# US 6,719,974 B1 · Heat shock protein-based vaccines and immunotherapies

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- Number: US 6,719,974 B1
- Document ID: US6719974B1
- Status: Granted
- Kind: B1
- Application number: 09/680,806
- Filed: 2000-10-05
- Granted: 2004-04-13
- Earliest claimed: 1995-08-18
- Assignees: Sloan-Kettering Institute for Cancer Research
- Inventors: James E. Rothman; Franz Ulrich Hartl; Mee H. Hoe; Alan Houghton; Yoshizumi Takeuchi; Mark Mayhew
- Web page: https://patentgrep.com/patent/US6719974B1

## CPC

- C07K 7/06: having 5 to 11 amino acids
- A61K 39/00119: {Melanoma antigens}
- A61K 39/02: Bacterial antigens
- A61K 39/12: Viral antigens
- A61K 47/646: {the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines}
- A61K 47/65: Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
- A61K 47/6901: {Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors}
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 14/47: from mammals
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/6043: {Heat shock proteins}
- A61K 2039/622: {non-covalent binding}
- A61K 2039/625: {binding through the biotin-streptavidin system or similar}
- C12N 2710/20034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- Y10S 530/828: Cancer
- 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)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The present invention relates to methods and compositions for inducing an immune response in a subject, wherein the subject is administered an effective amount of a heat shock protein complexed to a hybrid antigen comprising an antigenic domain and a heat shock protein-binding domain. These methods and compositions may be used in the treatment of infectious diseases and cancers.

## Claims

### Claim 1

A method of treating melanoma, comprising administering to a subject an effective amount of a complex of:
(a) a hybrid antigen comprising an antigenic domain of a tumor antigen from melanoma and a binding domain that noncovalently binds to a eukaryotic hsp70 and comprises a heptameric region having the sequence motif HyXHyXHyXHy (SEQ ID NO:29) where Hy represents a hydrophobic amino acid and X is any amino acid, wherein the antigenic domain and the binding domain are separated by a short peptide linker; and
(b) the eukaryotic hsp70;
wherein the hsp70 is from the same species as the subject;
wherein the antigenic domain is from a first source and the binding domain is from a second source different from the first source;
wherein the binding domain, the short peptide linker, and the antigenic domain are covalently bound in vitro; and
wherein the hybrid antigen and the hsp70 are combined in vitro under conditions wherein binding of the hybrid antigen to the hsp70 occurs to form a non-covalent hybrid antigen-hsp70 complex.

### Claim 2 (depends on 1)

The method of claim 1, wherein the hydrophobic amino acid is independently selected from the group consisting of tryptophan, leucine and phenylalanine.

### Claim 3 (depends on 1)

The method of claim 1, wherein the binding domain comprises the sequence His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO: 1).

### Claim 4 (depends on 1)

The method of claim 1, wherein the binding domain comprises the sequence Phe Trp Gly Leu Trp Pro Trp Glu (SEQ ID NO:4).

### Claim 5

A method of treating melanoma, comprising administering to a subject an effective amount of a complex of:
(a) a hybrid antigen comprising an antigenic domain of a tumor antigen from melanoma and a binding domain that comprises a hydrophobic peptide of 7-15 amino acids that non-covalently binds to a eukaryotic hsp70, wherein the antigenic domain and the binding domain are separated by a short peptide linker; and
(b) the eukaryotic hsp70;
wherein the hsp70 is from the same species as the subject;
wherein the antigenic domain is from a first source and the binding domain is from a second source different from the first source;
wherein the binding domain, the short peptide linker, and the antigenic domain are covalently bound in vitro; and
wherein the hybrid antigen and the hsp70 are combined in vitro under conditions wherein binding of the hybrid antigen to the hsp70 occurs to form a non-covalent hybrid antigen-hsp70 complex.

### Claim 6 (depends on 5)

The method of claim 5, wherein the binding domain comprises a heptameric region having the sequence motif HyXHyXHyXHy (SEQ ID NO:29) where Hy represents a hydrophobic amino acid and X is any amino acid.

### Claim 7 (depends on 6)

The method of claim 6, wherein the hydrophobic amino acid is independently selected from the group consisting of tryptophan, leucine and phenylalanine.

### Claim 8 (depends on 6)

The method of claim 6, wherein the binding domain comprises a region having the sequence His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO:1).

### Claim 9 (depends on 6)

The method of claim 6, wherein the binding domain comprises a region having the sequence Phe Trp Gly Leu Trp Pro Trp Glu (SEQ ID NO:4).

### Claim 10 (depends on 1, 5)

The method of claim 1 or 5, wherein the short peptide linker is Gly-Ser-Gly.

### Claim 11 (depends on 1, 5)

The method of claim 1 or 5, wherein the hsp70 is mammalian.

