# US 7,744,892 B2 · Optimized expression of HPV 52 L1 in yeast

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- Number: US 7,744,892 B2
- Document ID: US7744892B2
- Status: Granted
- Kind: B2
- Application number: 12/578,995
- Filed: 2009-10-14
- Published: 2010-02-11
- Granted: 2010-06-29
- Earliest claimed: 2004-03-24
- Assignees: Merck Sharp & Dohme Corp.
- Inventors: Janine T. Bryan; Michelle K. Brownlow; Loren D. Schultz; Kathrin U. Jansen
- Web page: https://patentgrep.com/patent/US7744892B2

## CPC

- C07K 14/005: from viruses
- A61P 31/12: Antivirals
- A61P 31/20: for DNA viruses
- A61P 35/00: Antineoplastic agents
- A61P 37/00: Drugs for immunological or allergic disorders
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 2039/5258: {Virus-like particles}
- C12N 2710/20022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2710/20023: Virus like particles [VLP]
- 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)
- 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)
- 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)

## Abstract

Synthetic DNA molecules encoding the HPV 52 L1 protein are provided. Specifically, the present invention provides polynucleotides encoding HPV 52 L1 protein, wherein said polynucleotides are codon-optimized for high level expression in a yeast cell. In alternative embodiments of the invention, the nucleotide sequence of the synthetic molecule is altered to eliminate transcription termination signals that are recognized by yeast. The synthetic molecules may be used to produce HPV 52 virus-like particles (VLPs), and to produce vaccines and pharmaceutical compositions comprising the HPV 52 VLPs. The vaccines of the present invention provide effective immunoprophylaxis against papillomavirus infection through neutralizing antibody and cell-mediated immunity and may also be useful for treatment of existing HPV infections.

## Claims

### Claim 1

An HPV52 virus-like particle (VLP) comprising recombinant HPV52 L1 protein comprising the sequence of SEQ ID NO: 2, wherein the recombinant HPV52 L1 protein is produced in yeast and wherein the HPV52 L1 protein is encoded by a HPV52 L1 nucleic acid molecule which is codon-optimized for high-level expression in a yeast cell.

### Claim 2 (depends on 1)

The HPV52 VLP of claim 1, wherein the codon-optimized nucleic acid molecule comprising the sequence of SEQ ID NO: 1.

### Claim 3

A method of producing an HPV52 virus-like particle (VLP), comprising:
(a) transforming a yeast cell with a DNA molecule encoding HPV52 L1 protein which comprises the sequence of SEQ ID NO: 2, wherein the DNA molecule is codon-optimized for high-level expression in a yeast cell;
(b) cultivating the transformed yeast cell under conditions that permit expression of the codon-optimized DNA molecule to produce a recombinant papillomavirus protein; and
(c) isolating the recombinant papillomavirus protein to produce the HPV52 VLP.

### Claim 4 (depends on 1)

A pharmaceutical composition comprising the HPV52 VLP of claim 1.

### Claim 5 (depends on 4)

A method for inducing an immune response in an animal comprising administering the pharmaceutical composition of claim 4 to the animal.

### Claim 6 (depends on 3)

The method of claim 3 wherein the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyvermycesfragilis, Kluyveromyces lactis, and Schizosaccharomyces pombe.

### Claim 7 (depends on 6)

The method of claim 6, wherein the yeast cell is Saccharomyces cerevisiae.

### Claim 8 (depends on 4)

The pharmaceutical composition of claim 4, further comprising VLPs of at least one additional HPV type.

### Claim 9 (depends on 8)

The pharmaceutical composition of claim 8 wherein the at least one additional HPV type is selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV55, HPV56, HPV58, HPV59, and HPV68.