Hyaluronidase HYAL1 mutant that exhibits activity at neutral pH

Abstract

The present invention provides a Hyal1 variant in which one or more of the amino acids adjacent to the catalytic amino acids are substituted with acidic or polar amino acids, a method for producing the Hyal1 variant, a nucleic acid, an expression vector, and a host cell applicable to the production of the Hyal1 variant, and a preparation or use of the Hyal1 variant, based on wild-type human hyaluronidase Hyal1 containing aspartic acid and glutamic acid as catalytic amino acids. Further, the present invention provides a Hyal1 variant in which one or more of the amino acids adjacent to the catalytic amino acids in the tertiary structure are substituted with basic amino acids, a method for producing the Hyal1 variant, a nucleic acid, an expression vector, and a host cell applicable to the production of the Hyal1 variant, and a preparation or use of the Hyal1 variant. The hyaluronidase Hyal1 variant of the present invention can effectively decompose hyaluronic acid even at neutral pH, and has the effect of high usability.

Description

Technical Field

[0001] The present invention is a technology for changing the working pH of human hyaluronidase, and specifically relates to a variant of human hyaluronidase Hyal1 that has a catalytic effect in the acidic to neutral pH range.

Background Art

[0002] Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid, and human hyaluronidases include Hyal1, Hyal2, Hyal3, Hyal4, and PH20.

[0003] Hyaluronidase PH20 is used in the subcutaneous injection form of clinical antibody therapeutic agents due to its property of hydrolyzing hyaluronic acid in the extracellular matrix and can be used to increase drug delivery to the subcutaneous area. However, currently, the hydrolysis of hyaluronic acid depends on one kind of enzyme, PH20. That is, among human hyaluronidases other than PH20, Hyal1 mainly has a catalytic effect under acidic conditions of pH 3 - 4, so its utilization is limited. For example, when applying Hyal1 to a subcutaneous site showing neutrality of pH 7.0 - 7.5, it is difficult to expect a hyaluronic acid decomposition effect. In addition, wild Hyal2, Hyal3, Hyal4, etc. are not actually used because their activities are weak in the neutral region.

[0004] Therefore, as a result of the inventor's research to improve the utilization of such wild - type human Hyal1, the present invention has been completed.

Prior Art Documents

Non - Patent Documents

[0005]

Non - Patent Document 1

[0006] The problem that this invention aims to solve is to provide a variant of human Hyal1 that can effectively hydrolyze hyaluronic acid at a neutral pH. [Means for solving the problem]

[0007] The human hyaluronidase Hyal1 mutant of the present invention has Asp129 and Glu131 as catalytic amino acids. In order for Hyal1 to be able to catalyze even at a neutral pH, the present invention replaces the catalytic amino acids with acidic or polar amino acids in the primary or tertiary structure of Hyal1.

[0008] Furthermore, in the tertiary structure of Hyal1, one of the amino acids adjacent to the catalytic amino acid is substituted with a basic amino acid. In this case, the substituted basic amino acid can form an ionic bond with an acidic or polar amino acid adjacent to the catalytic amino acid.

[0009] The catalytic amino acid of Hyal1 and the amino acids adjacent to it in the primary or tertiary structure are either amino acids located in a loop consisting of Ala132 or Ser76~Leu98 amino acids, or amino acids adjacent to acidic or polar amino acids in the tertiary structure of Hyal1 that may affect the activity of the catalytic amino acid.

[0010] Ala132 can be substituted with an acidic or polar amino acid. In this case, the acidic amino acid is Asp or Glu, and the polar amino acid is Ser, Thr, Asn, Gln, or Tyr. In the loop consisting of amino acids Ser76 to Leu98, the amino acid adjacent to the catalytic amino acid in the tertiary structure is Ser, Thr, or Pro, and these amino acids are substituted with Asp or Glu.

[0011] On the other hand, other adjacent amino acids that can affect the activity of the catalytic amino acid in the tertiary structure of Hyal1 are the acidic amino acid Asp206 and the polar amino acid Tyr210. Substitute the amino acid adjacent to Asp206 or Tyr210 with the basic amino acids Arg, Lys, or His.

[0012] For example, if Phe139 adjacent to Tyr210 is replaced with the basic amino acid Arg, an ionic bond is formed between Arg139 and Tyr210. Similarly, if Tyr210 adjacent to Asp206 is replaced with the basic amino acid His, an ionic bond is formed between Asp206 and His210. As a result, the catalytic amino acid can hydrolyze hyaluronic acid even at a neutral pH.

[0013] Furthermore, the present invention provides nucleic acids encoding the hyaluronidase Hyal1 variant.

[0014] Furthermore, the present invention provides a recombination expression vector containing the nucleic acid.

[0015] Furthermore, the present invention provides host cells transformed with the expression vector.

[0016] Furthermore, the present invention provides a method for producing a hyaluronidase Hyal1 variant, which includes the step of culturing a host cell.

[0017] In addition, the present invention provides a hyaluronic acid degrading agent containing the hyaluronidase Hyal1 variant.

[0018] Furthermore, the present invention provides a drug delivery agent containing the hyaluronidase Hyal1 variant.

