WO2018102585A1 - Immunothérapie personnalisée en association avec une immunothérapie ciblant des mutations de cancer récurrentes - Google Patents

Immunothérapie personnalisée en association avec une immunothérapie ciblant des mutations de cancer récurrentes Download PDF

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WO2018102585A1
WO2018102585A1 PCT/US2017/064016 US2017064016W WO2018102585A1 WO 2018102585 A1 WO2018102585 A1 WO 2018102585A1 US 2017064016 W US2017064016 W US 2017064016W WO 2018102585 A1 WO2018102585 A1 WO 2018102585A1
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cancer
fusion polypeptide
antigenic peptides
mutations
peptides
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PCT/US2017/064016
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Robert Petit
Kyle Perry
Michael F. PRINCIOTTA
Daniel J. O'connor
Brandon CODER
David BALLI
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Advaxis, Inc.
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Publication of WO2018102585A1 publication Critical patent/WO2018102585A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A61K2039/645—Dendrimers; Multiple antigen peptides

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • Methods and compositions are provided for cancer immunotherapy.
  • 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
  • each of the antigenic peptides in (a) comprises a different recurrent cancer mutation from a different cancer-associated protein.
  • each of the antigenic peptides in (a) comprises a different recurrent cancer mutation from a single type of cancer.
  • a recurrent cancer mutation immunotherapy composition 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 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
  • the first PEST-containing peptide comprises a bacterial secretion signal sequence
  • 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.
  • a recurrent cancer mutation immunotherapy composition 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; 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
  • each of the antigenic peptides in (a) comprises a different recurrent cancer mutation from a different cancer-associated protein.
  • each of the antigenic peptides in (a) comprises a different recurrent cancer mutation from a single type of cancer.
  • a recurrent cancer mutation immunotherapy composition 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 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 P
  • the first PEST-containing peptide comprises a bacterial secretion signal sequence
  • 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.
  • 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.
  • 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.
  • Figures 3A and 3B show Western blots of the Lmdda- ⁇ 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).
  • 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)
  • lane 3 is a negative control.
  • lane 1 is the ladder
  • lane 2 is the negative control
  • lane 3 is the WT1- tLLO-FLAG-Ub-heteroclitic phenylalanine minigene construct (construct #1).
  • Figure 4 shows colony PCR results for several Lm-minigene constructs expressing heteroclitic mutant WT1 peptides. Mutated residues are bolded and underlined.
  • 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.
  • 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.
  • FIGS 7A and 7B show IFN- ⁇ 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.
  • FIGS 8A and 8B show IFN- ⁇ 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.
  • FIG. 9 shows MC38 tumor volume in mice treated with LmddA-214 control, Lm
  • 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 intravenous dosing
  • IV intravenous dosing
  • FIG 11 shows CT26 tumor volume in mice treated with PBS control or Lm
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • FIG. 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.
  • Figure 20 shows IFN- ⁇ spot-forming cells (SFC) per 2xl0 5 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.
  • 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 (#).
  • 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.
  • FIG. 23A General overview of the tumor sequencing and DNA generation work stream.
  • FIG. 23B General overview of DNA cloning and immunotherapy
  • Figure 24 Diagram of a cluster of fully enclosed single use cell growth systems arranged for parallel manufacturing of personalized immunotherapy compositions.
  • FIG. 25 Detailed diagram of the inoculation and fermentation segments of fully enclosed single use cell growth system.
  • FIG. 26 Detailed diagram of the concentration segment of fully enclosed single use cell growth system.
  • FIG. 27 Detailed diagram of the diafiltration segment of fully enclosed single use cell growth system.
  • Figure 28 Detailed diagram of the product dispensation segment of fully enclosed single use cell growth system.
  • Figure 29 A Diagram of the process of using a serial selection of neo-epitopes in order to improve efficiency of immunotherapy.
  • Figure 29B Diagram of the process of using a parallel selection multiple neo- epitopes.
  • 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.
  • a delivery vector e.g., Listeria monocytogenes using output data containing all neo-antigens and patient HLA types.
  • 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.
  • Figure 32A shows the timeline for B 16F10 tumor experiments, including treatments with Lm Neo constructs.
  • Figure 32B shows tumor regression with Lmdd ⁇ A, Lm-Neo-12, and Lm-Neo- 20, with PBS used as a negative control.
  • 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.
  • 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).
  • 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.
  • Figure 35A shows tumor regression with LmddAllA, Lm-Neo-12, Lm-Neo-20, and Lm-Neo 30, with PBS used as a negative control.
  • 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.
  • 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.
  • Figure 37 shows the relative CD8 cell response in mice immunized with lung neo- epitope constructs.
  • 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.
  • 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.
  • 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.
  • FIG 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.
  • SFC spot-forming colonies
  • FIG 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • protein refers 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.
  • Proteins are said to have an "N-terminus” and a "C-terminus.”
  • N- terminus relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (-NH2).
  • C-terminus relates to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).
  • 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.
  • a chimeric molecule can be recombinantly expressed as a single-chain fusion protein.
  • 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.
  • 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.
  • 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.
  • discrete elements are referred to as being "upstream” or 5' of the "downstream” or 3' elements.
  • 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.
  • 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.
  • 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.
  • vector refers to a construct which is capable of delivering, and, optionally, expressing, one or more fusion polypeptides in a host cell.
  • extrachromosomal plasmid refers to a nucleic acid vector that is physically separate from chromosomal DNA (i.e., episomal or
  • 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.
  • 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.
  • 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.
  • the period can be at least 15 generations, 20
  • 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.
  • An "open reading frame” or “ORF” is a portion of a DNA which contains a sequence of bases that could potentially encode a protein.
  • an ORF can be located between the start-code sequence (initiation codon) and the stop-codon sequence (termination codon) of a gene.
  • 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
  • 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.
  • 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.
  • 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).
  • sequence 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue.
  • 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.
  • 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.
  • 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.
  • a "homologous" 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.
  • 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).
  • isolated 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • fragment when referring to a protein means a protein that is shorter or has fewer amino acids than the full length protein.
  • 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.
  • 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.
  • 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.
  • biological activities or functions can include, for example, the ability to elicit an immune response when administered to a subject.
  • biological activities or functions can also include, for example, binding to an interaction partner.
  • 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.
  • immunogenicity refers 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.
  • a molecule e.g., a protein, a nucleic acid, an antigen, or an organism
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mutation refers to the any change of the structure of a gene or a protein.
  • 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.
  • 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.
  • 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.
  • a "synonymous" mutation in a DNA is one that does not alter the amino acid sequence of a protein (due to codon degeneracy).
  • 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.
  • 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.
  • in vitro refers to artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube).
  • 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.
  • compositions or methods "comprising” or “including” one or more recited elements may include other elements not specifically recited.
  • a composition that "comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients.
  • Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.
  • 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.
  • an antigen or “at least one antigen” can include a plurality of antigens, including mixtures thereof.
  • 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.
  • 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.
  • 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.
  • recombinant fusion polypeptides from such compositions nucleic acids encoding such fusion proteins.
  • 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.
  • 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.
  • 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.
  • Lm-based vectors are a far superior platform for the generation of CD8+ dominant T cell responses compared to peptide vaccines.
  • 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.
  • 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.
  • 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.
  • 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.
  • TLRs toll- like receptors
  • PAMPs pathogen-associated molecular patterns
  • STING interferon genes
  • MDSCs myeloid derived suppressor cells
  • 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.
