide vlA (WT1-F)

733 Protein Heteroclitic WTl Peptide v2

734 Protein Heteroclitic WTl Peptide v3

735 Protein Heteroclitic WTl Peptide v5

736 Protein Heteroclitic WTl Peptide v8

737 Protein Heteroclitic WTl Peptide v4

738 Protein Heteroclitic WTl Peptide v7

739 Protein Heteroclitic WTl Peptide v9

740 Protein Heteroclitic WTl Peptide v6

741 Protein Heteroclitic WTl Peptide vlB (WT1-A1)

742 Protein WTl-FLAG-Ub-heteroclitic phenylalanine minigene construct

743 Protein Wild-Type WTl Peptide vl4 - WT1-427 long

744 Protein Wild-Type WTl Peptide vl5 - WT1-331 long

745 Protein Heteroclitic WTl Peptide vlD (WTl-122Al-long)

746 Protein Native WTl Peptide vlB

747 Protein Ubiquitin

748 Protein WTl-Pl-P2-P3-FLAG-Ub-heteroclitic tyrosine minigene construct

749 Protein Wild-Type WTl Peptide vl (Al)

750 Protein Wild-Type WTl Peptide v2

751 Protein Wild-Type WTl Peptide v3

752 Protein Wild-Type WTl Peptide v5

753 Protein Wild-Type WTl Peptide v8

754 Protein Wild-Type WTl Peptide v4

755 Protein Wild-Type WTl Peptide v7

756 Protein Wild-Type WTl Peptide v9

757 Protein Wild-Type WTl Peptide v6

758 Protein Adpgk + Dpagtl Insert

759 Protein Adpgk Minigene Insert

760 Protein Dpagtl Minigene Insert

761 Protein AH1 Heteroclitic Peptide

762 Protein FLAG Tag

763 Protein AR_T878A 21mer

764 Protein AR_L702H 21mer

765 Protein AR_W742C 21mer

766 Protein AR_H875Y 21mer

767 Protein AR_F877L 21mer

768 Protein AR_H875Y_T878A 24mer

769 Protein FGFR3_S249C 21mer

770 Protein RXRA_S427F 21mer

771 Protein FBXW7_R505G 21mer

772 Protein NFE2L2_E79K 21mer

773 Protein FGFR3_R248C 21mer

774 Protein ESRl_K303R 21mer

775 Protein ESR1_D538G 21mer

776 Protein ESR1_Y537S 21mer

777 Protein ESR1_Y537N 21mer

778 Protein ESR1_Y537C 21mer

779 Protein ESR1_E380Q 21mer

780 Protein SMAD4_R361C 21mer

781 Protein GNAS_R201C 21mer

782 Protein GN AS_R201 H 21 mer

783 Protein CTNNBl_S37F 21mer

784 Protein CTNNB1_S37C 21mer

785 Protein FBXW7_R505C 21mer

786 Protein IDH1_R132C 21mer

787 Protein IDH1_R132G 21mer SEQ ID Type Description NO

788 Protein IDHl_R132H 21mer

789 Protein IDH1_R132S 21mer

790 Protein IDH2_R172K 21mer

791 Protein CEACAM5_A0301 9mer

792 Protein MAGEA6_A0301 9mer

793 Protein CEACAM5_B0702 9mer

794 Protein MAGEA4_B0702 9mer

795 Protein GAGE1_B0702 9mer

796 Protein CEACAM5_A2402 9mer

797 Protein NYESOl_A0201 9mer

798 Protein CEACAM5_A0201 9mer

799 Protein STEAP1_A0201 9mer

800 Protein STEAP1_A2402 9mer

801 Protein RNF43_B0702 9mer

802 Protein SSX2_A0201 9mer

803 Protein SART3_A0201 9mer

804 Protein PAGE4_A0201 9mer

805 Protein PSMA_A2402 9mer

806 Protein PSA_A0301 9mer

807 Protein NUF2_A0201 9mer

808 Protein NUF2_A2402 9mer

809 Protein KLHL7_A2402 9mer

810 Protein MAGEA3_A2402 9mer

811 Protein GAGE1_A0301 9mer

812 Protein MAGEA3_A0301 9mer

813 Protein NYESOl_B0702 9mer

814 Protein MAGEA3_B0702 9mer

815 Protein PRAME_A0201 9mer

816 Protein hTERT_A0201_A2402 9mer

817 Protein MAGEA3_A0201_A2402 9mer

818 Protein SURVIVIN_A0201 9mer

819 Protein SURVIVIN_A2402 9mer

820 Protein CEACAM5_A0201 9mer

821 Protein Linker

822 Protein Linker

823 Protein Linker

824 Protein Linker

825 Protein Linker

826 Protein Linker

827 Protein Linker

828 Protein Linker

829 Protein Linker

830 Protein ZNF814_D404E

831 Protein KRTAP1-5_I88T

832 Protein KRTAP4-11_L161V

833 Protein HRAS_G13V

834 Protein TRIM48_Y192H

835 Protein PTEN_R130N

836 Protein POLE_V411L

837 Protein POLE_P286R

838 Protein PIK3CA_R88N

839 Protein PGM5_I98V

840 Protein MBOAT2_R43N

841 Protein KIAA2026_R574C

842 Protein FBXW7_R465C

843 Protein C12orf4_R335N SEQ ID Type Description NO

844 Protein ZBTB20_p.Pro692LeufsTer43

845 Protein XYLT2_p.Gly529AlafsTer78

846 Protein WNT16_p.Glyl67AlafsTerl7

847 Protein UBR5_p.Glu2121LysfsTer28

848 Protein TGFBR2_p.Glul50GlyfsTer35

849 Protein SVIL_p.Metl863TrpfsTer44

850 Protein RNF43_p.Gly659 ValfsTer41

851 Protein PLEKHA6_p.Val328TyrfsTerl72

852 Protein LARP4B_p.Thrl63HisfsTer47

853 Protein FHOD3_p.Ser336ValfsTerl38

854 Protein DOCK3_p.Prol852GlnfsTer45

855 Protein BMPR2_p.Asn583ThrfsTer44

856 Protein ARID 1 A_p. Asp 1850ThrfsTer33

857 Protein ADAM28_p.Asn75LysfsTerl5

858 Protein ACVR2A_p.Lys435GlufsTerl9

859 Protein NSCLC HOT EV02 EAAAK.G4S (A)

860 Protein NSCLC HOT G4S (A)

861 Protein NSCLC HOT EV02 EAAAK-G4S mix (A)

862 Protein NSCLC HOT EV02 EAAAK.i20 (A)

863 Protein NSCLC HOT EV02 G4S.i20 (A)

864 Protein NSCLC HOT EVO 2 G4S LS#1 (A)

865 Protein NSCLC HOT EVO 2 G4S LS#2 (A)

866 Protein PANC HOT EV02 EAAAK.G4S (A)

867 Protein PANC HOT G4S (A)

868 Protein PANC HOT EV02 EAAAK-G4S mix (A)

869 Protein PANC HOT EV02 EAAAK.i20 (A)

870 Protein PANC HOT EV02 G4S.i20 (A)

871 Protein ProStar EV02 EAAAK.G4S (A)

872 Protein ProStar EV02 G4S (A)

873 Protein ProStar EV02 EAAAK-G4S mix (A)

874 Protein ProStar EV02 EAAAK.i20 (A)

875 Protein ProStar EV02 G4S.i20 (A)

876 Protein ProStar EVO 2 G4S LS#1 (A)

877 Protein ProStar EVO 2 G4S LS#2 (A)

878 Protein Bladder HOT EV02 EAAAK.G4S (A)

879 Protein Bladder HOT G4S (A)

880 Protein Bladder HOT EV02 EAAAK-G4S mix (A)

881 Protein Bladder HOT EV02 EAAAK.i20 (A)

882 Protein Bladder HOT EV02 G4S.i20 (A)

883 Protein Breast HOT EV02 EAAAK.G4S (A)

884 Protein Breast HOT G4S (A)

885 Protein Breast HOT EV02 EAAAK-G4S mix (A)

886 Protein Breast HOT EV02 EAAAK.i20 (A)

887 Protein Breast HOT EV02 G4S.i20 (A)

888 Protein Bladder HOT EV02 EAAAK.G4S (B)

889 Protein Bladder HOT EV02 EAAAK.i20 (B)

890 Protein Bladder HOT EV02 EAAAK.G4S NUF minigene (B)

891 Protein Bladder HOT EV02 EAAAK.i20_NUF minigene (B)

892 Protein ProStar EV02 EAAAK.G4S (B)

893 Protein ProStar EV02 EAAAK.i20 (B)

894 Protein NSCLC HOT EV02 EAAAK.G4S (B)

895 Protein NSCLC HOT EV02 EAAAK.i20 (B)

896 Protein Uterine HOT EV02 EAAAK.G4S

897 Protein Uterine HOT EV02 EAAAK.i20

898 Protein Ovarian HOT EV02 EAAAK.G4S (C)

899 Protein Ovarian HOT EV02 EAAAK.i20 (C) SEQ ID Type Description NO

900 Protein LGG HOT EV02 EAAAK.G4S NUF minigene (C)

901 Protein LGG HOT EV02 EAAAK.i20_NUF minigene (C)

902 Protein CRC MSS EV02 EAAAK.G4S (C)

903 Protein CRC MSS EV02 EAAAK.i20 (C)

904 Protein Uterine A24 HOT

905 Protein NSCLC A24 HOT

906 Protein Prostar A24 HOT

907 Protein Breast A24 HOT

908 Protein Pancreas A24 HOT

909 Protein NSCLC HS + HC

910 Protein NSCLC HS + MG

911 Protein NSCLC HC + MG

912 Protein NSCLC HC only

913 Protein Prostar HS + HC

914 Protein Prostar HS + MG

915 Protein Prostar HC + MG

916 Protein Prostar HC only

917 Protein DNA Mismatch Repair HOT EV02 EAAAK.G4S

918 Protein Head & Neck HOT EV02 EAAAK.G4S

919 Protein Head & Neck HOT EV02 EAAAK.i20

920 Protein LLO Signal Sequence

921 Protein ActA Signal Sequence

922 Protein SIINFEKL Tag

923 DNA NSCLC HOT EV02 EAAAK.G4S

924 DNA NSCLC HOT G4S

925 DNA NSCLC HOT EV02 EAAAK-G4S mix

926 DNA NSCLC HOT EV02 EAAAK.i20

927 DNA NSCLC HOT EV02 G4S.i20

928 DNA NSCLC HOT EVO 2 G4S LS#1

929 DNA NSCLC HOT EVO 2 G4S LS#2

930 DNA NSCLC HOT EV02 EAAAK G4S

931 DNA NSCLC HOT G4S

932 DNA NSCLC HOT EV02 EAAAK-G4S mix

933 DNA NSCLC HOT EV02 EAAAK i20

934 DNA NSCLC HOT EV02 G4S i20

935 DNA NSCLC HOT EVO 2 G4S LS#1

936 DNA NSCLC HOT EVO 2 G4S LS#2

937 DNA NSCLC HOT EV02 EAAAK.G4S

938 DNA NSCLC HOT EV02 EAAAK.i20

939 DNA NSCLC HOT EV02 EAAAK G4S v2

940 DNA NSCLC HOT EV02 EAAAK i20 v2

941 DNA In house NSCLC HOT EV02 EAAAKi20

942 DNA ProStar EV02 EAAAK.G4S

943 DNA ProStar EV02 G4S

944 DNA ProStar EV02 EAAAK-G4S mix

945 DNA ProStar EV02 EAAAK.i20

946 DNA ProStar EV02 G4S.i20

947 DNA ProStar EVO 2 G4S LS#1

948 DNA ProStar EVO 2 G4S LS#2

949 DNA ProStar EV02 EAAAK.G4S

950 DNA ProStar EV02 G4S

951 DNA ProStar EV02 EAAAK-G4S mix

952 DNA ProStar EV02 EAAAK.i20

953 DNA ProStar EV02 G4S.i20

954 DNA ProStar EVO 2 G4S LS#1

955 DNA ProStar EVO 2 G4S LS#2 SEQ ID Type Description NO

956 DNA ProStar EV02 EAAAK.G4S

957 DNA ProStar EV02 EAAAK.i20

958 DNA ProStar EV02 EAAAK G4S v4

959 DNA ProStar EV02 EAAAK i20 v4

960 DNA In house NSCLC HOT EV02 EAAAKi20

961 DNA Bladder HOT Evo2 G4S

962 DNA Bladder HOT Evo2 G4S

963 DNA Bladder HOT EV02 EAAAK.G4S

964 DNA Bladder HOT G4S

965 DNA Bladder HOT EV02 EAAAK-G4S mix

966 DNA Bladder HOT EV02 EAAAK.i20

967 DNA Bladder HOT EV02 G4S.i20

968 DNA Bladder HOT EV02 EAAAK.G4S

969 DNA Bladder HOT G4S

970 DNA Bladder HOT EV02 EAAAK-G4S mix

971 DNA Bladder HOT EV02 EAAAK.i20

972 DNA Bladder HOT EV02 G4S.i20

973 DNA Bladder HOT EV02 EAAAK G4S v2

974 DNA Bladder HOT EV02 EAAAK i20 v2

975 DNA Bladder HOT EV02 EAAAK G4S NUF minigene v3

976 DNA Bladder HOT EV02 EAAAK i20 NUF minigene v3

977 DNA Bladder HOT EV02 EAAAK G4S NUF minigene v3

978 DNA Bladder HOT EV02 EAAAK i20 NUF minigene v3

979 DNA Breast HOT EV02 EAAAK.G4S

980 DNA Breast HOT G4S

981 DNA Breast HOT EV02 EAAAK-G4S mix

982 DNA Breast HOT EV02 EAAAK.i20

983 DNA Breast HOT EV02 G4S.i20

984 DNA Breast HOT EV02 EAAAK.G4S

985 DNA Breast HOT G4S

986 DNA Breast HOT EV02 EAAAK-G4S mix

987 DNA Breast HOT EV02 EAAAK.i20

988 DNA Breast HOT EV02 G4S.i20

989 DNA PANC HOT EV02 EAAAK G4S

990 DNA PANC HOT G4S

991 DNA PANC HOT EV02 EAAAK-G4S mix

992 DNA PANC HOT EV02 EAAAK i20

993 DNA PANC HOT EV02 G4S i20

994 DNA PANC HOT EV02 EAAAK G4S

995 DNA PANC HOT G4S

996 DNA PANC HOT EV02 EAAAK-G4S mix

997 DNA PANC HOT EV02 EAAAK i20

998 DNA PANC HOT EV02 G4S i20

999 DNA CRC MSS EV02 EAAAK G4S

1000 DNA CRC MSS EV02 EAAAK i20

1001 DNA CRC MSS EV02 EAAAK G4S

1002 DNA CRC MSS EV02 EAAAK i20

1003 Protein Lm-AHl 21mer Insert

1004 Protein Lm-AHl Minigene Insert

1005 Protein Lm-AHl HC Insert

1006 Protein AH1 Wild Type

1007 Protein SIINFEKL Peptide

1008 Protein Linker

1009 Protein CEACAM5_A0201 Native Peptide

1010 Protein CEACAM5_A0201 Native Peptide SEQ ID Type Description NO

1011 Protein CEACAM5_A0301 Native Peptide

1012 Protein CEACAM5_A2402 Native Peptide

1013 Protein CEACAM5_B0702 Native Peptide

1014 Protein GAGE1_A0301 Native Peptide

1015 Protein GAGE1_B0702 Native Peptide

1016 Protein hTERT_A0201_A2402 Native Peptide

1017 Protein KLHL7_A2402 Native Peptide

1018 Protein MAGEA3_A0201_A2402 Native Peptide

1019 Protein MAGEA3_A0301 Native Peptide

1020 Protein MAGEA3_A2402 Native Peptide

1021 Protein MAGEA3_B0702 Native Peptide

1022 Protein MAGEA4_B0702 Native Peptide

1023 Protein MAGEA6_A0301 Native Peptide

1024 Protein NUF2_A0201 Native Peptide

1025 Protein NUF2_A2402 Native Peptide

1026 Protein NYESOl_A0201 Native Peptide

1027 Protein NYESOl_B0702 Native Peptide

1028 Protein PAGE4_A0201 Native Peptide

1029 Protein PRAME_A0201 Native Peptide

1030 Protein PSA_A0301 Native Peptide

1031 Protein PSMA_A2402 Native Peptide

1032 Protein RNF43_B0702 Native Peptide

1033 Protein SART3_A0201 Native Peptide

1034-1052 DNA NSCLC KRAS G12C Sequences

1053-1071 DNA NSCLC EGFR L858R Sequences

1072-1090 DNA NSCLC KRAS G12D Sequences

1091-1109 DNA NSCLC U2AF S34F Sequences

1110-1128 DNA NSCLC BRAF V600E Sequences

1129-1147 DNA NSCLC KRAS G12V Sequences

1148-1166 DNA NSCLC PIK3CA E545K Sequences

1167-1185 DNA NSCLC TP53 R158L Sequences

1186-1204 DNA NSCLC KRAS G12A Sequences

1205-1223 DNA NSCLC EGFR L861Q Sequences

1224-1242 DNA NSCLC TP53 R273L Sequences

1243-1260 DNA Prostate SPOP F133V Sequences

1261-1278 DNA Prostate CHEK2 K373E Sequences

1279-1296 DNA Prostate RGPD8 PI 760 A Sequences

1297-1314 DNA Prostate ANKRD36C I634T Sequences

1315-1332 DNA Prostate ANKRD36C D629Y Sequences

1333-1350 DNA Prostate SPOP W131G Sequences

1351-1368 DNA Prostate ANKRD36C D626N Sequences

1369-1386 DNA Prostate SPOP F133L Sequences

1387-1404 DNA Prostate AR T878A Sequences

1405-1422 DNA Prostate AR L702H Sequences

1423-1440 DNA Prostate AR H875Y Sequences

1441-1458 DNA Prostate AR F877L Sequences

1459-1476 DNA Prostate AR H875Y_T878A Sequences

1477-1494 DNA Bladder PIK3CA E545K Sequences

1495-1512 DNA Bladder FGFR3 S249C Sequences

1513-1530 DNA Bladder TP53 R248Q Sequences

1531-1548 DNA Bladder PIK3CA E542K Sequences

1549-1566 DNA Bladder RXRA S427F Sequences

1567-1584 DNA Bladder FBXW7 R505G Sequences

1585-1602 DNA Bladder TP53 R280T Sequences

1603-1620 DNA Bladder NFE2L2 E79K Sequences

1621-1638 DNA Bladder FGFR3 R248C Sequences SEQ ID Type Description NO

1639-1656 DNA Bladder TP53 K132N Sequences

1657-1674 DNA Bladder TP53 R248W Sequences

1675-1692 DNA Bladder TP53 R175H Sequences

1693-1710 DNA Bladder TP53 R273C Sequences

1711-1720 DNA Breast PIK3CA E545K Sequences

1721-1730 DNA Breast PIK3CA E542K Sequences

1731-1740 DNA Breast PIK3CA H1047R Sequences

1741-1750 DNA Breast AKT1 E17K Sequences

1751-1760 DNA Breast PIK3CA H1047L Sequences

1761-1770 DNA Breast PIK3CA Q546K Sequences

1771-1780 DNA Breast PIK3CA E545A Sequences

1781-1790 DNA Breast PIK3CA E545G Sequences

1791-1800 DNA Breast ESR1 K303R Sequences

1801-1810 DNA Breast ESR1 D538G Sequences

1811-1820 DNA Breast ESR1 Y537S Sequences

1821-1830 DNA Breast ESR1 Y537N Sequences

1831-1840 DNA Breast ESR1 Y537C Sequences

1841-1850 DNA Breast ESR1 E380Q Sequences

1851-1860 DNA Pancreas KRAS G12C Sequences

1861-1870 DNA Pancreas KRAS G12D Sequences

1871-1880 DNA Pancreas U2AF1 S34F Sequences

1881-1890 DNA Pancreas KRAS G12V Sequences

1891-1900 DNA Pancreas TP53 R248Q Sequences

1901-1910 DNA Pancreas TP53 R248W Sequences

1911-1920 DNA Pancreas TP53 R175H Sequences

1921-1930 DNA Pancreas TP53 R273C Sequences

1931-1940 DNA Pancreas KRAS G12R Sequences

1941-1950 DNA Pancreas KRAS Q61H Sequences

1951-1960 DNA Pancreas TP53 R282W Sequences

1961-1970 DNA Pancreas TP53 R273H Sequences

1971-1980 DNA Pancreas TP53 G245S Sequences

1981-1990 DNA Pancreas SMAD4 R361C Sequences

1991-2000 DNA Pancreas GNAS R201C Sequences

2001-2010 DNA Pancreas GNAS R201H Sequences

2011-2014 DNA Colorectal KRAS G12C Sequences

2015-2018 DNA Colorectal KRAS G12D Sequences

2019-2022 DNA Colorectal BRAF V600E Sequences

2023-2026 DNA Colorectal KRAS G12V Sequences

2027-2030 DNA Colorectal PIK3CA E545K Sequences

2031-2034 DNA Colorectal TP53 R248W Sequences

2035-2038 DNA Colorectal TP53 R175H Sequences

2039-2042 DNA Colorectal TP53 R273C Sequences

2043-2046 DNA Colorectal PIK3CA H1047R Sequences

2047-2050 DNA Colorectal TP53 R282W Sequences

2051-2054 DNA Colorectal TP53 R273H Sequences

2055-2058 DNA Colorectal KRAS G13D Sequences

2059-2077 DNA NSCLC CEACAM5 A0301 Sequences

2078-2096 DNA NSCLC MAGEA6 A0301 Sequences

2097-2115 DNA NSCLC CEACAM5 B0702 Sequences

2116-2134 DNA NSCLC MAGEA4 B0702 Sequences

2135-2153 DNA NSCLC GAGE1 B0702 Sequences

2154-2172 DNA NSCLC CEACAM5 A2402 Sequences

2173-2191 DNA NSCLC NYESOl A0201 Sequences

2192-2210 DNA NSCLC CEACAM5 A0201 Sequences

2211-2228 DNA Prostate MAGEA4 B0702 Sequences

2229-2246 DNA Prostate STEAP1 A0201 Sequences SEQ ID Type Description NO

2247-2264 DNA Prostate STEAP1 A2402 Sequences

2265-2282 DNA Prostate SSX2 A0201 Sequences

2283-2300 DNA Prostate SART3 A0201 Sequences

2301-2318 DNA Prostate PAGE4 A0201 Sequences

2319-2336 DNA Prostate PSMA A2402 Sequences

2337-2354 DNA Prostate PSA A0301 Sequences

2355-2372 DNA Bladder GAGE1 B0702 Sequences

2373-2390 DNA Bladder NYESOl A0201 Sequences

2391-2408 DNA Bladder NUF2 A0201 Sequences

2409-2426 DNA Bladder NUF2 A2402 Sequences

2427-2444 DNA Bladder KLHL7 A2402 Sequences

2445-2462 DNA Bladder MAGEA3 A2402 Sequences

2463-2480 DNA Bladder GAGE1 A0301 Sequences

2481-2498 DNA Bladder MAGEA3 A0301 Sequences

2499-2516 DNA Bladder NYESOl B0702 Sequences

2517-2534 DNA Bladder MAGEA3 B0702 Sequences

2535-2544 DNA Breast CEACAM5 A0301 Sequences

2545-2554 DNA Breast CEACAM5 B0702 Sequences

2555-2564 DNA Breast CEACAM5 A2402 Sequences

2565-2574 DNA Breast CEACAM5 A0201 Sequences

2575-2584 DNA Breast STEAP1 A0201 Sequences

2585-2594 DNA Breast STEAP1 A2402 Sequences

2595-2604 DNA Breast RNFF43 B0702 Sequences

2605-2614 DNA Breast MAGEA3 A2402 Sequences

2615-2624 DNA Breast MAGE A3 A0301 Sequences

2625-2634 DNA Breast PRAME A0201 Sequences

2635-2644 DNA Breast hTERT A0201_A2402 Sequences

2645-2654 DNA Pancreas CEACAM5 A0301 Sequences

2655-2664 DNA Pancreas CEACAM5 B0702 Sequences

2665-2674 DNA Pancreas CEACAM5 A2402 Sequences

2675-2684 DNA Pancreas CEACAM5 A0201 Sequences

2685-2694 DNA Pancreas STEAP1 A0201 Sequences

2695-2704 DNA Pancreas STEAP1 A2402 Sequences

2705-2714 DNA Pancreas MAGEA3 A0301 Sequences

2715-2724 DNA Pancreas PRAME A0201 Sequences

2725-2734 DNA Pancreas hTERT A0201_A2402 Sequences

2735-2744 DNA Pancreas MAGEA3 A0201_A2402 Sequences

2745-2754 DNA Pancreas SURVIVIN A0201 Sequences

2755-2764 DNA Pancreas SURVIVIN A2402 Sequences

2765-2768 DNA Colorectal CEACAM5 A0301 Sequences

2769-2772 DNA Colorectal MAGEA6 A0301 Sequences

2773-2776 DNA Colorectal CEACAM5 B0702 Sequences

2777-2780 DNA Colorectal MAGEA4 B0702 Sequences

2781-2784 DNA Colorectal GAGE1 B0702 Sequences

2785-2788 DNA Colorectal CEACAM5 A2402 Sequences

2789-2792 DNA Colorectal NYESOl A0201 Sequences

2793-2796 DNA Colorectal STEAP1 A0201 Sequences

2797-2800 DNA Colorectal RNF43 B0702 Sequences

2801-2804 DNA Colorectal MAGEA3 A0201_A2402 Sequences

2805 DNA pAdvl34-MCS

2806 Protein C-terminal SIINFEKL and 6xHis AA sequence

2807 Protein H-2 D^b PSA₆₅-73

2808 Protein VvB8R

2809 Protein IAV PA

DNA/

2810-3408 Sequences Referenced in Examples 14-19

Protein SEQ ID Type Description

NO

3409-3426 DNA Prostate AR-W742C Sequences

3427 DNA NSCLC STEAP1 A0201 Sequence

3428 DNA NSCLC STEAP1 S2402 Sequence

3429 DNA NSCLC RNF43 B0702 Sequence

3430 DNA Prostate CEACAM5 B0702 Sequence

3431 DNA Prostate RNF43 B0702 Sequence

3432 DNA Bladder CEACAM5 A0301 Sequence

3433 DNA Bladder CEACAM5 A0201 Sequence

3434 DNA Bladder RNF43 B0702 Sequence

3435 DNA Bladder PRAME A0201 Sequence

3436 DNA NSCLC HS + HC

3437 DNA NSCLC HS + MG

3438 DNA NSCLC HS + MG

3439 DNA NSCLC HC + MG

3440 DNA NSCLC HC only

3441 DNA NSCLC HC only

3442 DNA Prostar HS + HC

3443 DNA Prostar HS + MG

3444 DNA Prostar HC + MG

3445 DNA Prostar HC + MG

3446 DNA Prostar HC only

3447 DNA NSCLC HOT EV02 EAAAK.G4S

3448 DNA NSCLC HOT EV02 EAAAK.i20

3449 DNA NSCLC HOT EV02 EAAAK G4S v2

3450 DNA NSCLC HOT EV02 EAAAK i20 v2

3451 DNA Prostar HC + MG

3452 DNA NSCLC HS + MG

EXAMPLES

Example 1. Design of ADXS-HOT Constructs for Tumor-Associated Proteins

[00802] We selected seven initial tumor-associated proteins with recurrent cancer mutations on which to focus preclinical development efforts for ADXS-HOT constructs (see Table 1). These seven tumor-associated proteins were selected because they have recurrent cancer mutations commonly presented in a large number of patients across multiple cancer types. Other ADXS-HOT constructs in production target commonly observed tumor drivers across multiple cancers, as well as additional mutated gene targets commonly observed in major cancer types like non- small cell lung cancer, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, and others. [00803] Table 1. Biomarker Expression Initial Selected Tumor Targets

% Expression as represented in the BROAD Institute's Tumor Portal dataset

[00804] For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed. In some cases, however, a peptide of a different length was used, such as 20 amino acids or 24 amino acids (e.g., with 9 amino acids flanking N- terminal and 10 amino acids flanking C-terminal, or with 10 amino acids flanking N-terminal and 13 amino acids flanking C-terminal). And in some cases, peptides comprising 2 or 3 recurrent cancer mutations were designed because of the close proximity of the mutated residues to each other in the protein. Examples of such peptides that are 23, 37, 39, or 53 amino acids in length are disclosed below.

[00805] The 21-mer peptides were designed to be fragments of the cancer-associated protein in which the recurrent cancer mutations occurs, including the recurrent cancer mutation and 10 amino acids of flanking sequence on each side. Antigenic peptides were scored by a Kyte and Doolittle hydropathy index with a 21 amino acid window, and peptides scoring above a cutoff of around 1.6 were excluded as they are unlikely to be secretable by Listeria monocytogenes. Constructs were designed with the peptides in multiple different orders generated by randomization. For each ordering of the peptides, constructs were designed with a 3xFLAG tag at the N-terminus and a SIINFEKL tag at the C-terminus, or with a 3xFLAG tag and a SIINFEKL tag at the C-terminus. Each ordering of the peptides was scored by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, and if any region for a particular ordering of peptides scored above a cutoff of around 1.6, the order of the peptides was reshuffled until the ordering of peptides resulted in a polypeptide with no regions scoring above the cutoff.

[00806] For the BRAF constructs, 8 recurrent cancer mutations were included in the constructs: G466E; G466V; G469A; G469R; G469S; G469V; V600E; and V600K. The reference wild type BRAF sequence is set forth in SEQ ID NO: 361. Constructs were designed with the peptides comprising the 8 recurrent cancer mutations in 4 different orders from N-terminal to C-terminal. Sequences for the constructs are found in SEQ ID NOS: 1- 24. The order of the hotspot mutation 21-mers in SEQ ID NOS: 1-6 is as follows:

BRAF\G469V; BRAF\G469R; BRAFW600E; BRAF\G469S; BRAF\G466V; BRAFW600K; BRAF\G469A; and BRAF\G466E. The order of the hotspot mutation 21-mers in SEQ ID NOS: 7-12 is as follows: BRAFW600K; BRAF\G469R; BRAF\G469V; BRAF\G466V;

BRAF\G466E; BRAFW600E; BRAF\G469A; and BRAF\G469S. The order of the hotspot mutation 21-mers in SEQ ID NOS: 13-18 is as follows: BRAF\G469V; BRAFW600K;

BRAF\G469S; BRAF\G466V; BRAF\G469A; BRAFW600E; BRAF\G466E; and

BRAF\G469R. The order of the hotspot mutation 21-mers in SEQ ID NOS: 19-24 is as follows: BRAFW600E; BRAFW600K; BRAF\G469A; BRAF\G469S; BRAF\G469R;

BRAF\G469V; BRAF\G466V; and BRAF\G466E. Examples of antigenic peptides includes in the constructs are provided in Table 2.

[00807] Table 2. BRAF Antigenic Peptides.

[00808] For the EGFR constructs, 16 recurrent cancer mutations were included in the constructs: R108K; A289V; G598V; E709A; E709K; G719A; G719C; G719S; L747P;

L747S; S768I; T790M; L833V/H835L; T833V; L858R; and L861Q. The reference wild type EGFR sequence is set forth in SEQ ID NO: 362. As indicated by the L833V/H835L and T833V mutations, position 833 can be "H" or "T" in different non-mutated versions of EGFR. In some constructs, the following 16 recurrent cancer mutations were included:

R108K; A289V; G598V; E709A; E709K; G719A; G719C; G719S; L747P; L747S; S768I; T790M; L833V/H835L; T833V; L858R; and L861Q. Constructs were designed with the peptides comprising the 8 recurrent cancer mutations in 4 different orders from N-terminal to C-terminal. Sequences for these constructs are set forth in SEQ ID NOS: 25-48. The order of the hotspot mutation 21-mers in SEQ ID NOS: 25-30 is as follows: EGFR\G719S;

EGFR\L747P; EGFR\G719C; EGFR\R108K; EGFR\S768I; (EGFR\L833V/H835L - 23- rnerj; EGFR\T833V; EGFR\E709A; EGFR\G598V; EGFR\T790M; EGFR\E709K;

EGFR\A289V; EGFR\L861Q; EGFR\G719A; EGFR\L747S; and EGFR\L858R. The order of the hotspot mutation 21-mers in SEQ ID NOS: 31-36 is as follows: EGFR\T790M;

EGFR\S768l; EGFR\G719C; EGFR\R108K; EGFR\L747P; EGFR\G719A; EGFR\L747S; EGFR\E709K; EGFR\T833V; EGFR\L861Q; EGFR\E709A; EGFR\L858R; EGFR\G598V; EGFR\A289V; (EGFR\L833V/H835L - 23-mer}; and EGFR\G719S. The order of the hotspot mutation 21-mers in SEQ ID NOS: 37-42 is as follows: EGFR\R108K; EGFR\T833V;

EGFR\L747S; EGFR\T790M; EGFR\G719C; EGFR\A289V; EGFR\L858R; EGFR\E709A; EGFR\G719S; EGFR\E709K; EGFR\G719A; EGFR\L747P; EGFR\G598V; EGFR\L861Q; EGFR\S768I; and (EGFR\L833V/H835L - 23-mer}. The order of the hotspot mutation 21- mers in SEQ ID NOS: 43-48 is as follows: EGFR\G719A; EGFR\L858R; EGFR\G719C; EGFR\A289V; EGFR\T790M; EGFR\S768I; EGFR\T833V; EGFR\G598V; EGFR\G719S; EGFR\L747S; EGFR\L747P; (EGFR\L833V/H835L - 23-mer}; EGFR\E709A;

EGFR\R108K; EGFR\L861Q; and EGFR\E709K. In other EGFR constructs, the following 11 recurrent cancer mutations were included: A289V; G598V; E709K; G719A; G719C; G719S; S768I; T790M; L833V/H835L; L858R; and L861Q. Sequences for these constructs are set forth in SEQ ID NOS: 229-235. The order of the hotspot mutation 21-mers in SEQ ID NOS: 229-235 is as follows: EGFRIA289V; EGFRIG598V; EGFRIE709K; EGFRIG719A; EGFRIS768I; EGFRIG719S; EGFRIL861Q; EGFRIT790M; EGFRIG719C;

{EGFRIL833V/H835L - 23-mer}; and EGFRIL858R. Examples of antigenic peptides includes in the constructs are provided in Table 3. [00809] Table 3. EGFR Antigenic Peptides.

[00810] For some of the PIK3CA constructs, 25 recurrent cancer mutations were included in the constructs: R38C; R38H; E81K; R88Q; R93Q; R93W; R108H; G118D; L334G; N345K; C420R; E453K; E542K; E545A; E545G; E545K; E545Q; Q546K; Q546R; E726K; M1043I; M1043V; H1047L; H1047R; and G1049R. The wild type PIK3CA reference sequence is set forth in SEQ ID NO: 363. Constructs were designed with the peptides comprising the 25 recurrent cancer mutations in 4 different orders from N-terminal to C- terminal. Sequences for the constructs are found in SEQ ID NOS: 49-72. The order of the hotspot mutation 21-mers in SEQ ID NOS: 49-54 is as follows: PIK3CA\M 1043V;

PIK3CA\E545G; PIK3CA\E726K; PIK3CA\Q546R; PIK3CA\L334G; PIK3CA\G1049R; PIK3CA\M1043I; PIK3CA\Q546K; PIK3CA\E542K; PIK3CA\R93Q; PIK3CA\H1047R; PIK3CA\R108H; PIK3CA\R93W; PIK3CA\E81K; PIK3CA\R38H; PIK3CA\N345K;

PIK3CA\R88Q; PIK3CA\G118D; PIK3CA\E545Q; PIK3CA\H1047L; PIK3CA\E545A; PIK3CA\E453K; PIK3CA\E545K; PIK3CA\R38C; and PIK3CA\C420R. The order of the hotspot mutation 21-mers in SEQ ID NOS: 55-60 is as follows: PIK3CA\E726K;

PIK3CA\E81K; PIK3CAM 1043V; PIK3CA\E545A; PIK3CA\E545K; PIK3CA\R38C;

PIK3CA\G118D; PIK3CAW93W; PIK3CA\E545G; PIK3CA\E542K; PIK3CA\G1049R; PIK3CA \N345K; PIK3CA\Q546K; PIK3CA\E453K; PIK3CA\C420R; PIK3CA\H1047L; PIK3CA\L334G; PIK3CA\E545Q; PIK3CA\R88Q; PIK3CA\H1047R; PIK3CA\M1043I; PIK3CA\R93Q; PIK3CA\R108H; PIK3CA\Q546R; and PIK3CA\R38H. The order of the hotspot mutation 21-mers in SEQ ID NOS: 61-66 is as follows: PIK3CA\R108H;

PIK3CAM 1043V; PIK3CA\R88Q; PIK3CA\R93W; PIK3CA\R38H; PIK3CA\H1047R;

PIK3CA\E545K; PIK3CA\M1043I; PIK3CA\Q546R; PIK3CA\E542K; PIK3CA \N345K; PIK3CA\R38C; PIK3CA\E545G; PIK3CA\E81K; PIK3CA\Q546K; PIK3CA\R93Q;

PIK3CA\E453K; PIK3CA\G1049R; PIK3CA\E545A; PIK3CA\C420R; PIK3CA\H1047L; PIK3CA\L334G; PIK3CA\G118D; PIK3CA\E726K; and PIK3CA\E545Q. The order of the hotspot mutation 21-mers in SEQ ID NOS: 67-72 is as follows: PIK3CA \N345K;

PIK3CA\R38H; PIK3CA\E545K; PIK3CA\G1049R; PIK3CA\H1047L; PIK3CA\E726K; PIK3CA\R88Q; PIK3CA\E81K; PIK3CA\R93Q; PIK3CA\E545Q; PIK3CA\L334G;

PIK3CA\R38C; PIK3CA\H1047R; PIK3CA\C420R; PIK3CA\R93W; PIK3CA\Q546K;

PIK3CA\M 1043V; PIK3CA\M1043I; PIK3CA\E545G; PIK3CA\E545A; PIK3CA\G118D; PIK3CA\E453K; PIK3CA\Q546R; PIK3CA\R108H; and PIK3CA\E542K. In other PIK3CA constructs, 17 recurrent cancer mutations were included in the constructs: R38H; E81K; R88Q; R108H; G118D; N345K; C420R; E542K; E545A; E545G; E545K; Q546K; Q546R; M1043I; H1047L; H1047R; and G1049R. Sequences for the constructs are found in SEQ ID NOS: 236-242. The order of the hotspot mutation 21-mers in SEQ ID NOS: 236-242 is as follows: PIK3CA\E542K; PIK3CA\E545K; PIK3CA\R88Q; PIK3CA\E545A;

PIK3CA\H1047R; PIK3CA\E545G; PIK3CA\H1047L; (PIK3CA\Q546K - 20-merJ;

PIK3CA\R38H; PIK3CA\E81K; PIK3CA\R108H; PIK3CA \N345K; PIK3CA\C420R;

PIK3CA\Q546R; PIK3CA\M 10431; PIK3CA\G118D; and PIK3CA\G1049R. In other PIK3CA constructs, 8 recurrent cancer mutations were included in the constructs: R88Q; E542K; E545A; E545G; E545K; Q546K; H1047L; and H1047. Sequences for the constructs are found in SEQ ID NOS: 243-249. The order of the hotspot mutation 21-mers in SEQ ID NOS: 243-249 is as follows: PIK3CA\E542K; PIK3CA\E545K; PIK3CAW88Q;

PIK3CA\E545A; PIK3CA\H1047R; PIK3CA\E545G; PIK3CA\H1047L; and

(PIK3CA\Q546K - 20-merJ. In other PIK3CA constructs, 9 recurrent cancer mutations were included in the constructs: R38H; E81K; R108H; G118D; N345K; C420R; Q546R; M1043I; and G1049R. Sequences for the constructs are found in SEQ ID NOS: 250-256. The order of the hotspot mutation 21-mers in SEQ ID NOS: 250-256 is as follows: PIK3CA\R38H; PIK3CA\E81K; PIK3CA\R108H; PIK3CA \N345K; PIK3CA\C420R; PIK3CA\Q546R; PIK3CA\M1043I; PIK3CA\G118D; and PIK3CA\G1049R. Examples of antigenic peptides includes in the constructs are provided in Table 4.