[0019] Furthermore, the present invention provides a preparation for intravenous injection containing the hyaluronidase Hyal1 variant.

[0020] In addition, the present invention provides a preparation for subcutaneous administration containing the hyaluronidase Hyal1 variant.

[0021] Furthermore, the present invention provides an ophthalmic preparation containing the hyaluronidase Hyal1 variant.

Advantages of the Invention

[0022] The hyaluronidase Hyal1 variant of the present invention can effectively degrade hyaluronic acid not only at acidic pH but also at neutral pH, and has the effect of high usability. That is, with the hyaluronidase of the present invention, hyaluronic acid degradation and drug delivery using the same are possible even at neutral pH, and it can be utilized in various preparations and applications such as preparations for subcutaneous administration, preparations for intravenous injection, and ophthalmic preparations. In addition, the nucleic acid, expression vector, host cell, and method of the present invention can effectively produce the hyaluronidase of the present invention.

Brief Description of the Drawings

[0023] [Figure 1] (A) shows the experimental results of examining whether the Hyal1 wild type and the Ala132 variant exhibit enzyme activity depending on pH. (B) shows the experimental results for confirming the expression of the Hyal1 wild type and the Ala132 variant in CHO-K1 cells. [Figure 2](A) shows the experimental results of confirming whether the Hyal1 wild type and the Ser77, Thr86 or Pro87 mutants exhibit enzymatic activity depending on pH. (B) shows the experimental results for confirming the expression of the Hyal1 wild type and the Ser77, Thr86 or Pro 87 mutants in CHO-K1 cells. [Figure 3] (A) is the experimental result of examining whether mutants with two or more substitutions together among Ala132, Ser77 or Thr86 exhibit enzymatic activity depending on pH. (B) shows the experimental results for confirming the expression of mutants with two or more substitutions together among Ala132, Ser77 or Thr86 in CHO-K1 cells. [Figure 4] It is a comparison of the enzymatic active site structures of human Hyal1 and human PH20. [Figure 5] (A) shows the experimental results of examining whether the Hyal1 wild type and the Phe139, Tyr210 or Phe139 / Ile225 mutants exhibit enzymatic activity depending on pH. (B) shows the experimental results for confirming the expression of the Hyal1 wild type and the Phe139, Tyr210 or Phe139 / Ile225 mutants in CHO-K1 cells. (C) shows the positions of Phe139, Asp206, Tyr210 and Ile225 adjacent to the catalytic amino acids in the tertiary structure of Hyal1.

Mode for Carrying Out the Invention

[0024] Hereinafter, the advantages and features of the present invention, and the methods for achieving them, will become clear by referring to the examples described in detail together with the accompanying drawings. However, the present invention is not limited to the examples disclosed below, and can be realized in various different forms. However, these examples are provided only to make the disclosure of the present invention complete, and to fully inform those with ordinary knowledge in the technical field to which the present invention belongs of the scope of the invention. The present invention is only defined by the scope of the claims.

[0025] Throughout this specification, “and / or” includes each of the components mentioned and all combinations of one or more of them. The terms used herein are for illustrative purposes only and are not intended to limit the invention. In this specification, the singular includes the plural unless otherwise specified in the text. As used herein, “comprises” and / or “comprising” does not exclude the presence or addition of one or more other components of the components mentioned.

[0026] One embodiment of the present invention is a hyaluronidase Hyal1 mutant (hereinafter referred to as "Hyal1 mutant"), which contains aspartic acid (e.g., Asp129) and glutamic acid (e.g., Glu131) as catalytic amino acids, in which one or more amino acids adjacent to the catalytic amino acids in the primary or tertiary structure are substituted with acidic or polar amino acids.

[0027] Another embodiment of the present invention, the Hyal1 mutant, has one of the amino acids adjacent to the catalytic amino acid in the tertiary structure of wild-type Hyal1 replaced with a basic amino acid. In this case, the substituted basic amino acid can ionically bond with an acidic or polar amino acid adjacent to the catalytic amino acid.

[0028] In other words, the hyaluronidase Hyal1 mutant, which is one embodiment of the present invention, corresponds to a mutant of wild-type human Hyal1. By substituting the catalytic amino acid with an amino acid adjacent to it in the primary or tertiary structure with a specific acidic amino acid or polar amino acid, or by substituting the amino acid adjacent to the catalytic amino acid in the tertiary structure with a basic amino acid, hyaluronic acid can be effectively decomposed even at a neutral pH, thereby increasing its usability.

[0029] In this specification, expressions such as "Ala132" or "A132," which consist of a three-letter or one-letter amino acid name and a number, refer to the amino acid at each position relative to the amino acid sequence of Sequence ID No. 1. For example, "Ala132" and "A132" both refer to Ala, the amino acid located at position 132 relative to the amino acid sequence of Sequence ID No. 1. Furthermore, in this specification, a variant of wild-type human hyaluronidase includes variants in which an amino acid is conservatively substituted at a specific amino acid position. In this case, "conservative substitution" means a variant modification that involves substituting one or more amino acids with amino acids having similar biological or biochemical properties that do not cause a loss of the biological or biochemical function of the variant in question. Specifically, a "conservatively substituted variant" may be a variant that has, for example, 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more of sequence homology to a Hyal1 variant consisting of an amino acid sequence selected from the group consisting of the amino acid sequences of Sequence ID No. 2 to Sequence ID No. 20, and has substantially the same function and / or effect.

