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CN-121991927-A - PH20 polypeptide variants, compositions thereof and related applications

CN121991927ACN 121991927 ACN121991927 ACN 121991927ACN-121991927-A

Abstract

The present application provides PH20 polypeptide variants having deletion mutations and/or substitution mutations relative to PH20 (36-482) molecules, as well as compositions and related uses thereof.

Inventors

  • AN ZHENMING
  • XIONG JIYUAN
  • SUN LIXIA
  • LIU SHENGBO
  • WANG GUIJIANG
  • WEN JIFU
  • FAN JIANMIN
  • WANG LE
  • WU HAI
  • LI DAOYUAN

Assignees

  • 齐鲁制药有限公司

Dates

Publication Date
20260508
Application Date
20260304
Priority Date
20251201

Claims (15)

  1. 1.A variant of a PH20 polypeptide which has been isolated from a human subject, The PH20 polypeptide variant comprises a deletion, insertion and/or substitution relative to the amino acid sequence of the PH20 (36-482) molecule wherein: The PH20 polypeptide variant has one or more deletion mutations selected from the group consisting of a G62-P67 deletion, a G62-F64 deletion, a K63 deletion, a M345-S347 deletion, a R346 deletion, a Q178 deletion, a V177-L179 deletion, a N176-S180 deletion and a Q479-Y482 deletion relative to the PH20 (36-482) molecule; And/or The PH20 polypeptide variant has a substitution mutation with respect to the PH20 (36-482) molecule selected from any of 1) T341 342 343 345 347 349 352 353 355 356 359D and I361T, 2) T341 344 345 347 348 350 353 355 358 359N and I361V, 3) T341 344 345 347 348 350 353 358 359K and I361V, and 4) T341 344 345 347T and M348K; Wherein the amino acid positions are based on the amino acid arrangement shown in SEQ ID NO. 11.
  2. 2. The PH20 polypeptide variant of claim 1, wherein the variant has a G62-P67 deletion and/or a Q479-Y482 deletion relative to the PH20 (36-482) molecule, and a substitution mutation of any one of the following groups: 1) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L Q, L353A, L354I, D355K, N356E, E D and I361T, and 2) T341D, I344N, M345T, S T, M348E, S350D, L353R, D82348 356D, M358W, E359N and I361V.
  3. 3. The variant of a PH20 polypeptide according to claim 1 wherein said variant has a G62-F64 deletion and/or a Q479-Y482 deletion relative to the PH20 (36-482) molecule and substitution mutations of T341S, I344N, M345T, S347T, M E, S350D, L E, L353R, D355Y, M358F, E359K and I361V.
  4. 4. The variant of a PH20 polypeptide according to claim 1 wherein said variant has a K63 deletion and/or a Q479-Y482 deletion relative to the PH20 (36-482) molecule and substitution mutations of T341D, I344N, M345T, S347T, M348E, S350D, L352Q, L R, D355S, N D, M358W, E359N and I361V.
  5. 5. The PH20 polypeptide variant of claim 1, wherein the variant has a deletion of M345-S347, a deletion of R346, and/or a deletion of Q479-Y482 relative to the PH20 (36-482) molecule.
  6. 6. The variant of a PH20 polypeptide according to claim 1, wherein said variant has any one of a Q178 deletion, a V177-L179 deletion and a N176-S180 deletion, and/or a Q479-Y482 deletion, relative to the PH20 (36-482) molecule, and has a substitution mutation of T341S, I344N, M345T, S347T, M K.
  7. 7. The PH20 polypeptide variant of claim 1, wherein the sequence of the PH20 polypeptide variant is set forth in any one of SEQ ID NOs 2-10 and 12.
  8. 8. A nucleic acid encoding the PH20 polypeptide variant of any one of claims 1-7.
  9. 9. A vector comprising the nucleic acid of claim 8.
  10. 10. A host cell comprising the nucleic acid of claim 8 or the vector of claim 9.
  11. 11. A pharmaceutical composition comprising the PH20 polypeptide variant of any one of claims 1-7 and a pharmaceutically acceptable carrier.
  12. 12. A pharmaceutical composition comprising the PH20 polypeptide variant of any one of claims 1-7 and an additional therapeutically active agent.
  13. 13. Use of a PH20 polypeptide variant according to any one of claims 1-7 in the manufacture of a medicament for the treatment of a disease, disorder or condition associated with hyaluronic acid, for the treatment of a disease or condition of hyaluronidase substrate accumulation, or for the delivery of a therapeutic agent or for increasing penetration of a chemotherapeutic agent into a solid tumor.
  14. 14. A method of making a PH20 polypeptide variant comprising culturing the host cell of claim 10.
  15. 15. The method of claim 14, comprising culturing the host cell of claim 10 under culture conditions suitable for secretory expression such that the host cell secretes the PH20 polypeptide variant.

