Novel hyaluronic acid-hydrolyzing enzyme variant and pharmaceutical composition comprising the same

Abstract

To provide a novel hyaluronidase PH20 variant or fragment thereof, exhibiting enhanced thermal stability, enzymatic activity, and expression rate compared to the mature wild-type PH20.SOLUTION: The present invention provides a hyaluronidase PH20 variant or fragment thereof, which comprises one or more amino acid residue substitutions in the region corresponding to an α-helix region and / or its linker region in a specific wild-type PH20 amino acid sequence, and selectively, additional N-terminal and / or C-terminal amino acid residue deletions.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to novel human hyaluronidase variants that have improved enzymatic activity and thermal stability of human hyaluronidase, a hyaluronan hydrolase. Specifically, the present invention relates to wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, preferably a hyaluronidase PH20 variant that includes a substitution of one or more amino acid residues at the α-helix region of mature wild-type PH20 consisting of amino acid residues L36 to S490, and / or a site corresponding to the junction site thereof, and optionally a portion of the amino acid residues at the N-terminus and / or C-terminus are deleted, or a fragment thereof, a method for producing the variant, and a pharmaceutical composition containing the variant. [Background technology]

[0002] Human skin is composed of the epidermis, dermis, and subcutaneous fat layer, and contains six types of glycosaminoglycans: hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratin sulfate.

[0003] Glycosaminoglycans have a structure in which disaccharide-type sugars are repeatedly linked. The length of the sugars varies from glycosaminoglycan to glycosaminoglycan, but can range from several hundred to several thousand. Among glycosaminoglycans, more than half of the hyaluronic acid present in the body is present in the skin. Hyaluronic acid is synthesized by hyaluronan synthase present in the cell membrane, and exists alone without being bound to proteoglycan, and is the only glycosaminoglycan without sulfate groups. Other glycosaminoglycans are bound to proteoglycans and have sulfate groups. Hyaluronic acid is formed by linking glucuronic acid and N-acetylglucosamine with β-1,3 bonds, and such disaccharides are repeated approximately 5,000 times. N-acetylglucosamine and glucuronic acid are linked with β-1,4 bonds. It is known that approximately one-third (5 g) of hyaluronic acid is broken down in the human body every day.

[0004] Hyaluronidase is an enzyme that breaks down hyaluronan located in the extracellular matrix. There are six known types of hyaluronidase in humans (Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20 / SPAM1). In humans, Hyal1 and Hyal2 are expressed in most tissues, and PH20 / SPAM1 (hereinafter also referred to as PH20) is expressed in the plasma membrane and acrosome membrane of sperm. However, HyalPS1 is not expressed as a pseudogene. Hyaluronidases are divided into three types based on the method by which they cleave hyaluronan. That is, they are divided into enzymes that cleave the β-1,4 bond between N-acetylglucosamine and glucuronic acid using HO (EC 3.2.1.35), enzymes that cleave the β-1,3 bond (EC 3.2.1.36), and bacterial hyaluronidases that cleave the β-1,4 bond without using HO (EC 4.2.99.1).

[0005] The catalytic amino acid residues of Hyal1 are D129 and E131, and it hydrolyzes hyaluronic acid by substrate-assisted catalysis. Hyal1 shows optimal activity at acidic pH 3-4, and has no enzymatic activity above pH 4.5. Unlike Hyal1, PH20 shows enzymatic activity over a wide pH range from pH 3 to 8.

[0006] Arming et al. revealed that the catalytic amino acids of PH20 are D111 and E113 (Arming et al., 1997). Since the first amino acid, Leu, was designated as number 1, the catalytic amino acid residues in the signal sequence of PH20 correspond to D146 and E148, respectively.

[0007] Hyaluronidase hydrolyzes hyaluronic acid, reducing the viscosity of hyaluronic acid in the extracellular matrix and increasing its permeability to tissues (skin). Since the subcutaneous part of the skin has a neutral pH of approximately 7.0 to 7.5, PH20 is used clinically among various hyaluronidases (Bookbinder et al., 2006). Examples of clinical uses of PH20 include its use as an eye relaxant and an additive to anesthesia injections in ophthalmic surgery, and its co-administration with antibody therapeutics when they are injected subcutaneously (Bookbinder et al., 2006). In addition, hyaluronic acid in the extracellular matrix of tumor cells is overexpressed in tumor cells, and PH20 is used to hydrolyze hyaluronic acid with PH20 to increase the accessibility of anticancer therapeutics to tumor cells. It is also used to promote the reabsorption of excess body fluids and blood in tissues.

[0008] PH20 was first identified in guinea pig sperm by Lathrop et al. and is known to be expressed in sperm of many other species. The human PH20 gene was cloned by Lin et al. and Gmachl et al. Human PH20 has the amino acid sequence of SEQ ID NO: 1, which is composed of 509 amino acid residues, and has 60% amino acid sequence identity with guinea pig PH20. Human PH20 enzyme is encoded by the SPAM1 (sperm adhesion molecule-1) gene and exists on the surface of the sperm cell membrane and on the inside of the acrosomal membrane in a form in which Ser490 of PH20 is bound to glycosylphosphatidylinositol (GPI). When sperm penetrate the cumulus layer of the egg, which is rich in hyaluronan, and penetrate into the egg, they hydrolyze hyaluronan using PH20. PH20 accounts for less than 1% of the protein mass in sperm and has six N-glycosylation sites (N82, N166, N235, N254, N368, and N393).

[0009] Currently, the most commonly used commercially available forms of PH20 are extracted from cow or sheep hyaluronidase, such as Amphadase® (bovine hyaluronidase) and Vitrase® (ovine hyaluronidase).

[0010] Bovine testicular hyaluronidase (BTH) is a form of wild-type bovine PH20 in which the signal peptide and 56 amino acids at the C-terminus have been removed during the post-translational modification process. BTH is also a glycoprotein, and its total components, including amino acids, are 5% mannose and 2.2% glucosamine. When animal-derived hyaluronidase is repeatedly administered to the human body in high doses, neutralizing antibodies can be produced. Animal-derived hyaluronidase contains other biological substances in addition to PH20, which can cause allergic reactions when administered to the human body (Bookbinder et al., 2006). In particular, the production and use of PH20 extracted from bovine animals is restricted due to concerns about mad cow disease. To address these issues, research on recombinant human PH20 protein has been progressing.

[0011] Recombinant human PH20 protein has been reported to be expressed in yeast (P. pastoris), DS-2 insect cells, animal cells, etc. The recombinant PH20 protein produced in insect cells and yeast differs from human PH20 in the N-glycosylation pattern during the post-translational modification process of the protein.

[0012] 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, and the catalytic domain has a (β / α)8 configuration in which eight α-helices and eight β-strands are repeated, characterizing the secondary structure of the protein (Chao et al., 2007). The EGF-like domain is conserved in all C-terminal differentially spliced ​​variants of Hyal1. The amino acid sequences of Hyal1 and PH20 are 35.1% identical, and the protein structure of PH20 has not yet been elucidated.

[0013] Recombinant human PH20 protein has been developed by Halozyme Therapeutic and is marketed under the trade name Hylenex (Bookbinder et al., 2006; Frost, 2007).

[0014] Mutation of the catalytic amino acids D146 and E148 of PH20 to asparagine (D146N) and glutamine (E148Q), respectively, resulted in no enzymatic activity (Arming et al., 1997). Substitution of R246 with glycine reduced the enzymatic activity by 90%, and substitution of E319 with glutamine and R322 with threonine abolished the enzymatic activity. A PH20 mutant with 36 amino acids deleted from the C-terminus (474-509 amino acid truncation) had 75% less enzymatic activity than wild-type PH20. This mutant was not secreted outside the cells and remained in HeLa cells. Deletion of 134 amino acids from the C-terminus of PH20 resulted in no enzymatic activity of PH20 and no secretion outside the cells. According to Frost et al., the C-terminal 477-483 region of PH20 is essential for soluble expression (Frost, 2007). The activity of full-length PH20 (1-509) or a PH20 mutant truncated at the C-terminus 467 was only 10% of that of a PH20 mutant truncated at any of the C-terminus 477-483 regions (Frost, 2007).

[0015] On the other hand, the thermostability and expression rate of recombinant PH20 are still insufficient, and there is a great demand for hyaluronidase with improved properties. Summary of the Invention [Problem to be solved by the invention]

[0016] The object of the present invention is to provide a novel hyaluronidase PH20 mutant or a fragment thereof that has improved thermostability, enzymatic activity and expression rate compared to wild-type PH20, preferably mature wild-type PH20.

[0017] Another object of the present invention is to provide a composition for treating cancer comprising the hyaluronidase PH20 mutant or a fragment thereof, and a method for treating cancer using the same. [Means for solving the problem]

[0018] To achieve the above objective, the present invention provides a hyaluronidase PH20 mutant, or a fragment thereof, which includes substitutions of one or more amino acid residues at sites corresponding to the alpha-helix region and / or its junction site in the amino acid sequence of wild-type PH20, preferably mature wild-type PH20, and optionally has a portion of amino acid residues deleted at the N-terminus and / or C-terminus.

