# US 7,465,454 B2 · Yeast-based vaccines as immunotherapy

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- Number: US 7,465,454 B2
- Document ID: US7465454B2
- Status: Granted
- Kind: B2
- Application number: 10/738,646
- Filed: 2003-12-16
- Published: 2004-08-12
- Granted: 2008-12-16
- Earliest claimed: 2002-12-16
- Assignees: GlobeImmune, Inc.
- Inventors: Alex Franzusoff; Donald Bellgrau
- Web page: https://patentgrep.com/patent/US7465454B2

## CPC

- A61K 47/46: Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
- A61K 39/0011: {Cancer antigens}
- A61K 39/001164: {GTPases, e.g. Ras or Rho}
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 35/04: specific for metastasis
- A61P 37/04: Immunostimulants
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/62: DNA sequences coding for fusion proteins
- A61K 2039/523: {expressing foreign proteins}
- A61K 2039/55588: {Adjuvants of undefined constitution}
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

Compositions and methods for treating and/or preventing a variety of diseases and conditions that are amenable to immunotherapy and, in one particular embodiment, compositions and methods for treating and/or preventing cancer in an animal are described. Specifically improvements related to the use of a yeast-based vaccine comprising a yeast vehicle and an antigen that is selected to elicit an antigen-specific cellular and humoral immune response in an animal, for use in prophylactic and/or therapeutic vaccination and the prevention and/or treatment of a variety of diseases and conditions are disclosed.

## Claims

### Claim 1

A method to increase survival or reduce tumor burden in an animal that has cancer, comprising administering to an animal a composition that increases survival of the animal or reduces tumor burden in the animal, wherein the composition comprises:
a) a yeast vehicle; and
b) a fusion protein expressed by the yeast vehicle, the fusion protein comprising:
i) at least one cancer antigen or an immunogenic domain thereof expressed by the animal's cancer; and
ii) a peptide linked to the N-terminus of the cancer antigen or immunogenic domain thereof the peptide consisting of between two and six amino acid residues that are heterologous to the cancer antigen or immunogenic domain thereof, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein, and wherein the peptide does not negatively impact an immune response against the cancer antigen or immunogenic domain thereof;
wherein the first six amino acids of the fusion protein consist of an amino acid sequence of M-X2—X3—X4—X5—X6;
(1) wherein M is methionine;
(2) wherein X2 is any amino acid except glycine, proline, lysine or arginine;
(3) wherein X3 is any amino acid except methionine, lysine or arginine;
(4) wherein X4 is any amino acid except methionine, lysine or arginine;
(5) wherein X5 is any amino acid except methionine, lysine or arginine; and
(6) wherein X6 is any amino acid except methionine.

### Claim 2 (depends on 1)

The method of claim 1, wherein X6 is a proline.

### Claim 3 (depends on 1)

The method of claim 1, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 4 (depends on 1)

The method of claim 1, wherein the fusion protein comprises at least two or more cancer antigens expressed by the cancer.

### Claim 5 (depends on 1)

The method of claim 1, wherein the fusion protein comprises at least one or more immunogenic domains of one or more cancer antigens expressed by the cancer.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cancer antigen is an antigen expressed by a cancer selected from the group consisting of: melanomas, squamous cell carcinoma, breast cancers, head and neck carcinomas, thyroid carcinomas, soft tissue sarcomas, bone sarcomas, testicular cancers, prostatic cancers, ovarian cancers, bladder cancers, skin cancers, brain cancers, angiosarcomas, hemangiosarcomas, mast cell tumors, primary hepatic cancers, lung cancers, pancreatic cancers, gastrointestinal cancers, renal cell carcinomas, hematopoietic neoplasias and metastatic cancers thereof.

### Claim 7 (depends on 1)

The method of claim 1, wherein the cancer antigen is wild-type or mutant protein encoded by a ras gene.

### Claim 8 (depends on 7)

The method of claim 7, wherein the cancer antigen is wild-type or mutant protein encoded by a ras gene selected from the group consisting of: K-ras, N-ras and H-ras genes.

### Claim 9 (depends on 7)

The method of claim 7, wherein the ras gene encodes a Ras protein with single or multiple mutations.

