Novel hyaluronic acid-hydrolyzing enzyme variant having improved stability and pharmaceutical composition comprising same

Abstract

The present application relates to a novel hyaluronase variant having improved stability and a pharmaceutical composition containing the same. The present application discloses novel PH20 variants or fragments thereof, human hyaluronidase having improved thermal stability and enzymatic activity wherein the hyaluronidase is an enzyme that hydrolyzes hyaluronic acid, in particular to PH20 variants or fragments thereof comprising one or more amino acid residue substitutions in a variant having the amino acid sequence of SEQ ID NO: 3, wherein one or more amino acid residues are selectively deleted at the N-terminus and / or C-terminus.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511402467.3 (22) Application Date 2021.01.25 (30) Priority Data 10-2020-0009046 2020.01.23 KR (62) Divisional Application Data 202180003323.4 2021.01.25 (71) Applicant Artergen Company Address Daejeon, South Korea (72) Inventors Park Soon-jae, Jung Hye-shin, Lee Seung-joo, Kim Kyu-wan, Song Hyeong-nam, Yoo Seon-ah, Lee Chang-woo (74) Patent Agency Beijing Anxin Fangda Intellectual Property Agency Co., Ltd. 11262 Patent Attorneys Luo Qun, Zhang Kuiyan (51) Int.Cl. C12N 9 / 26 (2006.01) (54) Title of Invention Novel Hyaluronidase Variants with Improved Stability and Pharmaceutical Compositions Containing the Thereof (57) Abstract This application relates to novel hyaluronidase variants with improved stability and pharmaceutical compositions containing the thereof. This application discloses novel PH20 variants or fragments thereof, human hyaluronidases with improved thermal stability and enzymatic activity, wherein the hyaluronidase is an enzyme that hydrolyzes hyaluronic acid, particularly relating to PH20 variants or fragments thereof containing one or more amino acid residue substitutions in variants having the amino acid sequence of SEQ ID NO:3, wherein one or more amino acid residues are selectively deleted at the N-terminus and / or C-terminus. Claims 1 page Description 87 pages Sequence Listing (electronic publication) Drawings 6 pages CN 121450619 A 2026.02.03 CN 1 21 45 06 19 A 1. A PH20 variant or fragment thereof, which contains amino acid residue substitution, deletion or insertion at at least one position in a variant containing the amino acid sequence of SEQ ID NO:3, and which has an aggregation temperature higher than that of wild-type PH20. 2. The PH20 variant or fragment of claim 1, wherein the PH20 variant or fragment thereof comprises a substitution of the amino acid residue at at least one position selected from R39, D65 to L68, N82, T84, I102 to I105, T132 to Y134, N166, L179 to T182, T185 to K187, V241 to K244, N266 to Q269, P271, V272, K290 to P292, Q311 to K314, G340 to N363, L441, S442, D451 to D453, D461, V463, and D461 to V463 in the variant having the amino acid sequence SEQ ID NO: 3. 3. The PH20 variant or fragment of claim 2, wherein the amino acid residue substitution comprises a substitution of the amino acid residue selected from R39, D65 to L68, N82, T84, I102 to I105, T132 to Y134, N166, L179 to T182, T185 to K187, V241 to K244, N266 to Q269, P271, V272, K290 to P292, Q311 to K314, G340 to N363, L441, S442, D451 to D453, D461, V463, and D461 to V463.R39K, D65A, E66A, P67A, L68A, N82A, T84N, I102A, D103A, S104A, S104N, I105A, I105Q, T132A, T132S, F133A, Y134A, N166A, N166K, L179A, L179S, L179I, L179F, S180T, S180A L181A, L181M, T182A, T185A, E186A, E186D, K187A, V241A, E242A, I243A, K244A, N266A, T267A, Q268A, Q268D, Q268I, Q268N, Q269A, P271A, V272A, K290A, I291A, I291G, I291L P292A, P292D, Q311A, V312A, L313A, L313P, L313M, K314A, G340Q, S341H, S341D, S341T, W342I, W342D, W342H, W342L, E343V, E343S, E343Y, E343Q, N344F, N344I, T345E, T345K T345S, R346M, R346F, R346L, R346T, R346S, R346A, T347Q, T347E, T347V, T347W, T347H, T347S, K348Q, K348F, K348D, K348T, K348E, K348M, E349L, E349W, E349A, S350Q, S350I S350D, S350T, S350E, S350N, Q352E, Q352G, Q352Y, Q352W, Q352T, A353E, A353Y, A353H, A353K, I354E, I354Q, I354S, I354V, I354A, I354N, I354T, I354R, I354W, I354L, K355Q K355H, K355D, E356M, E356F, E356I, E356L, E356Q, E356V, E356D, Y357W, Y357F, M358V, M358R, M358Y, M358L, D359K, D359V, D359Y, D359Q, D359T, D359S, D359E, T360Y, T360R T360L, T360D, T360S, T361M, T361E, T361H, T361L, T361D, T361I, L362A, N363M, N363E4. At least one of L441A, S442A, D451A, D451S, T452A, T452D, T452H, T452K, T452G, T452P, T452M, T452F, D453A, D461R, D461A, G462A, V463Y, and V463A. 5. The PH20 variant or fragment of any one of claims 1 to 3, further comprising the deletion of at least one amino acid residue at the C-terminus and / or N-terminus. 6. The PH20 variant or fragment of claim 4, wherein the at least one amino acid residue is deleted by cleaving from the N-terminus before an amino acid residue selected from M1 to P42. 7. The PH20 variant or fragment of claim 5, wherein the at least one amino acid residue is deleted by cleaving from the N-terminus before an amino acid residue of L36, N37, F38, R39, A40, P41, or P42. 7. The PH20 variant or fragment of claim 4, wherein the at least one amino acid residue is deleted by C-terminal cleavage following an amino acid residue selected from V455 to L509. 8. The PH20 variant or fragment of claim 7, wherein the at least one amino acid residue is deleted by C-terminal cleavage following an amino acid residue selected from V455 to S490. 9. The PH20 variant or fragment of claim 7, wherein the at least one amino acid residue is deleted by C-terminal cleavage following an amino acid residue of V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490. 10. The PH20 variant or fragment thereof as claimed in claim 4, wherein the at least one amino acid residue is deleted by cleavage from the N-terminus before the amino acid residue of F38 and from the C-terminus after the amino acid residue of F468. Claims 1 / 1 Page 2 CN 121450619 A Novel Hyaluronidase Variant with Improved Stability and Pharmaceutical Composition Containing the Thereof Related Application

[0001] This application is a divisional application of Chinese Patent Application No. 202180003323.4, filed on November 11, 2021, entitled "Novel Hyaluronidase Variant with Improved Stability and Pharmaceutical Composition Containing the Thereof". Technical Field

[0002] The present invention relates to a novel human PH20 variant or fragment thereof, which has increased enzymatic activity and thermal stability compared to human hyaluronidase, wherein the hyaluronidase is a hydrolytic agent for hyaluronic acid.Enzymes containing hyaluronic acid. This invention relates particularly to a PH20 variant or fragment thereof comprising one or more amino acid residues substituted, deleted, and / or inserted in a hyaluronidase variant having the amino acid sequence SEQ ID NO: 3, wherein one or more amino acid residues are selectively deleted from the N-terminus and / or C-terminus, a method for manufacturing said PH20 variant or fragment thereof, and a pharmaceutical composition comprising said PH20 variant or fragment thereof. Background Art

[0003] Human skin is composed of the epidermis, dermis, and subcutaneous fat layer, and contains six glycosaminoglycans. These glycosaminoglycans include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratin sulfate.

[0004] These glycosaminoglycans are composed of repeating disaccharide sugar units. The number of repeating disaccharide units in each glycosaminoglycan varies, ranging from hundreds to thousands. Among these glycosaminoglycans, hyaluronic acid accounts for more than half of the human body's composition in the skin. Hyaluronic acid is synthesized by hyaluronic acid synthase in cell membranes, exists independently without binding to proteoglycans, and is the only glycosaminoglycan that does not possess a sulfate group. Other glycosaminoglycans are bound to proteoglycans and possess sulfate groups. Hyaluronic acid is composed of glucuronic acid and N-acetylglucosamine linked by alternating β-1,4 and β-1,3 chains, and consists of 5000 repeating units of these disaccharides. It is known that approximately one-third (5 g) of hyaluronic acid in the human body is degraded daily.

[0005] Hyaluronidase is an enzyme that degrades hyaluronic acid in the extracellular matrix. Six known human hyaluronidase genes are: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20 / SPAM1. The human Hyal1 and Hyal2 genes are expressed in most tissues. PH20 / SPAM1 (hereinafter referred to as PH20) is expressed in the sperm plasma membrane and acrosomal membrane. However, HyalPS1 is a pseudogene.(pseudogene) was not expressed. Depending on the hyaluronic acid cleavage method, hyaluronidases are classified into three categories: enzymes that use water molecules (H2O) to cleave the β-1,4 chain between N-acetylglucosamine and glucuronic acid (EC 3.2.1.35); enzymes that use water molecules to cleave the β-1,3 chain between N-acetylglucosamine and glucuronic acid (EC 3.2.1.36); and bacterial hyaluronidases that do not use water molecules to cleave the β-1,4 chain (EC 4.2.99.1).

[0006] The catalytic amino acids of Hyal1 are D129 and E131, which hydrolyze hyaluronic acid through substrate-assisted catalysis. Hyal1 expresses optimal activity at acidic pH values ​​of 3 to 4, and is inactive at pH values ​​of 4.5 or above. Compared to Hyal1, PH20 exhibits activity across a wide pH range of 3 to 8.

[0007] Arming et al. identified the catalytic amino acids of PH20 as D111 and E113 (Arming et al., 1997). Arming et al. designated Leu as the first amino acid of PH20, in which signal peptides and the like are removed, and thus the catalytic amino acids of PH20 containing signal peptides correspond to D146 and E148, respectively.