[00811 ] Table 4. PIK3CA Antigenic Peptides.

PIK3CA Wild Type Mutated

1047WT: H1047R: EYFMKQMNDARHGGWTTKMDW

EYFMKQMNDAHHGGWTTKMDW(SEQ ID NO: (SEQ ID NO: 447)

422) H1047L: EYFMKQMNDALHGGWTTKMDW

(SEQ ID NO: 448)

1049WT: FMKQMNDAHHGGWTTKMDWIF G1049R: FMKQMNDAHHRGWTTKMDWIF (SEQ (SEQ ID NO: 423) ID NO: 449)

[00812] For the PIK3R1 constructs, 3 recurrent cancer mutations were included in the constructs: G376R; N564D; and K567E. The wild type PIK3R1 reference sequence is set forth in SEQ ID NO: 364. Constructs were designed with the peptides comprising the 3 recurrent cancer mutations in 2 different orders from N-terminal to C-terminal. Sequences for these constructs are set forth in SEQ ID NOS: 73-84. The order of the hotspot mutation 21-mers in SEQ ID NOS: 73-78 is as follows: PIK3RUG376R; PIK3R1 \N564D; and

PIK3R1 \K567E. The order of the hotspot mutation 21-mers in SEQ ID NOS: 79-84 is as follows: PIK3R1 \N564D; PIK3R1 \K567E; and PIK3RUG376R. Examples of antigenic peptides includes in the constructs are provided in Table 5.

[00813] Table 5. PIK3R Antigenic Peptides.

[00814] For PIK3CAIPIK3R1 combination constructs, 28 recurrent cancer mutations were included in the constructs: PIK3CAIR38C; PIK3CAIR38H; PIK3CAIE81K; PIK3CAIR88Q; PIK3CAIR93Q; PIK3CAIR93W; PIK3CAIR108H; PIK3CAIG118D; PIK3CAIL334G;

PIK3CAIN345K; PIK3CAIC420R; PIK3CAIE453K; PIK3CAIE542K; PIK3CAIE545A; PIK3CAIE545G; PIK3CAIE545K; PIK3CAIE545Q; PIK3CAIQ546K; PIK3CAIQ546R; PIK3CAIE726K; PIK3CAIM1043I; PIK3CAIM1043V; PIK3CAIH1047L; PIK3CAIH1047R; PIK3CAIG1049R; PIK3R1IG376R; PIK3R1IN564D; and PIK3R1IK567E. Constructs were designed with the peptides comprising the 28 recurrent cancer mutations in 4 different orders from N-terminal to C-terminal. Sequences for these constructs are set forth in SEQ ID NOS: 85-108. The order of the hotspot mutation 21-mers in SEQ ID NOS: 85-90 is as follows: PIK3CA\R38C; PIK3CA \N345K; PIK3CA\E726K; PIK3CA\E453K; PIK3CA\R93Q;

PIK3CA\H1047R; PIK3CA\E545A; PIK3CAM 1043V; PIK3R1 \N564D; PIK3R1 \K567E; PIK3CA\E81K; PIK3CA\R108H; PIK3CA\Q546R; PIK3CA\Q546K; PIK3CA\E545Q;

PIK3CA\G1049R; PIK3CA\C420R; PIK3CA\H1047L; PIK3CAW93W; PIK3CAW88Q; PIK3CAM1043I; PIK3CA\E545G; PIK3CA\G118D; PIK3CA\R38H; PIK3RUG376R;

PIK3CA\E542K; PIK3CA\E545K; and PIK3CA\L334G. The order of the hotspot mutation 21-mers in SEQ ID NOS: 91-96 is as follows: PIK3CA\R38C; PIK3CA\R108H;

PIK3CA\C420R; PIK3CA\R93Q; PIK3CA\E453K; PIK3CA\M 1043V; PIK3CA\H1047L; PIK3R1 \N564D; PIK3CA\E726K; PIK3CA\G118D; PIK3CA\Q546K; PIK3CA\Q546R; PIK3CA\E542K; PIK3CA\E545K; PIK3CA\G1049R; PIK3CA\M1043I; PIK3CA\L334G; PIK3R1 \K567E; PIK3CA\R38H; PIK3R1 \G376R; PIK3CA\R93W; PIK3CA\H1047R;

PIK3CA\E545G; PIK3CA\E81K; PIK3CA\R88Q; PIK3CA \N345K; PIK3CA\E545A; and PIK3CA\E545Q. The order of the hotspot mutation 21-mers in SEQ ID NOS: 97-102 is as follows: PIK3CA\R108H; PIK3CAM 1043V; PIK3CA\R88Q; PIK3CA\R93W;

PIK3CA\R38H; PIK3CA\H1047R; PIK3CA\E545K; PIK3CA\M1043I; PIK3CA\Q546R; PIK3CA\E542K; PIK3CA \N345K; PIK3CA\R38C; PIK3CA\E545G; PIK3CA\E81K;

PIK3CA\Q546K; PIK3CA\R93Q; PIK3CA\E453K; PIK3CA\G1049R; PIK3CA\E545A; PIK3CA\C420R; PIK3CA\H1047L; PIK3CA\L334G; PIK3CA\G118D; PIK3CA\E726K; and PIK3CA\E545Q. The order of the hotspot mutation 21-mers in SEQ ID NOS: 103-108 is as follows: PIK3CA\E545Q; PIK3CA\R93W; PIK3CA\H1047R; PIK3CA\G1049R;

PIK3CA \N345K; PIK3CA\Q546R; PIK3CA\E545K; PIK3CA\E453K; PIK3CA\L334G; PIK3CA\H1047L; PIK3R1 \G376R; PIK3CAM 1043V; PIK3CA\R88Q; PIK3CA\R38H; PIK3CA\G118D; PIK3R1 \K567E; PIK3CA\R38C; PIK3CA\E542K; PIK3CA\Q546K;

PIK3CA\E726K; PIK3CA\C420R; PIK3CA\E545A; PIK3CA\R93Q; PIK3R1 \N564D;

PIK3CA\R108H; PIK3CA\M1043I; PIK3CA\E545G; and PIK3CA\E81K.

[00815] For the PTEN constructs, 13 recurrent cancer mutations were included in the constructs: Y68H; Y88C; D92E; dell21-131; R130G; R130L; R130P; R130Q; C136Y; R142W; Y155C; R173H; and P246L. The wild type PTEN reference sequence is set forth in SEQ ID NO: 365. Constructs were designed with the peptides comprising the 13 recurrent cancer mutations in 4 different orders from N-terminal to C-terminal. Sequences for these constructs are set forth in SEQ ID NOS: 109-132. The order of the hotspot mutation 21-mers in SEQ ID NOS: 109-114 is as follows: PTEN\deltal21-131; PTEN\Y88C; PTEN\R130G; PTEN\Y155C; PTEN\D92E; PTEN\C136Y; PTEN\R130Q; PTEN\ Y68H; PTEN\R142W; PTEN\R173H; PTEN\R130L; PTEN\R130P; and PTEN\P246L. The order of the hotspot mutation 21-mers in SEQ ID NOS: 115-120 is as follows: PTEN\R130P; PTEN\R130G; PTEN\Y155C; PTEN\R130L; PTEN\C136Y; PTEN\deltal21-131; PTEN\P246L;

PTEN\D92E; PTEN\R173H; PTEN\ Y68H; PTEN\R130Q; PTEN\Y88C; and PTENW142W. The order of the hotspot mutation 21-mers in SEQ ID NOS: 121-126 is as follows: PTEMR130Q; PTEMR130G; PTEMdeltal 21-131; PTEMC136Y; PTEMR130L; PTEMP246L; PTEN\Y155C; PTEN\D92E; PTEN\R142W; PTEN\R130P; PTEN\Y88C;

PTEN\ Y68H; and PTEN\R173H. The order of the hotspot mutation 21-mers in SEQ ID NOS: 127-132 is as follows: PTEN\deltal21-131; PTEMC136Y; PTEN\ Y68H; PTEMR142W; PTEN\R173H; PTEN\R130L; PTEMP246L; PTEMR130G; PTEMR130P; PTEN\ Y88C; PTEMD92E; PTEMR130Q; and PTEMY155C. Examples of antigenic peptides includes in the constructs are provided in Table 6.

[00816] Table 6. PTEN Antigenic Peptides.

[00817] For the KRAS constructs, 20 recurrent cancer mutations were included in the constructs: G12A; G12C; G12D; G12R; G12S; G12V; G13C; G13D; G13R; G13S; G13V; L19F; Q61K; Q61H; Q61L; Q61R; K117N; A146T; A146V, and A164G. The wild type KRAS reference sequence is set forth in SEQ ID NO: 366. Constructs were designed with the peptides comprising the 20 recurrent cancer mutations in 4 different orders from N- terminal to C-terminal. Sequences for these constructs are set forth in SEQ ID NOS: 133- 156. The order of the hotspot mutation 21-mers in SEQ ID NOS: 133-138 is as follows: KRAS\Q61R; KRAS\Q61K; KRAS\Q61L; KRAS\Q61H; KRAS\L19F; KRAS\K117N;

KRAS\G12A; KRAS\A164G; RAS\G12D; KRAS\G13D; KRAS\G13S; KRAS\G12S;

KRAS\A146V; KRAS\G13R; KRAS\G13C; KRAS\G12C; KRAS\G12R; KRAS\G13V;

KRAS\G12V; and KRAS\A146T. The order of the hotspot mutation 21-mers in SEQ ID NOS: 139-144 is as follows: KRAS\Q61H; KRAS\K117N; KRAS\G13C; KRAS\G13R; KRAS\G12D; KRAS\G12S; KRAS\G12V; KRAS\G12A; KRAS\Q61K; KRAS\G13V; KRAS\G12C;

KRAS\L19F; KRAS\Q61R; KRAS\Q61L; KRAS\A146V; KRAS\A164G; KRAS\G12R;

KRAS\G13S; KRAS\A146T; and KRAS\G13D. The order of the hotspot mutation 21-mersin SEQ ID NOS: 145-150 is as follows: KRAS\G12D; KRAS\L19F; KRAS\A146V; KRAS\Q61H; KRAS\G12V; KRAS\A164G; KRAS\G12C; KRAS\Q61L; KRAS\A146T; KRAS\G13S;

KRAS\G12A; KRAS\G13V; KRAS\G13C; KRAS\G13D; KRAS\G12R; KRAS\G12S;

KRAS\Q61R; KRAS\Q61K; KRAS\G13R; and KRAS\K117N. The order of the hotspot mutation 21-mers in SEQ ID NOS: 151-156 is as follows: KRAS\G13V; KRAS\G13S;

KRAS\G12V; KRAS\G12R; KRAS\A146V; KRAS\G13D; KRAS\G12D; KRAS\K117N;

KRAS\Q61H; KRAS\G12C; KRAS\G13C; KRAS\A146T; KRAS\G12A; KRAS\Q61L;

KRAS\Q61K; KRAS\A164G; KRAS\G12S; KRAS\L19F; KRAS\G13R; and KRAS\Q61R. Examples of antigenic peptides includes in the constructs are provided in Table 7.

[00818] Table 7. KRAS Antigenic Peptides.

[00819] For some of the TP53 constructs, 33 recurrent cancer mutations were included in the constructs: Y107D; K132N; C141Y; V143A; V157F; Y163C; R175H; C176F; C176Y; H179R; H179W; H193R; I195T; V216M; Y220C; Y234C; Y234H; S241F; S242F; G245D; G245S; R248L; R248Q; R248W; R249S; R273C; R273H; R273L; P278L; P278S; R282G; R282W; and R337H. The wild type TP53 reference sequence is set forth in SEQ ID NO: 367. Constructs were designed with the peptides comprising the 33 recurrent cancer mutations in 4 different orders from N-terminal to C-terminal. Sequences for these constructs are set forth in SEQ ID NOS: 157-180. The order of the hotspot mutation 21-mers in SEQ ID NOS: 157-162 is as follows: TP53\H179W; TP53W273L; TP53W249S; TP53W248Q;

TP53\Y234H; TP53\G245D; TP53\Y220C; TP53\R248L; TP53\H193R; TP53\K132N;

TP53\S242F; TP53\Y234C; TP53\G245S; TP53\C176F; TP53\R282W; TP53W273H;

TP53\R282G; TP53\C141Y; TP53\R273C; TP53W216M; TP53\R337H; TP53\R248W;

TP53W143A; TP53U195T; TP53\P278S; TP53\S241F; TP53\C176Y; TP53\Y107D;

TP53\R175H; TP53\H179R; TP53W157F; TP53\P278L; and TP53\Y163C. The order of the hotspot mutation 21-mers in SEQ ID NOS: 163-168 is as follows: TP53\R248W;

TP53\R248L; TP53\Y220C; TP53\Y163C; TP53\G245D; TP53\Y107D; TP53\H179R;

TP53W216M; TP53\P278S; TP53\S241F; TP53\R273L; TP53\P278L; TP53\C176F;

TP53\C141Y; TP53\S242F; TP53\R249S; TP53W143A; TP53U195T; TP53\R273H;

TP53\R273C; TP53\R282G; TP53\H179W; TP53\R175H; TP53\R248Q; TP53\G245S;

TP53\H193R; TP53\R337H; TP53\R282W; TP53\Y234C; TP53W157F; TP53\Y234H;

TP53\C176Y; and TP53\K132N. The order of the hotspot mutation 21-mers in SEQ ID NOS: 169-174 is as follows: TP53\R248W; TP53\H179R; TP53\R273H; TP53\Y107D;

TP53\R337H; TP53\R282G; TP53W157F; TP53W143A; TP53\Y234H; TP53\Y220C;

TP53\R282W; TP53\R248L; TP53\S241F; TP53\H179W; TP53\R273C; TP53\C141Y;

TP53\R249S; TP53\P278L; TP53\G245S; TP53U195T; TP53\R175H; TP53\G245D;

TP53\R273L; TP53\K132N; TP53W216M; TP53\Y163C; TP53\C176F; TP53\S242F;

TP53\Y234C; TP53\H193R; TP53\R248Q; TP53\P278S; and TP53\C176Y. The order of the hotspot mutation 21-mers in SEQ ID NOS: 175-180 is as follows: TP53W143A;

TP53\R282W; TP53W157F; TP53\H179W; TP53\K132N; TP53\Y163C; TP53\C176Y;

TP53\G245D; TP53\Y220C; TP53\S242F; TP53\Y234C; TP53\R249S; TP53\H179R;

TP53\R273H; TP53\C141Y; TP53\R273L; TP53\P278S; TP53\C176F; TP53\R337H;

TP53\H193R; TP53\R273C; TP53\R282G; TP53\R175H; TP53\R248W; TP53\P278L;

TP53U195T; TP53\S241F; TP53\R248L; TP53\Y234H; TP53W216M; TP53\G245S;

TP53\Y107D; and TP53\R248Q. For other TP53 constructs, 23 recurrent cancer mutations were included in the constructs: Y107D; C141Y; V143A; V157F; Y163C; R175H; C176F; H193R; I195T; V216M; Y220C; Y234C; Y234H; G245D; G245S; R248Q; R248W; R249S; R273C; R273H; R273L; R282G; and R282W. Sequences for these constructs are set forth in SEQ ID NOS: 257-263. The order of the hotspot mutation 21-mers in SEQ ID NOS: 257- 263 is as follows: TP53\R248W; (TP53\R273H - 24-mer}; TP53W143A; TP53\R249S;

(TP53\R175H-TP53\H193R - 39-mer combined}; TP53\Y220C; (TP53\G245D - 20-merJ; TP53W248Q; TP53\R273C; TP53W282W; (TP53\Y107D - 20-merJ; {TP53\C141Y- TP53W157F - 37-mer combined}; (TP53\Y163C-TP53\C176F-TP53\I195T - 53-mer combined}; (TP53\V216M-TP53\Y234H - 39-mer combined}; TP53\G245S; TP53\R273L; TP53\Y234C; and TP53\R282G. For other TP53 constructs, 11 recurrent cancer mutations were included in the constructs: V143A; R175H; H193R; Y220C; G245D; R248Q; R248W; R249S; R273C; R273H; and R282W. Sequences for these constructs are set forth in SEQ ID NOS: 264-270. The order of the hotspot mutation 21-mers in SEQ ID NOS: 264-270 is as follows: TP53\R248W; TP53W273H; TP53W143A; TP53W249S; (TP53\R175H- TP53\H193R - 39-mer combined}; TP53\Y220C; (TP53\G245D - 20-mer}; TP53\R248Q; TP53\R273C; and TP53\R282 W. For other TP53 constructs, 12 recurrent cancer mutations were included in the constructs: Y107D; C141Y; V157F; Y163C; C176F; I195T; V216M; Y234C; Y234H; G245S; R273L; and R282G. Sequences for these constructs are set forth in SEQ ID NOS: 271-277. The order of the hotspot mutation 21-mers in SEQ ID NOS: 271- 277 is as follows: TP53\ Y107D; (TP53\C141Y-TP53\V157F - 37-mer combined};

(TP53\Y163C-TP53\C176F-TP53 195T - 53-mer combined}; (TP53\V216M-TP53\Y234H - 39-mer combined}; TP53\G245S; TP53\R273L; TP53\Y234C; and TP53\R282G. Other TP53 constructs were designed to comprise different combination of 17 recurrent cancer mutations. For other TP53 constructs, 17 recurrent cancer mutations were included in the constructs: Y107D; C141Y; V143A; Y163C; C176Y; H179R; H179W; H193R; V216M; Y234H;

S241F; G245D; R248Q; R248W; R273C; R273L; and P278S. Sequences for these constructs are set forth in SEQ ID NOS: 181-186. The order of the hotspot mutation 21-mers in SEQ ID NOS: 181-186 is as follows: TP53\S241F; TP53\G245D; TP53W143A;

TP53\P278S; TP53\R273C; TP53\C176Y; TP53\Y234H; TP53\R248W; TP53W216M;

TP53\R248Q; TP53\C141Y; TP53\Y163C; TP53\H193R; TP53\H179R; TP53\H179W;

TP53\Y107D; and TP53\R273L. For other TP53 constructs, 17 recurrent cancer mutations were included in the constructs: C141Y; R175H; H179R; H193R; V216M; Y234H; G245D; G245S; R248L; R248W; R273C; R273H; P278L; P278S; R282G; R282W;and R337H.

Sequences for these constructs are set forth in SEQ ID NOS: 193-198. The order of the hotspot mutation 21-mers in SEQ ID NOS: 193-198 is as follows: TP53\H193R; TP53\P278L; TP53\R273C; TP53W248W; TP53\H179R; TP53\P278S; TP53\R248L;

TP53W216M; TP53\R282G; TP53\R337H; TP53\R175H; TP53\Y234H; TP53\G245D; TP53\R273H; TP53\G245S; TP53W282W; and TP53\C141Y. For other TP53 constructs, 17 recurrent cancer mutations were included in the constructs: Y107D; C141Y; V143A; C176F; H179R; V216M; Y220C; S241F; S242F; G245S; R248L; R248W; R273L; P278L; P278S; R282G; and R282W. Sequences for these constructs are set forth in SEQ ID NOS: 205-210. The order of the hotspot mutation 21-mers in SEQ ID NOS: 205-210 is as follows:

TP53\P278S; TP53\C176F; TP53\H179R; TP53\R282G; TP53\S241F; TP53W273L;

TP53\P278L; TP53\C141Y; TP53\Y107D; TP53W248W; TP53W216M; TP53W282W;

TP53\S242F; TP53\Y220C; TP53W143A; TP53\G245S; and TP53\R248L. For other TP53 constructs, 17 recurrent cancer mutations were included in the constructs: Y107D; K132N; V143A; V157F; Y163C; R175H; C176Y; Y234C; Y234H; S241F; S242F; G245D; G245S; R273C; P278S; R282W; and R337H. Sequences for these constructs are set forth in SEQ ID NOS: 217-222. The order of the hotspot mutation 21-mers in SEQ ID NOS: 217-222 is as follows: TP53\C176Y; TP53W175H; TP53\G245D; TP53\R337H; TP53\S241F;

TP53\K132N; TP53W143A; TP53\P278S; TP53\R282W; TP53\Y163C; TP53\Y107D;

TP53\R273C; TP53\S242F; TP53\G245S; TP53W157F; TP53\Y234C; and TP53\ Y234H. Other TP 53 constructs were designed to comprise different combination of 16 recurrent cancer mutations. For other TP53 constructs, 16 recurrent cancer mutations were included in the constructs: K132N; V157F; R175H; C176F; I195T; Y220C; Y234C; S242F; G245S; R248L; R249S; R273H; P278L; R282G; R282W; and R337H. Sequences for these constructs are set forth in SEQ ID NOS: 187-192. The order of the hotspot mutation 21-mers in SEQ ID NOS: 187-192 is as follows: TP53\K132N; TP53\R282W; TP53\G245S;

TP53\Y234C; TP53\S242F; TP53\R175H; TP53\Y220C; TP53W157F; TP53\R282G;

TP53\C176F; TP53\R337H; TP53 195T; TP53\R249S; TP53\P278L; TP53\R273H; and TP53\R248L. For other TP53 constructs, 16 recurrent cancer mutations were included in the constructs: Y107D; K132N; V143A; V157F; Y163C; C176F; C176Y; H179W; I195T;

Y220C; Y234C; S241F; S242F; R248Q; R249S; and R273L. Sequences for these constructs are set forth in SEQ ID NOS: 199-204. The order of the hotspot mutation 21-mers in SEQ ID NOS: 199-204 is as follows: TP53\ Y107D; TP53\K132N; TP53\C176F; TP53\C176Y;

TP53\R273L; TP53\Y220C; TP53\R248Q; TP53W143A; TP53 195T; TP53\R249S;

TP53\S242F; TP53\Y234C; TP53\H179W; TP53W157F; TP53\Y163C; and TP53\S241F. For other TP53 constructs, 16 recurrent cancer mutations were included in the constructs: K132N; V157F; Y163C; R175H; C176Y; H179W; H193R; I195T; Y234C; Y234H; G245D; R248Q; R249S; R273C; R273H; and R337H. Sequences for these constructs are set forth in SEQ ID NOS: 211-216. The order of the hotspot mutation 21-mers in SEQ ID NOS: 211- 216 is as follows: TP53\R175H; TP53\H179W; TP53\R249S; TP53\Y234H; TP53 195T; TP53\R248Q; TP53\R273H; TP53\C176Y; TP53W157F; TP53\H193R; TP53\Y234C;

TP53\K132N; TP53\R273C; TP53\Y163C; TP53\G245D; and TP53\R337H. For other TP53 constructs, 16 recurrent cancer mutations were included in the constructs: C141Y; C176F; H179R; H179W; H193R; I195T; V216M; Y220C; R248L; R248Q; R248W; R249S; R273H; R273L; P278L; and R282G. Sequences for these constructs are set forth in SEQ ID NOS: 223-228. The order of the hotspot mutation 21-mers in SEQ ID NOS: 223-228 is as follows: TP53\C176F; TP53\R273L; TP53\H179R; TP53\R282G; TP53\Y220C; TP53 195T;

TP53\C141Y; TP53\R248L; TP53\R273H; TP53\H179W; TP53\H193R; TP53\R249S;

TP53W216M; TP53\P278L; TP53W248W; and TP53W248Q. Examples of antigenic peptides includes in the constructs are provided in Table 8.

[00820] Table 8. TP53 Antigenic Peptides.

[00821 ] Also in development are an additional set of constructs for cancer-associated proteins that are frequently mutated in certain high impact cancers in additional to those common across all cancers. These diseases include squamous and adenocarcinoma of the lung, colorectal cancer, breast cancer, ovarian cancer, and others.

[00822] Table 9. Biomarker Expression Additional Selected Tumor Targets

% Expression as represented in the BROAD Institute's Tumor Portal dataset

[00823] Out of these constructs several panels can be devised that cover shared mutated epitopes that are characteristic of most major types of cancers. Disease- specific panels being developed from ADXS-HOT constructs could include those in Table 10.

[00824] Table 10. Exemplary Panels.

% Expression as represented in the BROAD Institute's Tumor Portal dataset

[00825] Moreover, recurrent hotspot mutations are identified in more than eleven thousand human tumors, spanning more than 40 cancer types with 470 somatic substitution hotspots in 275 genes identified. See, e.g., Chang et al. (2016) Nat Biotechnol 34(2): 155- 163, herein incorporated by reference in its entirety for all purposes (providing a distribution of tumor types, the breakdown of known and classified hotspots, and the number of hotspots in each of 49 genes with two or more hotspots detected within a cohort). This landscape provides a great opportunity for the development of additional ADXS-HOT constructs to expand the number of "off the shelf treatments to broader cancer patient populations.

Example 2. Colorectal Cancer Immunotherapy Strategy: HOTSPOT Constructs

[00826] Oncogenesis of colorectal cancer (CRC) is driven by the acquisition and accumulation of somatic mutations. APC mutation is involved early in adenoma formation, followed by oncogenic mutation of KRAS that promotes the transition from intermediate adenomas to carcinomas with TP53 inactivation as a late event. Additional mutations can be acquired in PIK3CA, FBXW7, NRAS, and BRAF that contribute as tumor drivers and may confer (or be selected by) resistance to treatments like EGFR inhibition.

[00827] Recently, the advent of large-scale PCR-based sequencing has been used to depict the genomic landscape of CRC and a number of high-frequency mutated genes have been identified as "gene mountains" because of the commonality of shared mutations in these genes. They are comprised by somatic mutations occurring in critical tumor driver genes including APC, KRAS, TP53, FBXW7, PIK3CA, NRAS, and BRAF. Additional lower frequency shared mutation gene clusters have also been identified. However, the vast majority CRC tumors can be characterized by the incorporation one or more of representative mutations in these commonly observed shared tumor drive "gene mountains." Somatic mutations in these key tumor driver genes frequently occur in critical amino acid positions of the peptide that interfere with the function of the molecule in what can be described as mutational "hotspots." These types of shared mutations provide the opportunity to generate an immunotherapy that focuses on the majority of the commonly observed shared mutation epitopes in these tumor driver genes as opposed to neoantigens that are specific to an individual patient. As an example, the BRAF gene can exhibit a very well- characterized tumor- specific antigen associated with the somatic substitution at position 600 of V to E.

[00828] BRAF mutation is also known to be associated with shortened survival in patients with late- stage CRC. As a simple example, the vast majority of mutations that occur in BRAF are at amino acid position 600, represented as V600E, with the only other shared mutation that occurs with a significant frequency being BRAF G469V (or G469A). Thus, covering these three specific shared epitopes with an immunization could generate T cell responses against any somatic mutation that is likely to occur in BRAF in more than 99% of the cases. Patients identified with this high risk mutation could be treated with an

immunotherapy that targets this biomarker in an attempt to eliminate the cells associated with this prognosis.

[00829] A series Lm-LLO constructs are under development that will target the vast majority of tumor- specific epitopes that arise as a consequence of tumor- specific mutations in common tumor driver genes. These products will be based on our Lm-LLO platform, and each one is intended to cover > 99% of the potential mutations that are observed in a particular gene. The presence of key recurrent cancer mutations in tumor driver genes can be diagnosed through specific PCR-based kits or otherwise divulged through DNA or RNA sequencing. These constructs can be given in combinations if the patient has more than one recurrent cancer mutation simply by mixing the individual hot-spot constructs prior to administration. The application of these agents to colorectal cancer could be particularly useful since the commonly mutated genes have been clearly identified and most patients share a mutation in several of these tumor driver genes. These include, for example, mutations in APC, TP53, PIK3CA, KRAS, and BRAF.

[00830] Micro satellite instability (MSI) resulting from defects in DNA mismatch repair, causes a high mutational burden in 10-25% of sporadic (non-Lynch syndrome) CRC, but is also associated with better prognosis and has been response to checkpoint inhibition. Recent data suggest that these patients are more effectively treated with checkpoint inhibitor monotherapy. Therefore, the greatest medical need in CRC is for the 85-90% of patients with micro satellite stable (MSS) CRC.

[00831 ] Recent data suggest that MSS CRC can become sensitive to checkpoint inhibition treatment if the tumor becomes immunologically "hot" or infiltrated with lymphocytes associated with the expression of a TH-1 supportive microenvironment (ASCO 2016, oral presentation, abstract, met inhibition of MSS CRC followed by PD-1). Our Lm-LLO vectors have been found to contribute significant innate immune stimulation supporting TH-1 type T- cell immunity culminating in increased infiltration of T cells into solid tumor

microenvironments along with reduction in the suppressive ability of Tregs and MDSCs (Wallecha et al. (2013) J Immunother 36:468-476; Chen et al. (2014) Cancer Immunol Res 2(9):911-922; and Mkrtichyan et al. (2013) J Immunother Cancer 1: 15. doi: 10.1186/2051- 1426-1-15, each of which is herein incorporated by reference in its entirety for all purposes).

[00832] These effects could contribute collectively in altering the MSS CRC

microenvironment to make it "hot" if a tumor- specific target is presented. In addition, these constructs have been shown to induce epitope spreading. These treatments could be effective as monotherapy when targeting tumor- specific antigens that arise as a consequence of tumor- specific mutations in tumor driver genes, and could also greatly enhance their susceptibility to checkpoint inhibition treatment. In vitro studies of Lm-LLO constructs have demonstrated synergy in vitro and ongoing combination trials have demonstrated that they can be safely combined with checkpoint inhibitors.

[00833] Based on the known expression of recurrent cancer mutations in tumor driver genes for CRC, the development of a CRC specific ADXS-HOT treatment of MSS CRC would be directed against the following targets. The intention is to develop a panel of gene- specific constructs that could either be selected for combination treatment based on a diagnostic screen, or combined in a set combination strategy intended to be given to all MSS CRC patients. The CRC panel would include at a minimum the following tumor driver hotspot targeted constructs ("m" for mutated): mAPC (found in 76% of CRC patients), mTP53 (52% of patients), mRAS {KRASINRAS} (52% of patients {43%/9% of patients, respectively}), mPIK3CA (19% of patients), mBRAF (9% of patients).

[00834] There could be two treatment options developed from these constructs for CRC. One would be personalized for the patient based on expression of biomarkers from a

Nanostring or PCR-based diagnostic, or DNA or RNA sequencing.

[00835] Table 11. Expression of Driver Targets in Different Patients.

[00836] The other option is to give all patients with a common disease type the same combination mixture. For the personalized medicine approach, a combination of constructs from the panel would be assembled into a kit for a patient based on their biomarker testing results, and mixed together on site just prior to treatment. Additional targets can be added to the panel going forward. Several other tumor driver mutation target constructs will also be prepared to target genes that are frequently mutated in other diseases including squamous and adenocarcinoma of the lung, breast cancer and ovarian cancer. Some of these other constructs may also be useful in the CRC panel as they become available.

[00837] For the generalized common disease- specific mixture, all patients with the qualifying disease type would be given the same combination of constructs. For MSS CRC, this combination would include APC, TP53, PIK3CA, and RAS, and (potentially) BRAF. Since the somatic tumor driver mutations are found in CRC include mAPC in 76% of patients, mTP53 in 52% of patients, mRAS {KRAS/NRAS} in 52% of patients, and mPIK3CA in 19% of patients, there is a great likelihood that most patients would express anywhere from 2-4 or 2-5 of these representative mutated tumor driver genes, so multiple driver gene mutations would be targeted.

[00838] The potential also exists to use ADXS-HOT constructs as part of a combination treatment regimen either as several individual hotspot products together or in combination with other therapeutic cancer treatments. Similar to other of our Lm constructs, hotspot treatments can be given in combination or sequentially with other cancer treatments like checkpoint inhibitors, costimulatory agonists, radiation therapy, or personalized neoepitope immunization. The reason for this is that animal models and early data from clinical trials have shown that Lm-LLO immunotherapies have the potential for significant synergy with active immunotherapy agents, such as PD-1 and/or PD-Ll blocking antibodies.

[00839] The combination of an Lm-LLO-based vaccine with anti-PD- 1 antibody leads to increased antigen- specific immune responses and tumor-infiltrating CD8+ T cells, along with a decrease in immune suppressor cells (Tregs and MDSCs). The combination regimen led to synergistic activity, with significant inhibition of tumor growth and prolonged

survival/complete regression of tumors in treated animals. The combination of an Lm-LLO- based vaccine with blocking of PD-1/PD-L1 can lead to overall enhancement of the efficacy of anti-tumor immunotherapy over either agent alone. It was also shown that in vitro infection with Lm results in significant upregulation of surface PD-Ll expression on human monocyte-derived dendritic cells, which suggests the translational capacity of this finding.

[00840] Data presented at the American Association for Cancer Research Annual Meeting in 2016 (Sikora abstract, Advaxis reception data presentation) provided evidence supporting the upregulation of PD- 1 and activation of T cells by an Lm-LLO agent in human head and neck tumors. Data from the study showed increased immune activation within the tumor microenvironment, including upregulation of PD-1 and PD-Ll expression, reduction of Tregs and MDSCs, and infiltration of CD8+ and CD4+ T cells. These observations suggest potentially strong synergy with an anti-PD- 1 antibody (Wolf et al. (2013) J Immunol

190(6):2501-2509, herein incorporated by reference in its entirety for all purposes).

[00841 ] Preclinical data also suggest synergy with immune costimulatory agonists like Ox- 40 and GITR (Mkrtichyan et al. (2013) J Immunother Cancer 1: 15. doi: 10.1186/2051-1426- 1-15, herein incorporated by reference in its entirety for all purposes). Synergy of Lm-LLO vectors with radiation therapy has been demonstrated in preclinical models (Hannan et. al. (2012) Cancer Immunol Immunother 61(12):2227-2238, herein incorporated by reference in its entirety for all purposes) and has also been observed in ongoing veterinary trials in non- resected canine osteosarcoma. Lm-LLO treatments can also be given sequentially with chemotherapies provided there has been sufficient hematopoietic recovery. In addition, research to date shows there is no development of neutralizing antibodies with Lm vectors, so repeated treatments with a single Lm vector or simultaneous or sequential treatment with multiple vectors is possible.

[00842] ADXS-HOT immunotherapies as disclosed herein have the potential to revolutionize the treatment of cancer by providing highly efficacious, targeted attacks on hotspots with little to no impact on healthy cells. Tumor immunotherapies take advantage of the most effective cancer- fighting agents that nature has devised: the host's own immune cells. Successful application of these the ADXS-HOT CRC program in an effective regimen for MSS CRC has the potential to be developed into an effective immunotherapy option for this devastating disease where one currently does not exist.

Example 3. Design of Cancer- Type-Specific HOTSPOT Constructs

[00843] We selected five initial cancer types with recurrent cancer mutations on which to focus preclinical development efforts for ADXS-HOT constructs. These include luminal A breast cancer, colorectal adenocarcinoma, NSCLC adenocarcinoma, squamous cell cancer, and prostate cancer.

Luminal A Breast Cancer

[00844] A total of 11 hotspot mutations across 5 genes were selected for the luminal A breast cancer ADXS-HOT constructs. This panel of hotspot mutations covers 50.6% of all luminal A breast cancer patients.

[00845] Table 12. Exemplary Luminal A Breast Cancer Panel.

[00846] For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed. [00847] Table 13. Exemplary Luminal A Breast Cancer Panel 21-Mers.

[00848] The 21-mer peptides were designed to be fragments of the cancer-associated protein in which the recurrent cancer mutations occurs, including the recurrent cancer mutation and 10 amino acids of flanking sequence on each side. Antigenic peptides were scored by a Kyte and Doolittle hydropathy index with a 21 amino acid window, and peptides scoring above a cutoff of around 1.6 were excluded as they are unlikely to be secretable by Listeria monocytogenes. Constructs will be designed with the peptides in multiple different orders generated by randomization. Each ordering of the peptides will be scored by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, and if any region for a particular ordering of peptides scores above a cutoff of around 1.6, the order of the peptides will be reshuffled until the ordering of peptides resulted in a polypeptide with no regions scoring above the cutoff.

Colorectal Adenocarcinoma

[00849] A total of 17 hotspot mutations across 6 genes were selected for the colorectal adenocarcinoma ADXS-HOT constructs. This panel of hotspot mutations covers 42.8% of all colorectal cancer patients and 58% of micro satellite- stable patients. [00850] Table 14. Exemplary Colorectal Adenocarcinoma Panel.

[00851 ] For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed.

[00852] Table 15. Exemplary Colorectal Adenocarcinoma Panel 21-Mers.

[00853] The 21-mer peptides were designed to be fragments of the cancer-associated protein in which the recurrent cancer mutations occurs, including the recurrent cancer mutation and 10 amino acids of flanking sequence on each side. Antigenic peptides were scored by a Kyte and Doolittle hydropathy index with a 21 amino acid window, and peptides scoring above a cutoff of around 1.6 were excluded as they are unlikely to be secretable by Listeria monocytogenes. Constructs will be designed with the peptides in multiple different orders generated by randomization. Each ordering of the peptides will be scored by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, and if any region for a particular ordering of peptides scores above a cutoff of around 1.6, the order of the peptides will be reshuffled until the ordering of peptides resulted in a polypeptide with no regions scoring above the cutoff.

Lung Adenocarcinoma

[00854] A total of 30 hotspot mutations across 6 genes were selected for the lung adenocarcinoma (NSCLC) ADXS-HOT constructs. This panel of hotspot mutations covers 53.5% of all lung adenocarcinoma patients.

[00855] Table 16. Exemplary Lung Adenocarcinoma Panel.

[00856] For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed.

[00857] Table 17. Exemplary Lung Adenocarcinoma Panel 21-Mers.

[00858] The 21-mer peptides were designed to be fragments of the cancer-associated protein in which the recurrent cancer mutations occurs, including the recurrent cancer mutation and 10 amino acids of flanking sequence on each side. Antigenic peptides were scored by a Kyte and Doolittle hydropathy index with a 21 amino acid window, and peptides scoring above a cutoff of around 1.6 were excluded as they are unlikely to be secretable by Listeria monocytogenes. Constructs will be designed with the peptides in multiple different orders generated by randomization. Each ordering of the peptides will be scored by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, and if any region for a particular ordering of peptides scores above a cutoff of around 1.6, the order of the peptides will be reshuffled until the ordering of peptides resulted in a polypeptide with no regions scoring above the cutoff.

NSCLC Squamous Cell Cancer

[00859] A total of 60 hotspot mutations across 5 genes were selected for the NSCLC squamous cell cancer ADXS-HOT constructs. This panel of hotspot mutations covers 52.3% of all NSCLC squamous cancer patients.

[00860] Table 18. Exemplary NSCLC Squamous Cell Cancer Panel.

[00861 ] For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed.

[00862] Table 19. Exemplary NSCLC Squamous Cell Cancer Panel 21-Mers.

