Novel hyaluronic acid hydrolase mutant and pharmaceutical composition comprising it

Abstract

This invention relates to the field of protein engineering technology for enhancing the enzymatic activity and thermostability of human hyaluronidase as a hyaluronidase, and to a hyaluronidase PH20 mutant or fragment thereof, said hyaluronidase PH20 mutant or fragment thereof comprising: substitution of at least one amino acid residue in the wild-type PH20 amino acid sequence having SEQ ID NO:1 at an α-helix site and / or a site corresponding to its linking site; and selectively, additional deletion of an N-terminal amino acid residue and / or a C-terminal amino acid residue. In particular, the present invention relates to a PH20 mutant and a fragment thereof, wherein the wild-type PH20 having the sequence of SEQ ID NO:1 comprises: substitution of at least one residue selected from T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E and I361T; additionally, substitution of amino acids located at the α-helix site 8 and / or the connection site between the α-helix site 7 and the α-helix site 8; and deletion of some amino acids at the N-terminal site and the C-terminal site.

Description

[0001] This application is a divisional application of the invention patent application filed on July 25, 2019, with application number 201980023392.4 and invention title "Novel Hyaluronic Acid Hydrolase Mutant and Pharmaceutical Composition Containing the Same". Technical Field

[0002] This invention relates to novel human hyaluronidase variants that, compared to human hyaluronidase as an enzyme for hydrolyzing hyaluronic acid, exhibit increased enzymatic activity and thermostability; and more particularly to a hyaluronidase PH20 variant or fragment thereof, wherein the hyaluronidase PH20 variant or fragment thereof contains one or more amino acid residue substitutions in the α-helix region and / or the linker region of wild-type PH20 having the amino acid sequence of SEQ ID NO:1, preferably mature wild-type PH20 consisting of amino acid residues L36 to S490, and wherein one or more of the N-terminal or C-terminal amino acid residues are selectively deleted in the hyaluronidase PH20 variant or fragment thereof; a method for producing the hyaluronidase PH20 variant or fragment thereof; and a pharmaceutical composition comprising the hyaluronidase PH20 variant or fragment thereof. Background Technology

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

[0004] These glycosaminoglycans are composed of repeating disaccharide units. The number of disaccharide units varies in glycosaminoglycans, ranging from hundreds to thousands. Hyaluronic acid is present in the skin in more than half the amount found in the human body. Hyaluronic acid is synthesized by hyaluronic acid synthase, which is present in cell membranes, exists independently without being bound to proteoglycans, and is the only glycosaminoglycan without sulfate groups. Other glycosaminoglycans are bound to proteoglycans and have sulfate groups. Hyaluronic acid consists of glucuronic acid and N-acetylglucosamine linked by alternating β-1,4 and β-1,3 bonds, and is composed of approximately 5,000 repeating units of these disaccharides. It is known that approximately one-third (5g) of the hyaluronic acid in the human body is turned over daily.

[0005] Hyaluronidase is an enzyme that degrades hyaluronic acid, which is present in the extracellular matrix. Six types of hyaluronidase are known to exist in the human body: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20 / SPAM1. Human Hyal1 and Hyal2 are expressed in most tissues. PH20 / SPAM1 (hereinafter referred to as PH20) is expressed in the sperm plasma membrane and acrosomal membrane. However, HyalPS1 is not expressed because it is a pseudogene. Based on the method of cleaving hyaluronic acid, hyaluronidase is classified into three types: enzymes that cleave the β-1,4 bond between N-acetylglucosamine and glucuronic acid using H2O (EC 3.2.1.35); enzymes that cleave the β-1,3 bond between N-acetylglucosamine and glucuronic acid using H2O (EC 3.2.1.36); and bacterial hyaluronidase that cleaves the β-1,4 bond without using H2O (EC 4.2.99.1).

[0006] Hyal1 catalyzes the amino acids D129 and E131, which hydrolyze hyaluronic acid via substrate-assisted catalysis. Hyal1 exhibits optimal activity at acidic pH levels of 3 to 4, but shows no enzymatic activity at pH levels of 4.5 or higher. In contrast to Hyal1, PH20 exhibits enzymatic activity over a wide pH range of 3 to 8.

[0007] Arming et al. identified the catalytic amino acids of PH20 as D111 and E113 (Arming et al., 1997). Arming et al. labeled Leu as the first amino acid of the mature protein, and therefore the catalytic amino acids of the full-length PH20 with the signal peptide correspond to D146 and E148, respectively.

[0008] Hyaluronidase hydrolyzes hyaluronic acid, thereby reducing its viscosity in the extracellular matrix and increasing its permeability into tissues (skin). The subcutaneous region of the skin has a neutral pH of approximately 7.0 to 7.5. Therefore, among various types of hyaluronidase, PH20 is widely used in clinical practice (Bookbinder et al., 2006). Examples of PH20's clinical use include its use as an ophthalmospasm and anesthetic additive in ophthalmic surgery, and its co-administration with subcutaneously injected antibody therapeutics (Bookbinder et al., 2006). Furthermore, based on the overexpression of hyaluronic acid in tumor cells, PH20 is used to hydrolyze hyaluronic acid in the extracellular matrix of tumor cells, thereby increasing the accessibility of anticancer therapeutics to tumor cells. Additionally, it is used to promote the reabsorption of excess body fluids and blood in tissues.

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

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

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

[0012] Recombinant human PH20 proteins have been reported to be expressed in yeast (Pichia pastoris), DS-2 insect cells, and animal cells. The recombinant PH20 proteins produced in insect cells and yeast differ from human PH20 in their N-glycosylation patterns during post-translational modifications.

[0013] Among hyaluronidases, only the three-dimensional structures of Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidases (PDB ID: 1FCQ, 1FCU, 1FCV) have been determined. Hyal1 consists of two domains: a catalytic domain and an EGF-like domain. The catalytic domain is in the form of (β / α)8, where the α-helix and β-chain, which characterize the protein's secondary structure, are each repeated eight times (Chao et al., 2007). The EGF-like domain is completely conserved in variants in which the C-terminus of Hyal1 is spliced ​​differently. Hyal1 and PH20 share 35.1% amino acid sequence similarity, and the protein structure of PH20 has not yet been determined.

[0014] A recombinant human PH20 protein was developed by Halozyme Therapeutic, Inc. and marketed under the trade name... For Sale (Bookbinder et al., 2006; Frost, 2007).

[0015] When the catalytic amino acids D146 and E148 of PH20 are mutated to asparagine (D146N) and glutamine (E148Q), respectively, enzyme activity is absent (Arming et al., 1997). Furthermore, when R246 of PH20 is replaced with glycine, enzyme activity decreases by 90%, and when E319 is replaced with glutamine and R322 with threonine, enzyme activity disappears. Compared to wild-type PH20, a variant with 36 amino acids removed from the C-terminus (474-509 amino acid truncation) showed a 75% reduction in enzyme activity. This variant is not secreted extracellularly but remains in HeLa cells. When 134 amino acids are removed from the C-terminus of PH20, PH20 has no enzyme activity and is not secreted extracellularly. According to Frost et al., the C-terminal region 477-483 of PH20 is essential for soluble expression (Frost, 2007). The activity of the full-length PH20 (1 to 509) or the PH20 variant with its C-terminus truncated at position 467 is only 10% of that of the PH20 variant with its C-terminus truncated at one of positions 477 to 483 (Frost, 2007).

[0016] Meanwhile, recombinant PH20 still lacks sufficient thermostability or expression levels in recombinant cells. Therefore, there is a significant industrial demand for recombinant hyaluronidase with further improved biological and physicochemical characteristics. Summary of the Invention

[0017] Technical issues

[0018] One object of the present invention is to provide a hyaluronidase PH20 variant or a fragment thereof, which has improved thermostability, enzyme activity and expression level compared with wild-type PH20, preferably mature wild-type PH20.

[0019] Another object of the present invention is to provide a composition for treating cancer, the composition comprising the above-described hyaluronidase PH20 variant or a fragment thereof; and a method of treating cancer using the composition.

[0020] Technical solution

[0021] To achieve the above objectives, the present invention provides a hyaluronidase PH20 variant or a fragment thereof, wherein the hyaluronidase PH20 variant or the fragment thereof contains one or more amino acid residues substituted in the α-helix region and / or the linker region of the amino acid sequence corresponding to wild-type PH20, preferably mature wild-type PH20, and wherein one or more of the N-terminal or C-terminal amino acid residues are selectively deleted in the hyaluronidase PH20 variant or the fragment thereof.

[0022] The present invention also provides a composition for treating cancer, the composition comprising the above-described hyaluronidase PH20 variant or a fragment thereof; and a method of treating cancer using the composition. Attached Figure Description

[0023] Figure 1 A protein tertiary structure model of PH20 is shown. The protein tertiary structure of PH20 was modeled using Hyal1 (PDBID:2PE4) (Chao et al., 2007) as a template, and the protein crystal structure of Hyal1 was found in the Swiss-Model server (https: / / swissmodel.expasy.org / ).

[0024] Figure 1 A shows a tertiary structure model of the PH20 protein, indicating D146 and E148 as catalytic amino acids. The tertiary structure model of the PH20 protein consists of eight repeating sequences consisting of a β chain and an α helix.

[0025] Figure 1 B shows the eta(η)8 ring, in which the α-helix 8 of PH20 and G340 to I344, which form the linker region in the N-terminal region of the α-helix 8, are located. Residues G340, T341, L342, S343, and I344 are each shown.

[0026] Figure 1C shows the amino acid residues (C351, Y357, and N363) that interact with adjacent secondary structures in the α-helix 8 of PH20. C351 forms a disulfide bond with C60 in α-helix 1, Y357 hydrophobically interacts with F315 located between β-chain 7 and α-helix 7, and N363 forms a hydrogen bond with D69 residue in α-helix 1.

[0027] Figure 2 Protein expression levels were compared between wild-type (WT) and the variants constructed in this invention. WT and variants were expressed via transient transfection in ExpiCHO cells. The expression level of WT was 16.1 mg / L. Protein expression levels of variants HM1 and HM6 were higher than those of WT, with the highest expression levels observed in HM4 and HM7. The protein expression level of HM11, obtained by introducing additional amino acid substitutions (Y365F and I367L) into variant HM6, was reduced to 6.4 mg / mL.