[0019] The present invention also provides a composition for treating cancer comprising the hyaluronidase PH20 mutant or a fragment thereof according to the present invention, and a treatment method using the same. [Brief description of the drawings]

[0020] [Figure 1] Tertiary structure model of PH20 protein. Hyal1 (PDB ID: 2PE4), whose crystal structure has been revealed, was used as a template (Chao et al., 2007), and the tertiary structure of PH20 protein was modeled on the Swiss-Model server (https: / / swissmodel.expasy.org / ). Figure 1A shows the tertiary structure model of PH20 protein and the catalytic amino acids D146 and E148. The tertiary structure of PH20 protein is composed of eight repeated β-strands and α-helices. Figure 1B shows α-helix 8 of PH20 and the eta (η)8 loop in which residues G340-I344, which form the linking site toward the N-terminus of α-helix 8, are located. Residues G340, T341, L342, S343, and I344 are shown, respectively. Figure 1C shows the amino acid residues (C351, Y357, and N363) located in α-helix 8 of PH20 that interact with the adjacent secondary structures. C351 forms a disulfide bond with C60 located in α-helix 1, Y357 forms a hydrophobic interaction with F315 located between β-strand 7 and α-helix 7, and N363 forms a hydrogen bond with D69 residue located in α-helix 1. [Diagram 2]The protein expression levels of wild type (WT) PH20 and the mutants prepared in the present invention are compared. WT and the mutants were expressed in ExpiCHO cells by transient transfection. The expression level of WT is 16.1 mg / L. Mutant HM1 and the mutants based on HM6 showed higher protein expression levels than WT, with HM4 and HM7 showing the highest protein expression levels. HM11, which introduces additional amino acid substitutions (Y365F and I367L) in mutant HM6, showed a reduced protein expression of 6.4 mg / mL. [Diagram 3]The experimental results for mutants HM1 and HM6 are shown in Fig. 3A. SDS-PAGE results after purification of WT and mutants HM1 and HM6 were obtained. A HisTrap column and a Q Sepharose column were used for purification. The molecular weights of WT and mutants HM1 and HM6 are 70 kDa (Figure legend: M, molecular weight marker; CS, supernatant; FT, flow-through; Elution, elution fraction). Fig. 3B shows the enzyme activity values ​​of WT and mutants HM1 and HM6 measured by turbidimetric assay at pH 7.0. In the present invention, the enzyme activity measured by turbidimetric assay is shown as specific activity. Fig. 3C shows the enzyme activity of WT and mutants HM1 and HM6 measured by substrate-gel assay. After removing SDS at 4°C with 2.5% Triton X-100 (w / v), the enzyme reaction was carried out at 37°C for 1 to 4 hours. Mutant HM6 was renatured earlier than WT and mutant HM1, and hyaluronic acid was hydrolyzed in polyacrylamide gel. The white bands are the hyaluronic acid decomposed by WT and mutant proteins. Figure 3D shows the enzyme activity of WT and mutant HM1 at pH 5 to 8 by substrate-gel assay. WT and mutant HM1 showed activity in the pH range of 5 to 8, with the highest enzyme activity at pH 5.0. Mutant HM1 has a signal peptide of human serum albumin or human Hyal1. The white bands are the hyaluronic acid decomposed by WT and mutant proteins. Figure 3E shows the results of separation of WT, mutant HM1, and HM6 using a phenyl column. Mutants HM1 and HM6 elute earlier from the phenyl column than the WT. [Figure 4] The results are shown in Fig. 1. The final products of hyaluronic acid degradation by WT and mutant HM6 were analyzed after 10 minutes and 1 hour using an Amide-80 column. [Diagram 5]The experimental results of the amino acid mutation in the G340-I344 region of PH20 are shown in Fig. 5A. The results of SDS-PAGE of mutants HM7, HM8, HM9, HM10, and HM21 after HisTrap column purification are shown in Fig. 5B. The enzyme activity of WT and mutants HM6, HM8, HM9, HM10, HM21, and HM7 was measured by turbidimetric assay at pH 7.0. Fig. 5C shows the enzyme activity of WT and mutants HM6, HM8, HM9, HM10, HM21, and HM7 measured by substrate-gel assay. The bar graph at the bottom of Fig. 5C shows the enzyme activity level after staining the gel with Alcian blue and quantifying the bands. The white bands are the hyaluronic acid decomposed by the WT and mutant proteins. Figure 5D shows the results of analysis of WT and mutants HM8, HM9, HM10, HM21, and HM7 using a phenyl column, and Figure 5E shows the results of separation of WT and mutants HM6, HM8, HM9, HM10, HM21, and HM7 by isoelectric point value at pH 3 to 7 using IEF gel. [Figure 6] 6A shows the results of an experiment on mutant HM11. Figure 6A shows the results of protein purification using a HisTrap column for mutant HM11. Figure 6B shows the results of measuring the enzyme activity of WT and mutant HM11 using a turbidimetric assay at pH 7.0. [Figure 7]Experimental results for N-terminally truncated PH20 mutants HM40, HM13, HM41, HM24, HM42, and HM25. Figure 7A shows the results of protein purification using a HisTrap column for PH20 mutants HM40, HM13, HM41, HM24, HM42, and HM25. Figure 7B shows the expression levels of PH20 mutants HM40, HM13, HM41, HM24, HM42, HM25, HP61, and HP62 in ExpiCHO cells. Figure 7C shows the specific enzyme activity of PH20 mutants HM40, HM13, HM41, HM24, HM42, HM25, HP61, and HP62 at pH 7.0 by turbidimetric assay. Figure 7D shows the enzyme activity of PH20 mutants HM40, HM13, HM41, HM24, and HM42 by substrate-gel assay. The white bands indicate hyaluronan degraded by WT and mutant proteins. Figure 7E shows the results of analysis of WT and mutants HM40, HM13, HM41, HM24, and HM42 using a phenyl column. Figure 7F shows the change in particle size with increasing temperature for PH20 mutants HM40, HM13, HM41, and HM42. [Figure 8]The results are for the C-terminal truncation mutants HM14, HM15, and HM16, which were generated using HM6 as a template. Figure 8A shows the results of SDS-PAGE after HisTrap purification of mutants HM14, HM15, and HM16. WT and mutant HM6 were included as controls. Figure 8B shows the enzyme activity of WT and mutants HM6, HM14, HM15, and HM16 measured by turbidimetric assay at pH 7.0. Figure 8C shows the enzyme activity of WT and mutants HM6, HM14, HM15, and HM16 measured for 1, 2, and 4 hours by substrate-gel assay. The graph on the right of Figure 8C shows the enzyme activity in a bar graph after 1 hour of enzyme reaction, stained with Alcian blue. The white bands are the hyaluronan decomposed by WT and mutant proteins. Figure 8D shows the results of analysis of WT and mutants HM6, HM14, HM15, and HM16 using a phenyl column, and Figure 8E shows the results of separation of mutants HM14, HM15, and HM16 by their isoelectric points in the pH range of 3 to 7 using IEF gel. [Figure 9] The experimental results are for PH20 mutants HM19 and HM20, which were generated using HM10 as a template. Figure 9A shows the results of protein purification using a HisTrap column for PH20 mutants HM19 and HM20. Figure 9B shows the comparison of the enzyme activity of PH20 mutants HM19 and HM20 by turbidimetric assay at pH 7.0. Figure 9C shows the results of a substrate-gel assay in which WT, HM10, HM19, and HM20 were reacted at 37°C for 1 hour, followed by staining of SDS gel with Alcian blue stain. The white bands are hyaluronan decomposed by WT and mutant proteins. [Figure 10] The aggregation temperatures of WT and PH20 mutants were measured using a dynamic light scattering (DLS) device. The measurements were repeated three times, and the values ​​are shown as the mean ± SE. [Figure 11]The Stern-Volmer plots show the fluorescence change of tryptophan residues in WT and PH20 mutants upon addition of acrylamide (0-0.5M). Tryptophan in amino acids is excited at 295nm and emits maximum fluorescence at 340nm. Acrylamide is a small molecule that can penetrate into the protein structure and quench the fluorescence emission of tryptophan. The more flexible the protein structure, the greater the fluorescence quenching by acrylamide. F0 is the fluorescence value without acrylamide, and F is the fluorescence value with acrylamide (0-0.5M). The change in fluorescence measurement is shown as the ratio of F0 / F. Figure 11A shows the Stern-Volmer plots for WT and mutants HM1, HM4, HM6, and HM7. Figure 11B is a Stern-Volmer plot of WT and mutants HM14, HM15, and HM16. [Figure 12] The expression levels of HM10-based PH20 mutants in ExpiCHO cells are shown. Figure 12A shows the expression levels of each mutant in a graph. Figure 12B shows the expression levels of each mutant in a table. WT and PH20 mutants have a 6xHis-tag at the C-terminus, and the protein expression levels after HisTrap column purification are shown in mg / L. HM30 to HM33 mutants were not expressed in ExpiCHO cells. [Figure 13]Western blot results for cell culture fluid of mutants HM29, HM30, HM31, HM32, and HM33. HM10-based mutants HM29, HM30, HM31, HM32, and HM33 are truncated at the C-terminus at A467, C464, D461, C358, and C455. HM29 truncated at C-terminus A467 was expressed in ExpiCHO cells, but truncated at C-terminus C464 or shorter was not expressed in ExpiCHO cells. Primary antibody was rabbit anti-PH20 polyclonal antibody (Abcam) diluted 1:500. Secondary antibody was goat anti-rabbit IgG HRP diluted 1:2,000. [Figure 14] The experimental results are for C-terminal truncation mutants prepared using HM10 as a template. Figure 14A shows the results of measuring the enzyme activity of C-terminal truncation mutants prepared using HM10 as a template by turbidimetric assay at pH 7.0. Figure 14B shows the comparison of the enzyme activity of 17 PH20 mutants (HM43, HM44, HM45, HM20, HM19, HM35, HM36, HM37, HM38, HM39, HM47, HM48, HM49, HM50, HM51, HM52, HM10) prepared using HM10 as a template, depending on the C-terminal truncation site. Figure 14C shows the results of a substrate-gel assay of HM29, HM35, HM36, HM37, HM38, HM39, HM43, HM44, and HM45, which were generated using HM10 as a template, and stained SDS gels with Alcian blue stain after enzyme reaction at 37°C for 1 hour. The white bands represent hyaluronan decomposed by the WT and mutant proteins. [Figure 15]This shows SDS gels after the final column purification of protein HP34 (Fig. 15A) and HP46 (Fig. 15B) expressed in ExpiCHO cells. HP34 underwent four purification steps using Q Sepharose, Butyl HP, Heparin, and Blue Sepharose columns, and the SDS gel shows the results after purification using the Blue Sepharose column. HP46 underwent three purification steps using Q Sepharose, Butyl HP, and Heparin columns, and the SDS gel shows the results after purification using the Heparin column. [Figure 16] The enzymatic activity of HP34 and HP46, which are PH20 mutants without 6xHis-tag, was generated using HM21 as a template. Figure 16A shows the enzymatic activity of WT and mutants HM21, HP34, and HP46 measured by turbidimetric assay at pH 7.0. Figure 16B shows the enzymatic activity of HW2 and mutants HM21, HP34, and HP46 measured by Morgan-Elson assay at pH 5.3. Km: Michaelis-Menten constant, kcat: turnover number, kcat / Km: catalytic efficiency). [Figure 17]The results of the characterization of HM21-based mutants are shown in Figure 17A. The aggregation temperatures of HP34 and HP46, which are PH20 mutants without 6xHis-tags and were prepared using HM21 as a template, were measured by DLS. As a control, the aggregation temperatures of HW2 and HM21 were shown. Figure 17B shows the enzyme activity of HW2 and PH20 mutants HP20, HP34, and HP46 measured by substrate-gel assay for 1 hour. Figure 17C shows the results of substrate-gel assay after HW2 and HP46 samples were left at pH 3.0 and pH 7.0 for 14 hours, followed by SDS-PAGE, followed by removal of SDS with 2.5% Triton X-100 (w / v), and proceeding with the enzyme reaction at 37°C for 1 hour. Figure 17D shows the expression levels of PH20 mutants HM21, HM53, HM54, HM55, HM56, HP59, and HP60 in ExpiCHO cells. Figure 17E shows the specific enzyme activity of PH20 mutants HM21, HM53, HM54, HM55, HM56, HP59, and HP60 at pH 5.3 by turbidimetric assay. [Figure 18] These are the results of measuring the stimulating index of CD4+ T cells when treated with PH20 and the PH20 mutant at concentrations of 1.5 ng / mL and 15 ng / mL, respectively. [Figure 19] These are the results of measuring the stimulating index of CD8+ T cells when treated with PH20 and PH20 mutants at concentrations of 1.5 ng / mL and 15 ng / mL, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0022] The present invention provides a hyaluronidase PH20 mutant, or a fragment thereof, which comprises a substitution of one or more amino acid residues at the α-helix site and / or the junction site between α-helix 7 and α-helix 8 (S347-C381) in the amino acid sequence of wild-type PH20, preferably mature wild-type PH20, and optionally has a portion of amino acid residues at the N-terminus and / or C-terminus truncated and deleted.

