Novel hyaluronic acid hydrolase variants and pharmaceutical compositions containing the same

Abstract

The present invention relates to a protein engineering technology that enhances the enzyme activity of human hyaluronidase, a hyaluronic acid hydrolase, at neutral pH, and more particularly to a novel hyaluronidase mutant or section thereof, comprising one or more amino acid residue substitutions in or near the enzyme active site of the amino acid sequence of the natural hyaluronidase Hyal1 (SEQ ID NO: 1), and selectively cleaving additional N-terminal and / or C-terminal amino acid residues.

Description

Technical Field

[0001] The present invention relates to a novel human hyaluronidase variant which is a hyaluronic acid hydrolase with improved activity at neutral pH. Specifically, it involves substitution of one or more amino acid residues in or near the active site of a native hyaluronidase having an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4, and additionally a hyaluronidase variant or a fragment thereof in which the N-terminal or C-terminal amino acid residues are cleaved, a method for producing the same, and a pharmaceutical composition containing the same.

Background Art

[0002] Human skin is composed of the epidermis, dermis and subcutaneous fat layer, and there are six types of glycosaminoglycans in the skin. The glycosaminoglycans include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratan sulfate.

[0003] Glycosaminoglycans have a structure in which disaccharide-like sugars are repeatedly linked together. The length of the sugar varies depending on the glycosaminoglycan, ranging from several hundred to several thousand units. Of the glycosaminoglycans, hyaluronic acid accounts for more than half of the body's total sugar content, which is found in the skin. Hyaluronic acid is synthesized by hyaluronan synthase, which is present in the cell membrane, and exists independently without binding to proteoglycans. It is the only glycosaminoglycan that lacks a sulfate group. Other glycosaminoglycans are bound to proteoglycans and possess sulfate groups. Hyaluronic acid consists of glucuronic acid and N-acetylglucosamine linked by β-1,3 and β-1,4 bonds, with this disaccharide structure repeated approximately 5,000 times. It is known that in the human body, approximately one-third (5g) of hyaluronic acid is broken down every day.

[0004] Hyaluronidase is an enzyme that breaks down hyaluronic acid located in the extracellular matrix. Humans are known to have six types of hyaluronidase: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20 / SPAM1. In humans, Hyal1 and Hyal2 are hyaluronidases expressed in most tissues and are also known to be present in plasma. Natural PH20 / SPAM1 (hereinafter, PH20) is expressed in the cell membrane and acrosome membrane of sperm. However, HyalPS1 is a pseudogene and is not expressed. Natural human Hyal1 is expressed in intracellular lysosomes and in most tissues such as the liver, kidneys, and heart, and consists of the amino acid sequence of SEQ ID NO: 1. The mature form consists of amino acids F22-W435 from the SEQ ID NO: 1 sequence and is a protein with excellent enzymatic activity at acidic pH. Natural human Hyal2 is expressed in most tissues and consists of the amino acid sequence of SEQ ID NO: 2. The mature form consists of amino acids M21-G448 from the SEQ ID NO: 2 sequence and has enzymatic activity to hydrolyze high molecular weight hyaluronic acid to a size of approximately 20 kDa. Natural human Hyal3 is found in the skin, bone marrow, and testes, and consists of the amino acid sequence of SEQ ID NO: 3. The mature form consists of amino acids Q21-V417 from the SEQ ID NO: 3 sequence and has very weak enzymatic activity under in vitro conditions. Natural human Hyal4 is distributed in the placenta and muscles and consists of the amino acid sequence of SEQ ID NO: 4, and functions as a membrane protein. Natural human PH20 is expressed in the testes and sperm and consists of the amino acid sequence of SEQ ID NO: 5, while the mature form consists of amino acids L36-S490 of SEQ ID NO: 5 and exhibits excellent enzyme activity even at neutral pH.

[0005] Hyaluronidases can be classified into three types based on the method by which they cleave hyaluronic acid. These three types are: an enzyme that uses H2O to cleave the β-1,4 bond between N-acetylglucosamine and glucuronic acid (EC 3.2.1.35); an enzyme that cleaves the β-1,3 bond (EC 3.2.1.36); and a bacterial hyaluronidase that cleaves the β-1,4 bond without using H2O (EC 4.2.99.1).

[0006] The catalytic amino acids for Hyal1 are D129 and E131, and it hydrolyzes hyaluronic acid via a substrate-assisted catalysis mechanism. Hyal1 exhibits optimal activity at acidic pH 3-4 and has no enzymatic activity above pH 4.5. Unlike Hyal1, PH20 exhibits enzymatic activity over a wide pH range of 3-8.

[0007] Arming et al. revealed that the catalytic amino acids of PH20 are D111 and E113 (Arming et al., 1997). Arming et al. named Leu, the first amino acid of the mature PH20 protein, as "1," so the catalytic amino acids in full-length PH20 correspond to D146 and E148, respectively.

[0008] Hyaluronidase hydrolyzes hyaluronic acid, reducing its viscosity in the extracellular matrix and increasing the permeability of external substances to tissues (skin). Since the subcutaneous region of the skin has a neutral pH of approximately 7.0-7.5, PH20 is the most commonly used type of hyaluronidase in clinical practice (Bookbinder et al., 2006). Examples of PH20's clinical use include its use as an ocular relaxant and anesthetic injection additive in ophthalmic surgery, and its co-administration with antibody therapies during subcutaneous injections (Bookbinder et al., 2006). Furthermore, leveraging the characteristic of hyaluronidase overexpression in tumor cells, PH20 is used to hydrolyze hyaluronic acid in the extracellular matrix of tumor cells, thereby increasing the accessibility of anticancer agents to tumor cells. Furthermore, pH 20 is also used to promote the reabsorption of excess bodily fluids and blood within tissues.