NSCLC Squamous Cell Cancer Panel 21-Mers

Hotspot

Gene Sequence SEQ ID NO

Mutation

TP53 Y163C PGTRVRAMAICKQSQHMTEVV 659

TP53 R175G QSQHMTEVVRGCPHHERCSDS 660

TP53 C242F IHYNYMCNSSFMGGMNRRPIL 661

TP53 R273L NLLGRNSFEVLVCACPGRDRR 662

TP53 H179L MTEVVRRCPHLERCSDSDGLA 663

TP53 H193L SDSDGLAPPQLLIRVEGNLRV 664

TP53 H214R EYLDDRNTFRRSVVVPYEPPE 665

TP53 Y220C NTFRHSVVVPCEPPEVGSDCT 666

TP53 Y234C EVGSDCTTIHCNYMCNSSCMG 667

TP53 G245V NYMCNSSCMGVMNRRPILTII 668

TP53 L111Q KTYQGSYGFRQGFLHSGTAKS 669

TP53 T125P HSGTAKSVTCPYSPALNKMFC 670

TP53 K132R VTCTYSPALNRMFCQLAKTCP 671

TP53 C135W TYSPALNKMFWQLAKTCPVQL 672

TP53 C141W NKMFCQLAKTWPVQLWVDSTP 673

TP53 C176F SQHMTEVVRRFPHHERCSDSD 674

TP53 C176Y SQHMTEVVRRYPHHERCSDSD 675

TP53 H179R MTEVVRRCPHRERCSDSDGLA 676

TP53 H179Y MTEVVRRCPHYERCSDSDGLA 677

TP53 H193R SDSDGLAPPQRLIRVEGNLRV 678

TP53 I195S SDGLAPPQHLSRVEGNLRVEY 679

TP53 Y205C IRVEGNLRVECLDDRNTFRHS 680

TP53 R213G VEYLDDRNTFGHSVVVPYEPP 681

TP53 V216E LDDRNTFRHSEVVPYEPPEVG 682

TP53 Y234S EVGSDCTTIHSNYMCNSSCMG 683

TP53 Y236C GSDCTTIHYNCMCNSSCMGGM 684

TP53 M237I SDCTTIHYNYICNSSCMGGMN 685

TP53 G244C YNYMCNSSCMCGMNRRPILTI 686

TP53 G245S NYMCNSSCMGSMNRRPILTII 687

TP53 R248L CNSSCMGGMNLRPILTIITLE 688

TP53 R248P CNSSCMGGMNPRPILTIITLE 689

TP53 R248Q CNSSCMGGMNQRPILTIITLE 690

TP53 R248W CNSSCMGGMNWRPILTIITLE 691

TP53 R249G NSSCMGGMNRGPILTIITLED 692

TP53 R249S NSSCMGGMNRSPILTIITLED 693

TP53 R249W NSSCMGGMNRWPILTIITLED 694

TP53 G266V TLEDSSGNLLVRNSFEVRVCA 695

TP53 F270I SSGNLLGRNSIEVRVCACPGR 696

TP53 R273C NLLGRNSFEVCVCACPGRDRR 697

TP53 R273H NLLGRNSFEVHVCACPGRDRR 698 NSCLC Squamous Cell Cancer Panel 21-Mers

Hotspot

Gene Sequence SEQ ID NO

Mutation

TP53 R273P NLLGRNSFEVPVCACPGRDRR 699

TP53 R280I FEVRVCACPGIDRRTEEENLR 700

TP53 D281Y EVRVCACPGRYRRTEEENLRK 701

TP53 R282Q VRVCACPGRDQRTEEENLRKK 702

TP53 R282W VRVCACPGRDWRTEEENLRKK 703

[00863] The 21-mer peptides were designed to be fragments of the cancer-associated protein in which the recurrent cancer mutations occurs, including the recurrent cancer mutation and 10 amino acids of flanking sequence on each side. Antigenic peptides were scored by a Kyte and Doolittle hydropathy index with a 21 amino acid window, and peptides scoring above a cutoff of around 1.6 were excluded as they are unlikely to be secretable by Listeria monocytogenes. Constructs will be designed with the peptides in multiple different orders generated by randomization. Each ordering of the peptides will be scored by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, and if any region for a particular ordering of peptides scores above a cutoff of around 1.6, the order of the peptides will be reshuffled until the ordering of peptides resulted in a polypeptide with no regions scoring above the cutoff.

Prostate Cancer

[00864] A total of 21 hotspot mutations across 9 genes were selected for the prostate cancer panel. This panel of hotspot mutations covers 27.6% of all prostate cancer patients.

[00865] Table 20. Exemplary Prostate Cancer Panel.

[00866] For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed.

[00867] Table 21. Exemplary Prostate Cancer Panel 21-Mers.

[00868] The 21-mer peptides were designed to be fragments of the cancer-associated protein in which the recurrent cancer mutations occurs, including the recurrent cancer mutation and 10 amino acids of flanking sequence on each side. Antigenic peptides were scored by a Kyte and Doolittle hydropathy index with a 21 amino acid window, and peptides scoring above a cutoff of around 1.6 were excluded as they are unlikely to be secretable by Listeria monocytogenes. Constructs will be designed with the peptides in multiple different orders generated by randomization. Each ordering of the peptides will be scored by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, and if any region for a particular ordering of peptides scores above a cutoff of around 1.6, the order of the peptides will be reshuffled until the ordering of peptides resulted in a polypeptide with no regions scoring above the cutoff. Example 4. In Silico Methodology for ADXS_HOT Construct Design

[00869] Constructs were designed with peptides having hotspot mutations, heteroclitic peptides from tumor-associated antigen genes, and minigene constructs expressing a heteroclitic peptide. Additional constructs were designed to include these three elements alone or in any combination.

[00870] Hotspot mutations are somatic alterations that are recurrently altered across a large number of cancer patients. Many patients share common mutations in the functional domains of critical tumor driver genes that are the most frequently mutated or that are at least partially responsible for the creating a malignant phenotype. As described elsewhere herein, this mutational "sharing" across patients and tumor types creates an opportunity for the "off the shelf development of treatment constructs that target these common hotspots. Hotspots targets we included range in overall frequency from 16%-80% in an indication. As there are 12,500+ MHC class I HLA types, including target peptides to cover every possible Class I binder would allow us to be able to treat any potential patient that harbors the right

HLA/mutation combination. For example, by providing a 21mer hotspot target peptide having a hotspot mutation and 10 flanking amino acids from the cancer-associated protein on each side, the 21-mer target peptide will cover every 8mer, 9mer, lOmer, or 1 lmer peptide containing the hotspot missense mutation. By including every potential Class I epitope (8mer to 1 lmer), a hotspot panel could in principal cover any potential overlap with any of the known 12,500+ MHC class I molecules. Hotspots targets in ADXS_HOT constructs are designed to generate epitopes to virtually any of the 12,500+ identified HLA Class I alleles and are prioritized agnostic to in silico algorithms.

[00871 ] In addition to the hotspot peptides, heteroclitic sequences (i.e., sequence- optimized peptides) were designed to increase presentation by MHC Class I alleles.

Heteroclitic peptides were derived by altering peptides expressed by tumor-associated antigen genes, as these represent genes that are expressed in tumor tissue, but have minimal expression in normal, healthy tissue. In particular, the heteroclitic peptides were designed from cancer-associated proteins such as cancer testis antigens or oncofetal antigens (i.e., were designed from tumor-associated antigens). Cancer testis antigens (CTAs) are a large family of tumor-associated antigens expressed in human tumors of different histological origin but not in normal tissue, except for male germ cells. In cancer, these developmental antigens can be re-expressed and can serve as a locus of immune activation. Oncofetal antigens (OFAs) are proteins that are typically present only during fetal development but are found in adults with certain kinds of cancer. The tumor-restricted pattern of expression of CTAs and OFAs make them ideal targets for tumor- specific immunotherapy. The combination of multiple hotspot peptides and OFA/CTAs maximizes patient coverage. Most hotspot mutations and OFA/CTA proteins play critical roles in oncogenesis. Targeting both at once can

significantly impair cancer proliferation. Combining hotspot mutations with multi le OFA/CTAs peptides presents multiple high avidity targets in one treatment that are expressed in potentially all patients with the target disease. For example, constructs can be designed so that each patient expresses at least one target mutation. Hotspot peptides that the patient does not express do not elicit any immune response. Adding proprietary sequence-optimized peptides (i.e., heteroclitic peptides) can increase coverage up to 100% of patient population for an indication.

[00872] Heteroclitics were designed to the four most prevalent HLAs in North America from genes with up to 100% expression in a cancer type. The HLA types chosen included A0201, A0301, A2402, and B0702, which have frequencies of 47.8%, 20.6%, 20.6%, and 28.7%, respectively in Caucasian in North America, and frequencies of 16.8%, 23.8%, 8.9%, and 16.0% in African Americans in North America. This increases the odds of at least 1 peptide-MHC combination per patient. Heteroclitic sequences have been shown to be sufficient to prime a T cell response, to overcome central tolerance, and to elicit a successful cross-reactive immune response to the wild-type peptide. Addition of heteroclitic epitopes complements the hotspot mutation peptides in that total patient coverage within a cancer type approaches 100%. We therefore do not need to sequence a patient prior to treatment as we assume that they will express a tumor-associated antigen that we have designed heteroclitic peptides for to cover the most prevalent HLAs (HLA-A0201, HLA-A0301, HLA-A2402, and HLA-B0702).

[00873] Heteroclitic peptides to HLA-A0201 that had immunogenicity information from the literature were selected to be minigene epitopes in several constructs. Heteroclitic peptides to HLA-A2402 were also used in several constructs. Use of the minigene construct approach for the expression of specific MHC class I binding antigenic determinants in addition to the hotspot peptide approach and/or heteroclitic peptide approach disclosed herein allows for the highly efficient delivery of short peptide sequences to the antigen presentation pathway of professional antigen presenting cells (pAPC). A specific advantage of the minigene technology is that it bypasses the requirement for proteasome mediated degradation of larger proteins in order to liberate short peptide sequences that can be bound and presented on MHC class I molecules. This results in a much higher efficiency of peptide-MHC class I antigen presentation on the surface of the pAPC and, therefore, a much higher level of antigen expression for the priming of antigen specific T cell responses.

Hotspots

[00874] To identify recurrent somatic mutations "hotspots," publically available mutation databases were utilized. Databases included TCGA, ICGC, COSMIC, cBioportal, and so forth.

[00875] Mutation data were sub- stratified by disease indication type. In other words, all indication- specific samples were selected for mutation frequency calculations.

[00876] Recurrent somatic mutations included missense substitutions and INDELs resulting in in-frame and frameshift mutations.

[00877] Somatic mutations were rank-ordered within a specific-indication cohort based on frequency of the total number of mutation events observed across all samples.

[00878] Mutations occurring with frequencies below 1% were excluded.

[00879] Recurrent mutations with disease-indication frequencies equal to and above 1% were selected for panel.

[00880] Target peptides were generated for recurrent mutations. For missense

substitutions, the mutant amino acid was flanked by up to 10 wild-type amino acids immediately before and after missense mutation position. For frameshift substitutions, the predicted peptide sequence arising from out-of- frame INDEL substitution was generated from annotation transcript and up to 10 wild-type amino acids are added upstream of frameshift mutation position. For in-frame INDEL substitutions, up to 10 wild-type amino acid sequences before and after INDEL position were joined together.

[00881 ] Specific identifiers were generated for each hotspot target peptide that consist of the gene symbol (HGNC format) and mutation substitution information (HGVS format) separated by an underscore. For example, the substitution of glycine for aspartic acid at position 12 in KRAS would create a specific identifier of KRAS_G12D.

[00882] Target peptides were then subjected to BLAST analysis against the non-redundant protein sequences (nr) database for human. This step ensured that target peptide sequences generate from frameshift mutations did not represent known, wild-type sequences. For missense substations, this step ensured that flanking wild-type amino acids matched the known human reference proteome. Tumor-Associated Antigen Peptides (TAAPs) - Heteroclitic Mutations

[00883] A literature review was done to survey the genomic landscape of indication- specific tumor-associated antigens to generate a short-list of potential TAAs.

[00884] A second literature review was done to determine if short-list TAAs contained known immunogenic peptides that generate CD8+ T lymphocyte response. This approach focused primarily on MHC Class I epitopes consisting of 9 amino acids (9mer) from TAAs. This step identified potential TAAPs in 9mer format that bind to one of four HLAs types (HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, and HLA-B*07:02).

[00885] TAAPs were sequence optimized to enhance binding to MHC Class I molecules (aka heteroclitic peptide). To optimize binding to each HLA, the Peptide MHC Binding Motif and Amino Acid Binding Chart were assessed from the Immune Epitope Database and Analysis Resource (for example: iedb.org/MHCalleleid/143). The preferred amino acids at the anchor positions were inserted into the TAAP sequence (e.g., NUF2 - wild type:

YMMPVNSEV (SEQ ID NO: 725); and NUF2 - heteroclitic: YLMPVNSEV (SEQ ID NO: 726)).

[00886] The binding affinities of sequence-optimized TAAPs and wild-type TAAP sequences were then assessed using one of the following algorithms: NetMHC4.0 Server; NetMHCpan4.0 Server; and mhcflurry vO.2.0.

[00887] Sequence-optimized TAAPs were considered if predicting binding affinity to a specific HLA was equivalent or stronger than the wild-type TAAP sequence.

[00888] Selected sequence-optimized TAAPs were then screened for in vitro binding to specific HLAs using Prolmmune' s REVEAL assay. TAAPs with binding affinity >= 45% of the REVEAL assay's positive control peptide were considered binders.

[00889] Finally, the RNA expression level of TAAPs were measured in a specific- indication in TCGA RNAseqV2 dataset. The percentage of TCGA samples with normalized RNA expression reads greater than 0 were calculated. TAAPs with TCGA expression in a majority of samples were prioritized.

Example 5. Exemplary Protocol for Ligation of Insert into Vector, Transfection into Lm, Sequencing, PCR Confirmation, and Western Blot Confirmation of Lm Expression.

[00890] Synthesized DNA was received from an appropriate vendor (GENEWIZ,

GenScript, or others). The desired insert contained the restriction sites Xhol and Xmal on the flanking ends to allow for molecular manipulations. The vendor ligated the insert into a shuttle vector of their choice (typically a pUC vector). The insert must be cut out of the pUC vector and ligated into the pAdvl34 vector. Once this was completed, expression studies were performed in the LmddA strain.

Restriction Enzyme Digest of pAdvl34 Vector and Insert

[00891 ] Goal: To cut out the proper bands of both the pAdvl34 vector and the insert (in pUC or like shuttle vector) to ensure they had the correct sticky ends so that they could later be ligated together.

[00892] (1) Set up a restriction enzyme digest of 1.2 μg of DNA with the following reaction: DNA (-1.2 μg); Xhol; Xmal; lOx CutSmart buffer (final concentration of lx); and water (if needed). To the vector DNA only, added 1 μϊ_^ of CIP so that self-ligation was prevented.

[00893] (2) Quick mixed and spun of digests and left at 37°C for 2-3 hours.

[00894] (3) Added 6x loading dye to each digest to a final lx concentration.

[00895] (4) Loaded the entire digest on a 1% agarose gel.

[00896] (5) Loaded 10 μΐ_^ of an appropriate DNA ladder so that size may be monitored on the agarose gel.

[00897] (6) Ran the agarose gel at 120V for -45 minutes.

[00898] (7) Visualized and extracted the appropriate sized bands for each DNA sample from the agarose gel.

[00899] (8) Using a gel extraction kit (Zymo Clean Gel DNA Recovery Kit Cat. No. D4002/Zymo Research), purified the extracted bands.

[00900] (9) Measured the concentration of the purified DNA using a Nanodrop (or like, small-volume spectrophotometer.

[00901 ] (10) Loaded 1 μΐ_^ of the final purified DNA (+lx loading dye) on a 1% agarose gel alongside an appropriate DNA ladder.

[00902] (11) Ran the agarose gel at 120V for -45 minutes to ensure single, appropriately sized bands.

Ligation

[00903] Goal: To piece together the insert DNA with the pAdvl34 vector to obtain a fully circular piece of DNA including both insert and pAdvl34.

[00904] (1) Set up the ligation reaction: linearized (Xhol/Xmal cut) and purified pAdvl34 vector (50 ng); cut (Xhol/Xmal) and purified insert (100 ng); 2 μϊ_^ of lOx T4 ligase buffer (final concentration of lx); 1 μϊ_^ of T4 ligase; and water to a total volume of 20 μΐ_^. [00905] (2) Quick mixed and spun of ligation reactions.

[00906] (3) Incubated ligation reaction in a thermocycler with the following parameters: (Step 1) 22°C for 2 hrs; (Step 2) 16°C for 4 hrs; and (Step 3) 4°C overnight.

[00907] (4) Using a PCR purification kit (DNA Clean and Concentrator-5: Cat. No.

D4003/Zymo Research), purified the ligation reaction through a column to rid excess salts and enzymes. Followed the protocol provided in the kit but eluted with a final volume of 10 μϊ_^ of water.

Transformation

[00908] Goal: To allow the ligated plasmid product to gain entry into the E.coli MB2159 strain. Additionally, to allow > doubling of E.coli cells containing the plasmid.

[00909] (1) On ice, gently thawed 1 vial (75 μΐ. aliquot) of E.coli MB2159

electrocompetent cells.

[00910] (2) Added 5 μΐ, of purified ligation reaction to the thawed E. coli MB2159 electrocompetent cells.

[00911 ] (3) Transferred the cell suspension to a 1 mm electroporation cuvette and gently tapped to the bottom.

[00912] (4) Pulsed the cuvette lx with the following settings on an electroporator: V= 1800 V; R= 200 Ω; and C=25 μΡ.

[00913] (5) Immediately added 900 μΐ_^ SOC medium directly to the cuvette (gently pipette up and down a few times to resuspend cells).

[00914] (6) Transferred SOC medium with the electroporated cells to a 14 mL Falcon tube and grew shaking at 200 rpm for 1 hour at 37°C.

[00915] (7) Plated out 200 μΐ_^ of the cell suspension onto an LB plate.

[00916] (8) Incubated the plate at 37°C overnight.

[00917] (9) The following morning, picked colonies.

Clone Confirmation

[00918] Goal: To identify colonies that contain the pDNA.

[00919] (1) Prepared a PCR master mix to assess the number of colonies being examined for pDNA: 10 μΐ. Terra™ PCR Direct Red Dye Premix; 0.5 μΐ. Forward Primer (5' catcgatcactctgga (SEQ ID NO: 727)); 0.5 μί Reverse Primer (5' ctaactccaatgttacttg (SEQ ID NO: 728)); 9 μΐ_^ water; colony (added in step 2) for a total volume of 20 μΐ_^. [00920] (2) For each colony that needed to be assessed, performed the following steps: (a) picked up the colony with a pipette tip and re- streaked it on a fresh LB plate, trying to drag the colony around to obtain isolated colonies the following day; and (b) with some colony still on the tip, tapped/swirled it into the appropriate PCR reaction tube.

[00921 ] (3) Once finished with the colony restreaking, placed the new plate(s) at 37°C overnight.

[00922] (4) Ran the PCR reaction in a thermocycler with the following program settings:

98°C for 3 minutes; 98°C for 30 seconds; 58°C for 30 seconds (repeat for 34 cycles (35 cycles total)); 68°C for 2 minutes; 72°C for 5 minutes; and 4°C until ready to run.

[00923] (5) Loaded 10 μΐ_^ of PCR onto a 1% agarose gel, making sure to include 10 μΐ_^ of

1 kB+ DNA ladder in a separate lane.

[00924] (6) Ran the agarose gel at 120V for -45 minutes.

[00925] (7) Visualized the gel, looking for amplicons of the appropriate size. Those that were correct could be considered viable options for final pDNA constructs.

[00926] (8) The following day, removed the re-streaked plate(s) from the incubator in the morning and stored at 4°C until later that afternoon.

[00927] (9) In the late afternoon, picked 1 colony and inoculated it into 200 μΐ_^ of LB for each correct construct desired.

[00928] (10) The following morning, followed the midi prep directions for the

NucleoBond® Xtra Midi EF kit by Macherey-Nagel.

[00929] (11) Once the pDNA has been concentrated, measured the concentration using a Nanodrop spectrophotometer (or equivalent for small volumes). Ensured that the

concentration was -300 ng^L and the A260/280 ratio was -1.8.

[00930] (12) Sent the pDNA for Sanger sequencing to confirm 100% sequence match to reference.

Transformation into Listeria monocytogenes

[00931 ] Goal: To transform the plasmid DNA into the LmddA strain.

[00932] (1) On ice, gently thawed 1 vial (50 μΐ_^ aliquot) of LmddA electrocompetent cells.

[00933] (2) Added 500 μg of plasmid DNA to the thawed LmddA electrocompetent cells.

[00934] (3) Incubated on ice for 5 minutes.

[00935] (4) Transferred the cell suspension to a 1 mm electroporation cuvette and gently tapped to the bottom. [00936] (5) Pulsed the cuvette lx with the following settings on an electroporator: V = 1000 V; R = 400 Ω; and C = 25 μΡ.

[00937] (6) Immediately added 900 BHI + 0.5M sucrose directly to the cuvette (gently pipetted up and down a few times to resuspend cells).

[00938] (7) Transferred cell suspension to a 14 mL Falcon tube and grew shaking at 200 rpm for 1 hour at 30°C.

[00939] (8) Plated out 100 of the cell suspension onto a BHI + 100 g/mL

streptomycin plate.

[00940] (9) Incubated the plate at 37°C for -24 hours.

[00941 ] (10) Picked two colonies to restreak onto a new BHI + 100 μg/mL streptomycin plate to obtain single colony isolates.

[00942] (11) Incubated the plate at 37°C for -24 hours.

[00943] (12) The following evening, picked one isolated colony from the restruck plate (step 11 plate) and grew it up in 3 mL BHI + 100 μg/mL streptomycin at 30°C, stationary, overnight.

[00944] (13) The following morning, made a glycerol stock from the overnight culture by taking 500 μΐ_^ of culture, adding it to a cryovial, then adding 500 μΐ_^ of 50% glycerol. Mix well.

[00945] (14) Stored the glycerol stocks at -80°C. Quality Control on Glycerol Stocks

[00946] Goal: Ensure the pDNA that was transformed into the LmddA strain aligns to the correct sequence ID.

[00947] (1) Prepared a PCR master mix to assess the number of glycerol stocks being examined for pDNA insertion size: 10 μΐ_^ Terra™ PCR Direct Red Dye Premix; 0.5 μΐ_^

Forward Primer (5' catcgatcactctgga (SEQ ID NO: 727)); 5 μΐ_^ Reverse Primer (5' ctaactccaatgttacttg (SEQ ID NO: 728)); 9 μΐ_^ water; and glycerol stock material to a total volume of 20 μΐ_^.

[00948] (2) For each glycerol stock that needed to be assessed, performed the following steps: (a) with the glycerol stock on dry ice, took a pipette tip and scooped up a bit of material from the stock; and (b) tapped/swirled it into the appropriate PCR reaction tube.

[00949] (3) Ran the PCR reaction in a thermocycler with the following program settings: 98°C for 3 minutes; 98°C for 30 seconds; 58°C for 30 seconds (repeat for 34 cycles (35 cycles total)); 68°C for 2 minutes; 72°C for 5 minutes; and 4°C until ready to run. [00950] (4) Loaded 10 μΐ_^ of PCR onto a 1% agarose gel, making sure to include 10 μΐ_^ of

1 kB+ DNA ladder in a separate lane.

[00951 ] (5) Ran the agarose gel at 120V for -45 minutes.

[00952] (6) Visualized the gel, looking for amplicons of the appropriate size. This was the first step towards verification.

[00953] (7) Using a gel extraction kit (Zymo Clean Gel DNA Recovery Kit Cat. No. D4002/Zymo Research), purified the extracted bands, making sure to elute in water for the final step (otherwise, followed the kit's protocol).

[00954] (8) Sent the extracted DNA for Sanger sequencing to confirm sequence identify. This was the final step towards verification.

Lm Expression Studies

[00955] Goal: To visualize the amount of protein expression by our target antigen(s). This was assessed through the use of a FLAG tag at the 3' end of the construct. Loading was controlled by using an anti-p60 antibody.

[00956] (1) Streaked out the glycerol stock of interest onto a BHI + 100 μg/mL

streptomycin plate. Streaked so that single colonies would be able to be isolated the following day.

[00957] (2) Incubated the plate at 37°C for -24 hours.

[00958] (3) The following evening, inoculated one colony into 3 mL of TSB + 100 μg/mL streptomycin.

[00959] (4) Incubated the culture(s) at 37°C, shaking at 200 rpm, overnight.

[00960] (5) The following morning, added 1 mL of culture to a 1.5 mL tube.

[00961 ] (6) Centrifuged for 5 minutes at 6000g at 4°C.

[00962] (7) While spinning, prepared SDS-PAGE loading buffer: 10% 2-mercaptoethanol in 4xLaemmli buffer (example, 50 μΐ_^ BME into 450 μΐ_^ 4xLaemmli). Also, prechilled transfer tubes to 4°C.

[00963] (8) Transferred the 1 mL supernatant to a new, prechilled 1.5 mL tube. Avoided pellet.

[00964] (9) Prepared SDS-PAGE sample tubes at room temperature: (a) added 90 μΐ_^ of supernatant to a new 1.5 mL tube; (b) added 30 μΐ_^ of prepared Laemmli loading buffer; (c) added a LidLock to cap the tubes (prevents popping open during heating); (d) "boiled" the samples for 10 minutes (98°C worked fine); (e) while boiling, placed the remainder of the supernatant sample at -20°C for long term storage; and (f) let the heated samples sit for a few minutes prior to removing the LidLock (relieves pressure).

[00965] (10) Prepared the SDS-PAGE gels (one gel for anti-Flag and one for anti-p60): (a) removed the comb and tape across the bottom; (b) assembled the gels in the Mini-PROTEAN Tetra Cell (can hold up to 4 gels); (c) prepared running buffer (made lx running buffer by creating a 10% lOxTris-Gly/SDS buffer in water (need about 1.2L to run 4 gels

simultaneously); and (d) filled the inner and outer chamber with running buffer to the designated level.

[00966] (11) Loaded 7 μΐ of standard (ladder) to appropriate well(s).

[00967] (12) Loaded 14 μΐ of sample per well.

[00968] (13) Ran the system for -90 minutes: (a) ran at 90V until the dye front was into the gel (-10 minutes); and (b) increased to 120V until achieved appropriate separation (-1.5 hours).

[00969] (14) Once completed, cracked open the cassettes with the cassette opening lever by aligning with arrows on the cassette.

[00970] (15) Proceeded with transfer.

[00971 ] (16) Opened the appropriate number of Trans-Blot Turbo Midi Transfer Packs (PVDF) - one pack will transfer 2 of the above used gels ("mini gels").

[00972] (17) Placed the membrane and bottom stack into the transfer base of the Trans- Blot Turbo transfer system - used a roller to remove bubbles.

[00973] (18) Gently removed any excess acrylamide (very bottom of gel and top lanes) with a sharp instrument.

[00974] (19) Removed the trimmed gel(s) from the cassette and placed directly on top of the PVDF membrane that was sitting in the transfer base - used the roller to remove bubbles.

[00975] (20) Placed the top stack of prewet papers from the Trans-Blot Turbo Midi Transfer Pack on top of the gel(s) - used the roller to remove bubbles.

[00976] (21) Gently but firmly placed the top of the Trans-Blot Turbo transfer unit on top of the stack; then, while pushing down, locked the cassette lid into place.

[00977] (22) Placed the cassette into the Trans-Blot Turbo Transfer unit.

[00978] (23) Began the transfer.

[00979] (24) While the transfer was running, prepared 75mL of working iBind Solution for the next section (a 72 niL volume covers 4 mini gels - each iBind Flex machine can process 2 mini gels, so processing 4 gels needs 2 machines): added 59.25 mL water; added 750 μΐ_^ lOOx Additive; added 15 mL 5xBuffer; and kept at 4°C until use. [00980] (25) While the transfer was running, prepared antibody solutions (volumes shown per blot): (a) primary: added 2 mL of prepared iBind Flex solution to a 15 mL conical +

1: 1000 primary (2 μί)*; and (b) secondary: added 2 mL of prepared iBind Flex solution to a

15 mL conical + 1: 1000 secondary (2 μί)* (^dilutions may change based on antibody used - shown here are for a-Flag and a-p60 blots (all at 1:1000)).

[00981 ] (26) Once completed, opened the transfer cassette(s) and removed the

membrane(s) - placed into water, gently rocking until ready for blotting.

[00982] (27) Set up the correct reagent trays in the iBind Flex machine(s).

[00983] (28) Added an iBind Flex card to the machine(s).

[00984] (29) Pre-wet the card with 10 mL of prepared iBind Flex solution.

[00985] (30) Added an additional 1 mL of prepared iBind Flex solution to the area where each mini blot will lay immediately prior to placing membrane on the card.

[00986] (31) Using guide grids, placed blot(s) protein-side down in the appropriate area with low molecular weight closest to the stack.

[00987] (32) Rolled the blots to remove any air bubbles.

[00988] (33) Closed and pushed down the latch on the lid.

[00989] (34) Added the reagents to the reagent tray (volumes here are for mini gels): (a) Lane 1: 2 mL per blot of primary antibody (previously prepared in iBind Flex solution); (b) Lane 2: 2 mL per blot of prepared iBind Flex solution; (c) Lane 3: 2 mL per blot of secondary antibody (previously prepared in iBind Flex solution); and (d) Lane 4: 6 mL per blot of prepared iBind Flex solution.

[00990] (35) Closed the reagent cover and recorded the start time for the incubation.

[00991 ] (36) Let incubation proceed for at least 3 hours but up to O/N.

[00992] (37) Once time had passed, opened the lid to the reagent reservoir to ensure all liquid was gone.

[00993] (38) Removed the membranes and rinsed/stored in water.

[00994] (39) Discarded the iBind Flex card.

[00995] (40) Mixed equal parts of Super Signal West Dura Stable Peroxide Solution and Luminol/Enhancer Solution in a tube.

[00996] (41) Turned on the GE AI600. While it was warming up, added the developing reagent to the first blot: (a) decanted all water from the membrane; (b) added the membrane to the internal compartment of a plastic sheet protector; (c) added 1 mL of prepared developing solution directly to the membrane and placed the top of the sheet protector down on top of the solution/blot; eliminated any bubbles; and (d) let the membrane incubate for ~1- 5 minutes.

[00997] (42) Imaged the blot.

[00998] (43) Rinsed and saved blots at 4°C in water or discarded if not further needed.

Example 6. Proof of Concept: Therapeutic Efficacy of L n-KRAS_G12D Hotspot Constructs in a CT26 Challenge Studies.

[00999] This study determined the therapeutic efficacy of the Lm-KRAS G12D hotspot constructs in suppressing CT26 tumor growth. The KRAS_G12D mutation targeted here in the CT26 mouse model is identical to the human KRAS_G12D hotspot identified in many human tumor indications. Additionally, this study assessed the efficacy of the KRAS_G12D construct delivered as a non-minigene or delivered as a minigene construct. The

KRAS_G12D K^d and D^d constructs were designed using the hotspot heteroclitic design strategy used for predicting immunogenic 9mers that bind specific MHC alleles (MHC-I K^d and MHC-I D^d). However, these constructs were not given a heteroclitic mutation as the target naturally has a HOT spot mutation.

Treatment Schedule

[001000] Lm-KRAS_G12D hotspot vaccinations began as described in Table 1 and 2, followed with two boosts at one-week intervals. The details of the implantation and dosing schedules are given in Table 22.

[001001 ] Table 22. Treatments Schedule.

Experimental Details

[001002] Tumor Cell Line Expansion. CT26 cell line (mouse colon carcinoma cell line) was cultured in RPMI with 10% FBS. [001003] Tumor Inoculation. On Day 0, (14JUN17) CT26 cells will be trypsinized with 0.25% trypsin (IX) and washed twice with media at the appropriate concentration in PBS (5xl0⁵ cells/mouse). CT26 cells were implanted subcutaneously in the right flank of each mouse.

[001004] Treatment. Vaccine preparation was as follows: (a) thawed 1 vial form -80°C in 37°C water bath; (b) spun at 14,000 rpm for 2 min and discarded supernatant; (c) washed 2 times with 1 mL PBS and discarded PBS; and (d) re-suspended to a final concentration of 5xl0⁸ CFU/mL. Dosing started 4 days after tumor implantation.

[001005] Table 23. Construct Sequences.

Results and Conclusion

[001006] The KRAS_G12D hotspot mutation was able to significantly control tumor growth in the murine CT26 colorectal cancer model. See Figure 1. These data provided a strong proof of concept for targeting shared hotspot mutations. Additionally, the

KRAS_G12D hotspot used here was identical to the KRAS_G12D hotspot mutation identified in various human cancer indications.

[001007] Furthermore, the KRAS_G12D hotspot construct was able to effectively control tumor growth whether it was delivered as a 21mer or as a minigene. See Figure 1. The efficacy of the minigene constructs strongly supported the design strategy for predicting and selecting minigene constructs based on in silico predictive MHC binding algorithms. CT26 Challenge Study with Lm NSCLC HOT EV02 ΕΑΑΑΚΛ20 (B)

[001008] A similar experiment was performed to determine the therapeutic efficacy of the Lm NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) hotspot construct (fusion polypeptide insert sequence set forth in SEQ I DNO: 895) in suppressing CT26 tumor growth. It is known that KRAS mutations are frequent drivers of the linear and uniform evolution of spontaneous human cancers. The same KRAS gene is mutated in the CT26 colorectal mouse model (KRAS_G12D). Therefore, given that the Lm NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) hotspot construct contains the same KRAS_G12D mutational target as our KRAS_G12D_21mer, we hypothesized that the Lm NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) hotspot construct would suppress tumor growth in a similar fashion. Naive BALB/c mice were implanted with 300,000 CT26 colorectal tumor cells in the flank. Four days after tumor implantation, mice were immunized with all Lm-constructs (LmddA-H (Control), Lm NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) hotspot construct (indicated as HOT-Lung in Figure 49), and HOT-Lm KRAS_G12D construct), followed with a boost one week after initial immunization. The data shown in Figure 49 show the group tumor measurements. The data shown in Figure 49 clearly demonstrate that the Lm NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) hotspot construct can significantly suppress tumor progression compared to the control groups (Naive and LmddA- 274). There was not significant difference between the Lm NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) hotspot construct and the KRAS_G12D_21mer construct. The results demonstrate that the Lm NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) hotspot construct can significantly control tumor growth equivalent to the KRAS_G12D_21mer construct, highlighting its antitumor properties.

T Cell Data

[001009] BALB/c mice (n=4/group) were immunized at days 0 and 7 with the Lm-HOT KRAS_G12D-21mer construct, and spleens were harvested one week post final

immunization (day 14) to assess the cellular immune responses. Figures 39A and 39B demonstrate that the Lm-HOT KRAS_G12D therapy can induce antigen- specific T cell responses in non-tumor bearing mice. Lm-HOT therapy augmented effector T cell function over controls, as evidenced by increased levels of splenic KRAS-specific IFNg ELISpot responses. The induction of a TH1 responses is shown by the number of KRAS_G12D- specific IFNg spot-forming colonies (SFC) per million splenocytes determined by IFNg ELISpot assay. Splenocytes were stimulated for 18 hours using KRAS_G12D pooled peptides (15-mers overlapping by 9 amino acids; 2.5 μg/mL final concentration) spanning the entire KRAS G12D 21mer antigen target.

[001010] Figures 40A-40D show that Lm-HOT KRAS-G12D therapy can alter the tumor immune microenvironment. Naive BALB/c mice were implanted with 300,000 CT26 colorectal tumor cells in the flank. Four days after tumor implantation, mice were immunized with the HOT-Lm KRAS_G12D construct, followed with a boost one week after initial immunization. TILs from tumors of treated CT26 mice were harvested 14 days after tumor implantation. Vaccine therapy altered the tumor- infiltrating lymphocyte (TIL) composition in the CT26 colorectal tumor model. Vaccine therapy showed significantly increased infiltration of total CD45 population, the percentage of tumor-infiltrating CD8 T cells, and significantly reduced the percentage of tumor-infiltrating CD4 Tregs. Furthermore, Figure 40C shows that Lm-HOT constructs can drive KRAS-specific tumor-infiltrating T cell responses. Consequently, Lm-HOT therapy significantly suppressed tumor growth in the CT26 colorectal tumor model.

Example 7. Proof of Concept: Efficacy of Lm Heteroclitic WT1 Minigene Fusion Protein Constructs.

[00101 1 ] The peptide minigene expression system was used to assess unique heteroclitic minigenes targeting the Wilms tumor protein. This expression system was designed to facilitate cloning of panels of recombinant proteins containing distinct peptide moieties at the carboxy- terminus. This is accomplished by a simple PCR reaction utilizing a sequence encoding one of the Signal Sequence (SS)-Ubiquitin (Ub)-Antigenic Peptide constructs as a template. By using a primer that extends into the carboxy- terminal region of the Ub sequence and introducing codons for the desired peptide sequence at the 3' end of the primer, a new SS-Ub-Peptide sequence can be generated in a single PCR reaction. The 5' primer encoding the bacterial promoter and first few nucleotides of the signal sequence (e.g., LLO or ActAi-ioo secretion signal) can be the same for all constructs. The constructs generated using this strategy are represented schematically in Figures 2A and 2B.

[001012] One of the advantages of the minigene system is that it will be possible to load cells with multiple peptides using a single Listeria vector construct. Multiple peptides can be introduce into recombinant attenuated Listeria (e.g., Lmdda) using a modification of the single peptide expression system described above. A chimeric protein encoding multiple distinct peptides from sequential SS-Ub-Peptide sequences can be encoded in one insert. See, e.g., Figure 2B. Shine-Dalgarno ribosome binding sites can be introduced before each SS-Ub-Peptide coding sequence to enable separate translation of each of the peptide constructs. Figure 2B demonstrates a schematic representation of a construct designed to express three separate peptide antigens from one strain of recombinant Listeria.

[001013] To assess the expression of tLLO-WTl -heteroclitic fusion proteins by ADXS Lmdda Listeria constructs, unique heteroclitic minigenes targeting the Wilms Tumor 1 protein were generated in the pAdvl34 plasmid and transformed into Lmdda. The pAdvl34 tLLO plasmid encodes the N-terminal LLO fragment set forth in SEQ ID NO: 336. The tLLO-WTl heteroclitic fusion proteins comprise from N-terminal end to C-terminal end: the N-terminal LLO fragment set forth in SEQ ID NO: 336, followed by the FLAG tag set forth in SEQ ID NO: 762, followed by the ubiquitin sequence set forth in SEQ ID NO: 747, followed by a heteroclitic WT1 9-mer listed in Table 24, below.

[001014] Table 24. Heteroclitic WT1 Peptides.

[001015] The combined WT1- tLLO-FLAG-Ub-heteroclitic phenylalanine construct (construct #1) is set forth in SEQ ID NO: 742 (tLLO = 1-441; FLAG = 442-462; ubiquitin = 463-537; heteroclitic phenylalanine peptide = 538-546). One additional construct (Lmdda- WT1- tLLO-Pl-P2-P3 -FLAG-UB-heteroclitic tyrosine minigene construct) was generated that targets 3 WT1 peptides (P1-P2-P3; SEQ ID NOS: 743

(RSDELVRHHNMHQRNMTKL), 744 (PGCNKRYFKLS HLQMHS RKHTG) , and 745 (SGQAYMFPNAPYLPSCLES), respectively). Each 'P' peptide is comprised of 19-22 amino acids, sufficient in length to provide additional CD4 T helper epitopes. The three peptides are separated by linkers. The P3 peptide contains a heteroclitic mutation converting SGQARMFPNAPYLPSCLES (SEQ ID NO: 746) to SGQAYMFPNAPYLPSCLES (SEQ ID NO: 745). In addition to the heteroclitic P3 peptide, the Lmdda-ΨΎΙ- tLLO-Pl-P2-P3- FLAG-UB -heteroclitic tyrosine minigene construct contains a ubiquitin-YMFPNAPYL (SEQ ID NO: 741) moiety at the C-terminus. The combined WT1- tLLO-Pl-P2-P3-FLAG-UB- heteroclitic tyrosine minigene construct is set forth in SEQ ID NO: 748 (tLLO = 1-441; wild- type WT1 peptide vl4— WT1-427 long = 442-460; wild type WT1 peptide vl5— WT1-331 long = 466-487; heteroclitic WT1 peptide vlB— WTl-122Al-long = 493-511; FLAG = 512- 532; ubiquitin = 533-607; heteroclitic tyrosine peptide = 608-616). Each individual Lmdda construct was assayed by Western blot for tLLO-fusion protein expression of the unique heteroclitic WT1 minigene product.

