# US 11,897,927 B2 · Immunogenic compositions targeting recurrent cancer mutations and methods of use thereof

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- Number: US 11,897,927 B2
- Document ID: US11897927B2
- Status: Granted
- Kind: B2
- Application number: 16/348,274
- Filed: 2017-11-30
- Published: 2019-10-24
- Granted: 2024-02-13
- Earliest claimed: 2016-11-30
- Assignees: Advaxis, Inc.
- Applicants: Advaxis, Inc.
- Inventors: Robert Petit; Michael F. Princiotta; Brandon Coder; David Balli
- Web page: https://patentgrep.com/patent/US11897927B2

## CPC

- C07K 14/4748: {Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE}
- A61K 35/74: Bacteria (therapeutic use of a bacterial protein A61K38/00)
- A61K 39/0011: {Cancer antigens}
- A61K 39/001103: {Receptors for growth factors}
- A61K 39/001104: {Epidermal growth factor receptors [EGFR]}
- A61K 39/001107: {Fibroblast growth factor receptors [FGFR]}
- A61K 39/00115: {Apoptosis related proteins, e.g. survivin or livin}
- A61K 39/001157: {Telomerase or TERT [telomerase reverse transcriptase]}
- A61K 39/001186: {MAGE}
- A61K 39/001188: {NY-ESO}
- A61K 39/001189: {PRAME}
- A61K 39/001194: {Prostate specific antigen [PSA]}
- A61K 39/001195: {Prostate specific membrane antigen [PSMA]}
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- A61K 2039/523: {expressing foreign proteins}
- A61K 2039/55544: {Bacterial toxins}
- A61K 2039/55594: {from bacteria}
- A61K 2039/64: {characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units}
- A61K 2039/645: {Dendrimers; Multiple antigen peptides}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/43: containing a FLAG-tag
- C07K 2319/95: containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are recombinant fusion polypeptides 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. For example, provided herein are recombinant fusion polypeptides comprising two or more antigenic peptides (e.g., fused to a PEST-containing peptide), wherein each antigenic peptide comprises a recurrent cancer mutation, and wherein at least two of the antigenic peptides are fragments of the same cancer-associated protein. Also provided are nucleic acids encoding such fusion polypeptides, recombinant bacteria or Listeria strains comprising such fusion polypeptides or such nucleic acids, and cell banks comprising such recombinant bacteria or Listeria strains. Also provided herein are methods of generating such fusion polypeptides, such nucleic acids, and such recombinant bacteria or Listeria strains. Also provided are immunogenic compositions, pharmaceutical compositions, and vaccines comprising such fusion polypeptides, such nucleic acids, or such recombinant bacteria or Listeria strains. Also provided are methods of inducing an anti-tumor-associated-antigen immune response in a subject, methods of inducing an anti-tumor or anti-cancer immune response in a subject, methods of treating a tumor or cancer in a subject, methods of preventing a tumor or cancer in a subject, and methods of protecting a subject against a tumor or cancer using such recombinant fusion polypeptides, nucleic acids, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines.

## Claims

### Claim 1

A recombinant Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide comprising a PEST-containing peptide fused to two or more antigenic peptides,
wherein at least one antigenic peptide is from a cancer-associated protein and comprises a recurrent cancer mutation,
wherein at least one antigenic peptide is from a cancer-associated protein and comprises a heteroclitic mutation,
wherein the PEST-containing peptide comprises a bacterial secretion signal sequence, and the fusion polypeptide further comprises a ubiquitin protein fused to a carboxy-terminal antigenic peptide,
wherein the PEST-containing peptide, the 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 fusion polypeptide, and
wherein (i) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by all of the following genes: SPOP, CHEK2, RGPD8, ANKRD36C, and AR; or (ii) the antigenic peptides comprise heteroclitic mutations in proteins encoded by all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA.

### Claim 2 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the antigenic peptides comprise recurrent cancer mutations from proteins encoded by all of the following genes: SPOP, CHEK2, RGPD8, ANKRD36C, and AR.

### Claim 3 (depends on 2)

The recombinant Listeria strain of claim 2, wherein the antigenic peptides comprise all of the following recurrent cancer mutations: SPOP_F133V, CHEK2_K373E, RGPD8_P1760A, ANKRD36C_1634T, ANKRD36C_D629Y, SPOP_W131G, ANKRD36C_D626N, SPOP_F133L, AR_T878A, AR_L702H, AR_W742C, AR_H875Y, and AR_F877L.