  • 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.
  • tLLO listeriolysin O
  • 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.
  • MHC major histocompatibility complex
  • 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.
  • 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.
  • the peptides are manufactured by the bacteria right at the point of use for antigen processing.
  • 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.
  • 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).
  • specific epitopes e.g., T cell epitopes
  • multiple recurrent cancer mutations e.g., shared tumor driver hotspot mutations
  • cancer-associated genes e.g., key tumor driver genes.
  • 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.
  • 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.
  • 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.
  • 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.
  • the total spectrum of potential tumor driver gene missense mutations for solid tumors can be covered within the capacity of one Lm construct.
  • 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
  • RNA sequencing or another diagnostic biomarker procedure, on a biopsy or archived tumor tissue or DNA or RNA sequencing information that may already exist.
  • 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.
  • heteroclitic sequences i.e., sequence-optimized peptides
  • tumor-associated antigen genes e.g., from cancer testis antigens or oncofetal antigens
  • 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%.
  • HLA-A0201, HLA-A0301, HLA-A2402, and HLA- B0702 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).
  • minigene construct approach 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).
  • pAPC professional antigen presenting cells
  • 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.
  • 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.
  • 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).
  • CTAs cancer testis antigens
  • OFAs oncofetal antigens
  • tLLO fusion proteins tumor-associated antigen fusion proteins
  • 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.
  • 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.
  • somatic mutations cancer testis antigens
  • 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.
  • 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.
  • Patients with multiple mutations in cancer-associated 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).
  • a combination e.g., a single dosing regimen consisting of two or more immunotherapies
  • a combination kit or panel e.g., a single dosing regimen consisting of two or more immunotherapies
  • 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.
  • 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.
  • CRC colorectal cancer
  • 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.
  • 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.
  • 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.
  • 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.
  • ADXS-HOT approach has inherent advantages over personalized, neoepitope-targeted, patient- specific products for the treatment of cancer.
  • 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.
  • 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.
  • 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.
  • the personalized product can replace the ADXS-HOT regimen and/or add targeting of the personalized neoepitopes to the recurrent cancer mutations being targeted.
  • 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.
  • 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
  • Disease-specific panels can target the majority of patients with a specific disease that share common mutations.
  • 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.
  • 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.
  • 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.
  • hotspot treatments can be given in combination or sequentially with other cancer treatments like checkpoint inhibitors, costimulatory agonists, radiation therapy, or personalized neoepitope immunization.
  • other cancer treatments like checkpoint inhibitors, costimulatory agonists, radiation therapy, or personalized neoepitope immunization.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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
  • each of the antigenic peptides comprises a different recurrent cancer mutation from a different cancer- associated protein.
  • 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).
  • PEST-containing peptide e.g., PEST1- peptidel ; PEST2-peptide2
  • 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).
  • each of the antigenic peptides comprises a different recurrent cancer mutation from a single type of cancer.
  • 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.
  • 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.
  • 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
  • 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.
  • each of the antigenic peptides comprises a different recurrent cancer mutation from a different cancer-associated protein.
  • some or all of the fragments are non-contiguous fragments of the same cancer-associated protein.
  • each of the antigenic peptides comprises a different recurrent cancer mutation from a single type of cancer.
  • 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.
  • 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.
  • 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
  • each of the antigenic peptides comprises a different recurrent cancer mutation from a different cancer-associated protein.
  • 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).
  • Ub protein e.g., Ubl-peptidel ; Ub2-peptide2.
  • some or all of the fragments are noncontiguous fragments of the same cancer-associated protein.
  • each of the antigenic peptides comprises a different recurrent cancer mutation from a single type of cancer.
  • 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.
  • breast cancer e.g., ER+ breast cancer
  • uterine cancer e.g., ovarian cancer
  • low-grade glioma e.g., MSS colorectal cancer
  • head and neck cancer e.g., MSS colorectal cancer
  • Nucleic acids 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.
  • 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.
  • the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.
  • 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.
  • 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).
  • the secretion signal sequence is from a Listeria protein, such as an ActA 3 oo secretion signal or an ActAioo secretion signal.
  • 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.
  • the recombinant fusion polypeptides can comprise one or more tags.
  • 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.
  • tags can be located anywhere within the recombinant fusion polypeptide and in any order.
  • 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
  • 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).
  • CBP chitin binding protein
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • TRX thioredoxin
  • poly(NANP) poly(NANP
  • 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.
  • immune response can be monitored using a number of reagents including, for example, monoclonal antibodies and DNA or RNA probes specific for these tags.
  • 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.
  • 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.
  • the recombinant fusion polypeptide can be any molecular weight.
  • 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).
  • the recombinant fusion polypeptide is less than or no more than about 150 kDa or less than or no more about 130 kDa.
  • 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.
  • the recombinant fusion polypeptide is between about 50-150, 100-150, 50-125, or 100-125 kDa.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 is followed by two stop codons to ensure termination of protein synthesis.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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).
  • 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.
  • the antigenic peptides can be linked together in any manner.
  • the antigenic peptides can be fused directly to each other with no intervening sequence.
  • the antigenic peptides can be linked to each other indirectly via one or more linkers, such as peptide linkers.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • 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.
  • 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 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 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.
  • the fusion polypeptide can comprise any number of antigenic peptides.
  • 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.
  • 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.
  • the fusion polypeptide can include a single antigenic peptide.
  • 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.
  • the fusion polypeptide can include up to about 100, 10, 20, 30, 40, or 50 antigenic peptides.
  • 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.
  • 84 85, 86,
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • Each antigenic peptide can comprise a different (i.e., unique) recurrent cancer mutation.
  • two or more of the antigenic peptides in the fusion polypeptide can comprise the same recurrent cancer mutation.
  • 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).
  • the antigenic peptides comprise a different (i.e., unique) recurrent cancer mutation that is not present in any of the other antigenic peptides.
  • At least two of the antigenic peptides can comprise overlapping fragments of the same cancer-associated protein.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • breast cancer e.g., ER+ breast cancer
  • uterine cancer e.g., ovarian cancer
  • low-grade glioma e.g., MSS colorectal cancer
  • head and neck cancer e.g., MSS colorectal cancer
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • genes i.e., tumor driver genes
  • 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.
  • 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.
  • biologically active proteins for example, kinase domains or binding domains
  • interrupt active sites for example, phosphorylation sites
  • the cancer-associated protein can be any one of the following:
  • 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.
  • 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.
  • RAS e.g., KRAS
  • 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.
  • ACVR2A ACVR2A, ADAM28, AKT1, ANKRD36C, AR, ARID1A, BMPR2, BRAF,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA mutations, from N-terminal to C-terminal: R108H;
  • 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.
  • the fusion polypeptide can comprise antigenic peptides comprising the following PIK3CA mutations, from N-terminal to C-terminal: E542K; E545K; R88Q;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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;
  • 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;
  • 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 See, e.g., SEQ ID NOS: 97-102.
  • 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 PIK3R1IK567E
  • PIK3CAIR38C PIK3CAIE542K
  • PIK3CAIQ546K PIK3CAIE726K
  • PIK3CAIC420R PIK3CAIE545A; PIK3CAIR93Q; PIK3R1IN564D; PIK3CAIR108H; PIK3CAIM1043I; PIK3CAIE545G; and PIK3CAIE81K.