[0009] PH20 was first identified in guinea pig sperm by Lathrop et al. and is known to be expressed in the sperm of many other species. The human PH20 gene was cloned by Lin et al. and Gmachl et al. The natural human PH20 of Sequence ID No. 5 has a 60% amino acid sequence match with the guinea pig PH20 gene. The human PH20 enzyme is coded from the SPAM1 (sperm adhesion molecule-1) gene and exists on the surface of the sperm cell membrane and inside the acrosome membrane in the form of PH20 Ser490 bound to GPI (glycosylphosphatidylinositol). When sperm penetrate the cumulus layer of the egg, which is rich in hyaluronic acid, they use PH20 to hydrolyze the hyaluronic acid. PH20 is present in sperm at a concentration of less than 1% of the total protein content and has six N-glycation sites (N82, N166, N235, N254, N368, N393).

[0010] Currently, PH20 is widely used commercially in the form extracted from the testes of cattle and sheep. Examples of PH20 include amphadase, a bovine hyaluronidase, and vitrase, a sheep hyaluronidase.

[0011] BTH (Bovine testicular hyaluronidase) is a form of bovine PH20 obtained by removing the signal peptide and the 56 C-terminal amino acids during post-translational modification. BTH is also a glycoprotein, with mannose accounting for 5% and glucosamine for 2.2% of its total amino acid content. When animal-derived hyaluronidase is repeatedly administered to humans in high doses, neutralizing antibodies may be generated. Furthermore, animal-derived hyaluronidase contains other biomolecules besides PH20, which can cause allergic reactions when administered to humans (Bookbinder et al., 2006). In particular, the production and use of PH20 extracted from cattle are restricted due to concerns about mad cow disease. To address these issues, research has been conducted on recombinant human PH20 proteins.

[0012] Recombinant human PH20 proteins have been reported to be expressed in yeast (P. pastoris), DS-2 insect cells, and animal cells. In the case of recombinant PH20 proteins produced in insect cells and yeast, the N-glycation process during post-translational modification differs from that of human PH20.

[0013] Among hyaluronidases, the protein structures of Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidase (PDB ID: 1FCQ, 1FCU, 1FCV) have been elucidated. Hyal1 consists of two domains: a catalytic domain and an EGF-like domain. In the catalytic domain, the α-helix and β-strand, which are characteristic of the protein's secondary structure, are repeated eight times each, forming eight (β / α) morphologies (Chao et al., 2007). The EGF-like domain is conserved in all variants of Hyal1 in which the C-terminus is spliced ​​in a different way. The amino acid sequences of Hyal1 and PH20 are 35.1% identical, but the protein structure of PH20 has not yet been elucidated.

[0014] Recombinant human PH20 protein was developed by Halozyme Therapeutic and is sold under the trade name Hylenex (Bookbinder et al., 2006; Frost, 2007).

[0015] When the catalytic amino acids D146 and E148 of PH20 were mutated to asparagine (D146N) and glutamine (E148Q), respectively, no enzyme activity was observed (Arming et al., 1997). Furthermore, substituting R246 of PH20 with glycine reduced enzyme activity by 90%, and substituting E319 with glutamine and R322 with threonine eliminated enzyme activity. A mutant in which 36 amino acids were removed from the C-terminal region of PH20 (474-509 amino acid cleavage) showed a 75% reduction in enzyme activity compared to the natural PH20. This mutation did not result in extracellular secretion but persisted within HeLa cells. When 134 amino acids were removed from the C-terminal region of PH20, the enzyme activity of PH20 was absent, and it was not secreted extracellularly. According to Frost et al., the 477-483 region at the C-terminus of PH20 is essential for soluble expression (Frost, 2007). The activity of full-length PH20 (1-509) or PH20 mutants with the C-terminus cleaved at 467 (1-467) was only 10% of the activity of PH20 mutants with the C-terminus cleaved at one site between 477 and 483 (Frost, 2007).

[0016] On the other hand, recombinant PH20 still lacks sufficient thermal stability and expression rate, and there is a great demand for hyaluronidase with improved properties. In the human body, there is a potential risk of safety-related problems when a protein that exists only in the testes and sperm is used in skin or other tissues. To solve this, recombinant proteins using Hyal1, which is expressed in most tissues in the human body, are needed, but these enzymes are only active at acidic pH, which reduces their usability. [Prior art documents] [Non-patent literature]

[0017] [Non-Patent Document 1] 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. [Non-Patent Document 2] Bookbinder, LH, Hofer, A., Haller, MF, Zepeda, ML, Keller, GA, Lim, JE, Edgington, TS, Shepard, HM, Patton, JS, and Frost, GI (2006). A recombinant human enzyme for enhanced interstitial transport of therapeutics. J Control Release 114, 230-241. [Non-Patent Document 3] Chao, KL, 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. [Non-Patent Document 4] Frost, GI (2007). Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin Drug Deliv 4,427-440. [Overview of the project] [Problems that the invention aims to solve]

[0018] An object of the present invention is to provide a novel hyaluronidase variant or a fragment thereof that has a structure similar to mature native hyaluronidase and is active at neutral pH, the native hyaluronidase being preferably a mature native hyaluronidase.

[0019] Another object of the present invention is to provide a therapeutic composition containing the novel hyaluronidase variant or a fragment thereof and a treatment method using the same.

