# US 12,110,520 B2 · PH20 polypeptide variants, formulations and uses thereof

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- Number: US 12,110,520 B2
- Document ID: US12110520B2
- Status: Granted
- Kind: B2
- Application number: 18/068,418
- Filed: 2022-12-19
- Published: 2023-08-10
- Granted: 2024-10-08
- Earliest claimed: 2011-12-30
- Assignees: Halozyme, Inc.
- Applicants: Halozyme, Inc.
- Inventors: Ge Wei; H. Michael Shepard; Qiping Zhao; Robert James Connor
- Web page: https://patentgrep.com/patent/US12110520B2

## CPC

- C12N 9/2474: {Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 38/28: Insulins
- A61K 38/47: acting on glycosyl compounds (3.2), e.g. cellulases, lactases
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/10: Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
- A61P 3/04: Anorexiants; Antiobesity agents
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 27/02: Ophthalmic agents
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/47: from mammals
- C12Q 1/34: involving hydrolase
- C12Y 302/01035: Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
- 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)
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- G01N 2333/926: acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
- G01N 2333/928: acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Modified PH20 hyaluronidase polypeptides, including modified polypeptides that exhibit increased stability and/or increased activity, are provided. Also provided are compositions and formulations and uses thereof.

## Claims

### Claim 1

A modified PH20 polypeptide, comprising one or more amino acid modifications in an unmodified PH20 polypeptide, wherein:
the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7 and 32-66;
amino acid modifications are selected from the group consisting of amino acid replacements(s), deletion(s), and/or insertion(s);
the modified PH20 polypeptide comprises an amino acid replacement at a position corresponding to residue 324, with reference to amino acid positions set forth in SEQ ID NO: 3;
the replacement at the position corresponding to residue 324 is selected from the group consisting of A, D, H, M, N, R and S;
corresponding amino acid positions are identified by alignment of the PH20 polypeptide with the polypeptide having the amino acid sequence of SEQ ID NO: 3; and
the modified PH20 polypeptide has at least 91% sequence identity to a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7 and 32-66.

### Claim 2 (depends on 1)

The modified PH20 polypeptide of claim 1, wherein the modified PH20 polypeptide has at least 95% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, and 32-66.

### Claim 3 (depends on 1)

The modified PH20 polypeptide of claim 1 that has increased resistance to or stability in denaturing conditions compared to an unmodified PH20 polypeptide that does not contain the amino acid modification(s).

### Claim 4 (depends on 1)

The modified PH20 polypeptide of claim 1 that exhibits increased hyaluronidase activity compared to the unmodified PH20 polypeptide not containing the amino acid replacement at position 324.

### Claim 5 (depends on 1)

The modified PH20 polypeptide of claim 1 that is a soluble PH20 polypeptide.

### Claim 6 (depends on 1)

The modified PH20 polypeptide of claim 1, wherein the replacement at the position corresponding to residue 324 is D.

### Claim 7 (depends on 1)

The modified PH20 polypeptide of claim 1, wherein the replacement at the position corresponding to residue 324 is N or R.

### Claim 8 (depends on 1)

The modified PH20 polypeptide of claim 1, wherein the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 3 and 32-66.

### Claim 9 (depends on 7)

The modified PH20 polypeptide of claim 7, wherein the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 3 and 32-66.

### Claim 10 (depends on 6)

The modified PH20 polypeptide of claim 6, wherein the unmodified PH20 polypeptide consists of the amino acid sequence selected from among the group consisting of SEQ ID NO: 3 and 32-66.

### Claim 11 (depends on 1)

The modified PH20 polypeptide of claim 1, wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:35.

### Claim 12 (depends on 1)

The modified PH20 polypeptide of claim 1, wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:32.

### Claim 13 (depends on 6)

The modified PH20 polypeptide of claim 6, wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:35.

### Claim 14 (depends on 6)

The modified PH20 polypeptide of claim 6, wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:32.

### Claim 15 (depends on 1)

The modified PH20 polypeptide of claim 1, comprising a sequence of amino acids that exhibits at least 91% sequence identity to the sequence of amino acids selected from the group consisting of SEQ ID NO: 3, and 32-66 and that contains an amino acid replacement D at the residue corresponding to residue 324 with reference to SEQ ID NO: 3.