[0008] Hyaluronidase hydrolyzes hyaluronic acid, thereby reducing the viscosity of hyaluronic acid in the extracellular matrix and increasing its permeability into tissues (skin). The subcutaneous region of the skin has a neutral pH of approximately 7.0 to 7.5. Therefore, among the various types of hyaluronidase, PH20 is widely used clinically (Bookbinder et al., 2006). In clinical applications, PH20 is used as an ocular relaxant and anesthetic additive in ophthalmic surgery, and is also co-administered with subcutaneously injected antibody therapeutics (Bookbinder et al., 2006). Furthermore, based on the characteristic of hyaluronic acid overexpression in tumors, PH20 is used to hydrolyze hyaluronic acid in the extracellular matrix of tumor cells, thereby increasing the chance of antitumor therapeutics entering tumor cells. It is also used to promote the absorption of excess body fluids and blood in human tissues.

[0009] PH20 was first identified in guinea pig semen by Lathrop et al., and is also known to be expressed in sperm from different species. The human PH20 gene was cloned by Lin et al. and Gmachl et al. The human PH20 has the amino acid sequence SEQ ID NO: 1, consisting of 509 amino acid residues, and exhibits identity with 60% of the guinea pig PH20 gene.(identity). The human PH20 enzyme system is encoded by the SPAM1 (sperm adhesion molecule-1) gene, and PH20 Ser490 exists on the sperm cell membrane surface and in the acrosomal membrane in a form bound to glycosylphosphatidylinositol (GPI). When sperm penetrates the ovum through the hyaluronic acid-rich cumulus layer, sperm use PH20 to hydrolyze hyaluronic acid. The content of PH20 is equivalent to 1% or less of the protein content in sperm and has six N-glycosylation sites (N82, N166, N235, N254, N368, and N393).

[0010] Currently commercially available PH20 is obtained by extraction from the testes of cattle or sheep. Examples include Amphadase® (bovine hyaluronidase) and Vitrase® (sheep hyaluronidase).

[0011] Bovine testicular hyaluronidase (BTH) is obtained by removing the signal peptide and 56 amino acids from the C-terminus of wild-type bovine PH20 during post-translational modification. BTH is also a glycoprotein and, based on its total composition including amino acids, contains 5% mannose and 2.2% glucosamine. When animal-derived hyaluronidase is repeatedly administered to humans in high doses, neutralizing antibodies can be produced. Because animal-derived hyaluronidase contains other biological materials besides PH20, it may cause allergic reactions when administered to humans (Bookbinder et al., 2006). In particular, the manufacture and use of PH20 extracted from cattle may be limited due to concerns about mad cow disease. To overcome this problem, much research has been conducted on recombinant human PH20 proteins.

[0012] Recombinant human PH20 proteins have been found to be expressed in yeast (P. pastoris), DS-2 insect cells, and animal cells. The N-glycosylation morphology of PH20 recombinant proteins manufactured in insect cells and yeast differs from that of human PH20 during post-translational modification.

[0013] Hyaluronidases, whose protein structures have been identified as Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidase (PDB ID: 1FCQ, 1FCU, 1FCV). Hyal1 consists of two domains: a catalytic domain and an epidermal growth factor-like domain (EGF-like).The catalytic domain is in the form of (β / α)8, in which the α-helix and beta-strand, which characterize the secondary structure of the protein, are each repeated eight times (Chao et al., 2007). In variants of Hyal1 with different splicing methods at the C-terminus, the epidermal growth factor-like domain is completely retained. The amino acid sequences of Hyal1 and PH20 (page 2 / 87, CN 121450619 A) share 35.1% identity, and the protein structure of PH20 has not yet been discovered.

[0014] The recombinant protein of human PH20 was developed by Halozyme Therapeutic, Inc. and has been sold under the trade name Hylenex® (Bookbinder et al., 2006; Frost, 2007).

[0015] When the catalytic amino acids D146 and E148 of PH20 are mutated to asparagine (D146N) and glutamine (E148Q), respectively, no enzyme activity is observed (Arming et al., 1997). Furthermore, when R246 of PH20 is replaced by glycine, enzyme activity decreases by 90%, and when E319 is replaced by glycine, enzyme activity disappears. Compared to wild-type PH20, the PH20 variant with 36 amino acids removed from the C-terminus (truncated amino acids 474-509) exhibits a 75% reduction in enzyme activity. This mutant is not secreted extracellularly but resides in HeLa cells. The PH20 mutant with 134 amino acids removed from the C-terminus lacks enzyme activity and is not secreted extracellularly. According to Frost et al., the C-terminal region 477-483 of PH20 is essential for soluble expression (Frost, 2007). The activity of the full-length PH20 (1-509) or the PH20 variant truncated at position 467 at the C-terminus is only 10% of that of the PH20 variant truncated at one of the C-terminal positions 477-483 (Frost, 2007).

[0016] Recombinant PH20 is used in medicine as a carrier to facilitate the subcutaneous delivery of drugs, to reduce intraocular pressure in patients with ophthalmic diseases, to delay stenosis after surgery, as a dispersant to enhance the activity of chemotherapeutic drugs in diseases such as cancer, and as an adjuvant in surgery, etc.

[0017] Especially in the case of protein drugs, high-dose products with concentrations ranging from tens to hundreds of milligrams per milliliter (mL) have recently been developed, and therefore the application of recombinant PH20 as a carrier to facilitate subcutaneous delivery of protein drugs has also increased. Protein drugs may haveDue to its high concentration, recombinant PH20 exhibits increased viscosity and protein aggregation, leading to low physical stability. Furthermore, protein aggregation is irreversible, and small amounts of protein begin to aggregate, forming larger clumps (Schön et al., 2015). That is, co-administered recombinant PH20 aggregates, thereby reducing the stability of the protein drug.

[0018] Meanwhile, conventional recombinant PH20 remains insufficient in terms of thermal stability and expression levels. Therefore, there is a significant industrial demand for recombinant hyaluronidase with further improved biological and physicochemical properties. Reference Arming, S., Strobl, B., Wechselberger, C., and Kreil, G. (1997). In vitro mutagenesis of PH-20 hyaluronidase from human sperm. Eur. J. Biochem. 247, 810-814. Bookbinder, L.H., Hofer, A., Haller, M .F., Zepeda, M.L., Keller, G.A., Lim, J.E., Edgington, T.S., Shepard, H.M., Patton, J.S., and Frost, GI (2006). A recombinant human enzyme for enhanced interstitial transport of therapeutics. J. Control. Release 114, 230-241. Chao, K.L., Muthukumar, L., and Herzberg, O. (2007). Structure of human hyaluronidase‑1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesis. Biochemistry 46, 6911‑6920. Frost, G.I. (2007). Recombinant human Hyaluronidase (rHuPH20): an enabling platform forSubcutaneous drug and fluid administration. Expert Opin. Drug Deliv. 4, 427-440. Schön, A., Clarkson, BR, Siles, R., Ross, P., Brown, RK, Freire, E. (2015) Denatured state aggregation parameters derived from concentration. Specification 3 / 87 pages 5 CN 121450619 A dependence of protein stability. Anal. Chem. 488, 45-50 WO 2020 / 022791A (2020. 1. 30.) Summary of the Invention

[0020] Therefore, the present invention has been made in view of the above problems. One object of the present invention is to provide a PH20 variant or a fragment thereof, which has improved thermal stability, enzyme activity and expression level compared to wild-type PH20, especially mature wild-type PH20.

[0021] Another object of the present invention is to provide a composition comprising a PH20 variant or a fragment thereof.

[0022] According to one aspect of the invention, the above and other objectives can be achieved by providing a PH20 variant or a fragment thereof, wherein the PH20 variant or the fragment thereof comprises one or more amino acid residue substitutions, deletions and / or insertions in a hyaluronidase variant having the amino acid sequence SEQ ID NO: 3, wherein one or more amino acid residues are selectively deleted at the N-terminus or C-terminus.

[0023] According to another aspect of the invention, a composition for treating cancer and a method of using the same for treating cancer are provided, wherein the composition comprises a PH20 variant or a fragment thereof.

[0024] Efficacy of the Invention When expressed in CHO (ExpiCHO) cells, the PH20 variant or the fragment thereof according to the invention has an enhanced level of protein expression and an increased protein aggregation temperature of about 4 to 11.5°C, thus the PH20 variant or the fragment thereof according to the invention is efficiently produced and has higher thermal stability than mature wild-type PH20.

[0025] Furthermore, as shown by the substrate-gel assay, one of several tests for measuring hyaluronidase activity, the PH20 variant or fragment according to the invention has an effect on improving protein refolding, and therefore re-natures faster than mature wild-type PH20, regardless of the C-terminus.The cleavage site is reached, and the original enzyme activity is still preserved.

[0026] Furthermore, the PH20 variant or fragment thereof according to the present invention has low immunogenicity, which allows it to be repeatedly applied to the human body.

