Hyaluronidase hyal1 variant

Abstract

The present invention provides: a Hyal1 variant in which, in wild-type human hyaluronidase Hyal1 comprising glutamic acid 131 as a catalytic amino acid, at least one amino acid among amino acids adjacent to the catalytic amino acid, i.e., glutamic acid 131 in primary and tertiary structures is substituted with an acidic, polar or basic amino acid; a method for producing the Hyal1 variant; a nucleic acid applicable to the production of the Hyal1 variant; an expression vector; a host cell; and a Hyal1 variant preparation or use thereof. The hyaluronidase Hyal1 variant of the present invention can effectively degrade hyaluronic acid not only at acidic pH but also at neutral pH, and thus is highly applicable.

Description

Hyaluronidase HYAL1 mutant

[0001] The present invention relates to a technology for changing the working pH and enzyme activity of human hyaluronidase, and more specifically, to a variant of hyaluronidase Hyal1 that hydrolyzes hyaluronic acid in an acidic to neutral pH range and a Hyal1 variant with increased enzyme activity at acidic pH.

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

[0003] Hyaluronidase PH20, due to its property of hydrolyzing extracellular matrix hyaluronic acid, is clinically used in subcutaneous formulations of antibody therapeutics, potentially enhancing subcutaneous drug delivery. However, hyaluronic acid hydrolysis currently relies on a single enzyme, PH20. Among hyaluronidases other than PH20, Hyal1 primarily catalyzes action at acidic pH levels of 3 to 4, limiting its utility. For example, applying Hyal1 to a subcutaneous area with a neutral pH of 7.0 to 7.5 will likely result in little hyaluronic acid degradation. Wild-type Hyal2, Hyal3, and Hyal4 also exhibit weak activity in neutral pH levels, limiting their practical application.

[0004] In addition, although enzyme replacement therapy or gene therapy technology development is required to treat Hyal1 deficiency, there has been little research on increasing the enzymatic activity of Hyal1 at acidic pH.

[0005] Accordingly, the inventor of the present invention completed the present invention as a result of research to improve the usability of such wild-type Hyal1.

[0006] The problem to be solved by the present invention is to provide a mutant of Hyal1 capable of effectively hydrolyzing hyaluronic acid at acidic to neutral pH and a mutant of Hyal1 having higher enzymatic activity than wild-type Hyal1 at acidic pH.

[0007] The human hyaluronidase Hyal1 variant of the present invention has acidic amino acids Asp129 and Glu131 in the active site. Glu131 acts as a general acid-base catalyst, and Asp129 stabilizes the substrate during catalysis.

[0008] The present invention substitutes an amino acid adjacent to the catalytic amino acid Glu131 in the primary or tertiary structure of Hyal1 with an acidic or basic amino acid so that Hyal1 can perform catalytic action at neutral pH.

[0009] The amino acid adjacent to the catalytic amino acid Glu131 of Hyal1 in the primary or tertiary structure is an amino acid located in a loop composed of amino acids Ala132 or Ser76 to Leu98, or an amino acid adjacent to an acidic or polar amino acid that may affect the activity of the catalytic amino acid in the tertiary structure of Hyal1.

[0010] Ala132 can be substituted with an acidic, polar, or basic amino acid. The acidic amino acid is Asp or Glu, the polar amino acid is Ser, Thr, Asn, Gln, or Tyr, and the basic amino acid is His.

[0011] In the loop composed of amino acids Ser76 to Leu98, the catalytic amino acid Glu131 and the adjacent amino acids in the tertiary structure are Ser76, Ser77, Thr86, Pro87, or Thr88, and these amino acids are substituted with Asp or Glu.

[0012] In addition, in the tertiary structure of Hyal1, one of the amino acids adjacent to the catalytic amino acid Glu131 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 Glu131. For example, in the tertiary structure of Hyal1, adjacent amino acids that can affect the activity of the catalytic amino acid Glu131 are the acidic amino acid Asp206 and the polar amino acid Tyr210. 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 Hyal1 variant of the present invention can hydrolyze hyaluronic acid even at neutral pH.

[0013] The present invention also replaces an amino acid adjacent to the catalytic amino acid Glu131 in the primary structure or tertiary structure of Hyal1 with an acidic amino acid or replaces Ala132 with an amino acid larger than Ala in order to increase the enzymatic activity of Hyal1 at acidic pH.