### Claim 10 (depends on 1)

The method of claim 1, wherein the cancer antigen comprises fragments of at least 5-9 contiguous amino acid residues of a wild-type Ras protein containing amino acid positions 12, 13, 59 or 61 relative to the wild-type Ras protein, wherein the amino acid residues at positions 12, 13, 59 or 61 are mutated with respect to the wild-type Ras protein.

### Claim 11 (depends on 1)

The method of claim 1, wherein the cancer antigen consists of a fusion protein construct comprising multiple domains, wherein each domain consists of a peptide from an oncoprotein, the peptide consisting of at least 4 amino acid residues flanking either side of and including a mutated amino acid that is found in the protein, wherein the mutation is associated with tumorigenicity.

### Claim 12 (depends on 11)

The method of claim 11, wherein the fusion protein construct consists of at least one peptide that is fused in frame with another mutated cancer antigen, wherein the peptide is selected from the group consisting of:
a) a peptide comprising at least from positions 8-16 of SEQ ID NO:3, wherein the amino acid residue at position 12 with respect to SEQ ID NO:3 is mutated as compared to SEQ ID NO:3;
b) a peptide comprising at least from positions 9-17 of SEQ ID NO:3, wherein the amino acid residue at position 13 with respect to SEQ ID NO:3 is mutated as compared to SEQ ID NO:3;
c) a peptide comprising at least from positions 55-63 of SEQ ID NO:3, wherein the amino acid residue at position 59 with respect to SEQ ID NO:3 is mutated as compared to SEQ ID NO:3; and
d) a peptide comprising at least from positions 57-65 of SEQ ID NO:3, wherein the amino acid residue at position 61 with respect to SEQ ID NO:3 is mutated as compared to SEQ ID NO:3.

### Claim 13 (depends on 12)

The method of claim 12, wherein the mutated cancer antigen is a Ras protein comprising at least one mutation relative to a wild-type Ras protein sequence.

### Claim 14 (depends on 1)

The method of claim 1, wherein the yeast vehicle is selected from the group consisting of a whole yeast, a yeast spheroplast, a yeast cytoplast, a yeast ghost, and a subcellular yeast membrane extract or fraction thereof.

### Claim 15 (depends on 1)

The method of claim 1, wherein a yeast cell or yeast spheroplast used to prepare the yeast vehicle was transformed with a recombinant nucleic acid molecule encoding the fusion protein such that the fusion protein is recombinantly expressed by the yeast cell or yeast spheroplast.

### Claim 16 (depends on 15)

The method of claim 15, wherein the yeast cell or yeast spheroplast that recombinantly expresses the fusion protein is used to produce a yeast vehicle comprising a yeast cytoplast, a yeast ghost, or a subcellular yeast membrane extract or fraction thereof.

### Claim 17 (depends on 1)

The method of claim 1, wherein the yeast vehicle is from a non-pathogenic yeast.

### Claim 18 (depends on 1)

The method of claim 1, wherein the yeast vehicle is from a yeast selected from the group consisting of: Saccharomyces, Schizosaccharomyces, Kiuveromyces, Hansenula, Candida and Pichia.

### Claim 19 (depends on 1)

The method of claim 1, wherein Saccharomyces is S. cerevisiae.

### Claim 20 (depends on 1)

The method of claim 1, wherein the composition is administered to the respiratory tract.

### Claim 21 (depends on 1)

The method of claim 1, wherein the composition is administered by a parenteral route of administration.

### Claim 22 (depends on 1)

The method of claim 1, wherein the composition further comprises dendritic cells or macrophages, wherein the yeast vehicle expressing the fusion protein is delivered to dendritic cells or macrophages ex vivo and wherein the dendritic cell or macrophage containing the yeast vehicle expressing the fusion protein is administered to the animal.

### Claim 23 (depends on 22)

The method of claim 22, wherein the dendritic cell or the yeast vehicle has been additionally loaded with free antigen.

### Claim 24 (depends on 1)

The method of claim 1, wherein the animal has a cancer selected from the group consisting of brain cancer, lung cancer, breast cancer, melanoma, and renal cancer.

### Claim 25 (depends on 1)

The method of claim 1, wherein administration of the composition occurs after surgical resection of a tumor from the animal.

### Claim 26 (depends on 1)

The method of claim 1, wherein administration of the composition occurs after surgical resection of a tumor from the animal and after administration of non-myeloablative allogeneic stem cell transplantation.