### Claim 4 (depends on 3)

The recombinant Listeria strain of claim 3, wherein the antigenic peptides comprise all of the peptides set forth in SEQ ID NOS: 717, 707, 714, 704, 705, 715, 706, 716, and 763-768.

### Claim 5 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the antigenic peptides comprise heteroclitic mutations in proteins encoded by all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA.

### Claim 6 (depends on 5)

The recombinant Listeria strain of claim 5, wherein the antigenic peptides comprise all of the peptides set forth in SEQ ID NOS: 793, 794, and 799-806.

### Claim 7 (depends on 4)

The recombinant Listeria strain of claim 4, wherein the antigenic peptides comprise all of the peptides set forth in SEQ ID NOS: 717, 707, 714, 704, 705, 715, 706, 716, 763-768, 793, 794, and 799-806.

### Claim 8 (depends on 7)

The recombinant Listeria strain of claim 7, wherein one or more of the antigenic peptides comprising a recurrent cancer mutation are preceded by the linker set forth in SEQ ID NO: 316, and wherein one or more of the antigenic peptides comprising a heteroclitic mutation are preceded by the linker set forth in any one of SEQ ID NOS: 821-829.

### Claim 9 (depends on 8)

The recombinant Listeria strain of claim 8, wherein the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 893.

### Claim 10 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the carboxy-terminal antigenic peptide is from a cancer-associated protein and comprises a heteroclitic mutation.

### Claim 11 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the carboxy-terminal antigenic peptide is about 7-11, 8-10, or 9 amino acids in length.

### Claim 12

The recombinant Listeria strain of e4aim-2claim 1, wherein the carboxy-terminal antigenic peptide binds to one or more of the following HLA types: HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, and HLA-B*07:02.

### Claim 13 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the carboxy-terminal antigenic peptide is from a protein encoded by one of the following genes: STEAP1, CEACAM5, NYESO1, and NUF2.

### Claim 14 (depends on 13)

The recombinant Listeria strain of claim 13, wherein the carboxy-terminal antigenic peptide is selected from the peptides set forth in SEQ ID NOS: 796, 797, 798, 799, 800, and 807.

### Claim 15 (depends on 1)

The recombinant Listeria strain of claim 1, wherein each antigenic peptide is a fragment of a cancer-associated protein and is about 7-200 amino acids in length.

### Claim 16 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the fusion polypeptide comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 antigenic peptides or comprises between about 5-50, 10-40, or 20-30 antigenic peptides.

### Claim 17 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the fusion polypeptide comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic peptides comprising a recurrent cancer mutation or between about 5-30 or 10-20 antigenic peptides comprising a recurrent cancer mutation, and/or
wherein the fusion polypeptide comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic peptides comprising a heteroclitic mutation or between about 5-30 or 10-20 antigenic peptides comprising a heteroclitic mutation.

### Claim 18 (depends on 17)

The recombinant Listeria strain of claim 17, wherein the antigenic peptides comprising a recurrent cancer mutation are in tandem, and the antigenic peptides comprising a heteroclitic mutation are in tandem.

### Claim 19 (depends on 17)

The recombinant Listeria strain of claim 17, wherein the antigenic peptides comprising a recurrent cancer mutation and the antigenic peptides comprising a heteroclitic mutation are intermixed within the fusion polypeptide.

### Claim 20 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the two or more antigenic peptides are linked to each other via peptide linkers.

### Claim 21 (depends on 20)

The recombinant Listeria strain of claim 20, wherein the peptide linkers comprise flexibility linkers and/or rigidity linkers and/or immunoproteasome processing linkers, or wherein one or more of the linkers set forth in SEQ ID NOS: 313-316, 319, and 821-829 are used to link the two or more antigenic peptides.

### Claim 22 (depends on 21)

The recombinant Listeria strain of claim 21, wherein the peptide linker upstream of one or more of the antigenic peptides comprising a heteroclitic mutation is an immunoproteasome processing linker or is selected from the linkers set forth in SEQ ID NOS: 821-829.

### Claim 23 (depends on 1)

The recombinant Listeria strain of claim 1, wherein no region of the fusion polypeptide scores above a cutoff of around 1.6 when scored for hydropathy by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window.

### Claim 24 (depends on 1)

The recombinant Listeria strain of claim 1, wherein at least two of the antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein.

### Claim 25 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the recurrent cancer mutations in at least two of the antigenic peptides are from the same cancer-associated protein and do not occur naturally together.