[0014] The amino acids adjacent to the above catalytic amino acid Glu131 are Ser76, Ser77, Thr86, Pro87, Thr88, or Gly89 in the loop composed of amino acids Ser76 to Leu98.

[0015] Additionally, amino acids larger than Ala132 include all acidic amino acids, polar amino acids, basic amino acids, and hydrophobic amino acids, except for Gly, which is smaller than Ala.

[0016] In addition, the present invention provides a nucleic acid encoding the hyaluronidase Hyal1 variant.

[0017] Additionally, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0018] In addition, the present invention provides a host cell transformed with the expression vector.

[0019] In addition, the present invention provides a method for producing a hyaluronidase Hyal1 variant, which comprises a step of culturing a host cell.

[0020] In addition, the present invention provides a hyaluronic acid decomposing agent comprising the hyaluronidase Hyal1 variant.

[0021] In addition, the present invention provides a drug delivery agent comprising the hyaluronidase Hyal1 variant.

[0022] In addition, the present invention provides an intravenous injection preparation comprising the hyaluronidase Hyal1 variant.

[0023] In addition, the present invention provides a formulation for subcutaneous administration comprising the hyaluronidase Hyal1 variant.

[0024] In addition, the present invention provides an ophthalmic preparation comprising the hyaluronidase Hyal1 variant.

[0025] The hyaluronidase Hyal1 variant of the present invention can effectively decompose hyaluronic acid at acidic to neutral pH, and thus has the effect of high usability.

[0026] That is, the hyaluronidase of the present invention is capable of decomposing hyaluronic acid at neutral pH and delivering drugs using the same, and can be utilized in various formulations and uses such as formulations for subcutaneous administration, formulations for intravenous injection, and ophthalmic formulations.

[0027] In addition, since the Hyal1 variant of the present invention has higher enzyme activity than the Hyal1 wild type at acidic pH, the Hyal1 variant of the present invention can be developed as a treatment for Hyal1 deficiency using enzyme replacement therapy or gene therapy.

[0028] In addition, the hyaluronidase of the present invention can be effectively produced by the nucleic acid, expression vector, host cell, and method of the present invention.

[0029] Figure 1(A) shows the results of an experiment investigating whether the Hyal1 wild type and Ala132 mutant exhibit enzymatic activity depending on pH.

[0030] Figure 1(B) shows the results of a Western blot experiment to confirm the expression of Hyal1 wild type and Ala132 mutant in CHO-K1 cells.

[0031] Figure 1(C) shows the experimental results comparing the enzyme activities of the Hyal1 wild type and A132G mutant.

[0032] Figure 2(A) shows the results of an experiment confirming whether the Hyal1 wild type and Ser76 mutant exhibit enzymatic activity depending on pH.

[0033] Figure 2(B) shows the results of an experiment confirming whether the Hyal1 wild type and Ser77 mutant exhibit enzymatic activity depending on pH.

[0034] Figure 2(C) shows the results of an experiment to confirm whether the Hyal1 wild type and Tyr85, Thr86, Pro87, Thr88, or Gly89 mutants exhibit enzymatic activity. In Figures 2(A) and 2(C), the A132E mutant was used as a positive control.

[0035] Figure 3(A) shows the results of an experiment to investigate whether a mutant in which two or more of Ser77, Thr86, or Ala132 are substituted together exhibits enzyme activity depending on pH.

[0036] Figure 3(B) shows the results of a Western blot experiment to confirm the expression of a mutant in which two or more of Ser77, Thr86, or Ala132 are substituted together in CHO-K1 cells.

[0037] Figure 4 compares the active site structures of Hyal1 and PH20. The active sites of Hyal1 and PH20 share the acidic amino acids Asp and Glu (Asp129 and Glu131 in Hyal1, and Asp146 and Glu148 in PH20). The catalytic amino acid is Glu131 in Hyal1, and Glu148 in PH20.

[0038] Figure 5(A) shows the results of an experiment investigating whether the Hyal1 wild type and Phe139, Tyr210, or Phe139 / Ile225 mutants exhibit enzymatic activity depending on pH.

[0039] Figure 5(B) shows the results of a Western blot experiment to confirm the expression of Hyal1 wild type and Phe139, Tyr210, or Phe139 / Ile225 mutants in CHO-K1 cells.