[0023] In the present invention, the amino acid residue positions of each mutant correspond to the amino acid positions of wild-type PH20 having the amino acid sequence of SEQ ID NO:1.

[0024] In addition, in the present invention, "mature wild-type PH20" means a protein consisting of amino acid residues L36 to S490 of SEQ ID NO: 1, in which the signal peptide M1 to T35 and A491 to L509, which are not related to the actual function of PH20, are deleted in the amino acid sequence of wild-type PH20 of SEQ ID NO: 1.

[0025] [Table 1]

[0026] Specifically, the PH20 mutant or fragment thereof of the present invention is characterized in that it contains one or more mutations, preferably substitutions of amino acid residues, selected from the group consisting of T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E, and I361T in wild-type PH20 having the amino acid sequence of SEQ ID NO:1, and preferably contains one or more substitutions of amino acid residues selected from the group consisting of T341A, T341C, L354I, and N356E.

[0027] In the present invention, the term "PH20 mutant" is used in a concept that includes not only those that contain mutations, preferably substitutions, of some amino acid residues in the wild-type PH20 sequence, but also those in which the amino acid residue substitutions have been accompanied by deletion of some amino acid residues at the N-terminus or C-terminus, and is used in a concept that is substantially similar to the expression "PH20 mutant or fragment thereof."

[0028] In the present invention, based on Hyal1 (SEQ ID NO: 2), a human hyaluronidase with a known tertiary structure, the tertiary structure of PH20 protein located outside the active site was studied through protein structure modeling of human PH20. As a result, an attempt was made to enhance the thermal stability of the protein structure without affecting the catalytic activity of the enzyme by selecting amino acids located in the α-helix 8 site of PH20 and substituting them with the amino acid sequence of α-helix 8 of Hyal1. In particular, α-helix 8 is located on the outside in the tertiary structure of the PH20 protein, and has less interaction with adjacent α-helices or β-strands compared to other α-helices. According to the present invention, based on the experimental result that the enzymatic activity and protein aggregation temperature (Tagg.) at neutral pH are increased when the amino acid sequence of the α-helix 8 region and the junction between α-helix 7 and α-helix 8 of human PH20 is partially replaced with the amino acid sequence of the α-helix 8 region and the junction between α-helix 7 and α-helix 8 of Hyal1, which is highly hydrophilic, the present invention has found that it is possible to provide a new PH20 mutant, or a fragment thereof, which has increased enzymatic activity and thermal stability compared to wild-type PH20.

[0029] Thus, the PH20 mutant of the present invention comprises a substitution of one or more amino acid residues selected from the group consisting of T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E, and I361T in the amino acid sequence of wild-type PH20 (having the amino acid sequence of SEQ ID NO: 1), preferably a substitution of one or more amino acid residues selected from the group consisting of T341A, T341C, L354I, and N356E in the amino acid sequence of mature wild-type PH20 (having a sequence consisting of L36 to S490 in the amino acid sequence of SEQ ID NO: 1), The polypeptide is characterized in that one or more amino acid residues have been substituted at the α-helix site and / or the site corresponding to the junction thereof, preferably the α-helix 8 site (S347 to C381) and / or the junction site between α-helix 7 and α-helix 8 (A333 to R346), and more preferably at the sites corresponding to T341 to N363, T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361, or M345 to N363.

[0030] In particular, the PH20 mutant of the present invention may have the α-helix 8 site (S347 to C381) and / or the junction site between α-helix 7 and α-helix 8 (A333 to R346) of the wild-type PH20, preferably the mature wild-type PH20, substituted with some amino acid residues in the amino acid sequence of the corresponding site of Hyal1 having the sequence of SEQ ID NO: 2 (see Tables 2 and 3), but is not limited thereto.

[0031] [Table 2]

[0032] [Table 3]

[0033] More specifically, the novel PH20 variant or fragment thereof of the present invention comprises an amino acid substitution of L354I and / or N356E in the amino acid sequence of wild-type PH20, preferably mature wild-type PH20, Furthermore, it is preferred, but not limited to, that the substitutions include substitutions of amino acid residues at one or more positions selected between T341 and N363, particularly at one or more positions selected from the group consisting of T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361, and N363.

[0034] It is further preferred that the substitution of amino acid residues at one or more positions selected from the group consisting of T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361, and N363 is one or more selected from the group consisting of T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T, and N363G, but is not limited thereto.

[0035] Preferably, the novel PH20 variant or fragment thereof of the present invention is characterized in that it comprises one or more amino acid residue substitutions selected from the group consisting of M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, Furthermore, the present invention is characterized in that it contains one or more amino acid residue substitutions selected from the group consisting of, but not limited to, T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N, and N363G.

[0036] More preferably, the novel PH20 variant or fragment thereof of the present invention may be any one selected from the group consisting of, but not limited to: (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G; (d) T341G, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (e) T341A, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (f) T341C, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (g) T341D, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (h) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; and (i) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T.

[0037] In the present invention, an expression in which a one-letter amino acid residue name and a number are written together, such as "S347", means the amino acid residue at the corresponding position in the amino acid sequence of SEQ ID NO:1.

[0038] For example, "S347" means that the amino acid residue at position 347 in the amino acid sequence of SEQ ID NO:1 is a serine.

[0039] In addition, "S347T" means that the serine at position 347 of SEQ ID NO:1 has been replaced with threonine.

[0040] A PH20 mutant or fragment thereof according to the present invention is intended to include mutants or fragments thereof in which amino acid residues are conservatively substituted at specific amino acid residue positions.

[0041] As used herein, the term "conservative substitution" refers to a modification of a PH20 variant involving the replacement of one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the PH20 variant.

[0042] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined and are well known in the art. These families of amino acid residues include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0043] It is expected that the PH20 variants of the invention can make conservative amino acid substitutions and still retain activity.

[0044] Furthermore, the PH20 mutant or fragment thereof of the present invention is also interpreted as including a PH20 mutant or fragment thereof that has substantially the same function and / or effect as the PH20 mutant or fragment thereof of the present invention, and has an amino acid sequence homology of 80% or 85% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more.

[0045] The PH20 mutant or fragment thereof of the present invention has the effect of increasing expression in animal cells and increasing protein folding and thermostability compared to mature wild-type PH20, and furthermore, despite the increased thermostability, these enzymatic activities are increased or similar to those of mature wild-type PH20.

[0046] On the other hand, it is known that truncation of the C-terminal region of mature wild-type PH20 reduces the enzymatic activity. However, the PH20 mutants of the present invention maintain increased or similar enzymatic activity to that of mature wild-type PH20 despite the truncation of the C-terminus due to increased protein folding and thermal stability, and further maintain enzymatic activity even when up to five amino acids are truncated at the N-terminus, indicating that the N-terminal P41 residue plays an important role in protein expression and enzymatic activity.

[0047] Thus, the PH20 mutant or fragment thereof of the present invention is characterized by, but is not limited to, the substitution of some amino acid residues at the α-helix 8 site (S347-C381) and / or the junction site between α-helix 7 and α-helix 8 (A333-R346) of wild-type PH20, as well as the deletion of some amino acid residues at the C-terminus and / or N-terminus.

[0048] In one aspect, the PH20 mutant or fragment thereof of the present invention has a cleavage occurring before an amino acid residue selected from the group consisting of M1 to P42 at the N-terminus of the amino acid sequence of SEQ ID NO:1, preferably before an amino acid residue of L36, N37, F38, R39, A40, P41, or P42, resulting in the deletion of some amino acid residues at the N-terminus, and / or a cleavage occurring after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus, preferably after an amino acid residue selected from the group consisting of V455 to L509. is characterized in that the truncation occurs after an amino acid residue selected from the group consisting of V455 to S490, most preferably after the amino acid residue V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490, and some amino acid residues at the C-terminus are deleted.

[0049] The expression that cleavage occurs before an amino acid residue selected from the group consisting of M1 to P42 at the N-terminus means that cleavage and deletion occurs from M1 to P42 at the N-terminus to the amino acid residue immediately preceding the selected amino acid residue.

[0050] For example, the expression that cleavage occurs before amino acid residue L36, N37, F38, R39, A40, P41, or P42 means that in the sequence of SEQ ID NO:1, cleavage has been removed from M1 to T35, the amino acid residue immediately preceding L36, from M1 to L36, the amino acid residue immediately preceding N37, from M1 to N37, the amino acid residue immediately preceding F38, from M1 to F38, the amino acid residue immediately preceding R39, from M1 to R39, the amino acid residue immediately preceding A40, from M1 to A40, the amino acid residue immediately preceding P41, and from M1 to P41, the amino acid residue immediately preceding P42.

[0051] In addition, the expression that cleavage occurs after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus means that cleavage occurs and deletion occurs from the amino acid residue immediately following the amino acid residue selected from V455 to L509 at the C-terminus.