### Claim 12

A method of treating melanoma, comprising administering to a subject an effective amount of a complex of:
(a) a hybrid antigen comprising an antigenic domain of a tumor antigen from melanoma and a binding domain that noncovalently binds to a eukaryotic heat shock protein selected from the group consisting of hsp70, hsc70, and BiP, and comprises a heptameric region having the sequence motif HyXHyXHyXHy (SEQ ID NO:29) where Hy represents a hydrophobic amino acid and X is any amino acid, wherein the antigenic domain and the binding domain are separated by a short peptide linker; and
(b) the eukaryotic heat shock protein;
wherein the heat shock protein is from the same species as the subject;
wherein the antigenic domain is from a first source and the binding domain is from a second source different from the first source;
wherein the binding domain, the short peptide linker, and the antigenic domain are covalently bound in vitro; and
wherein the hybrid antigen and the heat shock protein are combined in vitro under conditions wherein binding of the hybrid antigen to the heat shock protein occurs to form a non-covalent hybrid antigen-heat shock protein complex.

### Claim 13 (depends on 12)

The method of claim 12, wherein the hydrophobic amino acid is independently selected from the group consisting of tryptophan, leucine and phenylalanine.

### Claim 14 (depends on 12)

The method of claim 12, wherein the binding domain comprises the sequence His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO:1).

### Claim 15 (depends on 12)

The method of claim 12, wherein the binding domain comprises the sequence Phe Trp Gly Leu Trp Pro Trp Glu (SEQ ID NO:4).

### Claim 16

A method of treating melanoma, comprising administering to a subject an effective amount of a complex of:
(a) a hybrid antigen comprising an antigenic domain of a tumor antigen from melanoma and a binding domain that comprises a hydrophobic peptide of 7-15 amino acids that non-covalently binds to a eukaryotic heat shock protein selected from the group consisting of hsp70, hsc70, and BiP, wherein the antigenic domain and the binding domain are separated by a short peptide linker; and
(b) the eukaryotic heat shock protein;
wherein the heat shock protein is from the same species as the subject;
wherein the antigenic domain is from a first source and the binding domain is from a second source different from the first source;
wherein the binding domain, the short peptide linker, and the antigenic domain are covalently bound in vitro; and
wherein the hybrid antigen and the heat shock protein are combined in vitro under conditions wherein binding of the hybrid antigen to the heat shock protein occurs to form a non-covalent hybrid antigen-heat shock protein complex.

### Claim 17 (depends on 16)

The method of claim 16, wherein the binding domain comprises a heptameric region having the sequence motif HyXHyXHyXHy (SEQ ID NO:29) where Hy represents a hydrophobic amino acid and X is any amino acid.

### Claim 18 (depends on 17)

The method of claim 17, wherein the hydrophobic amino acid is independently selected from the group consisting of tryptophan, leucine and phenylalanine.

### Claim 19 (depends on 17)

The method of claim 17, wherein the binding domain comprises a region having the sequence His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO:1).

### Claim 20 (depends on 17)

The method of claim 17, wherein the binding domain comprises a region having the sequence Phe Trp Gly Leu Trp Pro Trp Glu (SEQ ID NO:4).

### Claim 21 (depends on 12, 16)

The method of claim 12 or 16, wherein the short peptide linker is Gly-Ser-Gly.

### Claim 22 (depends on 12, 16)

The method of claim 12 or 16, wherein the heat shock protein is mammalian.

### Claim 23

A method of treating lymphoma, comprising administering to a subject an effective amount of a complex of:
(a) a hybrid antigen comprising an antigenic domain of a tumor antigen from lymphoma and a binding domain that noncovalently binds to a eukaryotic hsp70 and comprises a heptameric region having the sequence motif HyXHyXHyXHy (SEQ ID NO:29) where Hy represents a hydrophobic amino acid and X is any amino acid, wherein the antigenic domain and the binding domain are separated by a short peptide linker; and
(b) the eukaryotic hsp70;
wherein the hsp70 is from the same species as the subject;
wherein the antigenic domain is from a first source and the binding domain is from a second source different from the first source;
wherein the binding domain, the short peptide linker, and the antigenic domain are covalently bound in vitro; and
wherein the hybrid antigen and the hsp70 are combined in vitro under conditions wherein binding of the hybrid antigen to the hsp70 occurs to form a non-covalent hybrid antigen-hsp70 complex.

### Claim 24 (depends on 23)

The method of claim 23, wherein the hydrophobic amino acid is independently selected from the group consisting of tryptophan, leucine and phenylalanine.

### Claim 25 (depends on 23)

The method of claim 23, wherein the binding domain comprises the sequence His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO:1).

### Claim 26 (depends on 23)

The method of claim 23, wherein the binding domain comprises the sequence Phe Trp Gly Leu Trp Pro Trp Glu (SEQ ID NO:4).

