HYAL1 mutants that exhibit 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 catalyzes 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 formulation of clinical antibody therapeutics due to its property of hydrolyzing hyaluronic acid in the extracellular matrix and can be used to increase drug delivery subcutaneously. However, currently, the hydrolysis of hyaluronic acid depends on a single enzyme, PH20. That is, among human hyaluronidases other than PH20, Hyal1 mainly catalyzes 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 degradation 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 present 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 to be solved by the present invention is to provide a variant of human Hyal1 that can effectively hydrolyze hyaluronic acid at neutral pH. [Means for Solving the Problems]

[0007] The human hyaluronidase Hyal1 variant of the present invention has Asp129 and Glu131 as catalytic amino acids. The present invention substitutes an amino acid adjacent to the catalytic amino acid with an acidic or polar amino acid in the primary or tertiary structure of Hyal1 so that Hyal1 can exhibit catalytic activity even at neutral pH.

[0008] Further, in the tertiary structure of Hyal1, one of the amino acids adjacent to the catalytic amino acid is substituted with a basic amino acid. At this time, 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 thereto in the primary or tertiary structure are amino acids located in the loop consisting of the amino acids of Ala132 or Ser76 to Leu98, or amino acids adjacent to acidic or polar amino acids that can affect the activity of the catalytic amino acid in the tertiary structure of Hyal1.

[0010] Ala132 can be substituted with an acidic or polar amino acid. At this time, 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 the amino acids of 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, another adjacent amino acid that can affect the activity of the catalytic amino acid in the tertiary structure of Hyal1 is the acidic amino acid Asp206 and the polar amino acid Tyr210. Substitute the amino acid adjacent to Asp206 or Tyr210 with a basic amino acid Arg, Lys, or His.

[0012] For example, when Phe139 adjacent to Tyr210 is substituted with the basic amino acid Arg, an ionic bond is formed between Arg139 and Tyr210. Similarly, when Tyr210 adjacent to Asp206 is substituted 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 neutral pH.

[0013] Furthermore, the present invention provides a nucleic acid encoding the hyaluronidase Hyal1 mutant.

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

[0015] In addition, the present invention provides a host cell 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

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Mode for Carrying Out the Invention

[0024] Hereinafter, the advantages and features of the present invention, and the methods for achieving them, will become apparent 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, and the present invention is only defined by the scope of the claims.

[0025] "And / or" throughout this specification includes each and all combinations of the recited components. The terms used in this specification are for the purpose of describing embodiments and are not intended to limit the present invention. In this specification, the singular form also includes the plural form unless specifically stated otherwise in the context. As used herein, "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components to the recited components.

[0026] One embodiment of the present invention, the hyaluronidase Hyal1 variant (hereinafter referred to as "Hyal1 variant"), with respect to wild-type human Hyal1 containing as catalytic amino acids aspartic acid (e.g., Asp129) and glutamic acid (e.g., Glu131), refers to the substitution of one or more of the amino acids adjacent to the catalytic amino acids in the primary or tertiary structure with acidic or polar amino acids.

[0027] Another embodiment of the present invention, the Hyal1 variant, in the tertiary structure of wild Hyal1, one of the amino acids adjacent to the catalytic amino acid is substituted with a basic amino acid. At this time, the substituted basic amino acid can form an ionic bond with an acidic or polar amino acid adjacent to the catalytic amino acid.

[0028] That is, the hyaluronidase Hyal1 variant, which is one embodiment of the present invention, corresponds to a variant of wild-type human Hyal1. By substituting the amino acids adjacent to the catalytic amino acids in the primary or tertiary structure with specific acidic or polar amino acids, or substituting the amino acids adjacent to the catalytic amino acids in the tertiary structure with basic amino acids, hyaluronic acid can be effectively decomposed even at neutral pH, and the usability can be enhanced.