[0030] One embodiment of the present invention, the Hyal1 mutant, corresponds to a hyaluronidase capable of degrading hyaluronic acid in a wide pH range from acidic to neutral. In the hyaluronidase of one embodiment of the present invention, the wild-type human hyaluronidase is Hyal1, and Hyal1 consists of the amino acid sequence of SEQ ID NO: 1 and may include substitutions of one or more amino acids selected from the group consisting of S77D, S77E, T86D, T86E, P87E, A132D, A132E, F139R, Y210H, and F139R / I225D. That is, the mutant of one embodiment of the present invention, based on wild-type Hyal1 consisting of the amino acid sequence of SEQ ID NO: 1, may include substitutions of one or more amino acids selected from the group consisting of S77D, S77E, T86D, T86E, P87E, A132D, A132E, F139R, Y210H, and F139R / I225D.

[0031] Here, S77D means that the 77th serine molecule in SEQ ID NO: 1 has been replaced with aspartic acid.

[0032] Hyal1 mutants are mutations in some amino acids of the wild-type human Hyal1 sequence, specifically amino acid substitutions, and include one or more amino acid substitutions selected from the group consisting of S77D, S77E, T86D, T86E, P87E, A132D, A132E, F139R, Y210H, and F139R / I225D. Here, the substitution F139R / I225D means that the amino acid substitutions F139R and I225D are combined.

[0033] The amino acid sequence of wild-type human Hyal1 is shown in Table 1 below. Similar to the sequence shown in Table 1, Ala132 is located adjacent to the catalytic amino acids Asp129 and Glu131. Hyal1 mutants in which amino acids adjacent to the catalytic amino acids are substituted with specific amino acids appear to exhibit different characteristics from the wild type while maintaining hyaluronic acid hydrolase activity. In other words, as confirmed by experimental examples, substituting Ala132 with Asp or Glu allows for the hydrolysis of hyaluronic acid even at neutral pH.

[0034] In other words, by substituting Ala, the 132nd amino acid in the amino acid sequence of wild-type human Hyal1, with Asp or Glu, hyaluronic acid can be hydrolyzed even at a neutral pH. However, Hyal1 mutants in which Ala132 was substituted with other similar amino acids did not show such an effect. Therefore, it can be seen that, in particular, the Hyal1 mutant, which is one embodiment of the present invention, can effectively degrade hyaluronic acid even at a neutral pH.

[0035] Furthermore, Ser77 is located adjacent to the catalytic amino acid in the tertiary structure of Hyal1, and substituting Ser77 with Asp or Glu allows for the hydrolysis of hyaluronic acid at neutral pH. In other words, by substituting Ser, the 77th amino acid in the amino acid sequence of wild-type human Hyal1, with Asp or Glu, hyaluronic acid can be hydrolyzed even at neutral pH.

[0036] Furthermore, Thr86 and Pro87 are located adjacent to the catalytic amino acid in the tertiary structure of Hyal1, and substituting Thr86 with Asp or Glu, or Pro87 with Glu, also allows for the hydrolysis of hyaluronic acid at a neutral pH. In other words, by substituting Thr and Pro, which are the 86th and 87th amino acids respectively based on the amino acid sequence of wild-type human Hyal1, with Asp or Glu, hyaluronic acid can be hydrolyzed even at a neutral pH.

[0037] On the other hand, Asp206 and Tyr210 are located adjacent to the catalytic amino acid in the tertiary structure of Hyal1. In this case, when Phe139 adjacent to Tyr210 is substituted with a basic amino acid, an ionic bond is formed with Tyr210, and when the Tyr210 adjacent to Asp206 is substituted with a basic amino acid, an ionic bond is formed with Asp206.

[0038] Specifically, replacing Phe139 with Arg allows the hydroxyl group of Tyr210 to be dehydrogenated due to the ionic bond between Arg139 and Tyr210, thereby increasing the pKa value of the catalytic amino acid Glu131. Furthermore, substituting Phe139 and Ile225 together with Arg and Asp (F139R / I225D) stabilizes the positive charge of Arg139 through the ionic bond between Arg139 and Asp225, allowing Arg139 to again ionically bond with Tyr210 and dehydrogenate the hydroxyl group of Tyr210.

[0039] On the other hand, substituting Tyr210 with His reduces the pKa value of Asp206 due to the ionic bonding between Asp206 and His210, thereby increasing the pKa value of the catalytic amino acid Glu131.