### Claim 27 (depends on 1)

The method of claim 1, wherein administration of the composition occurs after surgical resection of a tumor from the animal, after administration of non-myeloablative allogeneic stem cell transplantation, and after allogeneic donor lymphocyte infusion.

### Claim 28

A method to inhibit tumor growth in an animal that has cancer, comprising administering to an animal a composition that inhibits tumor growth in the animal, wherein the composition comprises:
a) a yeast vehicle; and
b) a fusion protein expressed by the yeast vehicle, the fusion protein comprising:
i) at least one cancer antigen or an immunogenic domain thereof expressed by the animal's cancer; and
ii) a peptide linked to the N-terminus of the cancer antigen or immunogenic domain thereof the peptide consisting of between two and six amino acid residues that are heterologous to the cancer antigen or immunogenic domain thereof, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein, and wherein the peptide does not negatively impact an immune response against the cancer antigen or immunogenic domain thereof;
wherein the first six amino acids of the fusion protein consist of an amino acid sequence of M-X2—X3—X4—X5—X6;
(1) wherein M is methionine;
(2) wherein X2 is any amino acid except glycine, proline, lysine or arginine;
(3) wherein X3 is any amino acid except methionine, lysine or arginine;
(4) wherein X4 is any amino acid except methionine, lysine or arginine;
(5) wherein X5 is any amino acid except methionine, lysine or arginine; and
(6) wherein X6 is any amino acid except methionine.

### Claim 29 (depends on 1)

The method of claim 1, wherein the cancer antigen is EGF-R.

### Claim 30 (depends on 1)

The method of claim 1, wherein the cancer antigen is MART1.

### Claim 31 (depends on 1)

The method of claim 1, wherein the cancer antigen is VHL (von Hippel's Lindau protein).

### Claim 32 (depends on 1)

The method of claim 1, wherein the cancer antigen is a fusion protein comprising the following polypeptides, in any order:
a) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with a leucine as compared to a wild-type Ras; and
b) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 12, wherein the glycine at position 12 is substituted with a valine as compared to wild-type Ras; and
c) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with an arginine as compared to a wild-type Ras.

### Claim 33 (depends on 1)

The method of claim 1, wherein the cancer antigen is a fusion protein comprising the following polypeptides, in any order:
a) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with a leucine as compared to a wild-type Ras; and
b) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 12, wherein the glycine at position 12 is substituted with a cysteine as compared to wild-type Ras; and
c) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with an arginine as compared to a wild-type Ras.

### Claim 34 (depends on 1)

The method of claim 1, wherein the cancer antigen is a fusion protein comprising the following polypeptides, in any order:
a) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with a leucine as compared to a wild-type Ras; and
b) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 12, wherein the glycine at position 12 is substituted with an aspartic acid residue as compared to wild-type Ras; and
c) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with an arginine as compared to a wild-type Ras.

### Claim 35 (depends on 28)

The method of claim 28, wherein the cancer antigen is wild-type or mutant protein encoded by a ras gene.

### Claim 36 (depends on 28)

The method of claim 28, wherein the cancer antigen is a fusion protein comprising the following polypeptides, in any order:
a) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with a leucine as compared to a wild-type Ras; and
b) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 12, wherein the glycine at position 12 is substituted with a valine as compared to wild-type Ras; and
c) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with an arginine as compared to a wild-type Ras.

### Claim 37 (depends on 28)

The method of claim 28, wherein the cancer antigen is a fusion protein comprising the following polypeptides, in any order:
a) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with a leucine as compared to a wild-type Ras; and
b) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 12, wherein the glycine at position 12 is substituted with a cysteine as compared to wild-type Ras; and
c) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with an arginine as compared to a wild-type Ras.

### Claim 38 (depends on 28)

The method of claim 28, wherein the cancer antigen is a fusion protein comprising the following polypeptides, in any order:
a) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with a leucine as compared to a wild-type Ras; and
b) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 12, wherein the glycine at position 12 is substituted with an aspartic acid residue as compared to wild-type Ras; and
c) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with an arginine as compared to a wild-type Ras.

### Claim 39 (depends on 28)

The method of claim 28, wherein the cancer antigen is EGF-R.

### Claim 40 (depends on 28)

The method of claim 28, wherein the cancer antigen is MART1.