[0029] In this specification, an expression in which a three-letter or one-letter amino acid name and a number are described together, such as "Ala132" or "A132", means the amino acid at each position based on the amino acid sequence of SEQ ID NO: 1. For example, "Ala132" and "A132" indicate Ala, which is the amino acid located at the 132nd position, based on the amino acid sequence of SEQ ID NO: 1. In addition, in this specification, a mutant of wild-type human hyaluronidase also includes a mutant in which an amino acid is conservatively substituted at a specific amino acid position. At this time, "conservative substitution" means a modification of a mutant that includes substituting one or more amino acids with amino acids having similar biological or biochemical properties that do not cause loss of the biological or biochemical function of the corresponding mutant. Specifically, a "conservatively substituted mutant" may have, for example, 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence homology with a Hyal1 mutant consisting of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2 to 20, and may be a mutant having substantially the same function and / or effect.

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

[0031] Here, the meaning of S77D means that the 77th serine of SEQ ID NO: 1 is substituted with aspartic acid.

[0032] The Hyal1 variant is a variant in which some amino acids are mutated in the sequence of wild-type human Hyal1, specifically those in which the amino acids are substituted, and includes 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 meaning of the substitution of F139R / I225D means that the amino acid substitutions of F139R and I225D occur together.

[0033] The amino acid sequence of wild-type human Hyal1 is as shown in Table 1 below. Similar to the sequence described in Table 1, Ala132 is located adjacent to the catalytic amino acids Asp129 and Glu131. The Hyal1 variant in which the amino acid adjacent to the catalytic amino acid is substituted with a specific amino acid is considered to exhibit characteristics different from those of the wild type while maintaining hyaluronic acid hydrolase activity. That is, as confirmed from the experimental examples, when Ala132 is substituted with Asp or Glu, hyaluronic acid can be hydrolyzed even at neutral pH.

[0034] That is, by substituting Ala, which is the 132nd amino acid based on the amino acid sequence of wild-type human Hyal1, with Asp or Glu, hyaluronic acid can be hydrolyzed even at neutral pH. However, the Hyal1 variant in which Ala132 is substituted with other similar amino acids did not show such an effect. Therefore, it can be seen that, particularly according to the Hyal1 variant which is an embodiment of the present invention, hyaluronic acid can be effectively decomposed even at neutral pH.

[0035] Also, Ser77 is located adjacent to the catalytic amino acid in the tertiary structure of Hyal1, and when Ser77 is substituted with Asp or Glu, hyaluronic acid can be hydrolyzed at neutral pH. That is, by substituting Ser, which is the 77th amino acid based on the amino acid sequence of wild-type human Hyal1, with Asp or Glu, hyaluronic acid can be hydrolyzed even at neutral pH.

[0036] Moreover, Thr86 and Pro87 are located adjacent to the catalytic amino acids in the tertiary structure of Hyal1. When Thr86 is substituted with Asp or Glu, or Pro87 is substituted with Glu, hyaluronic acid can still be hydrolyzed at neutral pH. That is, by substituting Thr and Pro, which are the 86th and 87th amino acids based on the amino acid sequence of wild-type human Hyal1, with Asp or Glu, hyaluronic acid can be hydrolyzed even at neutral pH.

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

[0038] Specifically, when Phe139 is replaced with Arg, due to the ionic bond between Arg139 and Tyr210, the hydroxyl group of Tyr210 exists in a dehydrogenated state, and the pKa value of the catalytic amino acid Glu131 can be increased. Also, when Phe139 and Ile225 are each replaced with Arg and Asp together (F139R / I225D), the positive charge of Arg139 is stabilized by the ionic bond between Arg139 and Asp225, and Arg139 can again form an ionic bond with Tyr210 to dehydrogenate the hydroxyl group of Tyr210.

[0039] On the other hand, when Tyr210 is replaced with His, the pKa value of Asp206 is decreased due to the ionic bond between Asp206 and His210, and as a result, the pKa value of the catalytic amino acid Glu131 can be increased.