[001016] Construct #1 (Lmdda-WTl- tLLO-FLAG-Ub-heteroclitic phenylalanine minigene construct) and the Lmdda-WTl- tLLO-Pl-P2-P3-FLAG-UB -heteroclitic tyrosine minigene construct were assayed by Western blot for tLLO-fusion protein expression of the unique heteroclitic WT1 minigene product. Single colonies from plates containing Lm WT1 minigene constructs were used to inoculate an overnight culture in 6 mL of Brain Heart Infusion (BHI) broth in a dry shaking incubator at 37°C. The following day, 1: 10 dilution of the original overnight culture were re-suspended in 9 mL of fresh BHI and grown in the dry shaking incubator at 37°C until reaching an OD6oo=0.6. Cells were pelleted by 2- minute centrifugation at 13000 RPM. Sample supernatant were collected and run on SDS-PAGE. Samples were prepared by diluting 75 μΐ_^ of sample with 25 μΐ_^ of 4X LDS Sample Buffer (Cat#l 61-0747), boiled at 98°C for 10 minutes, placed on ice, and then centrifuged at max speed for 10 minutes at 4°C. 13 μΐ_^ of the sample was run on 4-15% precast protein gel (BioRad Cat#4561086). Protein gels were transferred using the Trans-Blot Turbo transfer apparatus (Cat#170-4155) and PVDF Midi transfer packs (Bio-Rad #170-4157). Blots were incubated with anti-FLAG monoclonal Antibody (Sigma F1804) or anti-LLO (Abeam ab200538) as primary and goat anti- mouse IgG-HRP conjugated (sc2005) as a secondary antibody. The blots were then incubated on iBind Flex (Invitrogen cat#1772866), washed, and then developed by Super Signal West Dura Extended Duration Substrate (ThermoFisher #34076); the images were developed on the Amersham Imager 600 (GE).

[001017] Expression and secretion of the unique tLLO-WTl -heteroclitic minigene fusion proteins was confirmed. Anti-Flag tag antibody Western blots of culture supernatant from construct #1 and the Lmdifa-WT1-Pl-P2-P3-YMFPNAPYL (SEQ ID NO: 741) Heteroclitic tyrosine + minigene construct are shown in Figures 3A and 3B, respectively. We were able to detect a protein band corresponding to the correct size and identity for each individual tLLO-WTl -heteroclitic minigene fusion protein. These data demonstrate the ability for heteroclitic peptides targeting multiple peptide fragments within the WT1 protein to be generated using the pAdvl34 plasmid and Lmdda Listeria strain. [001018] For constructs #2-9 in Table 24, each individual Lmdda construct was assa; colony PCR in order to detect plasmid DNA from each unique tLLO-fusion protein containing heteroclitic WT1 minigenes.

[001019] Table 25. Materials.

Procedure

[001020] The general colony PCR procedure that was used is as follows. Obtained plate with large colonies (generally, plates grown at 37°C for 24 hours work well for this procedure). Created master mix for PCR as follows.

Reagent Volume (μΐ.)

PCR water 16

DreamTaq lOx Buffer 2

Forward primer 0.5

Reverse primer 0.5

lOmM dNTPs 0.5

Dream Taq Polymerase 0.5

= 20

[001021 ] Aliquoted 20

of master mix into each PCR tube. Using a pipette tip (10-20 volume works best), scooped up a generous volume from one colony. Tapped the pipette tip into the PCR tube several times and swirled around to dislodge the bacteria. Ran the PCR reaction(s) in a thermocycler using the following PCR program.

Step Temp (°C) Time

1 94 2 minutes

2 94 30 seconds

3 55* 30 seconds

4 72 1 minute

repeat steps 2-4 an additional 29x

72 5 minutes

4 ∞ [001022] Removed PCR tubes from the thermocycler, added 4 μΐ_^ of 6X loading dye. Ran 10 μΐ_^ of each PCR reaction on a 1% agarose gel, alongside 10 μΐ_^ of the 1 kb+ DNA ladder. The primers added an additional 163 base pairs to the product. The forward primer bound 70 base pairs upstream of the 3' end of tLLO (includes the Xhol site). The reverse primer bound 93 base pairs downstream of the stop sites (includes the Xmal site).

[001023] Representative colony PCR results showing Lmdda strains containing pAdvl34 WTl-heteroclitic plasmids #2-9 from Table 24 are shown in Figure 4. We were able to detect a DNA band corresponding to the correct size and identity for each individual tLLO- WTl-heteroclitic minigene plasmid. These data demonstrate the ability for heteroclitic peptides targeting multiple peptide fragments within the WTl protein to be generated using the pAdvl34 plasmid and Lmdda Listeria strain, which indicates that such constructs can be used as therapeutic compositions to target WTl to create or enhance immune responses against WTl and WTl -expressing cancers and tumors.

[001024] To assess the generation of WTl -specific T cell responses in AAD mice using two different WTl constructs, ELISpots was performed to determine the desired vaccine-induced Ag-specific responses. The AAD mice (B6.Cg-Tg(HLA-A/H2-D)2Enge/J; The Jackson Laboratory - Stock No.: 004191) are transgenic mice that express an interspecies hybrid class I MHC gene, AAD, which contains the alpha- 1 and alpha-2 domains of the human HLA- A2.1 gene and the alpha-3 transmembrane and cytoplasmic domains of the mouse H- 2D^d gene, under the direction of the human HLA-A2.1 promoter. This transgenic strain enables the modeling of human T cell immune responses to HLA-A2 presented antigens, and may be useful in testing of vaccines for infectious diseases or cancer therapy. The immunization schedule is provided in Table 26. The mice that were used were female C57BL/6 mice aged 8-10 weeks.

[001025] Table 26. Immunization Schedule.

[001026] Vaccine Preparations. Briefly, each glycerol stock was streaked over required nutrient plate and grown overnight. A single colony was used for growth in an overnight culture of Brain Heart Infusion (BHI) broth under antibiotic selection. Overnight cultures were used at a 1 : 10 (vol/vol) dilution to inoculate fresh BHI broth. Bacteria were incubated in an orbital shaker for 1-3 hours at 37°C to mid-log phase, an OD of -0.6-0.7. Mice were infected with lxlO⁹ CFU Lm by i.p. inoculation in PBS.

[001027] ELISPOT. On day 18, mice were sacrificed by C0₂ asphyxiation in accordance with IACUC protocols, spleens were harvested, and splenocyte single-cell suspensions were plated on 96-well plates and stimulated with either the wild-type or heteroclitic peptide (Table 27). Similar experiments are done with other wild-type and heteroclitic peptide pairs (Table 28). An ELISPOT assay was used to enumerate antigen specific CD8 T Cells responding to either the wild-type or heteroclitic peptides. The full ELISPOT protocol was as per CTL immunospot (www.immunospot.com/resources/protocols/ELISPOT- protocol.htm).

[001028] Table 27. Wild-Type and Heteroclitic WT1 Peptides.

[001029] Table 28. Wild-Type and Heteroclitic WT1 Peptides.

[001030] A generic ELISPOT protocol is provided below.

[001031 ] DAY 0 (Sterile Conditions). Prepared Capture Solution by diluting the Capture Antibody according to specific protocol. Many cytokines benefit from pre- wetting the PVDF membrane with 70% ethanol for 30 sec and washing with 150

of PBS three times before adding 80 of the Capture Solution into each well. Incubated plate overnight at 4°C in a humidified chamber.

[001032] DAY 1 (Sterile Conditions). Prepared CTL-Test™ Medium by adding 1% fresh L-glutamine. Prepared antigen/mitogen solutions at 2X final concentration in CTL-Test™ Medium. Decanted plate with coating antibody from Day 0 and washed one time with 150 μί ΡΒ8. Plated antigen/mitogen solutions, 100 After thawing PBMC or isolating white blood cells with density gradient, adjusted PBMC to desired concentration in CTL- Test™ Medium, e.g., 3 million/mL corresponding to 300,000 cells/well (however, cell numbers can be adjusted according to expected spot counts since 100,000-800,000 cells/well will provide linear results). While processing PBMC and until plating, kept cells at 37°C in humidified incubator, 5-9% C0₂. Plated PBMC, 100 μΐ/ννεΐΐ using large orifice tips. Once completed, gently tapped the sides of the plate and immediately placed into a 37°C humidified incubator, 5-9% CO2. Incubated for 24-72 hours depending on your cytokine. Did not stack plates. Avoided shaking plates by carefully opening and shutting incubator door. Did not touch plates during incubation.

[001033] DAY2. Prepared Wash Solutions for the day: PBS, distilled water and Tween- PBS. Prepared Detection Solution by diluting Detection Antibody according to

specific protocol. Washed plate two times with PBS and then two times with 0.05% Tween- PBS, 200 μΐ/ννεΐΐ each time. Added 80 μΐ/ννεΐΐ Detection Solution. Incubated at RT, 2h. Prepared Tertiary Solution by diluting the Tertiary Antibody according to specific protocol. Washed plate three times with 0.05% Tween-PBS, 200 μίΛνεΙΙ. Added 80 μΙ,ΛνεΙΙ of Strep- AP Solution. Incubated at RT, 30 min. Prepared Developer Solution according to your specific protocol. Washed plate two times with 0.05% Tween-PBS, and then two times with distilled water, 200 μΐ/ννεΐΐ each time. Add Developer Solution, 80 μΐ/ννεΐΐ. Incubated at RT, 10-20 min. Stopp8d reaction by gently rinsing membrane with tap water, decanted, and repeated three times. Removed protective underdrain of the plate and rinsed back of plate with tap water. Air dried plate for 2 hours face-down in running hood or on paper towels for 24 hours on bench top. Scanned and counted plate.

[001034] HLA-A2 transgenic B6 mice were vaccinated as described, and splenocytes were stimulated ex vivo with specific WT1 peptides (RMFPNAPYL (SEQ ID NO: 749),

FMFPNAPYL (SEQ ID NO 732)) and analyzed by IFNg ELISpot assay. Heteroclitic vaccination (WTl-F minigene: FMFPNAPYL; SEQ ID NO: 732) induced Ag-specific T cell responses in immunized HLA2 transgenic mice. See Figure 5 and Figure 7B. In addition, heteroclitic vaccination elicited T cell responses that cross-reacted with the native WT1 tumor antigen (RMFPNAPYL; SEQ ID NO: 749). See Figure 5 and Figure 7A. The data demonstrated that vaccination with the WTl-F heteroclitic minigene vaccine can elicit T cells that are cross-reactive with the WTl-native tumor antigen (RMFPNAPYL; SEQ ID NO: 749). Overall, the data demonstrated that the heteroclitic minigene vaccine can elicit T cells that cross-react with the native tumor antigen. [001035] HLA-A2 transgenic B6 mice were vaccinated as described and splenocytes were harvested. The ability of T cells to produce IFNg in response to vaccine- specific

YMFPNAPYL peptide (SEQ ID NO: 741) or native WT1 peptide (RMFPNAPYL; SEQ ID NO: 749) was determined by IFNg ELISpot assay. Heteroclitic vaccination (WTl-AHl-Tyr minigene: YMFPNAPYL; SEQ ID NO: 741) induced Ag-specific T cell responses in immunized HLA2 transgenic mice. See Figure 6 and Figure 8B. In addition, heteroclitic vaccination elicited T cell responses that cross-react with the native WT1 tumor antigen (RMFPAPYL; SEQ ID NO: 749). See Figure 6 and Figure 8A.

Example 8. Proof of Concept: Therapeutic Efficacy of Lm MC38 Constructs in the MC38-Based Transplantable Colorectal Tumor Model Using C57BL/6 Female Mice.

[001036] This study investigated therapeutic efficacy of various MC38 constructs (non- minigenes and minigenes) in controlling established MC38 tumors. MC38 tumors are a tumor model using the MC38 cell line, which was derived from a mouse colon

adenocarcinoma. Tumor volume and survival were observed. Two mutations present in all MC38 tumor models were tested in 21mer non- minigene form and in minigene form. One mutation was in the mouse Adpgk gene (ADP-dependent glucokinase; UniProt Accession No. Q8VDL4), and one mutation was in the mouse Dpagtl gene (UDP-N-acetylglucosamine— dolichyl-phosphate N-acetylglucosaminephosphotransferase; UniProt Accession No.

P42867). These mutations were identified from Yadav et al. (2014) Nature 515(7528):572- 576, herein incorporated by reference in its entirety for all purposes.

Treatment Schedule

[001037] Once tumors were palpable (Day 8 - 15JUN17) mice were dosed once per week with various (Lm) MC38 constructs intraperitoneally (IP), for 3 consecutive weeks

(indefinitely).

[001038] Table 29. Treatments Schedule

Experimental Details

[001039] Tumor Cell Line Expansion. MC38 cells were split at 1:5 dilution and grown in a medium (IMDM Complete medium (c-RPMI); FBS at 10% (50 mL); Glutamax at 5 mL). On Day 0 (07JUN17), MC38 cells were cultured in IMDM and reached mid- to late-log phase of growth (~ 50% confluency). The cells were trypsinized with 0.25% trypsin (IX) for 2 minutes at RT. Trypsin was inhibited with 3 times volume complete media, and centrifuged at 1200rpm for 5 minutes. The pellet was re-suspended in media (without antibiotics) and counted with MoxiFlow by BC.

[001040] Tumor Inoculation. Cells were counted and re-suspended at a concentration of 2xl0⁵ cells/200uL/mouse. Tumor cells were injected subcutaneously in the right flank of each mouse.

[001041 ] Treatment. Vaccine preparation was as follows: (a) thawed 1 vial form -80°C in 37°C water bath; (b) spun at 14,000 rpm for 2 min and discarded supernatant; (c) washed 2 times with 1 mL PBS and discarded PBS; and (d) re-suspended to a final concentration of 5xl0⁸ CFU/mL.

[001042] Table 30. Construct Sequences.

Results and Conclusions

[001043] When mice were immunized with either Adpgk or Dpagtl as minigenes, MC38 tumor volume was significantly reduced compared to LmddA-274 empty vector control and compared to the combined Adpgk and Dpagtl mutations targeted in a non- minigene form. See Figure 9. Example 9. Proof of Concept: Therapeutic Efficacy of Non-Minigene and Minigene L n-AHl Constructs in a CT26 Challenge Study.

[001044] This study examined the therapeutic efficacy of Lm-AHl constructs, including minigene and non-minigene constructs, in suppressing CT26 tumor growth. The constructs express fusion polypeptides comprising wild type peptides from gp70. AHl refers to a bioactive nanomeric peptide derived from envelope glycoprotein 70 (gp70) of endogenous murine leukemia virus (MuLV), and is expressed by BALB/c-derived CT26 colorectal carcinomas. See, e.g., Scrimieri et al. (2013) Oncoimmunology 2(l l):e26889, herein incorporated by reference in its entirety for all purposes.

Treatment Schedule

[001045] Lm-AHl vaccination began 7-9 days after tumor implantation, followed with two boosts at one- week intervals. The details of the implantation and dosing schedules are given in Table 31.

[001046] Table 31. Treatments Schedule.

Experimental Details

[001047] Tumor Cell Line Expansion. CT26 cell line were cultured in RPMI with 10% FBS.

[001048] Tumor Inoculation. On Day 0, (14JUN17) CT26 cells will be trypsinized with 0.25% trypsin (IX) and washed twice with media at the appropriate concentration in PBS (5xl0⁵ cells/mouse). CT26 cells were implanted subcutaneously in the right flank of each mouse. [001049] Treatment. Vaccine preparation was as follows: (a) thawed 1 vial form -80°C in 37°C water bath; (b) spun at 14,000 rpm for 2 min and discarded supernatant; (c) washed 2 times with 1 mL PBS and discarded PBS; and (d) re-suspended to a final concentration of 5xl0⁸ CFU/mL.

[001050] Table 32. Construct Sequences.

Results and Conclusions

[001051 ] Here we demonstrated that the most effective route of administration for AH1 vaccines in the CT26 model is via IV dosing. Additionally, minigene constructs perform slightly better than the non-minigene counterparts, although this result is not statistically different. See Figures 10A and 10B. These data support efficacy of Lm constructs in both minigene and non-minigene form.

Example 10. Proof of Concept: Therapeutic Efficacy of Heteroclitic Lm-AHl

Constructs in a CT26 Challenge Study.

[001052] This study examined if Lm AHl-HC heteroclitic minigene vaccine could control or suppress CT26 tumor growth.

Treatment Schedule

[001053] Heteroclitic AHl-HC vaccination began as described in Table 33, followed with two boosts at one-week intervals with the recommended vaccine. [001054] Table 33. Treatments Schedule.

Experimental Details

[001055] Vaccine Dosing Details. AH1-HC refers to mice primed and boosted with heteroclitic AH1-HC vaccine.

[001056] Tumor Cell Line Expansion. CT26 cell line were cultured in RPMI with 10% FBS.

[001057] Tumor Inoculation. On Day 0, (14JUN17) CT26 cells will be trypsinized with 0.25% trypsin (IX) and washed twice with media at the appropriate concentration in PBS (3xl0⁵ cells/mouse). CT26 cells were implanted subcutaneously in the right flank of each mouse.

[001058] Treatment. Vaccine preparation was as follows: (a) thawed 1 vial form -80°C in 37°C water bath; (b) spun at 14,000 rpm for 2 min and discarded supernatant; (c) washed 2 times with 1 mL PBS and discarded PBS; and (d) re-suspended to a final concentration of 5xl0⁸ CFU/mL. Vaccine dosing began 3-4 days after tumor implantation.

[001059] Table 34. Construct Sequences.

Results and Conclusions

[001060] The Lm-AHl HC construct was able to significantly control tumor growth in the murine CT26 colorectal cancer model. See Figure 11. Example 11. Design and Expression of Cancer- Type-Specific HOTSPOT Constructs with Heteroclitic Peptides and Minigenes

[001061 ] We selected cancer types with recurrent cancer mutations on which to focus preclinical development efforts for ADXS-HOT constructs. These included no n- small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer (e.g., ER+ breast cancer), uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer (e.g., MSS colorectal cancer), head and neck cancer, and DNA mismatch repair deficient cancers.

Exemplary amino acid sequences for the constructs are provided throughout the Example. Exemplary nucleic acid sequences encoding such constructs are provided, for example, in SEQ ID NOS: 923-1002 and 3436-3452. Table 141 provides a summary of the constructs. The last column indicates the number of tumor-associated antigen (e.g., CTA/OFA) genes in the previous column that were expressed in at least 90% of The Cancer Genome Atlas (TCGA) patients for that indication. For example 3 TAA genes were expressed in over 90% of NSCLC patients. The rest of the TAA genes were expressed in < 90% of the population of TCGA NSCLC patients.

[001062] Table 141. Summary of ADXS-HOT Constructs.

Non-Small-Cell Lung Cancer (NSCLC) Hotspot/Heteroclitic/Minigene Constructs

[001063] A total of 11 hotspot mutations across 6 genes were selected as described in Example 4 and elsewhere herein for the NSCLC ADXS-HOT constructs. This panel of hotspot mutations covers 43% of all non-small cell lung cancer patients (i.e., 43% of non- small cell lung cancer patients will have at least one of the hotspot mutations from the panel). For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 35. The hotspot mutation in each is bolded and underlined. [001064] Table 35. Exemplary NSCLC Panel Hotspot 21-Mers.

[001065] A total of 11 peptides with heteroclitic mutations across 7 genes were selected for the NSCLC ADXS-HOT constructs. For each heteroclitic mutation, a peptide of 9 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 36. The heteroclitic mutation in each is as described in Table 140.

[001066] Table 36. Exemplary NSCLC Panel Heteroclitic 9-Mers.

[001067] The in silico predicted binding affinity and in vitro binding affinity of the heteroclitic 9-mer peptides are provided in Table 36B. The in silico predicted binding affinity is based on the NetMHC4.0 algorithm, which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values (nM); a lower number reflects stronger predicted binding affinity. The in vitro binding affinity was determined through a binding assay that determines the ability of each candidate peptide to bind to the indicated MHC class I alleles and stabilize the MHC-peptide complex by comparing the binding to that of a high affinity T cell epitope. Briefly, each peptide is incubated with its specific HLA molecule in an in vitro assay. Binding strength is compared against a known, immunogenic peptide for the same HLA molecule as a positive control with the positive control binding score set to 100%. The sequence- optimized binding score is normalized to the control peptide. That is, each peptide was given a score relative to the positive control peptide, which is a known T cell epitope with very strong binding properties. The score of the heteroclitic test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide. Peptides with scores greater than or equal to 45% of the positive control are considered binders. Also provided in Table 36B are the percent expression of each gene in patients with NSCLC (The Cancer Genome Atlas (TCGA) database), the HLA allele being tested, and whether the wild-type peptide corresponding to each heteroclitic peptide is known to be immunogenic. For a construct including each of the heteroclitic peptides in Table 36B, 100% of NSCLC patients with HLA type A*02:01 express at least one of the TAA genes, 100% of NSCLC patients with HLA type A*03:01 express at least one of the TAA genes, 100% of NSCLC patients with HLA type A*24:02 express at least one of the TAA genes, and 100% of NSCLC patients with HLA type B*07:02 express at least one of the TAA genes.

[001068] Table 36B. Binding Affinities of Heteroclitic 9-Mers to HLA.

# - NetMHC4.0

^Λ - % relative to positive control peptide binding

& - SEQ ID NO: 817 [001069] Constructs were designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide. In some constructs, the ubiquitin was fused to the CEACAM5_A0201 heteroclitic peptide. In some of the constructs, the ubiquitin was fused to the CEACAM5_A2402 heteroclitic peptide. In some constructs, the heteroclitic peptides are C-terminal to the hotspot peptides. In some constructs, the heteroclitic peptides are interspersed among the hotspot peptides. FLAG tags and SIINFEKL tags were also included upstream of the ubiquitin. The tLLO, hotspot peptide, heteroclitic peptide, and ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37. The VGKGGSGG linker (SEQ ID NO: 314) can be used, for example, as a longer linker after the tLLO and also before the tag sequences to provide additional space between the tLLO and the antigenic portion of the fusion peptide and before the tag sequences. It also can provide flexibility and to charge balance the fusion protein. The EAAAK linker (SEQ ID NO: 316) is a rigid/stiff linker that can be used to facilitate expression and secretion, for example, if the fusion protein would otherwise fold on itself. The GGGGS linker (SEQ ID NO: 313) is a flexible linker that can be used, for example, to add increased flexibility to the fusion protein to help facilitate expression and secretion. The "i20" linkers (e.g., SEQ ID NOS: 821-829) are immunoproteasome linkers that are designed, for example, to help facilitate cleavage of the fusion protein by the immunoproteasome and increase the frequency of obtaining the exact minimal binding fragment that is desired. These can be used, for example, around the heteroclitic peptide sequences because the exact minimal 8mer-to-l lmer desired to be generated is known.

Combinations of GGGGS and EAAAK linkers (SEQ ID NOS: 313 and 316, respectively) can be used, for example, to alternate flexibility and rigidity to help balance the construct for improved expression and secretion and to help facilitate DNA synthesis by providing more unique codons to choose from. Combinations of EAAAK linkers (SEQ ID NO: 316) and "i20" linkers can be used, for example, by providing the rigid EAAAK linker around the 2 lmer hotspot peptides and "i20" linkers around the heteroclitic sequences for which we know the exact 9mer desired to be generated. Combinations of GGGGS linkers (SEQ ID NO: 313) and "i20" linkers can be used, for example, by providing the flexible GGGGS linker around the 2 lmer hotspot peptides and "i20" linkers around the heteroclitic sequences for which we know the exact 9mer desired to be generated. [001070] Table 37. Linkers.

[001071 ] Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) include the following: (1) NSCLC HOT EV02 EAAAK. G4S (A) (SEQ ID NO: 859); (2) NSCLC HOT G4S (A) (SEQ ID NO: 860); (3) NSCLC HOT EV02 EAAAK-G4S mix (A) (SEQ ID NO: 861); (4) NSCLC HOT EV02 EAAAK. i20 (A) (SEQ ID NO: 862); (5) NSCLC HOT EV02 G4S.i20 (A) (SEQ ID NO: 863); (6) NSCLC HOT EVO 2 G4S LS#1 (A) (SEQ ID NO: 864); (7) NSCLC HOT EVO 2 G4S LS#2 (A) (SEQ ID NO: 865); (8) NSCLC HOT EV02 EAAAK. G4S (B) (SEQ ID NO: 894); (9) NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) (SEQ ID NO: 895); and (10) NSCLC A24 HOT (SEQ ID NO: 905). A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 38-47. For the (B) constructs, additional heteroclitic epitopes were added to complement the original hotspot mutation peptides so that total patient coverage within a cancer type approaches 100%. Any patient will therefore likely express a tumor-associated antigen that we have designed heteroclitic peptides for to cover the most prevalent HLAs (HLA-A0201, HLA-A0301, HLA-A2402, and HLA-B0702). For the A24 constructs, the 9- mer in the minigene was replaced by an A24 9mer. A24 (HLA-A2402) is the HLA type commonly found in Asia. For the LS constructs, the antigenic peptides in the fusion polypeptide were reordered based on hydrophobicity and charge. G4S, EAAAK, and i20 refer to inclusion of flexible linkers, rigid linkers, and immunoproteasome processing linkers, respectively. [001072] Table 38. Positions of Components of NSCLC HOT EV02 EAAAK.G4S (A)

Insert.

[001073] Table 39. Positions of Components of NSCLC HOT G4S (A) Insert.

[001074] Table 40. Positions of Components of NSCLC HOT EV02 EAAAK-G4S mix (A) Insert.

[001075] Table 41. Positions of Components of NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (A) Insert.

[001076] Table 42. Positions of Components of NSCLC HOT EV02 G4S.i20 (A)

Insert.

[001078] Table 44. Positions of Components of NSCLC HOT EVO 2 G4S LS#2 (A)

Insert.

[001079] Table 45. Positions of Components of NSCLC HOT EV02 EAAAK.G4S (B)

Insert.

[001082] Constructs were also designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides without a ubiquitin peptide (i.e., hotspot peptides plus heteroclitic peptides with no "minigene"). The tLLO, hotspot peptide and heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37. An exemplary fusion polypeptide insert sequence (i.e., the peptide sequence downstream of the tLLO) is NSCLC HS + HC (SEQ ID NO: 909). [001083] Constructs were also designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides with no heteroclitic peptides other than a ubiquitin peptide fused to a heteroclitic peptide at the C-terminal end (i.e., hotspot peptides plus "minigene" with no additional heteroclitic peptides). The tLLO, hotspot peptide, and ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37. An exemplary fusion polypeptide insert sequence (i.e., the peptide sequence downstream of the tLLO) is NSCLC HS + MG (SEQ ID NO: 910).

[001084] Constructs were also designed to encode a fusion polypeptide comprising tLLO fused to one or more heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide (i.e., heteroclitic peptides and "minigene" with no hotspot peptides). The tLLO, heteroclitic peptide, and ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37. An exemplary fusion polypeptide insert sequence (i.e., the peptide sequence downstream of the tLLO) is NSCLC HC + MG (SEQ ID NO: 911).

[001085] Constructs were also designed to encode a fusion polypeptide comprising tLLO fused to one or more heteroclitic peptides without any ubiquitin peptide and without any hotspot peptides (i.e., heteroclitic peptides with no "minigene" and with no hotspot peptides). The tLLO and heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37. An exemplary fusion polypeptide insert sequence (i.e., the peptide sequence downstream of the tLLO) is NSCLC HC only (SEQ ID NO: 912).

[001086] A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 48-51.

[001087] Table 48. Positions of Components of NSCLC HS + HC Insert.

[001088] Table 49. Positions of Components of NSCLC HS + MG Insert.

[001089] Table 50. Positions of Components of NSCLC HC + MG Insert.

21-29: CEACAM5_A0301 126-134: CEACAM5_A2402

239-259: FLAG

42-50: MAGEA6_A0301 147-155: NYESOl_A0201

260-279: Linker-SIINFEKL

63-71: CEACAM5_B0702 168-176: STEAP1_A0201

286-360: Ubiquitin

84-92: MAGEA4_B0702 189-197: STEAP1_A2402

361-369: CEACAM5_A0201_MINI 105-113: GAGE1 B0702 210-218: RNF43 B0702

[001090] Table 51. Positions of Components of NSCLC HC Only Insert.

21-29: CEACAM5_A0301

126-134: CEACAM5_A2402 210-218: RNF43_B0702

42-50: MAGEA6_A0301

147-155: NYESOl_A0201 239-259: FLAG

63-71: CEACAM5_B0702

168-176: STEAP1_A0201 260-279: Linker-SIINFEKL

84-92: MAGEA4_B0702

189-197: STEAP1 A2402 286-294: CEACAM5_A0201_MINI 105-113: GAGE1 B0702

[001091 ] To assess the expression of tLLO-antigenic-peptide fusion proteins by Lmdda Listeria constructs, the DNA constructs were generated as described elsewhere herein and transformed into Lmdda. Each individual Lmdda construct was assayed by Western blot for tLLO fusion polypeptide expression using an anti-FLAG antibody. Figures 12 and 18 show expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for various NSCLS constructs. The constructs visualized on these Western blots all fall between 103-125 kDa and are similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing. Figure 42 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the NSCLC HOT EV02

EAAAK.G4S (B) and NSCLC HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) constructs. The constructs visualized on these Western blots fall between 103-125 kDa and are similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing. Figure 47 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the NSCLC HS + MG construct. The expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing.

Prostate Cancer Hotspot/Heteroclitic/Minigene Constructs

[001092] A total of 14 hotspot mutations across 5 genes were selected as described in Example 4 and elsewhere herein for the prostate cancer ADXS-HOT constructs. This panel of hotspot mutations covers 16% of all prostate cancer patients (i.e., 16% of prostate cancer patients will have at least one of the hotspot mutations from the panel). For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. However, the RGPD_P1760A hotspot peptide was only 16 amino acids because the mutation is near the C-terminus of RGPD. In addition, the AR_H875Y_T878A double hotspot peptide, which includes two adjacent hotspot mutations, is 24 amino acids in length. The peptides are shown in Table 52. The hotspot mutation(s) in each is bolded and underlined.

[001093] Table 52. Exemplary Prostate Cancer Panel Hotspot Peptides.

[001094] A total of 10 peptides with heteroclitic mutations across 9 genes were selected for the prostate cancer ADXS-HOT constructs. For each heteroclitic mutation, a peptide of 9 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 53. The heteroclitic mutation in each is as described in Table

140.

[001095] Table 53. Exemplary Prostate Cancer Panel Heteroclitic 9-Mers.

[001096] The in silico predicted binding affinity and in vitro binding affinity of the heteroclitic 9-mer peptides are provided in Table 53B. The in silico predicted binding affinity is based on the NetMHC4.0 algorithm, which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values (nM); a lower number reflects stronger predicted binding affinity. The in vitro binding affinity was determined through a binding assay that determines the ability of each candidate peptide to bind to the indicated MHC class I alleles and stabilize the MHC-peptide complex by comparing the binding to that of a high affinity T cell epitope. Briefly, each peptide is incubated with its specific HLA molecule in an in vitro assay. Binding strength is compared against a known, immunogenic peptide for the same HLA molecule as a positive control with the positive control binding score set to 100%. The sequence- optimized binding score is normalized to the control peptide. That is, each peptide was given a score relative to the positive control peptide, which is a known T cell epitope with very strong binding properties. The score of the heteroclitic test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide. Peptides with scores greater than or equal to 45% of the positive control are considered binders. Also provided in Table 53B are the percent expression of each gene in patients with prostate cancer (The Cancer Genome Atlas database), the HLA allele being tested, and whether the wild-type peptide corresponding to each heteroclitic peptide is known to be immunogenic. For a construct including each of the heteroclitic peptides in Table 53B, 100% of prostate cancer patients with HLA type A*02:01 express at least one of the TAA genes, 100% of prostate cancer patients with HLA type A*03:01 express at least one of the TAA genes, 100% of prostate cancer patients with HLA type A*24:02 express at least one of the TAA genes, and 100% of prostate cancer patients with HLA type B*07:02 express at least one of the TAA genes.

[001097] Table 53B. Binding Affinities of Heteroclitic 9-Mers to HLA.

# - NetMHC4.0

^Λ - % relative to positive control peptide binding

[001098] Constructs were designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide. In some constructs, the ubiquitin was fused to the STEAP1_A0201 heteroclitic peptide. In some of the constructs, the ubiquitin was fused to the STEAP1_A2402 heteroclitic peptide. In some constructs, the heteroclitic peptides are C-terminal to the hotspot peptides. In some constructs, the heteroclitic peptides are interspersed among the hotspot peptides. FLAG tags and SIINFEKL tags were also included upstream of the ubiquitin. The tLLO, hotspot peptide, heteroclitic peptide, and ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37.

[001099] Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) include the following: (1) ProStar EV02 EAAAK.G4S (A) (SEQ ID NO: 871); (2) ProStar EV02 G4S (A) (SEQ ID NO: 872); (3) ProStar EV02 EAAAK- G4S mix (A) (SEQ ID NO: 873); (4) ProStar EV02 ΕΑΑΑΚ.Ϊ20 (A) (SEQ ID NO: 874); (5) ProStar EV02 G4S.i20 (A) (SEQ ID NO: 875); (6) ProStar EVO 2 G4S LS#1 (A) (SEQ ID NO: 876); (7) ProStar EVO 2 G4S LS#2 (A) (SEQ ID NO: 877); (8) ProStar EV02 EAAAK.G4S (B) (SEQ ID NO: 892); (9) ProStar EV02 EAAAK. i20 (B) (SEQ ID NO: 893); and (10) Prostar A24 HOT (SEQ ID NO: 906). A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 54-63. For the (B) constructs, additional heteroclitic epitopes were added to complement the original hotspot mutation peptides so that total patient coverage within a cancer type approaches 100%. Any patient will therefore likely express a tumor-associated antigen that we have designed heteroclitic peptides for to cover the most prevalent HLAs (HLA-A0201, HLA-A0301, HLA- A2402, and HLA-B0702). For the A24 construct, the 9-mer in the minigene was replaced by an A24 9mer. A24 (HLA-A2402) is the HLA type commonly found in Asia. For the LS constructs, the antigenic peptides in the fusion polypeptide were reordered based on hydrophobicity and charge. G4S, EAAAK, and i20 refer to inclusion of flexible linkers, rigid linkers, and immunoproteasome processing linkers, respectively.

[001100] Table 54. Positions of Components of ProStar EV02 EAAAK.G4S (A)

Insert.

[001102] Table 56. Positions of Components of ProStar EV02 EAAAK-G4S mix (A) Insert.

[001106] Table 60. Positions of Components of ProStar EVO 2 G4S LS#2 (A) Insert.

14-22: STEAP1_A2402 200-208: MAGEA4_B0702 382-402: CHEK2_K373E

28-36: PAGE4_A0201 214-234: AR_L702H 408-428: ANKRD36C_D629Y 42-62: ANKRD36C_D626N 240-263: 434-454: AR_F877L

AR_H875Y_T878A

68-88: AR_W742C 460-468: SART3_A0201

269-289: SPOP_F133V

94-114: SPOP_W131G 482-502: FLAG

295-315: AR_H875Y

120-128: PSMA_A2402 503-522: Linker-SIINFEKL

321-329: SSX2_A0201

134-154: ANKRD36C_I634T 528-602: Ubiquitin

335-355: AR_T878A

160-168: PSA_A0301 603-611:

361-376: RGPD8_P1760A STEAP1_A0201_MINI

174-194: SPOP F133L

[001107] Table 61. Positions of Components of ProStar EV02 EAAAK.G4S (B) Insert.

[001108] Table 62. Positions of Components of ProStar EV02 ΕΑΑΑΚ.Ϊ20 (B) Insert.

[001109] Table 63. Positions of Components of Prostar A24 HOT Insert.

[001110] Constructs were also designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides without a ubiquitin peptide (i.e., hotspot peptides plus heteroclitic peptides with no "minigene"). The tLLO, hotspot peptide and heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37. An exemplary fusion polypeptide insert sequence (i.e., the peptide sequence downstream of the tLLO) is Prostar HS + HC (SEQ ID NO: 913).

[001111 ] Constructs were also designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides other than the AR-H875Y-T878A with no heteroclitic peptides other than a ubiquitin peptide fused to a heteroclitic peptide at the C-terminal end (i.e., hotspot peptides plus "minigene" with no additional heteroclitic peptides). The tLLO, hotspot peptide, and ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37. An exemplary fusion polypeptide insert sequence (i.e., the peptide sequence downstream of the tLLO) is Prostar HS + MG (SEQ ID NO: 914).

[001112] Constructs were also designed to encode a fusion polypeptide comprising tLLO fused to one or more heteroclitic peptides and the AR-H875Y-T878A peptide, with the C- terminal heteroclitic peptide following a ubiquitin peptide (i.e., heteroclitic peptides and "minigene" with only the double hotspot peptide). The tLLO, heteroclitic peptide, and ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37. An exemplary fusion polypeptide insert sequence (i.e., the peptide sequence downstream of the tLLO) is Prostar HC + MG (SEQ ID NO: 915).

[001113] Constructs were also designed to encode a fusion polypeptide comprising tLLO fused to one or more heteroclitic peptides and the AR-H875Y-T878A peptide without any ubiquitin peptide and without any hotspot peptides other than the double hotspot AR-H875Y- T878A peptide (i.e., heteroclitic peptides with no "minigene" and with only the double hotspot peptide). The tLLO and heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37. An exemplary fusion polypeptide insert sequence (i.e., the peptide sequence downstream of the tLLO) is Prostar HC only (SEQ ID NO: 916).

[001114] A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 64-67.

[001115] Table 64. Positions of Components of Prostar HS + HC Insert.

14-34: SPOP_F133V 243-263 AR_L702H

459-467: PSA_A0301

40-60: CHEK2_K373E 269-289; AR_W742C

480-488: MAGEA4_B0702 66-81: RGPD8_P1760A 295-315 AR_H875Y

501-509: CEACAM5_B0702 87-107: ANKRD36C_I634T 321-341 AR_F877L

522-530: RNF43_B0702

113-133: ANKRD36C_D629Y 354-362 SSX2_A0201

543-566: AR_H875Y_T878A 139-159: SPOP_W131G 375-383; SART3_A0201

580-600: FLAG

165-185: ANKRD36C_D626N 396-404 PAGE4_A0201

601-620: Linker-SIINFEKL 191-211: SPOP_F133L 417-425 STEAP1_A2402

626-634: STEAP1_A0201_MINI 217-237: AR_T878A 438-446 PSMA_A2402

[001116] Table 65. Positions of Components of Prostar HS + MG Insert.

14-34: SPOP_F133V 165-185: ANKRD36C_D626N 321-341: AR_F877L

40-60: CHEK2_K373E 191-211: SPOP_F133L 350-370: FLAG

66-81: RGPD8_P1760A 217-237: AR_T878A 371-390: Linker-SIINFEKL

87-107: ANKRD36C_I634T 243-263: AR_L702H 396-470: Ubiquitin

113-133:

269-289: AR_W742C 471-479:

ANKRD36C_D629Y

295-315: AR_H875Y STEAP1 A0201 MINI

139-159: SPOP_W131G

[001117] Table 66. Positions of Components of Prostar HC + MG Insert.