### Claim 41 (depends on 28)

The method of claim 28, wherein the cancer antigen is VHL (von Hippel's Lindau protein).

### Claim 42 (depends on 28)

The method of claim 28, wherein the yeast vehicle is selected from the group consisting of a whole yeast, a yeast spheroplast, a yeast cytoplast, a yeast ghost, and a subcellular yeast membrane extract or fraction thereof.

### Claim 43 (depends on 28)

The method of claim 28, wherein a yeast cell or yeast spheroplast used to prepare the yeast vehicle was transformed with a recombinant nucleic acid molecule encoding the cancer antigen such that the cancer antigen is recombinantly expressed by the yeast cell or yeast spheroplast.

### Claim 44 (depends on 43)

The method of claim 43, wherein the yeast cell or yeast spheroplast that recombinantly expresses the cancer antigen is used to produce a yeast vehicle comprising a yeast cytoplast, a yeast ghost, or a subcellular yeast membrane extract or fraction thereof.

### Claim 45 (depends on 28)

The method of claim 28, wherein the yeast vehicle is from a non-pathogenic yeast.

### Claim 46 (depends on 28)

The method of claim 28, wherein the yeast vehicle is from a yeast selected from the group consisting of: Saccharomyces, Schizosaccharomyces, Kiuveromyces, Hansenula, Candida and Pichia.

### Claim 47 (depends on 46)

The method of claim 46, wherein Saccharomyces is S. cerevisiae.

### Claim 48

A method to increase survival or reduce tumor burden in an animal that has cancer, comprising administering to an animal a composition that increases survival of the animal or reduces tumor burden in the animal, wherein the composition comprises:
a) a yeast vehicle; and
b) a fusion protein expressed by the yeast vehicle, the fusion protein comprising:
i) at least one cancer antigen or an immunogenic domain thereof expressed by the animal's cancer; and
ii) a peptide comprising an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1), wherein said peptide is linked to the N-terminus of the cancer antigen or immunogenic domain thereof, the peptide consisting of between two and 200 amino acid residues that are heterologous to the cancer antigen or immunogenic domain thereof, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein, and wherein the peptide does not negatively impact an immune response against the cancer antigen or immunogenic domain thereof.

### Claim 49

A method to inhibit tumor growth in an animal that has cancer, comprising administering to an animal a composition that inhibits tumor growth in the animal, wherein the composition comprises:
a) a yeast vehicle; and
b) a fusion protein expressed by the yeast vehicle, the fusion protein comprising:
i) at least one cancer antigen or an immunogenic domain thereof expressed by the animal's cancer; and
ii) a peptide comprising an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1), wherein said peptide is linked to the N-terminus of the cancer antigen or immunogenic domain thereof, the peptide consisting of between two and 200 amino acid residues that are heterologous to the cancer antigen or immunogenic domain thereof, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein, and wherein the peptide does not negatively impact an immune response against the cancer antigen or immunogenic domain thereof.

### Claim 50

A method to increase survival, reduce tumor burden, or inhibit tumor growth in an animal that has cancer, comprising administering to an animal a composition that increases survival of the animal or reduces tumor burden in the animal, wherein the composition comprises:
a) a yeast vehicle; and
b) a fusion protein expressed by the yeast vehicle, the fusion protein comprising:
i) at least one cancer antigen or an immunogenic domain thereof expressed by the animal's cancer, wherein the cancer antigen is a mutant protein encoded by a ras gene selected from the group consisting of: K-ras, N-ras and H-ras genes; and
ii) a peptide linked to the N-terminus of the cancer antigen or immunogenic domain thereof, the peptide consisting of between two and 200 amino acid residues that are heterologous to the cancer antigen or immunogenic domain thereof, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein, and wherein the peptide does not negatively impact an immune response against the cancer antigen or immunogenic domain thereof;
wherein the first six amino acids of the fusion protein consist of an amino acid sequence of M-X2—X3—X4—X5—X6;
(1) wherein M is methionine;
(2) wherein X2 is any amino acid except glycine, proline, lysine or arginine;
(3) wherein X3 is any amino acid except methionine, lysine or arginine;
(4) wherein X4 is any amino acid except methionine, lysine or arginine;
(5) wherein X5 is any amino acid except methionine, lysine or arginine; and
(6) wherein X6 is any amino acid except methionine.

### Claim 51 (depends on 50)

The method of claim 50, wherein X6 is a proline.