[0040] The amino acid sequence of wild-type Hyal1 is shown in Table 1, the Ala132 mutant in Table 2, the loop mutant in Table 3, the Ala132 mutant and the loop mutant in Table 4, and the Phe139 and Tyr210 mutants in Table 5. [Table 1] [Table 2] JPEG0007894954000003.jpg231152JPEG0007894954000004.jpg231152JPEG0007894954000005.jpg203152 [Table 3] JPEG0007894954000007.jpg231152JPEG0007894954000008.jpg231152JPEG0007894954000009.jpg60152 [Table 4] JPEG0007894954000011.jpg231152JPEG0007894954000012.jpg231152JPEG0007894954000013.jpg60152 [Table 5] JPEG0007894954000015.jpg239152

[0041] Such Hyal1 variants can degrade hyaluronic acid in a wide pH range from acidic to neutral. Furthermore, because the Hyal1 variant, one embodiment of the present invention, can degrade hyaluronic acid in a pH range from acidic to neutral, it is possible to effectively deliver drugs to various sites. This is because when hyaluronic acid in the extracellular matrix is ​​hydrolyzed, its viscosity decreases, and its permeability to tissues (skin) increases. In particular, the subcutaneous region of the skin has a neutral pH of approximately 7.0 to 7.5, so permeability can be increased by the embodiment of the present invention, which can degrade hyaluronic acid at a neutral pH. Therefore, the drug delivery agent, one embodiment of the present invention, which includes the variant, can effectively deliver drugs to various sites.

[0042] In this case, the drug is a component exhibiting pharmacological activity, but is not limited to this; for example, it may be an ophthalmoscal, an anesthetic, an antibody therapy, or an anticancer drug. The drug may also constitute a composition together with a variant, which is one embodiment of the present invention. That is, the variant, which is one embodiment of the present invention, may be an example of a pharmaceutical composition, either by itself or together with the drug.

[0043] One embodiment of the present invention, the Hyal1 variant, can be included in formulations that can be applied to various sites. For example, one embodiment of the present invention, such as a subcutaneous formulation, an intravenous formulation, or an ophthalmic formulation, may contain the Hyal1 variant. In this case, the ophthalmic formulation may be an eye drop, which may be used to promote the diffusion of an anesthetic during ophthalmic surgery. Furthermore, the subcutaneous formulation may specifically be a subcutaneous injection. The subcutaneous injection may be used for hyaluronic acid hydrolysis in filler procedures. In addition, the intravenous formulation may be used to enhance the accessibility of anticancer drugs to tumor cells. This is because the intravenous formulation can move the Hyal1 variant along the bloodstream, hydrolyzing hyaluronic acid overexpressed on the surface of tumor cells and thereby enhancing the accessibility of anticancer drugs to tumor cells. Thus, one embodiment of the present invention, the Hyal1 variant, can be used not only for therapeutic purposes but also for cosmetic purposes. Therefore, one embodiment of the present invention can be a pharmaceutical formulation and / or a cosmetic formulation.

[0044] The dosage of the Hyal1 mutant, one embodiment of the present invention, is 10 ng / ml to 10 mg / ml per subcutaneous injection, preferably 10 ng / ml to 100 μg / ml. It can be administered once a day or in several divided doses. Such dosages may be based on an adult (60 kg body weight), but naturally, they may vary depending on body weight, physical condition, etc. The Hyal1 mutant, one embodiment of the present invention, is administered mainly by parenteral methods, for example, by subcutaneous injection, intravenous injection, or ophthalmic administration.

[0045] One embodiment of the present invention, the Hyal1 variant, can also be formulated with pharmaceutically acceptable excipients and manufactured in the form of injections, eye drops, transdermal patches, and the like.

[0046] Pharmacovigilant additives may be applied according to a variety of factors well known to those skilled in the art, such as the specific bioactive substance used, its concentration, stability, and intended bioavailability. Factors such as the disease and condition to be treated, the individual being treated, age, size, and general condition, and the location (nasal cavity, oral cavity, eyeball, local, transdermal, and muscular) should be considered, but are not limited to these. Pharmacovigilant additives commonly used for administering bioactive substances via routes other than oral administration include aqueous solutions containing D5W (5% glucose in water), dextrose, and physiological salts at a concentration of no more than 5% by volume. For local injection, various injectable hydrogels may be used to enhance the effect and increase the duration of action. Furthermore, pharmacovigilant additives may include additional components that can reinforce the stability of the active ingredient, such as preservatives and antioxidants. A variant, which is one embodiment of the present invention, can be formulated in appropriate methods of the art and may be preferably formulated according to each disease or condition, or according to the component.

[0047] An embodiment of the present invention, the Hyal1 mutant, can be stored in a physiological saline solution, freeze-dried into ampoules after the addition of mannitol or sorbitol, and then dissolved in physiological saline or the like when used for administration. In this case, the physiological saline solution may contain a buffer, stabilizer, and / or surfactant.

[0048] Furthermore, one embodiment of the present invention, a hyaluronic acid hydrolysis and / or drug delivery method, includes the step of administering a Hyal1 mutant, one embodiment of the present invention, to a mammal, including a human, that requires administration. In this case, the Hyal1 mutant administered may be an effective amount of the mutant.

[0049] Furthermore, one embodiment of the present invention is the use of the Hyal1 mutant, which is an embodiment of the present invention, for the production of hyaluronic acid hydrolyzing preparations or drug delivery preparations.