[0027] The above and other objects, features and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Figure 1 shows the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of various variants of the PH20 variant having the amino acid sequence of SEQ ID NO: 3. The following SDS-PAGE analysis results for each variant were obtained by purifying the animal cell culture medium expressing each variant by column chromatography and performing 10% SDS-PAGE analysis on the finally purified variants; More specifically, Figure 1A shows the SDS-PAGE results for variants HM98, HM99, HM130, HM143, HM71, HM100, HM131, HM72, HM101 and HM114; Figure 1B shows the SDS-PAGE results for variants HM63, HM102, HM115, HM64, HM103, HM116, HM125, HM132, HM65, HM133, HM144, HM104, and HM117; Figure 1C shows the SDS-PAGE results for variants HM66, HM105, HM134, HM76, HM106, HM135, HM136, and HM67; Figure 1D shows the SDS-PAGE results for variants HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM107, HM118, and HM90. (Instruction manual, page 4 / 87, 6 CN 121450619 A) The SDS-PAGE results for HM91, HM92, HM93, HM94, and HM95; Figure 1E shows the SDS-PAGE results for variants HM73, HM111, HM121, HM139, HM74, HM112, and HM140; Figure 1F shows the SDS-PAGE results for variants HM75, HM141, HM145, HM70, HM77, HM142, HM78, HM79, HM96, HM146, HM147, HM149, and HM150; Figure 2 shows the expression levels of mature wild-type PH20, Hyal2 variant, Hyal3 variant, and Hyal4 variant, where regions M345 to I361 of wild-type PH20 in Hyal2, Hyal3, and Hyal4 variants were replaced with Hyal2, Hyal3, and Hyal4 variants, respectively.The corresponding sequences of Hyal3 and Hyal4 are shown in Figure 3. Row CS of the SDS-PAGE sample represents the culture medium, row FT represents unbound impurities from the HisTag column, and row E represents the precipitate from the HisTag column. Figure 3 shows the results of SDS-PAGE analysis of various variants based on the PH20 variant with the amino acid sequence SEQ ID NO: 3. The following SDS-PAGE analysis results for each variant were obtained by purifying the animal cell culture medium expressing each variant using column chromatography and performing 10% SDS-PAGE analysis on the finally purified variants. Figure 3A shows the results for variants HM152, HM153, HM154, HM155, HM156, HM157, HM158, and HM159. Figure 3B shows the SDS-PAGE results for variants HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168, and HM169; Figure 3C shows the SDS-PAGE results for variants HM170, HM171, HM172, HM173, HM174, HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185, and HM186; Figure 3C shows the SDS-PAGE results for variants HM190, HM191, HM192, HM193, HM194, HM195, HM196, and HM197. Figure 3D shows the SDS-PAGE results for variants HM198, HM199, HM203, HM204, and HM205; Figure 3E shows the SDS-PAGE results for variants HM231, HM232, HM233, HM234, HM235, HM243, HM245, and HM246; Figure 3F shows the SDS-PAGE results for variants HM254, HM261, HM262, HM263, HM266, HM268, HM271, and HM275. SDS-PAGE results for HM276, HM279, HM280, HM287, and HM288; and Figure 4 shows the SDS-PAGE results confirming the thermostability of wild-type PH20 (L36-Y482) and the PH20 variants (F38-F468) with the amino acid sequence SEQ ID NO: 3, where rows A, B, C, and D show the results of the following SDS-PAGE analyses: regarding the initial wild-type PH20 (rows A and C) and in the reduced form.The images show the results of SDS-PAGE analysis of the following samples after storage at 42°C for seven days: the initial wild-type PH20 (lines E and G) and the PH20 variant of SEQ ID NO: 3 in the reduced (lines E and F) and non-reduced (lines G and H) forms.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. Generally, the names used herein are those known and commonly used in the art.

[0029] In this invention, when described as based on wild-type PH20, the positions of the amino acid residues of each variant are described with reference to the amino acid sequence of SEQ ID NO: 1, and when described as based on the PH20 variant having SEQ ID NO: 3, the positions of the amino acid residues of each variant are described with reference to the amino acid sequence of SEQ ID NO: 3.

[0030] The inventors of this case discovered a variant of hyaluronidase PH20 through prior research and filed a patent application (see WO 2020 / 022791A) on page 5 / 87 of the specification regarding this discovery. This variant of hyaluronidase PH20 contains one or more amino acid residues substituted in the region corresponding to the α-helix domain and / or its linker domain (preferably the α-helix 8 domain (S347 to C381) and / or the linker domain between α-helix 7 and α-helix 8 (A333 to R346)) of wild-type PH20 (preferably mature wild-type PH20) having the amino acid sequence of SEQ ID NO: 1. Optionally, one or more N-terminal and / or C-terminal amino acid residues are selectively cleaved or deleted. This variant of hyaluronidase PH20 exhibits superior efficacy compared to conventional wild-type PH20 or fragments thereof.

[0031] As used herein, the term “mature wild-type PH20” refers to a protein consisting of amino acid residues L36 to Y482 or L36 to S490 of SEQ ID NO: 1, wherein the amino acid residues lack M1 to T35, which form the signal peptide in the amino acid sequence of SEQ ID NO: 1 of wild-type PH20, and one of N483 to L509 or A491 to L509, which are not related to the actual enzymatic function of PH20.

[0032] Specifically, the inventors of this invention have found through prior research that when the amino acid positions corresponding to T341 to I361 (which are α-helix 8 domains (S347 to C381) and / or α-helix 7) are present in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, the signal peptide is formed.When the linker domain (A333 to R346) between the α-helix and α-helix 8 is replaced with amino acid residues of wild-type Hyal1 corresponding to the sequence of SEQ ID NO: 2, the expression efficiency and enzyme activity are improved, and the fragments with partial amino acid deletions at the N-terminus and C-terminus also show superior expression efficiency and high enzyme activity.

[0033] Table 1: Amino acid sequences of wild-type PH20 and wild-type Hyal1

[0034] As a result of their continued research, the inventors of this invention discovered that although variants of the sequence having SEQ ID NO: 3 contain additional substitutions, deletions, and / or insertions of amino acid residues, and more optionally, deletions of one or more amino acid residues at the N-terminus and / or C-terminus, variants of the sequence having SEQ ID NO: 3 still exhibit superior expression efficiency, high enzyme activity, and significantly improved protein aggregation temperature (Tagg) compared to wild-type PH20. The variants of the sequence having SEQ ID NO: 3 are constructed by substituting the amino acid regions T341 to I361 of wild-type PH20 corresponding to the amino acid sequence having SEQ ID NO: 1 with the amino acid sequence corresponding to wild-type Hyal1 having SEQ ID NO: 2. Based on this discovery, the present invention was completed.

[0035] A variant having the sequence of SEQ ID NO: 3 is constructed by substituting 15 amino acid residues, as described on page 6 / 87 of the specification, 8 CN 121450619 A, namely, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T in the wild-type PH20 having the amino acid sequence of SEQ ID NO: 1.

[0036] In this respect, the PH20 variant or fragment thereof according to the invention includes substitution, deletion, and / or insertion of one or more amino acid residues in the PH20 variant having the amino acid sequence of SEQ ID NO: 3, and optionally includes deletion of one or more amino acid residues at the N-terminus and / or C-terminus.

[0037] As described above, variants having the amino acid sequence SEQ ID NO: 3 are those in which the amino acid residues T341 to I361 of wild-type PH20 are replaced by the corresponding amino acid residues of wild-type Hyal1 (see Table 2). In previous studies, variants or fragments thereof having the amino acid sequence SEQ ID NO: 3 with amino acid residue deletions at the N-terminus and C-terminus were considered superior to variants of wild-type PH20 in terms of activity and stability.

[0038] Table 2: Amino acid sequences of PH20 variants with amino acid residues at positions T341 to I361 of wild-type PH20 replaced by amino acid residues corresponding to Hy11 (SEQ ID NO: 3)

[0039] Specifically, the PH20 variants or fragments thereof according to the present invention may contain one or more mutations, preferably substitutions, deletions and / or insertions of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 3, and have a higher protein aggregation temperature (Tagg) than wild-type PH20, wherein protein aggregation temperature is an indicator of protein stability. In addition, the PH20 variants according to the present invention do not contain wild-type PH20 of SEQ ID NO: 1.

[0040] The term “PH20 variant” as used herein is intended to include a variant that not only has mutations of one or more amino acid residues (preferably substitutions, deletions and / or insertions of one or more amino acid residues), but also deletions and substitutions, deletions and / or insertions of one or more amino acid residues at its N-terminus or C-terminus, and its use is substantially the same as the meaning of “PH20 variant or fragments thereof”.

[0041] Preferably, the PH20 variant according to the invention comprises amino acid residues selected from at least one of the following positions in a variant having the amino acid sequence of SEQ ID NO: 3: R39, D65 to L68, N82, T84, I102 to I105, T132 to Y134, N166, L179 to T182, T185 to K187, V241 to K244, N266 to Q269, P271, V272, K290 to P292, Q311 to K314, G340 to N363, L441, S442, D451 to D453, D461, V463, and D461 to V463, comprising substitution, deletion, and / or insertion of amino acid residues, and having a higher protein aggregation temperature (Tagg) than wild-type PH20.

[0042] The PH20 variant according to the present invention may contain mutations at 20 or fewer (preferably 17 or fewer, more ideally 15 or fewer) amino acid positions in the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0043] Preferably, the PH20 variant or fragment thereof according to the present invention is included in a variant having the amino acid sequence of SEQ ID NO: 3, selected from R39K, D65A, E66A, P67A, L68A, N82A, T84N, I102A, D103A, S104A, S104N, I105A, I105Q, T132A, T132S, F133A, Y134A, N166A, N166K, L179A, L179S, L179I, L179F, S180T,S180A, L181A, L181M, T182A, T185A, E186A, E186D, K187A, V241A, E242A, I243A, K244A, N266A, T267A, Q268A, Q268D, Q268I, Q268N, Q269A, P271A, V272A, K290A, I291A, I291G, I291L, P292A, P292D, Q311A, V312A, L313A, L313P, L313M, K314A, G340Q, S341H, S341D, Instruction Manual 7 / 87 pages 9 CN 121450619 A S341T, W342I, W342D, W342H, W342L, E343V, E343S, E343Y, E343Q, N344F, N344I, T345E, T345K, T345S, R346M, R346F, R346L, R346T, R346S, R346A, T347Q, T347E, T347V, T347W, T347H, T347S, K348Q, K348F, K348D, K348T, K348E, K348M, E349L, E349W, E349A, S350Q, S350I, S350D, S350T, S350E, S350N, Q352E, Q352G, Q352Y, Q352W, Q352T, A353E, A353Y, A353H, A353K, I354E, I354Q, I354S, I354V, I354A, I354N, I354T, I354R, I354W, I354L, K355Q, K355H, K355D, E356M, E356F, E356I, E356L, E356Q, E356V, E356D, Y357W, Y357F, M358V, M358R, M358Y, M358L, D359K, D359V, D359Y, D359Q, D359T, D359S, D359E, T360Y, T360R, T360L, T360D, T360S, T361M, T361E, T361H, T361L, T361D, T361I, L362A, N363M, N363E, L441A, S442A, D451A, D451S, T452A, T452D, T452H, T452K, T452G, T452P, T452M, The amino acid residues selected from T452F, D453A, D461R, D461A, G462A, V463Y, and V463A may be substituted, but are not limited thereto.