[0040] The amino acid sequence of wild-type Hyal1 is shown in Table 1, the Ala132 mutant is shown in Table 2, the amino acid sequence of the loop mutant is shown in Table 3, the amino acid sequence of the mutant in which one or more of Ala132 or the loop mutants are substituted together is shown in Table 4, and the amino acid sequences of the Phe139 and Tyr210 mutants are shown in Table 5, respectively. [Table 1] [Table 2] JPEG2025523398000004.jpg231152JPEG2025523398000005.jpg231152JPEG2025523398000006.jpg203152 [Table 3] JPEG2025523398000008.jpg231152JPEG2025523398000009.jpg231152JPEG2025523398000010.jpg60152 [Table 4] JPEG2025523398000012.jpg231152JPEG2025523398000013.jpg231152JPEG2025523398000014.jpg60152 [Table 5] JPEG2025523398000016.jpg239152

[0041] Such Hyal1 variants are capable of degrading hyaluronic acid in a wide pH range from acidic to neutral. Also, since the Hyal1 variant which is an embodiment of the present invention is capable of degrading hyaluronic acid in the pH range from acidic to neutral, it is also possible to effectively deliver drugs to various sites. This is because when the hyaluronic acid in the extracellular matrix is hydrolyzed, the viscosity of hyaluronic acid is decreased and the permeability to tissues (skin) is increased. In particular, since the subcutaneous site of the skin is neutral with a pH of about 7.0 to 7.5, the permeability can be increased by an embodiment of the present invention which can degrade hyaluronic acid at neutral pH. Therefore, a drug delivery agent which is an embodiment of the present invention contains a variant which is an embodiment of the present invention and can effectively deliver drugs to various sites.

[0042] At this time, the drug is a component showing pharmacological activity and can be, but is not limited to, for example, an eye relaxant, an anesthetic, an antibody therapeutic agent, an anticancer agent, etc. The drug may sometimes consist of a composition together with a variant which is an embodiment of the present invention. That is, a variant which is an embodiment of the present invention may sometimes be an example of a pharmaceutical composition by itself or together with a drug.

[0043] The Hyal1 variant according to an embodiment of the present invention can be included in a formulation applicable to various sites. For example, a formulation for subcutaneous administration, a formulation for intravenous injection, or an ophthalmic formulation according to an embodiment of the present invention may contain the Hyal1 variant according to an embodiment of the present invention. At this time, the ophthalmic formulation may be an eye drop, and the eye drop may be for promoting the diffusion of an anesthetic during ophthalmic surgery. Also, the formulation for subcutaneous administration may specifically be an injection for subcutaneous administration. The injection for subcutaneous administration may be for hyaluronic acid hydrolysis in the treatment of a filler. Furthermore, the formulation for intravenous injection may be for enhancing the accessibility of an anticancer agent to tumor cells. This is because the formulation for intravenous injection can move the Hyal1 variant according to an embodiment of the present invention along the blood to hydrolyze the hyaluronic acid overexpressed on the surface of tumor cells, thereby enhancing the accessibility of the anticancer agent to tumor cells. Thus, the Hyal1 variant according to an embodiment of the present invention is also used for cosmetic purposes in addition to therapeutic purposes. Therefore, the formulation according to an embodiment of the present invention may be a pharmaceutical formulation and / or a cosmetic formulation.

[0044] The dosage of the Hyal1 variant according to an embodiment of the present invention is 10 ng / ml to 10 mg / ml per injection during subcutaneous injection, preferably 10 ng / ml to 100 μg / ml. It is also possible to administer it once a day or divided into several times, and such a dosage may be based on an adult (body weight 60 kg), but it is natural that it may vary depending on body weight, physical condition, etc. The Hyal1 variant according to an embodiment of the present invention is mainly administered by a parenteral method, for example, by subcutaneous injection or intravenous injection, or by eye drop.

[0045] The Hyal1 variant according to an embodiment of the present invention can also be formulated with pharmaceutically acceptable additives and manufactured in the form of an injection, an eye drop, a patch for transdermal administration, etc.