  • SEQ ID NOS: 103-108 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.
  • 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;
  • the fusion polypeptide can comprise antigenic peptides comprising the following PTEN mutations, from N-terminal to C-terminal: dell21- 131; Y88C; R130G; Y155C; D92E;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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;
  • 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;
  • the mutations can be in any order.
  • 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.
  • antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: H179W; R273L; R249S; R248Q; Y234H; G245D;
  • 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;
  • Y234C V157F; Y234H; C176Y; and K132N. See, e.g., SEQ ID NOS: 163-166.
  • 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.
  • antigenic peptides comprising the following TP53 mutations, from N-terminal to C-terminal: R248W; H179R; R273H; Y107D; R337H; R282G; V
  • 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.
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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;
  • 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.
  • 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.
  • the recurrent cancer mutations can be from multiple cancer- associated proteins.
  • 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.
  • 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;
  • 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.
  • 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;
  • TP53IR248W TP53IR273C; TP53IR282W; TP53IR273H; TP53IR248Q; TP53IG245S;
  • 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.
  • 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.
  • 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 BRAF1IG466V
  • 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
  • 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.
  • TP53IC135W TP53IC141W
  • TP53IC176F TP53IC176Y
  • TP53IH179R TP53IH179Y
  • TP53IY236C TP53IM237I; TP53IG244C; TP53IG245S; TP53IR248L; TP53IR248P;
  • TP53IR248Q TP53IR248W; TP53IR249G; TP53IR249S; TP53IR249W; TP53IG266V;
  • TP53IR282Q TP53IR282Q
  • TP53IR282W PIK3CAIE545K
  • PIK3CAIE542K PIK3CAIH1047R
  • PIK3CAIE726K PIK3CAIC420R
  • NFE2L2IE79Q PIK3CAIC420R
  • NFE2L2IE79Q PIK3CAIC420R
  • NFE2L2IE79Q PIK3CAIC420R
  • NFE2L2IE79Q PIK3CAIC420R
  • NFE2L2IE79Q PIK3CAIC420R
  • NFE2L2IE79Q PIK3CAIE79Q
  • NFE2L2IR34Q NFE2L2IL30F
  • NFE2L2IG81S NFE2L2IG31A; NFE2L2ID29G; NFE2L2IG81V; CDKN2AID108Y;
  • 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.
  • two or more cancer associated proteins comprise proteins encoded by two or more or all of the following genes: ANKRD36C, SPOP, CHEK2,
  • 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.
  • ANKRD36CII645T ANKRD36CID629Y; ANKRD36CID629N; SPOPIW131G; SPOPIF133L; SPOPIF133V; SPOPIF133C; SPOPIW131R; SPOPIW131L; CHEK2IK373E; KRTAP4-11IM93V; KRTAP4-11IR51K; KRTAP4-11IL161V; RGPD8IP1760A;
  • 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.
  • 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:
  • Such mutations are associated with, for example, non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung 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.
  • 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 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,
  • 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.
  • 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.
  • 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 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_G
  • 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.
  • 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 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:
  • 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,
  • 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.
  • 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 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 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.
  • 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 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:
  • 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,
  • 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.
  • 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 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.
  • 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 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,
  • the 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.
  • 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 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,
  • 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.
  • 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 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,
  • the 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.
  • 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 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,
  • 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,
  • KRAS_G12D BRAF_V600E, PIK3CA_H1047R, TRIM48_Y192H, PTEN_R130N, POLE_V411L, POLE_P286R, PIK3CA_R88N, PGM5_I98V, MBOAT2_R43N,
  • 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.
  • 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.
  • 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.
  • a PEST containing peptide is not part of and is separate from the fusion
  • 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.
  • 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).
  • a PEST or PEST-like sequence can be identified using the PEST-find program.
  • a PEST-like sequence can be a region rich in proline (P), glutamic acid (E), serine (S), and threonine (T) residues.
  • the PEST-like sequence can be flanked by one or more clusters containing several positively charged amino acids.
  • 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.
  • P proline
  • D aspartate
  • E glutamate
  • S serine
  • T threonine residues.
  • 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
  • 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.
  • a PEST-like sequence must contain at least one P, at least one D or E, and at least one S or T.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • KENSISSMAPPASPPASPK SEQ ID NO: 321.
  • 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.
  • the PEST-like sequence can be from a Listeria species, such as from Listeria monocytogenes.
  • 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
  • Streptolysin O proteins from Streptococcus sp. also contain a PEST sequence.
  • 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).
  • 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.
  • compositions and methods disclosed herein is a listeriolysin O (LLO) peptide.
  • LLO listeriolysin O
  • 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%.
  • 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.
  • 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.
  • 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.
  • LLO 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.
  • 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.
  • the LLO peptide can be a full-length LLO protein or a truncated LLO protein or LLO fragment.
  • 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.
  • 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.
  • the LLO peptide can be a non-hemolytic LLO.
  • Other functions of LLO are known, as are methods and assays for evaluating LLO functionality.
  • 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.
  • LLO proteins or fragments have reduced hemolytic activity relative to wild type LLO or are non-hemolytic fragments.
  • 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.
  • 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.
  • 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).
  • 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.”
  • a mutant LLO protein can be created with an internal deletion comprising the cholesterol-binding domain.
  • the internal deletion can be a 1-11 amino acid deletion, an 11-50 amino acid deletion, or longer.
  • 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, 15
  • 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).
  • the mutated region can also be a fragment of the CBD or can overlap with a portion of the CBD.
  • 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.
  • 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.
  • 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.
  • ESLLMWITQCR antigen NY-ESO-1
  • the resulting LLO is termed "ctLLO.”
  • the mutated region can be replaced by a heterologous sequence.
  • 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, 15-50, 15-60, 15-70, 15-
  • an LLO peptide may have a deletion in the signal sequence and a mutation or substitution in the CBD.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.:
  • 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.
  • 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).
  • truncated ActA proteins comprise at least one PEST sequence (e.g., more than one PEST sequence).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • the truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 341 can be used.
  • 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.
  • 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.
  • 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.
  • the ActA fragment can be fused to a heterologous signal peptide.
  • SEQ ID NO: 349 sets forth an ActA fragment fused to an Hly signal peptide.
  • 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.
  • 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.
  • frequency of occurrence across multiple types of cancer e.g., occurrence in
  • 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.
  • 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;
  • mutations on function of the protein e.g., loss of function of a tumor suppressor protein; known cancer "driver” mutations; known chemotherapy resistance mutations.
  • one or more of nonsense mutations, deletion mutations, insertion mutations, frameshift mutations, or translocation mutations can be excluded.
  • somatic missense mutations are considered.
  • frameshift e.g., somatic frameshift mutations
  • both somatic missense and frameshift mutations are considered.
  • a set of recurrent cancer mutations can be selected based on one or more additional criteria.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • mutation data can be sub- stratified by disease indication type. Particular types of mutations can be selected for consideration.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • a certain hydropathy threshold which can be predictive of secretability in a particular bacteria of interest (e.g., Listeria monocytogenes).
  • 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.
  • 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.
  • 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.
  • 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.
  • HLA human leukocyte antigen
  • IED Immune Epitope Database
  • 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.
  • 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.
  • immunosuppressive epitopes e.g., T-reg epitopes, IL-10-inducing T helper epitopes, and so forth
  • a predicative algorithm for immunogenicity of the epitopes can be used to screen the antigenic peptides.