[0052] For example, references to cleavage occurring after the C-terminal amino acid residues V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 refer to the sequence of SEQ ID NO:1, respectively. In this context, this means that the amino acid residue following the amino acid residue V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 is cleaved and removed.

[0053] Preferably, the PH20 mutant or fragment thereof according to the present invention may be selected from the group consisting of the amino acid sequences of SEQ ID NOs: 60 to 115, but is not limited thereto.

[0054] Most preferably, the novel PH20 variant or fragment thereof according to the present invention is characterized in that it has the amino acid sequence of SEQ ID NO: 99. The novel PH20 variant having the amino acid sequence of SEQ ID NO: 99 is characterized in that it contains 15 amino acid residue substitutions, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and is truncated before the F38 residue at the N-terminus and after the F468 residue at the C-terminus.

[0055] In the PH20 mutants prepared in specific examples according to the present invention, the sequences of substituted or truncated amino acids are as shown in Table 11.

[0056] There have been no reported studies attempting to increase the enzymatic activity and thermal stability of PH20 by substituting the amino acids at the α-helix, which is a secondary structure that forms the tertiary structure of a protein, and its connecting site, as in the present invention. Previous studies have reported that the enzymatic activity of wild-type PH20 changes depending on the cleavage position of the amino acid residues located at the C-terminus. In the present invention, a specific α-helix forming the secondary structure of PH20 was replaced with the α-helix of another human hyaluronidase to produce a PH20 mutant that is more stable than wild-type PH20. These mutants are characterized by the fact that the substituted α-helix domain exhibits a different interaction with other parts of the secondary structure of PH20 from that of wild-type PH20, and thus exhibit a certain enzymatic activity regardless of the cleavage position at the C-terminus.

[0057] In a specific embodiment of the present invention, the new PH20 mutant or fragment thereof of the present invention, which has increased enzymatic activity and thermostability compared to mature wild-type PH20, is characterized in that in the amino acid sequence of wild-type PH20, preferably mature wild-type PH20, it contains one or more amino acid residue substitutions selected from the group consisting of T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E, and I361T, and some amino acids located at the α-helix 8 site (S347-C381) and / or the junction site between α-helix 7 and α-helix 8 (A333-R346) are replaced with other amino acids.

[0058] Specifically, the substitution of amino acid residues at α-helix 8 and at the junction between α-helix 7 and α-helix 8 includes substitution of some amino acid residues from the sections T341-N363, T341-I361, L342-I361, S343-I361, I344-I361, M345-I361, or M345-N363.

[0059] To confirm the effect of C-terminal truncation in PH20 mutants in which the junction sites of α-helix 8 and α-helix 7 and α-helix 8 were replaced, three PH20 mutants, HM6, HM10, and HM21, were selected as templates.

[0060] HM6 is a mutant in which the amino acids in the M345 to N363 section are replaced with the amino acid sequence of Hyal1 (M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T are replaced in SEQ ID NO:1), and does not contain an additional C-terminal truncation (i.e., the C-terminal amino acid residue is S490, as in mature wild-type PH20). Among the PH20 mutants of the present invention, HM6 is the mutant with the fewest replacements at the α-helix 8 site and the junction site between α-helix 7 and α-helix 8, HM10 is a mutant in which the amino acids in the L342 to I361 section are replaced with the amino acid sequence of Hyal1 (342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T are replaced in SEQ ID NO: 1), and does not contain additional C-terminal truncations. Among the PH20 mutants of the present invention, HM10 has an enzyme activity similar to that of mature wild-type PH20 and is the most thermostable mutant. HM21 is a mutant in which the amino acids in the T341 to I361 section are replaced with the amino acid sequence of Hyal1 (T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T are substituted in SEQ ID NO:1), and does not contain any additional C-terminal truncations. The enzyme activity at pH 7.0 is approximately twice as high as that of wild-type PH20.

[0061] The HM6-based PH20 mutants generated in the present invention were truncated at L36 at the N-terminus and truncated next to I465, F468, or P471 at the C-terminus, as shown in Table 4.

[0062] [Table 4]

[0063] The HM10-based PH20 mutants were commonly N-terminally truncated before residue F38 and C-terminally truncated after residues V455, C458, D461, C464, I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, or T488, as shown in Table 5.

[0064] [Table 5]

[0065] As shown by the examples of mutants using HM10 as a template, which contain substitutions of amino acid residues at the α-helix 8 site and the L342 to I361 sites corresponding to the junction between α-helix 7 and α-helix 8, and in which the N-terminus is truncated before the F38 residue and the C-terminus is truncated at I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, or T488, the PH20 mutants of the present invention exhibited enzyme activity similar to that of mature wild-type PH20 regardless of the truncation site at the C-terminus.

[0066] As shown in Table 6, the two HM21-based PH20 mutants have in common an N-terminus truncated before the F38 residue and a C-terminus truncated after the F468 or K470 residue.

[0067] [Table 6]

[0068] Surprisingly, using HM21, which has approximately twice the enzymatic activity of mature wild-type PH20, as a template, mutants containing amino acid residue substitutions in the T341-I361 section, which corresponds to the α-helix 8 site and the junction between α-helices 7 and α-helices 8, and in which the N-terminus is truncated before the F38 residue and the C-terminus is truncated after the F468 or K470 residues, maintained the high enzymatic activity of HM21 regardless of the C-terminal truncation site.

[0069] In a study by Frost et al., when the C-terminus of PH20 was truncated before 477 and shorter, the enzyme activity was reduced by 10% compared to a mutant whose C-terminus was truncated after 477 (Frost, 2007). In the present invention, however, when the amino acids at α-helix 8 and its connecting site of PH20 were substituted, the stability of the protein was increased, and the enzyme activity was maintained regardless of the truncated position of the C-terminus. This result is significant because it solves the problem of reduced enzyme activity due to truncation of the C-terminus of wild-type PH20.

[0070] In addition, in the present invention, the effects of amino acids at the N-terminus of PH20, which were unknown in previous studies, were investigated.

[0071] In order to confirm the effect of the N-terminal truncated amino acid in mutant HM6 (SEQ ID NO: 1 in which M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T are substituted, and no additional C-terminal truncation is included), some amino acid residues in the α-helix 8 site and the junction site between α-helix 7 and α-helix 8 (M345-I361) of wild-type PH20 were substituted with the corresponding amino acid residues in Hyal1 (SEQ ID NO: 1 in which M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T are substituted, and no additional C-terminal truncation is included), mutants in which amino acid residues L36 to V47 in the amino acid sequence of SEQ ID NO: 1 were substituted with FRGPLLPNR or amino acid residues L36 to A52 were substituted with FRGPLLPNRPFTTV were prepared using HM6 as a template. In addition, using HM6 as a template, mutants HM40, HM13, HM41, HM24, HM42, and HM25 were produced in which the N-terminus of the amino acid sequence of SEQ ID NO:1 was truncated before the N37, F38, R39, A40, P41, or P42 residues (see Table 7).

[0072] [Table 7]

[0073] As a result, when the N-terminus of HM6 was truncated before the N37, F38, R39, A40, or P41 residue, the enzyme activity was not significantly affected, but when the N-terminus was truncated before the P42 residue, the enzyme activity was significantly reduced, indicating that the N-terminal portion of PH20 after P41 is important for protein expression and enzyme activity. In addition, when the amino acids in the L36-V47 or L36-A52 section of the N-terminus of HM6 were replaced with amino acids of Hyal1, the protein was not expressed in ExpiCHO cells, indicating that the N-terminal portion is important for protein expression.

[0074] In addition, in the present invention, we attempted to improve expression of recombinant PH20 protein by using signal peptides from other proteins that are highly expressed in animal cells, rather than the signal peptide specific to PH20.

[0075] Thus, in yet another aspect, the novel PH20 mutant of the present invention is characterized in that, instead of the signal peptide of wild-type PH20 M1 to T35, the N-terminus further comprises a signal peptide derived from human growth hormone having the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA of SEQ ID NO: 3 as shown in Table 8, a signal peptide derived from human serum albumin having the amino acid sequence MKWVTFISLLFLFSSAYS of SEQ ID NO: 4, or a signal peptide derived from human Hyal1 having the amino acid sequence MAAHLLPICALFLTLLDMAQG of SEQ ID NO: 5, but is not limited thereto.

[0076] The above phrase "instead of the wild-type PH20 signal peptide of M1 to T35" means that part or all of the wild-type PH20 signal peptide is deleted and the wild-type PH20 signal peptide function cannot be performed, and is used to include cases where a part of the N-terminus is further deleted, for example, before residues N37, F38, R39, A40, P41, or P42, resulting in the deletion of the wild-type PH20 signal peptide and additional deletion of the N-terminus.

[0077] [Table 8]

[0078] In another aspect, the present invention relates to a composition for cancer treatment comprising the novel PH20 mutant of the present invention and a method for cancer treatment using the same.

[0079] Cancers or carcinomas treatable with the novel PH20 variants of the present invention are not particularly limited and include all solid and hematological cancers. Such cancers may be selected from the group consisting of, for example, skin cancers 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 tumors, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, bile duct cancer, testicular cancer, cervical cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma. Preferably, the cancers treatable with the compositions of the present invention may be selected from the group consisting of, for example, colorectal cancer, breast cancer, lung cancer, and kidney cancer, but are not limited thereto.

[0080] The composition may be a pharmaceutical composition. The pharmaceutical composition may further comprise a pharma- ceutically acceptable composition, which may be one or more of the following commonly used in the formulation of drugs: lactose, textrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto. The pharmaceutical composition may further comprise one or more of the following commonly used in the preparation of pharmaceutical compositions: diluent, excipient, lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, and preservative.

[0081] The pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. During oral administration, proteins or peptides are digested, so oral compositions should be formulated to coat the active agent or protect it from gastrointestinal degradation. The composition may also be administered by any device that can transport the active agent to target cells.

[0082] The pharmaceutical composition may be formulated in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, powder, granule, tablet or capsule, and may further contain a dispersing agent or stabilizer.

[0083] In particular, the composition for cancer treatment according to the present invention is characterized in that it is used for combined treatment with other anticancer agents.

[0084] Anticancer agents that can be used in combination therapy with the novel PH20 mutant of the present invention are preferably, but not limited to, chemical anticancer agents, antibody-type anticancer agents, RNAi, cell therapy agents, etc.