[0046] Pharmaceutically acceptable additives can be applied according to various factors well known to those skilled in the art, for example, the specific bioactive substance utilized, its concentration, stability, and intended bioavailability. Diseases, illnesses or conditions to be treated and individuals to be treated, age, size and general condition, and factors such as nasal cavity, oral cavity, eyeball, topical, transdermal and muscular, etc. should be considered, but are not limited to these. Pharmaceutically available additives generally used for administration of bioactive substances other than the oral administration route include D5W (5% glucose in water), aqueous solutions containing dextrose and physiological salts within 5% of the volume, etc. In the case of local injection, various injectable hydrogels can be used to enhance the effect and increase the duration. Furthermore, pharmaceutically available additives may include additional components that can reinforce the stability of active ingredients such as preservatives and antioxidants. The variant which is an embodiment of the present invention can be formulated by appropriate methods in the art and can be preferably formulated according to each disease or condition or according to the components.

[0047] The Hyal1 variant which is an embodiment of the present invention can be stored in a physiological saline aqueous solution, can be lyophilized in an ampule after addition of mannitol or sorbitol, and can be dissolved in physiological saline or the like when used for administration. At this time, a buffer, a stabilizer and / or a surfactant can be included in the physiological saline aqueous solution.

[0048] Furthermore, the hyaluronic acid degradation and / or drug delivery method which is an embodiment of the present invention includes the step of administering the Hyal1 variant which is an embodiment of the present invention to a mammal including a human in need of administration. At this time, the Hyal1 variant which is an embodiment of the present invention to be administered can be an effective amount of the variant.

[0049] Also, the use which is an embodiment of the present invention is for the production of a hyaluronic acid degradation preparation or a drug delivery preparation of the Hyal1 variant which is an embodiment of the present invention.

[0050] The Hyal1 variant, which is an embodiment of the present invention, can be produced by genetic engineering techniques. For example, a fusion gene encoding a fusion protein consisting of a fusion partner and the Hyal1 variant protein, which is an embodiment of the present invention, is produced via genetic engineering, and after transforming a host cell therewith, the fusion protein is expressed in the host cell in the form of a fusion protein, and then, using a proteolytic enzyme or a compound, the Hyal1 variant, which is an embodiment of the present invention, is cleaved and separated from the fusion protein to be produced in a desired protein form. Therefore, the Hyal1 variant, which is an embodiment of the present invention, can be effectively produced by the nucleic acid, expression vector, host cell and / or method, which are embodiments of the present invention. Specifically, the nucleic acid, which is an embodiment of the present invention, encodes the variant, which is an embodiment of the present invention. The nucleic acid can be present in a cell, cell lysate, or can also be present in a partially purified form or a substantially pure form. The nucleic acid can be, for example, DNA or RNA.

[0051] In addition, the expression vector which is an embodiment of the present invention contains the nucleic acid which is an embodiment of the present invention. For the expression of the Hyal1 variant which is an embodiment of the present invention, DNA encoding the Hyal1 variant which is an embodiment of the present invention is obtained by molecular biology techniques (for example, PCR amplification, cDNA cloning using a hybridoma expressing the Hyal1 variant which is an embodiment of the present invention), and such DNA is ligated to transcription and translation control sequences so as to operate and inserted into an expression vector to produce the expression vector which is an embodiment of the present invention. At this time, "ligated so as to operate" means that the gene encoding the Hyal1 variant which is an embodiment of the present invention is ligated into the vector so that the transcription and translation control sequences in the vector perform the intended function of regulating the transcription and translation of the gene encoding the Hyal1 variant which is an embodiment of the present invention. The expression vector and the expression control sequence are selected to be compatible with the host cell for expression to be used. The gene encoding the Hyal1 variant which is an embodiment of the present invention is inserted into the expression vector by a standard method (for example, ligation of a gene fragment encoding the Hyal1 variant which is an embodiment of the present invention and a complementary restriction enzyme site on the vector, or blunt end ligation when there is no restriction enzyme site at all). The recombinant expression vector has a regulatory sequence that controls the expression of the gene encoding the Hyal1 variant which is an embodiment of the present invention in the host cell. The "regulatory sequence" may include a promoter, an enhancer, and other expression control elements (for example, a polyadenylation signal) that control the transcription or translation of the gene encoding the Hyal1 variant which is an embodiment of the present invention. At this time, it goes without saying that the design of the expression vector may change depending on factors such as the selection of the host cell to be transformed and the expression level of the protein, and the regulatory sequence may be selected differently.