[0044] In this invention, a description consisting of a letter and number representing an amino acid residue code (e.g., S341) refers to an amino acid residue at each position in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

[0045] For example, “S341” means that the amino acid residue at position 341 in the amino acid sequence of SEQ ID NO: 3 is serine, while “S341H” means that the serine residue at position 341 in SEQ ID NO: 3 is replaced by histidine.

[0046] The PH20 variant or fragment thereof according to the invention is interpreted as comprising a variant or fragment thereof, wherein the amino acid residue at a specific amino acid residue position is conservatively substituted.

[0047] As used herein, the term “conservative substitution” refers to a modification of the PH20 variant that relates to the substitution of one or more amino acids with other amino acids having similar biochemical properties, wherein having similar biochemical properties means not causing loss of biological or biochemical function of the PH20 variant.

[0048] The term “conservative amino acid substitution” refers to the substitution of an amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined and are known in the field to which this invention pertains. This group includes amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids with nonpolar side chains (such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids with β (beta) side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0049] The PH20 variants or fragments thereof of the present invention have been found to retain their activity despite having conserved amino acid substitutions.

[0050] Furthermore, the PH20 variants or fragments thereof according to the present invention are interpreted as comprising PH20 variants and fragments thereof having substantially the same function and / or effect as the PH20 variants or fragments thereof according to the present invention, and having at least 80% or 85% similarity to the PH20 variants or fragments thereof.The amino acid sequence of the PH20 variant or fragment thereof according to the present invention is preferably at least 90% homologous, more preferably at least 95% homologous, and most preferably at least 99% homologous.

[0051] The PH20 variant or fragment thereof according to the present invention has increased expression and protein refolding rate, and thus has higher thermal stability than mature wild-type PH20. Furthermore, despite the increased thermal stability, the enzyme activity of the PH20 variant is still greater than or similar to that of wild-type PH20. Specification 8 / 87 pages 10 CN 121450619 A

[0052] Meanwhile, although mature wild-type PH20 with cleavage at the C-terminus is known to have reduced enzyme activity, the PH20 variant according to the present invention, despite the cleavage and deletion of one or more amino acid residues at the C-terminus and / or 1 to 7 (preferably 1 to 5) amino acid residues at the N-terminus, still exhibits similar or increased enzyme activity and expression efficiency, as well as a high protein aggregation temperature (Tagg) due to a faster protein refolding rate and its thermal stability.

[0053] Accordingly, the PH20 variant or fragment thereof according to the present invention is characterized in that it contains one or more amino acid mutations (preferably one or more amino acid substitutions, deletions and / or insertions in variants having the amino acid sequence of SEQ ID NO: 3, etc.) and one or more amino acid residues at the N-terminus and / or C-terminus are additionally deleted, but not limited thereto.

[0054] In one embodiment, the PH20 variant or fragment thereof according to the invention may be wherein the cleavage occurs before amino acid residues selected from M1 to P42 from the N-terminus, preferably before amino acid residues L36, N37, F38, R39, A40, P41 or P42 at the N-terminus in the amino acid sequence of SEQ ID NO: 3, so that one or more amino acid residues at the N-terminus are deleted, and / or the cleavage occurs after amino acid residues selected from V455 to L509 from the C-terminus, preferably after amino acid residues selected from V455 to S490, more preferably after amino acid residues selected from V455, D456, C458, D461, C464, I465, D466, A467, Following amino acid residues F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490, one or more amino acid residues at the C-terminus are deleted.

[0055] The phrase "the cleavage occurs before the amino acid residues selected from M1 to P42 from the N-terminus" means that a portion of amino acid residues immediately preceding the amino acid residues selected from M1 to P42 at the N-terminus are cleaved and deleted. The phrase "the cleavage occurs before M1" means that no cleavage occurs at the N-terminus.

[0056] For example, the phrase "the cleavage occurs before amino acid residues L36, N37, F38, R39, A40, P41, or P42" means that in the amino acid sequence SEQ ID NO: 3 according to the present invention, all amino acid residues immediately preceding L36 from M1 to T35, all amino acid residues immediately preceding N37 from M1 to L36, all amino acid residues immediately preceding F38 from M1 to N37, all amino acid residues immediately preceding R39 from M1 to F38, all amino acid residues immediately preceding A40 from M1 to R39, all amino acid residues immediately preceding P41 from M1 to A40, or all amino acid residues immediately preceding P42 from M1 to P41 are cleaved and deleted.

[0057] Furthermore, the phrase “the cleavage occurs after the amino acid residues selected from V455 to L509 from the C-terminus” means that a portion of the amino acid residues immediately following the amino acid residues selected from V455 to L509 at the C-terminus are cleaved and deleted.

[0058] For example, the phrase "cleavage occurring after amino acid residues V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490 at the C-terminus" means that in the amino acid sequence SEQ ID NO: 3 according to the present invention, after amino acid residues V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, ... The amino acid residues following P478, I480, Y482, A484, P486, T488, or S490 are cleaved, or all amino acid residues immediately following the selected amino acid residues up to L509 are deleted.

[0059] Preferably, the novel PH20 variant or fragment thereof according to the invention is characterized by comprising substitution, deletion, or insertion of amino acid residues at one or more positions in the variant having the amino acid sequence of SEQ ID NO: 3, truncation before F38 at the N-terminus, and truncation after F468 at the C-terminus.

[0060] More preferably, the novel PH20 variant or fragment thereof according to the invention may comprise an amino acid sequence selected from, but not limited to, the amino acid sequences of SEQ ID NO: 163 to 316.

[0061] The sequences of amino acids substituted or cleaved in the PH20 variant according to specific embodiments of the invention are shown in Table 6.

[0062] Furthermore, in the invention, attempts are made to use other signal peptides that are highly expressed in animal cells to increase the recombinant specification 9 / 87 pages 11 CN.121450619 A PH20 protein expression, instead of using the original PH20 signal peptide.

[0063] Therefore, in another embodiment, the novel PH20 variant according to the invention may be as shown in Table 3 below, wherein the N-terminus further comprises the human growth hormone signal peptide having the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA of SEQ ID NO: 4, the human serum albumin signal peptide having the amino acid sequence MKWVTFISLLFLFSSAYS of SEQ ID NO: 5, or the human Hyal1 signal peptide having the amino acid sequence MAAHLLPICALFLTLLDMAQG of SEQ ID NO: 6, in place of the wild-type PH20 signal peptide consisting of M1 to T35, but is not limited thereto.

[0064] The phrase "in place of the wild-type PH20 signal peptide consisting of M1 to T35" refers to the case where the signal peptide in the amino acid sequence SEQ ID NO: 3 is partially or completely deleted, and therefore its function is not performed. Furthermore, the above description refers to the case where a portion of the N-terminus is further deleted, for example, where a cleavage occurs before residues N37, F38, R39, A40, P41, or P42, causing the additional deletion of the N-terminus to occur together with the deletion of the wild-type PH20 signal peptide.

[0065] Table 3: Signal peptide sequence according to the invention

[0066] On the other hand, the present invention relates to a composition for treating cancer and a method of using said composition to treat cancer, wherein said composition comprises a novel PH20 variant or fragment thereof according to the invention.

[0067] Cancers or carcinomas that can be treated by the novel PH20 variant or fragment thereof according to the invention are not particularly limited, but include solid tumors and hematologic malignancies. The cancer may be selected from skin cancer, such as melanoma, liver cancer, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, bile duct cancer, testicular cancer, rectal cancer, head and neck cancer, ureteral cancer, osteosarcoma, neurocytoma, sarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma, but is not limited thereto. Preferably, the cancers that can be treated by the composition according to the present invention may be selected from colorectal cancer, breast cancer, lung cancer, and kidney cancer, but are not limited thereto.

[0068] The composition of the present invention may be a pharmaceutical composition. The pharmaceutical composition may further comprise a medically acceptable composition. The composition may contain a selection from lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, etc.Acacia), calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, wherein the substances contained in the above compositions are commonly used in the preparation of pharmaceuticals, but are not limited thereto. Furthermore, the pharmaceutical composition may contain a selection from diluents, excipients, lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions, and preservatives, wherein the substances contained in the above compositions are commonly used in the preparation of pharmaceuticals.

[0069] The pharmaceutical composition can be administered orally or non-orally. Non-oral administration methods include intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, local administration, nasal administration, intrapulmonary administration, rectal administration, etc. For oral administration, considering that peptides and proteins will decompose in the stomach, the active ingredients in the oral composition need to be formulated into a coated dosage form or a dosage form that can protect the active ingredients from decomposition in the stomach. Alternatively, the composition described in this specification (pages 10 / 87, CN 121450619 A) can be administered through any device through which the active ingredient can move to the target cells.

[0070] The pharmaceutical composition can be formulated as a liquid, suspension, syrup, or emulsion in an oil or aqueous medium, or as an extract, microparticle, powder, granule, tablet, or capsule, and may additionally contain dispersants or stabilizers for formulation purposes.

[0071] In particular, the composition for treating cancer according to the invention can be used in combination with other anticancer drugs.

[0072] Anticancer drugs that can be used in combination with the novel PH20 variant or fragment thereof according to the invention are preferably chemopreventive drugs, antibody-based anticancer drugs, biological anticancer drugs, RNAi or cell therapy agents, but are not limited thereto.

[0073] Preferably, the anticancer drug that can be used in combination with the novel PH20 variant or fragment thereof according to the invention is preferably an immuno-oncologic agent, and more preferably an immune checkpoint inhibitor, but is not limited thereto.

