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US-12618055-B2 - Hyaluronidase variants with improved stability and pharmaceutical composition comprising the same

US12618055B2US 12618055 B2US12618055 B2US 12618055B2US-12618055-B2

Abstract

Disclosed are novel PH20 variants or fragments thereof with improved thermal stability and enzymatic activity of human hyaluronidase, which is an enzyme that hydrolyzes hyaluronic acid, and more particularly novel PH20 variants or fragments thereof including one or more amino acid residue substitutions in the variant having the amino acid sequence of SEQ ID NO: 3, wherein one or more amino acid residues at the N-terminus and/or the C-terminus are optionally further deleted.

Inventors

  • Soon Jae Park
  • Hye-Shin Chung
  • Seung Joo Lee
  • Kyuwan KIM
  • HYUNG-NAM SONG
  • Sun-Ah You
  • Chang Woo Lee

Assignees

  • ALTEOGEN INC.

Dates

Publication Date
20260505
Application Date
20210125
Priority Date
20200123

Claims (20)

  1. 1 . A PH20 variant, wherein the amino acid sequence of the PH20 variant is SEQ ID NO: 3 with modifications consisting of: (a) an amino acid residue substitution of: Y357W, M358V, M358R, L362A, W342I, or E343V relative to SEQ ID NO: 3; (b) an N-terminus deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, M1 to F38, M1 to R39, or M1 to A40 of SEQ ID NO: 3; and (c) a C-terminus deletion, wherein the C-terminus of the PH20 variant ends with an amino acid residue selected from any one of amino acid residues I465 to S490 of SEQ ID NO: 3, and wherein the PH20 variant exhibits higher enzymatic activity compared to mature, wild-type human PH20 under the same conditions.
  2. 2 . The PH20 variant of claim 1 , wherein the amino acid residue substitution is Y357W, M358V, or M358R relative to SEQ ID NO: 3.
  3. 3 . The PH20 variant of claim 1 , wherein the amino acid residue substitution is L362A.
  4. 4 . The PH20 variant of claim 1 , wherein the amino acid residue substitution is W342I or E343V relative to SEQ ID NO: 3.
  5. 5 . The PH20 variant of claim 1 , wherein the amino acid sequence of the PH20 variant consists of the amino acid sequence of SEQ ID NO: 217, 171, or 207.
  6. 6 . The PH20 variant of claim 1 , wherein the amino acid sequence of the PH20 variant consists of the amino acid sequence of SEQ ID NO: 172.
  7. 7 . The PH20 variant of claim 1 , wherein the amino acid sequence of the PH20 variant consists of the amino acid sequence of SEQ ID NO: 198 or 173.
  8. 8 . A pharmaceutical composition comprising the PH20 variant of claim 1 , and one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions and preservatives.
  9. 9 . The pharmaceutical composition of claim 8 , wherein the pharmaceutical composition is formulated for subcutaneous injection.
  10. 10 . The pharmaceutical composition of claim 8 , wherein the pharmaceutical composition further comprises a protein drug and is formulated for subcutaneous injection.
  11. 11 . The pharmaceutical composition of claim 8 , wherein the pharmaceutical composition further comprises an anti-cancer drug and is formulated for subcutaneous injection.
  12. 12 . A PH20 variant comprising the amino acid sequence of SEQ ID NO: 217, 171, or 207.
  13. 13 . A PH20 variant comprising the amino acid sequence of SEQ ID NO: 172.
  14. 14 . A PH20 variant comprising the amino acid sequence of SEQ ID NO: 198 or 173.
  15. 15 . A pharmaceutical composition comprising the PH20 variant of claim 12 , and one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions and preservatives.
  16. 16 . The pharmaceutical composition of claim 15 , wherein the pharmaceutical composition is formulated for subcutaneous injection.
  17. 17 . The pharmaceutical composition of claim 15 , wherein the pharmaceutical composition further comprises a protein drug for treating a disease and is formulated for subcutaneous injection.
  18. 18 . The pharmaceutical composition of claim 15 , wherein the pharmaceutical composition further comprises an anti-cancer drug and is formulated for subcutaneous injection.
  19. 19 . A pharmaceutical composition comprising the PH20 variant of claim 13 , and one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions and preservatives.