### Claim 26 (depends on 1)

The recombinant Listeria strain of claim 1, wherein at least two of the antigenic peptides are overlapping fragments of the same cancer-associated protein.

### Claim 27 (depends on 26)

The recombinant Listeria strain of claim 26, wherein the recurrent cancer mutations in at least two of the antigenic peptides are from the same cancer-associated protein and occur at the same amino acid residue of the cancer-associated protein.

### Claim 28 (depends on 27)

The recombinant Listeria strain of claim 27, wherein two of the antigenic peptides comprise the same recurrent cancer mutation.

### Claim 29 (depends on 1)

The recombinant Listeria strain of claim 1, wherein each antigenic peptide comprising a recurrent cancer mutation comprises a different recurrent cancer mutation.

### Claim 30 (depends on 1)

The recombinant Listeria strain of claim 1, wherein each recurrent cancer mutation in the fusion polypeptide is a somatic frameshift mutation or a somatic missense mutation.

### Claim 31 (depends on 30)

The recombinant Listeria strain of claim 30, wherein each recurrent cancer mutation in the fusion polypeptide is a somatic missense mutation.

### Claim 32 (depends on 1)

The recombinant Listeria strain of claim 1, wherein one or more or all of the antigenic peptides comprising a recurrent cancer mutation have an equal number of amino acids flanking each side of the recurrent cancer mutation.

### Claim 33 (depends on 32)

The recombinant Listeria strain of claim 32, wherein the number of flanking amino acids on each side of the recurrent cancer mutation is at least 10 amino acids.

### Claim 34 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the antigenic peptides comprise the 2, 3, 4, 5, 6, 7, 8, 9, or 10 most common recurrent cancer mutations or recurrent somatic missense cancer mutations from a particular type of cancer.

### Claim 35 (depends on 1)

The recombinant Listeria strain of claim 1, wherein at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 35%, 50%, 60%, 70%, 80%, or 90% of patients with a particular type of cancer have a recurrent cancer mutation that is included in the combination of antigenic peptides in the fusion polypeptide.

### Claim 36 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the antigenic peptides comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different recurrent cancer mutations or recurrent somatic missense cancer mutations from a particular type of cancer, or wherein the antigenic peptides comprise about 2-80, 10-60, 10-50, 10-40, or 10-30 different recurrent cancer mutations or recurrent somatic missense cancer mutations from a particular type of cancer.

### Claim 37 (depends on 34)

The recombinant Listeria strain of claim 34, wherein the particular type of cancer is non-small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, low-grade glioma, colorectal cancer, or head and neck cancer.

### Claim 38 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the antigenic peptides are from two or more cancer-associated proteins.

### Claim 39 (depends on 38)

The recombinant Listeria strain of claim 38, wherein the two or more cancer-associated proteins are at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer-associated proteins, or wherein the two or more cancer-associated proteins are about 2-30, 2-25, 2-20, 2-15, or 2-10 cancer-associated proteins.

### Claim 40 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more of the following genes: ACVR2A, ADAM28, AKT1, ANKRD36C, AR, ARID1A, BMPR2, BRAF, CHEK2, C12orf4, CTNNB1, DOCK3, EGFR, ESR1, FBXW7, FGFR3, FHOD3, GNAS, HRAS, IDH1, IDH2, KIAA2026, KRAS\, KRTAP1-5, KRTAP4-11, LARP4B, MBOAT2, NFE2L2, PGM5, PIK3CA, PLEKHA6, POLE, PTEN, RGPD8, RNF43, RXRA, SMAD4, SPOP, SVIL, TGFBR2, TP53, TRIM48, UBR5, U2AF1, WNT16, XYLT2, ZBTB20, and ZNF814.

### Claim 41 (depends on 40)

The recombinant Listeria strain of claim 40, wherein:
(a) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS\, EGFR, U2AF1, BRAF, PIK3CA, and TP53;
(b) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: SPOP, CHEK2, RGPD8, ANKRD36C, and AR;
(c) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS\, U2AF1, TP53, SMAD4, and GNAS;
(d) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: PIK3CA, FGFR3, TP53, RXRA, FBXW7, and NFE2L2;
(e) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: PIK3CA, AKT1, and ESR1;
(f) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: PTEN, KRAS\, PIK3CA, CTNNB1, FBXW7, and TP53;
(g) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: TP53;
(h) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: TP53, PIK3CA, IDH1, IDH2, and EGFR;
(i) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: KRAS\, BRAF, PIK3CA, and TP53; or
(j) the antigenic peptides comprise recurrent cancer mutations from proteins encoded by one or more or all of the following genes: PIK3CA, CHEK2, RGPD8, ANKRD36C, TP53, ZNF814, KRTAP1-5, KRTAP4-11, and HRAS.