[0085] Preferably, the anti-cancer agent that can be used in combination therapy with the novel PH20 mutant of the present invention is an immune anti-cancer agent, preferably, but not limited to, an immune checkpoint inhibitor.

[0086] In another aspect, the invention relates to a nucleic acid encoding a PH20 variant or a fragment thereof according to the invention.

[0087] As used herein, a "nucleic acid" may be present in a cell, a cell lysate, or may be present in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. A nucleic acid of the invention may be, for example, DNA or RNA.

[0088] In another aspect, the invention relates to a vector comprising the nucleic acid. For expression of the PH20 variants or fragments thereof of the invention, DNA encoding the PH20 variants can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas expressing the PH20 variants) and the DNA can be "operably linked" to transcriptional and translational control sequences and inserted into an expression vector.

[0089] As used herein, "operably linked" can mean that the gene encoding the PH20 variant or fragment thereof is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the gene encoding the PH20 variant or fragment thereof. Expression vectors and expression control sequences are selected to be appropriate for the expression host cell used. The gene encoding the PH20 variant is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites on the gene fragment encoding the PH20 variant or fragment thereof and the vector, or by blunt end ligation if no restriction enzyme sites are present).

[0090] The recombinant expression vector also has a regulatory sequence that controls the expression of the gene encoding the PH20 variant in a host cell. A "regulatory sequence" can include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the gene encoding the PH20 variant or fragment thereof. One of ordinary skill in the art will recognize that different regulatory sequences may be selected depending on factors such as the choice of host cell to be transformed, the expression level of the protein, and the like, and may vary in the design of the expression vector.

[0091] In another aspect, 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.

[0092] Specifically, the host cell of the present invention may be a prokaryotic cell such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp. It may also be a eukaryotic cell such as a fungus such as Aspergillus sp., yeast such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa, other lower eukaryotic cells, and higher eukaryotic cells such as cells derived from insects.

[0093] The host cells may be derived from plants or mammals. Preferably, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT78 cells, HEK293 cells, etc. may be used, but are not limited thereto. Particularly preferably, CHO cells may be used.

[0094] The nucleic acid or the vector is transfected or transfected into a host cell. To "transfect" or "transfect", various techniques commonly used to introduce exogenous nucleic acid (DNA or RNA) into a prokaryotic or eukaryotic host cell 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 fragment thereof of the present invention. Expression vectors suitable for cell hosts include those derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retroviruses. Expression vectors that can be used for bacterial hosts include bacterial plasmids derived from Escherichia coli such as pET, pRSETR, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids with broader host range such as RP4, phage DNA exemplified by λgt10 and λgt11, highly diverse phage lambda derivatives such as NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. A useful expression vector for yeast cells is the 2μ plasmid and its derivatives, and a useful vector for insect cells is pVL941. A useful expression vector for yeast cells is the 2μm plasmid and its derivatives, and a useful vector for insect cells is pVL941.

[0095] In still another aspect, the present invention relates to a method for producing a PH20 mutant or a fragment thereof of the present invention, comprising the step of culturing a host cell to express the PH20 mutant or a fragment thereof of the present invention.

[0096] When a recombinant expression vector capable of expressing the PH20 variant or fragment thereof is introduced into a mammalian host cell, the PH20 variant or fragment thereof can be produced by culturing the host cell for a period of time sufficient to allow it to be expressed in the host cell, or, more preferably, to allow the PH20 variant or fragment thereof to be secreted into the culture medium in which the host cell is cultured.

[0097] In some cases, the expressed PH20 variant can be isolated from the host cell and purified to homogeneity. The isolation or purification of the PH20 variant can be carried out by a conventional isolation or purification method used for proteins, such as chromatography. The chromatography can be, for example, one or more combinations selected from the group consisting of affinity chromatography, ion exchange chromatography, or hydrophobic chromatography, but is not limited thereto. In addition to the chromatography, filtration, ultrafiltration, salting out, dialysis, etc. can also be used in combination.

[0098] The present invention will be described in more detail below with reference to examples. These examples are provided solely for the purpose of illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0099] Example 1. Construction of PH20 mutants To generate PH20 mutants, wild-type PH20 cDNA (clone ID: hMU002604) was purchased from Korea Human Gene Bank. Wild-type PH20 encodes amino acids L36 to S490. The PH20 gene was amplified by polymerase chain reaction (PCR) and inserted into the XhoI and NotI restriction enzyme sites of the pcDNA3.4-TOPO vector. For expression in ExpiCHO cells, one of the signal peptides from human growth hormone, human serum albumin, or human Hyal1 was used as a signal peptide instead of the inherent signal peptide of PH20. For protein purification using a HisTrap column, a 6xHis-tag DNA sequence was located at the 3´-end of the PH20 cDNA. The amino acid substitutions in the PH20 mutants were performed using PCR, and the amino acid substitutions were confirmed by DNA sequencing.

[0100] The list of primers used for cloning the PH20 mutants is summarized in Table 9, and their specific sequences are summarized in Table 10.

[0101] [Table 9] TIFF2025004002000011.tif255151

[0102] [Table 10] TIFF2025004002000013.tif253164TIFF2025004002000014.tif255160TIFF2025004002000015.tif154168

[0103] After searching for PH20 mutants with increased enzymatic activity and thermostability, we generated cDNAs of the PH20 mutants in a form without the 6xHis-tag.

[0104] The number of ExpiCHO cells was 6x10 6 When the total volume of PH20 was 100 / mL, the ExpiCHO cells were transfected with a plasmid in which the cDNA of wild-type or mutant PH20 was inserted into the pcDNA3.4-TOPO vector, using ExpiFectamine CHO Reagent. ExpiCHO Expression Medium (100-500 mL) was used as the cell culture medium. After transfection, the ExpiCHO cells were cultured with shaking at 130 rpm for a total of 6 days, during which they were cultured at 37°C for 1 day and then the temperature was lowered to 32°C for an additional 5 days. When the culture was completed, the cell supernatant was collected by centrifugation at 10,000 rpm for 30 minutes.

[0105] The recombinant wild-type and mutant PH20 proteins with 6xHis at the C-terminus produced from ExpiCHO cells were purified in three steps using an AKTA prime equipment, a HisTrap column, a Q Sepharose column, and a phenyl column.

[0106] For protein purification using a HisTrap column, buffer A (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl, 0.5 M imidazole) were prepared. Proteins were bound to the HisTrap column, and to remove non-specifically bound proteins, 5 CV (column volume) of buffer A was applied. After confirming that the conductivity was maintained constant, 5 CV of 20% buffer B was applied to elute the proteins. The eluted proteins were dialyzed using dialysis buffer (20 mM sodium phosphate, pH 7.5, 50 mM NaCl). 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. The protein was bound to a Q Sepharose column, and non-specifically bound proteins were removed by applying 5 CV of buffer A. The protein was then eluted by applying 5 CV of buffer B with a concentration gradient of 0 to 100%.

[0107] Buffer A (20 mM sodium phosphate, pH 7.0, 1.5 M (NH4)2SO4) and buffer B (20 mM sodium phosphate, pH 7.0) were prepared for protein purification using a phenyl column. Proteins were bound to the phenyl column, and 5 CV of buffer A was applied to remove non-specifically bound proteins. After that, 5 CV of buffer B was applied with a concentration gradient of 0-100% to elute the proteins.

[0108] The enzyme activities of wild-type and mutant PH20 were measured using turbidimetric assay, substrate-gel assay, and Morgan-Elson assay.

[0109] Turbidimetric assay is a method to measure the absorbance of precipitates formed when hyaluronic acid and albumin (BSA) are mixed. When hyaluronic acid is hydrolyzed by PH20, the absorbance decreases when mixed with albumin. Hyaluronidase PH20 (Sigma) is diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, and 60 Units / mL and prepared in each tube. Purified protein samples are dissolved in enzyme diluent buffer (20 mM TrisXHCl, pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and diluted to 100X, 300X, 600X, 1200X, and 2400X and prepared in each tube. In a new tube, dilute the 3 mg / mL hyaluronic acid solution 10 times to a concentration of 0.3 mg / mL, so that the volume of each tube is 180 μL. Add 60 μL of enzyme to the diluted hyaluronic acid solution, mix, and react at 37 ° C for 45 minutes. When the reaction is over, add 50 μL of the reacted enzyme and 250 μL of acidic albumin solution to each well of a 96-well plate, shake for 10 minutes, and then measure the absorbance at 600 nm using a spectrophotometer.

[0110] In the substrate-gel assay, proteins were electrophoresed on a 10% SDS gel (containing 0.17 mg / mL hyaluronic acid) for 1 hour, and then SDS was removed using 2.5% Triton X-100 (w / v) at 4°C for 2 hours. Then, enzyme reaction was performed for 1 to 4 hours at 37°C, the optimal temperature for pH 20, in 50 mM sodium phosphate, pH 7.0, 150 mM NaCl buffer, and proteins were stained using 0.5% Alcian blue reagent. Alcian blue reagent not bound to hyaluronic acid was removed using a de-staining solution. After taking a photograph of the Alcian blue-stained SDS gel, the bands were quantified.

[0111] Protein thermal stability was evaluated using dynamic light scattering (DLS) to measure aggregation temperature, and melting temperature (T) measured by real-time PCR using Sypro-Orange dye. m ), or by measuring enzyme activity after leaving it at a specific temperature for a certain period of time. Measurement of aggregation temperature using DLS is highly sensitive because it uses light scattering to measure the aggregation phenomenon of molecules, and is generally performed at the melting point (T m ) is lower in temperature.

[0112] In the PH20 mutants prepared in the present invention, the sequences of substituted or truncated amino acids are as shown in Table 11 below.

[0113] Among the mutants according to the present invention, the mutant with a 6xHis-tag attached to the C-terminus was named HM, the mutant without the 6xHis-tag was named HP, the matured wild-type PH20 (L36-S490) with a 6xHis-tag attached to the C-terminus was named WT, and the matured wild-type PH20 (L36-Y482) without the 6xHis-tag and truncated at the C-terminus after Y482 was named HW2.