  • these algorithms are at best 20% accurate in predicting which peptide will generate a T cell response.
  • the antigenic peptides can be screened for immunogenicity.
  • 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- ⁇ , 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.
  • the mutant amino acid can be flanked by, e.g., up to 10 wild-type amino acids immediately before and after missense mutation position.
  • 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.
  • 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.
  • 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.
  • 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.
  • ranges of linear antigenic peptides can be starting, for example, with about 50, 40, 30, 20, or 10 antigenic peptides per plasmid.
  • 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).
  • the order of the antigenic peptides can be generated using selected parameters, such as a predefined ranking of the antigenic peptides.
  • the combination of antigenic peptides or the entire fusion polypeptide can also be scored for hydrophobicity.
  • 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).
  • 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).
  • one or more linkers between antigenic peptides as disclosed elsewhere herein can be added or modified to change the hydrophobicity.
  • 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).
  • other suitable hydropathy plots or other appropriate scales could be used.
  • 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.
  • immunosuppressive epitopes e.g., T-reg epitopes, IL- 10- inducing T helper epitopes, and so forth
  • a nucleic acid encoding a candidate combination of antigenic peptides or fusion polypeptide can then be designed and optimized.
  • the sequence can be optimized for increased levels of translation, duration of expression, levels of secretion, levels of transcription, and any combination thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • Optimizing a sequence can also entail, for example, adapting the codon usage to the codon bias of host genes (e.g., Listeria monocytogenes genes).
  • host genes e.g., Listeria monocytogenes genes.
  • the codons below can be used for Listeria monocytogenes.
  • a nucleic acid encoding a fusion polypeptide can be generated and introduced into a delivery vehicle such as a bacteria strain or Listeria strain.
  • 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.
  • 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.
  • 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.
  • each of the antigenic peptides comprises a different heteroclitic mutation from a different cancer-associated protein.
  • each antigenic peptide is fused to its own PEST-containing peptide (e.g., PESTl-peptidel; PEST2-peptide2).
  • PEST-containing peptide e.g., PESTl-peptidel; PEST2-peptide2
  • 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).
  • 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.
  • each antigenic peptide e.g., from a cancer-associated protein
  • each antigenic peptide comprises a heteroclitic mutation
  • at least two of the antigenic peptides comprise different heteroclitic mutations and are fragments of the same cancer-associated protein
  • the fusion polypeptide does not comprise a PEST-containing peptide.
  • each of the antigenic peptides comprises a different heteroclitic mutation from a different cancer- associated protein.
  • some or all of the fragments are non-contiguous fragments of the same cancer-associated protein.
  • 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.
  • Ub ubiquitin
  • Ub ubiquitin
  • Nucleic acids 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.
  • 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.
  • the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.
  • 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.
  • 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).
  • the secretion signal sequence is from a Listeria protein, such as an ActA 3 oo secretion signal or an ActAioo secretion signal.
  • 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.
  • fusion polypeptides 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.
  • 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.
  • 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.
  • the recombinant fusion polypeptide can be any molecular weight.
  • 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).
  • the recombinant fusion polypeptide is less than or no more than about 150 kDa or less than or no more than about 130 kDa.
  • 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.
  • the recombinant fusion polypeptide is between about 50-150, 100-150, 50-125, or 100-125 kDa.
  • 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.
  • 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.
  • the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome.
  • 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).
  • 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.
  • 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 is followed by two stop codons to ensure termination of protein synthesis.
  • 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.
  • 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, 15
  • 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.
  • 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.
  • a heteroclitic antigenic peptide can be 9 amino acids in length.
  • 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.
  • a certain hydropathy threshold can be predictive of secretability in Listeria monocytogenes or another bacteria of interest.
  • 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.
  • 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.
  • the heteroclitic antigenic peptides can be linked together in any manner.
  • the heteroclitic antigenic peptides can be fused directly to each other with no intervening sequence.
  • the heteroclitic antigenic peptides can be linked to each other indirectly via one or more linkers, such as peptide linkers.
  • 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.
  • 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.
  • 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.
  • SEQ ID NOS SEQ ID NOS: 572-582
  • 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.
  • peptide linker sequences may contain Gly, Asn and Ser residues.
  • linker 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.
  • the fusion polypeptide can comprise any number of heteroclitic antigenic peptides.
  • 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.
  • the fusion polypeptide can comprise at least 3, 4,
  • the fusion polypeptide can include a single heteroclitic antigenic peptide.
  • 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.
  • the fusion polypeptide can include up to about 100, 10, 20, 30, 40, or 50 heteroclitic antigenic peptides.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • Each heteroclitic antigenic peptide can comprise a different (i.e., unique) heteroclitic mutation.
  • two or more of the heteroclitic antigenic peptides in the fusion polypeptide can comprise the same heteroclitic mutation.
  • two or more copies of the same heteroclitic antigenic polypeptide can be included in the fusion
  • the fusion polypeptide comprises two or more copies of the same heteroclitic antigenic peptide.
  • 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.
  • at least two of the heteroclitic antigenic peptides can comprise overlapping fragments of the same cancer-associated protein.
  • two or more of the heteroclitic antigenic peptides can comprise different heteroclitic mutations at the same amino acid residue of the cancer-associated protein.
  • 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.
  • heteroclitic antigenic peptides can be included that bind to one or more different HLA types.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • heteroclitic antigenic peptides are disclosed elsewhere herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • cancer-associated protein does not have to have cancer-specific, cancer-restricted, tumor-specific, or tumor-restricted expression.
  • proteins that are considered cancer- specific or cancer-restricted are cancer testis antigens or oncofetal antigens.
  • Cancer testis antigens CTAs
  • CTAs cancer testis antigens
  • Oncofetal antigens OFAs
  • OFAs Oncofetal antigens
  • 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).
  • YLMPVNSEV SEQ ID NO: 726
  • YMMPVNSEV SEQ ID NO: 725
  • a heteroclitic peptide can generate an immune response that recognizes the native peptide from which the heteroclitic peptide was derived.
  • 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.
  • 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.
  • MHC major histocompatibility complex
  • 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.
  • 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.
  • a heteroclitic peptide disclosed herein can bind to one or more HLA class II molecules.
  • 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.
  • a native or heteroclitic peptide disclosed herein can bind to one or more HLA class I molecules.
  • 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.
  • 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.
  • 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.
  • Heteroclitic peptides can comprise a mutation that enhances binding of the peptide to an HLA class II molecule relative to the corresponding native peptide.
  • heteroclitic peptides can comprise a mutation that enhances binding of the peptide to an HLA class I molecule relative to the corresponding native peptide.
  • 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).
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • PSSMs position specific scoring matrices
  • 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.
  • PSSMs for the prediction of peptide binders of different sizes are usually available for each MHC I molecule.
  • 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).
  • MHC class I epitopes are also well-known.
  • 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.
  • the MHC class I epitope can be identified using SYFPEITHI.
  • the MHC class I epitope can be identified using SVMHC.
  • 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).
  • 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).
  • the mutation can be in position 2 of the HLA class I binding motif (e.g., a mutation to leucine, valine, isoleucine, or methionine).
  • the mutation can be in position 6 of the HLA class I binding motif (e.g., to valine, cysteine, glutamine, or histidine).
  • 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.
  • 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.
  • a point mutation can be in a position selected from positions 4, 5, and 8.