[0028] Figure 3 Experimental results for variants HM1 and HM6 are shown.

[0029] Figure 3 Figure A shows the results of SDS-PAGE after purification of WT and variants HM1 and HM6. Purification was performed using HisTrap and QSepharose columns. The molecular weight of WT and variants HM1 and HM6 is approximately 70 kDa (Legend: M, molecular weight label; CS, supernatant; FT, flow rate; and elution, elution fraction).

[0030] Figure 3 B shows the enzyme activity values ​​of WT and variants HM1 and HM6 as measured by turbidity at pH 7.0. In this invention, enzyme activity values ​​measured by turbidity are expressed as specific activity.

[0031] Figure 3 C shows the enzyme activities of WT and variants HM1 and HM6 as measured by substrate-gel assay. Enzymatic reactions were carried out at 37°C for 1 to 4 h after SDS removal with 2.5% Triton X-100 (w / v) at 4°C. Variant HM6 renatured faster than WT and variant HM1, and therefore hydrolyzed hyaluronic acid more rapidly on polyacrylamide gels. White bands indicate hyaluronic acid degraded by WT and variant proteins.

[0032] Figure 3Figure D shows the enzyme activities of WT and variant HM1 as measured by substrate-gel assay at pH 5 to 8. WT and variant HM1 exhibited activity in the pH range of 5 to 8, with the highest enzyme activity at pH 5.0. Variant HM1 possesses the signal peptide of human serum albumin or human Hyal1. White bands indicate hyaluronic acid degraded by WT and variant proteins.

[0033] Figure 3 E shows the results of separating WT and variants HM1 and HM6 by a phenyl column. The variants eluted from the phenyl column faster than WT.

[0034] Figure 4 The results are shown in the final products of hyaluronic acid degraded by WT and variant HM6 after 10 min and 1 h by amide-80 column analysis.

[0035] Figure 5 The experimental results of the G340 to I344 amino acid mutation in PH20 are shown.

[0036] Figure 5 A shows the SDS-PAGE results of variants HM7, HM8, HM9, HM10, and HM21 after purification on a HisTrap column.

[0037] Figure 5 B shows the results of measuring the enzyme activity of WT and variants HM6, HM8, HM9, HM10, HM21 and HM7 by turbidity assay at pH 7.0.

[0038] Figure 5 C shows the results of measuring the enzyme activity of WT and variants HM6, HM8, HM9, HM10, HM21 and HM7 by substrate-gel assay. Figure 5 The bar graph at the bottom of C shows the level of enzyme activity obtained by quantitative banding after staining the gel with Asin Blue. The white bands show hyaluronic acid degraded by WT and variant proteins.

[0039] Figure 5 D shows the results of analysis of WT and variants HM8, HM9, HM10, HM21 and HM7 by phenyl column chromatography.

[0040] Figure 5 E shows the results of separating WT and variants HM6, HM8, HM9, HM10, HM21 and HM7 by IEF gel at pH 3 to 7 according to their isoelectric points.

[0041] Figure 6 Experimental results for variant HM11 are shown.

[0042] Figure 6A shows the results of protein purification using HisTrap column chromatography for variant HM11.

[0043] Figure 6 B shows the results of measuring the enzyme activity of WT and variant HM11 at pH 7.0 by turbidity assay.

[0044] Figure 7 Experimental results for the N-terminally truncated PH20 variants HM40, HM13, HM41, HM24, HM42, and HM25 are shown.

[0045] Figure 7 A shows the results of protein purification by HisTrap column chromatography for PH20 variants HM40, HM13, HM41, HM24, HM42, and HM25.

[0046] Figure 7 B shows the expression levels of PH20 variants HM40, HM13, HM41, HM24, HM42, HM25, HP61, and HP62 in ExpiCHO cells.

[0047] Figure 7 C shows the enzyme activities of the PH20 variants HM40, HM13, HM41, HM24, HM42, HM25, HP61, and HP62 as measured by turbidity determination at pH 7.0, and expressed as specific activities.

[0048] Figure 7 D shows the enzyme activities of PH20 variants HM40, HM13, HM41, HM24, and HM42 as measured by substrate-gel assay. White bands indicate hyaluronic acid degraded by WT and variant proteins.

[0049] Figure 7 E shows the results of analysis of WT and variants HM40, HM13, HM41, HM24 and HM42 by phenyl column chromatography.

[0050] Figure 7 F shows the changes in particle size of PH20 variants HM40, HM13, HM41, and HM42 as temperature increases.

[0051] Figure 8 Experimental results are shown for C-terminal truncated variants HM14, HM15, and HM16 constructed using HM6 as a template.

[0052] Figure 8 A shows the SDS-PAGE results of variants HM14, HM15, and HM16 after HisTrap purification. WT and variant HM6 are included as controls.

[0053] Figure 8 B shows the results of measuring the enzyme activity of WT and variants HM6, HM14, HM15 and HM16 at pH 7.0 by turbidity assay.

[0054] Figure 8 C shows the results of measuring the enzyme activity of WT and variants HM6, HM14, HM15 and HM16 for 1, 2 and 4 h by substrate-gel assay. Figure 8 The right-hand graph in C is a bar graph showing the enzyme activity measured after 1 hour of enzymatic reaction and staining with Asin Blue. The white bands indicate hyaluronic acid degraded by WT and variant proteins.

[0055] Figure 8 D shows the results of analysis of WT and variants HM6, HM14, HM15 and HM16 by phenyl column chromatography.

[0056] Figure 8 E shows the results of separating variants HM14, HM15, and HM16 according to their isoelectric points using IEF gels at pH 3 to 7.

[0057] Figure 9 Experimental results for the PH20 variants HM19 and HM20, constructed using HM10 as a template, are shown.

[0058] Figure 9 A shows the results of protein purification by HisTrap column chromatography for PH20 variants HM19 and HM20.

[0059] Figure 9 B shows the results of comparing the enzyme activities of the PH20 variants HM19 and HM20 at pH 7.0 by turbidity assay.

[0060] Figure 9 C shows the results of staining SDS gels with Aspen Blue after an enzymatic reaction at 37°C for 1 h for WT and variants HM10, HM19, and HM20, via substrate-gel assay. White bands indicate hyaluronic acid degraded by WT and variant proteins.

[0061] Figure 10 Results of measurements of the aggregation temperatures of the WT and PH20 variants using a dynamic light scattering (DLS) system are shown. Measurements were performed in triplicate and are expressed as mean ± SE values.

[0062] Figure 11Stern-Volmer plots are shown after measuring the fluorescence changes of tryptophan residues in WT and PH20 variants by adding acrylamide (0 to 0.5 M). Among the amino acids, tryptophan is excited at 295 nm and emits maximum fluorescence at 340 nm. Acrylamide is a small molecule that can penetrate protein structures and quench the fluorescence emission of tryptophan. Due to the greater flexibility of protein structures, the quenching effect of acrylamide is more pronounced. F0 is the fluorescence value in the absence of acrylamide, while F is the fluorescence value in the presence of acrylamide (0 to 0.5 M). The measured changes in fluorescence value are expressed as the ratio F0 / F.

[0063] Figure 11 A is the Stern-Volmer plot of WT and its variants HM1, HM4, HM6, and HM7.

[0064] Figure 11 B is the Stern-Volmer plot of WT and its variants HM14, HM15 and HM16.

[0065] Figure 12 The expression level of the HM10-based PH20 variant in ExpiCHO cells is shown.

[0066] Figure 12 A graphically illustrates the expression levels of each variant.

[0067] Figure 12 B shows the expression levels of the various variants in the table. The WT and PH20 variants have a 6xHis tag at the C-terminus, and the protein expression levels after HisTrap column purification are expressed in mg / L. The HM30 to HM33 variants were not expressed in ExpiCHO cells.

[0068] Figure 13 Western blot results for cell cultures of variants HM29, HM30, HM31, HM32, and HM33 are shown. The C-termini of HM10-based variants HM29, HM30, HM31, HM32, and HM33 were cleaved at A467, C464, D461, C358, or C455, respectively. C-terminally cleaved HM29 was expressed in ExpiCHO cells, but variants with a C-terminus cleaved at C464 or shorter were not expressed in ExpiCHO cells. The primary antibody was rabbit anti-PH20 polyclonal antibody (Abcam) diluted 1:500. The secondary antibody was goat anti-rabbit IgG HRP diluted 1:2,000.

[0069] Figure 14 Experimental results are shown for a C-terminal truncated variant constructed using HM10 as a template.

[0070] Figure 14 A shows the results of measuring the enzyme activity at pH 7.0 of the C-terminal cleavage variant constructed using HM10 as a template by turbidity assay.

[0071] Figure 14 B compared the enzyme activities dependent on the C-terminal cleavage site of 17 PH20 variants (HM43, HM44, HM45, HM20, HM19, HM35, HM36, HM37, HM38, HM39, HM47, HM48, HM49, HM50, HM51, HM52, and HM10) constructed using HM10 as a template.

[0072] Figure 14 C shows the results of substrate-gel assays for some of the PH20 variants (HM29, HM35, HM36, HM37, HM38, HM39, HM43, HM44, and HM45) constructed using HM10 as a template, after 1 h of enzymatic reaction at 37°C, stained with Aspen Blue. White bands indicate hyaluronic acid degraded by WT and variant proteins.

[0073] Figure 15 HP34 expressed in ExpiCHO cells is shown. Figure 15 A) and HP46 Figure 15 B) Protein purification process followed by a final column SDS gel. HP34 underwent a four-step chromatographic purification procedure consisting of Q Sepharose, Butyl HP, heparin, and Blue Sepharose columns, with the SDS gel obtained after the Blue Sepharose column chromatography. HP46 underwent a three-step chromatographic purification procedure consisting of Q Sepharose, Butyl HP, and heparin columns, with the SDS gel obtained after the heparin column chromatography.

[0074] Figure 16 The enzyme activities of the PH20 variants HP34 and HP46 without the 6xHis tag, constructed using HM21 as a template, are shown.