[0114] [Table 11] TIFF2025004002000017.tif255164TIFF2025004002000018.tif255164TIFF2025004002000019.tif255167 TIFF2025004002000020.tif255164TIFF2025004002000021.tif255166TIFF2025004002000022.tif255167 TIFF2025004002000023.tif255167TIFF2025004002000024.tif255166TIFF2025004002000025.tif255166 TIFF2025004002000026.tif255167TIFF2025004002000027.tif255164TIFF2025004002000028.tif255166 TIFF2025004002000029.tif255166TIFF2025004002000030.tif255164TIFF2025004002000031.tif255166 TIFF2025004002000032.tif255166TIFF2025004002000033.tif255166TIFF2025004002000034.tif255166 TIFF2025004002000035.tif255166TIFF2025004002000036.tif255164TIFF2025004002000037.tif255164 TIFF2025004002000038.tif255164TIFF2025004002000039.tif255166TIFF2025004002000040.tif115170

[0115] Example 2. Generation of PH20 mutants HM1 and HM6 As shown in Figure 1B, the amino acid sequence M345-N363 of PH20 corresponds to the α-helix 8 site and the junction site between α-helix 7 and α-helix 8 in the tertiary structure model of the protein. Among the amino acids of α-helix 8, C351 forms a disulfide bond with C60 of α-helix 1, Y357 forms a hydrophobic interaction with F315 of α-helix 7, and N363 forms a hydrogen bond with D69 of α-helix 1, stabilizing the secondary structure adjacent to α-helix 8 (Figure 1C).

[0116] In order to create mutants with higher enzyme activity and thermostability than WT by substituting amino acids located at the α-helix 8 site and the junction site between α-helix 7 and α-helix 8 of PH20, mutants HM1 in which 12 amino acids were substituted in the M345-N363 section, HM2 in which 7 amino acids were substituted in the Y365-V379 section, and HM3 in which 19 amino acids were substituted in the M345-V379 section were created. Among the amino acids located in α-helix 8 of PH20, C351 involved in disulfide bonds and Y357 involved in hydrophobic interactions were not substituted. The sequences of the substituted amino acids in mutants HM1, HM2, and HM3 are shown in Table 11. When ExpiCHO cells were transfected with the pcDNA3.4-TOPO plasmid containing the genes of mutants HM1, HM2, and HM3, mutant HM1 was expressed in the ExpiCHO cells (Figure 3A), while mutants HM2 and HM3 were not expressed. The expression of the proteins was confirmed not only by measuring the enzyme activity but also by Western blot using an antibody against human PH20 (Abcam, ab193009). The epitope of the antibody is the Q173-P222 section. This experimental result suggests that the amino acid substitution in the Y365-V379 section used to generate mutants HM2 and HM3 in the amino acid sequence of α-helix 8 had a serious effect on the protein structure, and mutant HM2 and HM3 proteins were not expressed. The expression level of mutant HM1 was 3.4 times higher than that of the wild type WT (Figure 2). Mutant HM1 was created by substituting N363 with glycine to remove the hydrogen bond between N363 of α-helix 8 and D69 of α-helix 1 (FIG. 1C). To restore the hydrogen bond between N363 and D69, mutant HM6 was created by substituting G363 with asparagine in HM1. The substituted amino acids in mutant HM6 are shown in Table 11.

[0117] Mutant HM6 was expressed in ExpiCHO cells, and its expression level was similar to that of mutant HM1 (3.4-fold higher than WT) (Fig. 2). When enzyme activity was measured by turbidimetric assay, mutant HM6 showed 1.3-fold higher enzyme activity than WT (Fig. 3B). In the substrate-gel assay, SDS is generally removed using 2.5% Triton X-100 (w / v) after SDS-PAGE, and the degree of hydrolysis of hyaluronic acid by hyaluronidase is measured using Alcian blue staining agent while reacting at 37℃ for 1-4 hours. When SDS is removed from the substrate gel, protein refolding occurs immediately, and it is known that the substrate does not affect protein folding. When the enzyme activity of WT, mutants HM1, and HM6 was measured by substrate-gel assay at 37℃ for 1 to 4 hours, HM6 showed higher enzyme activity than WT and mutant HM1 (Figure 3C). This result suggests that the protein refolding and renaturation of mutants HM1 and HM6 are faster than that of WT, and the mutants thus constructed show higher enzyme activity than WT. When the signal peptide of PH20 itself was used, the protein expression level in ExpiCHO cells was low. To solve this problem, the signal peptide sequence of human serum albumin or human Hyal1 was used. As shown in Figure 3C, when the signal peptide of human serum albumin and human Hyal1 were used as the signal peptide of mutant HM1, the protein expression increased, and there was no significant difference between the two types of signal peptides. When enzyme activity was measured by substrate-gel assay, WT having a signal peptide of human serum albumin and mutant HM1 having a signal peptide of human serum albumin or Hyal1 showed enzyme activity in the pH range of 5 to 8 (Figure 3D). In the present invention, the signal peptide of human serum albumin was used as the signal peptide of mutants created after HM1.

[0118] The thermal stability of the WT and mutants HM1 and HM6 was compared in terms of aggregation temperature, which was 46.5°C, 53.0°C, and 50.5°C, respectively, and the aggregation temperatures of mutants HM1 and HM6 were 6.5°C and 4.0°C higher than that of the WT (Fig. 10). The aggregation temperature measurements are consistent with the protein folding results shown by substrate-gel assay. These results are the first to suggest that the hydrogen bond formed by the N363 residue in α-helix 8 plays an important role in the thermal stability of the protein structure and enzymatic activity.

[0119] In addition, when the hydrophilic / hydrophobic properties of WT and mutants HM1 and HM6 were compared using a phenyl column, both HM1 and HM6 were eluted before WT. This suggests that mutants HM1 and HM6 have more hydrophilic properties than WT due to amino acid substitutions. However, unlike WT, mutants HM1 and HM6 eluted from the phenyl column showed two peaks and the same molecular weight when treated with PNGase F. This is thought to be due to the difference in N-glycosylation (Figure 3E).

[0120] Example 3. Generation of PH20 mutants HM4, HM7, HM8, HM9, HM10, HM11, HM12 In Example 2, the production of mutants HM1 and HM6, in which the substitution of amino acids in the M345-N363 and M345-I361 sections increased both the enzyme activity and thermostability of PH20, is considered to be a major breakthrough in terms of protein engineering. Therefore, based on the mutants HM1 and HM6, further extensions were made in the N-terminal and C-terminal directions to substitute additional amino acids.

[0121] First, mutants HM4 and HM7 were prepared by replacing the amino acids between G340 and I344 with G340V, T341S, L342W, S343E, and I344N, respectively, and containing the amino acids replaced in mutants HM1 and HM6. The sequences of the replaced amino acids in mutants HM4 and HM7 are shown in Table 11. Mutants HM4 and HM7 were expressed in ExpiCHO cells. The results of protein purification for HM7 are shown in FIG. 5A. Protein expression in HM4 and HM7 was increased by 6.3 times compared to WT, and the aggregation temperatures were increased by 10°C and 11.5°C, respectively. However, the enzyme activities of HM4 and HM7 measured by turbidimetric assay were only about 15% of those of WT (FIG. 5B). In general, there is a trade-off between enzyme activity and thermostability. In the present invention, the substitutions introduced in mutants HM1 and HM6 increased thermostability while maintaining enzyme activity, whereas in mutants HM4 and HM7, the enzyme activity decreased due to excessive increase in thermostability. From the viewpoint of protein engineering, the increase in aggregation temperature of 11.5°C in mutants HM4 and HM7 is a very significant result. When the flexibility of the protein structure was analyzed by Stern-Volmer plot, HM1, HM6, HM4, and HM7, which had substitutions in α-helix 8 and its connecting site, all had higher structural flexibility than WT (Figure 11A). This result suggests that the increase in local thermostability increased the overall flexibility of the protein structure.

[0122] The difference between mutants HM6 and HM7 is the amino acids between G340 and I344. To confirm the amino acids involved in the increased thermal stability of mutant HM7, mutant HM6 was used as a base to generate HM8 with I344N substituted, HM9 with S343 and I344N substituted, HM10 with L342W, S343E, and I344N substituted, and HM21 with T341S, L342W, S343E, and I344N substituted. The sequences of the substituted amino acids in mutants HM8, HM9, HM10, and HM21 are shown in Table 11. Mutants HM8, HM9, HM10, and HM21 were expressed in ExpiCHO cells (Figure 5A). Based on the mutant HM6, the I344N, S343E, and L342W mutations were introduced toward the N-terminus of α-helix 8, and the aggregation temperatures of the mutants HM8, HM9, and HM10 increased to 52.5°C, 53°C, and 55.5°C, respectively (Figure 10). However, the enzyme activities of the mutants HM8, HM9, and HM10 were similar to those of the WT (Figure 5B). This result suggests that the amino acid substitutions introduced in the mutants HM8, HM9, and HM10 had a local effect on the thermal stability of the enzyme, but did not have a large effect on the enzyme activity. However, HM21 showed a lower thermal stability than HM10, but showed a two-fold higher characteristic than the WT at pH 7.0. When the WT and each mutant were reacted with the substrate for 1 hour in a substrate-gel assay, the enzyme activity was shown to be in the following order: HM21>HM10>HM9>HM8>HM6>WT (Figure 5C).

[0123] When the physical properties of mutants HM7, HM8, HM9, HM10, and HM21 were examined using a phenyl column, these PH20 mutants were eluted earlier than the WT, indicating that they all have hydrophilic properties, but showed different main peak patterns depending on the amino acid substitution positions (Figure 5D).

[0124] In the case of mutant HM7, like mutants HM1 and HM6, two peaks appear in the phenyl column, indicating the existence of two different forms.

[0125] To confirm the migration patterns of the WT and mutants according to their isoelectric points, isoelectric focusing (IEF) analysis was performed (Figure 5E). In the IEF gel, the WT and mutants HM6 and HM8 showed similar migration patterns, while mutants HM9, HM10, HM21, and HM7, which contained the S343E mutation, migrated toward a more acidic region. This result indicates that the introduction of glutamic acid by the S344E substitution between G340 and I344 changes the isoelectric point of the protein.

[0126] Then, mutants HM11 and HM12 were prepared by substituting amino acids extending toward the C-terminus of α-helix 8 based on mutant HM6. The sequences of the substituted amino acids in mutants HM11 and HM12 are shown in Table 11. Mutant HM11 was expressed in ExpiCHO cells, but the expression level was lower than that of the wild type WT (Figure 2), and mutant HM12 was not expressed in ExpiCHO cells. Mutant HM11 showed 32% activity compared to WT (Figure 6B).