  20. 20 . The pharmaceutical composition of claim 19 , wherein the pharmaceutical composition is formulated for subcutaneous injection.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This is a U.S. national phase under 35 USC § 371 of International Patent Application No. PCT/KR2021/000943 filed Jan. 25, 2021, which in turn claims priority under 35 USC § 119 of Korean Patent Application No. 10-2020-0009046 filed Jan. 23, 2020. The disclosures of all such applications are hereby incorporated herein by reference in their respective entireties, for all purposes. REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “Q303377_SeqListing ST25.txt” created on Aug. 11, 2025 and is 630,784 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to novel human PH20 variants or fragments thereof having increased enzymatic activity and thermal stability compared to human hyaluronidase, which is an enzyme that hydrolyzes hyaluronic acid, and more particularly to PH20 variants or fragments thereof, which include one or more amino acid residue substitutions, deletions and/or insertions in hyaluronidase variants having the amino acid sequence of SEQ ID NO: 3, and optionally in which one or more amino acid residues are deleted from the N-terminus and/or C-terminus, a method for producing the same, and a pharmaceutical composition containing the same. Description of the Related Art The human skin is composed of the epidermis, the dermis, and a subcutaneous fat layer, and there are six types of glycosaminoglycans in the skin. These glycosaminoglycans include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratin sulfate. These glycosaminoglycans are composed of repeating disaccharide sugar units. The number of repeating disaccharide sugar units is different among glycosaminoglycans, but ranges from several hundreds to several thousands. Among the glycosaminoglycans, hyaluronic acid is present in the skin more than half of the amount in the body. Hyaluronic acid is synthesized by hyaluronan synthase present in the cell membrane, is present alone without binding to proteoglycans, and is the only glycosaminoglycan having no sulfate group. Other glycosaminoglycans bind to proteoglycans and have a sulfate group. Hyaluronic acid consists of glucuronic acid and N-acetylglucosamine, alternately linked via β-1,4 and β-1,3 bonds, and is composed of about 5,000 repeating units of these disaccharides. It is known that about one-third (5 g) of hyaluronic acid in the human body is degraded every day. Hyaluronidases are enzymes that degrade hyaluronic acid present in the extracellular matrix. Six hyaluronidase genes are known in humans: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20/SPAM1. Human Hyal1 and Hyal2 are expressed in most tissues. PH20/SPAM1 (hereinafter referred to as PH20) is expressed in the sperm plasma membrane and the acrosomal membrane. However, HyalPS1 is not expressed, because it is a pseudogene. Hyaluronidases are divided, depending on the method by which hyaluronic acid is cleaved, into three types: enzymes (EC 3.2.1.35) that cleave β-1,4 bonds between N-acetylglucosamine and glucuronic acid by the use of H2O; enzymes (EC 3.2.1.36) that cleave β-1,3 bonds between N-acetylglucosamine and glucuronic acid by the use of H2O; and bacterial hyaluronidases (EC 4.2.99.1) that cleave β-1,4 bonds without using H2O. The catalytic amino acids of Hyal1 are D129 and E131, which hydrolyze hyaluronic acid by substrate-assisted catalysis. Hyal1 exhibits optimum activity at an acidic pH of 3 to 4, and has no enzymatic activity at a pH of 4.5 or higher. In contrast to Hyal1, PH20 exhibits activity throughout a wide pH range of 3 to 8. Arming et al. identified that the catalytic amino acids of PH20 are D111 and E113 (Arming et al., 1997). Arming et al. designated Leu as the first amino acid of the PH20, from which a signal peptide or the like is removed, and thus the catalytic amino acids of the PH20 containing the signal peptide correspond to D146 and E148, respectively. Hyaluronidase hydrolyzes hyaluronic acid, thereby reducing the viscosity of hyaluronic acid in the extracellular matrix and increasing the permeability thereof into tissue (skin). The subcutaneous area of the skin has a neutral pH of about 7.0 to 7.5. Thus, among various types of hyaluronidases, PH20 is widely used in clinical practice (Bookbinder et al., 2006). In examples in which PH20 is used in clinical practice, PH20 is used as an eye relaxant and an anesthetic additive in ophthalmic surgery, and is also co-administered with an antibody therapeutic agent which is injected subcutaneously (Bookbinder et al., 2006). In addition, based on the property of hyaluronic acid, which is overexpressed in tumor cells, PH20 is used to hydrolyze hyaluronic acid in the extr