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

[0053] The nucleic acid or expression vector is transfected or transfected into the host cell. Various techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for "injection" or "transfection", such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection, etc. can be used. Various expression host / vector combinations can be utilized to express the Hyal1 mutant, which is an example of the present invention. Expression vectors compatible with eukaryotic hosts include, but are not limited to, expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. Expression vectors that can be used 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 having a wider host range such as RP4, phage DNA exemplified by very diverse phage lambda derivatives such as λgt10 and λgt11, NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. Expression vectors useful for yeast cells are 2μm plasmids and their derivatives. The vector useful for insect cells is pVL941.

[0054] In addition, the method for producing a Hyal1 variant, which is an embodiment of the present invention, can include the step of culturing a host cell, which is an embodiment of the present invention. When a recombinant expression vector capable of expressing a Hyal1 variant, which is an embodiment of the present invention, is introduced into a mammalian host cell, the variant, which is an embodiment of the present invention, can be produced by culturing the host cell for a period sufficient for the variant to be expressed in the host cell, more preferably for a period sufficient for the Hyal1 variant, which is an embodiment of the present invention, to be secreted into the culture medium in which the host cell is cultured.

[0055] If necessary, the expressed Hyal1 variant, which is an embodiment of the present invention, can be separated from the host cell and purified to be homogeneous. The separation or purification of the Hyal1 variant, which is an embodiment of the present invention, can be performed by separation and purification methods commonly used for ordinary proteins, such as chromatography. Chromatography can be, for example, one or more combinations selected from affinity chromatography, ion exchange chromatography, or hydrophobic chromatography, but is not limited thereto. In addition to chromatography, filtration, ultrafiltration, salting out, dialysis, etc. can also be used in combination.

[0056] Unless otherwise specified, the matters referred to in the Hyal1 variant, hyaluronic acid degrading agent, drug delivery agent, composition, use, formulation, and method, which are embodiments of the present invention, are applied to each other in the same way within the scope of identity as long as they do not conflict with each other.

[0057] Hereinafter, the present invention will be described in more detail with reference to Examples, Comparative Examples, and Production Examples. However, the following Examples and Production Examples are only for illustrating the present invention, and the content of the present invention is not limited by the following Examples and Production Examples.

[0058] Hereinafter, the reagents used in Examples and the like are commercially available products, and the best products were used. Unless otherwise specified, those purchased from Sigma-Aldrich were used.

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

Table 6

[0060] <Example 2>Confirmation of Enzyme Activity and Expression of Hyal1 Ala132 Mutant CHO-K1 cells (purchased from the Korean Cell Line Bank) were transformed using polyethylenimine (PEI, Sigma-Aldrich) or Lipofectamine3000 (Thermofisher) reagent with the plasmid inserted with the Hyal1 wild-type or mutant gene produced in Example 1. When the CHO-K1 cells grew to 90 - 95% of the area in a 6-well plate, 150 μL of DMEM medium containing 2 μg of plasmid DNA was mixed. After 30 minutes, the PEI-DNA mixture was carefully transferred to the 6-well plate and cultured in a 5% CO2 incubator.