[0127] Example 4. Generation of N-terminally truncated HM6-based mutants Although the C-terminal region of PH20 is already known to play an important role in the expression and enzyme activity of PH20, the role of amino acids at the N-terminal region of PH20 is not well known. To confirm how truncation of amino acids at the N-terminal region of PH20 affects enzyme activity, mutants HM40, HM13, HM41, HM24, HM42, and HM25 were prepared based on mutant HM6, with the N-terminus truncated at N37, F38, R39, A40, P41, and P42 (Table 11). Furthermore, HP61 and HP62 were prepared by modifying the amino acids at the N-terminus.

[0128] The mutants HM40, HM13, HM41, HM24, HM42, HP61, and HP62 were expressed in ExpiCHO cells, but HM25 was not expressed (Figures 7A and 7B). The N-terminally truncated PH20 mutants showed differences in enzyme activity depending on the starting position of the N-terminus. The mutants HM40, HM13, and HM41, which had 1 to 3 amino acids truncated, had similar enzyme activity compared to the template HM6, but the activity of HM24 and HM42, which had 4 to 5 amino acids truncated, was slightly reduced compared to HM6 (Figure 7C). However, HM25, which had 6 amino acids truncated, was not only hardly expressed in ExpiCHO, but also had a significantly low enzyme activity of 3.5 U / μg. HP61 and HP62, which had modifications to the N-terminal amino acids, showed no significant change in enzyme activity.

[0129] The enzyme activity of the N-terminal truncated mutants, measured by substrate-gel assay (1-hour reaction), was similar to that of the template HM6 in the cases of HM40, HM13, and HM41, but was reduced compared to that of HM6 in the cases of HM24 and HM42 (Fig. 7D). Analysis of HM25, in which six amino acids were truncated, was impossible due to the small amount of protein.

[0130] When the physical properties of mutants HM40, HM13, HM41, HM24, and HM42 were analyzed using a phenyl column, these mutants were eluted from the column before the WT, indicating hydrophilic properties (Figure 7E). This result is likely due to the hydrophilic properties of HM6, considering that mutants HM40, HM13, HM41, HM24, and HM42 were generated based on mutant HM6, and the properties of residues L36 to A40 are considered to be due to the hydrophilic properties of HM6.

[0131] The aggregation temperatures of the N-terminal truncated mutants measured by DLS differed for each mutant depending on the starting position of the amino acid (Figure 7F). Mutants HM40, HM13, HM41, and HM42 showed aggregation temperatures of 50°C or higher despite the N-terminus being truncated, and the characteristics of the template HM6 were maintained as they were. Among them, HM40 and HM42 showed aggregation temperatures 3 to 4°C higher than HM6, which is considered to have increased thermal stability. In addition, to confirm the effect of replacing amino acids located at the N-terminal site of PH20 on protein expression and enzyme activity, mutant HM17 in which the N-terminal amino acid sequence 36 to 47 (LNFRAPPVIPNV) of PH20 was replaced with FRGPLLPNR, and mutant HM18 in which the N-terminal amino acid sequence 36 to 52 (LNFRAPPVIPNVPFLWA) of PH20 was replaced with FRGPLLPNRPFTTV were prepared. The sequences of the replaced amino acids in mutants HM17 and HM18 are shown in Table 11. Mutants HM17 and HM18 were not expressed in ExpiCHO cells. Although truncations of up to five amino acids at the N-terminus showed protein expression and enzymatic activity, we suggest that substitutions of more amino acids, such as 36-47 or 36-52, affect protein folding.

[0132] Example 5. Generation of HM6-based mutants HM14, HM15, and HM16 with C-terminal truncations of PH20 The C-terminus of PH20 is known to be an important site in protein expression and enzyme activity. In the present invention, mutants HM14, HM15, and HM16 were prepared by truncating the C-terminal amino acids at I465, F468, and K471, respectively, based on mutant HM6. The sequences of the substituted amino acids in mutants HM14, HM15, and HM16 are shown in Table 11. Mutants HM14, HM15, and HM16 were expressed in ExpiCHO cells (Figure 8A), and the protein expression levels were in the order of HM16>HM15>HM14, and the more amino acids were truncated at the C-terminus, the lower the protein expression levels were (Figure 8A). However, the enzyme activity of mutants HM14, HM15, and HM16 was in the order of HM16>HM14(≫WT)>HM15 (Figure 8B). According to Frost et al., the C-terminal 477-483 region of PH20 is necessary for soluble expression, and when the C-terminus is truncated at C467, the enzyme activity is only 10% of that of a PH20 mutant truncated at C-terminus 477-483, and when the C-terminus is truncated before 467, there is no enzyme activity. However, the C-terminal truncated mutants (HM14, HM15, HM16) prepared in the present invention are based on the mutant HM6, and the substitution of amino acids in the M345-I361 section increases protein folding, which increases thermal stability. Therefore, even when the mutants are truncated after I465, F468, and P471 at the C-terminus, their enzyme activity is similar to that of the WT and there is no significant decrease in enzyme activity.

[0133] The structural flexibility of WT and mutants HM14, HM15, and HM16 was examined by fluorescence quenching with acrylamide (Figure 11B). Mutants HM14, HM15, and HM16 all had more flexible structures than WT. This result indicates that the C-terminal truncated mutants prepared based on mutant HM6 also maintain structural flexibility.

[0134] The enzyme activities of mutants HM14, HM15, and HM16 measured by turbidimetric assay were also confirmed by substrate-gel assay (FIG. 8C).

[0135] When the physical properties of the C-terminally truncated mutants were analyzed using a phenyl column, mutants HM14, HM15, and HM16 were all eluted earlier than WT and exhibited hydrophilic properties, with the hydrophilicity of the mutants being in the following order: mutant HM16>HM14>HM15 (Figure 8D).

[0136] Example 6. Generation of HM10-based mutants HM19 and HM20 containing amino acid truncations at the N- and C-terminus The PH20 mutants prepared in the present invention are based on HM6, and HM8, HM9, and HM10, which are additionally substituted with amino acids G340 to I344, showed superior performance in terms of protein expression level, enzyme activity, and thermal stability compared to WT. Among HM8, HM9, and HM10, HM10, which has high enzyme activity and thermal stability, was used as a base to prepare mutants HM19 and HM20, which are truncated at the N-terminus with F38 and at the C-terminus with K470 or F468. Both HM19 and HM20 were expressed in ExpiCHO cells and purified using a HisTrap column (Figure 9A). When the enzyme activity was measured by turbidimetric assay, HM19 and HM20 showed 10% higher enzyme activity than WT (Figure 9B). In the substrate-gel assay, HM19 and HM20 also showed higher enzyme activity than WT (Figure 9C).

[0137] Example 7. Characterization of HM10-Based PH20 Mutants The HM10-based C-terminal truncation mutants showed a tendency to decrease in expression in ExpiCHO cells as the C-terminus length became shorter, and were not expressed when the C-terminus length was C464 or less (Figure 12). C464 is essential because it forms a disulfide bond with C437 and is important for maintaining the protein structure.

[0138] Western blot experiments were performed to confirm whether the HM30, HM31, HM32, and HM33 mutants were not expressed in ExpiCHO when the C-terminus was truncated at 464 or shorter. As shown in Figure 13, the HM30, HM31, HM32, and HM33 mutants were not detected by Western blot.

[0139] The enzyme activity of HM10-based C-terminal truncation mutants measured by turbidimetric assay is shown in Figure 14A and Figure 14B. The C-terminal truncation mutants showed ±20% enzyme activity compared to WT. When the C-terminus was truncated after I480, the enzyme activity generally increased. In addition, HP19 and HP20, which are mutants in which the 6xHis-tag was removed from HM19 and HM20, showed a 23% and 9.6% decrease in enzyme activity, respectively, compared to when the 6xHis-tag was present, suggesting that the 6His-tag affects enzyme activity.

[0140] When the enzymatic activity of HM10-based C-terminal truncation mutants was measured by substrate gel assay, these mutants showed higher enzymatic activity than the WT and was similar to that of the template HM10, with no significant difference depending on the length of the C-terminus (Figure 14C).

[0141] Example 8. Characterization of HM21-Based PH20 Mutants Mutant HP34 was purified through four column steps (Figure 15A), and HP46 was purified through three column steps (Figure 15B). The yields of HP34 and HP46 were 1.73mg / L and 25.6mg / L, respectively. HP34 and HP46 are mutants without 6xHis-tag, and the purification process is different from that of mutants with 6xHis-tag, making it difficult to compare the protein expression levels.

[0142] In a turbidimetric assay, the activities of HP34 and HP46 were 45.6 U / μg and 47.2 U / μg, respectively, which were approximately two-fold higher than that of the WT and approximately 10% higher than that of the template, HM21 (FIG. 16A).

[0143] The kinetics of each mutant was measured by Morgan-Elson assay and are shown in Figure 16B. The catalytic efficiency (k cat / K m) is 1.7-2 times higher than that of wild-type HW2, which is consistent with the result that the specific activity is higher than that of WT. m ) was lower in these mutants than in HW2, which is considered to have increased affinity for the substrate. From these results, it can be concluded that the HM21, HP34, and HP46 mutants not only bind strongly to the substrate, but also have high efficiency in converting the substrate to the product. This property is the effect of substituting serine at T341. Substituting threonine at position 341 with amino acids such as alanine, glycine, and aspartic acid can be predicted to affect the enzyme activity.

[0144] The aggregation temperatures of HP34 and HP46 measured by DLS were 51.5°C and 51.0°C, respectively, which were similar to that of the template HM21 and showed approximately 5°C higher thermal stability than HW2 (Figure 17A). The activity of the mutant HP34 measured by substrate-gel assay was similar to that of HP20, while HP46 showed higher enzymatic activity than HP20. This suggests that the protein folding of HM21 as a template is superior to that of HM10 (Figure 17B).

[0145] When the wild-type HW2 and mutant HP46 were incubated overnight at pH 7.0 and pH 3.0, and their enzyme activities were compared by substrate-gel assay, HP46 showed high activity not only at pH 7.0 but also at pH 3.0, indicating excellent stability (Figure 17C).

[0146] HM53, HM54, HM55, HM56, HP59, and HP60 are HM21-based mutants that have a mutation in the amino acid at position 341. It was confirmed that the mutation in the amino acid at position 341 has various effects on the expression level and activity (FIGS. 17D and 17E).