[0050] An embodiment of the present invention, the Hyal1 mutant, can be produced by genetic engineering techniques. For example, a fusion gene encoding a fusion protein consisting of a fusion partner and the Hyal1 mutant protein of an embodiment of the present invention can be produced through genetic engineering, followed by transformation of host cells with this gene. After expression in the host cells in the form of the fusion protein, the Hyal1 mutant of an embodiment of the present invention can be cleaved and separated from the fusion protein using proteolytic enzymes or compounds to produce the desired protein form. Thus, an embodiment of the present invention, the Hyal1 mutant, can be effectively produced by an embodiment of the present invention, the nucleic acid, expression vector, host cells, and / or method. Specifically, an embodiment of the present invention encodes an embodiment of the mutant. The nucleic acid may be present in cells, cell lysates, or in a partially purified or substantially pure form. The nucleic acid may be, for example, DNA or RNA.

[0051] Furthermore, an expression vector according to one embodiment of the present invention includes a nucleic acid according to one embodiment of the present invention. For the expression of the Hyal1 mutant according to one embodiment of the present invention, the DNA encoding the Hyal1 mutant according to one embodiment of the present invention can be obtained by molecular biology techniques (e.g., PCR amplification, cDNA cloning using a hybridoma expressing the Hyal1 mutant according to one embodiment of the present invention), and such DNA can be functionally ligated to transcriptional and translational regulatory sequences and inserted into an expression vector to produce an expression vector according to one embodiment of the present invention. In this case, "functionally ligated" can mean that the gene encoding the Hyal1 mutant according to one embodiment of the present invention is ligated into the vector so that the transcriptional and translational regulatory sequences in the vector perform the intended function of regulating the transcription and translation of the gene encoding the Hyal1 mutant according to one embodiment of the present invention. The expression vector and expression regulatory sequences are selected to be compatible with the host cells used for expression. The gene encoding the Hyal1 mutant, which is one embodiment of the present invention, is inserted into the expression vector using a standard method (e.g., ligation of the gene fragment encoding the Hyal1 mutant, which is one embodiment of the present invention, and complementary restriction enzyme sites on the vector, or blunt terminal ligation if no restriction enzyme sites are present). The recombination expression vector has regulatory sequences that control the expression of the gene encoding the Hyal1 mutant, which is one embodiment of the present invention, in host cells. The "regulatory sequences" may include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals) that control the transcription or translation of the gene encoding the Hyal1 mutant, which is one embodiment of the present invention. Of course, the design of the expression vector may change depending on factors such as the selection of host cells to be transformed and the level of protein expression, by selecting different regulatory sequences.

[0052] Furthermore, a host cell, which is one embodiment of the present invention, may include a nucleic acid or an expression vector, which is one embodiment of the present invention. The host cell is not limited to these, but may be selected from the group consisting of, for example, animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0053] Nucleic acids or expression vectors are transfected or transfected into host cells. Various techniques of different types commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for "transfecting" or "transfecting" can be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. Various host / vector combinations can be used to express the Hyal1 variant, which is one embodiment of the present invention. Expression vectors adapted to eukaryotic hosts include, but are not limited to, SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus-derived regulatory sequences. Expression vectors usable in bacterial hosts include bacterial plasmids obtained from Escherichia coli such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9, and their derivatives; plasmids with a broader host range, such as RP4; phage DNA, which can be exemplified by a wide variety of phage lambda derivatives such as λgt10 and λgt11, NM989; and other DNA phages such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells are 2 μm plasmids and their derivatives. A useful vector for insect cells is pVL941.

[0054] Furthermore, a method for producing a Hyal1 mutant according to one embodiment of the present invention may include a step of culturing host cells according to one embodiment of the present invention. When a recombination expression vector capable of expressing a Hyal1 mutant according to one embodiment of the present invention is introduced into mammalian host cells, the mutant according to one embodiment of the present invention can be produced by culturing the host cells for a period of time sufficient for expression in the host cells, more preferably for a period of time sufficient for the Hyal1 mutant according to one embodiment of the present invention to be secreted into the culture medium in which the host cells are cultured.

[0055] If necessary, the expressed Hyal1 mutant, which is an embodiment of the present invention, can be isolated from host cells and purified to a homogeneous state. Isolation or purification of the Hyal1 mutant, which is an embodiment of the present invention, can be carried out by conventional protein isolation and purification methods, such as chromatography. Chromatography may be, but is not limited to, one or more selected combinations of affinity chromatography, ion exchange chromatography, or hydrophobic chromatography. In addition to chromatography, further methods such as filtration, ultrafiltration, salting out, and dialysis may also be used in combination.

[0056] Unless otherwise stated, the provisions relating to the Hyal1 variant, hyaluronic acid degrading agent, drug delivery agent, composition, use, formulation, and method of the present invention, as embodiments of the present invention, apply equally to each other to the extent of identity, provided they do not contradict each other.

[0057] The present invention will be described in more detail below with reference to examples, comparative examples, and manufacturing examples. However, the following examples and manufacturing examples are for illustrative purposes only, and the content of the present invention is not limited to the following examples and manufacturing examples.

[0058] The reagents used in the following examples are commercially available, and only the highest quality products were used. Unless otherwise specified, the reagents were purchased from Sigma-Aldrich.