[0075] Figure 16 A shows the results of measuring the enzyme activity of WT and variants HM21, HP34 and HP46 at pH 7.0 by turbidity assay.

[0076] Figure 16 B shows the results obtained through Morgan-Elson determination (K m Michaelis-Menten constant, k cat : Transformation number, and k cat / Km The results of measuring the enzyme activity of WT(HW2) and variants HM21, HP34 and HP46 at pH 5.3 were: Catalytic efficiency.

[0077] Figure 17 The characterization results of the PH20 variant based on HM21 are shown.

[0078] Figure 17 A shows the results of DLS measurements of the aggregation temperatures of the 6xHis-label-free PH20 variants HP34 and HP46 constructed using HM21 as a template. As a control, the aggregation temperatures of HW2 and HM21 are shown.

[0079] Figure 17 B shows the results of measuring 1-hour enzyme activity for HW2 and variants PH20 (HP20, HP34, and HP46) using a substrate-gel assay.

[0080] Figure 17 C shows the results of substrate-gel assays performed after the variant (HW2 and HP46) samples were allowed to stand at pH 3.0 and pH 7.0 for 14 h. After SDS-PAGE, SDS was removed with 2.5% Triton X-100 (w / v), and the enzymatic reaction was carried out at 37 °C for 1 h.

[0081] Figure 17 D shows the expression levels of PH20 variants HM21, HM53, HM54, HM55, HM56, HP59, and HP60 in ExpiCHO cells.

[0082] Figure 17 E shows the enzyme activity values ​​of the PH20 variants HM21, HM53, HM54, HM55, HM56, HP59 and HP60 at pH 5.3 as a representation of specific activity, measured by turbidity assay.

[0083] Figure 18 The results show the results of measuring the stimulation index of CD4+ T cells when treated with PH20 and its variant at concentrations of 1.5 ng / mL and 15 ng / mL, respectively.

[0084] Figure 19 The results show the results of measuring the stimulation index of CD8+ T cells when treated with PH20 and PH20 variant at concentrations of 1.5 ng / mL and 15 ng / mL, respectively. Detailed Implementation

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental methods described below are those well-known and frequently used in the art.

[0086] The present invention provides a hyaluronidase PH20 variant or a fragment thereof, wherein the hyaluronidase PH20 variant or the fragment thereof contains one or more amino acid residues substituted in the α-helix region and / or its linker region, preferably the α-helix 8 region (S347 to C381) and / or the linker region (A333 to R346) between the α-helix 7 and the α-helix 8 in the amino acid sequence corresponding to wild-type PH20, preferably mature wild-type PH20, and wherein one or more of the N-terminal or C-terminal amino acid residues are selectively cleaved or deleted in the hyaluronidase PH20 variant or the fragment thereof.

[0087] In this invention, the position of the amino acid residue in each variant corresponds to the amino acid position of wild-type PH20 having the sequence SEQ ID NO:1.

[0088] Furthermore, in this invention, "mature wild-type PH20" means a protein in the amino acid sequence of wild-type PH20 having the sequence of SEQ ID NO:1 consisting of amino acid residues L36 to S490 of SEQ ID NO:1, said protein lacking M1 to T35 for forming a signal peptide and A491 to L509 which are not related to the basic enzymatic function of PH20.

[0089] Table 1. Amino acid sequence of wild-type PH20 (SEQ ID NO:1)

[0090]

[0091] In particular, the PH20 variant or fragment thereof according to the present invention may contain one or more mutations in the wild-type PH20 having the amino acid sequence of SEQ ID NO:1, preferably selected from T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E and I361T, more preferably selected from the substitution of amino acid residues of T341A, T341C, L354I and N356E.

[0092] In this invention, the term "PH20 variant" is intended to include mutations in one or more amino acid residues in the amino acid sequence of wild-type PH20, preferably substitutions of one or more amino acid residues, and deletions and substitutions of one or more amino acid residues at the N-terminus or C-terminus; and is used to have substantially the same meaning as "PH20 variant or fragment thereof".

[0093] In this invention, the tertiary structure of human PH20 located outside the active site was investigated by modeling the protein structure of Hyal1 (SEQ ID NO:2), which is a human hyaluronidase (whose protein tertiary structure is known). As a result, amino acids located in the α-helix 8 region of PH20 were selected and substituted with the amino acid sequence of the α-helix 8 region of Hyal1, thereby attempting to enhance the thermal stability of the protein structure without affecting the catalytic activity of the enzyme. Specifically, because the α-helix 8 region is located outside the tertiary structure of PH20, its interaction with adjacent α-helices or β-chains is smaller than that with other α-helices of PH20. According to the invention, it has been found that when the amino acid sequences of the α-helix 8 region and the linker region between α-helix 7 and α-helix 8 of human PH20 are partially substituted with the amino acid sequences of the α-helix 8 region and the linker region between α-helix 7 and α-helix 8 of the highly hydrophilic Hyal1, the enzyme activity at neutral pH and the protein aggregation temperature (T0) are improved. agg. Based on these experimental results, it has been found that novel PH20 variants or fragments thereof can be provided, which have increased enzyme activity and thermostability compared to wild-type PH20.

[0094] Therefore, the PH20 variant according to the invention contains, in the amino acid sequence of wild-type PH20 (having the amino acid sequence of SEQ ID NO:1), preferably mature wild-type PH20 (having a sequence consisting of amino acid residues L36 to S490 in the amino acid sequence of SEQ ID NO:1), one or more amino acid residues selected from T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E and I361T, preferably selected from T341A, T341C, L354I and N356E.

[0095] The PH20 variant according to the invention also contains one or more amino acid residue substitutions in the α-helix region and / or its linker region corresponding to the amino acid sequence having SEQ ID NO:1 in the wild-type PH20, preferably the α-helix 8 region (S347 to C381) and / or the linker region between α-helix 7 and α-helix 8 (A333 to R346), more preferably the regions T341 to N363, T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361 or M345 to N363.

[0096] In particular, in the PH20 variant according to the invention, the α-helix 8 region (S347 to C381) and / or the linker region (A333 to R346) between the α-helix 7 and the α-helix 8 of wild-type PH20, preferably mature wild-type PH20, may be replaced by one or more amino acid residues of the corresponding region of Hyal1 having the sequence of SEQ ID NO:2 (see Tables 2 and 3), but is not limited thereto.

[0097] Table 2. Amino acid sequence of wild-type Hyal1 (SEQ ID NO:2)

[0098]

[0099] Table 3. Comparison of α-helix and amino acid sequences between PH20 and Hyal1

[0100]

[0101]

[0102] More particularly, the novel PH20 variants or fragments thereof according to the invention preferably contain substitutions of amino acid residues L354I and / or N356E in the amino acid sequence of wild-type PH20, preferably mature wild-type PH20.

[0103] It also includes, but is not limited to, amino acid residue substitutions at one or more positions selected from T341 to N363, particularly at one or more positions selected from T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361, and N363.

[0104] More preferably, the substitution of amino acid residues at one or more positions selected from T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361, and N363 may be the substitution of one or more amino acid residues selected from T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T, and N363G, but is not limited thereto.

[0105] Preferably, the novel PH20 variant or fragment thereof according to the present invention may contain one or more amino acid residues substituted from M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T.

[0106] It may also contain one or more amino acid residues selected from T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N, and N363G, but is not limited thereto.

[0107] More preferably, the novel PH20 variant or fragment thereof according to the present invention may contain any amino acid residue substitution selected from the group consisting of, but not limited to:

[0108] (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;

[0109] (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;

[0110] (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T and N363G;

[0111] (d) T341G, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;

[0112] (e) T341A, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;

[0113] (f) T341C, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;

[0114] (g) T341D, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;

[0115] (h)I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; and

[0116] (i) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T.

[0117] In this invention, a representation described by a single-letter amino acid residue code and a number (such as "S347") refers to an amino acid residue at a position in the amino acid sequence of SEQ ID NO:1.

[0118] For example, “S347” means that the amino acid residue at position 347 in the amino acid sequence of SEQ ID NO:1 is serine.

[0119] In addition, “S347T” means that the serine at position 347 of SEQ ID NO:1 is replaced by threonine.

[0120] The PH20 variants according to the invention are interpreted as including variants or fragments thereof, wherein amino acid residues at specific amino acid residue positions are conservatively substituted.

[0121] As used herein, the term “conservative substitution” refers to a modification of the PH20 variant involving the substitution of one or more amino acids with amino acids having similar biochemical properties, which does not result in the loss of the biological or biochemical function of the PH20 variant.

[0122] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by 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 to which this invention pertains. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0123] It is foreseeable that the PH20 variant or fragment of the present invention, despite having conserved amino acid substitutions, can still maintain its activity.

[0124] Furthermore, the PH20 variants or fragments thereof according to the present invention are interpreted as including PH20 variants or fragments thereof that have substantially the same function and / or effect as the PH20 variants or fragments thereof according to the present invention, and have at least 80% or 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% amino acid sequence homology with the PH20 variants or fragments thereof according to the present invention.

[0125] Compared to mature wild-type PH20, the PH20 variant according to the invention exhibits increased expression levels and protein refolding rates, thereby increasing high thermal stability. Furthermore, despite the increased thermal stability, the enzyme activity of the PH20 variant is increased more significantly or similarly to that of mature wild-type PH20.

[0126] Furthermore, while mature wild-type PH20 variants with C-terminal deletions exhibit reduced enzyme activity, based on this invention, C-terminal-deleted PH20 variants show similar or increased enzyme activity due to faster protein refolding and thermal stability. Moreover, the PH20 variants of this invention maintain enzyme activity when up to five amino acid residues are missing from the N-terminus. This indicates that the N-terminal P41 is important for both protein expression and enzyme activity.

[0127] Therefore, the PH20 variant or fragment thereof according to the present invention is characterized in that the PH20 variant or fragment thereof contains one or more amino acid residues substituted in the α-helix 8 region (S347 to C381) and / or the linker region (A333 to R346) between α-helix 7 and α-helix 8 in the amino acid sequence of wild-type PH20, and additionally omits one or more of the N-terminal and / or C-terminal amino acid residues, but is not limited thereto.