[0147] Example 9. In vitro immunogenicity analysis of PH20 mutants Biopharmaceuticals, which have a larger molecular weight than low molecular weight chemical synthetic substances, have the risk of causing unexpected immune reactions when they are introduced into the human body. The secondary and tertiary structures of large molecular weight biomaterials have contact surfaces with the outside, which are created by folding and interaction with adjacent domains, and can provide epitopes to the immune system in the human body, thereby promoting immune reactions to the biomaterial. Such immune reactions can produce anti-drug antibodies (ADA), which can inhibit the effectiveness of the drug, induce hypersensitivity reactions to the drug, or promote clearance of the drug in the human body. Therefore, immune reactions to drugs can affect the results of clinical trials and can induce serious abnormal reactions when used for a long period of time. Various factors can affect such immune reactions, and they can be induced by reactions specific to the drug itself or the disease, or by factors depending on the dosage method or each patient. Factors related to the drug itself include similarity / dissimilarity between the biopharmaceutical and peptides in the human body, posttranslational modification, impurities, aggregate formation, characteristics of the dosage form, etc. Factors related to each patient include gender, reactivity with other drugs being taken, and genetic factors based on HLA (human leukocyte antigen) type.

[0148] These immunogenic responses are driven by CD4+ T cells and CD8+ T cells that recognize antigenic determinants regardless of the cause of the immune response. Due to the diversity of HLA class II genes in each individual, the antigenic determinants of CD4+ T cells vary from person to person. Therefore, the reactivity of CD4+ T cells in the blood of each healthy donor to biopharmaceuticals can be a very important criterion for evaluating the immune reactivity that may occur during the clinical process. CD4+ T cells are activated when they recognize antigens presented by antigen presenting cells (APCs) via their own type II MHC (major histocompatibility complex). Activated CD4+ T cells release cytokines to activate macrophages, cytotoxic T cells, and B cells, resulting in the production of large amounts of antibodies. In contrast, CD8+ T cells have direct cytotoxicity and directly eliminate cells infected with antigens, damaged cells, or cells that have lost their original functions. CD8+T cells have T cell receptors and can recognize peptides of specific antigens attached to type I MHC molecules located on the surface of all cells. CD8+T cells can also be activated by recognizing antigens presented by antigen-presenting cells, and this activation can be further promoted by CD4+T cell cytokines. Therefore, by measuring the activation level of CD4+T cells and CD8+T cells of a new biomaterial in vitro, it is possible to predict the immunogenic response that may be induced in the clinical course. In this example, CD4+T cells and CD8+T cells were isolated from PBMCs, and then treated with PH20 and PH20 mutant (HP46) as control substances at 1.5ng / mL and 15ng / mL, respectively, and the distribution of activated CD4+T cells and CD8+T cells was measured to predict the immunogenicity of the PH20 mutant by comparing it with the control substance. The activation level of each T cell was measured using the Stimulating Index, and the Stimulating Index (SI) is defined as follows:When the SI value is 2 or more, it can be determined that the activation is at a significant level.

[0149] Stimulating Index (SI) = (T cell activation after treatment with test sample) / (T cell activation after treatment with vehicle)

[0150] Since immunogenic responses may vary depending on the HLA type, to measure responses with a wider variety of HLA types, experiments were performed using T cells isolated from PBMCs received from 10 healthy donors. The HLA types of the 10 PBMCs used are shown in Table 12 below.

[0151] [Table 12]

[0152] The results of measuring the activation levels of CD4+ and CD8+ T cells in PH20 and the PH20 mutant are summarized in Table 13. Looking at the results of measuring the activation levels of CD4+ and CD8+ T cells, both PH20 and the PH20 mutant are judged to have relatively low levels of activation. PH20 was measured to have a CD4+ T cell activation level of 2 or higher in two experiments, while the PH20 mutant was not measured to have CD4+ T cell activation. In the case of CD8+ T cells, PH20 was measured to have activation in one experiment, and the PH20 mutant was also measured to have activation in one experiment. However, PH20 was measured to have an SI value of 2 or higher at both 1.5 ng / mL and 15 ng / mL, while the PH20 mutant was measured to have an SI value of less than 2 at 1.5 ng / mL and an SI value of more than 2 at 15 ng / mL (see Figures 18 and 19).

[0153] Therefore, at low concentrations, the activation of CD8+ T cells was observed to be low in the PH20 mutant, and it is considered that the activation of CD8+ T cells is higher in PH20 than in the PH20 mutant. The conclusions derived from the above results are as follows: 1) PH20 and PH20 mutants showed relatively low activation of CD4+ and CD8+ T cells. 2) The PH20 mutant is judged to have a lower ability to activate CD4+ T cells and CD8+ T cells than PH20.

[0154] These results predict that PH20 variants are less likely to induce immunogenic responses in the clinic than PH20.

[0155] [Table 13] [Industrial Applicability]

[0156] The PH20 mutant or fragment thereof of the present invention has the advantage that, when expressed in ExpiCHO cells, the protein expression level is increased compared to mature wild-type PH20, the protein aggregation temperature is increased to about 4 to 11.5°C, and thus the mutant has high thermostability and can be efficiently produced.

[0157] In addition, the PH20 mutant or fragment thereof of the present invention has the effect of improving protein refolding and being renatured more quickly than mature wild-type PH20 in the results of a substrate-gel assay, one of the experiments measuring hyaluronidase activity, and has the effect of maintaining the original enzyme activity regardless of the C-terminal truncation site.

[0158] Furthermore, the PH20 mutant or fragment thereof of the present invention has low immunogenicity and can be administered repeatedly to the human body.

[0159] <References> Arming,S.,Strobl,B.,Wechselberger,C.,andKreil,G.(1997).Invitromutagenesis ofPH-20hyaluronidasefromhumansperm.EurJBiochem247,810-814. 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.,andFrost,G.I.(2006).Arecombinant humanenzymeforenhancedinterstitialtransportoftherapeutics.JControlRelease 114,230-241. Chao,K.L.,Muthukumar,L.,andHerzberg,O.(2007).Structureofhuman hyaluronidase-1,ahyaluronanhydrolyzingenzymeinvolvedintumorgrowthand angiogenesis.Biochemistry46,6911-6920. Frost,G.I.(2007).Recombinanthumanhyaluronidase(rHuPH20):anenabling platformforsubcutaneousdrugandfluidadministration.ExpertOpinDrugDeliv4, 427-440.

Claims

1. A method for producing a PH20 mutant, comprising culturing a host cell containing a recombinant expression vector, The recombinant expression vector comprises a nucleic acid encoding the PH20 variant, and the amino acid sequence of the PH20 variant is (i) substitutions of amino acid residues M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T in SEQ ID NO:1; (ii) a deletion of the N-terminus of M1-T35, M1-L36, M1-N37, M1-F38, M1-R39, M1-A40, or M1-P41 in SEQ ID NO: 1; and (iii) a C-terminal deletion of the PH20 mutant, the C-terminus of which ends with an amino acid residue selected from any one of amino acid residues I465 to S490 in SEQ ID NO:1: The method for producing the above-mentioned compound, which is SEQ ID NO: 1 having the modifications consisting of:

2. The method described in claim 1, wherein the C-terminus of the PH20 mutant ends with an amino acid residue of I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490 in SEQ ID NO:

1.

3. A method for producing a PH20 mutant, comprising culturing a host cell containing a recombinant expression vector, The recombinant expression vector comprises a nucleic acid encoding the PH20 variant, and the amino acid sequence of the PH20 variant is (i) substitutions of amino acid residues M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T in SEQ ID NO:1, and substitutions of one or more amino acid residues selected from the group consisting of T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N, and N363G in SEQ ID NO:1; (ii) a deletion of the N-terminus of M1-T35, M1-L36, M1-N37, M1-F38, M1-R39, M1-A40, or M1-P41 in SEQ ID NO: 1; and (iii) a C-terminal deletion of the PH20 mutant, the C-terminus of which ends with an amino acid residue selected from any one of amino acid residues I465 to S490 in SEQ ID NO:1: The method for producing the above-mentioned compound, which is SEQ ID NO: 1 having the modifications consisting of:

4. The substitution of the amino acid residue in SEQ ID NO:1 is selected from the following amino acid residue substitution group: (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G; (d) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; and (e) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T: The method of claim 3, comprising the substitution of an amino acid residue in any one of:

5. A method for producing a PH20 mutant, comprising culturing a host cell containing a recombinant expression vector, The recombinant expression vector comprises a nucleic acid encoding the PH20 variant, and the amino acid sequence of the PH20 variant is (i) substitutions of amino acid residues T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T in SEQ ID NO:1; (ii) a deletion of the N-terminus of M1-T35, M1-L36, M1-N37, M1-F38, M1-R39, or M1-A40 in SEQ ID NO: 1; and (iii) a C-terminal deletion of the PH20 mutant, the C-terminus of which ends with an amino acid residue selected from any one of amino acid residues I465 to S490 in SEQ ID NO:1: The method for producing the above-mentioned compound, which is SEQ ID NO: 1 having the modifications consisting of:

6. The N-terminal deletion is a deletion of amino acid residues M1 to T35, M1 to L36, M1 to N37, or M1 to F38 in SEQ ID NO: 1, The method of claim 5, wherein the C-terminus of the PH20 variant ends with an amino acid residue of I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490 in SEQ ID NO:

1.

7. A method for producing a PH20 mutant, comprising culturing a host cell containing a recombinant expression vector, The recombinant expression vector comprises a nucleic acid encoding the PH20 variant, and the amino acid sequence of the PH20 variant is SEQ ID NO: 60, 63, 64, 65, 66, 67, 68, 69, 71, 72, 73, 74, 77, 78, 79, 80, 82, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 112, 113, 114, or 115. The method for producing the above.

8. The method described in claim 7, wherein the amino acid sequence of the PH20 variant consists of the amino acid sequence of SEQ ID NO: 60, 64, 66, 67, 68, 71, 72, 74, 77, 78, 79, 87, 88, 89, 91, 93, 94, 97, 99, 101, 102, 103, 104, or 105.

9. The method described in claim 7, wherein the amino acid sequence of the PH20 variant consists of the amino acid sequences of SEQ ID NOs: 64, 66, 67, 71, 74, 77, 78, 79, 87, 88, 93, 94, 97, 99, 101, 102, 103, 104, and 105.

10. The method described in claim 7, wherein the amino acid sequence of the PH20 mutant consists of the amino acid sequence of SEQ ID NO:99.

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