### Claim 42 (depends on 41)

The recombinant Listeria strain of claim 41, wherein:
(a) the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: KRAS\_G12C, EGFR_L858R, KRAS\_G12D, U2AF1_S34F, BRAF_V600E, KRAS\_G12V, PIK3CA_E545K, TP53_R158L, KRAS\_G12A, EGFR_L861Q, and TP53_R273L;
(b) the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: SPOP_F133V, CHEK2_K373E, RGPD8_P1760A, ANKRD36C_I634T, ANKRD36C_D629Y, SPOP_W131G, ANKRD36C_D626N, SPOP_F133L, AR_T878A, AR_L702H, AR_W742C, AR_H875Y, and AR_F877L;
(c) the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: KRAS\_G12C, KRAS\_G12D, U2AF1_S34F, KRAS\_G12V, TP53_R248Q, TP53_R248W, TP53_R175H, TP53_R273C, KRAS\_G12R, KRAS\_Q61H, TP53_R282W, TP53_R273H, TP53_G245S, SMAD4_R361C, GNAS_R201C, and GNAS_R201H;
(d) the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: PIK3CA_E545K, FGFR3_S249C, TP53_R248Q, PIK3CA_E542K, RXRA_S427F, FBXW7_R505G, TP53_R280T, NFE2L2_E79K, FGFR3_R248C, TP53_K132N, TP53_R248W, TP53_R175H, and TP53_R273C;
(e) the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: PIK3CA_E545K, PIK3CA_E542K, PIK3CA_H1047R, AKT1_E17K, PIK3CA_H1047L, PIK3CA_Q546K, PIK3CA_E545A, PIK3CA_E545G, ESR1_K303R, ESR1_D538G, ESR1_Y537S, ESR1_Y537N, ESR1_Y537C, and ESR1_E380Q;
(f) the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: PTEN_R130G, PTEN_R130Q, KRAS\_G12D, KRAS\_G12V, PIK3CA_H1047R; PIK3CA\R88Q, PIK3CA_E545K, PIK3CA_E542K, CTNNB1_S37F, KRAS\_G13D, CTNNB1_S37C, PIK3CA_H1047L, PIK3CA_G118D, KRAS\_G12A, FBXW7_R505C, and TP53_R248W;
(g) the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: TP53_R248Q, TP53_R248W, TP53_R175H, TP53_R273C, TP53_R282W, TP53_R273H, TP53_Y220C, TP53_1195T, TP53_C176Y, TP53_H179R, TP53_S241F, and TP53_H193R;
(h) the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: TP53_R273L, TP53_R273C, TP53_R273H, PIK3CA_G118D, IDH1_R132C, IDH1_R132G, IDH1_R132H, IDH1_R132S, IDH2_R172K, PIK3CA_E453K, and EGFR\G598V;
(i) the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: KRAS\_G12C, KRAS\_G12D, BRAF_V600E, KRAS\_G12V, PIK3CA_E545K, TP53_R248W, TP53_R175H, TP53_R273C, PIK3CA_H1047R, TP53_R282W, TP53_R273H, and KRAS\_G13D; or
(j) the antigenic peptides comprise one or more or all of the following recurrent cancer mutations: PIK3CA_E545K, CHEK2_K373E, RGPD8_P1760A, ANKRD36C_I634T, TP53 R248Q, PIK3CA_E542K, TP53_R248W, TP53_R175H, PIK3CA_H1047R, TP53_R282W, TP53_R273H, TP53_G245S, TP53_Y220C, ZNF814_D404E, KRTAP1-5_I88T, KRTAP4-11_L161V, and HRAS_G13V.

### Claim 43 (depends on 42)

The recombinant Listeria strain of claim 42 wherein:
(a) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 486, 405, 489, 643, 377, 491, 437, 631, 487, 406, and 566;
(b) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 717, 707, 714, 704, 705, 715, 706, 716, and 763-768;
(c) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 486, 489, 643, 491, 560, 559, 542, 565, 490, 498, 569, 563, 558, and 780-782;
(d) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 437, 769, 560, 436, 770, 771, 642, 772, 773, 535, 559, 542, and 565;
(e) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 437, 436, 447, 585, 448, 442, 438, 439, and 774-779;
(f) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 471, 470, 489, 491, 447, 427, 437, 436, 783, 492, 784, 448, 431, 487, 785 and 559;
(g) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 560, 559, 542, 565, 569, 563, 552, 549, 545, 546, 555, and 548;
(h) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 566, 565, 563, 431, 786-790, 435, and 393;
(i) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 486, 489, 377, 491, 437, 559, 542, 565, 447, 569, 563, and 492; or
(j) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 437, 707, 714, 704, 560, 436, 559, 542, 447, 569, 563, 558, 552, and 830-833.