Means for Solving the Problems

[0020] To achieve the above object, the present invention provides a novel hyaluronidase variant or a fragment thereof that has improved activity at neutral pH, which contains substitutions of one or more amino acid residues in the amino acid sequence of native hyaluronidase, preferably mature native hyaluronidase, at the enzyme active site and / or substrate binding site and / or in the vicinity thereof, and additionally has a part of the amino acid residues at the N-terminus and / or C-terminus cleaved.

[0021] The present invention also provides a composition for cancer treatment containing the novel hyaluronidase variant or a fragment thereof according to the present invention and a treatment method using the same.

[0022] The present invention relates to a novel hyaluronidase variant or a fragment thereof that contains substitutions of one or more amino acid residues at the enzyme active site, substrate binding site and / or adjacent site of native hyaluronidase and exhibits hyaluronidase activity at neutral pH.

[0023] The present invention also relates to a novel hyaluronidase variant or a fragment thereof, wherein the native hyaluronidase is mature human native hyaluronidase.

[0024] The present invention also relates to a novel hyaluronidase variant or a fragment thereof, wherein the mature human native hyaluronidase is in a state where the signal peptide has been removed from human native hyaluronidase.

[0025] The present invention also relates to a novel hyaluronidase variant or a fragment thereof, wherein the native hyaluronidase is human native Hyal1.

[0026] Preferably, human native Hyal1 or a fragment thereof may have the amino acid sequence of the sequence of SEQ ID NO: 1, or may have the amino acid sequence of F22 - W435 in the sequence of SEQ ID NO: 1, but is not limited thereto.

[0027] The present invention also relates to a novel hyaluronidase variant or a fragment thereof, wherein the novel hyaluronidase variant exhibits hyaluronidase activity at neutral pH.

[0028] The present invention relates to a novel hyaluronidase variant or a fragment thereof, wherein the enzyme active site is D129 and / or E131 of SEQ ID NO: 1.

[0029] The present invention also relates to a novel hyaluronidase variant or a fragment thereof, wherein the amino acid at one or more positions selected from the group consisting of Q41, W42, G63, S76, S77, Q78, T81, F93, R108, F110, Q111, A115, A116, A132, F139, D142, T143, R148, V170, Q172, G177, A181, Q188, R191, Y210, F212, L213, S214, N216, T218, Q220, Q228, P249, V251, Q263, A267, Q288, D292, T293, T294, H296, H305, L307, L356, R372, L379, G396, A403, M412, E415 and K417 in the amino acid sequence of SEQ ID NO: 1, or at one or more positions corresponding to the above amino acids in the amino acid sequence of mature human Hyal1, is substituted.

[0030] Preferably, the present invention relates to a novel hyaluronidase mutant or section thereof, in which an amino acid is substituted at one or more positions selected from the group consisting of S76, S77, Q78, A132, D142, T143, Y210, F212, L213, P249, V251, Q288, and D292 in the amino acid sequence of Sequence ID No. 1, or at one or more positions in the amino acid sequence of mature human Hyal1 that correspond to the aforementioned amino acids.

[0031] More preferably, the present invention relates to a novel hyaluronidase mutant or section thereof, wherein the amino acid substitution includes an amino acid residue substitution at D142 or P249 in the amino acid sequence of SEQ ID NO: 1, and is further substituted at one or more positions selected from the group consisting of S77, Q78, A132, D142, T143, Y210, F212, P249 and V251, or at one or more amino acid positions in the amino acid sequence of mature human Hyal1 that correspond to the aforementioned amino acids.

[0032] Specifically, the present invention relates to the amino acid sequence of SEQ ID NO: Q41E, W42F, G63R, S76V, S77D, Q78R, T81Y, F93N, R108K, F110K, Q111K, A115F, A116Y, A132E, F139R, D142K, T143P, R148K, V170K, Q172K, G177K, A181D, Q188K, R191K, Y210H, F212Y, L213K, S214K, N216G, T218N, Q220S, Q228R, P249N, V This relates to a novel hyaluronidase mutant or section thereof, comprising one or more amino acid substitutions selected from the group consisting of 251Q, Q263R, A267R, Q288R, D292T, T293D, T294Q, H296K, H305Y, L307F, L356K, R372K, L379Y, G396K, A403K, M412F, E415K, and K417Y, or an amino acid substitution at one or more amino acid positions in the amino acid sequence of mature human Hyal1 that correspond to the aforementioned amino acids.

[0033] Preferably, the present invention relates to a novel hyaluronidase mutant or section thereof, comprising one or more amino acid substitutions selected from the group consisting of S76V, S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, L213K, P249N, V251Q, Q288R, and D292T in the amino acid sequence of Sequence ID No. 1, or an amino acid substitution at one or more amino acid positions in the amino acid sequence of mature human Hyal1 that correspond to the aforementioned amino acids.

[0034] More preferably, the present invention relates to a novel hyaluronidase mutant or section thereof, comprising a substitution of the amino acid residue D142K or P249N in the amino acid sequence of SEQ ID NO: 1, and one or more amino acid substitutions selectively selected from the group consisting of S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, P249N and V251Q, or further comprising an amino acid substitution at one or more amino acid positions in the amino acid sequence of mature human Hyal1 that correspond to the aforementioned amino acids.

[0035] Furthermore, the present invention relates to a composition for the treatment of diseases, preferably cancer, comprising the novel hyaluronidase variant described above or a section thereof.

[0036] Furthermore, the present invention relates to nucleic acids that encode the novel hyaluronidase variants or sections thereof described above.

[0037] Furthermore, the present invention relates to a recombinant expression vector containing the nucleic acid.

[0038] Furthermore, the present invention relates to host cells transformed with the recombinant expression vector.