[0061] Forty-eight hours after transfection, the cell culture medium was collected and centrifuged at 10,000×g to obtain the supernatant. The enzyme activities of Hyal1 wild type and mutants were measured by the substrate-gel assay method. Specifically, after electrophoresis using a 10% SDS gel containing hyaluronic acid (1.0 mg / mL), SDS was removed using a buffer containing Triton X-100 (3% Triton X-100, 50 mM Tris, 100 mM NaCl, pH 7.5) for 2 hours at 4°C. Phosphate buffers containing 100 mM NaCl and adjusted to pH 4, 5, 6, and 7 using disodium phosphate and monosodium phosphate were prepared respectively. Each SDS gel was placed in the phosphate buffer at pH 4-7, and the enzyme reaction was allowed to proceed for 4-17 hours while shaking at 50 rpm at 37°C. After the enzyme reaction, the hyaluronic acid in the SDS gel was stained with a 1.0% Alcian blue staining agent. Since the present invention aims to examine the enzyme activity of the Hya1 mutant at neutral pH, pH 4 was used as the control group, and the enzyme activities at pH 6 and pH 7 were examined.

[0062] Figure 1(A) shows the experimental results of confirming whether the Hyal1 mutant exhibits enzyme activity depending on pH. Since hyaluronic acid hydrolyzed by the Hyal1 mutant in the substrate-gel assay method is not stained by Alcian blue, it 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 the respective Hyal1 mutants.

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

[0064] In addition, to examine protein expression in CHO-K1 cells, Western blotting was performed after protein electrophoresis as in the experiment of Fig. 1(A). Specifically, after transferring a 10% SDS gel to a nitrocellulose membrane at 100 V for 1 hour, the expression of wild-type Hyal1 and Ala132 mutants was examined using a Hyal1 monoclonal antibody (1D10, Santa Cruze Biotechnology, Dallas, TX, US) and an anti-mouse IgG secondary antibody (m-IgGk BP-HRP, Santa Cruze Biotechnology). The results are shown in Fig. 1(B). Fig. 1(B) shows the experimental results for confirming the expression of Hyal1 mutants. In Fig. 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 the respective mutants. As shown in Fig. 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 such results, it is inferred that the negative result of the A132Y mutant shown in Fig. 1(A) is due to the lack of protein expression.

[0065] The Hyal1 variant of the present invention hydrolyzes hyaluronic acid by general acid-base catalysis. Since the carboxyl group (-COOH) of the catalytic amino acid Glu131 serves to provide a positive charge to the Hydroxyl leaving group, the protonation of GLU131 is important for the catalytic reaction. The results in Figure 1(A) mean that when the catalytic amino acid Glu131 and the amino acids adjacent to it in the tertiary structure are replaced with acidic amino acids, the pKa value of Glu131 increases and Glu131 can serve as a positive charge provider at acidic to neutral pH. However, even though they are the same acidic amino acids, the enzyme activity of A132E was higher than that of A132D at pH 7. Therefore, it can be seen that, particularly according to the Hyal1 variant A132E which is an embodiment of the present invention, hyaluronic acid can be effectively decomposed even at neutral pH.

[0066] <Example 3>Confirmation of Enzyme Activity and Expression of Hyal1 Loop Mutants To examine the enzyme activities of the prepared Hyal1 loop mutants (S77D, S77E, T86D, T86E, and P87E) in the pH range of 4 to 7, CHO-K1 cells were transformed using the PEI or Lipofectamaine method as described in Example 1 with a plasmid inserted with the gene of the Hyal1 loop mutant. 48 hours after transformation, the cell culture medium was collected and centrifuged at 10,000×g to obtain the supernatant. The enzyme activities of Hyal1 WT and the loop mutants contained in the supernatant were examined by the Substrate-gel assay method. The enzyme reaction was carried out at 37°C for 4 to 17 hours using a phosphate buffer in the pH range of 4 to 7, and the amount of hydrolyzed hyaluronic acid was examined using a 1.0% Alcian blue staining agent. The results are shown in Figure 2(A). Figure 2(A) is the experimental result of confirming whether Hyal1 WT and the loop mutants exhibit enzyme activity depending on pH. In Figure 2(A), Control represents the control group transfected with only the vector, WT represents wild-type Hyal1, and S77D, S77E, T86D, T86E, and P87E represent the respective Hyal1 mutants.