  • different residues in HLA class II binding sites can be mutated.
  • an HLA class II motif anchor residue can be modified.
  • the PI position, the P2 position, the P6 position, or the P9 position can be mutated.
  • theP4 position, the P5 position, the P10 position, the PI 1 position, the P12 position, or the P13 position can be mutated.
  • 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.
  • 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.
  • the cancer-associated protein can be any one of the cancer- associated proteins listed elsewhere herein.
  • 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.
  • 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.
  • the heteroclitic antigenic peptides can be from multiple cancer-associated proteins (e.g., two or more cancer-associated proteins).
  • 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).
  • NSCLC non-small cell lung 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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).
  • heteroclitic antigenic peptides examples 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • a PEST containing peptide is not part of and is separate from the fusion
  • 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).
  • a PEST-like sequence such as an LLO peptide
  • PEST-containing peptides are disclosed in more detail elsewhere herein.
  • 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.
  • 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.
  • 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.
  • the sequence of the heteroclitic antigenic peptide can then be optimized to enhance binding to MHC Class I molecules.
  • 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)).
  • 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
  • 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.
  • 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.
  • hydropathy index threshold value 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
  • 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).
  • Target peptides can then be sequence optimized to enhance binding to MHC Class I molecules (aka heteroclitic peptide).
  • 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:
  • 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.
  • 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.
  • 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.
  • Nucleic acids 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.
  • 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.
  • 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.
  • additional antigenic peptides from cancer-associated proteins e.g., comprising a recurrent cancer mutation and/or a heteroclitic mutation
  • they can be fused directly to each other or linked via a peptide linker.
  • 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.
  • 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.
  • the recombinant fusion polypeptides can comprise one or more tags as disclosed elsewhere herein.
  • 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).
  • 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.
  • 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.
  • the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome.
  • 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).
  • 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.
  • 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.
  • Some exemplary antigenic peptides for inclusion in minigene constructs include those in the table below.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an antigenic peptide can be 9 amino acids in length.
  • 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
  • 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.
  • 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.
  • the antigenic peptides can be linked together in any manner.
  • the antigenic peptides can be fused directly to each other with no intervening sequence.
  • the antigenic peptides can be linked to each other indirectly via one or more linkers, such as peptide linkers.
  • 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.
  • 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.
  • 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.
  • 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.
  • the signal sequence can be an LLO signal sequence.
  • An exemplary LLO signal sequence is set forth in SEQ ID NO: 920.
  • 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.
  • 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).
  • the secretion signal sequence is from a Listeria protein, such as an ActA 3 oo 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.
  • 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.
  • 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.
  • 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.
  • the recombinant fusion polypeptides can comprise recurrent cancer mutation antigenic peptides only, heteroclitic antigenic peptides only, or minigene construct antigenic peptides only.
  • the recombinant fusion polypeptides can comprise both recurrent cancer mutation antigenic peptides and heteroclitic antigenic peptides but no minigene construct antigenic peptides.
  • the recombinant fusion polypeptides can comprise both recurrent cancer mutation antigenic peptides and minigene construct antigenic peptides but no heteroclitic antigenic peptides.
  • the recombinant fusion polypeptides can comprise both heteroclitic antigenic peptides and minigene construct antigenic peptides but no recurrent cancer mutation antigenic peptides.
  • 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.
  • 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.
  • 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).
  • antigenic peptides examples include Suitable antigenic peptides. Examples of antigenic peptides comprising recurrent cancer mutations are disclosed elsewhere herein.
  • 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).
  • each antigenic peptide is fused to its own secretion sequence and Ub protein (e.g., Ubl-peptidel ; Ub2-peptide2).
  • Ub protein e.g., Ubl-peptidel ; Ub2-peptide2.
  • suitable antigenic peptides are disclosed elsewhere herein.
  • antigenic peptides comprising heteroclitic mutations are disclosed elsewhere herein.
  • 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.
  • the antigenic peptides can be in tandem (e.g., Ub-peptidel-peptide2).
  • 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).
  • suitable antigenic peptides are disclosed elsewhere herein.
  • antigenic peptides comprising recurrent cancer mutations are disclosed elsewhere herein.
  • antigenic peptides comprising heteroclitic mutations are disclosed elsewhere herein.
  • 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.
  • 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.
  • 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.
  • the fusion polypeptide can comprise any number of antigenic peptides.
  • 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.
  • 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.
  • the fusion polypeptide can include a single antigenic peptide.
  • 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.
  • the fusion polypeptide can include up to about 100, 10, 20, 30, 40, or 50 antigenic peptides.
  • 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.
  • 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.
  • 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.
  • the antigenic peptides can be from any number of cancer-associated proteins.
  • 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.
  • 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.
  • the antigenic peptides comprising a recurrent cancer mutation can be in tandem
  • the antigenic peptides comprising a heteroclitic mutation can be in tandem.
  • the antigenic peptides comprising a recurrent cancer mutation and the antigenic peptides comprising a heteroclitic mutation can be intermixed within the fusion polypeptide.
  • 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.
  • 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).
  • 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.
  • 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 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 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.
  • the recombinant fusion polypeptide can be any molecular weight.
  • 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).
  • the recombinant fusion polypeptide is less than or no more than about 150 kDa or less than or no more than about 130 kDa.
  • 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.
  • the recombinant fusion polypeptide is between about 50-150, 100-150, 50-125, or 100-125 kDa.
  • 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.
  • 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.
  • the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome.
  • 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).
  • 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.
  • 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 is followed by two stop codons to ensure termination of protein synthesis.
  • 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.
  • the antigenic peptides can be from multiple cancer-associated proteins (e.g., two or more cancer-associated proteins).
  • 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,
  • the mutations are associated with, for example, non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung 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.
  • 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.
  • 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.
  • 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.
  • the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by CEACAM5.
  • the minigene antigenic peptide can comprise SEQ ID NO: 798 or SEQ ID NO: 796.
  • 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 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.
  • 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,
  • the 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.
  • 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.
  • 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.
  • the cancer- associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAPl.
  • the minigene antigenic peptide can comprise SEQ ID NO: 799 or SEQ ID NO: 800.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by CEACAM5.
  • the minigene antigenic peptide can comprise SEQ ID NO: 798 or SEQ ID NO: 796.
  • 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 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.
  • 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:
  • TP53_R248W TP53_R175H
  • 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.
  • 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.
  • 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.
  • the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by NYESOl or NUF2.
  • the minigene antigenic peptide can comprise SEQ ID NO: 797 or SEQ ID NO: 800.
  • 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
  • a breakdown of the amino acids positions of the individual components in each construct is provided in Tables 78-86.
  • 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,
  • 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.
  • 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.
  • 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.
  • the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAP1.
  • the minigene antigenic peptide can comprise SEQ ID NO: 799 or SEQ ID NO: 800.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAP1.
  • the minigene antigenic peptide can comprise SEQ ID NO: 799 or SEQ ID NO: 800.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAP1.
  • the minigene antigenic peptide can comprise SEQ ID NO: 799.
  • 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 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.
  • 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,
  • the 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.
  • 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.
  • 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.
  • the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by NUF2.
  • the minigene antigenic peptide can comprise SEQ ID NO: 807.
  • 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 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • the cancer-associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAP1.
  • the minigene antigenic peptide can comprise SEQ ID NO: 799.
  • 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 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • the cancer- associated protein from which the minigene antigenic peptide is generated can comprise protein encoded by STEAPl.