[0039] Furthermore, the present invention relates to a host cell characterized in that the host cell is selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0040] Furthermore, the present invention relates to a method for producing a novel hyaluronidase variant or a section thereof, which includes the step of culturing the host cells. [Brief explanation of the drawing]

[0041] [Figure 1] This diagram shows the superimposed protein structures of hyaluronidase Hyal1 and hyaluronidase PH20. A confirms that the active sites of Hyal1 and PH20 are identical, B confirms that the binding sites for the substrate hyaluronic acid are identical in Hyal1 and PH20, and C identifies amino acids in the peripheral regions of the active sites and / or substrate binding sites of Hyal1 and PH20 that are closely involved in enzyme activity and / or substrate binding. [Figure 2] The results of quantitative analysis using SDS-PAGE on samples prepared and purified from recombinant hyaluronidase Hyal1 and Hyal1-E131H mutants are described in Example 1. A is the SDS-PAGE result for the purified sample, B is the quantitative curve created using BSA, and C is the quantitative result for purified samples 1 and 2. [Figure 3] This figure shows the results of measuring the enzyme activity of recombinant hyaluronidase Hyal1 and recombinant hyaluronidase mutant Hyal1-E131H at different pH levels. (A) Results of measuring the enzyme activity of Hyal1 at different pH levels. (B) Results of measuring the enzyme activity of Hyal1-E131H at different pH levels. [Modes for carrying out the invention]

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by experts skilled in the art to which the invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0043] The present invention provides a hyaluronidase mutant or section thereof in which the amino acid sequence of a natural hyaluronidase, preferably a mature natural hyaluronidase, includes the substitution of one or more amino acid residues at a site corresponding to the active site and / or its linking site, preferably at D129, E131 and / or adjacent sites of Hyal1, and additionally, a portion of the N-terminal and / or C-terminal amino acid residues are cleaved.

[0044] In the present invention, the positions of amino acid residues in each mutant follow the amino acid positions of the native hyaluronidase Hyal1 according to Sequence ID No. 1.

[0045] Furthermore, in the present invention, "mature natural hyaluronidase" means a protein consisting of amino acid residues F22 to W435 of SEQ ID NO: 1, in which signal peptides M1 to G21 are deleted from the amino acid sequence of the natural hyaluronidase Hyal1 of SEQ ID NO: 1.

[0046] In this invention, based on the protein tertiary structure of the human hyaluronidase Hyal1 (SEQ ID NO: 1), we selected amino acids located at the active sites D129 and E131 and their vicinity, and attempted to mutate them so that the enzyme could catalyze even at neutral pH. In particular, the E131H mutant showed a tendency for increased activity at pH 6 (see Figure 3). In this invention, we investigated the possibility of providing new hyaluronidase mutants or sections thereof that are different from the natural pH 20 and its mutants, through various mutations of the active site and / or substrate binding site and / or adjacent sites of the human hyaluronidase Hyal1.

[0047] Accordingly, the novel hyaluronidase variant according to the present invention is characterized in that, in the natural hyaluronidase Hyal1 (hyaluronidase having the amino acid sequence of SEQ ID NO: 1), preferably in the mature natural hyaluronidase Hyal1 (a protein consisting of F22 to W435 in the amino acid sequence of SEQ ID NO: 1), the active site D129 and / or E131 and / or some amino acid residues adjacent thereto are substituted with other amino acid residues, but is not limited thereto.

[0048] In the case of hyaluronidase Hyal1, the X-ray diffraction crystal structure has been elucidated, and the protein structure has been deposited in the public database Protein Data Bank (https: / / www.rcsb.org / ) with ID number: 2PE4. Furthermore, while there is no X-ray diffraction crystal structure for hyaluronidase PH20, which is active at neutral pH, the protein structure estimated by DeepMind (USA) and the European Bioinformatics Laboratory (EMBL-EBI) has been deposited in the public database Alphafold Protein Database (https: / / alphafold.ebi.ac.uk / ). Structural comparison studies of the active sites of these proteins are conducted as shown in Figure 1. Through this, possible amino acid substitution mutants are secured, and the objectives of this invention are achieved by conducting subsequent studies on these mutants.

[0049] Table 1 below shows the amino acid sequences of natural human Hyal1, Hyal2, Hyal3, Hyal4, and PH20. [Table 1]

[0050] In this invention, expressions such as "E131," which consist of a single-letter amino acid residue name and a number, refer to the amino acid residue at each position in the amino acid sequence specified by the sequence number. For example, "E131" in sequence number 1 means that the amino acid residue at position 131 in the amino acid sequence is glutamic acid.

[0051] Furthermore, "E131H" in Sequence ID No. 1 means that the 131st amino acid, glutamic acid, in Sequence ID No. 1 has been replaced with histidine.

[0052] In addition to D129 and E131, Y75, Y202, Y247, and W321 are known to participate in the active site of hyaluronidase Hyal1 (Chao KL2007), and these are conserved in another hyaluronidase, PH20. Furthermore, the amino acids involved in the binding of the substrate, hyaluronic acid, are also conserved in both proteins. These amino acids in the active site, the site involved in substrate binding, and the amino acids near these two sites can influence the pH environment of enzyme activity.

[0053] The novel hyaluronidase variants according to the present invention can be modified at any position in the amino acid sequence of Hyal1 of SEQ ID NO: 1, but preferably Q41, W42, G63, S76, S77, Q78, T81, F93, R108, F110, Q111, A115, A116, A132, F139, D142, T143, R148, V170, Q172, G177, A181, Q188, R191, Y210, F212, L213, S214, N216, T218, Q220, Q228, P249, V25 Mutations are possible, but not limited to, at one or more positions selected from the group consisting of 1, Q263, A267, Q288, D292, T293, T294, H296, H305, L307, L356, R372, L379, G396, A403, M412, E415 and K417, preferably at one or more positions selected from the group consisting of S76, S77, Q78, A132, D142, T143, Y210, F212, L213, P249, V251, Q288 and D292, particularly amino acid substitutions.

