PH20 polypeptide variants, formulations and uses thereof

Patent metadata

Field	Value
Publication number	US-9447401-B2
Application number	US-201213694731-A
Country	US
Kind code	B2
Filing date	Dec 28, 2012
Priority date	Dec 30, 2011
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

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.

First claim

The invention claimed is:

1. A modified PH20 polypeptide that exhibits increased stability in the presence of a phenolic preservative, comprising an amino acid replacement in an unmodified PH20 polypeptide, wherein:
the amino acid replacement confers the increased stability;
the amino acid replacement is replacement with proline at a position corresponding to position 204 in a PH20 polypeptide with reference to amino acid residue positions set forth in SEQ ID NO:3;
corresponding amino acid positions are identified by alignment of the modified PH20 polypeptide with the polypeptide whose sequence is set forth in SEQ ID NO:3;
the modified PH20 polypeptide has at least 85% sequence identity to the sequence of amino acids set forth in SEQ ID NO: 3 or 7; and
stability is increased compared to the unmodified polypeptide without the amino acid replacement, wherein stability is measured by resistance to denaturation in the presence of a phenolic preservative.

2. The modified PH20 polypeptide of claim 1 , wherein the unmodified PH20 polypeptide is a soluble PH20 polypeptide that consists of the sequence of amino acids set forth in any of SEQ ID NOS: 3, 7 and 32-66.

3. The modified PH20 polypeptide of claim 1 , wherein:
the unmodified PH20 polypeptide consists of the sequence of amino acids set forth in SEQ ID NO:3 or 7; and
the modified PH20 polypeptide has at least 95% sequence identity to SEQ ID NO:3.

4. The modified PH20 polypeptide of claim 1 , that has only up to 10 amino acid replacements compared to the polypeptide of SEQ ID NO: 7 or 3.

5. The modified PH20 polypeptide of claim 1 , wherein the modified PH20 polypeptide exhibits at least 15% of the hyaluronidase activity in the presence of the phenolic preservative(s) for at least 4 hours compared to the hyaluronidase activity in the absence of the phenolic preservative(s), wherein the activity is compared under the same conditions except for the presence of the phenolic preservative(s).

6. The modified PH20 polypeptide of claim 1 , wherein the modified PH20 polypeptide is stable in the presence of an anti-microbially effective amount of one or more phenolic preservatives.

7. The modified PH20 polypeptide of claim 6 , wherein the anti-microbially effective amount is a total amount of one or more phenolic preservative agents as a percentage (%) of mass concentration (w/v) that is from about 0.05% to 0.6%.

8. The modified PH20 polypeptide of claim 1 , wherein the phenolic preservative is selected from among one or more of phenol, metacresol (m-cresol), benzyl alcohol and a paraben.

9. The modified PH20 polypeptide of claim 1 , wherein the preservative is a phenolic preservative that is m-cresol, phenol, or a combination of m-cresol and phenol.

10. The modified PH20 polypeptide of claim 1 , further comprising at least one amino acid replacement at the amino acid position selected from the group consisting of 58, 10, 12, 20, 22, 26, 34, 36, 46, 50, 52, 68, 70, 74, 82, 83, 84, 86, 97, 127, 131, 138, 142, 143, 144, 166, 169, 174, 193, 195, 196, 205, 206, 213, 219, 234, 237, 238, 240, 249, 261, 267, 277, 279, 291, 309, 310, 314, 315, 317, 318, 347, 367, 375, 376, 399, 401, 407, 416, 419, 421, 431, 433, 439, 440, 443 and 445 with reference to amino acid positions set forth in SEQ ID NO:3, wherein amino acid position is identified by alignment of the modified PH20 polypeptide with SEQ ID NO:3.

11. The modified PH20 polypeptide of claim 10 , comprising at least one amino acid replacement selected from the group consisting of: G at a position corresponding to position 10; K at a position corresponding to position 12; S at a position corresponding to position 20; T at a position corresponding to position 22; M at a position corresponding to position 26; W at a position corresponding to position 34; N at a position corresponding to position 36; L at a position corresponding to position 46; M at a position corresponding to position 50; T at a position corresponding to position 52; S at a position corresponding to position 52; C at a position corresponding to position 58; K at a position corresponding to position 58; R at a position corresponding to position 58; N at a position corresponding to position 58; Y at a position corresponding to position 58; P at a position corresponding to position 58; H at a position corresponding to position 58; P at a position corresponding to position 68; V at a position corresponding to position 70; E at a position corresponding to position 74; L at a position corresponding to position 82; N at a position corresponding to position 82; V at a position corresponding to position 83; Q at a position corresponding to position 83; S at a position corresponding to position 83; G at a position corresponding to position 83; N at a position corresponding to position 84; A at a position corresponding to position 86; K at a position corresponding to position 86; E at a position corresponding to position 97; L at a position corresponding to position 97; R at a position corresponding to position 127; R at a position corresponding to position 131; L at a position corresponding to position 138; K at a position corresponding to position 142; N at a position corresponding to position 142; P at a position corresponding to position 142; S at a position corresponding to position 142; T at a position corresponding to position 142; G at a position corresponding to position 143; K at a position corresponding to position 143; T at a position corresponding to position 144; Q at a position corresponding to position 166; T at a position corresponding to position 166; L at a position corresponding to position 169; G at a position corresponding to position 174; N at a position corresponding to position 174; Q at a position corresponding to position 193; T at a position corresponding to position 195; N at a position corresponding to position 195; E at a position corresponding to position 196; R at a position corresponding to position 196; A at a position corresponding to position 205; E at a position corresponding to position 205; I at a position corresponding to position 206; A at a position corresponding to position 213; I at a position corresponding to position 219; M at a position corresponding to position 234; T at a position corresponding to position 237; H at a position corresponding to position 238; Q at a position corresponding to position 240; V at a position corresponding to position 249; A at a position corresponding to position 26t; K at a position corresponding to position 261; T at a position corresponding to position 267; K at a position corresponding to position 277; H at a position corresponding to position 279; V at a position corresponding to position 279; V at a position corresponding to position 291; E at a position corresponding to position 309; Q at a position corresponding to position 310; Y at a position corresponding to position 314; Y at a position corresponding to position 315; N at a position corresponding to position 317; W at a position corresponding to position 317; D at a position corresponding to position 318; G at a position corresponding to position 347; A at a position corresponding to position 367; R at a position corresponding to position 375; R at a position corresponding to position 376; V at a position corresponding to position 399; E at a position corresponding to position 401; A at a position corresponding to position 407; L at a position corresponding to position 416; K at a position corresponding to position 419; H at a position corresponding to position 421; E at a position corresponding to position 431; T at a position corresponding to position 433; V at a position corresponding to position 433; C at a position corresponding to po

Assignees

• Wei Ge
• Shepard H Michael
• Zhao Qiping
• Connor Robert James
• Halozyme Inc

Inventors

• Wei Ge
• Shepard H Michael
• Zhao Qiping
• Connor Robert James

Classifications

• A61P3/10

  for hyperglycaemia, e.g. antidiabetics · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P27/02

  Ophthalmic agents · CPC title

• A61P3/04

  Anorexiants; Antiobesity agents · CPC title