[0040] Figure 5(C) shows the positions of Phe139, Asp206, Tyr210, and Ile225 adjacent to the catalytic amino acid Glu131 in the tertiary structure of Hyal1.

[0041] Hereinafter, the advantages and features of the present invention, and the methods for achieving them, will be clarified by referring to the embodiments described in detail below together with the attached drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0042] Throughout this specification, the term "and / or" includes each and every one or more of the mentioned elements and any combination thereof. The terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting of the invention. As used herein, the singular also includes the plural unless specifically stated otherwise. As used herein, the terms "comprises" and / or "comprising" do not exclude the presence or addition of one or more other elements.

[0043] A hyaluronidase Hyal1 variant (hereinafter referred to as “Hyal1 variant”), which is an embodiment of the present invention, refers to a variant in which at least one of the amino acids adjacent to the catalytic amino acid in the primary structure or tertiary structure is substituted with an acidic, polar, or basic amino acid, based on wild-type human Hyal1 including Glu131 as a catalytic amino acid. That is, a hyaluronidase Hyal1 variant, which is an embodiment of the present invention, corresponds to a variant of wild-type Hyal1, and by substituting the catalytic amino acid Glu131 and the amino acid adjacent to the primary structure or tertiary structure with an acidic, polar, or basic amino acid, hyaluronic acid can be effectively decomposed at an acidic to neutral pH, thereby increasing usability.

[0044] In this specification, expressions such as “Ala132” or “A132” in which a three-letter or one-letter amino acid name and a number are written together mean the amino acid at each position based on the amino acid sequence of SEQ ID NO: 1. For example, “Ala132” and “A132” represent Ala, the amino acid at position 132 based on the amino acid sequence of SEQ ID NO: 1. In addition, in this specification, a variant of wild-type hyaluronidase also means a variant in which an amino acid is conservatively substituted at a specific amino acid position. In this case, “conservative substitution” means a modification of a variant 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 variant. Specifically, a "conservatively substituted variant" may be a variant having a sequence identity of, for example, 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more, with a Hyal1 variant comprising an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 26, and having substantially the same function and / or effect.

[0045] The Hyal1 variant, which is an embodiment of the present invention, corresponds to a hyaluronidase capable of decomposing hyaluronic acid in a wide pH range from acidic to neutral. In the hyaluronidase, which is an embodiment of the present invention, the wild-type hyaluronidase is Hyal1, and Hyal1 consists of the amino acid sequence of SEQ ID NO: 1 and may include a substitution of one or more amino acids selected from the group consisting of S76D, S76E, S77D, S77E, Y85D, T86D, T86E, P87D, P87E, G89D, A132D, A132E, A132G, A132H, A132N, A132S, F139R, Y210H, and F139R / I225D. That is, the variant, which is an embodiment of the present invention, may include a substitution of one or more amino acids selected from the group consisting of S76D, S76E, S77D, S77E, Y85D, T86D, T86E, P87D, P87E, T88D, G89D, A132D, A132E, A132G, A132H, A132N, A132S, F139R, Y210H and F139R / I225D, based on the wild-type Hyal1 having the amino acid sequence of SEQ ID NO: 1.

[0046] Here, the meaning of S76D is that the 76th Ser of sequence number 1 is substituted with Asp.

[0047] Hyal1 variants are those in which some amino acids are changed, specifically amino acid substitutions, in the sequence of wild-type Hyal1, and include one or more amino acid substitutions selected from the group consisting of S76D, S76E, S77D, S77E, Y85D, T86D, T86E, P87D, P87E, T88D, G89D, A132D, A132E, A132G, A132H, A132N, A132S, F139R, Y210H, and F139R / I225D. Here, the meaning of the substitution of F139R / I225D means that the amino acid substitutions of F139R and I225D are performed together.

[0048] The amino acid sequence of wild-type Hyal1 is shown in Table 1 below. As shown in the sequence described in Table 1, Ala132 is located adjacent to the catalytic amino acid Glu131. Hyal1 mutants in which the amino acid adjacent to the catalytic amino acid is substituted with a specific amino acid appear to exhibit different characteristics from the wild-type while maintaining activity as a hyaluronic acid hydrolase. That is, as confirmed in the experimental examples, substituting Ala132 with Asp or Glu enables hydrolysis of hyaluronic acid even at neutral pH.