[0128] In one aspect, the PH20 variant or fragment thereof according to the invention can be a variant or fragment thereof in which cleavage occurs in a sequence selected from SEQ ID. The cleavage occurs before amino acid residues M1 to P42 of the amino acid sequence of NO:1, preferably before amino acid residues L36, N37, F38, R39, A40, P41, or P42 at the N-terminus, such that one or more amino acid residues at the N-terminus are deleted, and / or after amino acid residues selected from V455 to L509 at the C-terminus, preferably after amino acid residues selected from V455 to S490, most preferably after 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, such that one or more amino acid residues at the C-terminus are deleted.

[0129] The statement "The cleavage occurs before the amino acid residues selected from M1 to P42 at the N-terminus" means that the amino acid residue immediately preceding the amino acid residues selected from M1 to P42 at the N-terminus is cleaved and deleted.

[0130] For example, the statement "the cleavage occurs before amino acid residues L36, N37, F38, R39, A40, P41, or P42" means, respectively, cleaving and removing all amino acid residues from M1 to T35 immediately preceding L36, all amino acid residues from M1 to L36 immediately preceding N37, all amino acid residues from M1 to N37 immediately preceding F38, all amino acid residues from M1 to F38 immediately preceding R39, all amino acid residues from M1 to R39 immediately preceding A40, all amino acid residues from M1 to A40 immediately preceding P41, or all amino acid residues from M1 to P41 immediately preceding P42.

[0131] Furthermore, the statement "the cleavage occurs after the amino acid residues selected from V455 to L509 at the C-terminus" means that the amino acid residues immediately following the amino acid residues selected from V455 to L509 at the C-terminus are cleaved and deleted.

[0132] For example, the statement "the cleavage occurs after the 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 at the C-terminus" means that the SEQ ID is cleaved and removed. The amino acid residues following 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 in the amino acid sequence of NO:1.

[0133] Preferably, the novel PH20 variant or fragment thereof according to the present invention may be selected from the amino acid sequences of SEQ ID NO:60 to 115, but is not limited thereto.

[0134] Most preferably, the novel PH20 variant or fragment thereof according to the present invention may have the amino acid sequence of SEQ ID NO:99. The novel PH20 variant or fragment thereof having the amino acid sequence of SEQ ID NO:99 may contain 15 amino acid substitutions of T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, as well as a deletion before F38 at the N-terminus and a truncation after F468 at the C-terminus.

[0135] The sequences of amino acids that are substituted or cleaved in the PH20 variant constructed according to a specific embodiment of the invention are shown in Table 11.

[0136] As disclosed in this invention, there have been no previously reported studies focusing on increasing the enzymatic activity and thermostability of PH20 through amino acid substitutions of the α-helix and its linker region, which are secondary structures forming the tertiary structure of the protein. Previous studies have reported that the enzymatic activity of wild-type PH20 varies depending on the cleavage position of the amino acid residues located at the C-terminal region. However, in this invention, a specific α-helix forming the secondary structure of PH20 is substituted with the α-helix of other hyaluronidases, thereby constructing PH20 variants with higher stability than wild-type PH20. These variants can be variants in which the interaction between the substituted α-helix domain and the portion forming the secondary structure of other PH20 exhibits a pattern different from that of wild-type PH20, indicating that the variant has consistent enzymatic activity regardless of the C-terminal cleavage position.

[0137] In a particular embodiment, a novel PH20 variant or fragment thereof according to the invention, having increased enzyme activity and thermostability compared to mature wild-type PH20, may be a variant or fragment thereof comprising substitutions of one or more amino acid residues selected from the following: T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E, and I361T, wherein one or more amino acids located in the α-helix 8 region (S347 to C381) and / or the linker region (A333 to R346) between α-helix 7 and α-helix 8 in the amino acid sequence of wild-type PH20, preferably mature wild-type PH20, are substituted with other amino acids.

[0138] Specifically, the amino acid substitutions in the linker region between α-helix 7 and α-helix 8 include the substitution of one or more amino acid residues in the region consisting of amino acid residues T341 to N363, T341 to I361, L342 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361, or M345 to N363.

[0139] To examine the effects of C-terminal truncation in PH20 variants where the junction region between α-helix 8 and α-helix 7 and α-helix 8 is replaced, three PH20 variants (HM6, HM10, and HM21) were selected as templates.

[0140] HM6 is a variant in which the amino acids in the M345 to N363 region are replaced by the amino acid sequence of Hyal1 (M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T in SEQ ID NO:1 are replaced). Furthermore, HM6 is a variant in which the substitution of the α-helix 8 and the α-helix 7 at the junction with α-helix 8 is the least substituted variant of the PH20 variant according to the invention, which does not contain additional C-terminal cleavage (i.e., where the C-terminal amino acid residues are in the form of S490, like mature wild-type PH20).

[0141] HM10 is a variant in which the amino acids in the L342 to I361 region are replaced by the amino acid sequence of Hyal1 (L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T in SEQ ID NO:1 are replaced), and in the PH20 variant according to the invention without additional C-terminal truncation, HM10 has the highest thermostability while having enzymatic activity similar to that of mature wild-type PH20.

[0142] HM21 is a variant in which the amino acids in the T341 to I361 regions are replaced by the amino acid sequence of Hyal1 (T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T in SEQ ID NO:1 are replaced), and in the PH20 variant according to the invention without additional C-terminal truncation, said HM21 has an enzyme activity at pH 7.0 that is about twice as high as that of wild-type PH20.

[0143] As shown in Table 4 below, the HM6-based PH20 variant constructed in this invention has an N-terminus starting at L36 and a C-terminus terminating at I465, F468, or P471.

[0144] Table 4. C-terminal amino acid-trunculated PH20 variants constructed using HM6 as a template

[0145]

[0146] As shown in Table 5 below, the HM10-based PH20 variants typically have an N-terminus cleaved before the F38 residue and a C-terminus cleaved after the V455, C458, D461, C464, I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, or T488 residues.

[0147] Table 5. C-terminal amino acid-trunculated PH20 variants constructed using HM10 as a template

[0148]

[0149]

[0150] As exemplified by variants containing amino acid substitutions in regions L342 to I361 corresponding to the α-helix 8 region and the linker region between α-helix 7 and α-helix 8 of HM10 as template, wherein the N-terminus is cleaved before the F38 residue and the C-terminus is cleaved at I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, or T488, the PH20 variants according to the invention exhibit enzymatic activity similar to mature wild-type PH20 regardless of the C-terminal cleavage location.

[0151] As shown in Table 6 below, the two HM21-based PH20 variants typically have an N-terminus cleaved before the F38 residue and a C-terminus cleaved after the F468 or K470 residue.

[0152] Table 6. C-terminal amino acid-trunculated PH20 variants constructed using HM21 as a template

[0153]

[0154] Variants were constructed using HM21 as a template, exhibiting approximately twice the enzyme activity of the mature wild-type PH20. These variants contain amino acid substitutions in the T341 to I361 regions corresponding to the α-helix 8 region and the linker region between α-helix 7 and α-helix 8, and in these variants, the N-terminus is cleaved before the F38 residue, and the C-terminus is cleaved after either F468 or K470. Surprisingly, these variants maintained the high enzyme activity of HM21 regardless of the C-terminal cleavage position.

[0155] In a study by Frost et al., when the length of PH20 was shortened due to cleavage before the 477th amino acid position, the enzyme activity decreased to about 10% of that of variants with a C-terminus cleaved after position 477. However, in this invention, when one or more amino acids in the α-helix 8 and its linker region of PH20 are substituted, the enzyme activity is maintained due to increased protein stability, regardless of the C-terminal cleavage position. This result is significant because it addresses the problem of reduced enzyme activity in wild-type PH20 due to C-terminal truncation.

[0156] Furthermore, the role of the N-terminal amino acid of PH20 was investigated in this invention, which was previously unknown.

[0157] To examine the effect of the N-terminal cleavage site in the HM6 variant, one or more amino acid residues in the region (M345 to I361) corresponding to the α-helix 8 region and the linker region between α-helix 7 and α-helix 8 of wild-type PH20 were replaced by amino acid residues in the corresponding α-helix 8 region and the linker region between α-helix 7 and α-helix 8 of Hyal1 (replaced by M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D or I361T in SEQ ID NO:1 and without additional C-terminal cleavage). Using HM6 as a template, variants were constructed in which amino acid residues L36 to V47 in SEQ ID NO:1 were replaced by FRGPLLPNR or amino acid residues L36 to A52 in SEQ ID NO:1 were replaced by FRGPLLPNR. In addition, using HM6 as a template, variants HM40, HM13, HM41, HM24, HM42, and HM25 were constructed, wherein the N-terminus of the amino acid sequence of SEQ ID NO:1 was cleaved before residues N37, F38, R39, A40, P41, or P42 (see Table 7).

[0158] Table 7. Variants based on HM6-based N-terminal amino acid cleavage

[0159]

[0160] The results showed that enzyme activity was not significantly affected when the N-terminus of HM6 was cleaved before residues N37, F38, R39, A40, or P41; however, enzyme activity was significantly reduced when the N-terminus was cleaved before residue P42, indicating that the N-terminal region after P41 in PH20 is important for protein expression and enzyme activity. Furthermore, when one or more amino acids in the L36 to V47 or L36 to A52 region of the HM6 N-terminus were substituted with amino acids from Hyal1, the variant protein was not expressed in ExpiCHO cells, indicating that the N-terminal region is important for protein expression.

[0161] Furthermore, in this invention, an attempt was made to increase the expression of recombinant PH20 protein in animal cells by using signal peptides of other proteins instead of the original signal peptide of PH20.

[0162] Therefore, in another aspect, the novel PH20 variant according to the invention can be a variant in which the N-terminus further comprises, as shown in Table 8 below, the human growth hormone signal peptide having the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA of SEQ ID NO:3, the human serum albumin signal peptide having the amino acid sequence MKWVTFISLLFLFSSAYS of SEQ ID NO:4, or the human Hyal1 signal peptide having the amino acid sequence MAAHLLPICALFLTLLDMAQG of SEQ ID NO:5, in place of the wild-type PH20 signal peptide composed of M1 to T35, but is not limited thereto.