[0074] Furthermore, the present invention relates to a method of treating cancer by combining the novel PH20 variant or fragment thereof with other anticancer drugs, especially the aforementioned anticancer drugs.

[0075] On the other hand, the present invention relates to nucleic acids encoding PH20 variants or fragments thereof.

[0076] As used herein, nucleic acids may be present in cells, in cell lysates, or in partially or substantially purified forms. When referring to nucleic acids, “isolated” or “substantially purified” means nucleic acids (e.g., other nucleic acids or proteins) that have been purified by standard techniques and thus separated from other cellular components or other contaminants, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art. The nucleic acids of the present invention may be DNA or RNA.

[0077] In yet another aspect, the present invention relates to recombinant expression vectors containing nucleic acids. For the expression of the PH20 variant or fragment thereof according to the present invention, the DNA encoding the PH20 variant or fragment thereof can be obtained by standard meristem techniques (such as polymerase chain reaction (PCR) amplification or using a cDNA clone of a hybridoma expressing the PH20 variant), and the DNA can be inserted into an expression vector to be "operatively linked" with transcription and translation control sequences.

[0078] As used herein, the term "operatively linked" is intended to mean that the gene encoding the PH20 variant or fragment thereof is ligated into the vector such that the transcription and translation control sequences perform the intended function of regulating the transcription and translation of the gene encoding the PH20 variant or fragment thereof. The selected expression vector and expression control sequences are compatible with the expression host cells used. The gene encoding PH20 is inserted into the expression vector by standard methods (such as ligating a complementary restriction enzyme site on the gene fragment encoding the PH20 variant or fragment thereof to the vector, or, if no restriction enzyme site is present, performing blunt-end ligation)).

[0079] Furthermore, the recombinant expression vector carries a regulatory sequence that controls the expression of the gene encoding a PH20 variant or a fragment thereof in the host cell. This "regulatory sequence" is intended to include a promoter, enhancer, and other expression control elements (such as polyadenylation signals) that control the transcription and translation of the gene encoding the PH20 variant or a fragment thereof. Those skilled in the art will understand that the design of the expression vector includes regulatory...The choice of sequence may depend on factors such as the selection of the host cell to be transformed, the desired level of protein expression, etc.

[0080] In another aspect, the present invention relates to host cells comprising nucleic acids or vectors. Host cells according to the invention are preferably selected from animal cells, plant cells, yeast, *Escherichia coli*, and insect cells, but are not limited thereto.

[0081] Specifically, host cells according to the invention comprise prokaryotic cells, fungi, yeast, and eukaryotic cells. Prokaryotic cells are, for example, *Escherichia coli*, *Bacillus subtilis*, *Streptomyces sp.*, *Pseudomonas sp.*, *Proteus mirabilis*, or *Staphylococcus*. Fungi are, for example, *Aspergillus sp.*. Yeasts include, for example, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces sp.*, and *Neurospora crass*. Eukaryotic cells include, for example, lower eukaryotic cells and other higher eukaryotic cells (such as insect cells).

[0082] Furthermore, the host cells used in this invention can be obtained from plants or mammals. Preferably, examples of host cells include, but are not limited to: monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cells, HuT 78 cells, and HEK293 cells. More preferably, CHO cells can be used.

[0083] Nucleic acids or vectors are transfected into the host cells. Transfection can be performed using various techniques commonly used to introduce foreign nucleic acids (DNA or RNA) into prokaryotic or eukaryotic cells, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. This is to express the H20 variant or fragment recombinant expression vector of the present invention in host cells.Various combinations can be used. Preferred expression vectors for eukaryotic cells include gene expression regulatory sequences, receptors with broad host range, phage DNA such as various phage λ derivatives (e.g., λgt10, λgt11, and NM989), and other DNA phages. Gene expression regulatory sequences are derived from, but not limited to, SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors that can be used for bacterial hosts include bacterial receptors derived from E. coli, such as pET, pRSET, pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9, and their derivatives. Receptors with broad host range are, for example, RP4. Other DNA phages include, for example, M13 and filamentous single-stranded DNA phages. Expression vectors that can be used in yeast cells can be 2-μm receptors and their derivatives. Expression vectors for insect cells include pVL941.

[0084] In another aspect, the present invention relates to a method for producing a PH20 variant or a fragment thereof, the method comprising culturing host cells according to the invention and expressing a PH20 variant or a fragment thereof.

[0085] When a recombinant expression vector capable of expressing a PH20 variant or a fragment thereof is introduced into a mammalian host cell, the PH20 variant or a fragment thereof can be produced by culturing the host cell for a period of time, so that the PH20 variant or a fragment thereof is expressed in the host cell, wherein the period of time is preferably a period of time during which the PH20 variant is secreted into the culture medium during the culture of the host cell.

[0086] In another embodiment, the expressible PH20 variant or a fragment thereof is isolated and purified from the host cell. The isolation and purification of the PH20 variant or a fragment thereof can be performed using conventional methods for the isolation / purification of proteins (such as chromatography). Chromatography may include, but is not limited to, one or more combinations selected from affinity chromatography, ion exchange chromatography, and hydrophobic chromatography. Besides chromatography, filtration, ultrafiltration, salting out, dialysis, etc., can also be used.

[0087] To confirm the industrial applicability of the enzyme, it is necessary to analyze the enzyme's catalytic reaction rate. The types of enzyme reactions include active sites with fixed reactivity.Enzymatic reactions involving sites and enzyme reactions with multiple active sites exhibiting various reactivity. It is known that the catalytic reaction rate of enzymes with fixed active sites, such as hyaluronidase, follows the Michaelis-Menten rate formula. Specification 12 / 87 pages 14 CN 121450619 A

[0088] Michaelis-Menten enzyme kinetics is based on the assumption that enzyme reactions are two-stage reaction systems, which include a reversible reaction stage in which an enzyme (E)-substrate (S) complex [ES] is formed, and an irreversible reaction stage in which the ES complex dissociates to produce the product (P). In this case, kf, kr, and kcat are the rate constants of the reaction in each direction (Alan Fersht (1977) Enzyme Structure and Mechanism).

[0089]

[0090] An enzyme reaction is considered to be in a pseudo-steady state if the process of generating the ES complex from the substrate reaction reaches equilibrium rapidly, or if the enzyme concentration is sufficiently reduced to satisfy d[ES] / dt≒0. This pseudo-steady state is achieved by maintaining a sufficiently high substrate concentration. Since the rate formulas assume rapid equilibrium and pseudo-steady state are derived in the same way, in most experiments, a pseudo-steady state is assumed where the substrate concentration is initially higher than the enzyme concentration.

[0091] When the assumptions are that "the amount of enzyme is constant before and after the reaction" and "when the chemical reaction reaches chemical equilibrium, the reaction rate to obtain the product is equal to the rate at which the product decomposes again", the reaction rate of the final product can be expressed by the Michaelis-Menten rate formula as follows. In this case, KM = (kr + kcat) / kf, and Vmax = kcat[E]0.

[0092]

[0093] The Lineweaver-Burk equation is used to experimentally analyze enzyme reaction rates using the Michaelis-Menten rate formula. This equation shows the relationship between the reciprocal of the reaction rate 1 / V measured in the experiment and the reciprocal of the substrate concentration 1 / [S] given in the experiment. This equation is a statistical verification of the linear equation, demonstrating that the enzyme reaction follows the Michaelis-Menten rate formula, and KM and Vmax can be calculated using this equation.

[0094] Enzymes that catalyze chemical reactions have a transition state after binding to the substrate at their active site, and the high activation energy required to reach the transition state is reduced through repeated binding with the substrate. The equilibrium constant used to reach this transition state is proportional to kcat / KM. Here, 1 / KM is an exponent of the extent to which the enzyme-substrate complex is produced by binding the enzyme to the substrate and the extent to which the enzyme-substrate complex remains unchanged and is not decomposed, while kcat is the product concentration from the enzyme-substrate complex.The equilibrium constant obtained by the substrate complex. Therefore, kcat / KM can be regarded as an indicator of how much product can be obtained from the substrate and enzyme, i.e., the catalytic efficiency of the enzyme.

[0095] The industrial applicability of hyaluronidase is proportional to its catalytic efficiency. In particular, the catalytic efficiency of hyaluronidase plays an important role when the enzyme is injected subcutaneously together with a polymeric pharmacologically active substance. In the case of a variant according to the invention having a higher kcat / KM than the wild type PH20, when hyaluronidase combined with the polymeric pharmacologically active substance is injected subcutaneously, the hyaluronic acid therein is rapidly decomposed and thus the polymeric pharmacologically active substance can be rapidly dispersed. In addition, when the variant according to the invention has a larger kcat than the wild type PH20, the maximum reaction rate Vmax increases at the same enzyme concentration, thereby providing the excellent effect of decomposing a larger amount of hyaluronic acid and dispersing the polymeric pharmacologically active substance over a wider area in the same time.

[0096] Therefore, in order to verify the enzyme properties of the PH20 variant according to the present invention, the enzyme reaction rate of each variant was analyzed, and its Vmax (maximum enzyme reaction rate), KM (Vmax at a substrate concentration of 50%), kcat (substrate conversion rate), and kcat / KM (enzyme catalytic efficiency) were compared in Example 4. The above results show that the PH20 variant according to the present invention is better than the wild-type PH20. Specification 13 / 87 pages 15 CN 121450619 A Examples

[0097] In the following, the present invention will be described in more detail with reference to the examples. However, it will be apparent to those skilled in the art that these examples are provided only as examples of the present invention and should not be construed as limiting the scope of the present invention.