[0054] More preferably, the present invention relates to a novel hyaluronidase mutant or section thereof, wherein the substitution of one or more amino acid residues includes a substitution of an amino acid residue at D142 or P249 in the amino acid sequence of SEQ ID NO: 1, and further amino acids are substituted at one or more positions selected from the group consisting of S77, Q78, A132, D142, T143, Y210, F212, P249 and V251.

[0055] More preferably, the novel hyaluronidase variant according to the present invention is one in which the amino acid sequence of the natural hyaluronidase Hyal1 of SEQ ID NO: Q41E, W42F, G63R, S76V, S77D, Q78R, T81Y, F93N, R108K, F110K, Q111K, A115F, A116Y, A132E, F139R, D142K, T143P, R148K, V170K, Q172K, G177K, A181D, Q188K, R191K, Y210H, F212Y, L213K, S214K, N216G, T218N, Q220S, Q228R, P249N, V251Q, Q Mutants are possible, but not limited to, those comprising one or more amino acid substitutions selected from the group consisting of 263R, A267R, Q288R, D292T, T293D, T294Q, H296K, H305Y, L307F, L356K, R372K, L379Y, G396K, A403K, M412F, E415K, and K417Y, more preferably one or more amino acid substitutions selected from the group consisting of S76V, S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, L213K, P249N, V251Q, Q288R, and D292T.

[0056] More preferably, the novel hyaluronidase variant according to the present invention includes a substitution of the amino acid residue D142K or P249N in the amino acid sequence of SEQ ID NO: 1, and may further include one or more amino acid substitutions selectively selected from the group consisting of S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, P249N, and V251Q.

[0057] The hyaluronidase variants according to the present invention are interpreted to include each variant in which an amino acid residue is conservatively substituted at the specific amino acid residue position.

[0058] In this specification, “conservative substitution” means a variant modification comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not cause a loss of the biological or biochemical function of the variant.

[0059] "Conservative amino acid substitution" is the substitution of an amino acid residue with an amino acid residue having a similar side chain. The categories of amino acid residues having similar side chains are defined in the art and are well known. These categories include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0060] The hyaluronidase variants of the present invention are expected to retain their activity even when they have conservative amino acid substitutions.

[0061] The novel hyaluronidase variant according to the present invention may be, but is not limited to, a section in which some amino acids are deleted, for example, a section in which a signal peptide is deleted or removed.

[0062] Preferably, human natural Hyal1 or its slices have the amino acid sequence of Sequence ID No. 1, or the amino acid sequence of F22 to W435 in Sequence ID No. 1, and additionally include the aforementioned amino acid substitutions, but are not limited thereto.

[0063] In other aspects, the present invention can provide a disease treatment composition comprising the hyaluronidase variant of the present invention and a disease treatment method using the same.

[0064] The disease treatment composition may be a pharmaceutical composition. The pharmaceutical composition may further contain a pharmaceutically acceptable carrier, which may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., and may not be limited thereto. Furthermore, the pharmaceutical composition may further contain one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives, which are commonly used in the manufacture of pharmaceutical compositions.

[0065] The pharmaceutical composition may be administered orally or parenterally. Parenteral administration may include intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, endothelial infusion, local infusion, intranasal infusion, intrapulmonary infusion, and intrarectal infusion. When administered orally, since proteins or peptides are digested, the oral composition may be formulated to coat the active agent or protect it from digestion in the stomach. The composition may also be administered by any device that allows the active substance to move to target cells.

[0066] The pharmaceutical composition may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or it may be in the form of an extract, powder, granule, tablet, or capsule, and may further contain a dispersant or stabilizer for dosage form.

[0067] In particular, the therapeutic composition according to the present invention is characterized by being used alone or in combination with other therapeutic agents.

[0068] In other words, the present invention relates to a hyaluronidase variant or a nucleic acid coding for such variant.

[0069] The nucleic acids used herein may be present in cells or cell lysates, or in partially purified or substantially pure forms. If the nucleic acids are purified from other cellular components or other contaminants, such as nucleic acids or proteins from other cells, according to standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art, they are “isolated” or “substantially purified.” The nucleic acids of the present invention may be, for example, DNA or RNA.

[0070] In further aspects, the present invention relates to a vector comprising the nucleic acid. For the expression of a hyaluronidase variant or section thereof according to the present invention, DNA encoding the hyaluronidase variant can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the hyaluronidase variant), and the DNA can be "operationally bound" to transcriptional and translational regulatory sequences and inserted into an expression vector.

[0071] As used herein, the term “binding to function” may mean that the gene coding the hyaluronidase variant or its section is ligated into the vector such that the transcriptional and translational regulatory sequences within the vector perform the intended function of regulating the transcription and translation of the gene coding the hyaluronidase variant or its section. The expression vector and expression regulatory sequences are selected to be compatible with the host cells used for expression. The gene coding the hyaluronidase variant is inserted into the expression vector by a standard method (e.g., ligation of the gene fragment coding the hyaluronidase variant or its section and a complementary restriction enzyme site on the vector, or, if no restriction enzyme site is present, blunt-terminus ligation).