[0163] The designation “replacing the wild-type PH20 signal peptide composed of M1 to T35” indicates a partial or complete deletion of the wild-type PH20 signal peptide; therefore, it does not perform its function. Furthermore, the designation is intended to include cases where a portion of the N-terminus is further deleted, for example, where the cleavage occurs prior to residues N37, F38, R39, A40, P41, or P42, such that the additional deletion of the N-terminus occurs concurrently with the deletion of the wild-type PH20 signal peptide.

[0164] Table 8. Amino acid sequences of signal peptides from human growth hormone, human serum albumin, or human Hyal1.

[0165]

[0166] In another aspect, the present invention provides a composition for treating cancer comprising a novel PH20 variant according to the invention, and a method of treating cancer using said composition.

[0167] There are no particular limitations on the cancers or carcinomas that can be treated with the novel PH20 variant according to the invention, but both solid cancers and hematologic cancers are included. The cancers may be selected from, but are not limited to, liver cancer, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal carcinoma, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, ureteral cancer, osteosarcoma, neurocytoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma. Preferably, the cancers that can be treated with the composition according to the invention may be selected from, but are not limited to, colorectal cancer, breast cancer, lung cancer, and kidney cancer.

[0168] The compositions of the present invention can be pharmaceutical compositions. The pharmaceutical compositions may also contain pharmaceutically acceptable components. Components commonly used in pharmaceutical formulations may be selected from, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. Furthermore, the pharmaceutical compositions may also contain one or more selected from diluents, excipients, lubricants, wetting agents, sweeteners, aromatic compounds, emulsifiers, suspensions, and preservatives.

[0169] The pharmaceutical compositions of the present invention can be administered orally or parenterally. Parenteral administration can be performed via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, rectal administration, etc. For oral administration, considering the digestion of peptides and proteins in the stomach, it is necessary to formulate the active ingredients in the composition into a coated dosage form or a dosage form that protects the active ingredients from disintegration in the stomach. Alternatively, the compositions of the present invention can be administered via any device through which the active ingredients can move to the target cells.

[0170] The pharmaceutical composition may be formulated as a solution, suspension, syrup or emulsion in an oil or aqueous medium, or as an extract, grain, suppository, powder, granule, tablet or capsule, and may additionally include dispersants or stabilizers for formulation purposes.

[0171] In particular, the composition for treating cancer according to the present invention can be used in combination with other anticancer drugs.

[0172] Anticancer drugs that can be used in combination with the novel PH20 variant according to the invention are preferably chemoanticancer drugs, antibody-based anticancer drugs, biological anticancer drugs, RNAi or cell therapy agents, but are not limited thereto.

[0173] Preferably, the anticancer drug that can be used in combination with the novel PH20 variant according to the invention is preferably an immunotumor agent, more preferably an immune checkpoint inhibitor, but not limited thereto.

[0174] In another respect, the present invention relates to nucleic acids encoding PH20 variants or fragments thereof.

[0175] Nucleic acids as used herein may be present in cells, in cell lysates, or in partially purified or substantially pure forms. When referring to nucleic acids, “isolated” or “substantially pure” means those that have been purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) using standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. The nucleic acids of this invention may be DNA or RNA.

[0176] In another aspect, the present invention relates to recombinant expression vectors containing said nucleic acids. To express the PH20 variant or a fragment thereof according to the invention, DNA encoding the PH20 variant can be obtained by standard molecular biology techniques (e.g., PCR amplification using a hybridoma expressing the PH20 variant or cDNA cloning), and said DNA can be inserted into an expression vector such that it is "operably linked" to transcriptional and translational control sequences.

[0177] As used herein, the term "operably ligated" is intended to mean ligating a gene encoding a variant of PH20 or a fragment thereof into a vector such that the transcriptional and translational control sequence performs its intended function of regulating the transcription and translation of the gene encoding a variant of PH20 or a fragment thereof. The expression vector and expression control sequence are selected to be compatible with the host cells used for expression. The gene encoding PH20 is inserted into the expression vector using standard methods (e.g., ligating the gene fragment encoding a variant of PH20 or a fragment thereof to a complementary restriction enzyme site on the vector, or, if no restriction enzyme site is present, blunt-end ligation).

[0178] Furthermore, the recombinant expression vector carries a regulatory sequence that controls the expression of the gene encoding the PH20 variant in the host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signaling) that control the transcription or translation of the gene encoding the PH20 variant or a fragment thereof. Those skilled in the art will understand that the design of the expression vector (including the selection of the regulatory sequence) may depend on factors such as the selection of the host cell to be transformed and the desired protein expression level.

[0179] In another aspect, the present invention relates to host cells comprising the nucleic acid or the vector. The host cells according to the invention are preferably selected from animal cells, plant cells, yeast, Escherichia coli (E. coli), and insect cells, but are not limited thereto.

[0180] In particular, the host cells according to the invention include prokaryotic cells such as *Escherichia coli*, *Bacillus subtilis*, *Streptomyces* sp., *Pseudomonas* sp., *Proteus mirabilis*, or *Staphylococcus* sp.; fungi such as *Aspergillus* sp., yeasts such as *Pichia pastoris*, *Saccharomyces cerevisiae*, and *Schizosaccharomyces* sp., and *Neurosporacrassa*; and eukaryotic cells such as lower eukaryotic cells and other higher eukaryotic cells (such as insect cells).

[0181] Furthermore, the host cells used in this invention can be derived from plants or mammals. Preferably, examples of the host cells include, but are not limited to, monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, young hamster kidney (BHK) cells, MDCK, myeloma cells, HuT 78 cells, and HEK293 cells. More preferably, CHO cells can be used.

[0182] The nucleic acid or the vector is transfected into host cells. Transfection can be performed using various techniques commonly used to introduce foreign nucleic acids (DNA or RNA) into prokaryotic or eukaryotic cells (e.g., electrophoresis, calcium phosphate precipitation, DEAE-glucan transfection, or lipid transfection). Various combinations of recombinant expression vectors and host cells can be used to express the PH20 variant or fragments thereof of the present invention. Preferred expression vectors for eukaryotic cells include gene expression regulatory sequences derived from, but not limited to, SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retroviruses. Expression vectors that can be used for bacterial hosts include bacterial plasmids such as pET, pRSET, pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9, and their derivatives obtained from *Escherichia coli*; plasmids with broad host range, such as RP4; phage DNA, exemplified by various phage λ derivatives, such as λgt10, λgt11, and NM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phages. Expression vectors that can be used in yeast cells include 2-μm plasmids and their derivatives. Expression vectors for insect cells include pVL941.

[0183] In another aspect, the present invention relates to a method for generating a PH20 variant or a fragment thereof, the method comprising the steps of culturing host cells and expressing a PH20 variant or a fragment thereof according to the invention.

[0184] When a recombinant expression vector capable of expressing a PH20 variant or a fragment thereof is introduced into a mammalian host cell, the PH20 variant or a fragment thereof can be generated by culturing the host cell for a period of time to allow the PH20 variant or a fragment thereof to be expressed in the host cell, preferably by culturing for a period of time to allow the PH20 variant to be secreted into the culture medium during the host cell culture process.

[0185] In some cases, expressed PH20 variants can be isolated and purified from host cells. The isolation or purification of PH20 variants can be performed using conventional protein isolation / purification methods (e.g., chromatography). The chromatography may include, but is not limited to, one or more combinations selected from affinity chromatography, ion exchange chromatography, and hydrophobic chromatography. In addition to chromatography, combinations of filtration, ultrafiltration, salting out, dialysis, etc., can also be used to isolate and purify antibodies.

[0186] Example

[0187] The invention will be described in further detail below with reference to embodiments. It will be apparent to those skilled in the art that these embodiments are for illustrative purposes only and should not be construed as limiting or changing the scope of the invention.

[0188] Example 1. Construction of the PH20 variant

[0189] To construct the PH20 variant, wild-type PH20 cDNA (clone ID: hMU002604) was purchased from the Korean Human Gene Bank. Wild-type PH20 encodes amino acids from L36 to S490. The PH20 gene was amplified by polymerase chain reaction (hereinafter referred to as PCR) and inserted into the XhoI and NotI restriction enzyme sites of the pcDNA3.4-TOPO vector. For expression in ExpiCHO cells, the signal peptide of human growth hormone, human serum hormone, or human Hyal1 was used as the signal peptide instead of the original PH20 signal peptide. For protein purification using a HisTrap column, the 6xHis-tagged DNA sequence was located at the 3' end of the PH20 cDNA. Amino acid substitutions for the PH20 variant were performed using PCR and confirmed by DNA sequencing.

[0190] The list of primers used to clone the PH20 variant is summarized in Table 9 below, and the specific sequences of the primers are summarized in Table 10 below.

[0191] Table 9. List of primers used for cloning PH20 variants

[0192]

[0193]

[0194]

[0195] Table 10. Primer sequences used for cloning PH20 variants

[0196]

[0197]

[0198]

[0199] Following the discovery of the PH20 variant with increased enzyme activity and thermostability, a 6xHis-free cDNA of the PH20 variant was also constructed.

[0200] When the cell density of ExpiCHO cells reached 6×10 6 At a concentration of 100-500 mL, plasmids containing wild-type or variant PH20 cDNA inserted into the pcDNA3.4-TOPO vector were transfected into ExpiCHO cells using ExpiFectamine CHO reagent. ExpiCHO expression medium (100-500 mL) was used as the cell culture medium. Following transfection, ExpiCHO cells were cultured with shaking at 130 rpm for a total of 6 days, during which time cells were cultured at 37°C for 1 day and then further cultured at a lower temperature of 32°C for 5 days. After culture, the cell supernatant was collected by centrifugation at 10,000 rpm for 30 min.

[0201] Recombinant proteins of wild-type PH20 and variant PH20, generated in ExpiCHO cells, were purified in three steps using the AKTA primer system (using HisTrap column, Q agarose column, and phenyl column, respectively).

[0202] 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. The protein was bound to the HisTrap column, and the column was washed with 5 column volumes (CV) of buffer A to remove non-specifically bound proteins. Confirming that the conductivity remained constant, the column was washed with 5 CV of 20% buffer B to elute the protein. The eluted protein was dialyzed against 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 the column was washed with 5CV buffer A to remove non-specifically bound proteins, followed by washing with 5CV buffer B at a concentration gradient from 0 to 100% to elute the protein.