21-29: SSX2_A0201 126-134: PSA_A0301

247-267: FLAG

42-50: SART3_A0201 147-155: MAGEA4_B0702

268-287: Linker-SIINFEKL 63-71: PAGE4_A0201 168-176: CEACAM5_B0702

293-367: Ubiquitin

84-92: STEAP1_A2402 189-197: RNF43_B0702

368-376: STEAP1 A0201 MINI 105-113: PSMA_A2402 210-233: AR_H875Y_T878A

[001118] Table 67. Positions of Components of Prostar HC Only Insert.

21-29: SSX2_A0201

126-134: PSA_A0301 210-233: AR_H875Y_T878A 42-50: SART3_A0201

147-155: MAGEA4_B0702 247-267: FLAG

63-71: PAGE4_A0201

168-176: CEACAM5_B0702 268-287: Linker-SIINFEKL 84-92: STEAP1_A2402

189-197: RNF43 B0702 292-300: STEAP1_A0201_MINI 105-113: PSMA A2402 [001119] To assess the expression of tLLO-antigenic-peptide fusion proteins by Lmdda Listeria constructs, the DNA constructs were generated as described elsewhere herein and transformed into Lmdda. Each individual Lmdda construct was assayed by Western blot for tLLO fusion polypeptide expression using an anti-FLAG antibody. Figures 13 and 19 show expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for various prostate cancer constructs. The constructs visualized on these Western blots all fall between 103-125 kDa and are similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing. Figure 43 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the ProStar EV02 EAAAK.G4S (B) and ProStar EV02 ΕΑΑΑΚ.Ϊ20 (B) constructs. The constructs visualized on these Western blots fall between 103-125 kDa and are similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing. Figure 48 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the ProStar HS + HC, ProStar HS + MG, ProStar HC + MG, and ProStar HC only constructs. The expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing.

Pancreatic Cancer Hotspot/Heteroclitic/Minigene Constructs

[001120] A total of 16 hotspot mutations across 5 genes were selected as described in Example 4 and elsewhere herein for the pancreatic cancer ADXS-HOT constructs. This panel of hotspot mutations covers 87% of all pancreatic cancer patients (i.e., 87% of pancreatic cancer patients will have at least one of the hotspot mutations from the panel). For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 68. The hotspot mutation in each is bolded and underlined. [001121 ] Table 68. Exemplary Pancreatic Cancer Panel Hotspot Peptides.

[001122] A total of 12 peptides with heteroclitic mutations across 6 genes were selected for the pancreatic cancer ADXS-HOT constructs. For each heteroclitic mutation, a peptide of 9 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 69. The heteroclitic mutation in each is as described in Table 140.

[001123] Table 69. Exemplary Pancreatic Cancer Panel Heteroclitic 9-Mers.

[001124] The in silico predicted binding affinity and in vitro binding affinity of the heteroclitic 9-mer peptides are provided in Table 69B. The in silico predicted binding affinity is based on the NetMHC4.0 algorithm, which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values (nM); a lower number reflects stronger predicted binding affinity. The in vitro binding affinity was determined through a binding assay that determines the ability of each candidate peptide to bind to the indicated MHC class I alleles and stabilize the MHC-peptide complex by comparing the binding to that of a high affinity T cell epitope. Briefly, each peptide is incubated with its specific HLA molecule in an in vitro assay. Binding strength is compared against a known, immunogenic peptide for the same HLA molecule as a positive control with the positive control binding score set to 100%. The sequence- optimized binding score is normalized to the control peptide. That is, each peptide was given a score relative to the positive control peptide, which is a known T cell epitope with very strong binding properties. The score of the heteroclitic test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide. Peptides with scores greater than or equal to 45% of the positive control are considered binders. Also provided in Table 69B are the percent expression of each gene in patients with pancreatic cancer (The Cancer Genome Atlas database), the HLA allele being tested, and whether the wild-type peptide corresponding to each heteroclitic peptide is known to be immunogenic. For a construct including each of the heteroclitic peptides in Table 69B, 100% of pancreatic cancer patients with HLA type A*02:01 express at least one of the TAA genes, 98% of pancreatic cancer patients with HLA type A*03:01 express at least one of the TAA genes, 100% of pancreatic cancer patients with HLA type A*24:02 express at least one of the TAA genes, and 98% of pancreatic cancer patients with HLA type B*07:02 express at least one of the TAA genes.

[001125] Table 69B. Binding Affinities of Heteroclitic 9-Mers to HLA.

# - NetMHC4.0

^Λ - % relative to positive control peptide binding

[001127] Constructs were designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide. In some constructs, the ubiquitin was fused to the CEACAM5_A0201 heteroclitic peptide. In some of the constructs, the ubiquitin was fused to the CEACAM5_A2402 heteroclitic peptide. The heteroclitic peptides were C- terminal to the hotspot peptides. FLAG tags and SIINFEKL tags were also included upstream of the ubiquitin. The tLLO, hotspot peptide, heteroclitic peptide, and

ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37.

[001128] Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) include the following: (1) PANC HOT EV02 EAAAK.G4S (A) (SEQ ID NO: 866); (2) PANC HOT G4S (A) (SEQ ID NO: 867); (3) PANC HOT EV02 EAAAK-G4S mix (A) (SEQ ID NO: 868); (4) PANC HOT EV02 ΕΑΑΑΚ.Ϊ20 (A) (SEQ ID NO: 869); (5) PANC HOT EV02 G4S.i20 (A) (SEQ ID NO: 870); and (6) Pancreas A24 HOT (SEQ ID NO: 908). A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 70-75. For the A24 construct, the 9-mer in the minigene was replaced by an A24 9mer. A24 (HLA-A2402) is the HLA type commonly found in Asia. G4S, EAAAK, and i20 refer to inclusion of flexible linkers, rigid linkers, and immunoproteasome processing linkers, respectively.

[001129] Table 70. Positions of Components of PANC HOT EV02 EAAAK.G4S (A) Insert.

14-34: KRAS_G12D 300-320: TP53_G245S 500-508: STEAP1_A2402

40-60: KRAS_G12V 326-346: U2AF1_S34F 514-522: CEACAM5_A2402 66-86: KRAS_G12R 352-372: SMAD4_R361C 528-536: CEACAM5_B0702 92-112: KRAS_Q61H 378-398: GNAS_R201C 542-550:

MAGEA3_A0201_A2402

118-138: TP53_R175H 404-424: GNAS_R201H

556-564: SURVIVIN_A0201 144-164: TP53_R282W 430-438: STEAP1_A0201

570-578: SURVIVIN_A2402 170-190: TP53_R273H 444-452: PRAME_A0201

196-216: KRAS_G12C 458-466: 592-612: FLAG

hTERT_A0201_A2402 613-632: Linker-SIINFEKL 222-242: TP53_R248Q

472-480: CEACAM5_A0301 639-713: Ubiquitin

248-268: TP53_R273C

486-494: MAGEA3 A0301 714-722:

274-294: TP53_R248W CEAC AM5_A0201_MINI

[001130] Table 71. Positions of Components of PANC HOT G4S (A) Insert.

14-24: KRAS_G12D 300-320: TP53_G245S 500-508: STEAP1_A2402

40-60: KRAS_G12V 326-346: U2AF1_S34F 514-522: CEACAM5_A2402 66-86: KRAS_G12R 352-372: SMAD4_R361C 528-536: CEACAM5_B0702 92-112: KRAS_Q61H 378-398: GNAS_R201C 542-550:

MAGEA3_A0201_A2402

118-138: TP53_R175H 404-424: GNAS_R201H

556-564: SURVIVIN_A0201 144-164: TP53_R282W 430-438: STEAP1_A0201

570-578: SURVIVIN_A2402 170-190: TP53_R273H 444-452: PRAME_A0201

196-216: KRAS_G12C 458-466: 592-612: FLAG

hTERT_A0201_A2402 613-632: Linker-SIINFEKL 222-242: TP53_R248Q

472-480: CEACAM5_A0301 639-713: Ubiquitin

248-268: TP53_R273C

486-494: MAGEA3_A0301 714-722:

274-294: TP53 R248W CEAC AM5_A0201_MINI

[001131 ] Table 72. Positions of Components of PANC HOT EV02 EAAAK-G4S mix (A) Insert.

14-34: KRAS_G12D 300-320: TP53_G245S 500-508: STEAP1_A2402

40-60: KRAS_G12V 326-346: U2AF1_S34F 514-522: CEACAM5_A2402 66-86: KRAS_G12R 352-372: SMAD4_R361C 528-536: CEACAM5_B0702 92-112: KRAS_Q61H 378-398: GNAS_R201C 542-550:

MAGEA3_A0201_A2402

118-138: TP53_R175H 404-424: GNAS_R201H

556-564: SURVIVIN_A0201 144-164: TP53_R282W 430-438: STEAP1_A0201

570-578: SURVIVIN_A2402 170-190: TP53_R273H 444-452: PRAME_A0201

196-216: KRAS_G12C 458-466: 592-612: FLAG

hTERT_A0201_A2402 613-632: Linker-SIINFEKL

222-242: TP53_R248Q

472-480: CEACAM5_A0301 639-713: Ubiquitin

248-268: TP53_R273C

486-494: MAGEA3_A0301 714-722:

274-294: TP53 R248W CEACAM5 A0201 MINI [001132] Table 73. Positions of Components of PANC HOT EV02 ΕΑΑΑΚ.Ϊ20 (A) Insert.

14-24: KRAS_G12D 300-320: TP53_G245S 542-550: STEAP1_A2402

40-60: KRAS_G12V 326-346: U2AF1_S34F 563-571: CEACAM5_A2402

66-86: KRAS_G12R 352-372: SMAD4_R361C 584-592: CEACAM5_B0702

92-112: KRAS_Q61H 378-398: GNAS_R201C 605-613:

MAGEA3_A0201_A2402

118-138: TP53_R175H 404-424: GNAS_R201H

626-634: SURVIVIN_A0201

144-164: TP53_R282W 437-445: STEAP1_A0201

647-655: SURVIVIN_A2402

170-190: TP53_R273H 458-466: PRAME_A0201

87: 676-696: FLAG

196-216: KRAS_G12C 479-4

hTERT_A0201_A2402 697-716: Linker-SIINFEKL

222-242: TP53_R248Q

500-508: CEACAM5_A0301 723-797: Ubiquitin

248-268: TP53_R273C

521-529: MAGEA3 A0301 798-806:

274-294: TP53 R248W CEACAM5 A0201 MINI

[001133] Table 74. Positions of Components of PANC HOT EV02 G4S.i20 (A) Insert.

14-24: KRAS_G12D 300-320: TP53_G245S 542-550: STEAP1_A2402

40-60: KRAS_G12V 326-346: U2AF1_S34F 563-571: CEACAM5_A2402

66-86: KRAS_G12R 352-372: SMAD4_R361C 584-592: CEACAM5_B0702

92-112: KRAS_Q61H 378-398: GNAS_R201C 605-613:

MAGEA3_A0201_A2402

118-138: TP53_R175H 404-424: GNAS_R201H

626-634: SURVIVIN_A0201

144-164: TP53_R282W 437-445: STEAP1_A0201

647-655: SURVIVIN_A2402

170-190: TP53_R273H 458-466: PRAME_A0201

676-696: FLAG

196-216: KRAS_G12C 479-487:

hTERT_A0201_A2402 697-716: Linker-SIINFEKL

222-242: TP53_R248Q

500-508: CEACAM5_A0301 723-797: Ubiquitin

248-268: TP53_R273C

521-529: MAGEA3 A0301 798-806:

274-294: TP53_R248W CEAC AM5_A0201_MINI

[001134] Table 75. Positions of Components of Pancreas A24 HOT Insert.

14-24: KRAS_G12D 300-320: TP53_G245S 542-550: STEAP1_A2402

40-60: KRAS_G12V 326-346: U2AF1_S34F 563-571: CEACAM5_A0201

66-86: KRAS_G12R 352-372: SMAD4_R361C 584-592: CEACAM5_B0702

92-112: KRAS_Q61H 378-398: GNAS_R201C 605-613:

MAGEA3_A0201_A2402

118-138: TP53_R175H 404-424: GNAS_R201H

626-634: SURVIVIN_A0201

144-164: TP53_R282W 437-445: STEAP1_A0201

647-655:SURVIVIN_A2402

170-190: TP53_R273H 458-466: PRAME_A0201

196-216: KRAS_G12C 479-487: 676-696: FLAG

hTERT_A0201_A2402 697-716: Linker-SIINFEKL

222-242: TP53_R248Q

500-508: CEACAM5_A0301 723-797: Ubiquitin

248-268: TP53_R273C

521-529: MAGEA3 A0301 798-806: CEACAM5_A2402

274-294: TP53_R248W MINI

[001135] To assess the expression of tLLO-antigenic-peptide fusion proteins by Lmdda Listeria constructs, the DNA constructs were generated as described elsewhere herein and transformed into Lmdda. Each individual Lmdda construct was assayed by Western blot for tLLO fusion polypeptide expression using an anti-FLAG antibody. Figure 17 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the PANC HOT EV02 EAAK G4S construct. The construct visualized on these Western blots falls between 103-125 kDa and is similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing. Figure 45 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the PANC HOT EV02 EAAAK.G4S, PANC HOT EV02 EAAAK-G4S mix, PANC HOT EV02 ΕΑΑΑΚ.Ϊ20, and PANC HOT EV02 G4S.i20 constructs. The constructs visualized on these Western blots fall between 103-135 kDa and are similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing.

Bladder Cancer Hotspot/Heteroclitic/Minigene Constructs

[001136] A total of 13 hotspot mutations across 6 genes were selected as described in Example 4 and elsewhere herein for the bladder cancer ADXS-HOT constructs. This panel of hotspot mutations covers 43% of all bladder cancer patients (i.e., 43% of bladder cancer patients will have at least one of the hotspot mutations from the panel). For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 76. The hotspot mutation in each is bolded and underlined.

[001137] Table 76. Exemplary Bladder Cancer Panel Hotspot Peptides.

[001138] A total of 14 peptides with heteroclitic mutations across 8 genes were selected for the bladder cancer ADXS-HOT constructs. For each heteroclitic mutation, a peptide of 9 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 77. The heteroclitic mutation in each is as described in Table 140.

[001139] Table 77. Exemplary Bladder Cancer Panel Heteroclitic 9-Mers.

[001140] The in silico predicted binding affinity and in vitro binding affinity of the heteroclitic 9-mer peptides are provided in Table 77B. The in silico predicted binding affinity is based on the NetMHC4.0 algorithm, which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values (nM); a lower number reflects stronger predicted binding affinity. The in vitro binding affinity was determined through a binding assay that determines the ability of each candidate peptide to bind to the indicated MHC class I alleles and stabilize the MHC-peptide complex by comparing the binding to that of a high affinity T cell epitope. Briefly, each peptide is incubated with its specific HLA molecule in an in vitro assay. Binding strength is compared against a known, immunogenic peptide for the same HLA molecule as a positive control with the positive control binding score set to 100%. The sequence- optimized binding score is normalized to the control peptide. That is, each peptide was given a score relative to the positive control peptide, which is a known T cell epitope with very strong binding properties. The score of the heteroclitic test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide. Peptides with scores greater than or equal to 45% of the positive control are considered binders. Also provided in Table 77B are the percent expression of each gene in patients with bladder cancer (The Cancer Genome Atlas database), the HLA allele being tested, and whether the wild-type peptide corresponding to each heteroclitic peptide is known to be immunogenic. For a construct including each of the heteroclitic peptides in Table 77B, 100% of bladder cancer patients with HLA type A*02:01 express at least one of the TAA genes, 100% of bladder cancer patients with HLA type A*03:01 express at least one of the TAA genes, 100% of bladder cancer patients with HLA type A*24:02 express at least one of the TAA genes, and 100% of bladder cancer patients with HLA type B*07:02 express at least one of the TAA genes.

[001141 ] Table 77B. Binding Affinities of Heteroclitic 9-Mers to HLA.

# - NetMHC4.0

^Λ - % relative to positive control peptide binding

[001142] Constructs were designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide. In some constructs, the ubiquitin was fused to the NYESOl_A0201 heteroclitic peptide. In some of the constructs, the ubiquitin was fused to the NUF2_A0201 heteroclitic peptide. The heteroclitic peptides were C- terminal to the hotspot peptides. FLAG tags and SIINFEKL tags were also included upstream of the ubiquitin. The tLLO, hotspot peptide, heteroclitic peptide, and

ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37.

[001143] Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) include the following: (1) Bladder HOT EV02 EAAAK.G4S (A) (SEQ ID NO: 878); (2) Bladder HOT G4S (A) (SEQ ID NO: 879); (3) Bladder HOT EV02 EAAAK-G4S mix (A) (SEQ ID NO: 880); (4) Bladder HOT EV02 ΕΑΑΑΚ.Ϊ20 (A) (SEQ ID NO: 881); (5) Bladder HOT EV02 G4S.i20 (A) (SEQ ID NO: 882); (6) Bladder HOT EV02 EAAAK.G4S (B) (SEQ ID NO: 888); (7) Bladder HOT EV02 ΕΑΑΑΚ.Ϊ20 (B) (SEQ ID NO: 889); (8) Bladder HOT EV02 EAAAK.G4S NUF minigene (B) (SEQ ID NO: 890); and (9) Bladder HOT EV02 ΕΑΑΑΚ.Ϊ20 NUF minigene (B) (SEQ ID NO: 891). A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 78-86. For the (B) constructs, additional heteroclitic epitopes were added to complement the original hotspot mutation peptides so that total patient coverage within a cancer type approaches 100%. Any patient will therefore likely express a tumor-associated antigen that we have designed heteroclitic peptides for to cover the most prevalent HLAs (HLA-A0201, HLA-A0301, HLA-A2402, and HLA-B0702). G4S, EAAAK, and i20 refer to inclusion of flexible linkers, rigid linkers, and immunoproteasome processing linkers, respectively.

[001144] Table 78. Positions of Components of Bladder HOT EV02 EAAAK.G4S (A)

Insert.

[001146] Table 80. Positions of Components of Bladder HOT EV02 EAAAK-G4S mix

(A) Insert.

[001149] Table 83. Positions of Components of Bladder HOT EV02 EAAAK.G4S (B)

Insert.

[001151 ] Table 85. Positions of Components of Bladder HOT EV02 EAAAK.G4S NUF minigene (B) Insert.

[001152] Table 86. Positions of Components of Bladder HOT EV02 EAAAK.i20_NUF minigene (B) Insert.

[001153] To assess the expression of tLLO-antigenic-peptide fusion proteins by Lmdda Listeria constructs, the DNA constructs were generated as described elsewhere herein and transformed into Lmdda. Each individual Lmdda construct was assayed by Western blot for tLLO fusion polypeptide expression using an anti-FLAG antibody. Figures 14 and 15 show expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for various bladder cancer constructs. The constructs visualized on these Western blots all fall between 103-125 kDa and are similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing. Figure 41 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the Bladder HOT EV02

EAAAK.G4S, Bladder HOT G4S, Bladder HOT EAAAK-G4S mix, Bladder HOT EV02 ΕΑΑΑΚ.Ϊ20, and Bladder HOT EV02 G4S.i20 constructs. The constructs visualized on these Western blots fall between 103-125 kDa and are similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing. Figure 46 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the Bladder HOT EV02 EAAAK.G4S (B), Bladder HOT EV02 EAAAK.G4S NUF minigene (B), and Bladder HOT EV02 ΕΑΑΑΚ.Ϊ20 NUF minigene (B) constructs. The constructs visualized on these Western blots fall between 103-135 kDa and are similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing.

Breast Cancer Hotspot/Heteroclitic/Minigene Constructs

[001154] A total of 14 hotspot mutations across 3 genes were selected as described in Example 4 and elsewhere herein for the breast cancer ADXS-HOT constructs. This panel of hotspot mutations covers 47% of all estrogen-receptor-positive (ER+) breast cancer patients (i.e., 47% of ER+ breast cancer patients will have at least one of the hotspot mutations from the panel). For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 87. The hotspot mutation in each is bolded and underlined.

[001155] Table 87. Exemplary Breast Cancer Panel Hotspot Peptides.

[001156] A total of 11 peptides with heteroclitic mutations across 6 genes were selected for the breast cancer ADXS-HOT constructs. For each heteroclitic mutation, a peptide of 9 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 88. The heteroclitic mutation in each is as described in Table

140.

[001157] Table 88. Exemplary Breast Cancer Panel Heteroclitic 9-Mers.

[001158] The in silico predicted binding affinity and in vitro binding affinity of the heteroclitic 9-mer peptides are provided in Table 88B. The in silico predicted binding affinity is based on the NetMHC4.0 algorithm, which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values (nM); a lower number reflects stronger predicted binding affinity. The in vitro binding affinity was determined through a binding assay that determines the ability of each candidate peptide to bind to the indicated MHC class I alleles and stabilize the MHC-peptide complex by comparing the binding to that of a high affinity T cell epitope. Briefly, each peptide is incubated with its specific HLA molecule in an in vitro assay. Binding strength is compared against a known, immunogenic peptide for the same HLA molecule as a positive control with the positive control binding score set to 100%. The sequence- optimized binding score is normalized to the control peptide. That is, each peptide was given a score relative to the positive control peptide, which is a known T cell epitope with very strong binding properties. The score of the heteroclitic test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide. Peptides with scores greater than or equal to 45% of the positive control are considered binders. Also provided in Table 88B are the percent expression of each gene in patients with breast cancer (The Cancer Genome Atlas database), the HLA allele being tested, and whether the wild-type peptide corresponding to each heteroclitic peptide is known to be immunogenic. For a construct including each of the heteroclitic peptides in Table 88B, 100% of breast cancer patients with HLA type A*02:01 express at least one of the TAA genes, 95% of breast cancer patients with HLA type A*03:01 express at least one of the TAA genes, 100% of breast cancer patients with HLA type A*24:02 express at least one of the TAA genes, and 100% of breast cancer patients with HLA type B*07:02 express at least one of the TAA genes.

[001159] Table 88B. Binding Affinities of Heteroclitic 9-Mers to HLA.

# - NetMHC4.0

^Λ - % relative to positive control peptide binding

[001160] Constructs were designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide. In some constructs, the ubiquitin was fused to the STEAP1_A0201 heteroclitic peptide. In some of the constructs, the ubiquitin was fused to the STEAP1_A2402 heteroclitic peptide. The heteroclitic peptides were C- terminal to the hotspot peptides. FLAG tags and SIINFEKL tags were also included upstream of the ubiquitin. The tLLO, hotspot peptide, heteroclitic peptide, and

ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37.

[001161 ] Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) include the following: (1) Breast HOT EV02 EAAAK.G4S (A) (SEQ ID NO: 883); (2) Breast HOT G4S (A) (SEQ ID NO: 884); (3) Breast HOT EV02 EAAAK-G4S mix (A) (SEQ ID NO: 885); (4) Breast HOT EV02 ΕΑΑΑΚ.Ϊ20 (A) (SEQ ID NO: 886); (5) Breast HOT EV02 G4S.i20 (A) (SEQ ID NO: 887); and (6) Breast A24 HOT (SEQ ID NO: 907). A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 89-94. For the A24 construct, the 9-mer in the minigene was replaced by an A24 9mer. A24 (HLA-A2402) is the HLA type commonly found in Asia. G4S, EAAAK, and i20 refer to inclusion of flexible linkers, rigid linkers, and immunoproteasome processing linkers, respectively.

[001162] Table 89. Positions of Components of Breast HOT EV02 EAAAK.G4S (A)

Insert.

[001164] Table 91. Positions of Components of Breast HOT EV02 EAAAK-G4S mix (A) Insert.

[001165] Table 92. Positions of Components of Breast HOT EV02 ΕΑΑΑΚ.Ϊ20 (A) Insert.

[001166] Table 93. Positions of Components of Breast HOT EV02 G4S.i20 (A) Insert.

[001167] Table 94. Positions of Components of Breast A24 HOT Insert.

14-34: PIK3CA_H1047R 274-294: ESR1_Y537S 490-498: STEAP1_A0201

40-60: PIK3CA_E545K 300-320: ESR1_Y537N 511-519:

CEACAM5_A2402

66-86: PIK3CA_E542K 326-346: ESR1_Y537C

532-540: MAGEA3_A2402 92-112: AKT1_E17K 352-372: ESR1_E380Q

553-561:

118-138: PIK3CA_H1047L 385-393: CEACAM5_A0201 CEACAM5_B0702

144-164: PIK3CA_Q546K 406-414: PRAME_A0201 574-582: RNF43_B0702

170-190: PIK3CA_E545A 427-435: 603-623: FLAG

hTERT_A0201_A2402

196-216: PIK3CA_E545G 624-643: Linker-SIINFEKL

448-456: CEACAM5_A0301

222-242: ESR1_K303R 650-724: Ubiquitin

469-477: MAGEA3 A0301

248-268: ESR1_D538G 725-733: STEAP1_A2402

MINI

[001168] To assess the expression of tLLO-antigenic-peptide fusion proteins by Lmdda Listeria constructs, the DNA constructs were generated as described elsewhere herein and transformed into Lmdda. Each individual Lmdda construct was assayed by Western blot for tLLO fusion polypeptide expression using an anti-FLAG antibody. Figure 16 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the Breast HOT G4S construct. Figure 38 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the Breast HOT EV02 EAAAK.G4S, Breast HOT G4S, Breast HOT EV02 EAAAK-G4S mix, Breast HOT EV02 ΕΑΑΑΚ.Ϊ20, and Breast HOT EV02 G4s.i20 constructs. The constructs visualized on these Western blots fall between 103-130 kDa and are similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing.

Uterine Cancer Hotspot/Heteroclitic/Minigene Constructs

[001169] A total of 16 hotspot mutations across 6 genes were selected as described in Example 4 and elsewhere herein for the uterine cancer ADXS-HOT constructs. This panel of hotspot mutations covers 64% of all uterine cancer patients (i.e., 64% of uterine cancer patients will have at least one of the hotspot mutations from the panel). For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table

95. The hotspot mutation in each is bolded and underlined.

[001170] Table 95. Exemplary Uterine Cancer Panel Hotspot 21-Mers.

[001171 ] A total of 14 peptides with heteroclitic mutations across 8 genes were selected for the uterine cancer ADXS-HOT constructs. For each heteroclitic mutation, a peptide of 9 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 96. The heteroclitic mutation in each is as described in Table 140.

[001172] Table 96. Exemplary Uterine Cancer Panel Heteroclitic 9-Mers.

[001173] The in silico predicted binding affinity and in vitro binding affinity of the heteroclitic 9-mer peptides are provided in Table 96B. The in silico predicted binding affinity is based on the NetMHC4.0 algorithm, which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values (nM); a lower number reflects stronger predicted binding affinity. The in vitro binding affinity was determined through a binding assay that determines the ability of each candidate peptide to bind to the indicated MHC class I alleles and stabilize the MHC-peptide complex by comparing the binding to that of a high affinity T cell epitope. Briefly, each peptide is incubated with its specific HLA molecule in an in vitro assay. Binding strength is compared against a known, immunogenic peptide for the same HLA molecule as a positive control with the positive control binding score set to 100%. The sequence- optimized binding score is normalized to the control peptide. That is, each peptide was given a score relative to the positive control peptide, which is a known T cell epitope with very strong binding properties. The score of the heteroclitic test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide. Peptides with scores greater than or equal to 45% of the positive control are considered binders. Also provided in Table 96B are the percent expression of each gene in patients with uterine cancer (The Cancer Genome Atlas (TCGA) database), the HLA allele being tested, and whether the wild-type peptide corresponding to each heteroclitic peptide is known to be immunogenic. For a construct including each of the heteroclitic peptides in Table 96B, 100% of uterine cancer patients with HLA type A*02:01 express at least one of the TAA genes, 83% of uterine cancer patients with HLA type A*03:01 express at least one of the TAA genes, 100% of uterine cancer patients with HLA type A*24:02 express at least one of the TAA genes, and 100% of uterine cancer patients with HLA type B*07:02 express at least one of the TAA genes.

[001174] Table 96B. Binding Affinities of Heteroclitic 9-Mers to HLA.

# - NetMHC4.0

^Λ - % relative to positive control peptide binding

¹ - SEQ ID NO: 798

² - SEQ ID NO: 820

[001175] Constructs were designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide. In some constructs, the ubiquitin was fused to the STEAP1_A0201 heteroclitic peptide. In some of the constructs, the ubiquitin was fused to the STEAP_A2402 heteroclitic peptide. FLAG tags and SIINFEKL tags were also included upstream of the ubiquitin. The tLLO, hotspot peptide, heteroclitic peptide, and ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37.

[001176] Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) include the following: (1) Uterine HOT EV02 EAAAK.G4S (SEQ ID NO: 896); (2) Uterine HOT EV02 EAAAK. i20 (SEQ ID NO: 897); and (3) Uterine A24 HOT (SEQ ID NO: 904). A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 97-99. For the A24 construct, the 9-mer in the minigene was replaced by an A24 9mer. A24 (HLA-A2402) is the HLA type commonly found in Asia. G4S, EAAAK, and i20 refer to inclusion of flexible linkers, rigid linkers, and immunoproteasome processing linkers, respectively.

[001177] Table 97. Positions of Components of Uterine HOT EV02 EAAAk.G4S

Insert.

[001179] Table 99. Positions of Components of Uterine A24 HOT Insert.

Ovarian Cancer Hotspot/Heteroclitic/Minigene Constructs

[001180] A total of 12 hotspot mutations across 1 gene were selected as described in Example 4 and elsewhere herein for the ovarian cancer ADXS-HOT constructs. This panel of hotspot mutations covers 25% of all ovarian cancer patients (i.e., 25% of ovarian cancer patients will have at least one of the hotspot mutations from the panel). For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table

100. The hotspot mutation in each is bolded and underlined.

[001181 ] Table 100. Exemplary Ovarian Cancer Panel Hotspot 21-Mers.

[001182] A total of 14 peptides with heteroclitic mutations across 8 genes were selected for the ovarian cancer ADXS-HOT constructs. For each heteroclitic mutation, a peptide of 9 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 101. The heteroclitic mutation in each is as described in Table 140.

[001183] Table 101. Exemplary Ovarian Cancer Panel Heteroclitic 9-Mers.

[001184] The in silico predicted binding affinity and in vitro binding affinity of the heteroclitic 9-mer peptides are provided in Table 101B. The in silico predicted binding affinity is based on the NetMHC4.0 algorithm, which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values (nM); a lower number reflects stronger predicted binding affinity. The in vitro binding affinity was determined through a binding assay that determines the ability of each candidate peptide to bind to the indicated MHC class I alleles and stabilize the MHC-peptide complex by comparing the binding to that of a high affinity T cell epitope. Briefly, each peptide is incubated with its specific HLA molecule in an in vitro assay. Binding strength is compared against a known, immunogenic peptide for the same HLA molecule as a positive control with the positive control binding score set to 100%. The sequence- optimized binding score is normalized to the control peptide. That is, each peptide was given a score relative to the positive control peptide, which is a known T cell epitope with very strong binding properties. The score of the heteroclitic test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide. Peptides with scores greater than or equal to 45% of the positive control are considered binders. Also provided in Table 101B are the percent expression of each gene in patients with ovarian cancer (The Cancer Genome Atlas (TCGA) database), the HLA allele being tested, and whether the wild-type peptide corresponding to each heteroclitic peptide is known to be immunogenic. For a construct including each of the heteroclitic peptides in Table 101B, 100% of ovarian cancer patients with HLA type

A*02:01 express at least one of the TAA genes, 83% of ovarian cancer patients with HLA type A*03:01 express at least one of the TAA genes, 100% of ovarian cancer patients with HLA type A*24:02 express at least one of the TAA genes, and 100% of ovarian cancer patients with HLA type B*07:02 express at least one of the TAA genes.

[001185] Table 101B. Binding Affinities of Heteroclitic 9-Mers to HLA.

# - NetMHC4.0

^Λ - % relative to positive control peptide binding

¹ - SEQ ID NO: 798

² - SEQ ID NO: 820

[001186] Constructs were designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide. The ubiquitin was fused to the

STEAP1_A0201 heteroclitic peptide. FLAG tags and SIINFEKL tags were also included upstream of the ubiquitin. The tLLO, hotspot peptide, heteroclitic peptide, and

ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37.

[001187] Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) include the following: (1) Ovarian HOT EV02 EAAAK.G4S (C) (SEQ ID NO: 898); and (2) Ovarian HOT EV02 ΕΑΑΑΚ.Ϊ20 (C) (SEQ ID NO: 899). A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 102-103. G4S, EAAAK, and i20 refer to inclusion of flexible linkers, rigid linkers, and immunoproteasome processing linkers, respectively.

[001188] Table 102. Positions of Components of Ovarian HOT EV02 EAAAK.G4S

(C) Insert.

Low- Grade Glioma (LGG) Hotspot/Heteroclitic/Minigene Constructs

[001190] A total of 11 hotspot mutations across 5 genes were selected as described in Example 4 and elsewhere herein for the low-grade glioma (LGG) ADXS-HOT constructs. This panel of hotspot mutations covers 80% of all low-grade glioma patients (i.e., 80% of low-grade glioma patients will have at least one of the hotspot mutations from the panel). For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 104. The hotspot mutation in each is bolded and underlined. [001191 ] Table 104. Exemplary LGG Panel Hotspot 21-Mers.

[001192] A total of 10 peptides with heteroclitic mutations across 8 genes were selected for the LGG ADXS-HOT constructs. For each heteroclitic mutation, a peptide of 9 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 105. The heteroclitic mutation in each is as described in Table 140.

[001193] Table 105. Exemplary LGG Panel Heteroclitic 9-Mers.

[001194] The in silico predicted binding affinity and in vitro binding affinity of the heteroclitic 9-mer peptides are provided in Table 105B. The in silico predicted binding affinity is based on the NetMHC4.0 algorithm, which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values (nM); a lower number reflects stronger predicted binding affinity. The in vitro binding affinity was determined through a binding assay that determines the ability of each candidate peptide to bind to the indicated MHC class I alleles and stabilize the MHC-peptide complex by comparing the binding to that of a high affinity T cell epitope. Briefly, each peptide is incubated with its specific HLA molecule in an in vitro assay. Binding strength is compared against a known, immunogenic peptide for the same HLA molecule as a positive control with the positive control binding score set to 100%. The sequence- optimized binding score is normalized to the control peptide. That is, each peptide was given a score relative to the positive control peptide, which is a known T cell epitope with very strong binding properties. The score of the heteroclitic test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide. Peptides with scores greater than or equal to 45% of the positive control are considered binders. Also provided in Table 105B are the percent expression of each gene in patients with low-grade glioma (LGG) (The Cancer Genome Atlas (TCGA) database), the HLA allele being tested, and whether the wild-type peptide corresponding to each heteroclitic peptide is known to be immunogenic. For a construct including each of the heteroclitic peptides in Table 105B, 100% of LGG patients with HLA type A*02:01 express at least one of the TAA genes, 43% of LGG patients with HLA type A*03:01 express at least one of the TAA genes, 100% of LGG patients with HLA type A*24:02 express at least one of the TAA genes, and 100% of LGG patients with HLA type

B*07:02 express at least one of the TAA genes.

[001195] Table 105B. Binding Affinities of Heteroclitic 9-Mers to HLA.

# - NetMHC4.0

^Λ - % relative to positive control peptide binding

[001196] Constructs were designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide. The ubiquitin was fused to the

NUF2_A0201 heteroclitic peptide. FLAG tags and SIINFEKL tags were also included upstream of the ubiquitin. The tLLO, hotspot peptide, heteroclitic peptide, and ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37.

[001197] Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) include the following: (1) LGG HOT EV02 EAAAK. G4S NUF minigene (C) (SEQ ID NO: 900); and (2) LGG HOT EV02 EAAAK. i20_NUF minigene (C) (SEQ ID NO: 901). A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 106-107. G4S, EAAAK, and 120 refer to inclusion of flexible linkers, rigid linkers, and immunoproteasome processing linkers, respectively.

[001198] Table 106. Positions of Components of LGG HOT EV02 EAAAK.G4S NUF minigene (C) Insert.

Colorectal Cancer (CRC) Hotspot/Heteroclitic/Minigene Constructs

[001200] A total of 12 hotspot mutations across 4 genes were selected as described in Example 4 and elsewhere herein for the colorectal cancer (CRC) ADXS-HOT constructs. This panel of hotspot mutations covers 58% of all micro satellite stable (MSS) colorectal cancer patients (i.e., 58% of MSS colorectal cancer patients will have at least one of the hotspot mutations from the panel). For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 108. The hotspot mutation in each is bolded and underlined.

[001201 ] Table 108. Exemplary CRC Panel Hotspot 21-Mers.

[001202] A total of 10 peptides with heteroclitic mutations across 8 genes were selected for the CRC ADXS-HOT constructs. For each heteroclitic mutation, a peptide of 9 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 109. The heteroclitic mutation in each is as described in Table 140.

[001203] Table 109. Exemplary CRC Panel Heteroclitic 9-Mers.

[001204] The in silico predicted binding affinity and in vitro binding affinity of the heteroclitic 9-mer peptides are provided in Table 109B. The in silico predicted binding affinity is based on the NetMHC4.0 algorithm, which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values (nM); a lower number reflects stronger predicted binding affinity. The in vitro binding affinity was determined through a binding assay that determines the ability of each candidate peptide to bind to the indicated MHC class I alleles and stabilize the MHC-peptide complex by comparing the binding to that of a high affinity T cell epitope. Briefly, each peptide is incubated with its specific HLA molecule in an in vitro assay. Binding strength is compared against a known, immunogenic peptide for the same HLA molecule as a positive control with the positive control binding score set to 100%. The sequence- optimized binding score is normalized to the control peptide. That is, each peptide was given a score relative to the positive control peptide, which is a known T cell epitope with very strong binding properties. The score of the heteroclitic test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide. Peptides with scores greater than or equal to 45% of the positive control are considered binders. Also provided in Table 109B are the percent expression of each gene in patients with colorectal cancer (The Cancer Genome Atlas database), the HLA allele being tested, and whether the wild-type peptide corresponding to each heteroclitic peptide is known to be immunogenic. For a construct including each of the heteroclitic peptides in Table 109B, 100% of colorectal cancer patients with HLA type A*02:01 express at least one of the TAA genes, 98% of colorectal cancer patients with HLA type A*03:01 express at least one of the TAA genes, 100% of colorectal cancer patients with HLA type A*24:02 express at least one of the TAA genes, and 98% of colorectal cancer patients with HLA type B*07:02 express at least one of the TAA genes.

[001205] Table 109B. Binding Affinities of Heteroclitic 9-Mers to HLA.

# - NetMHC4.0

^Λ - % relative to positive control peptide binding

[001206] Constructs were designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide. The CEACAM5_B0702 was included twice. The ubiquitin was fused to the STEAP1_A0201 heteroclitic peptide. FLAG tags and SIINFEKL tags were also included upstream of the ubiquitin. The tLLO, hotspot peptide, heteroclitic peptide, and ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37.

[001207] Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) include the following: (1) CRC MSS EV02 EAAAK.G4S (C) (SEQ ID NO: 902); and (2) CRC MSS EV02 ΕΑΑΑΚ.Ϊ20 (C) (SEQ ID NO: 903). A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 110-111. G4S, EAAAK, and i20 refer to inclusion of flexible linkers, rigid linkers, and immunoproteasome processing linkers, respectively.