[0059] <Example 1> Production of Hyal1 mutant The human Hyal1 gene (Clone ID: hMU005315) was purchased from the Korean Human Gene Bank. The human Hyal1 gene was amplified using the pCMV-SPORT6-human Hyal1 plasmid via PCR (Bioneer, AllInOneCycler PCR system). The amplified gene was inserted into a vector (named pSGHV1 vector) obtained by removing the human growth hormone (hGH) and TEV protease cleavage sites from the pSGHV0 vector using restriction enzymes Xho I and Not I. At this time, Ni 2+ — For protein purification using a column, the Hyal1 cDNA was configured to have a DNA sequence of six His residues at its 3' end. Hyal1 mutants were also prepared using a PCR reactor, and the DNA sequences of the prepared Hyal1 mutants were confirmed by DNA sequencing. The primers used for PCR are listed in Table 6. [Table 6] JPEG0007894954000017.jpg236152JPEG0007894954000018.jpg51152

[0060] <Example 2> Confirmation of enzyme activity and expression of Hyal1 Ala132 mutant CHO-K1 cells (purchased from the Korea Cell Line Bank) were transformed with a plasmid containing the Hyal1 wild-type or mutant gene prepared in Example 1, using polyethylenimine (PEI, Sigma-Aldrich) or Lipofectamine 3000 (Thermofisher) reagent. When the CHO-K1 cells had grown to 90-95% of the area in a 6-well plate, 150 μL of DMEM medium containing 2 μg of plasmid DNA was mixed in. After 30 minutes, the PEI-DNA mixture was carefully transferred to a 6-well plate and cultured in a 5% CO2 incubator.

[0061] 48 hours after transformation, the cell culture medium was collected and centrifuged at 10,000 × g to obtain the upper layer. The enzyme activity of Hyal1 wild-type and mutant cells was measured by a substrate-gel assay. Specifically, electrophoresis was performed using a 10% SDS gel containing hyaluronic acid (1.0 mg / mL), and then SDS was removed using a buffer containing Triton X-100 (3% Triton X-100, 50 mM Tris, 100 mM NaCl, pH 7.5) at 4°C for 2 hours. Phosphate buffers containing 100 mM NaCl were prepared, with pH adjusted to 4, 5, 6, and 7 using disodium phosphate and monosodium phosphate. Each SDS gel was placed in a phosphate buffer at pH 4–7, and the enzymatic reaction was carried out for 4–17 hours at 37°C with shaking at 50 rpm. After the enzymatic reaction, the hyaluronic acid on the SDS gel was stained with 1.0% Alcian blue stain. The purpose of this invention is to investigate the enzyme activity of the Hya1 mutant at neutral pH. Therefore, pH 4 was used as the control group, and the enzyme activity at pH 6 and pH 7 was investigated.

[0062] Figure 1(A) shows the experimental results confirming whether the Hyal1 mutant exhibits enzyme activity depending on pH. Hyaluronic acid hydrolyzed by the Hyal1 mutant using the Substrate-gel assay method is not stained with Alcian blue and appears as a white band. In Figure 1(A), Control represents the control group transfected with only the vector, WT represents wild-type Hyal1, and A132E, A132N, A132D, A132Y, A132S, and A132H represent their respective Hyal1 mutants.

[0063] As shown in Figure 1(A), at pH 4, all mutants excluding A132Y hydrolyzed hyaluronic acid and showed enzymatic activity. However, at pH 6, the A132E, A132D, and A132S mutants showed enzymatic activity, and at pH 7, only A132E and A132D showed enzymatic activity. Of these Hyal1 mutants, A132E showed the highest enzymatic activity at pH 7. A132N and A132S, which were substituted with polar amino acids, showed higher enzymatic activity than the wild type at pH 4, but at pH 6, only A132S showed partial enzymatic activity. The basic amino acid His showed enzymatic activity at pH 6, but not at pH 7.

[0064] Furthermore, to investigate protein expression in CHO-K1 cells, Western blotting was performed after protein electrophoresis, as shown in the experiment in Figure 1(A). Specifically, after transferring a 10% SDS gel through a nitrocellulose membrane at 100V for 1 hour, the expression of Hyal1 wild-type and Ala132 mutant cells was examined using a Hyal1 monoclonal antibody (1D10, Santa Cruze Biotechnology, Dallas, TX, US) and an ant-mouse IgG secondary antibody (m-IgGk BP-HRP, Santa Cruze Biotechnology). The results are shown in Figure 1(B). Figure 1(B) shows the experimental results to confirm Hyal1 mutant expression. In Figure 1(B), Control represents the control group transfected with only the vector, WT represents wild-type Hyal1, and A132E, A132N, A132D, A132Y, A132S, and A132H represent their respective mutants. As shown in Figure 1(B), WT, A132E, A132N, A132D, A132S, and A132H mutant proteins were detected with the Hyal1 antibody, but the A132Y mutant was not detected. From these results, it can be inferred that the negative result for the A132Y mutant shown in Figure 1(A) is due to protein non-expression.