[0098] Example 1: Construction of PH20 variant The PH20 variant was constructed by purchasing cDNA (clone ID: hMU002604) of wild-type PH20 from the Korean Human Gene Bank. Wild-type PH20 encodes amino acids from L36 to S490. The PH20 gene is amplified via polymerase chain reaction (PCR) and inserted into the restriction enzyme sites XhoI and NotI of the pcDNA3.4-TOPO vector. For expression in ExpiCHO cells, signal peptides of human growth hormone, human serum hormone, or human Hyal1 are used as a signal peptide, rather than the original PH20 signal peptide. For protein purification using HisTrap columns, the His-tag DNA sequence is located at the 3' end of the PH20 cDNA. Amino acids of the PH20 variant.The substitution lines were performed using PCR, and the amino acid substitution lines were confirmed by DNA sequencing.

[0099] The list of primers used to clone the PH20 variant is summarized in Table 4 below, and the specific primer sequences are summarized in Table 5 below.

[0100] Table 4: List of primers for cloning the PH20 variant according to the present invention (Page 14 / 87, CN 121450619 A) (Page 15 / 87, CN 121450619 A) (Page 16 / 87, CN 121450619 A) (Page 17 / 87, CN 121450619 A) (Page 18 / 87, CN 121450619 A)

[0101] Table 5: Primer sequences for cloning the PH20 variant (Page 19 / 87, CN 121450619 A) (Page 20 / 87, CN 121450619 A) (Page 21 / 87, CN 121450619 A) (Page 21 / 87, CN 121450619 A) (Page 22 / 87, CN 121450619 A) (Page 22 / 87, CN 121450619 A) (Page 23 / 87, CN 121450619 A) (Page 24 / 87, CN 121450619 A) (Page 23 / 87, CN 121450619 A) (Page 25 / 87, CN 121450619 A) 121450619 A Specification 24 / 87 Page 26 CN 121450619 A Specification 25 / 87 Page 27 CN 121450619 A

[0102] After finding a PH20 variant with increased enzyme activity and thermostability, cDNA of the PH20 variant without the PH20 variant was also constructed.

[0103] The PH20 variant was constructed using cDNA of the PH20 variant as follows.

[0104] The expression of the variant was performed using the ExpiCHO expression system. When the cell density of ExpiCHO cells reached 6 x 10⁶ / mL, plasmids containing wild-type PH20 or PH20 variant cDNA containing the inserted pcDNA3.4-TOPO vector were transfected into ExpiCHO cells using ExpiFectamine CHO reagent (transfection reagent). As the cell culture medium, ExpiCHO expression culture medium (100 to 500 mL) was used. Following transfection, ExpiCHO cells were cultured for a total of 6 days with shaking at 130 rpm, during which time the cells were cultured at 37°C for 1 day and then further cultured at 32°C for 5 days. After completion of culture, the cell suspension was collected by centrifugation at 10,000 rpm for 30 minutes.

[0105] Recombinant proteins of wild-type PH20 and PH20 variants with C-terminal His-tag (produced in ExpiCHO cells) were transfected...Purification was performed using a three-stage column chromatography method with the AKTA primer system (GE Healthcare Systems), depending on the variant. This three-stage column chromatography was carried out using HisTrap HP columns – Q Sepharose columns – phenyl HP columns and Q Sepharose columns – HisTrap HP columns – butyl HP columns, respectively.

[0106] Purification using HisTrap HP columns, Q Sepharose columns, and phenyl HP columns was performed as follows. For protein purification using HisTrap HP columns, buffer A (20 mM sodium phosphate, pH 7.5, 0.5 M sodium chloride (NaCl)) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M sodium chloride (NaCl), 0.5 M imidazole) were prepared. The protein was bound to the HisTrap HP column, which was then washed with 5 column volumes (CV) of buffer A to remove non-specifically bound proteins. After confirming that the conductivity remained constant, the column was then washed with 5 CV of 20% buffer B to elute the protein. The washed protein was then dialyzed with dialysis buffer (20 mM sodium phosphate, pH 7.5, 50 mM sodium chloride). For protein purification using a Q Sepharose column, buffer A (20 mM sodium phosphate, pH 7.5) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M sodium chloride) were prepared. The protein was bound to the Q Sepharose column, and the column was washed with 5 CV of buffer A to remove non-specifically bound proteins, followed by washing with 5 CV of buffer B at a concentration gradient from 0% to 100%. For protein purification using a phenyl HP column, buffer A (20 mM sodium phosphate, pH 7.0, 1.5 M ammonium sulfate ((NH4)2SO4)) and buffer B (20 mM sodium phosphate, pH 7.0) were prepared. The protein was bound to the phenyl column, and the column was washed with 5 mV of buffer A to remove non-specifically bound proteins, and then washed with 5 mV of buffer B at a concentration gradient from 0 to 100% to wash the protein.

[0107] Purification using Q Sepharose columns, HisTrap HP columns, and butyl HP columns was performed as follows. For protein purification using a Q Sepharose column, buffer A (20 mM sodium phosphate (NaPi), 15 mM sodium chloride, pH 8.0) and buffer B (20 mM sodium phosphate, pH 7.0) were prepared.A solution of 500 mM sodium phosphate and 500 mM sodium chloride (pH 8.0) was prepared. To adjust the pH and conductivity of the culture medium to match those of buffer A, the pH was titrated to 8 using 1 M Tris buffer (tris(hydroxymethyl)aminomethane) buffer, and the conductivity was adjusted to 5 mS / cm or less by adding water (PW). The culture medium was then filtered through a membrane with 0.22 μm pores. The protein was bound to a Q Sepharose column, which was washed with 5 CV of buffer A to remove non-specifically bound proteins, followed by a wash with 5 CV of buffer B to wash the target protein. For protein purification using a HisTrap HP column, buffer A (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.5) and buffer B (20 mM sodium phosphate, 500 mM sodium chloride, 500 mM imidazole, pH 7.5) were prepared. The protein was bound to the HisTrap HP column, which was then washed with 10 CV of 7% buffer B to remove non-specifically bound proteins, followed by a 3 CV wash with 40% buffer B to wash the protein. For protein purification using a butyl HP column, buffer A (20 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0) and buffer B (20 mM sodium phosphate, pH 7.0) were prepared. 3 M ammonium sulfate and the protein sample to be loaded onto the column were mixed in a 1:1 ratio, and the resulting mixture was then filtered through a membrane with 0.22 μm pores. The protein sample was bound to a butyl HP column, which was washed with 5 CV buffer A to remove impurities. The target protein was then washed with buffer B in a linear concentration gradient from 0 to 100% and dialyzed using dialysis buffer (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.0). The variants according to the invention were purified using the method proposed in this invention, and 10% SDS-PAGE analysis was performed on each purified product, and the results are shown in Figures 1 and 3.

[0108] The enzyme activity of wild-type PH20 and the PH20 variant was measured by a turbidity assay.

[0109] The turbidity assay is a method for measuring the absorbance of the precipitate formed when hyaluronic acid is mixed with albumin (BSA). When hyaluronic acid is hydrolyzed with pH 20, the absorbance of the precipitate formed when mixed with albumin decreases. Turbidity testing is typically performed as follows: Hyaluronidase pH 20 (Sigma) is diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, and 60 units.(units) / mL and prepared in individual test tubes. Purified protein samples were dissolved in enzyme dilution buffer (20 mM Tris-HCl, pH 7.0, 77 mM sodium chloride, 0.01% (w / v) fetal bovine serum albumin), diluted to 100X, 300X, 600X, 1200X, and 2400X, and prepared in individual test tubes. In new test tubes, a 3 mg / mL hyaluronic acid solution was diluted 10-fold to a concentration of 0.3 mg / mL to make the volume in each tube 180 μL. 60 μL of enzyme was added to the diluted hyaluronic acid solution and mixed, and allowed to react at 37°C for 45 minutes. After the reaction was complete, 50 μL of the reacted enzyme and 250 μL of acidic albumin solution were added to each well of the 96-well dish and shaken for 10 minutes. The absorbance was then measured at 600 nm using a spectrophotometer.

[0110] Methods for measuring the thermal stability of proteins include measuring the aggregation temperature via dynamic light scattering (DLS), measuring the melting point temperature (Tm) in real-time PCR using Sypro-Orange dye, and measuring enzyme activity after the protein has been placed at a predetermined temperature for a predetermined time. In the method of measuring the aggregation temperature via DLS, the aggregation of molecules is measured through light scattering, thus providing high sensitivity and the aggregation temperature is typically lower than the melting point temperature of the protein. Since each variant was prepared as a solution of the same concentration (0.2 mg / mL) and then measured, the physical properties of each variant could be compared by using the resulting value as the aggregation temperature (Philo, JS (2009) Cur. Pharm. Biotech. 10, 359-372).

[0111] The amino acid sequence of the PH20 variant constructed in this invention by substituting or cleaving amino acids from the PH20 variant having the sequence SEQ ID NO: 3 is shown in Table 6 below.

[0112] In this invention, experiments were conducted on variants in which six histidine residues for protein purification were added to the C-terminus of the sequence shown in Table 6. This addition at the C-terminus was found not to affect enzyme activity or protein stability. Variants according to the invention are named as a combination of HM and sequence number, and the variants according to Example 3 are named “Hyal2-variant”, “Hyal3-variant”, and “Hyal4-variant”.

