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 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 among amino acid replacements, deletions, and/or insertions;
the modified PH20 polypeptide comprises a modification at a position corresponding to position 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 amino acid sequence 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 NO: 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 NO: 3, 7 and 32-66.
3. The modified PH20 polypeptide of claim 1 that has increased resistance or stability in denaturing conditions compared to an unmodified PH20 polypeptide that does not contain the amino acid modification(s).
4. The modified PH20 polypeptide of claim 2 that is a soluble PH20 polypeptide.
5. The modified PH20 polypeptide of claim 1 , wherein the modification at the position corresponding to position 309 is selected from among replacement with Lat 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 4 that has increased hyaluronidase activity compared to an unmodified PH20 polypeptide that does not contain the amino acid modification(s).
7. The modified PH20 polypeptide of claim 5 , wherein the modification at the position corresponding to position 309 is replacement with N.
8. 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.
9. 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 NO: 3 and 32-66.
10. 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.
11. The modified PH20 polypeptide of claim 1 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO: 35.
12. The modified PH20 polypeptide of claim 1 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO: 32.
13. The modified PH20 polypeptide of claim 7 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO: 35.
14. The modified PH20 polypeptide of claim 7 , wherein the unmodified PH20 polypeptide consists of the amino acid sequence of SEQ ID NO: 32.
15. The modified PH20 polypeptide of claim 1 , comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 3 and 32-66 and that contains the amino acid replacement L, N, E, G, H, M, Q, R, S, T or V at the position corresponding to position 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 exhibits at least 91% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 3 and 32-66 and that contains the amino acid replacement N at the position corresponding to position 309.
17. The modified PH20 polypeptide of claim 1 that comprises one or more post-translational modifications of the polypeptide selected from among glycosylation, sialylation, albumination, famesylation, carboxylation, hydroxylation, and phosphorylation.
18. The modified PH20 polypeptide of claim 1 that is glycosylated.
19. 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.
20. The modified PH20 polypeptide of claim 1 that is conjugated to a polymer.
21. The modified PH20 polypeptide of claim 20 , wherein the polymer is dextran or polyethylene glycol (PEG).
22. 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.
23. A chimeric polypeptide, comprising the modified PH20 polypeptide of claim 6 .
24. A pharmaceutical composition, comprising the modified PH20 polypeptide of claim 1 .
25. The modified PH20 polypeptide of claim 5 , comprising the amino acid sequence of SEQ ID NO: 581, or an amino acid sequence that exhibits at least 91% sequence identity to the amino acid sequence of SEQ ID NO: 581 and that contains the amino acid replacement N at the position corresponding to position 309.
26. The modified PH20 polypeptide of claim 5 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.
27. The modified PH20 polypeptide of claim 5 , 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.
28. 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, 7 and 32-66.
29. A pharmaceutical composition, consisting of the modified PH20 polypeptide of claim 1 .
30. The pharmaceutical composition of claim 29 , further comprising a therapeutically active agent formulated in the composition or in a separate composition.
31. The pharmaceutical composition of claim 30 , wherein the therapeutically active agent is a polypeptide, a protein, a nucleic acid, a drug, a small molecule, or an organic molecule.
32. The pharmaceutical composition of claim 30 , wherein the therapeutically active agent is selected from among 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, an 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.
33. The pharmaceutical composition of claim 30 , wherein the therapeutically active agent is an antibody.
34. A method for treating a hyaluronan-associated disease or condition, comprising administering to a subject a modified PH20 polypeptide of claim 1 .
35. The method of claim 34 , wherein the hyaluronan-associated disease or condition is an inflammatory disease or a tumor or cancer.
36. The method of claim 35 , wherein hyaluronan-associated disease or condition is a solid tumor.
37. A method for increasing delivery of a therapeutic agent to a subject, comprising:
administering a modified PH20 polypeptide of claim 1 to the subject; and administering a therapeutic agent, wherein the modified PH20 polypeptide and
the therapeutic agent are administered in separate compositions or in the same composition.
38. The method of claim 37 , wherein administration of the therapeutic agent and PH20 polypeptide is by subcutaneous administration.
39. The method of claim 37 , wherein:
the modified PH20 polypeptide and the therapeutic agent are administered in separate compositions; and
the modified PH20 polypeptide is administered before the therapeutic agent.
40. The method of claim 37 , wherein the therapeutic agent is an antibody.