Description

PH20 polypeptide variants, compositions thereof and related applications This patent application claims priority from the chinese invention patent application filed on 1 month 2025, month 12, application number 2025117934863, and is incorporated herein in its entirety. Technical Field The application relates to the technical field of protein engineering. In particular, the application provides PH20 polypeptide variants and compositions thereof and related uses. Background Hyaluronic Acid (HA) is also called Hyaluronic acid or Hyaluronic acid, is a glycosaminoglycan naturally occurring in living bodies, and is formed by beta (1, 4) -N-acetyl-D-glucosamine and beta (1, 3) -D-glucuronic acid, two monosaccharides are connected by beta-1, 3-glycosidic bond to form a disaccharide unit, and the disaccharide unit is connected by beta-1, 4 glycosidic bond to form polysaccharide. Hyaluronic acid is widely present in animal connective tissue, such as joints, cartilage, skin, vitreous, and the like. In addition to hyaluronic acid, human skin contains six kinds of glycosaminoglycans, such as chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, etc., and hyaluronic acid exists alone but is not combined with proteoglycan, and is the only glycosaminoglycan without sulfate groups. Hyaluronidases (Hyaluronidase, HAase) refer to a class of glycosidases capable of degrading hyaluronic acid and part of glycosaminoglycans (Glycosaminoglycan, GAGs) into disaccharides or small-molecule oligosaccharides. Hyaluronidase was first discovered by Duran et al in 1929 to be derived from mammalian testis extracts, and was then referred to as a "diffusion factor". Hyaluronidase has various classification modes according to enzyme source, optimal reaction pH value, enzyme specificity, amino acid sequence homology and the like. Where hyaluronidases are classified into eukaryotic, prokaryotic and viral hyaluronidases depending on the source of the enzyme. The human genome contains 6 hyaluronidases, HYAL-1, HYAL-2, HYAL-3, HYAL-4, HYAL-P1 and PH20 (Spermadhesion molecular, spam 1), with a similarity between 33% and 42%. PH20 is a multifunctional protein located on the membrane of mammalian sperm and on the membrane of lysozyme-derived acrosomes. Such glycosphingomyelin inositol (GPI) anchor proteins promote fertilization by effectively hydrolyzing hyaluronic acid in the extracellular matrix of the cumulus. The PH20 on the sperm surface is effective at neutral PH, whereas the PH20 on the acrosome membrane exhibits hyaluronidase activity at both acidic and neutral PH. Among all naturally occurring mammalian hyaluronidases, the PH20 protein is the most active enzyme under physiological conditions. Hyaluronidases are widely used in a variety of fields. Such as plastic surgery, ophthalmic surgery, medical, oncological treatment, dermatological, gynecological, etc. There are 3 uses approved by the FDA in the united states that (1) enhance anesthetic effects in ophthalmic surgery. (2) additives for subcutaneous perfusate. (3) promote reabsorption of contrast agent upon urography. When subcutaneously injected, PH20 breaks down HA in the extracellular matrix within a few minutes, thereby decreasing the HA barrier in the subcutaneous space, increasing tissue permeability and thus promoting rapid subcutaneous absorption of large doses of solution. Most of the PH20 products on the market today are extracted from animal tissue (e.g. bovine testis hyaluronidase) and, due to their low purity, typically have an enzyme activity of only about 700U/mg and less than 1% of total protein, and contaminants such as proteases and immunoglobulins may cause IgE-mediated allergic reactions. In contrast, recombinant human hyaluronidase is considered to be of high purity and low immunogenicity. It is engineered by human main hyaluronidase PH20 protein, which is naturally expressed in human body and has enzyme activity in neutral and acidic environments. Although research is underway to develop various therapeutic agents in the form of subcutaneous injection using human PH20, human PH20 itself has problems of low expression level and poor stability. The inventor finds that the wild-type human PH20 has the problems of low expression level, poor stability, product breakage, end truncation, easy oxidation and the like, wherein the recombinant human PH20, namely PH20 (36-482), has the characteristic that after being expressed, the C terminal end of the recombinant human PH20 is easy to generate differently formed truncated variants, and the recombinant human PH20 is specifically expressed as one or more truncated variant forms of 'QIFY truncated', 'IFY truncated', 'FY truncated' and 'Y truncated'. Although the four truncated forms have been demonstrated to have no effect on the enzymatic activity of the product, their variability during the culture process leads to fluctuations in the ratio of the truncated forms between different culture batches, wh