[0067] As shown in Fig. 2(A), Hyal1 WT showed enzymatic activity by hydrolyzing hyaluronic acid at pH 4, while all the loop mutants (S77D, S77E, T86D, T86E, and P87E) showed enzymatic activity in the entire pH range of 4 - 7. From these results, it can be seen that Hyal1 mutants with Ser77, Thr86, and Pro87 at the loop site of wild-type human Hyal1 substituted with Asp or Glu can effectively degrade hyaluronic acid even at neutral pH. In particular, according to the Hyal1 mutant which is an embodiment of the present invention, S77D and S77E, and P87E had higher enzymatic activity at pH 7 than the T86D and T86E mutants. Fig. 2(B) shows the experimental results for confirming the expression of Hyal1 mutants. In Fig. 2(B), it can be seen that Hyal1 WT and the mutants were normally expressed in CHO-K1 cells.

[0068] <Example 4> Confirmation of Enzymatic Activity and Expression of Hyal1 Ala132 or Mixed Mutants with Substituted Loop Amino Acids Among the Ala132 mutants, the most active A132E mutant and one or more of the loop mutants were mutated together to examine the enzymatic activity in the pH range of 4 - 7. To examine the enzymatic activity of the prepared Hyal1 mutants (S77D / A132E, S77E / A132E, S77D / T86D, S77D / T86E, and S77D / T86D / A132E) 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 inserted with the genes of these Hyal1 mutants. 48 hours after transformation, the cell culture supernatant was collected and centrifuged at 10,000×g to obtain the supernatant. The enzymatic activity of the Hyal1 mutants contained in the supernatant was examined by the Substrate-gel assay method. The enzymatic reaction was carried out at 37°C for 4 - 17 hours using a phosphate buffer in the pH range of 4 - 7, and the amount of hydrolyzed hyaluronic acid was examined using a 1.0% Alcian blue staining agent.

[0069] As shown in Fig. 3(A), the double or triple mutants showed enzyme activity by hydrolyzing hyaluronic acid not only at pH 4 but also at pH 7. In particular, at pH 7, a difference in the degree of activity was observed, and the S77D / A132E mutant showed the best activity.

[0070] Fig. 3(B) shows the experimental results for confirming the expression of the Hyal1 mutants. In Fig. 3(B), it can be seen that the Hyal1 mutants were normally expressed in CHO-K1 cells.

[0071] <Example 5> Comparison of the Protein Tertiary Structures of Human Hyal1 and PH20 To compare the positions of the mutant amino acids of Hyal1 introduced in the present invention with the amino acids of PH20, the tertiary structures of human Hyal1 and PH20 were compared. The tertiary structure model of human PH20 was constructed 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 Fig. 4. As shown in Fig. 4, in human Hyal1, it can be seen that Ser76 and Ser77, Thr86 and Pro87 are located adjacent to the catalytic amino acid Glu131. In the tertiary structure model of human PH20, it can be seen that Asp94, Asp103, and Glu149 are located adjacent to the catalytic amino acid Glu148.

[0072] Therefore, it is considered that the Hyal1 mutants of the present invention have the effect of effectively decomposing hyaluronic acid even at neutral pH, in which the amino acids adjacent to the catalytic amino acid in the tertiary structure of human Hyal1 are replaced with acidic amino acids, similar to PH20 having enzyme activity at neutral pH.

[0073] <Example 6> Confirmation of the Enzyme Activity and Expression of the F139R and Y210H Mutants Another way to increase the pKa value of the catalytic amino acid Glu131 of Hyal1 is to substitute the amino acid adjacent to Glu131 with a basic amino acid, thereby decreasing the pKa value of an 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 decreases 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. Also, 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, thereby promoting the dehydrogenation of the hydroxyl group of Tyr210 more than in the F139R single mutant in the ionic bond between Arg139 and Tyr210. Further, substituting the polar amino acid Tyr210 with the basic amino acid His decreases the pKa value of Asp206 due to the ionic bond between Asp206 and His210, and as a result, the pKa value of the catalytic amino acid Glu131 can be increased.

