PH20 polypeptide variants, formulations and uses thereof
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.
1. A modified PH20 polypeptide, comprising one or more amino acid replacements in an unmodified PH20 polypeptide, wherein:
the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7 and 32-66;
the modified PH20 polypeptide exhibits increased hyaluronidase activity compared to the unmodified PH20 polypeptide not containing the amino acid replacement(s);
the amino acid replacement(s) confer(s) the increased hyaluronidase activity;
an amino acid replacement that confers the increased hyaluronidase activity is at a position corresponding to residue 309, with reference to amino acid positions of SEQ ID NO:3;
corresponding amino acid positions are identified by alignment of the PH20 polypeptide with the polypeptide having the sequence of amino acids of SEQ ID NO:3; and
the modified PH20 polypeptide has at least 91% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7 and 32-66.
2. 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 NOs: 3, 7, and 32-66.
3. The modified PH20 polypeptide of claim 2 that is a soluble PH20 polypeptide.
4. The modified PH20 polypeptide of claim 2 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 32-66.
5. The modified PH20 polypeptide of claim 1 , wherein the replacement is selected from among L at a position corresponding to position 309; N at a position corresponding to position 309; E at a position corresponding to position 309; G at a position corresponding to position 309; H at a position corresponding to position 309; and M at a position corresponding to position 309.
6. The modified PH20 polypeptide of claim 5 , wherein the replacement at the position corresponding to position 309 is N.
7. The modified PH20 polypeptide of claim 6 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 32-66.
8. The modified PH20 polypeptide of claim 6 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:35.
9. The modified PH20 polypeptide of claim 6 , wherein:
the unmodified PH20 polypeptide consists of the amino acid sequence set forth in SEQ ID NO:32; and
the amino acid sequence of the modified PH20 polypeptide has at least 95% sequence identity to SEQ ID NO:32.
10. The modified PH20 polypeptide of claim 6 , wherein:
the unmodified PH20 polypeptide consists of the amino acid sequence set forth in SEQ ID NO:35; and
the amino acid sequence of the modified PH20 polypeptide has at least 95% sequence identity to SEQ ID NO:32.
11. 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 NOs: 3 and 32-66.
12. The modified PH20 polypeptide of claim 1 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence SEQ ID NO:35.
13. The modified PH20 polypeptide of claim 1 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO:32.
14. The modified PH20 polypeptide of claim 1 , wherein the modified PH20 polypeptide exhibits increased hyaluronidase activity that is at least 120% of the hyaluronidase activity compared to the unmodified PH20 polypeptide not containing the amino acid replacement or replacements.
15. The modified PH20 polypeptide of claim 1 , comprising the amino acid sequence selected from the group consisting of SEQ ID NOs:576-586, or an amino acid sequence that exhibits at least 91% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs:3 and 32-66 and that contains the amino acid replacement L, N, E, G, H, M, Q, R, S, T or V at a position corresponding to residue 309.
16. The modified PH20 polypeptide of claim 1 , comprising the amino acid sequence of SEQ ID NO:581, or an amino acid sequence that has at least 91% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 32-66 and that contains the amino acid replacement N at a position corresponding to residue 309.
17. The modified PH20 polypeptide of claim 1 that further comprises one or more modifications selected from among glycosylation, sialylation, albumination, farnesylation, carboxylation, hydroxylation, and phosphorylation.
18. The modified PH20 polypeptide of claim 1 that further comprises an N-acetylglucosamine moiety linked to each of at least three asparagine (N) residues.
19. The modified PH20 polypeptide of claim 1 that is conjugated to a polymer.
20. The modified PH20 polypeptide of claim 19 , wherein the polymer is dextran or polyethylene glycol (PEG).
21. The modified PH20 polypeptide of claim 1 , further comprising a heterologous signal sequence, wherein the unmodified PH20 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 32-66.
22. A chimeric polypeptide, comprising the modified PH20 polypeptide of claim 1 .
23. A pharmaceutical composition, comprising the modified PH20 polypeptide of claim 1 .
24. The modified PH20 polypeptide of claim 1 , comprising the amino acid sequence of SEQ ID NO:581, or an amino acid sequence that exhibits at least 95% sequence identity to the amino acid sequence of SEQ ID NO:581 and that contains the amino acid replacement N at a position corresponding to residue 309.
25. The modified PH20 polypeptide of claim 1 , further comprising a signal sequence, wherein the unmodified PH20 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 32-66.