[0065] The Hyal1 mutant of the present invention hydrolyzes hyaluronic acid by general acid-base catalysis. The carboxyl group (-COOH) of the catalytic amino acid Glu131 plays a role in providing a positive molecule to the hydroxyl leaving group, so protonation of GLU131 is important for the catalytic reaction. The results in Figure 1(A) show that substituting an amino acid adjacent to the catalytic amino acid Glu131 in the tertiary structure with an acidic amino acid increases the pKa value of Glu131, meaning that Glu131 can play a role in providing a positive molecule at acidic to neutral pH. However, even with the same acidic amino acid, A132E showed even higher enzyme activity at pH 7 than A132D. Therefore, it can be seen that the Hyal1 mutant A132E, in particular, which is one embodiment of the present invention, can effectively decompose hyaluronic acid even at neutral pH.

[0066] <Example 3> Confirmation of enzyme activity and expression of Hyal1 loop mutant To investigate the enzyme activity of the fabricated Hyal1 loop mutants (S77D, S77E, T86D, T86E, and P87E) in the pH range of 4–7, CHO-K1 cells were transformed using the PEI or Lipofectamaine method as described in Example 1, with plasmids containing the Hyal1 loop mutant genes inserted. 48 hours after transformation, the cell culture medium was collected and centrifuged at 10,000 × g to obtain the upper layer. The enzyme activity of Hyal1 WT and loop mutants in the upper layer was examined using a substrate-gel assay at 37°C and pH range of 4–7 using phosphate buffer for 4–17 hours, and the amount of hydrolyzed hyaluronic acid was examined using 1.0% Alcian blue stain. The results are shown in Figure 2(A). Figure 2(A) shows the experimental results confirming whether Hyal1 WT and loop mutants exhibit enzyme activity at different pH levels. In Figure 2(A), Control represents the control group transfectioned with the vector only, WT represents wild-type Hyal1, and S77D, S77E, T86D, T86E, and P87E represent their respective Hyal1 variants.

[0067] As shown in Figure 2(A), Hyal1 WT showed enzymatic activity by hydrolyzing hyaluronic acid at pH 4, but all loop mutants (S77D, S77E, T86D, T86E, and P87E) showed enzymatic activity in the entire pH range from 4 to 7. These results indicate that Hyal1 mutants, in which Ser77, Thr86, and Pro87 in the loop region of wild-type human Hyal1 are replaced with Asp or Glu, can effectively degrade hyaluronic acid even at neutral pH. In particular, when using Hyal1 mutants, which are one embodiment of the present invention, S77D, S77E, and P87E showed higher enzymatic activity at pH 7 than the T86D and T86E mutants. Figure 2(B) shows the experimental results to confirm Hyal1 mutant expression. In Figure 2(B), it can be seen that Hyal1 WT and mutants were normally expressed in CHO-K1 cells.

[0068] <Example 4> Confirmation of enzyme activity and expression of mixed mutants in which Hyal1 Ala132 or loop amino acids are substituted. Among the Ala132 mutants, the most active A132E mutant and one or more loop mutants were mutated together, and their enzyme activity in the pH range of 4-7 was investigated. To investigate the enzyme activity in the pH range of 4-7 of the constructed Hyal1 mutants (S77D / A132E, S77E / A132E, S77D / T86D, S77D / T86E, and S77D / T86D / A132E), CHO-K1 cells were transformed using the PEI or Lipofectamaine method as described in Example 1 with plasmids into which the genes of these Hyal1 mutants were inserted. 48 hours after transformation, the cell culture medium was collected and centrifuged at 10,000 × g to obtain the supernatant. The enzyme activity of the Hyal1 mutant contained in the upper layer of the substrate-gel assay was investigated by enzymatic reaction with phosphate buffer at 37°C and pH 4–7 for 4–17 hours, and the amount of hydrolyzed hyaluronic acid was examined using 1.0% Alcian blue staining.

[0069] As shown in Figure 3(A), the double and triple mutants exhibited enzyme activity by hydrolyzing hyaluronic acid not only at pH 4 but also at pH 7. In particular, differences in activity levels were observed at pH 7, with the S77D / A132E mutant showing the best activity.

[0070] Figure 3(B) shows the experimental results to confirm the expression of the Hyal1 mutant. In Figure 3(B), it can be seen that the Hyal1 mutant was expressed normally in CHO-K1 cells.

[0071] <Example 5> Comparison of the tertiary structures of human Hyal1 and PH20 proteins To compare the positions of the mutant amino acids in Hyal1 introduced in this invention with those of PH20, the tertiary structures of human Hyal1 and PH20 were compared. The tertiary structure model of human PH20 was fabricated using Swiss-Model based on the crystal structure of human Hyal1 (PDB code 2PE4). The structures of human Hyal1 and PH20 were compared using the PyMol program, and the results are shown in Figure 4. As shown in Figure 4, in human Hyal1, Ser76 and Ser77, Thr86 and Pro87 are located adjacent to the catalytic amino acid Glu131. In the tertiary structure model of human PH20, Asp94, Asp103, and Glu149 are located adjacent to the catalytic amino acid Glu148.

[0072] Therefore, it appears that the Hyal1 variant of the present invention exhibits the effect of effectively degrading hyaluronic acid even at a neutral pH, as PH20 exhibits enzymatic activity at a neutral pH, in which the amino acid adjacent to the catalytic amino acid in the tertiary structure of human Hyal1 is substituted with an acidic amino acid.