[0203] For protein purification using a phenyl column, 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. The protein was bound to the phenyl column, and the column was washed with 5CV buffer A to remove non-specifically bound proteins, followed by washing with 5CV buffer B at a concentration gradient from 0 to 100% to elute the proteins.

[0204] The enzyme activities of wild-type PH20 and variant PH20 were measured by turbidity assay, substrate-gel assay and Morgan-Elson assay.

[0205] Turbidity assay is a method for measuring the absorbance of the precipitate formed when hyaluronic acid is mixed with albumin (BSA). The absorbance of the precipitate formed when hyaluronic acid is hydrolyzed with pH 20 decreases when mixed with albumin. Hyaluronidase pH 20 (Sigma) was diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, and 60 units / mL and prepared in each tube. Purified protein samples were dissolved in enzyme dilution buffer (20 mM Tris·HCl (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 new tubes, a 3 mg / mL hyaluronic acid solution was diluted 10-fold to a concentration of 0.3 mg / mL, resulting in a volume of 180 μL per tube. 60 μL of enzyme was added to a diluted hyaluronic acid solution and mixed, and the mixture was allowed to react at 37 °C for 45 min. After the reaction was complete, 50 μL of the reacted enzyme and 250 μL of acidic albumin solution were added to each well of a 96-well plate and shaken for 10 min. The absorbance at 600 nm was then measured using a spectrophotometer.

[0206] In substrate-gel assays, proteins were electrophoresed on a 10% SDS gel (containing 0.17 mg / mL hyaluronic acid) for 1 h, followed by SDS removal at 4 °C with 2.5% Triton X-100 (w / v) for 2 h. Subsequently, enzymatic reactions were carried out in buffer (50 mM sodium phosphate (pH 7.0), 150 mM NaCl) at 37 °C (optimal temperature for pH 20) for 1 to 4 h, and the proteins were stained with 0.5% acinblue reagent. Unbound acinblue reagent was removed using a destaining solution. The SDS gel stained with acinblue was imaged, and the bands were then quantified.

[0207] The thermal stability of the protein was measured using the following methods: aggregation temperature by dynamic light scattering (DLS), and melting temperature (T) by real-time PCR using Sypro-Orange dye. m Methods such as those for measuring enzyme activity after placing a protein at a predetermined temperature for a predetermined time are used. In the method of measuring aggregation temperature by DLS, light scattering is used to measure molecular aggregation, thus providing high sensitivity, and the aggregation temperature is typically lower than the melting temperature of the protein.

[0208] The sequences of amino acids that have been substituted or cleaved in the PH20 variant constructed in this invention are shown in Table 11 below.

[0209] In the variants according to the invention, the variant having a 6xHis tag attached to the C-terminus of PH20 is named HM; the variant without the 6xHis tag is named HP; the mature PH20 (L36 to S490) having a 6xHis tag attached to the C-terminus is named WT; and the mature wild-type PH20 (L36 to Y482) having a C-terminus cut off after Y482 and without the 6xHis tag is named HW2.

[0210] Table 11. Amino acid sequences and substitution / cleavage characteristics of the PH20 variant according to the present invention.

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229] Example 2. Construction of PH20 variants HM1 and HM6

[0230] like Figure 1 As shown in B, amino acid residues M345 to N363 of PH20 correspond to the α-helix 8 region and the linker region between α-helix 7 and α-helix 8 in the protein tertiary structure model. In 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, thereby stabilizing the secondary structure adjacent to α-helix 8. Figure 1 C).

[0231] To construct variants with higher enzymatic activity and thermostability than WT by substituting amino acids in the α-helix 8 region and the linker region between α-helix 7 and α-helix 8 of PH20, the following variants were constructed: variant HM1, in which 12 amino acids in the M345 to N363 region were substituted; variant HM2, in which 7 amino acids in the Y365 to V379 region were substituted; and variant HM3, in which 19 amino acids in the M345 to V379 region were substituted. Among the amino acids in the α-helix 8 region of PH20, C351, which is involved in disulfide bonding, and Y357, which is involved in hydrophobic interactions, were not substituted. The substituted amino acid sequences in variants HM1, HM2, and HM3 are shown in Table 11 above. When ExpiCHO cells were transfected with the pcDNA3.4-TOPO plasmid containing the genes of variants HM1, HM2, or HM3, variant HM1 was expressed in ExpiCHO cells. Figure 3 A), while variants HM2 and HM3 were not expressed in cells. Protein expression was confirmed not only by measuring enzyme activity but also by Western blot analysis using an antibody against human PH20 (Abcam, ab193009). The epitopes of the antibody were in the Q173 to P222 region. These results indicate that amino acid substitutions in the Y365 to V379 region of the α-helix 8 amino acid sequence used to construct variants HM2 and HM3 severely affected protein structure; therefore, variants HM2 and HM3 were not expressed. Variant HM1 was expressed at a level 3.4 times higher than wild-type WT. Figure 2 Variant HM1 is a variant in which the hydrogen bond between N363 of α-helix 8 and D69 of α-helix 1 is removed by replacing N363 with glycine. Figure 1 C). To restore the hydrogen bond between N363 and D69, G363 in HM1 was replaced with asparagine, thus constructing variant HM6. The substituted amino acids in variant HM6 are shown in Table 11 above.

[0232] Variant HM6 was expressed in ExpiCHO cells, and its expression level was similar to that of variant HM1 (the expression level was 3.4 times higher than that of WT). Figure 2When enzyme activity was measured by turbidity assay, the enzyme activity of variant HM6 was shown to be 1.3 times higher than that of WT. Figure 3 B). In substrate-gel assays, SDS is typically removed with 2.5% Triton X-100 (w / v) after SDS-PAGE, and the enzymatic reaction is carried out at 37°C for 1 to 4 hours, during which time the extent of hyaluronidase hydrolysis of hyaluronic acid is measured using Aspen Blue dye. It is known that protein folding occurs immediately upon removal of SDS from the substrate gel, and the substrate does not affect protein folding. When the enzyme activity of WT and variants HM1 and HM6 was measured by substrate-gel assay at 37°C for 1 to 4 hours, HM6 exhibited higher enzyme activity than WT and variant HM1. Figure 3 C). These results indicate that the proteins of variants HM1 and HM6 fold and refold faster than those of WT, and the constructed variants exhibit higher enzymatic activity than WT. When the signal peptide of PH20 itself is used, the expression level of the protein in ExpiCHO cells is low, and to address this issue, the signal peptide sequence of human serum albumin or human Hyal1 is used. Figure 3 As shown in Figure C, when each of the signal peptides of human serum albumin and human Hyal1 was used as the signal peptide of variant HM1, the expression of the protein increased, and there was no significant difference between the two signal peptides. When enzyme activity was measured by substrate-gel assay, WT with the human serum albumin signal peptide and variant HM1 with either the human serum albumin or Hyal1 signal peptide exhibited enzyme activity in the pH range of 5 to 8. Figure 3 D). In this invention, the signal peptide of human serum albumin is used as the signal peptide of a variant constructed after HM1.

[0233] When comparing the thermal stability of WT and variants HM1 and HM6 based on their aggregation temperatures, the aggregation temperatures were 46.5℃, 53.0℃, and 50.5℃, respectively. This indicates that the aggregation temperatures of variants HM1 and HM6 are 6.5℃ and 4.0℃ higher than those of WT, respectively. Figure 10 The results of the aggregation temperature measurements were consistent with the protein refolding results shown in the substrate-gel assay. These results demonstrate for the first time that hydrogen bonds formed by the N363 residue of α-helix 8 play an important role in the thermal stability and enzyme activity of PH20.

[0234] Furthermore, when comparing the hydrophilic / hydrophobic properties of WT and the HM1 and HM6 variants using phenyl column chromatography, HM1 and HM6 eluted earlier than WT. This indicates that variants HM1 and HM6 possess more hydrophilic properties than WT due to amino acid substitution. However, unlike WT, variants HM1 and HM6 eluted from the phenyl column showed two peaks and exhibited the same molecular weight upon treatment with PNGase F. This reveals a difference caused by N-glycosylation. Figure 3 E).

[0235] Example 3. Construction of PH20 variants HM4, HM7, HM8, HM9, HM10, HM11 and HM12

[0236] It is believed that the construction of variants HM1 and HM6 in Example 2, with the substitution of amino acids in the M345 to N363 and M345 to I361 regions, resulted in an increase in both the enzymatic activity and thermostability of PH20, representing a significant advancement in protein engineering. Therefore, based on variants HM1 and HM6, other amino acids in the N-terminal and C-terminal directions were additionally substituted.

[0237] First, variants HM4 and HM7 were constructed, containing the substituted amino acids found in variants HM1 and HM6, with the amino acid between G340 and I344 further substituted by G340V, T341S, L342W, S343E, and I344N. The sequences of the substituted amino acids in variants HM4 and HM7 are shown in Table 11 above. Variants HM4 and HM7 were expressed in ExpiCHO cells. The protein purification results of HM7 were obtained in... Figure 5 As shown in Figure A, HM4 and HM7 showed a 6.3-fold increase in protein expression compared to WT, and exhibited increases in aggregation temperature of 10°C and 11.5°C, respectively, compared to WT. However, the enzyme activities of HM4 and HM7, as measured by turbidity assay, were approximately 15% of those of WT. Figure 5 B). Enzyme activity and thermostability are usually traded off, but in this invention, the substitutions introduced in variants HM1 and HM6 showed increased thermostability while maintaining enzyme activity, while the enzyme activity of variants HM4 and HM7 decreased due to excessive increases in thermostability. For protein engineering purposes, the 11.5°C increase in aggregation temperature observed in variants HM4 and HM7 is a significant result. Stern-Volmer plot analysis of protein structural flexibility showed that HM1, HM6, HM4, and HM7, obtained by substituting α-helix 8 and its linker region, all exhibited higher structural flexibility than the WT variant. Figure 11 A). This result indicates that increased local thermal stability leads to increased flexibility of the overall protein structure.