### Claim 16 (depends on 1)

The modified PH20 polypeptide of claim 1 that is C-terminally truncated, whereby the polypeptide is soluble.

### Claim 17 (depends on 1)

The modified PH20 polypeptide of claim 1 that comprises one or more post-translational modifications of the polypeptide selected from the group consisting of glycosylation, sialylation, albumination, farnesylation, carboxylation, hydroxylation, and phosphorylation.

### Claim 18 (depends on 17)

The modified PH20 polypeptide of claim 17, wherein the post-translational modification is glycosylation.

### Claim 19 (depends on 18)

The modified PH20 polypeptide of claim 18, wherein the polypeptide is a glycoprotein that comprises an N-acetylglucosamine moiety linked to each of at least three asparagine (N) residues.

### Claim 20 (depends on 1)

The modified PH20 polypeptide of claim 1 that is conjugated to a polymer.

### Claim 21 (depends on 20)

The modified PH20 polypeptide of claim 20, wherein the polymer is dextran or polyethylene glycol (PEG).

### Claim 22 (depends on 1)

The modified PH20 polypeptide of claim 1, further comprising a heterologous signal sequence, wherein the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 3 and 32-66.

### Claim 23 (depends on 1)

A chimeric polypeptide, comprising the modified PH20 polypeptide of claim 1.

### Claim 24 (depends on 1)

A pharmaceutical composition, comprising the modified PH20 polypeptide of claim 1.

### Claim 25 (depends on 6)

The modified PH20 polypeptide of claim 6, wherein:
the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:32; and
the amino acid sequence of the modified PH20 polypeptide has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:32.

### Claim 26 (depends on 6)

The modified PH20 polypeptide of claim 6, wherein:
the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:35; and
the amino acid sequence of the modified PH20 polypeptide has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:35.

### Claim 27 (depends on 24)

The pharmaceutical composition of claim 24, further comprising a therapeutically active agent formulated in the same composition or in a separate composition.

### Claim 28 (depends on 27)

The pharmaceutical composition of claim 27, wherein the therapeutically active agent is a polypeptide, a protein, a nucleic acid, a drug, a small molecule, or an organic molecule.

### Claim 29 (depends on 27)

The pharmaceutical composition of claim 27, wherein the therapeutically active agent is selected from the group consisting of a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amoebicidal agent, a trichomonacidal agent, an anti-Parkinson agent, an anti-malarial agent, an anticonvulsant agent, an anti-depressant agent, an antiarthritics agent, an anti-fungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasite agent, an antihistamine agent, an alpha-adrenergic agonist agent, an alpha blocker agent, an anesthetic agent, a bronchial dilator agent, a biocide agent, a bactericide agent, a bacteriostat agent, a beta adrenergic blocker agent, a calcium channel blocker agent, a cardiovascular drug agent, a contraceptive agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent, an electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contractant agent, an ophthalmic agent, a parasympathomimetic agent, a psychic energizer agent, a sedative agent, a sympathomimetic agent, a tranquilizer agent, a urinary agent, a vaginal agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent, and a sleep inducer.

### Claim 30 (depends on 27)

The pharmaceutical composition of claim 27, wherein the therapeutically active agent is an antibody.

### Claim 31 (depends on 1)

A method for treating a hyaluronan-associated disease or condition, comprising administering to a subject a modified PH20 polypeptide of claim 1.

### Claim 32 (depends on 31)

The method of claim 31, wherein the hyaluronan-associated disease or condition is an inflammatory disease or a tumor or cancer.

### Claim 33 (depends on 32)

The method of claim 32, wherein the hyaluronan-associated disease or condition is a solid tumor.

### Claim 34 (depends on 1)

The modified PH20 polypeptide of claim 1 that is further modified by conjugation to a moiety selected from the group consisting of a multimerization domain, a toxin, a detectable label, and a drug.

### Claim 35 (depends on 34)

The modified PH20 polypeptide of claim 34, wherein the modified PH20 polypeptide is conjugated to a multimerization domain that is an Fc domain.