[001208] Table 110. Positions of Components of CRC MSS EV02 EAAAK.G4S (C) Insert.

14-34: BRAF_V600E 248-268: TP53_R273C 410- ■418: CEACAM5_A2402

40-60: KRAS_G12D 274-294: TP53_R282W 424- ■432: NYESOl_A0201

66-86: KRAS_G12V 300-320: TP53_R273H 438- ■446: CEACAM5_B0702

92-112: TP53_R175H 326-334: 452- ■460: RNF43_B0702

MAGEA3_A0201_A2402

118-138: KRAS_G13D 474- ■494: FLAG

340-348: CEACAM5_A0301

144-164: PIK3CA_E545K 495- ■514: Linker-SIINFEKL

354-362: MAGEA6_A0301

170-190: KRAS_G12C 520- ■594: Ubiquitin

196-216: 368-376: CEACAM5_B0702

595- ■603: STEAP1_A0201_MINI

PIK3CA_H1047R 382-390: MAGEA4_B0702

222-242: TP53 R248W 396-404: GAGE 1 B0702 [001209] Table 111. Positions of Components of CRC MSS EV02 ΕΑΑΑΚ.Ϊ20 (C) Insert.

14-34: BRAF_V600E 248-268: TP53_R273C 459-467: CEACAM5_A2402

40-60: KRAS_G12D 274-294: TP53_R282W 480-488: NYESOl_A0201

66-86: KRAS_G12V 300-320: TP53_R273H 501-509: CEACAM5_B0702

92-112: TP53_R175H 333-341: 522-530: RNF43_B0702

MAGEA3_A0201_A2402

118-138: KRAS_G13D 551-571: FLAG

354-362: CEACAM5_A0301

144-164: PIK3CA_E545K 572-591: Linker-SIINFEKL

375-383: MAGEA6_A0301

170-190: KRAS_G12C 598-672: Ubiquitin

196-216: 396-404: CEACAM5_B0702

673-681: STEAP1 A0201 MINI

PIK3CA_H1047R 417-425: MAGEA4_B0702

222-242: TP53 R248W 438-446: GAGE 1 B0702

[001210] To assess the expression of tLLO-antigenic-peptide fusion proteins by Lmdda Listeria constructs, the DNA constructs were generated as described elsewhere herein and transformed into Lmdda. Each individual Lmdda construct was assayed by Western blot for tLLO fusion polypeptide expression using an anti-FLAG antibody. Figure 44 shows expression and secretion of the tLLO fusion polypeptide into supernatant by Lmdda for the CRC MSS EV02 EAAAK.G4S and CRC MSS EV02 ΕΑΑΑΚ.Ϊ20 constructs. The constructs visualized on these Western blots fall between 103-125 kDa and are similarly sized to the majority of constructs found within this filing. While these sizes indicate fairly large proteins, the expression data shown demonstrate the ability of the LmddA strain to express and secrete the HOT constructs at levels that should be more than sufficient for antigen processing.

Head and Neck Cancer Hotspot/Heteroclitic/Minigene Constructs

[001211 ] A total of 17 hotspot mutations across 9 genes were selected as described in Example 4 and elsewhere herein for the head and neck cancer ADXS-HOT constructs. This panel of hotspot mutations covers 34% of all head and neck cancer patients (i.e., 34% of head and neck cancer patients will have at least one of the hotspot mutations from the panel). For each recurrent cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. However, the

RGPD8_P1760A peptide was 16 amino acid in length because the hotspot mutation was near the C-terminus of the protein. The peptides are shown in Table 112. The hotspot mutation in each is bolded and underlined. [001212] Table 112. Exemplary Head and Neck Cancer Panel Hotspot 21-Mers.

[001213] A total of 10 peptides with heteroclitic mutations across 6 genes were selected for the head and neck cancer ADXS-HOT constructs. For each heteroclitic mutation, a peptide of 9 amino acids in length was designed as described in Example 4 and elsewhere herein. The peptides are shown in Table 113. The heteroclitic mutation in each is as described in

Table 140.

[001214] Table 113. Exemplary Head and Neck Cancer Panel Heteroclitic 9-Mers.

[001215] The in silico predicted binding affinity and in vitro binding affinity of the heteroclitic 9-mer peptides are provided in Table 113B. The in silico predicted binding affinity is based on the NetMHC4.0 algorithm, which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values (nM); a lower number reflects stronger predicted binding affinity. The in vitro binding affinity was determined through a binding assay that determines the ability of each candidate peptide to bind to the indicated MHC class I alleles and stabilize the MHC-peptide complex by comparing the binding to that of a high affinity T cell epitope. Briefly, each peptide is incubated with its specific HLA molecule in an in vitro assay. Binding strength is compared against a known, immunogenic peptide for the same HLA molecule as a positive control with the positive control binding score set to 100%. The sequence- optimized binding score is normalized to the control peptide. That is, each peptide was given a score relative to the positive control peptide, which is a known T cell epitope with very strong binding properties. The score of the heteroclitic test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide. Peptides with scores greater than or equal to 45% of the positive control are considered binders. Also provided in Table 113B are the percent expression of each gene in patients with head and neck cancer (The Cancer Genome Atlas database), the HLA allele being tested, and whether the wild-type peptide corresponding to each heteroclitic peptide is known to be immunogenic. For a construct including each of the heteroclitic peptides in Table 113B, 100% of head and neck cancer patients with HLA type A*02:01 express at least one of the TAA genes, 100% of head and neck cancer patients with HLA type A*03:01 express at least one of the TAA genes, 100% of head and neck cancer patients with HLA type A*24:02 express at least one of the TAA genes, and 100% of head and neck cancer patients with HLA type B*07:02 express at least one of the TAA genes.

[001216] Table 113B. Binding Affinities of Heteroclitic 9-Mers to HLA.

# - NetMHC4.0

^Λ - % relative to positive control peptide binding

[001217] Constructs were designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides and one or more or all of the heteroclitic peptides, with the C-terminal heteroclitic peptide following a ubiquitin peptide. The ubiquitin was fused to the

STEAP1_A0201 heteroclitic peptide. FLAG tags and SIINFEKL tags were also included upstream of the ubiquitin. The tLLO, hotspot peptide, heteroclitic peptide, and

ubiquitin/heteroclitic peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37.

[001218] Exemplary fusion polypeptide insert sequences (i.e., the peptide sequence downstream of the tLLO) include the following: (1) Head & Neck HOT EV02 EAAAK.G4S (SEQ ID NO: 918); and (2) Head & Neck HOT EV02 ΕΑΑΑΚ.Ϊ20 (SEQ ID NO: 919). A breakdown of the amino acids positions of the individual components in each construct is provided in Tables 114-115. G4S, EAAAK, and i20 refer to inclusion of flexible linkers, rigid linkers, and immunoproteasome processing linkers, respectively.

[001219] Table 114. Positions of Components of Head & Neck HOT EV02 EAAAK.G4S Insert.

14-34: PIK3CA_E545K 274-294: ANKRD36C_I634T 493-501: CEACAM5_A0201

40-60: PIK3CA_E542K 300-320: KRTAP1-5_I88T 507-515: PRAME_A0201

66-86: TP53_R248Q 326-346: KRTAP4-11_L161V 521-529:

hTERT_A0201_A2402

92-112: TP53_R175H 352-372: TP53_G245S

535-543: STEAP1_A2402

118-138: PIK3CA_H1047R 378-398: TP53_R273H

549-557: CEACAM5_A2402

144-164: CHEK2_K373E 404-424: HRAS_G13V

563-571: NYESOl_B0702

170-190: TP53_R282W 430-445: RGPD8_P1760A

585-605: FLAG

196-216: TP53_Y220C 451-459: CEACAM5_A0301

606-625: Linker-SIINFEKL

222-242: TP53_R248W 465-473: CEACAM5_B0702

632-706: Ubiquitin

248-268: ZNF814 D404E 479-487: MAGEA4 B0702

707-715: STEAP1_A0201_MINI

[001220] Table 115. Positions of Components of Head & Neck HOT EV02

ΕΑΑΑΚ.Ϊ20 Insert.

14-34: PIK3CA_E545K 274-294: ANKRD36C_I634T 521-529: CEACAM5_A0201

40-60: PIK3CA_E542K 300-320: KRTAP1-5_I88T 542-550: PRAME_A0201

66-86: TP53_R248Q 326-346: KRTAP4-11_L161V 563-571:

hTERT_A0201_A2402

92-112: TP53_R175H 352-372: TP53_G245S

584-592: STEAP1_A2402

118-138: PIK3CA_H1047R 378-398: TP53_R273H

605-613: CEACAM5_A2402

144-164: CHEK2_K373E 404-424: HRAS_G13V

626-634: NYESOl_B0702

170-190: TP53_R282W 430-445: RGPD8_P1760A

655-675: FLAG

196-216: TP53_Y220C 458-466: CEACAM5_A0301

676-695: Linker-SIINFEKL

222-242: TP53_R248W 479-487: CEACAM5_B0702

702-776: Ubiquitin

248-268: ZNF814 D404E 500-508: MAGEA4 B0702

777-785: STEAP1_A0201_MINI dMMR Hotspot Constructs

[001221 ] DNA mismatch repair is a biological mechanism that identifies and repairs genetic mismatches during DNA replication. Deficient DNA Mismatch Repair (dMMR) results in the inability to repair DNA mismatches arising during replication. Patients with dMMR generally develop micro satellite instability high (MSI-H) due to frequent errors during DNA replication that give rise to high mutation rates. These high mutation tumors are excellent targets for immunotherapy as each mutation can be presented by the MHC system and recognized as foreign by a T cell. dMMR is associated with loss of four proteins: PMS2, MLHl, MSH6, and MSH2.

[001222] To design the dMMR panel, we selected all patients in TCGA across numerous tumor types. We filtered for patients that harbored a mutation in PMS2, MLHl, MSH6 and MSH2, as patients with mutations in those genes are more likely to be MSI. The frequency of all somatic mutations within this cohort were then calculated, and the top mutation with frequencies above 4.5% were selected.

[001223] A total of 28 hotspot mutations across 26 genes were selected as described in Example 4 and elsewhere herein for the DNA mismatch repair deficient (dMMR) ADXS- HOT constructs. This panel of hotspot mutations covers 54% of all DNA mismatch repair deficient cancer patients (i.e., 54% of DNA mismatch repair deficient cancer patients will have at least one of the hotspot mutations from the panel). For each recurrent missense cancer mutation included in the constructs, a peptide of 21 amino acids in length was designed as described in Example 4 and elsewhere herein. For each frameshift mutation included in the constructs, longer peptides were designed to include the predicted peptide sequence arising from the out-of- frame INDEL substitution. The peptides are shown in

Table 116. The hotspot mutation in each is bolded and underlined.

[001224] Table 116. Exemplary dMMR Panel Hotspot Peptides.

dMMR Panel Hotspot Peptides

SEQ ID

Gene Hotspot Mutation Sequence

NO

LOOWVNLRRGYPRLKTFGVPLGSILCLAGSLST MAPTPPSTPMIISTTROECGRRASVPCRPMIGSA

p.Val328TyrfsTerl7 RPGPWRTSAMPSAMGVALPTSCESGRSPPATG

PLEKHA6 851

2 GRMPPSGSOAPPGSOSIMMSWMPPLAPCAACP

CSPAPTLCPAHPARAPTAVPAFTPLSAHPVPVLS

GCHLAVRTSMLTLLPM

OEDPREVLKKHWNSAYLGRTLLVTCILYHRWI

LARP4B p.Thrl63HisfsTer47 852

VTSMCOSORWLTSTTSRSSALMWT

DGDETTEPPPVGAGTGGGPACVPVAEASTGAW TAEGAAGTRCRASRAPCRPPPVPAPSOLPASSP

p.Ser336ValfsTerl3

FHOD3 TKCEICVKNTAILAITLITPODPHLDPVCPPPPH 853

8

HPSHPHRRPGWKGHHRVVFSHHPSGSTKSHW OORERGGGRREKKGCRE

p.Prol852GlnfsTer4 AFHHPLGDTPOPSLPGPCASLLSTLSOPPPOAPS

DOCK3 854

5 OVWTAATLRCPAVPAAACPP

SSTPLTIGEKTEIOLTMNDSKHKLESPALKOVSP

BMPR2 p.Asn583ThrfsTer44 855

ASPPTOOPOTPODSROVLA

p.Aspl850ThrfsTer3 GLLHWRIGGGTPLSISRPTSRAROSCCLPGLTHP

ARID 1 A 856

3 AHOPLGSM

ADAM28 p.Asn75LysfsTerl5 GKIAVLYLKKKOEPPCTRLHGNIL 857

ACVR2A p.Lys435GlufsTer!9 LEDMOEVVVHEACFKRLLAETCWNGNAL 858

[001225] A construct was designed to encode a fusion polypeptide comprising tLLO fused to the hotspot peptides. A SIINFEKL tag was also included. The tLLO and hotspot peptide components of the fusion polypeptides were joined by various linkers selected from those in Table 37. The sequence of the fusion polypeptide insert (i.e., the peptide downstream of the tLLO) is set forth in SEQ ID NO: 917 (DNA Mismatch Repair HOT EV02 EAAAK.G4S). A breakdown of the amino acids positions of the individual components in each construct is provided in Table 117. G4S and EAAAK refer to inclusion of flexible linkers and rigid linkers, respectively.

[001226] Table 117. Positions of Components of DNA Mismatch Repair HOT EV02 EAAAK.G4S Insert.

Example 12. Proof of Concept: Generation of Immunity with Low-Expressing Lm Constructs.

[001227] This study evaluated the immune response for constructs with extremely low or below the limit of detection Western blot results in C57BL/6 mice after immunization with Lm constructs. This assay examined the generation of SIINFEKL-specific immunity in mice immunized with 10 different Lm SIINFEKL-tagged constructs. Each Lm construct had a SIINFEKL tag at the C-terminus of the fusion protein (tLLO + antigens). SIINFEKL- specific immune response was detected by ex vivo stimulation of splenocytes with SIINFEKL peptide and detection of IFNy-positive cells with the ELISPOT assay. The details of immunization schedule and strains are given in Tables 118 and 119.

Treatment Schedule

[001228] Table 118. Immunization Schedule.

[001229] Table 119. Identification of Vaccine.

Vaccine Preparations

[001230] Vaccine preparation was as follows (all prepared with BHI media): (a) thawed 1 vial form -80°C in 37°C water bath; (b) spun at 14,000 rpm for 2 min and discarded supernatant; (c) washed 2 times with 1 mL PBS and discarded PBS; and (d) re-suspended to an appropriate final concentration of 5x10^s CFU/mL.

[001231 ] Table 120. Materials.

Preparing Isolated Splenocytes

[001232] (1) Harvested spleens from experimental and controls using sterile forceps and scissors. Transported in 15 mL tubes containing complete RPMI to the lab.

[001233] (2) Poured the spleens into a sterile Petri dish.

[001234] (3) Disrupted the spleens in cRPMI using the back of plunger from a 3 mL syringe.

[001235] (4) Transferred cells in the medium to a 15 mL tube, for 1 or 2 spleens or 50 mL tubes if more than two spleens.

[001236] (5) Pelleted cells at 1,000 RPM for 5 min at RT. [001237] (6) Discarded supernatant, re-suspended cells in the remaining wash buffer gently, and added 2 mL RBC lysis buffer per spleen to the cell pellet. Mixed cells gently with lysis buffer by tapping the tube and waited for 1 min.

[001238] (7) Immediately added 10 mL of c-RPMI medium to the cell suspension to deactivate lysis buffer.

[001239] (8) Spun cells at 1,000 for 5 min at RT.

[001240] (9) Passed the cells through a cell strainer and wash them one more time with 10 mL c-RPMI.

[001241 ] (10) Resuspended cell pellet in 25 mL of c-RPMI.

[001242] (11) Counted cells using hemocytometer and check the viability by PI staining. Each spleen yielded 1-2 x 10^s cells.

[001243] (12) Divided the cells for pentamer staining and ELISpot. ELISPOT Protocol

[001244] The ELISpot peptides used included peptide #1 (SIINFEKL - SEQ ID NO: 1007) and peptide #2 (PSA-9 - irrelevant peptide control and background subtraction).

[001245] On Dayl, the CTL immunospot protocol for plate coating was followed.

[001246] Antigen solutions were then set up as follows: (a) created 1 mM OVA-8

(SIINFEKL): 1: 10 dilution of 10 mM OVA-21 stock into IMDM. IE 5 μί:45 μ (b) created 2 μΜ Antigen Solution: added 2 μί/ητΕ of the 1 mM solution to the antigen solution, ~ 8 μΐ_^ of 1 mM OVA-8 to 4 mL antigen solution; (c) created 1 mM PSA-9 (irrelevant peptide): 1: 10 dilution of 10 mM PSA-9 stock into IMDM. IE 5 μL·Λ5 μ (d) created 2 μΜ Antigen Solution: added 2 μί/ητΕ of the 1 mM solution to the antigen solution, ~ 8 μΐ_^ of 1 mM PSA- 9 to 4 mL antigen solution; (e) created IX PMA/Iono: 1: 100 dilution lOOx PMA stock to antigen solution: ~ 4 μϊ_^ to 396 μΐ_^ antigen solution; and (f) added 100 μΐ_^ of each antigen solution to the appropriate wells.

[001247] Splenocytes were prepared as follows: (a) washed ~ 2xl0⁶ of each splenocyte. Resuspend in CTL test medium to concentration of 2xl0⁶/mL (~1 mL CTL medium); and (b) added 100 μΐ_^ of each splenocyte to appropriate well.

[001248] The ELISPOT protocol was then followed per kit instructions (i.e., incubated at 37°C overnight).

[001249] On Day2, the ELISPOT protocol was followed as provided below.

[001250] DAY 0 (Sterile Conditions). Prepared Capture Solution by diluting the Capture

Antibody according to specific protocol. Many cytokines benefit from pre- wetting the PVDF membrane with 70% ethanol for 30 sec and washing with 150 μΐ_^ of PBS three times before adding 80 μΐ_^ of the Capture Solution into each well. Incubated plate overnight at 4°C in a humidified chamber.

[001251 ] DAY 1 (Sterile Conditions ). Prepared CTL-Test™ Medium by adding 1 % fresh L-glutamine. Prepared antigen/mitogen solutions at 2X final concentration in CTL-Test™ Medium. Decanted plate with coating antibody from Day 0 and washed one time with 150 μί ΡΒ8. Plated antigen/mitogen solutions, 100

After thawing PBMC or isolating white blood cells with density gradient, adjusted PBMC to desired concentration in CTL- Test™ Medium, e.g., 3 million/mL corresponding to 300,000 cells/well (however, cell numbers can be adjusted according to expected spot counts since 100,000-800,000 cells/well will provide linear results). While processing PBMC and until plating, kept cells at 37°C in humidified incubator, 5-9% C0₂. Plated PBMC, 100 μΐ/ννεΐΐ using large orifice tips. Once completed, gently tapped the sides of the plate and immediately placed into a 37°C humidified incubator, 5-9% CO2. Incubated for 24-72 hours depending on your cytokine. Did not stack plates. Avoided shaking plates by carefully opening and shutting incubator door. Did not touch plates during incubation.

[001252] DAY2. Prepared Wash Solutions for the day: PBS, distilled water and Tween- PBS. Prepared Detection Solution by diluting Detection Antibody according to

specific protocol. Washed plate two times with PBS and then two times with 0.05% Tween- PBS, 200 μΐ/ννεΐΐ each time. Added 80 μΐ/ννεΐΐ Detection Solution. Incubated at RT, 2h. Prepared Tertiary Solution by diluting the Tertiary Antibody according to specific protocol. Washed plate three times with 0.05% Tween-PBS, 200 μΙ,ΛνεΙΙ. Added 80 μΙ,ΛνεΙΙ of Strep- AP Solution. Incubated at RT, 30 min. Prepared Developer Solution according to your specific protocol. Washed plate two times with 0.05% Tween-PBS, and then two times with distilled water, 200 μΐ/ννεΐΐ each time. Add Developer Solution, 80

Incubated at RT, 10-20 min. Stopped reaction by gently rinsing membrane with tap water, decanted, and repeated three times. Removed protective underdrain of the plate and rinsed back of plate with tap water. Air dried plate for 2 hours face-down in running hood or on paper towels for 24 hours on bench top. Scanned and counted plate.

Results and Conclusions

[001253] The 10 constructs with the lowest fusion protein expression levels to date (April 2017) were selected for immunization into mice. Even though the constructs were barely detectable or in some cases would have been considered "negative" for fusion protein expression by western blot, we were able to detect murine T cell responses targeting the SIINFEKL tag within the fusion protein. See Figure 20. In Figure 20, ex vivo splenocytes were stimulated with the minimal SIINFEKL peptide, which specifically binds MHC-I (H-2 K^b OVA₂57-264) in order to detect SIINFEKL specific CD8⁺ T cells. Subsequent testing revealed that the first control in Figure 20 was not a true negative control. Rather, it was a second positive control due to contamination with a construct that expressed SIINFEKL. However, all of the samples exhibited a positive T cell response.

[001254] These data demonstrate that Lm constructs with extremely low levels of fusion protein expression (or possibly non-detectable by western blot) are still capable of eliciting a T cell response following immunization with the Lm construct. In addition, these data demonstrate that Lm constructs with fusion protein expression (even low levels) elicit T cell responses following immunization with the Lm construct.

[001255]

Example 13. Inclusion of Linkers and Spacers in tLLO Fusion Proteins.

[001256] This study examined whether flanking antigenic peptides with defined linker sequences improves expression of the tLLO-antigenic-peptide fusion proteins. Secretion of the tLLO-antigenic-peptide fusion protein was detected by Western blot using anti-FLAG antibody. The preclinical data generated in these studies demonstrate that the inclusion of defined linker sequences significantly improves the expression and secretion of the tLLO- antigenic-peptide fusion protein and drastically improves the ability to generate constructs at a much higher rate of success.

[001257] In designing fusion polypeptides with multiple 21-mers (e.g., 21-mers from cancer-associated proteins with 10 amino acids flanking either side of a hotspot mutation), the sequential order of the unique 21-mers can be randomly generated in order to minimize the likelihood that the junction of two proximal 21-mers may interfere with Lm expression and secretion. However, the random assembly method may not always be dependable enough to generate constructs that express and secrete the tLLO fusion protein, as determined by anti-FLAG Western blot, with a 100% rate of success. The more 21-mers added to a construct using the "beads on a string" method significantly increases the odds that two or more 21-mer sequences will interact with one another in a way that significantly reduces, or even eliminates, the expression and secretion of the tLLO fusion protein.

[001258] Here, we describe a new plasmid design strategy used to significantly improve the expression and secretion of Lm constructs. We found that the limitations associated with the random assembly design strategy can be significantly mitigated by joining unique 21-mers by defined amino acid sequences known as linkers. Linkers, also known as spacers, are short amino acid sequences ranging from 2 to greater than 25 residues generated in nature to separate domains within a protein. There are many different classes of linkers with specific characteristics; our study focused on linkers that were designed to add rigidity, flexibility, or enhance proteasomal cleavage. The table below describes suitable linkers that were evaluated in Lm constructs:

[001259] Table 121. Linkers.

[001260] The data described in this report detail the construction of Lm constructs with a combination of rigid, flexible, and proteasomal linkers and demonstrates that expression and secretion are enhanced when linkers are present.

Materials and Methods

[001261 ] Construct Design. To generate sample constructs, target sequences were scored for hydropathy using the Kyte/Doolittle methodology with a window size of 21 amino acids (web.expasy.org/protscale/?_ga=l.215352275.536452039.1486395060), with all target sequences scoring greater than or equal to 1.6 being excluded from use as they are unlikely to be secretable by Listeria monocytogenes. All remaining target sequences were then reverse- translated into L. monocytogenes codon-optimized sequences using OPTIMIZER

(genomes.urv.es/OPTIMIZER/). Sample construct inserts were designed by concatamerizing 20 target sequences and adding a FLAG (DYKDHDGDYKDHDIDYKDDDK (SEQ ID NO: 762)) SIINFEKL tag-encoding sequences to the 3' end to generate construct inserts.

Individual insert suitability was then reconfirmed to contain no peaks at or above 1.6 by hydropathy as above using a 21-amino acid sliding window across the entirety of the insert sequence. [001262] Western Blot Screen for tLLO Fusion Polypeptide Secretion. Single colonies from plates containing Lm constructs were used to inoculate an overnight culture in 6 niL of BHI in a dry shaking incubator at 37°C. The following day, 1: 10 dilutions of the original overnight culture were resuspended in 9 mL of fresh BHI and grown in the dry shaking incubator at 37°C until reaching an OD6oo=0.6. Cells were pelleted by 2 minute

centrifugation at 13000 RPM. Sample supernatants were collected and run on SDS-PAGE. Samples were prepared by diluting 75

of sample with 25 of 4X LDS Sample Buffer (Cat#l 61-0747), boiled at 98°C for 10 minutes, placed on ice, and then centrifuged at max speed for 10 minutes at 4°C. 13

of the sample was run on 4-15% precast protein gel (BioRad Cat#4561086). Protein gels were transferred using the Trans-Blot Turbo transfer apparatus (Cat#170-4155) and PVDF Midi transfer packs (Bio-Rad #170-4157). Blots were incubated with anti-FLAG monoclonal Antibody (Sigma F1804) or Anti-LLO (Abeam ab200538) as primary and Goat Anti- mouse IgG-HRP conjugated (sc2005) as a secondary antibody. The blots were then incubated on iBind Flex (Invitrogen cat#1772866), washed, and then developed by Super Signal West Dura Extended Duration Substrate (ThermoFisher #34076); the images were developed on the Amersham Imager 600 (GE).

Results

[001263] Peptides comprising mutations from the murine mc38 tumor cell line were designed, and individual 21-mers were scored for hydropathy as per Kyte and Doolittle. The 21-mers that passed the hydropathy filter cut off were arranged either sequentially as 15 "beads-on-a-string" with a SIINFEKL minigene and a FLAG-Tag moiety added at the C- terminus (Figure 21) or with various permutations and combinations of linkers flanking each individual 21-mer (Figure 21). The addition of these C-terminal moiety tags allows for the monitoring of expression, secretion, processing and presentation, and immunogenicity of the tLLO fusion protein. Longer and slightly charged spacers (Figure 21, *) were used right after tLLO to provide additional separation between the TAA cassette and the N-terminus and C- terminus of the fusion protein as well as maintaining the net positive charge of the tLLO secretion signal. Flanking linkers (Figure 21, ^A) were used to separate individual 21-mers. Rigid, flexible, and proteasomal enhancing linkers were generated and tested in hundreds of Lm constructs. Finally, an immunoproteasome linker was placed between 3XFLAG and SIINFEKL, which allows for more efficient proteasomal processing of the C-terminal SIINFEKL tag. The immunoproteasome linker was comprised of a 12 amino acid cleavage motif, 6 C-terminal followed by 6 N-terminal, that is preferentially cleaved by the 20S subunit of the immunoproteasome. See, e.g., Toes et al. (2001) J. Exp. Med. 194(1): 1-12, herein incorporated by reference in its entirety for all purposes. The inclusion of proteasomal enhancing linkers allow for the directed liberation of a desired amino acid sequence (e.g., SIINFEKL (SEQ ID NO: 1007)) with a higher frequency than would be expected by stochastic proteasomal C-terminal cleavage and N-terminal degradation alone (Figure 21, #).

[001264] In order to evaluate the effect linkers and spacers have on the expression and secretion of the tLLO fusion protein, we generated hundreds of Lm constructs, each with unique combinations of flexible, rigid, and proteasome-enhancing linkers and spacers.

Figure 22 depicts 10 such constructs that were assayed by Western blot to determine the relative effect of linkers on expression, secretion, and overall rate of success regarding Lm product generation. The culture supernatant from the MT15 construct that was designed using only the random assembly strategy (Figure 22-Lane 2) did not yield detectible levels of tLLO fusion protein and ultimately failed the screening process. However, we could clearly detect the correct size tLLO fusion protein from a construct designed with the same randomly assembled MT15 construct (Lane 2), but with the inclusion of flexible long spacers and flexible linkers (Lane 3); this construct passed the overall screening process. In all cases, the inclusion of long spacers significantly improved the levels of detectable tLLO fusion protein compared to the base MT15 construct. Furthermore, the addition of flanking linkers to each corresponding long-spacer construct significantly enhanced tLLO fusion protein expression (data not shown) and secretion (e.g. Lane 4 vs. Lane 5). Additionally, adding flanking linkers removed the need for the brute-force random assembly design strategy. Constructs with significant differences in the levels of secreted tLLO fusion protein, resulting from only an alternate ordering of the 21-mers, showed no significant differences in tLLO fusion protein expression or secretion when designed with flanking linkers separating each unique 21-mer, regardless of 21-mer order (data not shown).

[001265] In conclusion, by incorporating spacers and linkers into the Lm construct, we can significantly improve the expression and secretion of the tLLO fusion protein. Additionally, the incorporation of linkers and spacers into Lm construct design significantly reduces the need for generating multiple randomly assembled constructs in order to increase the odds of producing a full Lm construct (e.g., that targets all desired hotspot mutations).

[001266] By adding linkers and spacers we can significantly enhance the expression and secretion of the tLLO fusion protein as well as the overall success rate of generating a construct that secretes a detectable level of tLLO fusion protein. From the hundreds of Lm constructs made with linkers and spacers, we have been able to detect tLLO fusion protein by Western blot in all cases. This is a significant improvement over the success rate of constructs designed without linkers and spacers. While we have identified effective linker and spacer combinations that yield significantly improved tLLO expression and secretion, the linkers identified in this study are not exhaustive. Collectively, these data demonstrate the feasibility of adding linkers and spacers to the Lm platform.

Example 14: Fully Enclosed Single Use Cell Growth System

[001267] The innovative system leverages readily available bioprocessing components and technologies arranged in a unique configuration to grow the engineered Lm bacteria, concentrate the fermentation broth, wash and purify the cells, exchange the fermentation media for formulation buffer, and dispense the patient- specific doses into ready-to-use IV bags using a single fully enclosed system. This type of system provides a complete segregation and control of each patient's immunotherapy. This system is particularly well suited for integration in the overall work stream of identification and clinical use of personalized neo-epitope targeting immunotherapeutics. See Figures 23A-23B.

[001268] The custom designed system is assembled using single use bioprocessing bags, patient IV bags, sampling bags, tubing, filters, quick connectors, and sensors. Its small footprint allows manufacture for an individual patient but can be replicated to manufacture product for multiple patients in parallel. See Figure 24. The entire assembly is comprised of 4 sections: (1) Inoculation and Fermentation; (2) Concentration; (3) Diafiltration; and (4) Drug Product Fill. Since the system has a fully enclosed fluid flow path and is sterilized prior to use, final formulated immunotherapies are dispensed directly into IV bags, frozen and shipped to the healthcare center. Therefore, this eliminates the need for the typical fill/finish and packaging involved when dispensing into vials or pre-filled syringes. This addresses the expectation for rapid turnaround and delivery to the patient.

[001269] The Inoculation and Fermentation section of the assembly is filled with growth media and warmed to the specified temperature. See Figure 25. The cell bank is then inoculated into either a single use rocking style bag fermentor or into a single use agitated bioreactor vessel. Once the bacteria grow to a specific density, the Concentration section of the assembly is used to remove the fermentation media and concentrate the batch using a hollow fiber filter. See Figure 26. A wash/formulation buffer bag is connected to the Diafiltration section of the assembly and the bacterial cells are washed/purified, the remaining media are exchanged with formulation buffer via a cross flow filtration in the hollow fiber filter, and the product is diluted to the final concentration. See Figure 27. Finally, the batch is aliquoted into sterile single use IV bags and sampling bags for QC testing using the Drug Product Fill section of the assembly. See Figure 28. The patient- specific immunotherapy is supplied frozen in a small volume parenteral IV bag containing a pure culture strain of the live attenuated engineered Lm bacteria at a specified concentration. Prior to patient administration, the IV bag is thawed, cells re-suspended, and the required dose withdrawn with a syringe and added to the larger infusion IV bag.

[001270] Several fully enclosed assemblies are used in parallel to manufacture personalized immunotherapeutic compositions either for several patients or for a single patient. See

Figures 29A-29B. In order to increase throughput, additional rockers or agitated vessel bioreactors systems are added to the processing train, as required. See e.g. Figure 24.

[001271 ] The fully enclosed design of the growth system allow complete quality control of immunotherapeutic compositions while in the process of manufacture, resulting in additional time savings. A full analytical control strategy is implemented in parallel with growing Listeria delivery vector. See Table 122. Thus the dispensed product is ready for immediate delivery to the patient with no additional testing required.

[001272] Table 122. Analytical Control Strategy

Example 15: Construction of a Neoepitope Expression Vector

[001273] Constructing an Lm vector comprising one or more neoepitopes is performed using the steps detailed below.

Whole Genome Sequencing

[001274] First, comparative whole genome sequencing including locating nonsynonymous mutations present in approximately >20% of tumor cells is performed and the results are provided in FASTA format. Matched normal/tumor samples from whole exomes are sequenced by an outside vendor, and output data is given in the preferred FASTA format listing all neoantigens as 21-amino acid sequence peptides (e.g., peptide having 10 non- mutant amino acids on either side of a mutant amino acid). Also included are patient HLA types.

[001275] DNA and RNA from a biological sample obtained from human tissue (or any non- human animal) are extracted in triplicates. Another source of neoantigens could be from sequencing metastases or circulating tumor cells. They may contain additional mutations that are not resident in the initial biopsy but could be included in the vector to specifically target cytotoxic T cells (CTCs) or metastases that have mutated differently than the primary biopsy that was sequenced. Triplicates of each sample are sequenced by DNA exome sequencing. In brief, 3 μg purified genomic DNA (gDNA) are fragmented to about 150-200 bp using an ultrasound device. Fragments are end repaired, 5' phosphorylated, 3' adenylated, and then Illumina paired end adapters are ligated to the gDNA fragments according to the

manufacturer's instructions. Enriched pre capture and flow cell specific sequences are added using Illumina PE PCR primers. About 500 ng of adapter ligated, PCR enriched gDNA fragments are hybridized to biotinylated exome (human exome or any other non-human animal exome such as mouse, guinea pig, rat, dog, or sheep). RNA library baits for 24 hr at 65°C. Hybridized gDNA/RNA bait complexes are then removed using streptavidin coated magnetic beads, washed and the RNA baits cleaved off. These eluted gDNA fragments are PCR amplified and then sequenced on an Illumina sequencing apparatus.

RNA Gene Expression Profiling (RNA-Seq)

[001276] Barcoded mRNA-seq cDNA libraries are prepared in triplicates from a total of about 5 μg of total RNA, then, in brief, mRNAs are isolated and fragmented. Following, mRNA fragments are converted to cDNA and connected to specific Illumina adaptors, clustered and sequenced according to standard illumine protocol. The output sequence reads are aligned to a referenced sequence (RefSeq). Genome alignments and transcriptome alignments are made. Reads are also aligned to exon-exon junctions. Expression values are determined by intersecting read coordinates with those of RefSeq transcripts, counting overlapping exon and exon junction reads, and normalized to standard normalizing units such as RPKM expression units (Reads which map per Kilobase of transcript per Million mapped reads).

Detecting Mutations

[001277] Fragments of isolated gDNA from a disease or condition bearing tissue sample are aligned to referenced matched gDNA of a healthy tissue by vendor available software such as Samtools, GATK, or Somatic Sniper.

[001278] About 10 flanking amino acids on each side of the detected mutation are incorporated to accommodate class 1 MHC-1 presentation, in order to provide at least some of the different HLA TCR reading frames.

[001279] Table 123 shows a sample list of 50 neoepitope peptides wherein each mutation is indicated by a bolded and underlined amino acid letter and is flanked by 10 amino acids on each side providing a 21 amino acid peptide neoepitope.

[001280] Table 123. Neoepitope Peptides.

Name Sequence SEQ ID NO:

MUT27 VELCPGNKYEMRRHGTTHSLV 2836

MUT28 GIDKLTQLKKPFLVNNKINKI 2837

MUT29 GTTILNCFHDVLSGKLSGGS 2838

MUT30 PSFQEFVDWENVSPELNSTDQ 2839

MUT31 PALVEEYLERGNFVANDLDWL 2840

MUT32 ELKACKPNGKRNPYCEVSMGS 2841

MUT33 SPFPAAVILRDALHMARGLKY 2842

MUT34 QQLDTYILKNVVAFSRTDKYR 2843

MUT35 SFVGQTRVLMINGEEVEETEL 2844

MUT36 AFFINFIAIYHHASRAIPFGT 2845

MUT37 GLALPNNYCDVCLGDSKINKK 2846

MUT38 EGQISIAKYENCPKDNPMYYC 2847

MUT39 NFKRKRVAAFO_KNLIEMSELE 2848

MUT40 KMKGELGMMLILQNVIQKTTT 2849

MUT41 SIECKGIDKEINESKNTHLDI 2850

MUT42 ELEAAIETVVCTFFTFAGREG 2851

MUT43 SLSHREREQMKATLNYEDHCF 2852

MUT44 HIKAFDRTFANNPGPMVVFAT 2853

MUT45 ITSNFVIPSEYWVEEKEEKQK 2854

MUT46 GLVTFQAFIDVMSRETTDTDT 2855

MUT47 HLLGRLAAIVGKQVLLGRKVV 2856

MUT48 HWNDLAVIPAGVVHNWDFEPR 2857

MUT49 SMDHKTGTIAMQNTTQLRSRY 2858

MUT50 QPLRRLVLHVVSAAQAERLAR 2859

[001281 ] Output FASTA file is used to design patient- specific constructs, either manually or by programmed script according to one or more of criteria detailed below. The programmed script automates the creation of the personalized plasma construct containing one or more neoepitopes for each subject using a series of protocols. See Figure 30. The output FASTA file is inputted, and, after running the protocols, the DNA sequence of an Lm vector including one or more neoepitopes is outputted. The software program is useful for creating personalized immunotherapy for each subject.

Prioritization of Neoepitopes for Incorporation into Constructs.

[001282] Neoepitopes are scored by a Kyte and Doolittle hydropathy index 21 amino acid window, and all scoring above cutoff (around 1.6) are excluded as they are unlikely to be secretable by Listeria monocytogenes. The remaining 21 -amino acid long peptides are then scored for their ability to bind patient HLA (for example by using IED, Immune epitope database and analysis source, iedb.org/) and ranked by best MHC binding score from each 21 -amino acid sequence peptide. Cutoffs may be different for different expression vectors such as Salmonella.

[001283] Determination of the number of constructs vs. mutational burden are performed to determine efficiency of expression and secretion of neoepitopes. Ranges of linear neoepitopes are tested, starting with about 50 epitopes per vector. In certain cases, constructs will include at least one neoepitope per vector. The number of vectors to be used is determined considering for example the efficiency of translation and secretion of multiple epitopes from a single vector, and the MOI needed for each Lm vector harboring specific neoepitopes, or in reference to the number of neoepitopes. Another consideration can be by predefining groups of known tumor-associated mutations/mutations found in circulating tumor cells/known cancer "driver" mutations/known chemotherapy resistance mutations and giving them priority in the 21 -amino acid sequence peptide selection. This can be accomplished by screening identified mutated genes against the COSMIC (Catalogue of somatic mutations in cancer, cancer.Sanger.ac.uk) or Cancer Genome Analysis or other similar cancer-associated gene database. Further, screening for immunosuppressive epitopes (T-reg epitopes, IL-10 inducing T helper epitopes, etc.) can be utilized to deselect neoepitopes or to avoid immunosuppressive influences on the vector. Selected codons are codon optimized to efficient translation and secretion according to specific Listeria strain. Example for codons optimized for L. monocytogenes is presented in Table 124.