### Claim 44 (depends on 1)

The recombinant Listeria strain of claim 1, wherein each antigenic peptide comprising a heteroclitic mutation is about 7-11, 8-10, or 9 amino acids in length.

### Claim 45 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the antigenic peptides comprising a heteroclitic mutation 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.

### Claim 46 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more of the following genes: CEACAM5, GAGE1, hTERT, KLHL7, MAGEA3, MAGEA4, MAGEA6, NUF2, NYESO1, PAGE4, PRAME, PSA, PSMA, RNF43, SART3, SSX2, STEAP1, and SURVIVIN.

### Claim 47

The recombinant Listeria strain of claim/5, wherein:
(a) the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESO1, STEAP1, and RNF43;
(b) the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA;
(c) the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, STEAP1, MAGEA3, PRAME, hTERT, and SURVIVIN;
(d) the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, GAGE1, NYESO1, RNF43, NUF2, KLHL7, MAGEA3, and PRAME;
(e) the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, STEAP1, RNF43, MAGEA3, PRAME, and hTERT;
(f) the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, PRAME, hTERT, STEAP1, RNF43, NUF2, KLHL7, and SART3;
(g) the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, STEAP1, RNF43, SART3, NUF2, KLHL7, PRAME, and hTERT;
(h) the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, MAGEA6, STEAP1, RNF43, SART3, NUF2, KLHL7, and hTERT;
(i) the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESO1, STEAP1, RNF43, and MAGEA3; or
(j) the antigenic peptides comprise heteroclitic mutations in proteins encoded by one or more or all of the following genes: CEACAM5, MAGEA4, STEAP1, NYESO1, PRAME, and hTERT.

### Claim 48 (depends on 47)

The recombinant Listeria strain of claim 47, wherein:
(a) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 791-801;
(b) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 793, 794, and 799-806;
(c) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 791, 793, 796, 798-800, 812, and 815-819;
(d) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 791, 795, 797, 798, 801, and 807-815;
(e) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 791, 793, 796, 798-801, 810, 812, 815, and 816;
(f) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 820, 791, 793, 798, 815, 816, 799, 796, 801, 807-809, 803, and 800;
(g) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 791, 793, 796, 798-801, 803, 807-809, 815, 816, and 820;
(h) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 791, 792, 799-801, 803, 807-809, and 816;
(i) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 791-797, 799, 801, and 817; or
(j) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 791, 793, 794, 796, 798-800, 813, 815, and 816.

### Claim 49 (depends on 1)

The recombinant Listeria strain of claim 1, wherein:
(a) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 486, 405, 489, 643, 377, 491, 437, 631, 487, 406, 566, and 791-801;
(b) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 717, 707, 714, 704, 705, 715, 706, 716, 763-768, 793, 794, and 799-806;
(c) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 486, 489, 643, 491, 560, 559, 542, 565, 490, 498, 569, 563, 558, 780-782, 791, 793, 796, 798-800, 812, and 815-819;
(d) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 437, 769, 560, 436, 770, 771, 642, 772, 773, 535, 559, 542, 565, 791, 795, 797, 798, 801, and 807-815;
(e) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 437, 436, 447, 585, 448, 442, 438, 439, 774-779, 791, 793, 796, 798-801, 810, 812, 815, and 816;
(f) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 471, 470, 489, 491, 447, 427, 437, 436, 783, 492, 784, 448, 431, 487, 785, 559, 820, 791, 793, 798, 815, 816, 799, 796, 801, 807-809, 803, and 800;
(g) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 560, 559, 542, 565, 569, 563, 552, 549, 545, 546, 555, 548, 791, 793, 796, 798-801, 803, 807-809, 815, 816, and 820;
(h) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 566, 565, 563, 431, 786-790, 435, 393, 791, 792, 799-801, 803, 807-809, and 816;
(i) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 486, 489, 377, 491, 437, 559, 542, 565, 447, 569, 563, 492, 791-797, 799, 801, and 817; or
(j) the antigenic peptides comprise one or more or all of the peptides set forth in SEQ ID NOS: 437, 707, 714, 704, 560, 436, 559, 542, 447, 569, 563, 558, 552, 830-833, 791, 793, 794, 796, 798-800, 813, 815, and 816.