[0072] Furthermore, the recombinant expression vector has a regulatory sequence that controls the expression of the gene coding the hyaluronidase variant in the host cell. The "regulatory sequence" may include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals) that control the transcription or translation of the gene coding the hyaluronidase variant or its section. A typical engineer can recognize that the design of the expression vector may change depending on factors such as the selection of the host cell to be transformed and the level of protein expression, by selecting different regulatory sequences.

[0073] In further aspects, the present invention relates to a host cell comprising the nucleic acid or the vector. The host cell according to the present invention is preferably selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells, but is not limited thereto.

[0074] Specifically, the host cells according to the present invention may be prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, or Staphylococcus sp. Furthermore, the host cells according to the present invention may be eukaryotic cells such as fungi like Aspergillus sp., yeasts like Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp., and Neurospora crassa, other lower eukaryotic cells, and cells of higher eukaryotes such as insect cells.

[0075] Furthermore, the host cells according to the present invention may be derived from plants or mammals. Preferably, the host cells according to the present invention include monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT78 cells, and HEK293 cells, but are not limited thereto, and CHO cells may be used in particular.

[0076] The nucleic acid or vector is transfected into a host cell. For "transfecting" or "transfecting," a variety of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells can be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. A variety of expression host / vector combinations can be used to express the PH20 variant or its section according to the present invention. Suitable expression vectors for eukaryotic hosts include, but are not limited to, SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus-derived expression regulatory sequences. Expression vectors usable in bacterial hosts include pET, pRSET, pBluescript, pGEX2T, pUC vectors, bacterial plasmids obtained from Escherichia coli such as col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids with a broader host range such as RP4, phage DNA which can be exemplified by a wide variety of phage lambda derivatives such as λ and λNM989, and other DNA phages such as M13 and filamentous single-strand DNA phages. Useful expression vectors for yeast cells are 2°C plasmids and their derivatives. A useful vector for insect cells is pVL941.

[0077] In another aspect, the present invention relates to a method for producing a hyaluronidase variant or a section thereof according to the present invention, comprising the step of culturing host cells to express a hyaluronidase variant or a section thereof according to the present invention.

[0078] When a recombinant expression vector capable of expressing the hyaluronidase mutant or a section thereof is introduced into mammalian host cells, the hyaluronidase mutant or the mutant thereof can be produced by culturing the host cells for a period of time sufficient for expression in the host cells, or more preferably, for a period of time sufficient for the hyaluronidase mutant to be secreted into the culture medium in which the host cells are cultured.

[0079] Depending on the circumstances, the expressed hyaluronidase variant can be uniformly purified after isolation from host cells. The isolation or purification of the hyaluronidase variant can be performed using conventional protein isolation or purification methods, such as chromatography. This chromatography may be, but is not limited to, one or more combinations selected from, for example, affinity chromatography, ion exchange chromatography, or hydrophobic chromatography. In addition to chromatography, methods such as filtration, ultrafiltration, salting out, and dialysis may be used in combination. [Examples]

[0080] The present invention will be described in more detail below through examples. It will be obvious to those ordinary in the art that these examples are for illustrative purposes only and that the scope of the present invention should not be construed as being limited by these examples. [Example 1] Production of hyaluronidase Hyal1 and its variants [Example 1-1.] Cloning of hyaluronidase Hyal1 and its variants To produce recombinant Hyal1, we purchased the cDNA of native Hyal1 (clone ID: hMU005315) from the Human Gene Bank of Korea. Mature native Hyal1 encodes amino acids F22 to W435. After amplifying the Hyal1 gene using polymerase chain reaction (PCR), we inserted it into the XhoI and NotI restriction enzyme sites of the pcDNA3.4-TOPO vector. For expression in ExpiCHO cells, we used the signal peptide of human Hyal1. Additionally, to purify the protein using a HisTrap column, we positioned a 6xHis tag DNA sequence at the 3' end of the Hyal1 cDNA. The amino acid substitution method for the Hyal1 mutant was used, and DNA sequencing was used to confirm the amino acid substitutions.

[0081] Table 2 lists the primers used for Hyal1 cloning, and Table 3 details their specific sequences.

[0082] [Table 2]

[0083] [Table 3] JPEG2026511560000005.jpg50153

[0084] [Examples 1-2.] Purification of hyaluronidase Hyal1 and Hyal1-E131H mutants Hyal1 recombinant protein and E131H mutant produced in ExpiCHO cells were purified using AKTA prime (Cytiva, USA) via the following three-step column chromatography procedure.

[0085] Tris was added to the culture supernatant and titrated to pH 10.0, then injected into a Blue Sepharose 6 Fast Flow resin equilibrated with buffer A (10 mM glycine, pH 10). After washing off impurities with buffer A, the target protein was eluted with buffer B (10 mM glycine, 1 M NaCl, pH 10). Next, the fraction collected for purification of the CM-650M column was dialyzed to buffer C (50 mM NaPi, 0.1% Triton X-100, pH 6.0).

[0086] The fraction collected using a Blue Sepharose 6 fast-flow column was injected into CM-650M resin equilibrated with buffer D (50 mM NaPi, 0.1% Triton X-100, pH 6.0). After washing off impurities with buffer D, the target protein was eluted using buffer E (50 mM NaPi, 0.1% Triton X-100, 0.5 M NaCl, pH 6.0). Next, the fraction collected for purification of the DEAE Sepharose fast-flow column was dialyzed to buffer F (10 mM NaPi, pH 7.0).