[001284] Table 124. Listeria monocytogenes Preferred Codon Table

[001285] The remaining 21 -amino acid peptide neoepitopes are assembled into a pAdvl34- MCS (SEQ ID NO: 2805) plasmid, or optionally into pAdvl34, exchanging the LLO-E7 cassette to create the tLLO-neoepitope-tag fusion polypeptide. The compatible insert as an amino acid sequence and the whole insert are rechecked by Kyte and Doolittle test to confirm no hydropathy problems across the whole construct. If needed, the insert order is rearranged or the problem 21 -amino acid sequence peptides are removed from the construct.

[001286] The construct amino acid sequence is reverse translated into the corresponding DNA sequence for DNA synthesis/cloning into pAdvl34-MCS (SEQ ID NO: 2805; multi- cloning site by outside vendor is at residues 2400-2453). Individual 21-amino acid peptides sequences and the SIINFEKL-6xHis tag DNA sequences (for example SEQ ID NO: 2806) are optimized for expression and secretion in L. monocytogenes while the 4x glycine linker sequences are one of eleven preset DNA sequences (Gl-Gl 1, SEQ ID NO: 572-582). Linker sequence codons are varied to avoid excess repetition to better enable DNA synthesis. Examples of the different sequence codons (Gl-Gl l, SEQ ID NO: 572-582) for 4x gly linkers are presented in Table 125.

[001287] Table 125. 4x Glycine Linker DNA and Terminal Tag Sequences

[001288] Each neoepitope is connected with a linker sequence to the following neoepitope encoded on the same vector. The final neoepitope in an insert is fused to a TAG sequence followed by a stop codon. The TAG fused is set forth in SEQ ID NO: 2806, a C-terminal SIINFEKL and 6xHis amino acid sequence. The TAG allows for easy detection of the tLLO- neoepitope during for example secretion from the Lm vector or when testing constructs for affinity to specific T-cells, or presentation by antigen-presenting cells. The linker is a 4Xglycine DNA sequence, selected from a group comprising Gl-Gl l (SEQ ID NO: 572- 582) or any combination thereof.

[001289] If there are more usable 21 -amino acid peptides than can fit into a single plasmid, the different 21-amino acid peptides are designated into 1^st, 2^nd, etc. constructs by priority rank as needed/desired. The priority of assignment to one of multiple vectors composing the entire set of desired neoepitopes can be determined based on factors like relative size, priority of transcription, and overall hydrophobicity of the translated polypeptide.

[001290] One exemplary construct structure disclosed herein comprises a nucleic acid sequence encoding an N-terminal LLO fragment fused to one or more 21-mer neoepitope amino acid sequences flanked by a linker sequence and followed by at least one second neoepitope flanked by another linker and terminated by a SIINFEKL-6xHis tag-and 2 stop codons closing the open reading frame: pH/ >-tLLO-21mer #l-4x glycine linker Gl-21mer #2-4x glycine linker G2-...-SIINFEKL-6xHis tag-2x stop codon. The construct' s expression can be driven by an hly gene promoter sequence or other suitable promoter sequence. Each 21-mer neoepitope sequence may also be fused to an immunogenic polypeptide such as a tLLO, truncated ActA, or a PEST amino acid sequence as disclosed herein. [001291 ] Different linker sequences can be distributed between and among the neoepitopes for minimizing repeats. This reduces possible secondary structures, thereby allowing efficient transcription, translation, secretion, maintenance, or stabilization of the plasmid including the insert within the Lm recombinant strain population.

[001292] DNA synthesis can be achieved by ordering a nucleotide sequence from a vendor comprising the construct including the open reading frame comprising the PEST-containing peptide (e.g., tLLO or tActA or ActA) fused to at least one neoepitope. Additionally or alternatively, multiple neoepitopes can be separated by one or more linker 4xglycine sequences. Additionally or alternatively, inserts can be constructed to comprise the desired sequence by molecular biology techniques such as by sewing PCR with specific overlapping primers and specific primers, or ligating different nucleotide sequences by an appropriate enzyme (e.g., ligase), optionally following dissection by restriction enzymes. Selected DNA inserts can also be synthesized by other known techniques (e.g., PCR, DNA replication, bio- replication, oligonucleotide chemical synthesis) and cloned into a plasmid. The plasmid can then transfected or conjugated into an Lm strain. Additionally or alternatively, the insert is integrated into a phage vector and inserted into an Lm strain by phage infection.

Confirmation of the construct can be performed utilizing techniques such as bacterial colony PCR with insert specific primers, or purifying the plasmid and sequencing at least a portion comprising the insert.

Example 16: Expression of Neoepitopes from Neoepitope Expression Vectors

[001293] As cancer is driven by mutations, the capability of providing a comprehensive map of somatic mutations in individual tumors provides a powerful tool to better understand and intervene against cancer. Human cancers carry tens to hundreds of non-synonymous mutations. See, e.g., Castle et al. (2012) Cancer Res. 72(5): 1081-1091, herein incorporated by reference in its entirety for all purposes. However, shared mutations among patients are rare, and the great majority of mutations are patient-specific, which has hindered exploitation of the mutanome for the development of broadly applicable drugs.

[001294] In this example, neoepitope expression vectors were constructed as in Example 14 based on approximately 200 non-synonymous mutations identified in no n- small-cell lung cancer tissue that are not present in healthy lung tissue. Tissues came from UMassMed cancer center of excellence tissue bank (www.umassmed.edu/ccoe/core-services/tissue-and- tumor-bank/banked-tumor-by-organ-of-origin). Others typically screen based on predictive algorithms for immunogenicity of the epitopes. These algorithms are at best 20% accurate in predicting which peptide will generate a T cell response. This is done because they cannot include all 200 mutations. Here, all mutations could be included, so no screening/predictive algorithms were used. Screening was performed for hydrophobicity, to determine what is likely to be secretable by the Lm strain (i.e., not too hydrophobic). The non-synonymous mutations (neoepitopes) are provided in Table 126.

[001295] Table 126. Non-Synonymous Mutations

SEQ ID NO SEQ ID NO

Hydropathy for Amino for

Name

Score Acid Nucleotide

Sequence Sequence

>ADRBllp.E250Klnonsynonymous SNV [mutant] -0.248 2924 2925

>DOCKllp.W103Clnonsynonymous SNV

-1.024

[mutant] 2926 2927

>KRTAP5-3lp.S166Clnonsynonymous SNV

0.143

[mutant] 2928 2929

>OR51Sllp.A58Vlnonsynonymous SNV [mutant] 0.624 2930 2931

>DCDC5lp.R345Klnonsynonymous SNV [mutant] 0.905 2932 2933

>OR5M8lp.E82Qlnonsynonymous SNV [mutant] -0.01 2934 2935

>ORlSllp.G154Vlnonsynonymous SNV [mutant] 1.243 2936 2937

>MS4A6Alp.S22Clnonsynonymous SNV [mutant] -0.305 2938 2939

>MS4A4Alp.W32Llnonsynonymous SNV

-0.01

[mutant] 2940 2941

>MAP3Kl llp.S300Clnonsynonymous SNV

0.362

[mutant] 2942 2943

>PCNXL3lp.V1269Llnonsynonymous SNV

0.871

[mutant] 2944 2945

>RCEllp.E22Qlnonsynonymous SNV [mutant] -0.343 2946 2947

>CARNSllp.L351Vlnonsynonymous SNV

1.014

[mutant] 2948 2949

>PDE2Alp.D232Hlnonsynonymous SNV [mutant] 0.357 2950 2951

>MYO7Alp.D2029Ylnonsynonymous SNV

-0.481

[mutant] 2952 2953

>FAT3lp.I3772Mlnonsynonymous SNV [mutant] -0.186 2954 2955

>FUT4lp.E298Qlnonsynonymous SNV [mutant] -0.981 2956 2957

>B3GATllp.D122Nlnonsynonymous SNV

-0.319

[mutant] 2958 2959

>CACNAlClp.P1820Tlnonsynonymous SNV

-1.352

[mutant] 2960 2961

>SLC2A3lp.G109Elnonsynonymous SNV

1.052

[mutant] 2962 2963

>SLC01Cllp.G396Alnonsynonymous SNV

0.957

[mutant] 2964 2965

>NELL2lp.S596Ilnonsynonymous SNV [mutant] -0.7 2966 2967

>KMT2Dlp.E2866Klnonsynonymous SNV

-0.424

[mutant] 2968 2969

>PAN2lp.E630Qlnonsynonymous SNV [mutant] -0.49 2970 2971

>LRIG3lp.I341Vlnonsynonymous SNV [mutant] 0.033 2972 2973

>ZDHHC17lp.D154Nlnonsynonymous SNV

0.181

[mutant] 2974 2975

>OTOGLIp.L43Vlnonsynonymous SNV [mutant] -0.79 2976 2977

>PPFIA2lp.S16Rlnonsynonymous SNV [mutant] -1.443 2978 2979

>ALDHlL2lp.K754Nlnonsynonymous SNV

-0.367

[mutant] 2980 2981

>ATP8A2lp.E680Qlnonsynonymous SNV

0.348

[mutant] 2982 2983

>MTUS2lp.W145Rlnonsynonymous SNV

-0.681

[mutant] 2984 2985

>MTUS2lp.T550Klnonsynonymous SNV [mutant] -0.552 2986 2987

>BRCA2lp.K2750Nlnonsynonymous SNV

0.31

[mutant] 2988 2989

>NBEAIp.E2100Qlnonsynonymous SNV [mutant] -0.19 2990 2991

>RAB20lp.S52Clnonsynonymous SNV [mutant] -0.8 2992 2993

>F7lp.A429Tlnonsynonymous SNV [mutant] 0.162 2994 2995

>NPAS3lp.G35Rlnonsynonymous SNV [mutant] -1.71 2996 2997 SEQ ID NO SEQ ID NO

Hydropathy for Amino for

Name

Score Acid Nucleotide

Sequence Sequence

>DDX24lp.T554Klnonsynonymous SNV [mutant] -0.695 2998 2999

>DYNC 1H1 Ip. V2568Ilnonsynonymous SNV

-0.257

[mutant] 3000 3001

>KIF26Alp.A254Slnonsynonymous SNV [mutant] 0.795 3002 3003

>HERC2lp.S319Clnonsynonymous SNV [mutant] -0.105 3004 3005

>MTMR10lp.E387Klnonsynonymous SNV

0.076

[mutant] 3006 3007

>ARHGAPl lAlp.E36Dlnonsynonymous SNV

-1.067

[mutant] 3008 3009

>SLC27A2lp.Y500Nlnonsynonymous SNV

-0.652

[mutant] 3010 3011

>PRTGIp.N908Slnonsynonymous SNV [mutant] -0.181 3012 3013

>ALDHlA2lp.S22Llnonsynonymous SNV

0.49

[mutant] 3014 3015

>CHTF18lp.A858Tlnonsynonymous SNV

-0.938

[mutant] 3016 3017

>IFT140lp.R1404Wlnonsynonymous SNV

-0.49

[mutant] 3018 3019

>SNX29lp.D644Elnonsynonymous SNV [mutant] -0.743 3020 3021

>EEF2Klp.D425Glnonsynonymous SNV [mutant] -1.514 3022 3023

>QPRTIp.R102Wlnonsynonymous SNV [mutant] 0.671 3024 3025

>CD2BP2lp.S49Glnonsynonymous SNV [mutant] -2.076 3026 3027

>PMFBPllp.L835Plnonsynonymous SNV

-1.405

[mutant] 3028 3029

>PLCG2lp.S1192Clnonsynonymous SNV

-0.3

[mutant] 3030 3031

>ADAD2lp.G44Alnonsynonymous SNV [mutant] -0.238 3032 3033

>ZNF469lp.G680Dlnonsynonymous SNV

-0.162

[mutant] 3034 3035

>MYH13lp.M80Ilnonsynonymous SNV [mutant] -0.71 3036 3037

>TRPV2lp.Q199Hlnonsynonymous SNV [mutant] -0.114 3038 3039

>LRRC75Alp.Q199Elnonsynonymous SNV

-1.243

[mutant] 3040 3041

>ATXN7L3lp.L249Vlnonsynonymous SNV

-0.2

[mutant] 3042 3043

>HOXB2lp.P91Qlnonsynonymous SNV [mutant] -0.938 3044 3045

>MPOIp.F508Llnonsynonymous SNV [mutant] -0.676 3046 3047

>TRIM37lp.R192Wlnonsynonymous SNV

0.243

[mutant] 3048 3049

>CHMPlBlp.Q146Hlnonsynonymous SNV

-0.29

[mutant] 3050 3051

>SEHlLlp.D118Nlnonsynonymous SNV [mutant] -0.071 3052 3053

>KCTDllp.S137Llnonsynonymous SNV [mutant] -0.548 3054 3055

>EVI5Llp.P715Slnonsynonymous SNV [mutant] 0.1 3056 3057

>KEAPllp.E218Klnonsynonymous SNV [mutant] -0.371 3058 3059

>MRIllp.G69Elnonsynonymous SNV [mutant] 1.338 3060 3061

>ZNF257lp.T304Nlnonsynonymous SNV [mutant] -1.019 3062 3063

>VSTM2Blp.E161Qlnonsynonymous SNV

-0.414

[mutant] 3064 3065

>DMKNIp.D93Hlnonsynonymous SNV [mutant] 0.224 3066 3067

>BCKDHAIp.E238Klnonsynonymous SNV

0.086

[mutant] 3068 3069

>CEACAM16lp.S155Rlnonsynonymous SNV

-0.752

[mutant] 3070 3071 SEQ ID NO SEQ ID NO

Hydropathy for Amino for

# Name

Score Acid Nucleotide

Sequence Sequence

107 >NKPDllp.Q125Elnonsynonymous SNV [mutant] -0.338 3072 3073

>EXOC3L2lp.R39Llnonsynonymous SNV

108 -0.267

[mutant] 3074 3075

109 >CAl llp.Q282Hlnonsynonymous SNV [mutant] -0.748 3076 3077

110 >NLRP8lp.R781Slnonsynonymous SNV [mutant] 0.11 3078 3079

111 >ZNF470lp.F462Llnonsynonymous SNV [mutant] -0.557 3080 3081

112 >ZNF586lp.R56Tlnonsynonymous SNV [mutant] -0.314 3082 3083

>ZSCANllp.Q134Plnonsynonymous SNV

113 -0.419

[mutant] 3084 3085

114 >TPOIp.A90Elnonsynonymous SNV [mutant] -0.171 3086 3087

115 >LTBPllp.V937Llnonsynonymous SNV [mutant] 0.748 3088 3089

116 >AFF3lp.E31Qlnonsynonymous SNV [mutant] -2.981 3090 3091

117 >CKAP2Llp.K30Nlnonsynonymous SNV [mutant] -1.19 3092 3093

>MY07Blp.A1791Vlnonsynonymous SNV

118 -0.843

[mutant] 3094 3095

>TANCllp.K906Mlnonsynonymous SNV

119 0.648

[mutant] 3096 3097

>SLC4A10lp.G309Vlnonsynonymous SNV

120 0.186

[mutant] 3098 3099

121 >SCN2Alp.S661Clnonsynonymous SNV [mutant] 0.824 3100 3101

122 >SP9lp.T14Klnonsynonymous SNV [mutant] 0.329 3102 3103

123 >TTNIp.C8217Ylnonsynonymous SNV [mutant] -0.695 3104 3105

>HECW2lp.D1350Hlnonsynonymous SNV

124 -0.405

[mutant] 3106 3107

>PARD3Blp.Y789Clnonsynonymous SNV

125 -0.819

[mutant] 3108 3109

126 >DIS3L2lp.P77Slnonsynonymous SNV [mutant] 0.114 3110 3111

127 >LZTS3lp.E465Klnonsynonymous SNV [mutant] 0.081 3112 3113

128 >KCNGllp.R205Hlnonsynonymous SNV [mutant] -1.376 3114 3115

>COL20Allp.N255Dlnonsynonymous SNV

129 -0.119

[mutant] 3116 3117

>BRWDl lp.A2213Vlnonsynonymous SNV

130 -0.586

[mutant] 3118 3119

131 >DSCAMIp.F271Llnonsynonymous SNV [mutant] -0.043 3120 3121

>KRTAP10-4lp.S221Tlnonsynonymous SNV

132 0.048

[mutant] 3122 3123

133 >NEFHIp.V446Alnonsynonymous SNV [mutant] -0.729 3124 3125

134 >SFIllp.T128Alnonsynonymous SNV [mutant] -0.21 3126 3127

>POLR3Hlp.R149Clnonsynonymous SNV

135 -0.062

[mutant] 3128 3129

136 >STABllp.S681Rlnonsynonymous SNV [mutant] -0.352 3130 3131

>SLC25A26lp.S82Llnonsynonymous SNV

137 -0.219

[mutant] 3132 3133

138 >EPHA6lp.V196Llnonsynonymous SNV [mutant] -0.462 3134 3135

>PLXNAllp.E607Klnonsynonymous SNV

139 -0.619

[mutant] 3136 3137

>DNAJC13lp.K514Ilnonsynonymous SNV

140 -0.433

[mutant] 3138 3139

141 >ESYT3lp.K496Nlnonsynonymous SNV [mutant] -0.905 3140 3141

>GPR149lp.R145Glnonsynonymous SNV

142 0.09

[mutant] 3142 3143

143 >PDCD10lp.E68Qlnonsynonymous SNV [mutant] -0.033 3144 3145

144 >MECOMIp.A78Tlnonsynonymous SNV [mutant] -0.39 3146 3147 SEQ ID NO SEQ ID NO

Hydropathy for Amino for

# Name

Score Acid Nucleotide

Sequence Sequence

>KIAA0226lp.R150Klnonsynonymous SNV

145 -0.176

[mutant] 3148 3149

146 >MSXllp.S92Llnonsynonymous SNV [mutant] 0.043 3150 3151

147 >LIMCHllp.Q60Hlnonsynonymous SNV [mutant] -0.648 3152 3153

>PTPN13lp.Q2276Hlnonsynonymous SNV

148 0

[mutant] 3154 3155

149 >PDHA2lp.C179Ylnonsynonymous SNV [mutant] 0.2 3156 3157

>EXOSC9lp.L266Flnonsynonymous SNV

150 -0.11

[mutant] 3158 3159

>TBClD9lp.E837Qlnonsynonymous SNV

151 -0.005

[mutant] 3160 3161

152 >FGGIp.G294Elnonsynonymous SNV [mutant] -0.757 3162 3163

>SLC9A3lp.E821Qlnonsynonymous SNV

153 -0.662

[mutant] 3164 3165

154 >NSUN2lp.F48Llnonsynonymous SNV [mutant] -0.6 3166 3167

155 >SPEF2lp.F1436Slnonsynonymous SNV [mutant] -0.495 3168 3169

156 >ITGA2lp.P43Tlnonsynonymous SNV [mutant] -0.162 3170 3171

157 >IPOl llp.N30Slnonsynonymous SNV [mutant] 0.076 3172 3173

158 >NR2Fllp.V380Mlnonsynonymous SNV [mutant] 0.833 3174 3175

>SLC04Cllp.K663Nlnonsynonymous SNV

159 0.781

[mutant] 3176 3177

160 >WDR55lp.F237Llnonsynonymous SNV [mutant] -0.114 3178 3179

>PCDHA9lp.T662Slnonsynonymous SNV

161 0.286

[mutant] 3180 3181

>PCDHGA12lp.K590Mlnonsynonymous SNV

162 -0.138

[mutant] RS AEPGYLVTM V V AVDRDSGQ 3182 3183

>SLC6A7lp.D151Hlnonsynonymous SNV

163 0.162

[mutant] 3184 3185

164 >TCOFllp.P566Slnonsynonymous SNV [mutant] -0.048 3186 3187

165 >LCP2lp.P138Hlnonsynonymous SNV [mutant] -2.005 3188 3189

166 >KCNIPllp.I19Mlnonsynonymous SNV [mutant] 0.448 3190 3191

>BTN3Allp.A186Slnonsynonymous SNV

167 -0.39

[mutant] 3192 3193

>ZBTB12lp.C266Slnonsynonymous SNV

168 -0.348

[mutant] 3194 3195

>CYP21A2lp.E295Klnonsynonymous SNV

169 -1.167

[mutant] 3196 3197

170 >TREM2lp.E202Vlnonsynonymous SNV [mutant] 0.633 3198 3199

171 >TTKIp.L309Flnonsynonymous SNV [mutant] -1.219 3200 3201

>SYNCRIPIp.D284Hlnonsynonymous SNV

172 -0.667

[mutant] 3202 3203

173 >HS3ST5lp.R82Glnonsynonymous SNV [mutant] -1.048 3204 3205

174 >RFX6lp.Y802Clnonsynonymous SNV [mutant] -1.086 3206 3207

>SYNEllp.T5594Alnonsynonymous SNV

175 -0.438

[mutant] 3208 3209

>CYP2Wllp.R328Hlnonsynonymous SNV

176 -0.729

[mutant] 3210 3211

177 >GNAT3lp.E216Qlnonsynonymous SNV [mutant] 0.433 3212 3213

>SEMA3Clp.Y141Clnonsynonymous SNV

178 -0.752

[mutant] 3214 3215

179 >PCLOIp.E1590Dlnonsynonymous SNV [mutant] -1.938 3216 3217

180 >SAMD9lp.I635Vlnonsynonymous SNV [mutant] -0.114 3218 3219

181 >COG5lp.A54Glnonsynonymous SNV [mutant] 0.11 3220 3221

182 >CBLLllp.S471Flnonsynonymous SNV [mutant] -1.19 3222 3223 SEQ ID NO SEQ ID NO

Hydropathy for Amino for

# Name

Score Acid Nucleotide

Sequence Sequence

183 >FOXP2lp.S139Clnonsynonymous SNV [mutant] -0.129 3224 3225

184 >MDFICIp.L78Flnonsynonymous SNV [mutant] -0.786 3226 3227

>IMPDHllp.P138Llnonsynonymous SNV

185 -0.148

[mutant] 3228 3229

186 >TRIM24lp.T772Slnonsynonymous SNV [mutant] -0.376 3230 3231

187 >CASP2lp.A338Slnonsynonymous SNV [mutant] -1.533 3232 3233

188 >ASIC3lp.L531Qlnonsynonymous SNV [mutant] -0.41 3234 3235

189 >INSIGllp.L126Flnonsynonymous SNV [mutant] -0.09 3236 3237

190 >TNKSIp.I1189Flnonsynonymous SNV [mutant] -0.205 3238 3239

191 >CHD7lp.Q1704Elnonsynonymous SNV [mutant] -0.748 3240 3241

>ZFHX4lp.T3413Alnonsynonymous SNV

192 -0.381

[mutant] 3242 3243

193 >CNGB3lp.L227Hlnonsynonymous SNV [mutant] 1.152 3244 3245

194 >HAS2lp.M118Vlnonsynonymous SNV [mutant] 0.11 3246 3247

195 >LRRC6lp.D453Nlnonsynonymous SNV [mutant] -1.286 3248 3249

196 >PLECIp.E1404Dlnonsynonymous SNV [mutant] -1.4 3250 3251

197 >RORBIp.R372Glnonsynonymous SNV [mutant] 0.267 3252 3253

>SPATA3 lC2lp. A1072Elnonsynonymous SNV

198 -1.09

[mutant] 3254 3255

199 >SVEPllp.R146Slnonsynonymous SNV [mutant] -0.571 3256 3257

200 >REX04lp.T241Slnonsynonymous SNV [mutant] 0.581 3258 3259

>RPS6KA3lp.P617Llnonsynonymous SNV

201 -0.081

[mutant] 3260 3261

>SMClAlp.K402Qlnonsynonymous SNV

202 -1.81

[mutant] 3262 3263

203 >MSNIp.K211Nlnonsynonymous SNV [mutant] -0.395 3264 3265

204 >TEXl llp.S163Clnonsynonymous SNV [mutant] -0.148 3266 3267

>SERPINA7lp.K290Rlnonsynonymous SNV

205 -0.695

[mutant] 3268 3269

206 >IL9Rlp.V287Elnonsynonymous SNV [mutant] -1.505 3270 3271

Reagents

[001296] The following reagents were used to test the lung permutation constructs:

Bacteria: Lmdda constructs tagged grown overnight in BHI

Cell lines: DC2.4

2% trypsin in HBSS

RPMI 10%FBS glutamax

FACS Buffer (PBS 2% FBS)

Cell counting solution

Gentamicin antibiotic

25D-APC conjugated antibody 100X Harvesting Antigen Presenting Cells

[001297] In some experiments, murine dendritic DC2.4 cells were stimulated with 20 ng/mL recombinant mouse IFN gamma for 48 hours. Media was removed and collected into two 50 mL conical tubes per flask. A volume of 10 mL 2% trypsin HBSS solution was added to the flask to remove residual FBS and was decanted into the two 50 mL collection tubes equally (5 mL each). A volume of 10 mL 2% trypsin HBSS solution was added to the flask to coat, and adherence was checked under a microscope, and a 5 min incubation followed at 37°C. The suspension was collected into the two 50-mL collection tubes (5 mL each) and spun for 5 minutes at 1200 rpm. The supernatant was discarded, and the pellet was resuspended in tube 1 with 25 mL RPMI 10%FBS glutamax solution. This was then decanted into the second collection tube to combine the two tubes into one.

[001298] Tubes (1.5 mL) were then labeled for counting. A volume of 135 μΐ_^ counting solution and a volume of 15 μΐ_^ cells was added, and the cells were incubated for 2 minutes at room temperature. The DC2.4 cells were then set up for infection in 24-well plates. The 24- well plates were incubated overnight at 37°C (5% C0₂). The plates were then spun for 5 seconds at 2000 rpm, supernatant was removed, and 1 mL fresh c-RPMI was added to each well.

Infection

[001299] The cells were then infected with Lmdda-PSA-Survivin-tag expressing vectors (grown overnight dry 37). Total Lmdda-neo construct cfu were lxl0⁹/mL. The Lmdda was spun down in 1.5 mL and resuspended in 1 mL room temperature RPMI- 10% FBS media. A volume of the Lmdda was added to the DC2.4 wells to reach the correct MOI for 2xl0⁶ cells (MOI: 10 = 20 μΐ_^ Lmdda). The plate was then spun at 1200 rpm for 15 minutes and placed in an incubator at 37°C with 5% CO2 for a four hour infection. To stop Lmdda killing of the cells, 10 μg/mL gentamicin was added after 1 hour of the incubation.

Staining with 25D-APC (SIINFEKL) and Flow Cytometry

[001300] After four hours of infection, the plate was spun for 30 seconds at 2000 rpm, and the supernatant was discarded. To block the cells were resuspended in 200 μΐ_^ 2.4G2 and transferred to a 96-well plate for 10 minute on ice. The cells were washed with FACS buffer (PBS + 2% FBS). Staining master mix was then added, and the cells were vortexed and placed on ice for 20 minutes. The cells were then washed with FACS buffer and resuspended in approximately 300 μΐ_^ FACS buffer (depending on size of pellet/cell number). The samples were then run on the flow cytometer for detection of 25D-APC.

Experiment 1

[001301 ] Table 127. Samples Tested for Detection of 25D-APC.

Sample # Lung Construct Order of Neoepitopes

101-120 H (SEQ ID NO: 3309) (peptide

27 101-120

sequence only)

121-140 H (SEQ ID NO: 3311) (peptide

28 121-140

sequence only)

141-160 H (SEQ ID NO: 3313) (peptide

29 141-160

sequence only)

30 No Infection

31 Unstained

[001302] An "H" at the end of the construct (such as "121-140 H") indicates that the peptide sequence is identical to the construct lacking the H, but the underlying nucleotide sequence which resulted in the same peptide sequence was modified.

[001303] Table 128. Detection of 25D-APC.

[001304] Detection of the C-terminal SIINFEKL tag with the 25D-APC conjugated antibody is shown in Table 128. As indicated in Table 128, the SVN-tag and PSMA-tag positive controls showed high levels of positive staining, whereas the SVN-no tag, the no infection, and the unstained negative controls were below the limit of detection. Similarly, samples 4-7, 10, 12, 16, 20-23, 25, and 27-29 showed high levels of positive staining. This demonstrates confirmation that the neoantigens express and secrete in antigen-presenting cells upon infection.

Experiment 2

[001305] The above was repeated in a second experiment with additional lung neoepitope constructs, as indicated in Table 129. In this experiment, the tag was moved to different locations within the lung constructs.

[001306] Table 129. Samples Tested for Detection of 25D-APC.

[001307] Table 130. Detection of 25D-APC.

[001308] Detection of the C-terminal SIINFEKL tag with the 25D-APC conjugated antibody is shown in Table 130. As indicated in Table 130, the SVN-tag positive control showed high levels of positive staining, whereas the no tag, the no infection, and the unstained negative controls were below the limit of detection. Similarly, samples 3, 7, and 8 showed high levels of positive staining. This demonstrates confirmation that the neoantigens express and secrete in antigen-presenting cells upon infection.

Experiment 3

[001309] The above was repeated in a third experiment with additional lung constructs, as indicated in Table 131. In these lung constructs, the tag was moved to different locations in the construct.

[001310] Table 131. Samples Tested for Detection of 25D-APC.

[001312] Detection of the C-terminal SIINFEKL tag with the 25D-APC conjugated antibody is shown in Table 132. As indicated in Table 132, the PSA Survivin and the Minigene positive controls showed high levels of positive staining, whereas the no tag and the no infection negative controls were below the limit of detection. Similarly, samples 4 and 5-12 showed high levels of positive staining. This demonstrates confirmation that the neoantigens express and secrete in antigen-presenting cells upon infection. [001313] Figure 31 shows surface K^b-SIINFEKL on DC2.4 cells infected with Lm constructs with SIINFEKL at various positions. The graph depicts a summary of the raw 25D data, depicting that the SIINFEKL tag identifies a secreted neoepitope whether

SIINFEKL is located at the C-terminus, the N-terminus, or in between. The last five bars correspond with the following constructs: 2712 SIINFEKL-121-140-6xHIS; 2712 121-125- SIINFEKL-126-140-6xHIS; 2712 121-130-SIINFEKL-131-140-6xHIS; 2712 121-135- SIINFEKL-136-140-6xHIS; and 2712 121-140-SIINFEKL-6xHIS, respectively.

Experiment 4

[001314] The above was repeated in a fourth experiment with additional Lmdda constructs, as indicated in Table 133.

[001315] Table 133. Samples Tested for Detection of 25D-APC.

[001316] Table 134. Detection of 25D-APC.

[001317] Detection of the C-terminal SIINFEKL tag with the 25D-APC conjugated antibody is shown in Table 134. As indicated in Table 134, the SVN positive control showed high levels of positive staining, whereas the no tag negative control showed a low level of staining. Similarly, samples 43-12, 15, 18, 19, 21, 22, 24, and 27-30 showed high levels of positive staining. This demonstrates confirmation that the neoantigens express and secrete in antigen-presenting cells upon infection. Figure 36 shows the effects of randomization of the order of neoepitopes on presentation and secretion of the neoepitopes. Ordering 1 thru 20 sequentially does not secrete. However, randomizing the entire order, or breaking down individual pieces, or randomizing those pieces results in successful secretion. Likewise, ordering 21-40 sequentially does not secrete. Individual regions of that 20'mer (1- 5, 6-10) do not work, and other regions work (16-20). However, randomizing individual regions results in the successful secretion of each individual region. Example 17: Therapeutic Effects of Lm Neoantigen Constructs in B16F10 Murine Melanoma Model

[001318] After non-synonymous mutations are identified in cancer cells that are not present in corresponding healthy cells, major efforts are typically invested to determine the mutational functional impact, such as cancer driver versus passenger status, to form a basis for selecting therapeutic targets. However, little attention has been devoted to either define the immunogenicity of these mutations or characterize the immune responses they elicit. From the immunologic perspective, mutations may be particularly potent vaccination targets, as they can create neoantigens that are not subject to central immune tolerance. When attention has been devoted to define the immunogenicity of these mutations or characterize the immune responses they elicit, efforts are typically directed to narrowing down the non- synonymous mutations to a single mutation to be included in a peptide for immunization. For example, in Castle et al., 962 non-synonymous point mutations were identified in B 16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Fifty of these mutations were selected based on selection criteria including low false discovery rate (FDR) confident value, location in an expressed gene, and predicted immunogenicity. Out of these 50, only 16 were found to elicit immune responses in immunized mice, and only 11 of the 16 induced an immune response preferentially recognizing the mutated epitope. Two of the mutations were then found to induce tumor growth inhibition. See, e.g., Castle et al. (2012) Cancer Res. 72(5): 1081-1091, herein incorporated by reference in its entirety for all purposes. In the constructs described in the following experiments, however, our data suggest that Neo 20 and Neo 30 are better at controlling tumor growth. In our constructs, Neo-12 contains the 12 most immunogenic epitopes. Neo- 12 contains both tumor controlling epitopes (Mut30 and Mut44, as disclosed above in Table 123). Neo 20 contains Mut30- Mut2-Mut3-Mut3-Mut4...Mutl9). Neo 30 contains Mut30-Mut2-Mut3...Mut-29). Neo 20 and Neo 30 only contain one of the tumor controlling epitopes identified by Castle (Mut30), and then they contain both immunogenic and non-immunogenic epitopes. Despite not having multiple tumor controlling epitopes, and containing many non-tumor controlling and even non-immunogenic epitopes.

Experiment 1

[001319] To determine therapeutic response generated by Lm neoantigen constructs, a tumor regression study was designed to examine the therapeutic effects of such constructs on tumor growth in the B 16F10 C57B1/6 murine melanoma model. Specifically, Lm neoantigen vectors were designed with 12 neoantigens (Lm-Castle 12, containing Mut30, Mut5, Mutl7, Mut20, Mut22, Mut24, Mut25, Mut44, Mut46, Mut48, and Mut50) or 20 neoantigens (Lm- Castle 20, containing Mut30, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, MutlO, Mutl l, Mutl2, Mutl3, Mutl4, Mutl5, Mutl6, Mutl7, Mutl8, Mutl9, and Mut20) identified by Castle et al. See, e.g., Castle et al. (2012) Cancer Res. 72(5): 1081-1091, herein incorporated by reference in its entirety for all purposes.

[001320] Tumor Cell Line Expansion. The B 16F10 melanoma cell line was cultured in c- RPMI containing 10% FBS (50 mL) and IX Glutamax (5 mL). The c-RPMI media includes the following components:

RPMI 1640 450 mL

FCS 50 mL

HEPES 5 mL

NEAA 5 mL

L-Glutamine 5 mL

Na-Pyruvate 5 mL

Pen/step 5 mL

2-ME (14.6M) 129

[001321 ] Tumor Inoculation. On Day 0, B 16F10 cells were trypsinized and washed twice with media. Cells were counted and re-suspended at a concentration of 1 x 10⁵ cells/200

of PBS for injection. B 16F10 cells were then implanted subcutaneously in the right flank of each mouse. Mice were vaccinated on Day 3 of the study. Tumors were measured and recorded twice per week until reaching a size of 12 mm in diameter. Once tumors met sacrifice criteria, mice were euthanized, and tumors were excised and measured.

[001322] Immunotherapy Treatment. On Day 3, immunotherapies and treatments began. Groups were treated with Lm (IP), and boosted twice. Details are listed in Table 135.

[001323] Table 135. Treatment Schedule.

[001324] Immunotherapy Treatment Preparation.

1. PBS ONLY - 200 pL/mouse IP.

2. LmddA-214 (Titer: 1.5 x 10⁹ CFU/mL)

a. Thaw 1 vial from -80°C in 37°C water bath.

b. Spin at 14, 000 rpm for 2 min and discard supernatant.

c. Wash 2 times with 1 mL PBS and discard PBS.

d. Re-suspend in PBS to a final concentration of 5x10^s CFU/mL.

3. Lm-Castle 12 (Titer: 1.59 x 10⁹ CFU/mL and Lm-Castle 20 (Titer: 1.6 x 10⁹ CFU/mL) a. Thaw 1 vial from -80°C in 37°C water bath.

b. Spin at 14, 000 rpm for 2min and discard supernatant.

c. Wash 2 times with 1 mL PBS and discard PBS.

d. Re-suspend in PBS to a final concentration of 5x10^s CFU/mL.

[001325] As shown in Figure 32B, growth of tumors was inhibited by Lm-Neo 12 and Lm- Neo 20 as compared with the control groups (PBS and LmddAH ). LmddA274 is the listeria control, and is an empty vector. It includes the truncated LLO (tLLO), however no neoepitopes are attached. In addition, Lm-Neo 20, which contained 20 neoantigens, inhibited tumor growth to a greater extent than Lm-Neo 12, which contained 12 neoantigens.

Likewise, Lm-Neo 20 and Lm-Neo 12 each result in increased survival time when compared with the control groups, with Lm-Neo 20 providing the greatest protective effect (Figure 32C). These data show that vaccination with Lm carrying neoepitopes is able to confer antitumoral effects, and increasing the number of neoepitopes increases the antitumoral effects. Experiment 2

[001326] To further compare therapeutic responses generated by different Lm neoantigen constructs, a tumor regression study was designed to examine the therapeutic effects of such constructs on tumor growth in the B 16F10 C57B1/6 murine melanoma model. Specifically, Lm neoantigen vectors were designed with 12 neoantigens (Lm-Castle 12), 20 neoantigens (Lm-Castle 20), or 39 neoantigens (Lm-Castle 39; no linker, no 20-29 (Lm-Castle 30)) identified by Castle et al. See, e.g., Castle et al. (2012) Cancer Res. 72(5): 1081-1091, herein incorporated by reference in its entirety for all purposes.

[001327] Tumor Cell Line Expansion. The B 16F10 melanoma cell line was cultured in c- RPMI containing 10% FBS (50 mL) and IX Glutamax (5 mL).

[001328] Tumor Inoculation. On Day 0, B 16F10 cells were trypsinized and washed twice with media. Cells were counted and re-suspended at a concentration of 1 x 10⁵ cells/200 μί. of PBS for injection. B 16F10 cells were then implanted subcutaneously in the right flank of each mouse. Mice were vaccinated on Day 4 of the study. Tumors were measured and recorded twice per week until reaching a size of 1500 mm³ in volume. Once tumors met sacrifice criteria, mice were euthanized, and tumors were excised and measured.

[001329] Immunotherapy Treatment. On Day 4, immunotherapies and treatments began. Animals were treated once every 7 days until the end of the study. Groups were treated with either PBS, LmddMl , Lm-Castle 12, Lm-Castle 20, Lm-Castle 39 no linker no 20-29, detailed in Table 136.

[001330] Table 136. Treatment Schedule.

(no link no

20-29)

(also called

Lm Castle

30)

(SEQ ID

NO: 3401)

[001331 ] Immunotherapy Treatment Preparation.

1. PBS ONLY - 200 pL/mouse IP.

2. LmddA-214 (Titer: 1.7 x 10⁹ CFU/mL)

a. Thaw 1 vial from -80°C in 37°C water bath.

b. Spin at 14,000 rpm for 2 min and discard supernatant.

c. Wash 2 times with 1 mL PBS and discard PBS.

d. Re-suspend in PBS to a final concentration of 5x10^s CFU/mL.

3. Lm-Castle 12 (Titer: 1.59 x 10⁹ CFU/mL and Lm-Castle 20 (Titer: 1.6 x 10⁹ CFU/mL) and Lm-Castle 39 )Titer: 1 x 10⁹ CFU/mL)

a. Thaw 1 vial from -80°C in 37°C water bath.

b. Spin at 14,000 rpm for 2min and discard supernatant.

c. Wash 2 times with 1 mL PBS and discard PBS.

d. Re-suspend in PBS to a final concentration of 5x10^s CFU/mL.