### Claim 52 (depends on 50)

The method of claim 50, wherein the peptide comprises an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 53 (depends on 50)

The method of claim 50, wherein the ras gene encodes a Ras protein with single or multiple mutations.

### Claim 54 (depends on 50)

The method of claim 50, wherein the cancer antigen comprises fragments of at least 5-9 contiguous amino acid residues of a wild-type Ras protein containing amino acid positions 12, 13, 59 or 61 relative to the wild-type Ras protein, wherein the amino acid residues at positions 12, 13, 59 or 61 are mutated with respect to the wild-type Ras protein.

### Claim 55 (depends on 50)

The method of claim 50, wherein the cancer antigen consists of a fusion protein construct comprising multiple domains, wherein each domain consists of a peptide from a Ras protein, the peptide consisting of at least 4 amino acid residues flanking either side of and including a mutated amino acid that is found in the protein, wherein the mutation is associated with tumorigenicity.

### Claim 56 (depends on 50)

The method of claim 50, wherein the cancer antigen consists of a fusion protein construct consisting of at least one peptide from a Ras protein that is fUsed in frame with another mutated cancer antigen, wherein the peptide is selected from the group consisting of:
a) a peptide comprising at least from positions 8-16 of SEQ ID NO:3, wherein the amino acid residue at position 12 with respect to SEQ ID NO:3 is mutated as compared to SEQ ID NO:3;
b) a peptide comprising at least from positions 9-17 of SEQ ID NO:3, wherein the amino acid residue at position 13 with respect to SEQ ID NO:3 is mutated as compared to SEQ ID NO:3;
c) a peptide comprising at least from positions 55-63 of SEQ ID NO:3, wherein the amino acid residue at position 59 with respect to SEQ ID NO:3 is mutated as compared to SEQ ID NO:3; and
d) a peptide comprising at least from positions 57-65 of SEQ ID NO:3, wherein the amino acid residue at position 61 with respect to SEQ ID NO:3 is mutated as compared to SEQ ID NO:3.

### Claim 57 (depends on 56)

The method of claim 56, wherein the mutated cancer antigen is a Ras protein comprising at least one mutation relative to a wild-type Ras protein sequence.

### Claim 58 (depends on 50)

The method of claim 50, wherein the cancer antigen is a fusion protein comprising the following polypeptides, in any order:
a) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with a leucine as compared to a wild-type Ras; and
b) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 12, wherein the glycine at position 12 is substituted with a valine as compared to wild-type Ras; and
c) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with an arginine as compared to a wild-type Ras.

### Claim 59 (depends on 58)

The method of claim 58, wherein the peptide comprises an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 60 (depends on 50)

The method of claim 50, wherein the cancer antigen is a fusion protein comprising the following polypeptides, in any order:
a) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with a leucine as compared to a wild-type Ras; and
b) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 12, wherein the glycine at position 12 is substituted with a cysteine as compared to wild-type Ras; and
c) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with an arginine as compared to a wild-type Ras.

### Claim 61 (depends on 60)

The method of claim 60, wherein the peptide comprises an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 62 (depends on 50)

The method of claim 50, wherein the cancer antigen is a fusion protein comprising the following polypeptides, in any order:
a) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with a leucine as compared to a wild-type Ras; and
b) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 12, wherein the glycine at position 12 is substituted with an aspartic acid residue as compared to wild-type Ras; and
c) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the glutamic acid at position 61 is substituted with an arginine as compared to a wild-type Ras.

### Claim 63 (depends on 62)

The method of claim 62, wherein the peptide comprises an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 64 (depends on 50)

The method of claim 50, wherein the yeast vehicle is selected from the group consisting of a whole yeast, a yeast spheroplast, a yeast cytoplast, a yeast ghost, and a subcellular yeast membrane extract or fraction thereof.

### Claim 65 (depends on 50)

The method of claim 50, wherein a yeast cell or yeast spheroplast used to prepare the yeast vehicle was transformed with a recombinant nucleic acid molecule encoding the cancer antigen such that the cancer antigen is recombinantly expressed by the yeast cell or yeast spheroplast.

### Claim 66 (depends on 65)

The method of claim 65, wherein the yeast cell or yeast spheroplast that recombinantly expresses the cancer antigen is used to produce a yeast vehicle comprising a yeast cytoplast, a yeast ghost, or a subcellular yeast membrane extract or fraction thereof.