[0238] The difference between variants HM6 and HM7 lies in the amino acid between G340 and I344. To identify the amino acids involved in the increased thermostability of variant HM7, the following variants were constructed based on variant HM6: 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 variants HM8, HM9, HM10, and HM21 are shown in Table 11 above. Variants HM8, HM9, HM10, and HM21 are expressed in ExpiCHO cells (…). Figure 5 A). When I344N, S343E, and L342W are introduced into the N-terminal directional loop of α-helix 8 based on variant HM6, the aggregation temperatures of variants HM8, HM9, and HM10 increase to 52.5℃, 53℃, and 55.5℃, respectively. Figure 10 However, variants HM8, HM9, and HM10 maintained enzyme activities similar to those of WT. Figure 5 B). This result indicates that the amino acid substitutions introduced in variants HM8, HM9, and HM10 have a local effect on the enzyme's thermostability but no significant effect on enzyme activity. However, at pH 7.0, HM21 showed decreased thermostability compared to HM10, but approximately 2-fold higher enzyme activity compared to WT. When WT and each variant were reacted with the substrate for 1 h in a substrate-gel assay, enzyme activity decreased in the order HM21 > HM10 > HM9 > HM8 > HM6 > WT. Figure 5 C).

[0239] When the physical properties of variants HM7, HM8, HM9, HM10, and HM21 were examined using phenyl column chromatography, these PH20 variants eluted earlier than WT, indicating that these variants are all hydrophilic. However, the patterns of the main peaks at the amino acid substitution sites showed differences. Figure 5 D).

[0240] Variant HM7 also showed two peaks in phenyl column chromatography, just like variants HM1 and HM6, indicating the existence of two different types.

[0241] To examine the migration patterns of WT and variants based on their isoelectric points, isoelectric point focusing (hereinafter referred to as IEF) analysis was performed. Figure 5E). On IEF gels, WT, along with variants HM6 and HM8, showed similar migration patterns, while variants HM9, HM10, HM21, and HM7, containing the S343E mutation, migrated to more acidic regions. This result suggests that the isoelectric point of the protein changes due to the introduction of glutamate via the S343E substitution of the amino acid between G340 and I344.

[0242] Furthermore, based on variant HM6, other amino acids in the C-terminal region of α-helix 8 were substituted, thus constructing variants HM11 and HM12. The sequences of the substituted amino acids in variants HM11 and HM12 are shown in Table 11 above. Variant HM11 was expressed in ExpiCHO cells, but its expression level was lower than that of WT cells. Figure 2 Furthermore, variant HM12 was not expressed in ExpiCHO cells. Variant HM11 exhibited 32% of the activity equivalent to WT ( Figure 6 B).

[0243] Example 4. Construction of the PH20 variant based on the N-terminal amino acid truncation of HM6

[0244] The C-terminal region of PH20 is well known to play an important role in PH20 expression and enzyme activity, but the role of the N-terminal region is not well understood. To examine the effect of amino acid cleavage at the N-terminal region of PH20 on enzyme activity, variants HM40, HM13, HM41, HM24, HM42, and HM25 (Table 11) with N-terminal cleavage at N37, F38, R39, A40, P41, or P42 were constructed based on variant HM6. In addition, HP61 and HP62, modified with N-terminal amino acids, were also constructed.

[0245] Variants HM40, HM13, HM41, HM24, HM42, HP61, and HP62 were expressed in ExpiCHO cells, but HM25 was not expressed. Figure 7 A and Figure 7 B). N-terminal truncated PH20 variants showed differences in enzyme activity depending on the N-terminal initiation position. Variants HM40, HM13, and HM41 (with one to three amino acids cleaved) showed similar enzyme activity to the template HM6, but HM24 and HM42 (with four to five amino acids cleaved) showed slightly lower activity than HM6. Figure 7 C). However, HM25, with six amino acids cleaved, was poorly expressed in ExpiCHO cells, and its enzyme activity was also significantly low (3.5 U / μg). The enzyme activity of HP61 and HP62, which had modifications at the N-terminal amino acids, was not significantly altered.

[0246] Regarding the enzyme activities of N-terminally cleaved PH20 variants measured by substrate-gel assay (reaction 1 h), the enzyme activities of HM40, HM13, and HM41 were similar to those of the HM6 variant, but the enzyme activities of HM24 and HM42 were lower than those of HM6. Figure 7 D). Due to the small amount of protein produced, it was impossible to analyze the HM25, which contained six amino acids that were cleaved.

[0247] When the physical properties of variants HM40, HM13, HM41, HM24, and HM42 were analyzed using phenyl column chromatography, these variants eluted from the column earlier than WT, indicating that these variants possess hydrophilic properties. Figure 7 E). This result indicates that, when considering the characteristics of residues L36 to A40, the variants HM40, HM13, HM41, HM24, and HM42 constructed based on variant HM6 retain the hydrophilic properties of HM6.

[0248] The aggregation temperature of N-terminal cleaved PH20 variants, as measured by DLS, varies between variants depending on the amino acid initiation position. Figure 7 F). Although the N-terminal amino acid residues of variants HM40, HM13, HM41, and HM42 were cleaved, they exhibited aggregation temperatures of 50 °C or higher, indicating that the characteristics of template HM6 remained intact. Among these variants, variants HM40 and HM42 exhibited aggregation temperatures 3 °C to 4 °C higher than HM6, indicating improved thermostability. Furthermore, to examine the effect of N-terminal amino acid substitutions of PH20 on protein expression and enzyme activity, the following variants were constructed: variant HM17, in which N-terminal amino acid residues 36 to 47 (LNFRAPPVIPNV) of PH20 were substituted with FRGPLLPNR; and variant HM18, in which N-terminal amino acid residues 36 to 52 (LNFRAPPVIPNVPFLWA) of PH20 were substituted with FRGPLLPNRPFTTV. The sequences of the substituted amino acids in variants HM17 and HM18 are shown in Table 11 above. Variants HM17 and HM18 were not expressed in ExpiCHO cells. This indicates that even when up to five amino acids at the N-terminus are cleaved, the variant exhibits protein expression and enzyme activity; however, substitution of more amino acid residues (such as 36 to 47 residues or 36 to 52 residues) does affect protein folding.

[0249] Example 5. Construction of HM6-based variants HM14, HM15, and HM16, based on the C-terminal amino acid truncation of PH20.

[0250] The C-terminal region of PH20 is known to play an important role in protein expression and enzyme activity. In this invention, variants HM14, HM15, and HM16 were constructed based on variant HM6, wherein the C-terminal amino acids were cleaved at I465, F468, and K471, respectively. The sequences of the substituted amino acids in these variants HM14, HM15, and HM16 are shown in Table 11 above. These variants HM14, HM15, and HM16 were expressed in ExpiCHO cells (…). Figure 8 A), and its protein expression levels are in the order HM16>HM15>HM14, which indicates that protein expression levels decrease with increasing number of cleaved C-terminal amino acids. Figure 8 A). However, the enzyme activities of variants HM14, HM15, and HM16 are in the order HM16 > HM14 (≈WT) > HM15. Figure 8 (B) According to Frost et al., the C-terminal region 477-483 of PH20 is essential for soluble expression, and when the C-terminus is cleaved at residue 467, the enzymatic activity of the variant is only 10% of that of the PH20 variant whose C-terminus is cleaved at residues 477-483, and when the C-terminus is cleaved before residue 467, the variant has no enzymatic activity. However, the C-terminal cleaved variants HM14, HM15, and HM16, constructed based on variant HM6 in this invention, exhibit increased protein folding due to amino acid substitutions in the M345-I361 region, and therefore increased thermostability. Consequently, even when the C-terminus is cleaved after I465, F468, or P471, these variants exhibit enzymatic activity similar to that of WT, and their enzymatic activity is not significantly reduced.

[0251] The structural flexibility of WT and its variants HM14, HM15 and HM16 was examined using fluorescence quenching with acrylamide. Figure 11 B). Variants HM14, HM15, and HM16 are all structurally more flexible than WT. This result indicates that the C-terminal-cut variant constructed using variant HM6 also retains its structural flexibility.

[0252] The enzyme activities of variants HM14, HM15, and HM16, measured by turbidity assay, were also confirmed in substrate-gel assays. Figure 8 C).

[0253] When the physical properties of the C-terminal cleaved variants were analyzed using phenyl column chromatography, variants HM14, HM15, and HM16 eluted earlier than WT, indicating that they are hydrophilic. Furthermore, the hydrophobicity of these variants was in the order HM16 > HM14 > HM15. Figure 8 D).

[0254] Example 6. Construction of HM10-based variants HM19 and HM20 containing N-terminal and C-terminal amino acid cleavages

[0255] The PH20 variant constructed in this invention is based on HM6, and HM8, HM9, and HM10 (where amino acid residues G340 to I344 are additionally substituted) exhibit better performance than WT in terms of protein expression levels, enzyme activity, and thermostability. Based on HM10, which exhibits high enzyme activity and thermostability among variants HM8, HM9, and HM10, variants HM19 and HM20 were constructed, each possessing an N-terminus cleaved at F38 and a C-terminus cleaved at F468. Both HM19 and HM20 were expressed in ExpiCHO cells and purified using HisTrap column chromatography. Figure 9 A). When the enzyme activity of these variants was measured by turbidity assay, HM19 and HM20 exhibited 10% higher enzyme activity than WT. Figure 9 B). In substrate-gel assays, HM19 and HM20 also exhibited higher enzyme activity than WT ( Figure 9 C).

[0256] Example 7. Characterization of the PH20 variant based on HM10

[0257] The expression levels of HM10-based C-terminal truncated variants in ExpiCHO cells showed a trend of decreasing with decreasing C-terminal region length, and these variants were not expressed when the C-terminus was truncated at C464 or shorter. Figure 12 C464 is essential because it forms a disulfide bond with C437 and is important for maintaining protein structure.

[0258] To examine whether the variant was not expressed in ExpiCHO cells when the C-terminus was cleaved at residue 464 or shorter, Western blot analysis was performed. Figure 13 As shown, HM30, HM31, HM32 and HM33 variants were not detected in the protein blot.