  • the minigene antigenic peptide can comprise SEQ ID NO: 799.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the antigenic peptides can be selected in step (c), for example, if they are below a hydropathy threshold predictive of secretability in Listeria monocytogenes.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the recombinant fusion polypeptide is between about 50-150, 100-150, 50-125, or 100-125 kDa.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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) .
  • Nucleic acids 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.
  • 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.
  • the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.
  • 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.
  • 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).
  • the secretion signal sequence is from a Listeria protein, such as an ActA 3 oo secretion signal or an ActAioo secretion signal.
  • 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.
  • the recombinant fusion polypeptides can comprise one or more tags.
  • 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.
  • 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.
  • 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.
  • the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome.
  • 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).
  • 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.
  • 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 is followed by two stop codons to ensure termination of protein synthesis.
  • 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.
  • 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-
  • 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.
  • 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
  • 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.
  • Each antigenic peptide can comprise, for example, a single neoepitope comprising a single cancer- specific mutation.
  • an antigenic peptide can comprise two or more neoepitopes or two or more cancer- specific mutations.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • TCR HLA T-cell receptor
  • this does not necessarily need to be the case.
  • 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).
  • the antigenic peptides can be linked together in any manner.
  • the antigenic peptides can be fused directly to each other with no intervening sequence.
  • the antigenic peptides can be linked to each other indirectly via one or more linkers, such as peptide linkers.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • "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.
  • the fusion polypeptide can comprise any number of antigenic peptides.
  • 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.
  • 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.
  • the fusion polypeptide can include a single antigenic peptide or neoepitope.
  • 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
  • the fusion polypeptide can include up to about 100, 10, 20, 30, 40, or 50 antigenic peptides or neoepitopes.
  • 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 anti
  • Each antigenic peptide can comprise a different (i.e., unique) neoepitope.
  • two or more of the antigenic peptides in the fusion polypeptide can comprise the same neoepitope.
  • 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).
  • polypeptides comprise a different (i.e., unique) neoepitope that is not present in any of the other antigenic peptides.
  • at least two of the antigenic peptides can comprise overlapping fragments of the same protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • T-reg T- regulatory
  • Neoepitopes can comprise a single mutation or two or more mutations.
  • 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.
  • 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.
  • Neoepitopes can be identified by whole genome sequencing, exome sequencing transcriptome sequencing, T-cell receptor sequencing, or any other means.
  • genome refers to the total amount of genetic information in the chromosomes of an organism
  • exome refers to the coding regions of the genome
  • transcriptome refers to the set of all mRNA molecules.
  • Any suitable sequencing method can be used.
  • next generation sequencing (NGS) technologies can be used.
  • 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.
  • NGS 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.
  • 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.
  • Such a personalized immunotherapy uses neoepitopes within mutated and variant antigens (neoantigens) that are specific to a particular subject's cancer or tumor.
  • 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.
  • 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.
  • 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.
  • incisional biopsy in which only a sample of tissue is removed
  • excisional biopsy in which an entire lump or suspicious area is removed
  • needle biopsy in which a sample of tissue or fluid is removed with a needle.
  • core biopsy When a wide needle is used, the procedure is called a core biopsy.
  • fine-needle aspiration biopsy the procedure is called a fine-needle aspiration biopsy.
  • 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).
  • healthy biological samples consist essentially or entirely of normal, healthy cells and can be used in comparison to a cancer sample.
  • the samples are of the same type (e.g., both blood or both sera).
  • the cancer sample comprises cells
  • 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).
  • the normal or healthy biological sample can be obtained at the same time.
  • 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.
  • 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.
  • CTC cytotoxic T cells
  • 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.
  • 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.
  • 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).
  • one or more of nonsense mutations, deletion mutations, insertion mutations, frameshift mutations, or translocation mutations can be excluded.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • a certain hydropathy threshold which can be predictive of secretability in a particular bacteria of interest (e.g., Listeria monocytogenes).
  • 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
  • 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.
  • 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,
  • the cutoff can vary, for example, depending on the genus or species of the bacteria being used to deliver the fusion polypeptide.
  • 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.
  • 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.
  • immunosuppressive epitopes e.g., T-reg epitopes, IL- 10- inducing T helper epitopes, and so forth
  • a predicative algorithm for immunogenicity of the epitopes can be used to screen the antigenic peptides or cancer- specific neoepitopes.
  • these algorithms are at best 20% accurate in predicting which peptide will generate a T cell response.
  • no screening/predictive algorithms are used.
  • 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- ⁇ , 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.
  • antigenic peptides comprising the one or more neoepitopes can be screened for an immunogenic response.
  • 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.
  • 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.
  • 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 51 Cr-release assay or a 3 H-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).
  • 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- ⁇ ⁇ see, e.g., US 8,962,319 and EP 177432, each of which is herein
  • a T-cell immune response may be assayed by a 51 Cr 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 51 Cr release assay, using a peptide/polypeptide comprising one or more neoepitopes as the target.
  • 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).
  • 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.
  • ranges of linear antigenic peptides can be starting, for example, with about 50, 40, 30, 20, or 10 antigenic peptides per plasmid.
  • 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).
  • the order of the antigenic peptides can be generated using selected parameters, such as a predefined ranking of the antigenic peptides.
  • the combination of antigenic peptides or the entire fusion polypeptide can also be scored for hydrophobicity.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • immunosuppressive epitopes e.g., T-reg epitopes, IL- 10- inducing T helper epitopes, and so forth
  • a nucleic acid encoding a candidate combination of antigenic peptides or fusion polypeptide can then be designed and optimized.
  • the sequence can be optimized for increased levels of translation, duration of expression, levels of secretion, levels of transcription, and any combination thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • Optimizing a sequence can also entail, for example, adapting the codon usage to the codon bias of host genes (e.g., Listeria monocytogenes genes).
  • host genes e.g., Listeria monocytogenes genes.
  • the codons below can be used for Listeria monocytogenes.
  • a nucleic acid encoding a fusion polypeptide can be generated and introduced into a delivery vehicle such as a bacteria strain or Listeria strain.
  • 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.
  • the bacteria or Listeria strain can be cultured and characterized to confirm expression and secretion of the fusion polypeptide comprising the antigenic peptides.
  • 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
  • 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 ne
  • 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 ne
  • 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
  • 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;
  • an immunogenic assay for testing the T-cell immunogenicity and/or binding of candidate peptides comprising one or more neo-epitopes;
  • analytic equipment, and associated software for sequencing and analyzing nucleic acid sequences, peptide amino acid sequences and T-cell receptor amino acid sequences;
  • 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 immuno
  • 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.
  • 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.
  • the recombinant Listeria strain can be any Listeria strain.
  • 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.
  • the recombinant listeria strain is a strain of the species Listeria monocytogenes. Examples of Listeria monocytogenes strains include the following: L.
  • 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.
  • 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
  • L. monocytogenes CS-LOOOl delta actA; delta MB
  • L. monocytogenes CS-L0002 delta actA; delta IplA
  • 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.
  • L. monocytogenes DP-L4038 delta actA; LLO L461T
  • L. monocytogenes DP- L4384 LLO S44A; LLO L461T
  • a L. monocytogenes strain with an IplAl deletion encoding lipoate protein ligase LplAl
  • IplAl deletion encoding lipoate protein ligase LplAl
  • 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.