[0087] Each fraction collected on the CM-650M column was injected into DEAE Sepharose fast-flow resin equilibrated with buffer G (5 mM K2HPO4, pH 7.1). In this step, after sample loading was completed in FT (Flow-through) mode, buffer G was additionally flowed to ensure that all target proteins in the column were recovered. Subsequently, each impurity was stripped using buffer H (5 mM K2HPO4, 1 M NaCl, pH 7.1). The DEAE Sepharose fast-flow column was purified twice using the fractions (#4-6, #8-14) collected in the CM-650M step, and the samples secured in the FT interval of each step were named Sample 1 and Sample 2. Each sample collected in the purification stage was subjected to electrophoresis at 150 Volt for 1 hour using a 10% acrylamide gel.

[0088] Samples 1 and 2, collected using a DEAE Sepharose fast-flow column, were dialyzed into buffer I (20 mM NaPi, 77 mM NaCl, pH 7.0) and concentrated using a stird cell. The concentrated samples were electrophoresed together with bovine serum albumin (BSA) as a standard on a 10% acrylamide gel, and the concentration of the target protein was calculated using band intensity obtained from the CS analysis program [Figure 2].

[0089] [Examples 1-3] Purification of HIS-labeled hyaluronidase Hyal1 and its variants.

[0090] Hyal1 mutant polypeptides with a His tag at the C-terminus, produced from ExpiCHO cells, underwent a two-step purification process using AKTA prime equipment or similar equipment (GE Healthcare), employing Q Sepharose for anion exchange column chromatography and HisTrap HP columns for His-tagged affinity chromatography.

[0091] 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 NaCl) were prepared. Proteins were bound to the Q Sepharose column, and nonspecifically bound proteins were removed by passing buffer A through the column at a rate of 5 CV. Then, the proteins were eluted by passing buffer B through the column at a rate of 5 CV with a concentration gradient from 0% to 100%.

[0092] For protein purification using a HisTrap HP column, buffer A (20 mM sodium phosphate, 500 mM NaCl, pH 7.5) and buffer B (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5) were prepared. After binding the protein sample to the HisTrap HP column, 7% buffer B was passed through for 7 CV to remove nonspecifically bound proteins, and 40% buffer B was passed through for 3 CV to elute the target protein. The column eluate was dialyzed using dialysis buffer (20 mM sodium phosphate, 100 mM NaCl, pH 7.0).

[0093] [Example 2] Measurement of hyaluronidase mutant activity Hyaluronidase activity was measured by turbidiometry, a method that measures the absorbance of the turbidity generated by the precipitate formed when hyaluronic acid is mixed with albumin (BSA). When hyaluronic acid is hydrolyzed by a pH20 polypeptide, the turbidity / absorbance of the precipitate formed when mixed with albumin decreases. This analysis is generally performed at pH 5.3 as follows: Hyaluronidase standards with known activity (units) were diluted to 6 units / mL, 8 units / mL, 10 units / mL, 12 units / mL, 15 units / mL, and 20 units / mL and prepared in separate test tubes. The purified protein samples were diluted in buffer (20 mM sodium phosphate, pH 5.3, 77 mM sodium chloride, and 0.01% (w / v) bovine serum albumin) by adjusting the dilution factors to fall within the standard curve range. 50 μl of diluted sample was dispensed into each well of a 96-well plate and incubated at 37°C for 10 minutes. An additional 50 μl of 0.06% hyaluronic acid was dispensed into each well. The 0.06% hyaluronic acid was dissolved in 300 mM sodium phosphate buffer, pH 5.3. The sample and 0.06% hyaluronic acid were reacted at 37°C for 45 minutes. After the reaction, 40 μl of the enzyme-substrate reaction solution was dispensed into 200 μl of acidic albumin solution and left at room temperature for 19 minutes. The absorbance was then measured at 600 nm using a spectrophotometer. The acidic albumin solution was a solution of 0.1% albumin (BSA) dissolved in 24 mM sodium acetate, 79 mM acetic acid, and a pH 3.75 buffer. The measured absorbance values ​​of the sample were converted to activity using a standard curve based on the activity standard.

[0094] When the above process was performed at pH 7.0, the protein sample buffer used was 20 mM sodium phosphate, pH 7.0, 77 mM sodium chloride, and 0.01% (w / v) bovine serum albumin. For the 0.06% hyaluronic acid aqueous solution, a solution prepared by dissolving it in 20 mM sodium phosphate buffer, pH 7.0, and 70 mM sodium chloride was used, and the same process was carried out.

[0095] While this type of activity measurement is also possible using culture medium, in this case, values ​​below 300 units / mL are unreliable. Therefore, the limit of quantification (LOQ) was set to 300 units / mL, and values ​​below this were displayed as no activity or 0. Furthermore, when measuring activity using purified protein samples, the limit of quantification (LOQ) was set to 15 units / μg.

[0096] [Example 3] Analysis of activity analysis results of hyaluronidase mutants

[0097] The pH-dependent enzyme activity of the natural Hyal1 (SEQ ID NO: 1) and the Hyal1-E131H mutant containing the E131H amino acid substitution was investigated.

[0098] The results are shown in Figure 3. Figure 3A shows the results of measuring the enzyme activity of Hyal1 at different pH levels. Figure 3B shows the results of measuring the enzyme activity of the Hyal1-E131H mutant at different pH levels. It was confirmed that the enzyme activity of Hyal1 is high at acidic pH levels but almost absent at neutral pH levels. Furthermore, in the case of the Hyal1-E131H mutant, the enzyme activity increases at neutral pH levels compared to acidic pH levels, but it shows relatively very low enzyme activity, indicating low industrial utility.

[0099] Based on these results, the design of mutants was extended to mutants near the enzyme active site, and the present invention was carried out using this combination. The characteristics and sequences of the novel hyaluronidase Hyal1 mutant according to the present invention are shown in Tables 4 and 5.