[0259] Enzyme activity of the HM10-based C-terminal truncated variant, measured by turbidity assay, was... Figure 14 A and Figure 14 As shown in B, the C-terminally truncated PH20 variant exhibited ±20% enzyme activity compared to WT. Overall, enzyme activity increased when the C-terminus was cleaved after I480. Furthermore, variants HP19 and HP20, obtained by removing the 6xHis tag from HM19 and HM20, respectively, showed a 23% and 9.6% decrease in enzyme activity, compared to the presence of the 6xHis tag. This indicates that the 6xHis tag has an effect on enzyme activity.

[0260] When the enzyme activity of HM10-based C-terminal truncated variants was measured by substrate-gel assay, these variants exhibited higher enzyme activity than WT and showed similar enzyme activity to the template HM10, indicating that the difference in enzyme activity depending on the length of the C-terminal region was not significant. Figure 14 C).

[0261] Example 8. Characterization of the PH20 variant based on HM21

[0262] The variant HP34 was purified by a four-step column chromatography method. Figure 15 A), and the variant HP46 was purified by three-step column chromatography. Figure 15 B). The amounts of HP34 and HP46 produced were 1.73 mg / L and 25.6 mg / L, respectively. HP34 and HP46 are variants without the 6xHis tag, and the purification process for these variants differs from that for variants with the 6xHis tag, making it difficult to compare the expression levels of the proteins.

[0263] In turbidity assays, the activities of HP34 and HP46 were 45.6 U / μg and 47.2 U / μg, respectively, which are approximately twice the activity of WT and approximately 10% higher than the activity of template HM21. Figure 16 A).

[0264] The kinetics of each variant were measured using Morgan-Elson assays, and the results were obtained in Figure 16 The catalytic efficiencies (kJ) of HP34 and HP46 are shown in section B. cat / K m The catalytic efficiency is 1.7 to 2 times higher than that of wild-type HW2. This result is consistent with the results showing that the specific activity is higher than that of WT. The Michaelis constant (K) of these variants is... m The values ​​below HW2 indicate increased substrate affinity for these variants. These results lead to the conclusion that the HM21, HP34, and HP46 variants bind strongly to the substrate and possess the property of efficiently converting the substrate into the product. This property is attributed to the effect of replacing T341 with serine. The effect of such substitution on enzyme activity can be predicted when threonine at position 341 is replaced by an amino acid such as alanine, glycine, aspartic acid, etc.

[0265] The aggregation temperatures of HP34 and HP46, measured by DLS, were 51.5℃ and 51.0℃, respectively, similar to those of template HM21 and approximately 5℃ higher than that of HW2. This indicates that these variants are thermally stable. Figure 17A). The enzyme activity of HP20, measured by substrate-gel assay, was similar to that of HP20, while HP46 exhibited higher enzyme activity than HP20, indicating that HM21, as a template, folds better than HM10. Figure 17 B).

[0266] Wild-type HW2 and variant HP46 were incubated overnight at pH 7.0 and pH 3.0, respectively, and their enzyme activities were compared by substrate-gel assay. The results showed that HP46 exhibited high activity not only at pH 7.0 but also at pH 3.0, indicating excellent stability. Figure 17 C).

[0267] HM53, HM54, HM55, HM56, HP59, and HP60 are variants of HM21 with a mutation at amino acid position 341. The mutation at position 341 was confirmed to have multiple effects on the expression levels and activities of the variants. Figure 17 D and Figure 17 E).

[0268] Example 9. In vitro immunogenicity assay of the PH20 variant

[0269] Biopharmaceuticals with molecular weights higher than those of low-molecular-weight synthetic chemicals pose a risk of triggering unintended immune responses upon entering the human body. External contact surfaces, created through folding or interaction with adjacent domains in the secondary or tertiary structures of high-molecular-weight biomaterials, can promote immune responses to the biomaterials by providing epitopes to the human immune system. Such immune responses can generate anti-drug antibodies (ADAs), which can inhibit the effectiveness of the drug, induce hypersensitivity to the drug, or promote drug clearance from the body. Therefore, immune responses to the drug can affect the outcomes of clinical trials and may cause serious adverse reactions with prolonged use. These immune responses can be influenced by a variety of factors and may be triggered by a specific response to the drug itself or a disease, or by factors depending on the method of drug administration or the individual patient. Factors caused by the drug itself include the similarity or dissimilarity of the biopharmaceutical to human peptides, post-translational modifications, impurities, aggregate formation, and formulation characteristics. Factors varying between individual patients include sex, responsiveness to other medications taken, and genetic factors depending on human leukocyte antigen (HLA) type.

[0270] This immunogenic response is triggered by either CD4+ T cells or CD8+ T cells that recognize epitopes, regardless of the cause of the immune response. Due to the diversity of HLA class II genes in individuals, the epitopes of CD4+ T cells differ between individuals, and therefore the responsiveness of CD4+ T cells in each blood sample from a healthy donor to biopharmaceuticals may be a very important criterion for assessing potential immune responses during clinical proceedings. CD4+ T cells are activated by antigen-presenting cells (APCs) that recognize antigens presented via their type II MHC (major histocompatibility complex). Activated CD4+ T cells release cytokines that activate macrophages, cytotoxic T cells, and B cells, leading to high levels of antibody production. Conversely, CD8+ T cells are directly cytotoxic and directly remove antigen-infected, damaged, or dysfunctional cells. CD8+ T cells possess T cell receptors that recognize specific antigenic peptides that bind to type I MHC molecules located on the surface of each cell. CD8+ T cells can also be activated by recognizing antigens presented by antigen-presenting cells, and this activation can be further enhanced by cytokines from CD4+ T cells. Therefore, when the activation levels of CD4+ T cells and CD8+ T cells on neomaterials are measured in vitro, induced immunogenic responses in the clinical process can be predicted. In this embodiment, to predict the immunogenicity of the PH20 variant compared to the control, CD4+ T cells and CD8+ T cells were isolated from PBMCs and then treated with control PH20 and the PH20 variant (HP46) at 1.5 ng / mL and 15 ng / mL, respectively. The distribution of activated CD4+ T cells and CD8+ T cells was then measured. The activation level of each T cell type was measured using a stimulation index, and the stimulation index (SI) is defined as follows:

[0271] Stimulation Index (SI) = (T cell activation level after treatment with test sample) / (T cell activation level after treatment with mediator)

[0272] If a cell's SI value is 2 or greater, the cell can be considered significantly activated. Immunogenic responses can vary depending on HLA type. Therefore, experiments were conducted using T cells isolated from PBMCs from 10 healthy donors to measure responses across more different HLA types. The HLA types of the 10 PBMCs used are shown in Table 12 below.

[0273] Table 12. HLA types of the tested PBMCs

[0274]

[0275]

[0276] The results of measuring the activation levels of CD4+ and CD8+ T cells treated with PH20 and its variant are summarized in Table 13 below. When reviewing the results of measuring CD4+ and CD8+ T cell activation levels, it becomes clear that both PH20 and the PH20 variant showed relatively low activation levels. With PH20, CD4+ T cell activation levels were measured to be 2 or higher in both experiments, while with the PH20 variant, no CD4+ T cell activation was detected. With PH20, CD8+ T cell activation was detected in one experiment, and with the PH20 variant, it was also detected in one experiment. However, with PH20, SI values ​​were measured to be 2 or higher at both 1.5 ng / mL and 15 ng / mL; but with the PH20 variant, SI values ​​were 2 or lower at 1.5 ng / mL and 2 or higher at 15 ng / mL (see Table 13). Figure 18 and Figure 19 ).

[0277] Therefore, at lower concentrations, low activation levels of CD8+ T cells were observed in the presence of the PH20 variant, and it was determined that PH20 induced higher activation levels of CD8+ T cells than the PH20 variant. The conclusions drawn from these results are as follows:

[0278] 1) The activation levels of CD4+ T cells and CD8+ T cells obtained through PH20 and its variants were relatively low; and

[0279] 2) The likelihood of activation of CD4+ T cells and CD8+ T cells obtained through the PH20 variant is lower than that obtained through PH20.

[0280] Based on these results, the PH20 variant is expected to be less likely to trigger an immunogenic response during clinical trials than the PH20 variant.

[0281] Table 13. Stimulation Index (SI) Measured from In Vitro Immunogenicity Assay Results

[0282]

[0283]

[0284] Beneficial effects

[0285] When expressed in CHO (ExpiCHO) cells, the PH20 variant or fragment thereof according to the invention exhibits increased protein expression levels and shows an increase in protein aggregation temperature of approximately 4°C-11.5°C compared to mature wild-type PH20, enabling them to be produced efficiently while possessing high thermal stability.

[0286] Furthermore, as a substrate-gel assay, one of the tests for measuring hyaluronidase activity, the PH20 variant or fragments according to the invention exhibit improved protein refolding, allowing them to renature faster than mature wild-type PH20 and maintain their original enzyme activity regardless of the C-terminal cleavage position.

[0287] Furthermore, the PH20 variants or fragments thereof according to the present invention have low immunogenicity, which allows them to be repeatedly administered to the human body.

[0288] References

[0289] Arming,S.,Strobl,B.,Wechselberger,C.,and Kreil,G.(1997).In vitromutagenesis of PH-20hyaluronidase from human sperm.Eur J Biochem 247,810-814.

[0290] Bookbinder, LH, Hofer, A., Haller, MF, Zepeda, ML, Keller, GA, Lim, JE, Edgington, TS, Shepard, HM, Patton, JS, and Frost, GI (2006). Arecombinant human enzyme for enhanced interstitial transport of therapeutics. J Control Release 114, 230-241.

[0291] Chao,KL,Muthukumar,L.,and Herzberg,O.(2007).Structure of humanhyaluronidase-1,a hyaluronan hydrolyzing enzyme involved in tumor growth andangiogenesis.Biochemistry 46,6911-6920.

[0292] Frost, GI (2007). Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin DrugDeliv 4, 427-440. Sequence Listing (Free Text)

[0293] Attached electronic files.

Claims

1. A PH20 variant, wherein the amino acid sequence of the PH20 variant consists of the amino acid sequence of SEQ ID NO:

99.

2. A composition comprising the PH20 variant of claim 1.

Images