  • the Listeria strain is L. monocytogenes EGD-e (see GenBank
  • 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.
  • the recombinant bacteria or Listeria can have wild-type virulence, can have attenuated virulence, or can be avirulent.
  • 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.
  • 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.
  • the recombinant Listeria strain lacks antibiotic resistance genes.
  • such recombinant Listeria strains can comprise a plasmid that does not encode an antibiotic resistance gene.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • promoters/regulatory sequences useful for driving constitutive expression of a gene include, for example, an hly, hlyA, actA, prfA, and p60 promoters of Listeria, the Streptococcus bac promoter, the
  • 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.
  • 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.
  • 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 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
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • an integrase gene and attachment site of a bacteriophage e.g., U153 or PSA listeriophage
  • 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.
  • 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.
  • 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(-)).
  • auxotrophic for essential enzymes including, for example, D-alanine racemase is used (e.g., Lm dal(-)dat(-)).
  • 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.
  • 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).
  • an endogenous actA sequence encoding an ActA protein
  • an endogenous hly sequence encoding an LLO protein
  • 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.
  • 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.
  • Selection of recombinant bacteria or Listeria strains can be achieved by any means.
  • 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.
  • 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.
  • transformed auxotrophic bacteria 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.
  • the metabolic enzyme e.g., amino acid metabolism gene
  • a temperature-sensitive plasmid can be used to select recombinants or any other known means for selecting recombinants.
  • 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.
  • 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.
  • the lethal dose at which 50% of inoculated animals survive 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
  • Attenuation can be accomplished by any known means.
  • 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).
  • mutation includes any type of mutation or
  • 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.
  • 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).
  • LmddA Listeria monocytogenes
  • LmddA Lm dal(-)dat(-) actA
  • LmddA Lm dal(-)dat(-) actA
  • Lm prfA(-) 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • a bacteria or Listeria strain can also be an auxotrophic strain having a mutation in a metabolic gene.
  • the strain can be deficient in one or more endogenous amino acid metabolism genes.
  • the generation of auxotrophic strains of Listeria deficient in D-alanine 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.
  • 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.
  • 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.
  • DAP diaminopimelic acid
  • Cysteine synthase A ⁇ cysK Cysteine synthase A ⁇ cysK
  • vitamin-B 12 independent methionine synthase t
  • 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.
  • 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
  • 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).
  • an atp synthase gene e.g., atpC, atpD-2, aptG, atpA-2, and so forth.
  • Attenuated Listeria strains can be deficient in endogenous phoP, aroA, aroC, aroD, or plcB.
  • 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
  • a molybdenum 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.
  • 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.
  • 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.
  • the amino acid can be used in cell wall biogenesis.
  • the metabolic enzyme is a synthetic enzyme for D-glutamic acid, a cell wall component.
  • 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.
  • 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
  • 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
  • An attenuated Listeria strain can be generated by mutation of other metabolic enzymes, such as a tRNA synthetase.
  • the metabolic enzyme can be encoded by the trpS gene, encoding tryptophanyltRNA synthetase.
  • the host strain bacteria can be A(trpS aroA), and both markers can be contained in an integration vector.
  • metabolic enzymes include aspartate aminotransferase, histidinol-phosphate aminotransferase (GenBank Accession No. NP_466347), or the cell wall teichoic acid glycosylation protein GtcA.
  • 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.
  • 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.
  • the metabolic enzyme can be any other Listeria metabolic enzyme or any other Listeria monocytogenes metabolic enzyme.
  • 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).
  • 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.
  • 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.
  • the complementing gene can be extrachromosomal or can be integrated into the bacteria or Listeria genome.
  • 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.
  • the auxotrophic Listeria strain can comprise an integrative plasmid (i.e., integration vector) comprising a nucleic acid encoding a metabolic enzyme.
  • integrative plasmids can be used for integration into a Listeria chromosome.
  • the episomal plasmid or the integrative plasmid lacks an antibiotic resistance marker.
  • the metabolic gene can be used for selection instead of or in addition to an antibiotic resistance gene.
  • transformed auxotrophic bacteria 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the PrfA protein can be any other gram-positive PrfA protein or any other PrfA protein.
  • 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.
  • 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.
  • the recombinant bacteria strain can be stored in a frozen cell bank or stored in a lyophilized cell bank.
  • a cell bank can be, for example, a master cell bank, a working cell bank, or a Good Manufacturing Practice (GMP) cell bank.
  • GMP Good Manufacturing Practice
  • 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
  • Such cell banks can be intended for production of clinical-grade material or can conform to regulatory practices for human use.
  • 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.
  • 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.
  • 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.
  • Recombinant bacteria strains can also be from a batch of vaccine doses, from a frozen stock, or from a lyophilized stock.
  • 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.
  • 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.
  • 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.
  • 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.
  • the culture e.g., the culture of a Listeria vaccine strain that is used to produce a batch of Listeria vaccine doses
  • the culture 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.
  • 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.
  • additives include, for example, mannitol, DMSO, sucrose, or any other colligative additive or additive with anti-freeze properties.
  • the nutrient medium utilized for growing a culture of a bacteria strain can be any suitable nutrient medium.
  • suitable media include, for example, LB; TB; a modified, animal-product-free Terrific Broth; or a defined medium.
  • the step of growing can be performed by any known means of growing bacteria.
  • 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.
  • a constant pH is maintained during growth of the culture (e.g. in a batch fermenter).
  • 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.
  • 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.
  • a constant temperature can be maintained during growth of the culture.
  • the temperature can be maintained at about 37°C or at 37°C.
  • 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.
  • a constant dissolved oxygen concentration can be maintained during growth of the culture.
  • 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.
  • Methods for lyophilization and cryopreservation of recombinant bacteria strains are known.
  • a Listeria culture can be flash-frozen in liquid nitrogen, followed by storage at the final freezing temperature.
  • 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.
  • the storage temperature of the culture can be, for example, between -20 and - 80°C.
  • the temperature can be significantly below -20°C or not warmer than - 70°C.
  • 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.
  • the temperature can be colder than 70°C or colder than -80°C.