[0100] As shown in Table 4, the novel hyaluronidase Hyal1 variant according to the present invention exhibits excellent enzymatic activity under pH 5.3 and pH 7.0 conditions. In particular, it was confirmed that it has extremely excellent enzymatic activity when it contains the amino acid substitution D142K, or further contains one or more amino acid substitutions selected from the group consisting of S77D, Q78R, A132E, T143P, Y210H, F212Y, P249N, and V251Q.

[0101] [Table 4]

[0102] [Table 5] JPEG2026511560000008.jpg244140JPEG2026511560000009.jpg245140JPEG2026511560000010.jpg245140JPEG2026511560000011.jpg147140 [Industrial applicability]

[0103] The natural hyaluronidase variant according to the present invention has a structure similar to that of mature natural PH20 and exhibits the effect of increased protein expression and enzyme activity at neutral pH.

Claims

1. A novel hyaluronidase mutant or section thereof that exhibits hyaluronidase activity, comprising amino acid residue substitutions at one or more positions in the enzymatic active site, substrate binding site, and / or adjacent sites of a natural hyaluronidase.

2. The novel hyaluronidase variant or section thereof according to claim 1, wherein the aforementioned natural hyaluronidase is mature human natural hyaluronidase.

3. The novel hyaluronidase mutant or section thereof according to claim 1, characterized in that the mature human native hyaluronidase is a form of human native hyaluronidase in which the signal peptide is deleted.

4. The novel hyaluronidase variant or section thereof according to claim 1, wherein the aforementioned natural hyaluronidase is human natural Hyal1.

5. The novel hyaluronidase variant or section thereof according to claim 4, wherein the natural hyaluronidase consists of the amino acid sequence F22 to W435 of the sequence of SEQ ID NO: 1, or the sequence of SEQ ID NO:

1.

6. The novel hyaluronidase mutant described above exhibits hyaluronidase activity at a neutral pH, or a section thereof, according to any one of claims 1 to 5.

7. The novel hyaluronidase mutant or section thereof according to claim 1, wherein the enzyme active site is D129 and / or E131 of SEQ ID NO:

1.

8. Among the amino acid sequences of Sequence ID No. 1, Q41, W42, G63, S76, S77, Q78, T81, F93, R108, F110, Q111, A115, A116, A132, F139, D142, T143, R148, V170, Q172, G177, A181, Q188, R191, Y210, F212, L213, S214, N216, T218, Q220, Q228, P249, V251, Q263, A267, Q288, D2 A novel hyaluronidase variant or section thereof according to claim 1, wherein one or more amino acids are substituted at positions selected from the group consisting of 92, T293, T294, H296, H305, L307, L356, R372, L379, G396, A403, M412, E415, and K417, or one or more amino acids are substituted at positions in the amino acid sequence of mature human Hyal1 that correspond to the aforementioned amino acids.

9. A novel hyaluronidase variant or section thereof according to claim 8, wherein one or more amino acids are substituted at positions selected from the group consisting of S76, S77, Q78, A132, D142, T143, Y210, F212, L213, P249, V251, Q288, and D292, or one or more amino acids are substituted at amino acid positions corresponding to the aforementioned amino acids in the amino acid sequence of mature human Hyal1.

10. A novel hyaluronidase mutant or section thereof according to claim 8, comprising an amino acid residue substitution at D142 or P249, wherein an amino acid is further substituted at one or more positions selected from the group consisting selectively of S77, Q78, D142, A132, T143, Y210, F212, P249 and V251, or at one or more amino acid positions in the amino acid sequence of mature human Hyal1 that correspond to the aforementioned amino acid.

11. Q41E, W42F, G63R, S76V, S77D, Q78R, T81Y, F93N, R108K, F110K, Q111K, A115F, A116Y, A132E, F139R, D142K, T143P, R148K, V 170K, Q172K, G177K, A181D, Q188K, R191K, Y210H, F212Y, L213K, S214K, N216G, T218N, Q220S, Q228R, P249N, V251Q, Q263R, A novel hyaluronidase variant or section thereof according to claim 8, comprising one or more amino acid substitutions selected from the group consisting of A267R, Q288R, D292T, T293D, T294Q, H296K, H305Y, L307F, L356K, R372K, L379Y, G396K, A403K, M412F, E415K, and K417Y, or an amino acid substitution at one or more positions in the amino acid sequence of mature human Hyal1 corresponding to the aforementioned amino acids.

12. A novel hyaluronidase variant or section thereof according to claim 11, comprising one or more amino acid substitutions selected from the group consisting of S76V, S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, L213K, V251Q, P249N, Q288R, and D292T, or an amino acid substitution at one or more positions in the amino acid sequence of mature human Hyal1 corresponding to the aforementioned amino acids.

13. A novel hyaluronidase variant or section thereof according to claim 11, comprising the amino acid substitution of D142K or P249N, further comprising one or more amino acid substitutions selectively selected from the group consisting of S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, P249N and V251Q, or further comprising amino acid substitutions at one or more positions in the amino acid sequence of mature human Hyal1 corresponding to the aforementioned amino acids.

14. A therapeutic composition comprising a novel hyaluronidase variant or a section thereof as described in any one of claims 1 to 13.

15. A nucleic acid encoding a novel hyaluronidase variant or a section thereof, as described in any one of claims 1 to 13.

16. A recombinant expression vector comprising the nucleic acid described in claim 15.

17. Host cells transformed with the recombinant expression vector according to claim 16.

18. The host cell according to claim 17, characterized in that the host cell is selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells.

19. A method for producing a novel hyaluronidase mutant or a section thereof, comprising the step of culturing the host cells described in claim 18.

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