### Claim 27

A method of treating lymphoma, comprising administering to a subject an effective amount of a complex of:
(a) a hybrid antigen comprising an antigenic domain of a tumor antigen from lymphoma and a binding domain that comprises a hydrophobic peptide of 7-15 amino acids that non-covalently binds to a eukaryotic hsp70, wherein the antigenic domain and the binding domain are separated by a short peptide linker; and
(b) the eukaryotic hsp70;
wherein the hsp70 is from the same species as the subject;
wherein the antigenic domain is from a first source and the binding domain is from a second source different from the first source;
wherein the binding domain, the short peptide linker, and the antigenic domain are covalently bound in vitro; and
wherein the hybrid antigen and the hsp70 are combined in vitro under conditions wherein binding of the hybrid antigen to the hsp70 occurs to form a non-covalent hybrid antigen-hsp70 complex.

### Claim 28 (depends on 27)

The method of claim 27, wherein the binding domain comprises a heptameric region having the sequence motif HyXHyXHyXHy (SEQ ID NO:29) where Hy represents a hydrophobic amino acid and X is any amino acid.

### Claim 29 (depends on 28)

The method of claim 28, wherein the hydrophobic amino acid is independently selected from the group consisting of tryptophan, leucine and phenylalanine.

### Claim 30 (depends on 28)

The method of claim 28, wherein the binding domain comprises a region having the sequence His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO:1).

### Claim 31 (depends on 28)

The method of claim 28, wherein the binding domain comprises a region having the sequence Phe Trp Gly Leu Trp Pro Trp Glu (SEQ ID NO:4).

### Claim 32 (depends on 23, 27)

The method of claim 23 or 27, wherein the short peptide linker is Gly-Ser-Gly.

### Claim 33 (depends on 23, 27)

The method of claim 23 or 27, wherein the hsp70 is mammalian.

### Claim 34

A method of treating lymphoma, comprising administering to a subject an effective amount of a complex of:
(a) a hybrid antigen comprising an antigenic domain of a tumor antigen from lymphoma and a binding domain that noncovalently binds to a eukaryotic heat shock protein selected from the group consisting of hsp70, hsc70, and BiP, and comprises a heptameric region having the sequence motif HyXHyXHyXHy (SEQ ID NO:29) where Hy represents a hydrophobic amino acid and X is any amino acid, wherein the antigenic domain and the binding domain are separated by a short peptide linker; and
(b) the eukaryotic heat shock protein;
wherein the heat shock protein is from the same species as the subject;
wherein the antigenic domain is from a first source and the binding domain is from a second source different from the first source;
wherein the binding domain, the short peptide linker, and the antigenic domain are covalently bound in vitro; and
wherein the hybrid antigen and the heat shock protein are combined in vitro under conditions wherein binding of the hybrid antigen to the heat shock protein occurs to form a non-covalent hybrid antigen-heat shock protein complex.

### Claim 35 (depends on 36)

The method of claim 36, wherein the hydrophobic amino acid is independently selected from the group consisting of tryptophan, leucine and phenylalanine.

### Claim 36 (depends on 34)

The method of claim 34, wherein the binding domain comprises the sequence His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO:1).

### Claim 37 (depends on 36)

The method of claim 36, wherein the binding domain comprises the sequence Phe Trp Gly Leu Trp Pro Trp Glu (SEQ ID NO:4).

### Claim 38

A method of treating lymphoma, comprising administering to a subject an effective amount of a complex of:
(a) a hybrid antigen comprising an antigenic domain of a tumor antigen from lymphoma and a binding domain that comprises a hydrophobic peptide of 7-15 amino acids that non-covalently binds to a eukaryotic heat shock protein selected from the group consisting of hsp70, hsc70, and BiP, wherein the antigenic domain and the binding domain are separated by a short peptide linker; and
(b) the eukaryotic heat shock protein;
wherein the heat shock protein is from the same species as the subject;
wherein the antigenic domain is from a first source and the binding domain is from a second source different from the first source;
wherein the binding domain, the short peptide linker, and the antigenic domain are covalently bound in vitro; and
wherein the hybrid antigen and the heat shock protein are combined in vitro under conditions wherein binding of the hybrid antigen to the heat shock protein occurs to form a non-covalent hybrid antigen-heat shock protein complex.

### Claim 39 (depends on 38)

The method of claim 38, wherein the binding domain comprises a heptameric region having the sequence motif HyXHyXHyXHy (SEQ ID NO:29) where Hy represents a hydrophobic amino acid and X is any amino acid.

### Claim 40 (depends on 39)

The method of claim 39, wherein the hydrophobic amino acid is independently selected from the group consisting of tryptophan, leucine and phenylalanine.

### Claim 41 (depends on 39)

The method of claim 39, wherein the binding domain comprises a region having the sequence His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO:1).

### Claim 42 (depends on 39)

The method of claim 39, wherein the binding domain comprises a region having the sequence Phe Trp Gly Leu Trp Pro Trp Glu (SEQ ID NO:4).

### Claim 43 (depends on 36, 38)

The method of claim 36 or 38, wherein the short peptide linker is Gly-Ser-Gly.

### Claim 44 (depends on 36, 38)

The method of claim 36 or 38, wherein the heat shock protein is mammalian.