[0073] <Example 6> Confirmation of enzyme activity and expression of F139R and Y210H mutants Another method to increase the pKa value of the catalytic amino acid Glu131 in Hyal1 is to substitute the amino acid adjacent to Glu131 with a basic amino acid, thereby decreasing the pKa value of the acidic or polar amino acid that forms an ionic bond with this basic amino acid. In the tertiary structure of Hyal1, Phe139, Asp206, Ile225, and Tyr210 are located adjacent to the catalytic amino acid. Substituting the hydrophobic amino acid Phe139 with Arg reduces the pKa value of Tyr210 due to the ionic bond between Arg139 and Tyr210, thereby increasing the pKa value of the catalytic amino acid Glu131. Furthermore, substituting the hydrophobic amino acids Phe139 and Ile225 with Arg and Asp, respectively, strengthens the positive charge of Arg139 due to the ionic bond between Arg139 and Asp225, which may promote dehydrogenation of the hydroxyl group of Tyr210 in the ionic bond between Arg139 and Tyr210 compared to the F139R single mutant. Furthermore, substituting the polar amino acid Tyr210 with the basic amino acid His reduces the pKa value of Asp206 due to the ionic bond between Asp206 and His210, thereby increasing the pKa value of the catalytic amino acid Glu131.

[0074] To investigate the enzyme activity of the fabricated Hyal1 mutants (F139R, F139R / I225D, and Y210H) in the pH range of 4–7, CHO-K1 cells were transformed using PEI or Lipofectamaine methods as described in Example 1, with plasmids containing the genes of these Hyal1 mutants inserted. 48 hours after transformation, the cell culture medium was collected and centrifuged at 10,000 × g to obtain the upper layer. The enzyme activity of Hyal1 WT and the mutants in the upper layer was examined using a substrate-gel assay by reacting them enzymatically with phosphate buffer at 37°C and pH range of 4–7 for 4–17 hours, and the amount of hydrolyzed hyaluronic acid was examined using 1.0% Alcian blue stain.

[0075] In Figure 5(A), Control represents the control group transfection with the vector only, WT represents wild-type Hyal1, and F139R, Y210H, and F139R / I225D represent their respective Hyal1 mutants. WT showed enzyme activity only at pH 4, while F139R, Y210H, and F139R / I225D mutants showed enzyme activity at both pH 4 and pH 7. The F139R / I225D double mutant showed higher activity at pH 6 than the F139R single mutant, but at pH 7, the enzyme activity of the single and double mutants was similar.

[0076] Figure 5(B) shows the experimental results to confirm the expression of the Hyal1 mutant. In Figure 5(B), it can be seen that the Hyal1 mutant was expressed normally in CHO-K1 cells.

[0077] Figure 5(C) shows the tertiary structure of Hyal1 with the aforementioned amino acids indicated. Asp206 and Tyr210 are located adjacent to the catalytic amino acid Glu131.

[0078] From the above results, it can be seen that the Hyal1 variant of the present invention can effectively degrade hyaluronic acid not only at acidic pH but also at neutral pH, demonstrating its high applicability. In other words, because the Hyal1 variant of the present invention can degrade hyaluronic acid even at neutral pH, drug delivery using it is possible, and it can be utilized in various formulations or applications such as subcutaneous formulations, intravenous formulations, and ophthalmic formulations.

[0079] Furthermore, these results demonstrate that Hyal1 variants can be effectively produced using the nucleic acid, expression vector, host cell, and method of the present invention.

[0080] <Manufacturing Example 1> Liquid Formulation Manufacturing One μg of the Hyal1 mutant protein prepared in Examples 2-6 was dissolved in PBS to prepare one mL of solution. The prepared solution was filled into an ampoule or sterile container to produce an injectable solution or eye drops. The preparations thus produced can be used as hyaluronic acid degraders, drug delivery agents, etc. In particular, the injectable solution can be used as a subcutaneous or intravenous injection preparation, and the eye drops can be used as an ophthalmic preparation.

Claims

1. Based on the wild-type human hyaluronidase Hyal1, which consists of the amino acid sequence of Sequence ID No. 1, It contains only one or more amino acid substitutions selected from the group consisting of S77D, S77E, T86D, T86E, P87E, A132D, A132E, F139R, and Y210H. A hyaluronidase Hyal1 variant characterized by the following:

2. A nucleic acid encoding the hyaluronidase Hyal1 variant according to claim 1.

3. An expression vector comprising the nucleic acid described in claim 2.

4. Host cells transformed with the expression vector described in claim 3.

5. A method for producing a hyaluronidase Hyal1 mutant, comprising the step of culturing the host cells described in claim 4.

6. A hyaluronic acid degrading agent comprising the hyaluronidase Hyal1 variant described in claim 1.

7. A drug delivery agent comprising the hyaluronidase Hyal1 variant as described in claim 1.

8. A subcutaneous formulation containing the hyaluronidase Hyal1 variant described in claim 1.

9. A formulation for intravenous injection containing the hyaluronidase Hyal1 variant described in claim 1.

10. An ophthalmic formulation comprising the hyaluronidase Hyal1 variant described in claim 1.

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