### Claim 67 (depends on 50)

The method of claim 50, wherein the yeast vehicle is from a non-pathogenic yeast.

### Claim 68 (depends on 50)

The method of claim 50, wherein the yeast vehicle is from a yeast selected from the group consisting of: Saccharomyces, Schizosaccharomyces, Kiuveromyces, Hansenula, Candida and Pichia.

### Claim 69 (depends on 68)

The method of claim 68, wherein Saccharomyces is S. cerevisiae.

### Claim 70

A method to increase survival, reduce tumor burden, or inhibit tumor growth in an animal that has cancer, comprising administering to an animal a composition that increases survival of the animal or reduces tumor burden in the animal, wherein the composition comprises:
a) a yeast vehicle; and
b) a fusion protein expressed by the yeast vehicle, the fusion protein comprising:
i) at least one cancer antigen or an immunogenic domain thereof expressed by the animal's cancer, wherein the cancer antigen is EGF-R; and
ii) a peptide linked to the N-terminus of the cancer antigen or immunogenic domain thereof, the peptide consisting of between two and 200 amino acid residues that are heterologous to the cancer antigen or immunogenic domain thereof, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein, and wherein the peptide does not negatively impact an immune response against the cancer antigen or immunogenic domain thereof
wherein the first six amino acids of the fusion protein consist of an amino acid sequence of M-X2—X3—X4—X5—X6;
(1) wherein M is methionine;
(2) wherein X2 is any amino acid except glycine, proline, lysine or arginine;
(3) wherein X3 is any amino acid except methionine, lysine or arginine;
(4) wherein X4 is any amino acid except methionine, lysine or arginine;
(5) wherein X5 is any amino acid except methionine, lysine or arginine; and
(6) wherein X6 is any amino acid except methionine.

### Claim 71 (depends on 70)

The method of claim 70, wherein X6 is a proline.

### Claim 72 (depends on 70)

The method of claim 70, wherein the peptide comprises an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 73 (depends on 70)

The method of claim 70, wherein the yeast vehicle is selected from the group consisting of a whole yeast, a yeast spheroplast, a yeast cytoplast, a yeast ghost, and a subcellular yeast membrane extract or fraction thereof.

### Claim 74 (depends on 70)

The method of claim 70, wherein a yeast cell or yeast spheroplast used to prepare the yeast vehicle was transformed with a recombinant nucleic acid molecule encoding the cancer antigen such that the cancer antigen is recombinantly expressed by the yeast cell or yeast spheroplast.

### Claim 75 (depends on 74)

The method of claim 74, wherein the yeast cell or yeast spheroplast that recombinantly expresses the cancer antigen is used to produce a yeast vehicle comprising a yeast cytoplast, a yeast ghost, or a subcellular yeast membrane extract or fraction thereof.

### Claim 76 (depends on 70)

The method of claim 70, wherein the yeast vehicle is from a non-pathogenic yeast.

### Claim 77 (depends on 70)

The method of claim 70, wherein the yeast vehicle is from a yeast selected from the group consisting of: Saccharomyces, Schizosaccharomyces, Kiuveromyces, Hansenula, Candida and Pichia.

### Claim 78 (depends on 77)

The method of claim 77, wherein Saccharomyces is S. cerevisiae.

### Claim 79

A method to increase survival, reduce tumor burden, or inhibit tumor growth in an animal that has cancer, comprising administering to an animal a composition that increases survival of the animal or reduces tumor burden in the animal, wherein the composition comprises:
a) a yeast vehicle; and
b) a fusion protein expressed by the yeast vehicle, the fusion protein comprising:
i) at least one cancer antigen or an immunogenic domain thereof expressed by the animal's cancer, wherein the cancer antigen is VHL (von Hippel's Lindau protein); and
ii) a peptide linked to the N-terminus of the cancer antigen or immunogenic domain thereof, the peptide consisting of between two and 200 amino acid residues that are heterologous to the cancer antigen or immunogenic domain thereof, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein, and wherein the peptide does not negatively impact an immune response against the cancer antigen or immunogenic domain thereof
wherein the first six amino acids of the fusion protein consist of an amino acid sequence of M-X2—X3—X4—X5—X6;
(1) wherein M is methionine;
(2) wherein X2 is any amino acid except glycine, proline, lysine or arginine;
(3) wherein X3 is any amino acid except methionine, lysine or arginine;
(4) wherein X4 is any amino acid except methionine, lysine or arginine;
(5) wherein X5 is any amino acid except methionine, lysine or arginine; and
(6) wherein X6 is any amino acid except methionine.