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

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

[0076] Figure 5(B) shows the experimental results for confirming the expression of the Hyal1 mutants. In Figure 5(B), it can be seen that the Hyal1 mutants were normally expressed in CHO-K1 cells.

[0077] Figure 5(C) shows the tertiary structure of Hyal1 with the 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 mutants of the present invention can effectively degrade hyaluronic acid not only at acidic pH but also at neutral pH, indicating high usability. That is, since the Hyal1 mutants of the present invention can degrade hyaluronic acid even at neutral pH, drug delivery using them is possible, and they can be used in various formulations or applications such as subcutaneous administration formulations, intravenous injection formulations, and ophthalmic formulations.

[0079] Also, from the above results, it can be seen that the nucleic acid, expression vector, host cell, and method of the present invention can effectively produce Hyal1 mutants.

[0080] <Production Example 1> Preparation of Liquid Preparation 1 μg of the Hyal1 mutant protein produced in Examples 2 to 6 was dissolved in PBS to produce 1 mL of a solution. The resulting solution was filled into an ampoule for injection or a sterile container to produce an injection solution or an eye drop. The formulation thus produced can be utilized as a hyaluronic acid degrading agent, a drug delivery agent, etc. In particular, the injection solution can be utilized as a formulation for subcutaneous administration or a formulation for intravenous injection, and the eye drop can be utilized as an ophthalmic formulation.

Claims

1. A hyaluronidase Hyal1 variant that exhibits activity at neutral pH.

2. The hyaluronidase Hyal1 variant contains aspartic acid (Asp) and glutamic acid (Glu) as catalytic amino acids, based on the wild-type human hyaluronidase Hyal1 consisting of the amino acid sequence of SEQ ID NO:

1. At least one of the amino acids adjacent to the catalytic amino acids in the primary or tertiary structure is substituted with an acidic or polar amino acid, or One or more of the amino acids adjacent to the catalytic amino acids in the tertiary structure are substituted with basic amino acids. The hyaluronidase Hyal1 variant that exhibits activity at neutral pH according to Claim 1.

3. The -ase Hyal1 variant contains one or more amino acid substitutions selected from the group consisting of S76D, S77D, T86D, P87E, A132D, and A132E, based on the wild-type human hyaluronidase Hyal1. The hyaluronidase Hyal1 variant that exhibits activity at neutral pH according to Claim 2.

4. The substituted basic amino acid forms an ionic bond with an acidic or polar amino acid adjacent to the catalytic amino acid. The hyaluronidase Hyal1 variant that exhibits activity at neutral pH according to Claim 2.

5. The hyaluronidase Hyal1 variant contains amino acid substitutions selected from the group consisting of F139R, Y210H, and F139R / I225D, where F139R / I225D means that F139R and I225D are substituted together. The hyaluronidase Hyal1 variant that exhibits activity at neutral pH according to Claim 2.

6. A nucleic acid encoding the hyaluronidase Hyal1 variant according to Claim 1.

7. A recombinant expression vector containing the nucleic acid according to Claim 6.

8. A host cell transformed with the expression vector according to Claim 7.

9. A method for producing a hyaluronidase Hyal1 variant, including the step of culturing the host cell according to Claim 8.

10. A hyaluronic acid degrading agent containing the hyaluronidase Hyal1 variant according to Claim 1.

11. A drug delivery agent containing the hyaluronidase Hyal1 variant according to Claim 1.

12. A preparation for subcutaneous administration containing the hyaluronidase Hyal1 variant according to Claim 1.

13. An intravenous preparation comprising the hyaluronidase Hyal1 variant according to claim 1.

14. An ophthalmic preparation comprising the hyaluronidase Hyal1 variant according to claim 1.

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