[0113] Table 6: Amino acid sequences and substitution / cleavage characteristics of the PH20 variant according to the present invention (Pages 27 / 87, CN 121450619 A, Pages 28 / 87, CN 121450619 A, Pages 29 / 87, CN 121450619 A, Pages 31 / 87, CN 121450619 A, Pages 30 / 87, CN 121450619 A, Pages 31 / 87, CN 121450619 A, Pages 32 / 87, CN 121450619 A, Pages 33 / 87, CN 121450619 A, Pages 33 / 87, CN 121450619 A, Pages 34 / 87, CN 121450619 A, Pages 35 / 87, CN 121450619 A, Pages 36 / 87, CN 121450619 A, Pages 37 / 87, CN 121450619 A, Pages 38 / 87, CN 121450619 A, Pages 37 / 87, CN 121450619 A) 121450619 A Instruction Manual 38 / 87 pages 40 CN 121450619 A Instruction Manual 39 / 87 pages 41 CN 121450619 A Instruction Manual 40 / 87 pages 42 CN 121450619 A Instruction Manual 41 / 87 pages 43 CN 121450619 A Instruction Manual 42 / 87 pages 44 CN 121450619 A Instruction Manual 43 / 87 pages 45 CN 121450619 A Instruction Manual 44 / 87 pages 46 CN 121450619 A Instruction Manual 45 / 87 pages 47 CN 121450619 A Instruction Manual 46 / 87 pages 48 CN 121450619 A Instruction Manual 47 / 87 pages 49 CN 121450619 A Instruction Manual 48 / 87 pages 50 CN 121450619 A Instruction Manual 49 / 87 pages 51 CN 121450619 A Instruction Manual 50 / 87 pages 52 CN 121450619 A Instruction Manual 51 / 87 pages 53 CN 121450619 A Instruction Manual 52 / 87 pages 54 CN 121450619 A Instruction Manual 53 / 87 pages 55 CN 121450619 A Instruction Manual 54 / 87 pages 56 CN 121450619 A Instruction Manual 55 / 87 pages 57 CN 121450619 A Instruction Manual 56 / 87 pages 58 CN 121450619 A Instruction ManualPages 57 / 87, 59 CN 121450619 A, Instruction Manual; Pages 58 / 87, 60 CN 121450619 A, Instruction Manual; Pages 59 / 87, 61 CN 121450619 A, Instruction Manual; Pages 60 / 87, 62 CN 121450619 A, Instruction Manual; Pages 61 / 87, 63 CN 121450619 A, Instruction Manual; Pages 62 / 87, 64 CN 121450619 A, Instruction Manual; Pages 63 / 87, 65 CN 121450619 A, Instruction Manual; Pages 64 / 87, 66 CN 121450619 A, Instruction Manual; Pages 65 / 87, 67 CN 121450619 A, Instruction Manual; Pages 66 / 87, 68 CN 121450619 A, Instruction Manual; Pages 67 / 87, 69 CN 121450619 A Instruction manual pages 68 / 87, 70 CN 121450619 A; Instruction manual pages 69 / 87, 71 CN 121450619 A; Instruction manual pages 70 / 87, 72 CN 121450619 A; Instruction manual pages 71 / 87, 73 CN 121450619 A; Instruction manual pages 72 / 87, 74 CN 121450619 A; Instruction manual pages 73 / 87, 75 CN 121450619 A; Instruction manual pages 74 / 87, 76 CN 121450619 A; Instruction manual pages 75 / 87, 77 CN 121450619 A; Instruction manual pages 76 / 87, 78 CN 121450619 A; Instruction manual pages 77 / 87, 79 CN 121450619 A; Instruction manual pages 78 / 87, 80 CN 121450619 A

[0114] Example 2: The properties of the PH20 variant according to the present invention were further investigated through the study of the variant, wherein the variant comprises cleavage at the N-terminus and C-terminus based on the amino acid sequence of SEQ ID NO: 3. The aggregation temperature shown in Table 7 is the result of analysis of the expression level and activity of the prepared variant.

[0115] The expression level and specific activity were analyzed by turbidity testing as described in Example 1. The test results are shown. At this time, based on the limit of quantification (LOQ) set for each activity after purification in the culture medium, the activity in the culture medium exceeding 300 units / mL was marked as ">LOQ", while the activity after purification exceeding 15Units / μg are labeled ">LOQ". In the opposite case, the inequality sign changes. The performance and limits of quantitation of the activity assays, and the test results based thereon, are shown in Table 7. The aggregation temperature of wild-type PH20 (L36-Y482) of SEQ ID NO: 1 is 46.5°C, while that of the PH20 variant (F38-F468) of SEQ ID NO: 3 is 51°C.

[0116] Table 7: Performance, specific activity and aggregation temperature of the PH20 variants according to the present invention 79 / 87 pages 81 CN 121450619 A 80 / 87 pages 82 CN 121450619 A 81 / 87 pages 83 CN 121450619 A 82 / 87 pages 84 CN 121450619 A 83 / 87 pages 85 CN 121450619 A 84 / 87 pages 86 CN 121450619 A

[0117] As can be seen from Table 7 above, among the multiple variants having the amino acid sequence SEQ ID NO: 3, there are a total of 133 variants with a single amino acid residue substitution (i.e., HM63, HM64, HM65, HM66, HM67, HM69, HM70, HM71, HM72, HM73, HM74, HM75, HM76, HM77, HM78, HM79, HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM90, HM91, HM92, HM93, HM94, HM95, HM97, HM98, HM99, HM100, HM101, HM102, HM103, HM104, HM105, HM106, HM107, HM110, HM111, HM112, HM114, HM115, HM116, HM117, HM118, HM121, HM125, HM126, HM130, HM131, HM132, HM133, HM134, HM135, HM136, HM138, HM139, HM140, HM141, HM142, HM143, HM144, HM145, HM152, HM153, HM154, HM155, HM156, HM157, HM158, HM159, HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168, HM169, HM170, HM171,HM172, HM173, HM174, HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185, HM186, HM190, HM191, HM192, HM193, HM194, HM195, HM196, HM197, HM198, HM199, HM203, HM204, HM205, HM208, HM210, HM211, HM212, HM213, HM214, HM216, HM217, HM218, HM219, HM220, HM231, HM232, HM233, HM234, HM235, HM243, HM245 and HM246 are variants that retain activity in the obtained purified fraction after purification and have an aggregation temperature of 48 to 58°C, thus exhibiting good thermal stability. There are a total of 65 variants (namely HM63, HM64, HM65, HM66, HM67, HM69, HM70, HM71, HM72, HM73, HM74, HM75, HM76, HM77, HM78, HM79, HM82, HM83, HM84, HM85, HM86, HM88, HM89, HM90, HM91, HM92, HM93, HM94, HM95, HM98, HM99, HM100, HM101, HM102). HM103, HM104, HM105, HM106, HM107, HM110, HM111, HM112, HM114, HM115, HM116, HM117, HM118, HM121, HM125, HM126, HM130, HM131, HM132, HM133, HM134, HM135, HM136, HM138, HM139, HM140, HM141, HM142, HM143, HM144 and HM145) are variants in which one of the multiple substitution sites in the sequence of SEQ ID NO: 3 of PH20 is mutated and has an aggregation temperature of 48 to 58°C. Among them, there are a total of 68 variants (HM97, HM152, HM153, HM154, HM155, HM156, HM157, HM158, HM159, HM160, HM161, HM162, HM163, HM164, HM165, HM166, HM167, HM168, HM169, HM170, HM171, HM172, HM173, HM174, ...).HM175, HM176, HM177, HM178, HM179, HM180, HM181, HM182, HM183, HM184, HM185, HM186, HM190, HM191, HM192, HM193, HM194, HM195, HM196, HM197, HM198, HM199, HM203, HM204, HM205, HM208, HM210, HM211, HM212, HM213, HM214, HM216, HM217, HM218, HM219, HM220, HM231, HM232, HM233, HM234, HM235, HM243, HM245, and HM246 are variants of PH20 with a mutation at a position other than the substitution site in SEQ ID NO: 3 and an aggregation temperature of 48 to 56°C.

[0118] Therefore, it can be understood that, regardless of the substitution site, the variants of PH20 with a substitution at a position in SEQ ID NO: 3 (pages 85 / 87, CN 121450619 A) have a higher aggregation temperature than the wild-type PH20 (L36-Y482) of SEQ ID NO: 1. However, HM174, HM208, HM210, and HM211 were found to have activity below 300 units / mL (LOQ) in culture medium, but above 15 units / μg (LOQ) after purification. In this case, when the activity of the variant is measured only in the culture medium, the characteristics of the variant itself cannot be accurately analyzed.

[0119] Furthermore, as shown in Table 7 above, among the variants having the amino acid sequence SEQ ID NO: 3, HM146, HM147, HM149, HM262, and HM263 retain the same mutations as the variants having the amino acid sequence SEQ ID NO: 3, which are substitutions of amino acid residues, but also include cleavage at the N-terminus and C-terminus, indicating that the performance and activity of the protein in the variants having the amino acid sequence SEQ ID NO: 3 are not affected by further cleavage at the N-terminus and C-terminus. These variants have an aggregation temperature of 49°C to 53°C, which is not significantly different from the variants of SEQ ID NO: 3, indicating that the physical properties of the variants are also not affected by further cleavage at the N-terminus and C-terminus.

[0120] Furthermore, among the variants having the amino acid sequence SEQ ID NO: 3, there are a total of 13 variants (i.e., HM96, HM150, HM254, HM261, HM266, HM268, HM271, HM275, HM276, HM279, HM280, HM287 and HM288, which are listed in the table above).The variants in column 7 (including variants with one or more amino acid substitutions and cleavages) successfully represented the protein, retaining enzyme activity and exhibiting an aggregation temperature of 48°C to 59°C. This indicates that even with multiple substitutions, the protein's activity and physical properties are still preserved. However, multiple substitutions exhibit unpredictable enzyme activity and aggregation temperature, which are unpredictable only when the characteristic combination of enzyme activity and aggregation temperature is obtained through each single substitution with the same composition.

[0121] Example 3: Activity analysis of variants with sequence substitutions Hyal2, Hyal3 and Hyal4 The amino acid sequences of Hyal2 (TTSTETCQYLKDYLTRL), Hyal3 (SSSEEECWHLHDYLVDT) and Hyal4 (TASKANCTKVKQFVSSD) are the corresponding parts of hyaluronidase in the human body except for Hyal1, replacing positions M345 to I361 in the amino acid sequence of wild-type PH20 of SEQ ID NO: 1, to study how the stability of the protein changes.

[0122] Variants constructed by replacing positions M345 to I361 of the mature wild-type PH20 (L36-S490) with the corresponding sequences Hyal2, Hyal3, and Hyal4 were designated as “Hyal2-variant,” “Hyal3-variant,” and “Hyal4-variant,” respectively.