  • immunogenic compositions 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne des systèmes comprenant des compositions d'immunothérapie ciblant des mutations de cancer récurrentes et des compositions d'immunothérapie ciblant des néo-épitopes personnalisées. L'invention concerne également des compositions d'immunothérapie ciblant des mutations de cancer récurrentes comprenant une souche de Listeria recombinante comprenant un acide nucléique comprenant un cadre de lecture ouvert codant pour un polypeptide de fusion recombinant comprenant un ou plusieurs peptides antigéniques (par ex. fusionnés à un peptide comportant PEST) provenant de protéines associées au cancer. Les peptides antigéniques peuvent comprendre Un ou plusieurs ou la totalité des peptides antigéniques comprenant une mutation de cancer récurrente, des peptides antigéniques comprenant une mutation hétéroclitique, ou des peptides antigéniques fusionnés à une protéine d'ubiquitine. L'invention concerne également des compositions d'immunothérapie ciblant des mutations de cancer récurrentes comprenant une souche de Listeria recombinante comprenant un acide nucléique comprenant un cadre de lecture ouvert codant pour un polypeptide de fusion, le polypeptide de fusion comprenant un peptide comportant PEST fusionné à deux ou plus peptides antigéniques, chacun des peptides antigéniques comprenant une mutation de cancer récurrente, et au moins deux des peptides antigéniques comprenant différentes mutations de cancer récurrentes et étant des fragments de la même protéine associée au cancer. L'invention concerne également des compositions d'immunothérapie personnalisées comprenant une souche de Listeria recombinante comprenant un acide nucléique comprenant un cadre de lecture ouvert codant pour un polypeptide de fusion, le polypeptide de fusion comprenant un peptide comportant PEST fusionné à un ou plusieurs peptides antigéniques, chacun des peptides antigéniques comprenant un néo-épitope spécifique au cancer comprenant une mutation spécifique au cancer détectée dans un échantillon cancéreux provenant d'un sujet, mais non dans un échantillon biologique sain provenant du sujet. L'invention concerne également des polypeptides de fusion recombinants, des acides nucléiques codant pour des polypeptides de fusion, et des procédés de production de ces compositions. L'invention concerne également des procédés d'induction d'une réponse immunitaire anti-antigène associée à une tumeur chez un sujet, des procédés d'induction d'une réponse immunitaire antitumorale ou anticancéreuse chez un sujet, des procédés de traitement d'une tumeur ou d'un cancer chez un sujet, des procédés de prévention d'une tumeur ou d'un cancer chez un sujet, et des procédés de protection d'un sujet contre une tumeur ou un cancer à l'aide de ces compositions d'immunothérapie, des polypeptides de fusion recombinants, des acides nucléiques, ou des bactéries recombinantes ou des souches de Listeria.
PCT/US2017/064016 2016-11-30 2017-11-30 Immunothérapie personnalisée en association avec une immunothérapie ciblant des mutations de cancer récurrentes WO2018102585A1 (fr)

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US201762583288P 2017-11-08 2017-11-08
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US11446369B2 (en) 2007-05-10 2022-09-20 Advaxis, Inc. Compositions and methods comprising KLK3 or FOLH1 antigen
US10258679B2 (en) 2014-04-24 2019-04-16 Advaxis, Inc. Recombinant Listeria vaccine strains and methods of producing the same
US10900044B2 (en) 2015-03-03 2021-01-26 Advaxis, Inc. Listeria-based compositions comprising a peptide minigene expression system and methods of use thereof
US11702664B2 (en) 2015-03-03 2023-07-18 Advaxis, Inc. Listeria-based compositions comprising a peptide minigene expression system and methods of use thereof
US11897927B2 (en) 2016-11-30 2024-02-13 Advaxis, Inc. Immunogenic compositions targeting recurrent cancer mutations and methods of use thereof
US10881730B2 (en) 2017-02-01 2021-01-05 Modernatx, Inc. Immunomodulatory therapeutic MRNA compositions encoding activating oncogene mutation peptides
US11504421B2 (en) 2017-05-08 2022-11-22 Gritstone Bio, Inc. Alphavirus neoantigen vectors
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US11179339B2 (en) 2017-09-19 2021-11-23 Advaxis, Inc. Compositions and methods for lyophilization of bacteria or listeria strains
US11666644B2 (en) 2018-09-04 2023-06-06 Treos Bio Limited Peptide vaccines
CN109438570A (zh) * 2018-11-28 2019-03-08 生命谷（海南）生物科技股份有限公司 肿瘤相关基因fgfr3突变短肽及其应用
CN109438570B (zh) * 2018-11-28 2021-07-20 生命谷（海南）生物科技股份有限公司 肿瘤相关基因fgfr3突变短肽及其应用
US20200222478A1 (en) * 2019-01-10 2020-07-16 Janssen Biotech, Inc. Prostate neoantigens and their uses
US11793843B2 (en) 2019-01-10 2023-10-24 Janssen Biotech, Inc. Prostate neoantigens and their uses
WO2020146773A1 (fr) * 2019-01-11 2020-07-16 University Of Miami Procédés d'utilisation d'une protéine de fusion d'il-2/cd25
US11591619B2 (en) 2019-05-30 2023-02-28 Gritstone Bio, Inc. Modified adenoviruses
WO2021101965A1 (fr) * 2019-11-18 2021-05-27 Epivax Oncology, Inc. Vaccins améliorés à base de néo-épitope et méthodes de traitement du cancer
CN111072763A (zh) * 2019-12-23 2020-04-28 维塔恩(广州)医药有限公司 肿瘤相关基因gnas突变相关抗原短肽及其应用
CN111057135A (zh) * 2019-12-23 2020-04-24 维塔恩(广州)医药有限公司 肿瘤相关基因fbxw7突变相关抗原短肽及其应用
WO2022009051A1 (fr) * 2020-07-06 2022-01-13 Janssen Biotech, Inc. Procédé de détermination de la réactivité à un traitement du cancer de la prostate
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WO2022032196A3 (fr) * 2020-08-06 2022-04-07 Gritstone Bio, Inc. Cassettes de vaccin à plusieurs épitopes
WO2022051449A3 (fr) * 2020-09-04 2022-04-14 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs des lymphocytes t reconnaissant les mutations r175h ou y220c dans p53
GB2614166A (en) * 2020-09-04 2023-06-28 Us Health T cell receptors recognizing R273C or Y220C mutations in P53
US11235039B1 (en) 2020-11-20 2022-02-01 Think Therapeutics, Inc. Immunogenic compositions comprising nucleic acids for RAS peptides
US11464837B2 (en) 2020-11-20 2022-10-11 Think Therapeutics, Inc. Immunogenic compositions comprising nucleic acids for RAS peptides
US11058751B1 (en) 2020-11-20 2021-07-13 Think Therapeutics, Inc. Compositions for optimized RAS peptide vaccines
US11161892B1 (en) 2020-12-07 2021-11-02 Think Therapeutics, Inc. Method of compact peptide vaccines using residue optimization
US11673936B2 (en) 2020-12-07 2023-06-13 Think Therapeutics, Inc. Method of compact peptide vaccines using residue optimization
US11421015B2 (en) 2020-12-07 2022-08-23 Think Therapeutics, Inc. Method of compact peptide vaccines using residue optimization
CN113173986B (zh) * 2021-03-24 2022-01-07 深圳市新靶向生物科技有限公司 一种与肺癌驱动基因突变相关的抗原肽及其应用
CN113173986A (zh) * 2021-03-24 2021-07-27 深圳市新靶向生物科技有限公司 一种与肺癌驱动基因突变相关的抗原肽及其应用
US11672850B2 (en) 2021-04-28 2023-06-13 Think Therapeutics, Inc. Compositions and method for optimized peptide vaccines using residue optimization
US11464842B1 (en) 2021-04-28 2022-10-11 Think Therapeutics, Inc. Compositions and method for optimized peptide vaccines using residue optimization
WO2022261514A3 (fr) * 2021-06-11 2023-02-09 RNAimmune, Inc. Vaccins à arnm tp53 et pan-ras multiplexés contre le cancer
WO2024050380A3 (fr) * 2022-08-29 2024-04-11 Flagship Pioneering Innovations Vi, Llc Détection d'une fusion ou d'une délétion génétique qui résulte en l'expression d'un néo-antigène
CN116970058A (zh) * 2023-09-22 2023-10-31 成都朗谷生物科技股份有限公司 针对tp53基因r249s突变的肿瘤新抗原多肽及其应用
CN116970058B (zh) * 2023-09-22 2023-12-15 成都朗谷生物科技股份有限公司 针对tp53基因r249s突变的肿瘤新抗原多肽及其应用

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