### Claim 80 (depends on 1)

The method of claim 1, wherein the yeast vehicle is a whole yeast.

### Claim 81 (depends on 28)

The method of claim 28, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 82 (depends on 28)

The method of claim 28, wherein the yeast vehicle is a whole yeast.

### Claim 83 (depends on 50)

The method of claim 50, wherein the yeast vehicle is a whole yeast.

### Claim 84 (depends on 50)

The method of claim 50, wherein the peptide consists of between two and six amino acids.

### Claim 85 (depends on 50)

The method of claim 50, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 86 (depends on 50)

The method of claim 50, wherein the cancer antigen is a fusion protein comprising the following polypeptides, in any order:
a) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the amino acid at position 61 is mutated as compared to a wild-type Ras; and
b) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 12, wherein the amino acid at position 12 is mutated as compared to wild-type Ras; and
c) a polypeptide comprising a mutated Ras protein or immunogenic domain thereof comprising position 61 of Ras, wherein the amino acid at position 61 is mutated as compared to a wild-type Ras.

### Claim 87 (depends on 86)

The method of claim 86, wherein the peptide consists of between two and six amino acids.

### Claim 88 (depends on 86)

The method of claim 86, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 89 (depends on 86)

The method of claim 86, wherein the yeast vehicle is a whole yeast.

### Claim 90 (depends on 50)

The method of claim 50, wherein the cancer antigen comprises at least one mutated Ras protein or immunogenic domain thereof comprising a mutation at position 12 of Ras as compared to a wild-type Ras.

### Claim 91 (depends on 90)

The method of claim 90, wherein the peptide consists of between two and six amino acids.

### Claim 92 (depends on 90)

The method of claim 90, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 93 (depends on 90)

The method of claim 90, wherein the yeast vehicle is a whole yeast.

### Claim 94 (depends on 50)

The method of claim 50, wherein the cancer antigen comprises at least one mutated Ras protein or immunogenic domain thereof comprising a mutation at position 61 of Ras as compared to a wild-type Ras.

### Claim 95 (depends on 94)

The method of claim 94, wherein the peptide consists of between two and six amino acids.

### Claim 96 (depends on 94)

The method of claim 94, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 97 (depends on 94)

The method of claim 94, wherein the yeast vehicle is a whole yeast.

### Claim 98 (depends on 94)

The method of claim 94, wherein the cancer antigen further comprises at least one mutated Ras protein or immunogenic domain thereof comprising a mutation at position 12 of Ras as compared to a wild-type Ras.

### Claim 99 (depends on 98)

The method of claim 98, wherein the peptide consists of between two and six amino acids.

### Claim 100 (depends on 98)

The method of claim 98, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 101 (depends on 98)

The method of claim 98, wherein the yeast vehicle is a whole yeast.

### Claim 102 (depends on 58)

The method of claim 58, wherein the peptide consists of between two and six amino acids.

### Claim 103 (depends on 58)

The method of claim 58, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 104 (depends on 58)

The method of claim 58, wherein the yeast vehicle is a whole yeast.

### Claim 105 (depends on 60)

The method of claim 60, wherein the peptide consists of between two and six amino acids.

### Claim 106 (depends on 60)

The method of claim 60, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 107 (depends on 60)

The method of claim 60, wherein the yeast vehicle is a whole yeast.

### Claim 108 (depends on 62)

The method of claim 62, wherein the peptide consists of between two and six amino acids.

### Claim 109 (depends on 62)

The method of claim 62, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 110 (depends on 62)

The method of claim 62, wherein the yeast vehicle is a whole yeast.

### Claim 111 (depends on 70)

The method of claim 70, wherein the peptide consists of between two and six amino acids.

### Claim 112 (depends on 70)

The method of claim 70, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

### Claim 113 (depends on 79)

The method of claim 79, wherein the peptide consists of between two and six amino acids.

### Claim 114 (depends on 79)

The method of claim 79, wherein the peptide consists of an amino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).