[0123] The Hyal2-variant, Hyal3-variant, and Hyal4-variant were constructed, and their thermal stability was analyzed (see Figure 2). The results showed that the aggregation temperature of the Hyal2-variant, as measured by DLS, was 48°C, which is 1.5°C higher than the 46.5°C aggregation temperature of the wild-type PH20, indicating increased thermal stability.

[0124] Furthermore, to confirm whether these variants were expressed in ExpiCHO cell culture, they were purified using the same method as with HisTrap columns, and the degree of protein expression was compared by SDS-PAGE analysis. The results showed that the Hyal3 variant exhibited the highest degree of expression, followed by the Hyal2 and Hyal4 variants.

[0125] Example 4, thermal stability analysis of the variants according to the invention was performed by SDS-PAGE analysis to confirm the thermal stability of the variants according to the invention. Purified wild-type PH20 of SEQ ID NO: 1 (L36-Y482) and purified protein of the PH20 variant according to the invention, SEQ ID NO: 3 (F38-F468), were stored at 42°C for 7 consecutive days, followed by 10% SDS-PAGE analysis under both reduced and non-reduced conditions (Figure 4).

[0126] The results showed that wild-type PH20 (L36-Y482) aggregated (row G in Figure 4), while SEQ ID NO: 3...The PH20 variants (F38-F468) did not aggregate (row H in Figure 4). This difference in aggregation was found to be due to the difference in aggregation temperature between the two proteins. Accordingly, the variants according to the invention are considered to exhibit higher thermal stability and are expected to be widely used in industry due to their higher aggregation temperature compared to wild-type PH20.

[0127] Example 5: Enzyme kinetics analysis of variants according to the invention Specification 86 / 87 pages 88 CN 121450619 A In order to analyze the enzyme kinetics of the variants according to the invention, the enzyme activity was measured by the Morgan-Elson method (Takahashi, T. et al. (2003), Anal. Biochem. 322:257-263). The Morgan-Elson method is a colorimetric method used to analyze the red substance (at 545 nm) generated by the reduction reaction of N-acetyl-D-glucosamine (GlcNAc), produced by the hydrolysis of hyaluronic acid with para-dimethylaminobenzaldehyde (DMAB) by hyaluronidase. It is an Ehrlich reagent. N-acetyl-D-glucosamine (GlcNAc, Sigma) is diluted to 0.25, 0.50, 0.75, 1.00, or 1.25 mM in a dilution buffer (0.1 M sodium phosphate, 0.1 M sodium chloride, 1.5 mM saccharic acid 1,4-lactone, pH 5.35). In each test tube, the diluted N-acetyl-D-glucosamine is reduced with tetraborate, followed by the addition of DMAB to induce the colorimetric reaction. After the reaction, absorbance was measured at 545 nm to generate a standard reaction curve for GlcNAc. Hyaluronic acid, as the substrate, was diluted in each tube with dilution buffer to 0.54, 0.65, 0.87, 1.23, or 2.17 μM, and hyaluronidase was added. The reaction was then terminated by incubation at 37°C for 5 minutes and heating at 100°C for 5 minutes. The resulting sample was reduced by treatment with tetraborate, and DMAB was added to induce a colorimetric reaction. After the reaction, absorbance was measured at 545 nm, and enzyme activity was measured using the standard reaction curve of GlcNAc described above. Enzyme kinetics of wild-type PH20 of SEQ ID NO: 1 and the PH20 variant according to the present invention were analyzed.The analysis was performed using this method. Linearity of the Lineweaver-Burk curve was detected as a result, indicating that the PH20 variants according to the invention follow the Michaelis-Menten enzyme kinetic equation.

[0128] Table 8 shows the Vmax (maximum enzyme reaction rate), KM (50% substrate concentration), kcat (substrate conversion rate), and kcat / KM (enzyme catalytic efficiency) obtained from the enzyme kinetic analysis results of wild-type PH20 (L36-Y482) of SEQ ID NO: 1, PH20 variants (F38-F468) of SEQ ID NO: 3, HM261, and HM268. It can be seen that as the KM value decreases, the substrate binding capacity of the enzyme increases, while as the kcat value increases, the substrate conversion rate of the enzyme increases. Therefore, the kcat / KM (enzyme catalytic efficiency) of each PH20 variant is higher than that of wild-type PH20. Furthermore, the kcat of each of SEQ ID NO: 3, HM261 and HM268 is greater than that of the wild-type PH20 of SEQ ID NO: 1, and therefore the substrate conversion efficiency of the enzyme is greater than that of the wild-type PH20 of SEQ ID NO: 1. Thus, the industrial availability of each PH20 variant is greater than that of the wild-type PH20.

[0129] Table 8: Results of enzyme kinetic analysis of the PH20 variant according to the present invention. (Pages 87 / 87, CN 121450619 A, Figure 1A, Figure 1B, Figure 1 / 6, CN 121450619 A, Figure 1C, Figure 1D, Figure 2 / 6, CN 121450619 A, Figure 1E, Figure 1F, Figure 2, Figure 3 / 6, CN 121450619 A, Figure 3A, Figure 3B, Figure 3C, Figure 4 / 6, CN 121450619 A, Figure 3D, Figure 3E, Figure 3F, Figure 5 / 6, CN 121450619 A, Figure 4, Figure 6 / 6, CN 121450619 A) The present application relates to a novel hyaluronic acid-hydrolyzing enzyme variant having improved stability and pharmaceutical composition comprising the same. The present application discloses a novel PH20 variant or afragment thereof, the variant having improved enzyme activity and thermal stability of human hyaluronidase, which is a hyaluronic acid-hydrolyzing enzyme, and relates to a novel PH20 variant, or a fragment thereof, the variant having a substitution of one or more amino acid residues in a variant that has an amino acid sequence of SEQ ID NO: 3, and, optionally, having an additional deletion of N-terminal and / or C- terminal amino acid residues.

Claims

1. A PH20 variant or a fragment thereof, comprising an amino acid residue substitution, deletion, or insertion in at least one position in a variant comprising the amino acid sequence of SEQ ID NO: 3, and having an aggregation temperature higher than that of wild-type PH20.

2. The PH20 variant or a fragment thereof of claim 1, wherein the PH20 variant or a fragment thereof comprises the amino acid residue substitution in at least one position in the variant having the amino acid sequence of SEQ ID NO: 3 selected from the group consisting of R39, D65 to L68, N82, T84, I102 to I105, T132 to Y134, N166, L179 to T182, T185 to K187, V241 to K244, N266 to Q269, P271, V272, K290 to P292, Q311 to K314, G340 to N363, L441, S442, D451 to D453, D461, V463, and D461 to V463.

3. The PH20 variant or a fragment thereof of claim 2, wherein the amino acid residue substitution comprises a substitution selected from the group consisting of R39K, D65A, E66A, P67A, L68A, N82A, T84N, I102A, D103A, S104A, S104N, I105A, I105Q, T132A, T132S, F133A, Y134A, N166A, N166K, L179A, L179S, L179I, L179F, S180T, S180A, L181A, L181M, T182A, T185A, E186A, E186D, K187A, V241A, E242A, I243A, K244A, N266A, T267A, Q268A, Q268D, Q268I, Q268N, Q269A, P271A, V272A, K290A, I291A, I291G, I291L, P292A, P292D, Q311A, V312A, L313A, L313P, L313M, K314A, G340Q, S341H, S341D, S341T, W342I, W342D, W342H, W342L, E343V, E343S, E343Y, E343Q, N344F, N344I, T345E, T345K, T345S, R346M, R346F, R346L, R346T, R346S, R346A, T347Q, T347E, T347V, T347W, T347H, T347S, K348Q, K348F, K348D, K348T, K348E, K348M, E349L, E349W, E349A, S350Q, S350I, S350D, S350T, S350E, S350N, Q352E, Q352G, Q352Y, Q352W, Q352T, A353E, A353Y, A353H, A353K, I354E, I354Q, I354S, I354V, I354A, I354N, I354T, I354R, I354W, I354L, K355Q, K355H, K355D, E356M, E356F, E356I, E356L, E356Q, E356V, E356D,at least one of Y357W, Y357F, M358V, M358R, M358Y, M358L, D359K, D359V, D359Y, D359Q, D359T, D359S, D359E, T360Y, T360R, T360L, T360D, T360S, T361M, T361E, T361H, T361L, T361D, T361I, L362A, N363M, N363E, L441A, S442A, D451A, D451S, T452A, T452D, T452H, T452K, T452G, T452P, T452M, T452F, D453A, D461R, D461A, G462A, V463Y, and V463A.

4. The PH20 variant or a fragment thereof of any one of claims 1 to 3, further comprising a deletion of at least one amino acid residue at the C-terminus and / or the N-terminus.

5. The PH20 variant or a fragment thereof of claim 4, wherein the at least one amino acid residue is deleted by cleavage from the N-terminus before an amino acid residue selected from the group consisting of M1 to P42.

6. The PH20 variant or a fragment thereof of claim 5, wherein the at least one amino acid residue is deleted by cleavage from the N-terminus before an amino acid residue of L36, N37, F38, R39, A40, P41, or P42.

7. The PH20 variant or a fragment thereof of claim 4, wherein the at least one amino acid residue is deleted by cleavage from the C-terminus after an amino acid residue selected from the group consisting of V455 to L509.

8. The PH20 variant or a fragment thereof of claim 7, wherein the at least one amino acid residue is deleted by cleavage from the C-terminus after an amino acid residue selected from the group consisting of V455 to S490.

9. The PH20 variant or a fragment thereof of claim 7, wherein the at least one amino acid residue is deleted by cleavage from the C-terminus after an amino acid residue of V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490.

10. The PH20 variant or a fragment thereof of claim 4, wherein the at least one amino acid residue is deleted by cleavage from the N-terminus before an amino acid residue of F38 and from the C-terminus after an amino acid residue of F468.

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