### Claim 50 (depends on 49)

The recombinant Listeria strain of claim 49, wherein:
(a) the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 859, 860, 861, 862, 863, 864, 865, 894, 895, and 905;
(b) the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 871, 872 873, 874, 875, 876, 877, 892, 893, and 906;
(c) the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 866, 867, 868, 869, 870, and 908;
(d) the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 878, 879, 880, 881, 882, 888, 889, 890, and 891;
(e) the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 883, 884, 885, 886, 887, and 907;
(f) the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 896, 897, and 904;
(g) the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 898 and 899;
(h) the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 900 and 901;
(i) the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 902 and 903; or
(j) the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 918 and 919.

### Claim 51 (depends on 1)

The recombinant Listeria strain of claim 1,
wherein the fusion polypeptide has a molecular weight of no more than about 150 kDa or no more than about 125 kDa.

### Claim 52 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the fusion polypeptide further comprises one or more peptide tags N-terminal and/or C-terminal to the combination of the two or more antigenic peptides, wherein the one or more peptide tags comprise one or both of the following: FLAG tag and SIINFEKL (SEQ ID NO: 1007) tag.

### Claim 53 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the PEST-containing peptide is on the N-terminal end of the fusion polypeptide.

### Claim 54 (depends on 53)

The recombinant Listeria strain of claim 53, wherein the PEST-containing peptide is an N-terminal fragment of LLO.

### Claim 55 (depends on 54)

The recombinant Listeria strain of claim 54, wherein the N-terminal fragment of LLO has the sequence set forth in SEQ ID NO: 336.

### Claim 56 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the nucleic acid is in an episomal plasmid.

### Claim 57 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the nucleic acid does not confer antibiotic resistance upon the recombinant Listeria strain.

### Claim 58 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the recombinant Listeria strain is an attenuated, auxotrophic Listeria strain.

### Claim 59 (depends on 58)

The recombinant Listeria strain of claim 58, wherein the attenuated, auxotrophic Listeria strain comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes.

### Claim 60 (depends on 59)

The recombinant Listeria strain of claim 59, wherein the one or more endogenous genes comprise act4, dal, and dat.

### Claim 61 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the nucleic acid comprises a second open reading frame encoding a metabolic enzyme.

### Claim 62 (depends on 61)

The recombinant Listeria strain of claim 61, wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid aminotransferase enzyme.

### Claim 63 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the fusion polypeptide is expressed from an hly promoter.

### Claim 64 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the recombinant Listeria strain is a recombinant Listeria monocytogenes strain.

### Claim 65 (depends on 1)

The recombinant Listeria strain of claim 1, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

### Claim 66 (depends on 1)

An immunogenic composition comprising the recombinant Listeria strain of claim 1.

### Claim 67 (depends on 66)

The immunogenic composition of claim 66, wherein the immunogenic composition comprises a combination of two or more recombinant Listeria strains, wherein each recombinant Listeria strain comprises a different set of antigenic peptides or the same set of antigenic peptides in a different order.

### Claim 68 (depends on 67)

The immunogenic composition of claim 67, wherein each recombinant Listeria strain comprises a different set of antigenic peptides.

### Claim 69 (depends on 68)

The immunogenic composition of claim 68, wherein the combination of recombinant Listeria strains comprises about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-120, 120-140, 140-160, 160-180, 180-200, 200-220, 220-240, 240-260, 260-280, or 280-300 different antigenic peptides.

### Claim 70 (depends on 66)

The immunogenic composition of claim 66, wherein the immunogenic composition further comprises an adjuvant.

### Claim 71 (depends on 70)

The immunogenic composition of claim 70, wherein the adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, an unmethylated CpG-containing oligonucleotide, or a detoxified listeriolysin O protein.

### Claim 72 (depends on 1)

A method of inducing an immune response against a tumor or cancer in a subject, comprising administering to the subject the recombinant Listeria strain of claim 1.

### Claim 73 (depends on 1)

A cell bank comprising one or more recombinant Listeria strains as in claim 1.

### Claim 74 (depends on 73)

The cell bank of claim 73, wherein the cell bank is a frozen cell bank or a lyophilized cell bank.