[001332] Harvesting Details. The spleen from each mouse was collected in an individual tube containing 5 mL of c-RPMI medium. Detailed steps are described below. All tumors were excised and measured at termination of the study.

1. Harvest spleens using sterile forceps and scissors.

2. Mash each spleen in wash medium (RPMI only) using two glass slides or the back of plunger from a 3 mL syringe.

3. Transfer cells in the medium to a 15 mL tube.

4. Pellet cells at 1,000 RPM for 5 min at room temperature.

5. Discard supernatant, re-suspend cells in the remaining wash buffer gently, and add 2 mL RBC lysis buffer per spleen to the cell pellet. Mix cells gently with lysis buffer by tapping the tube and wait for 1 min.

6. Immediately add 10 mL of c-RPMI medium to the cell suspension to deactivate the lysis buffer.

7. Spin cells at 1,000 for 5 min at room temperature. 8. Pass the cells through a cell strainer and wash them one more time with 10 mL c- RPMI.

9. Count cells using hemocytometer/moxi flow and check the viability by Trypan blue staining. Each spleen should yield -1-2 x 10^s cells.

10. Divide the cells for staining.

11. Follow immudex dextramer staining protocol: with the one exception of adding the cell surface antibodies (CD8, CD62L) in 2.4G2 instead of staining buffer

( w w w . immudex. co m/media/ 12135/tf 1003.03_general_staining_procedure_mhc_d extramer.pdf).

[001333] CD8+ T Cell Response. 25D assays were done as explained above to measure expression and secretion of the Lm-Neo 20 construct in antigen presenting cells. Figure 33A is a positive control (PSA-Survivin-SIINFEKL), Figure 33B is a negative control (PSA- Survivin without SIINFEKL), and Figure 33C is the Lm-Neo 20 (with SIINFEKL tag at C- terminus). As indicated in Figures 33A-33C, the Lm-Neo 20 expresses and is secreted, but only at low levels compared to the positive control. However, despite these low secretion levels, a specific CD8+ T cell response to SIINFEKL was observed. Figure 34 shows the SIINFEKL-specific CD8+ T cell response to the "low secretion" Lm-Neo 20 construct. As shown in Figure 34, approximately 20% of the CD8+ T cells are specific for antigens in the Lm Neo 20 construct.

[001334] Antitumor Effects. As shown in Figure 35A, growth of tumors was inhibited by Lm-Neo 12, Lm-Neo 20, and Lm-Neo 30 as compared with the control groups (PBS and LmddAH ). In addition, Lm-Neo 30, which contained 30 neoantigens, inhibited tumor growth to a greater extent than Lm-Neo 20, which contained 20 neoantigens, which inhibited tumor growth to a greater extent than Lm-Neo 12, which contained 12 neoantigens.

Likewise, Lm-Neo 30, Lm-Neo 20, and Lm-Neo 12 each result in increased survival time when compared with the control groups, with Lm-Neo 30 providing the greatest protective effect and Lm-Neo 20 providing the next greatest protective effect (Figure 32C). These data show that vaccination with Lm carrying neoepitopes is able to confer antitumoral effects, and increasing the number of neoepitopes increases the antitumoral effects.

Example 18: Neoepitope-Specific Immunity in Mice Immunized with Lm Neoantigen Constructs

[001335] An experiment was designed to evaluate the generation of neoepitope and signal peptide- specific responses in C57BL/6 mice after immunization with r M2, 1-20, 81-100, 101-120, 121-140, Lm 2712#1 & Lm 2712#3 neoepitope constructs. The neoepitope and signal peptide- specific immune response will be detected by pentamer staining using the known T cell H-2 D^b PSA₆₅-73 (HCIRNKSVI (SEQ ID NO: 2807)), VvB8R (TSYKFESV (SEQ ID NO: 2808)), IAV PA (SSLENFRAYV (SEQ ID NO: 2809)) as well as neoepitope peptide- specific responses as evaluated by intracellular cytokine staining for IFN-γ. The details of immunization schedule and strains are given in Table 137.

[001336] Table 137. Immunization Schedule.

[001337] Each of these constructs target mutations from the same 2712 Lung sample used in all of the lung construct experiments. rM2 is a random order of 20 21 'mers from the 200 non-synonymous mutation pool. 1-20 includes the first 20 non-synonymous mutations, in that order. The same applies for 81-100, 101-120, and 121-140. 2712#1 includes 50 21-mers (1-50). 2712 #3 includes 50 21-mers (101-150).

[001338] Immunotherapy Preparation.

1. Thaw 1 vial from -80°C in 37°C water bath.

2. Spin at 14,000 rpm for 2 min and discard supernatant.

3. Wash 2 times with 1 mL PBS and discard PBS.

4. Re-suspend in PBS to appropriate final concentration.

[001339] Preparing Isolated Splenocytes.

1. Harvest spleens using sterile forceps and scissors.

2. Mash each spleen in wash medium (RPMI only) using two glass slides or the back of plunger from a 3 mL syringe.

3. Transfer cells in the medium to a 15 mL tube.

4. Pellet cells at 1,000 RPM for 5 min at room temperature.

5. Discard supernatant, re-suspend cells in the remaining wash buffer gently, and add 2 mL RBC lysis buffer per spleen to the cell pellet. Mix cells gently with lysis buffer by tapping the tube and wait for 1 min. 6. Immediately add 10 mL of c-RPMI medium to the cell suspension to deactivate the lysis buffer.

7. Spin cells at 1,000 for 5 min at room temperature.

8. Pass the cells through a cell strainer and wash them one more time with 10 mL c- RPMI.

9. Count cells using hemocytometer/moxi flow and check the viability by Trypan blue staining. Each spleen should yield -1-2 x 10^s cells.

10. Divide the cells for pentamer staining and ELISpot.

ELISPOT for IFN Gamma

[001340] Day 1. Tubes to prepare: PMA (dilute 1: 1000 in complete medium). Cells were prepared as mentioned below (5 x 10⁶/ml) for each mouse).

1) Prepare complete medium (with BME) 100 mL

2) Grp 1/ mouse 1 cells (5X10⁶/mL of complete medium with BME) 2 mL

3) Grp 2/ mouse 1 cells (5X10⁶/mL of complete medium with BME) 2 mL

4) Grp 3/ mouse 1 cells (5X10⁶/mL of complete medium with BME) 2 mL

5) Grp 1/ mouse 1 cells (5X10⁴/mL of complete medium with BME) 2 mL

6) Grp 2/ mouse 1 cells (5X10⁴/mL of complete medium with BME) 2 mL

7) Grp 3/ mouse 1 cells (5X10⁴/mL of complete medium with BME) 2 mL

8) No peptide (Medium from tube 1) 3 mL

9) E7 peptide (a of 1 mM peptide/mL of medium from tube 1) - 16 mL

10) PMA (add 10

μg/mL stock/mL of medium from tube 1) + lonomycin (add 1 lL of 1 mg/mL stock/mL of medium from tube 1) 5 mL

[001341 ] Stimulation with Peptide:

a) Wash plate 4 times with sterile PBS (200 uL/well).

b) Add 200 μίΛνεΙΙ of complete medium. Incubate for at least 30 min at RT.

c) Remove the medium and add the cell suspension (100 μίΛνεΙΙ) + stimulants (100 μίΛνεΙΙ) as planned in the table.

d) Wrap the plate in aluminum foil and incubate the plate at 37°C, 5% C0₂ for 24h.

[001342] Day 2. Detection of Spots.

a) Remove cells by emptying the plate and wash 5 times with PBS (200 μίΛνεΙΙ). b) Add 100 μΐ/ννεΐΐ of diluted R4-6A2-biotin and incubate for 2 h at room temperature. c) Wash 5 times with PBS (200 L well).

d) Add 100 μΐ/ννεΐΐ of diluted Streptavidin-ALP and incubate for 1 h at room temperature. e) Wash 5 times with PBS (200 L well).

f) Add 100 nL/well of filtered (0.45μm filter) ready to use substrate solution (BCIP/NBT) and develop until distinct spots emerge.

g) Stop color development by washing extensively in tap water.

h) Leave the plate to dry and count spots the next day.

[001343] Results.

[001344] Table 138 details whether we were able to detect secretion of the 21'mers via 25D assay.

[001345] Table 138. Lung Immunogenicity Summary.

[001346] Figure 37 summarizes the SIINFEKL-specific CD8 T cell response in mice immunized with the various constructs. An immune response against SIINFEKL (e.g., surrogate tag for the neoepitope 21-mer amino acid chain being secreted into the host and the host generating an immune response against the 21-mer amino acid chain), was detected in all constructs, except the 2712 #1 50-21-mer construct.

[001347] Of importance, 3 constructs (r M 2, 1-20 & 2712 #3) that did not screen as a positive screener via 25D assay did in fact generate an in vivo immune response (although the response is less pronounced than constructs that screened positive by 25D). Additionally, an immune response to constructs up to 50 21-mers was able to be generated. Example 19: Testing of 27-mers

[001348] A 25D assay was performed, as described above, using neoepitope constructs comprised of 27 amino acid "27-mers." The results are shown below in Table 139 and show that the constructs may be composed of oligomers other than 21-mers.

[001349] Table 139: Detection of 25D-APC (Assay Using 27-mers).

Claims

We claim:

1. A method for inducing an immune response against a tumor or cancer in a subject or treating a tumor or cancer in a subject, comprising:

(a) administering a recurrent cancer mutation immunotherapy composition to the subject, wherein the recurrent cancer mutation immunotherapy composition comprises a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST-containing peptide fused to two or more first antigenic peptides, wherein at least one antigenic peptide is from a cancer-associated protein and comprises a recurrent cancer mutation, and at least one antigenic peptide is from a cancer-associated protein and comprises a heteroclitic mutation; and

(b) administering a personalized immunotherapy composition to the subject, wherein the personalized immunotherapy composition comprises a second recombinant Listeria strain comprising a second nucleic acid comprising a second open reading frame encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises a second PEST-containing peptide fused to one or more second antigenic peptides, wherein each of the second antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject.

2. The method of claim 1, wherein the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by all of the following genes: KRAS, EGFR, U2AF1, BRAF, PIK3CA, and TP53.

3. The method of claim 2, wherein the antigenic peptides in the first fusion polypeptide comprise all of the following recurrent cancer mutations: KRAS_G12C, EGFR_L858R, KRAS_G12D, U2AF1_S34F, BRAF_V600E, KRAS_G12V,

PIK3CA_E545K, TP53_R158L, KRAS_G12A, EGFR_L861Q, and TP53_R273L.

4. The method of claim 3, wherein the antigenic peptides in the first fusion polypeptide comprise all of the peptides set forth in Table 35.

5. The method of claim 1, wherein the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESOl, STEAP1, and RNF43.

6. The method of claim 5, wherein the antigenic peptides comprise all of the peptides set forth in Table 36.

7. The method of claim 4 or 6, wherein the antigenic peptides in the first fusion polypeptide comprise all of the peptides set forth in Tables 35 and 36.

8. The method of claim 7, wherein one or more of the antigenic peptides in the first fusion polypeptide comprising a recurrent cancer mutation are preceded by the linker set forth in SEQ ID NO: 316, and wherein one or more of the antigenic peptides in the first fusion polypeptide comprising a heteroclitic mutation are preceded by the linker set forth in any one of SEQ ID NOS: 821-829.

9. The method of claim 8, wherein the first fusion polypeptide comprises the sequence set forth in SEQ ID NO: 895.

10. The method of claim 1, wherein the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by all of the following genes: SPOP, CHEK2, RGPD8, ANKRD36C, and AT?.

11. The method of claim 10, wherein the antigenic peptides in the first fusion polypeptide comprise all of the following recurrent cancer mutations: SPOP_F133V, CHEK2_K373E, RGPD8_P1760A, ANKRD36C_I634T, ANKRD36C_D629Y,

SPOP_W131G, ANKRD36C_D626N, SPOP_F133L, AR_T878A, AR_L702H, AR_W742C, AR_H875Y, and AR_F877L.

12. The method of claim 11, wherein the antigenic peptides in the first fusion polypeptide comprise all of the peptides set forth in Table 52.

13. The method of claim 1, wherein the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA.

14. The method of claim 13, wherein the antigenic peptides in the first fusion polypeptide comprise all of the peptides set forth in Table 53.

15. The method of claim 12 or 14, wherein the antigenic peptides in the first fusion polypeptide comprise all of the peptides set forth in Tables 52 and 53.

16. The method of claim 15, wherein one or more of the antigenic peptides in the first fusion polypeptide comprising a recurrent cancer mutation are preceded by the linker set forth in SEQ ID NO: 316, and wherein one or more of the antigenic peptides in the first fusion polypeptide comprising a heteroclitic mutation are preceded by the linker set forth in any one of SEQ ID NOS: 821-829.

17. The method of claim 16, wherein the first fusion polypeptide comprises the sequence set forth in SEQ ID NO: 893.

18. The method of claim 1, wherein the recurrent cancer mutation immunotherapy composition is administered prior to the personalized immunotherapy composition.

19. The method of claim 1, wherein the recurrent cancer mutation immunotherapy composition is administered subsequent to the personalized immunotherapy composition.

20. The method of claim 1, wherein the recurrent cancer mutation immunotherapy composition and the personalized immunotherapy composition are administered concurrently.

21. The method of any one of claims 1 and 18-20, wherein the recurrent cancer mutation immunotherapy composition is administered with an adjuvant and/or the personalized immunotherapy composition is administered with an adjuvant.

22. The method of any one of claims 1 and 18-21, wherein the subject has a cancer associated with one or more recurrent cancer mutations in one or more cancer- associated proteins, and the first recombinant Listeria strain comprises antigenic peptides comprising one or more recurrent cancer mutations associated with the cancer.

23. The method of any one of claims 1 and 18-22, wherein the method comprises screening the subject for and identifying at least one of the one or more recurrent cancer mutations prior to the administering the recurrent cancer mutation immunotherapy composition, wherein the first recombinant Listeria strain comprises antigenic peptides comprising the at least one of the one or more recurrent cancer mutations identified in the subject.

24. The method of any one of claims 1 and 18-22, wherein the method does not comprise screening the subject for and identifying recurrent cancer mutations prior to administering the recurrent cancer mutation immunotherapy composition.

25. The method of any one of claims 1 and 18-24, further comprising generating the personalized immunotherapy composition for the subject.

26. The method of claim 25, wherein the personalized immunotherapy composition is generated concurrently with administering the recurrent cancer mutation immunotherapy composition to the subject.

27. The method of claim 25, wherein the personalized immunotherapy composition is generated subsequent to administering the recurrent cancer mutation immunotherapy composition to the subject.

28. The method of claim 25, wherein the personalized immunotherapy composition is generated prior to administering the recurrent cancer mutation immunotherapy composition to the subject.

29. The method of any one of claims 25-28, wherein generating the personalized immunotherapy composition comprises:

(a) comparing one or more open reading frame sequences or mRNA sequences from the cancer sample with one or more open reading frame sequences or mRNA sequences from the healthy biological sample, wherein the comparing identifies one or more cancer- specific neoepitopes, each comprising a different cancer- specific mutation;

(b) selecting a set of cancer- specific neoepitopes to include in the second nucleic acid and designing the second nucleic acid; and

(c) transforming a Listeria strain with the second nucleic acid.

30. The method of claim 29, further comprising obtaining the cancer sample from the subject and/or obtaining the healthy biological sample from the subject.

31. The method of claim 29 or 30, wherein the cancer sample and/or the healthy biological sample comprise a tissue, cells isolated from blood, cells isolated from sputum, cells isolated from saliva, or cells isolated from cerebrospinal fluid.

32. The method of any one of claims 29-31, wherein the open reading frame sequences are compared, and the open reading frame sequences are determined using exome sequencing.

33. The method of any one of claims 29-31, wherein the mRNA sequences are compared, and the mRNA sequences are determined using transcriptome sequencing.

34. The method of any one of claims 29-31, wherein the comparing comprises use of a screening assay or screening tool and associated digital software for comparing one or more open reading frames in nucleic acid sequences, wherein the associated digital software comprises access to a sequence database that allows screening of mutations within open reading frames for identification of the immunogenic potential of the one or more cancer- specific neoepitopes.

35. The method of any one of claims 29-34, wherein step (b) comprises designing an antigenic peptide for each of the one or more cancer- specific neoepitopes.

36. The method of claim 35, wherein each antigenic peptide comprises a different cancer- specific mutation and flanking sequence on each side.

37. The method of claim 36, wherein each antigenic peptide includes at least about 10 flanking amino acids on each side

38. The method of any one of claims 35-37, wherein step (b) comprises scoring the each antigenic peptide and selecting an antigenic peptide if it scores below a hydropathy threshold predictive of secretability in Listeria monocytogenes.

39. The method of claim 38, wherein the scoring is by a Kyte and Doolittle hydropathy index 21 amino acid window, and any antigenic peptides scoring above a cutoff of about 1.6 are excluded or are modified to score below the cutoff.

40. The method of any claim 38 or 39, wherein every identified cancer- specific neoepitope for which an antigenic peptide can be designed that scores below the threshold is selected for inclusion in the second nucleic acid in the second recombinant Listeria strain or in the second nucleic acid and one or more additional nucleic acids for transforming one or more additional recombinant Listeria strains.

41. The method of any one of claims 29-40, wherein designing the second nucleic acid in step (b) comprises determining an order for the cancer- specific neoepitopes in the second fusion polypeptide.

42. The method of claim 41, wherein the order is selected using randomization.

43 The method of any one of claims 29-42, wherein designing the second nucleic acid in step (b) comprises scoring the hydropathy of the second fusion polypeptide, and either reordering the cancer- specific neoepitopes or removing problematic cancer- specific neoepitopes if any region of the second fusion polypeptide scores above a selected hydropathy index threshold value.

44. The method of claim 43, wherein the second fusion polypeptide is scored by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window, and wherein the threshold value is about 1.6.

45. The method of any one of claims 29-44, wherein designing the second nucleic acid in step (b) comprises codon optimizing the second nucleic acid for expression and secretion in Listeria monocytogenes.

46. The method of any one of claims 29-45, wherein the transforming is accomplished using a plasmid or a phage vector.

47. The method of any one of claims 29-46, further comprising culturing and characterizing the transformed recombinant Listeria strain to confirm expression and/or secretion of the second fusion polypeptide.

48. The method of any one of claims 1 and 18-47, wherein the first PEST- containing peptide comprises a bacterial secretion signal sequence, and the first fusion polypeptide further comprises a ubiquitin protein fused to a carboxy-terminal antigenic peptide, wherein the first PEST-containing peptide, the two or more first antigenic peptides, the ubiquitin, and the carboxy-terminal antigenic peptide are arranged in tandem from the amino-terminal end to the carboxy-terminal end of the first fusion polypeptide.

49. The method of claim 48, wherein the carboxy-terminal antigenic peptide is from a cancer-associated protein and comprises a heteroclitic mutation.

50. The method of claim 48 or 49, wherein the carboxy-terminal antigenic peptide is about 7-11, 8-10, or 9 amino acids in length.

51. The method of any one of claims 48-50, wherein the carboxy-terminal antigenic peptide binds to one or more of the following HLA types: HLA-A*02:01, HLA- A*03:01, HLA-A*24:02, and HLA-B*07:02.

52. The method of any one of claims 48-51, wherein the carboxy-terminal antigenic peptide is from a protein encoded by one of the following genes: STEAP1,

CEACAM5, NYESOl, and NUF2.

53. The method of claim 52, wherein the carboxy-terminal antigenic peptide is selected from the peptides set forth in SEQ ID NOS: 796, 797, 798, 799, 800, and 807.

54. The method of any one of claims 1 and 18-53, wherein each antigenic peptide in the first fusion polypeptide is a fragment of a cancer-associated protein and is about 7-200 amino acids in length.

55. The method of any one of claims 1 and 18-54, wherein the first fusion polypeptide comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 antigenic peptides or comprises between about 5-50, 10-40, or 20-30 antigenic peptides.

56. The method of any one of claims 1 and 18-55, wherein the first fusion polypeptide comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic peptides comprising a recurrent cancer mutation or between about 5-30 or 10-20 antigenic peptides comprising a recurrent cancer mutation, and/or

wherein the first fusion polypeptide comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic peptides comprising a heteroclitic mutation or between about 5-30 or 10-20 antigenic peptides comprising a heteroclitic mutation.

57. The method of claim 56, wherein the antigenic peptides comprising a recurrent cancer mutation in the first fusion polypeptide are in tandem, and the antigenic peptides comprising a heteroclitic mutation in the first fusion polypeptide are in tandem.

58. The method of claim 56, wherein the antigenic peptides comprising a recurrent cancer mutation and the antigenic peptides comprising a heteroclitic mutation are intermixed within the first fusion polypeptide.

59. The method of any one of claims 1 and 18-58, wherein the two or more antigenic peptides in the first fusion polypeptide are linked to each other via peptide linkers.

60. The method of claim 59, wherein the peptide linkers comprise flexibility linkers and/or rigidity linkers and/or immunoproteasome processing linkers, or wherein one or more of the linkers set forth in SEQ ID NOS: 310-319 and 821-829 are used to link the two or more antigenic peptides.

61. The method of claim 60, wherein the peptide linker upstream of one or more of the antigenic peptides comprising a heteroclitic mutation is an immunoproteasome processing linker or is selected from the linkers set forth in SEQ ID NOS: 821-829.

62. The method of any one of claims 1 and 18-61, wherein no region of the first fusion polypeptide scores above a cutoff of around 1.6 when scored for hydropathy by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window.

63. The method of any one of claims 1 and 18-62, wherein at least two of the antigenic peptides in the first fusion polypeptide comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein.

64. The method of any one of claims 1 and 18-63, wherein the recurrent cancer mutations in at least two of the antigenic peptides in the first fusion polypeptide are from the same cancer-associated protein and do not occur naturally together.

65. The method of any one of claims 1 and 18-64, wherein at least two of the antigenic peptides in the first fusion polypeptide are overlapping fragments of the same cancer-associated protein.

66. The method of claim 65, wherein the recurrent cancer mutations in at least two of the antigenic peptides in the first fusion polypeptide are from the same cancer- associated protein and occur at the same amino acid residue of the cancer-associated protein.

67. The method of claim 66, wherein two of the antigenic peptides in the first fusion polypeptide comprise the same recurrent cancer mutation.

68. The method of any one of claims 1 and 18-66, wherein each antigenic peptide comprising a recurrent cancer mutation in the first fusion polypeptide comprises a different recurrent cancer mutation.

69. The method of any one of claims 1 and 18-68, wherein each recurrent cancer mutation in the first fusion polypeptide is a somatic frameshift mutation or a somatic mis sense mutation.

70. The method of claim 69, wherein each recurrent cancer mutation in the first fusion polypeptide is a somatic missense mutation.

71. The method of any one of claims 1 and 18-70, wherein one or more or all of the antigenic peptides comprising a recurrent cancer mutation in the first fusion polypeptide have an equal number of amino acids flanking each side of the recurrent cancer mutation.

72. The method of claim 71, wherein the number of flanking amino acids on each side of the recurrent cancer mutation is at least 10 amino acids.

73. The method of any one of claims 1 and 18-72, wherein the antigenic peptides in the first fusion polypeptide comprise the 2, 3, 4, 5, 6, 7, 8, 9, or 10 most common recurrent cancer mutations or recurrent somatic missense cancer mutations from a particular type of cancer.

74. The method of any one of claims 1 and 18-73, wherein at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 35%, 50%, 60%, 70%, 80%, or 90% of patients with a particular type of cancer have a recurrent cancer mutation that is included in the combination of antigenic peptides in the first fusion polypeptide.

75. The method of any one of claims 1 and 18-74, wherein the antigenic peptides in the first fusion polypeptide comprise at least about 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 recurrent cancer mutations or recurrent somatic missense cancer mutations from a particular type of cancer, or wherein the antigenic peptides in the first fusion polypeptide comprise about 2-80, 10-60, 10-50, 10-40, or 10-30 different recurrent cancer mutations or recurrent somatic missense cancer mutations from a particular type of cancer.

76. The method of any one of claims 73-75, wherein the particular type of cancer is no n- small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer, or head and neck cancer.

77. The method of any one of claims 1 and 18-76, wherein the antigenic peptides in the first fusion polypeptide are from two or more cancer-associated proteins.

78. The method of claim 77, wherein the two or more cancer-associated proteins are at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer-associated proteins, or wherein the two or more cancer-associated proteins are about 2-30, 2-25, 2-20, 2-15, or 2-10 cancer- associated proteins.

79. The method of any one of claims 1 and 18-78, wherein the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more of the following genes: ACVR2A, ADAM28, AKTl, ANKRD36C, AR, ARID1A, BMPR2, BRAF, CHEK2, C12orf4, CTNNB1, DOCK3, EGFR, ESR1, FBXW7, FGFR3, FHOD3, GNAS, HRAS, IDH1, IDH2, KIAA2026, KRAS, KRTAP1-5, KRTAP4-11, LARP4B, MBOAT2, NFE2L2, PGM5, PIK3CA, PLEKHA6, POLE, PTEN, RGPD8, RNF43, RXRA, SMAD4, SPOP, SVIL, TGFBR2, TP53, TRIM48, UBR5, U2AF1, WNT16, XYLT2, ZBTB20, and ZNF814.

80. The method of claim 79, wherein:

(a) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS, EGFR, U2AF1, BRAF, PIK3CA, and TP53;

(b) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: SPOP, CHEK2, RGPD8, ANKRD36C, and AR;

(c) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS, U2AF1, TP53, SMAD4, and GNAS; (d) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes:

PIK3CA, FGFR3, TP53, RXRA, FBXW7, and NFE2L2;

(e) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes:

PIK3CA, AKT1, and ESR1;

(f) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: PTEN, KRAS, PIK3CA, CTNNB1, FBXW7, and TP53;

(g) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: TP53;

(h) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: TP53, PIK3CA, IDH1, IDH2, and EGFR;

(i) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS, BRAF, PIK3CA, and TP53; or

(]) the antigenic peptides in the first fusion polypeptide comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes:

PIK3CA, CHEK2, RGPD8, ANKRD36C, TP53, ZNF814, KRTAP1-5, KRTAP4-11, and HRAS.

81. The method of claim 80, wherein:

(a) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: KRAS_G12C, EGFR_L858R, KRAS_G12D, U2AF1_S34F, BRAF_V600E, KRAS_G12V, PIK3CA_E545K,

TP53_R158L, KRAS_G12A, EGFR_L861Q, and TP53_R273L;

(b) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: SPOP_F133V, CHEK2_K373E, RGPD8_P1760A, ANKRD36C_I634T, ANKRD36C_D629Y, SPOP_W131G,

ANKRD36C_D626N, SPOP_F133L, AR_T878A, AR_L702H, AR_W742C, AR_H875Y, and AR_F877L;

(c) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: KRAS_G12C, KRAS_G12D, U2AF1_S34F, KRAS_G12V, TP53_R248Q, TP53_R248W, TP53_R175H, TP53_R273C, KRAS_G12R, KRAS_Q61H, TP53_R282W, TP53_R273H, TP53_G245S, SMAD4_R361C, GNAS_R201C, and GNAS_R201H;

(d) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: PIK3CA_E545K, FGFR3_S249C, TP53_R248Q, PIK3CA_E542K, RXRA_S427F, FBXW7_R505G, TP53_R280T,

NFE2L2_E79K, FGFR3_R248C, TP53_K132N, TP53_R248W, TP53_R175H, and

TP53_R273C;

(e) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: PIK3CA_E545K, PIK3CA_E542K, PIK3CA_H1047R, AKT1_E17K, PIK3CA_H1047L, PIK3CA_Q546K, PIK3CA_E545A, PIK3CA_E545G, ESR1_K303R, ESR1_D538G, ESR1_Y537S, ESR1_Y537N,

ESR1_Y537C, and ESR1_E380Q;

(f) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: PTEN_R130G, PTEN_R130Q, KRAS_G12D, KRAS_G12V, PIK3CA_H1047R; PIK3CA_R88Q, PIK3CA_E545K, PIK3CA_E542K, CTNNB 1_S37F, KRAS_G13D, CTNNB 1_S37C, PIK3CA_H1047L, PIK3CA_G118D, KRAS_G12A, FBXW7_R505C, and TP53_R248W;

(g) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: TP53_R248Q, TP53_R248W, TP53_R175H, TP53_R273C, TP53_R282W, TP53_R273H, TP53_Y220C, TP53_I195T, TP53_C176Y, TP53_H179R, TP53_S241F, and TP53_H193R;

(h) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: TP53_R273L, TP53_R273C, TP53_R273H, PIK3CA_G118D, IDH1_R132C, IDH1_R132G, IDH1_R132H,

IDH1_R132S, IDH2_R172K, PIK3CA_E453K, and EGFR_G598V;

(i) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: KRAS_G12C, KRAS_G12D, BRAF_V600E, KRAS_G12V, PIK3CA_E545K, TP53_R248W, TP53_R175H,

TP53_R273C, PIK3CA_H1047R, TP53_R282W, TP53_R273H, and KRAS_G13D; or

(]) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the following recurrent cancer mutations: PIK3CA_E545K, CHEK2_K373E, RGPD8_P1760A, ANKRD36C_I634T, TP53_R248Q, PIK3CA_E542K, TP53_R248W, TP53_R175H, PIK3CA_H1047R, TP53_R282W, TP53_R273H, TP53_G245S, TP53_Y220C, ZNF814_D404E, KRTAP1-5_I88T, KRTAP4-11_L161V, and HRAS_G13V.

82. The method of claim 81, wherein:

(a) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 35;

(b) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 52;

(c) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 68;

(d) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 76;

(e) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 87;

(f) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 95;

(g) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 100;

(h) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 104;

(i) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 108; or

(]) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 112.

83. The method of any one of claims 1 and 18-82, wherein each antigenic peptide comprising a heteroclitic mutation in the first fusion polypeptide is about 7-11, 8-10, or 9 amino acids in length.

84. The method of any one of claims 1 and 18-83, wherein the antigenic peptides comprising a heteroclitic mutation in the first fusion polypeptide bind to one or more or all of the following HLA types: HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, and HLA- B*07:02.

85. The method of any one of claims 1 and 18-84, wherein the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more of the following genes: CEACAM5, GAGEl, hTERT, KLHL7, MAGEA3, MAGEA4, MAGEA6, NUF2, NYESOl, PAGE4, PRAME, PSA, PSMA, RNF43, SART3, SSX2, STEAPl, and SURVIVIN.

86. The method of claim 85, wherein:

(a) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes:

CEACAM5, MAGEA6, MAGEA4, GAGEl, NYESOl, STEAPl, and RNF43;

(b) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes:

CEACAM5, MAGEA4, STEAPl, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA;

(c) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes:

CEACAM5, STEAPl, MAGEA3, PRAME, hTERT, and SURVIVIN;

(d) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes:

CEACAM5, GAGEl, NYESOl, RNF43, NUF2, KLHL7, MAGEA3, and PRAME;

(e) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes:

CEACAM5, STEAPl, RNF43, MAGEA3, PRAME, and hTERT;

(f) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes:

CEACAM5, PRAME, hTERT, STEAPl, RNF43, NUF2, KLHL7, and SART3;

(g) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes:

CEACAM5, STEAPl, RNF43, SART3, NUF2, KLHL7, PRAME, and hTERT;

(h) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes:

CEACAM5, MAGEA6, STEAPl, RNF43, SART3, NUF2, KLHL7, and hTERT;

(i) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes:

CEACAM5, MAGEA6, MAGEA4, GAGEl, NYESOl, STEAPl, RNF43, and MAGEA3; or (j) the antigenic peptides in the first fusion polypeptide comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, MAGEA4, STEAP1, NYESOl, PRAME, and hTERT.

87. The method of claim 86, wherein:

(a) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 36;

(b) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 53;

(c) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 69;

(d) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 77;

(e) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 88;

(f) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 96;

(g) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 101 ;

(h) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 105;

(i) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 109; or

(j) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Table 113.

88. The method of any one of claims 1 and 18-87, wherein:

(a) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 35 and 36;

(b) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 52 and 53;

(c) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 68 and 69; (d) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 76 and 77;

(e) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 87 and 88;

(f) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 95 and 96;

(g) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 100 and 101;

(h) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 104 and 105;

(i) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 108 and 109; or

(]) the antigenic peptides in the first fusion polypeptide comprise one or more or all of the peptides set forth in Tables 112 and 113.

89. The method of claim 88, wherein:

(a) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 859, 860, 861, 862, 863, 864, 865, 894, 895, and 905;

(b) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 871, 872, 873, 874, 875, 876, 877, 892, 893, and 906;

(c) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 866, 867, 868, 869, 870, and 908;

(d) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 878, 879, 880, 881, 882, 888, 889, 890, and 891;

(e) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 883, 884, 885, 886, 887, and 907;

(f) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 896, 897, and 904;

(g) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 898 and 899;

(h) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 900 and 901;

(i) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 902 and 903; or (j) the first fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 918 and 919.

90. The method of any one of claims 1 and 18-89, wherein the first fusion polypeptide has a molecular weight of no more than about 150 kDa or no more than about 125 kDa.

91. A method for inducing an immune response against a tumor or cancer in a subject or treating a tumor or cancer in a subject, comprising:

(a) administering a recurrent cancer mutation immunotherapy composition to the subject, wherein the recurrent cancer mutation immunotherapy composition comprises a first recombinant Listeria strain comprising a first nucleic acid comprising a first open reading frame encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first PEST-containing peptide fused to two or more first antigenic peptides, wherein each of the first antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the first antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein; and

(b) administering a personalized immunotherapy composition to the subject, wherein the personalized immunotherapy composition comprises a second recombinant Listeria strain comprising a second nucleic acid comprising a second open reading frame encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises a second PEST-containing peptide fused to one or more second antigenic peptides, wherein each of the second antigenic peptides comprises a cancer- specific neoepitope comprising a cancer- specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject.

92. The method of claim 91, wherein the recurrent cancer mutations in at least two of the antigenic peptides are from the same cancer-associated protein and do not occur naturally together.

93. The method of claim 91 or 92, wherein at least two of the antigenic peptides are overlapping fragments of the same cancer-associated protein.

94. The method of claim 93, wherein the recurrent cancer mutations in at least two of the antigenic peptides are from the same cancer-associated protein and occur at the same amino acid residue of the cancer-associated protein.

95. The method of any one of claims 91-94, wherein one or more of the recurrent cancer mutations in the fusion polypeptide is a somatic missense mutation.

96. The method of any one of claims 91-95, wherein one or more of the recurrent cancer mutations in the fusion polypeptide is a somatic frameshift mutation.

97. The method of any one of claims 91-96, wherein the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS, BRAF, PIK3CA, TRIM48, PTEN, POLE, PGM5, MBOAT2,

KIAA2026, FBXW7, C12orf4, ZBTB20, XYLT2, WNT16, UBR5, TGFBR2, SVIL, RNF43, PLEKHA6, LARP4B, FHOD3, DOCK3, BMPR2, ARID1A, ADAM28, and ACVR2A.

98. The method of claim 97, wherein the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: TRIM48_Y192H, PTEN_R130N, POLE_V411L, POLE_P286R, PIK3CA_H1047R, PIK3CA_R88N, PGM5_I98V,

MBOAT2_R43N, KRAS_G12D, KIAA2026_R574C, FBXW7_R465C, C12orf4_R335N, BRAF_V600E, ZBTB20_p.Pro692LeufsTer43, XYLT2_p.Gly529AlafsTer78,

WNT16_p.Glyl67AlafsTerl7, UBR5_p.Glu2121LysfsTer28,

TGFBR2_p.Glu 150GlyfsTer35, S VIL_p.Met 1863TrpfsTer44, RNF43_p.Gly659ValfsTer41 , PLEKHA6_p.Val328TyrfsTerl72, LARP4B_p.Thrl63HisfsTer47,

FHOD3_p.Ser336ValfsTerl38, DOCK3_p.Prol852GlnfsTer45,

BMPR2_p.Asn583ThrfsTer44, ARIDlA_p.Aspl850ThrfsTer33,

ADAM28_p.Asn75LysfsTerl5, and ACVR2A_p.Lys435GlufsTerl9.

99. The method of claim 98, wherein the antigenic peptides comprise one or more or all of the peptides set forth in Table 116.

100. The method of claim 99, wherein the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NO: 917.

101. The method of any preceding claim, wherein the neoepitope in each of or some of the one or more second antigenic peptides comprises a linear neoepitope, a conformational neoepitope, a solvent-exposed neoepitope, or a combination thereof.

102. The method of any preceding claim, wherein the neoepitope in each of or some of the one or more second antigenic peptides comprises a T-cell epitope.

103. The method of any preceding claim, wherein each of the second antigenic peptides is about 5-100, 15-50, or 21-27 amino acids in length.

104. The method of any preceding claim, wherein each of the second antigenic peptides comprises a cancer- specific mutation flanked on each side by an equal number of amino acids.

105. The method of any preceding claim, wherein each of the second antigenic peptides comprises a cancer- specific mutation flanked on each side by at least 10 or at least 13 amino acids.

106. The method of any preceding claim, wherein the one or more second antigenic peptides comprise at least 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 antigenic peptides or 2-40 antigenic peptides.

107. The method of claim 106, wherein the second antigenic peptides are linked to each other via peptide linkers.

108. The method of claim 107, wherein one or more of the linkers set forth in SEQ ID NOS: 313-316, 319, and 821-829 are used to link the second antigenic peptides.

109. The method of any preceding claim, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the neoepitopes in the subject formed by nonsynonymous, somatic, cancer- specific mutations or formed by nonsynonymous, somatic, missense, cancer- specific mutations are included in the second fusion polypeptide.

110. The method of any preceding claim, wherein each of the second antigenic peptides comprises a different neoepitope or a different cancer- specific mutation.

111. The method of any preceding claim, wherein each cancer- specific mutation in the second fusion polypeptide is a somatic missense mutation.

112. The method of any preceding claim, wherein the first and/or second fusion polypeptide further comprises one or more peptide tags N-terminal and/or C-terminal to the combination of the two or more antigenic peptides, wherein the one or more peptide tags comprise one or both of the following: FLAG tag and SIINFEKL tag.

113. The method of any preceding claim, wherein the first and/or second PEST-containing peptide is on the N-terminal end of the first and/or second fusion polypeptide.

114. The method of claim 113, wherein the first and/or second PEST- containing peptide is an N-terminal fragment of LLO.

115. The method of claim 114, wherein the N-terminal fragment of LLO has the sequence set forth in SEQ ID NO: 336.

116. The method of any preceding claim, wherein the first and/or second nucleic acid is in an episomal plasmid.

117. The method of any preceding claim, wherein the first and/or second nucleic acid does not confer antibiotic resistance upon the first and/or second recombinant Listeria strain.

118. The method of any preceding claim, wherein the first and/or second recombinant Listeria strain is an attenuated, auxotrophic Listeria strain.

119. The method of claim 118, wherein the attenuated, auxotrophic Listeria strain comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes.

120. The method of claim 119, wherein the one or more endogenous genes comprise actA, dal, and dat.

121. The method of any preceding claim, wherein the first and/or second nucleic acid comprises an open reading frame encoding a metabolic enzyme.

122. The method of claim 121, wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid aminotransferase enzyme.

123. The method of any preceding claim, wherein the first and/or second fusion polypeptide is expressed from an hly promoter.

124. The method of any preceding claim, wherein the first and/or second recombinant Listeria strain is a recombinant Listeria monocytogenes strain.

125. The method of any preceding claim, wherein the first and/or second recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the first and/or second nucleic acid is in an episomal plasmid and comprises an open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

126. A system for use in cancer immunotherapy in a subject, comprising:

(a) the recurrent cancer mutation immunotherapy composition of any preceding claim; and

(b) the personalized immunotherapy composition of any preceding claim.