[0049] That is, by substituting Ala, the 132nd amino acid in the amino acid sequence of wild-type Hyal1, with Asp or Glu, hyaluronic acid can be hydrolyzed even at neutral pH. However, Hyal1 mutants in which Ala132 is substituted with other similar amino acids did not exhibit such effects.

[0050] Therefore, it can be seen that when Ala132 is substituted with Asp or Glu in the Hyal1 mutant, which is an example of the present invention, hyaluronic acid can be effectively decomposed even at neutral pH.

[0051] In addition, Ser76 or Ser77 is located adjacent to the catalytic amino acid Glu131 in the tertiary structure of Hyal1, and substituting Ser76 with Asp or substituting Ser77 with Asp or Glu allows for hydrolysis of hyaluronic acid at neutral pH. That is, by substituting Ser, the 76th amino acid in the amino acid sequence of wild-type Hyal1, with Asp, or substituting Ser, the 77th amino acid, with Asp or Glu, hyaluronic acid can be hydrolyzed even at neutral pH.

[0052] In addition, Thr86, Pro87, or Thr88 are located adjacent to the catalytic amino acid Glu131 in the tertiary structure of Hyal1, and substituting Thr86, Pro87, or Thr88 with Asp or Glu also allows for hydrolysis of hyaluronic acid at neutral pH. That is, by substituting Thr, Pro, and Thr, which are amino acids at positions 86, 87, and 88 in the amino acid sequence of wild-type Hyal1, with Asp or Glu, hyaluronic acid can be hydrolyzed even at neutral pH.

[0053] Meanwhile, Asp206 and Tyr210 are located adjacent to the catalytic amino acid Glu131 in the tertiary structure of Hyal1. Substituting Phe139, adjacent to Tyr210, with a basic amino acid, or substituting Tyr210, adjacent to Asp206, with a basic amino acid, allows for the hydrolysis of hyaluronic acid at neutral pH.

[0054] Specifically, when Phe139 is substituted with Arg, the hydroxyl group of Tyr210 can be dehydrogenated by the ionic bond between Arg139 and Tyr210, thereby increasing the pKa value of the catalytic amino acid Glu131. In addition, when Phe139 and Ile225 are substituted together with Arg and Asp, respectively (F139R / I225D), the positive charge of Arg139 is stabilized by the ionic bond between Arg139 and Asp225, and Arg139 can then form an ionic bond with Tyr210 to dehydrogenate the hydroxyl group of Tyr210.

[0055] Meanwhile, when Tyr210 is replaced with His, the pKa value of Asp206 decreases due to the ionic bond between Asp206 and His210, and as a result, the pKa value of the catalytic amino acid Glu131 can increase due to the repulsion between negative charges.

[0056] Meanwhile, in an embodiment of the present invention, a mutant in which the catalytic amino acid Glu131 of Hyal1 and adjacent Ser76, Ser77, Thr86, Pro87, Thr88, and Gly89 are substituted with acidic amino acids such as Asp or Glu has an increased enzymatic activity than wild-type Hyal1 at pH 4, and can effectively hydrolyze hyaluronic acid.

[0057] Furthermore, the Ala132 mutant can exhibit increased enzyme activity at pH 4 compared to the wild-type Hyal1 not only by substitution of acidic amino acids but also by substitution of polar amino acids (Asn, Ser), and basic amino acids (His). In particular, the A132H mutant can exhibit enzyme activity not only at pH 4 but also at pH 6.

[0058] The amino acid sequence of wild-type Hyal1 is shown in Table 1, the amino acid sequence of 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 mutants in which one or more of Ala132 or the loop mutant is substituted together is shown in Table 4, and the amino acid sequences of the Phe139 and Tyr210 mutants are shown in Table 5, respectively.

[0059] 서열번호아미노산 서열야생형 인간 Hyal11MAAHLLPICALFLTLLDMAQGFRGPLLPNRPFTTVWNANTQWCLERHGVDVDVSVFDVVANPGQTFRGPDMTIFYSSQLGTYPYYTPTGEPVFGGLPQNASLIAHLARTFQDILAAIPAPDFSGLAVIDWEAWRPRWAFNWDTKDIYRQRSRALVQAQHPDWPAPQVEAVAQDQFQGAARAWMAGTLQLGRALRPRGLWGFYGFPDCYNYDFLSPNYTGQCPSGIRAQNDQLGWLWGQSRALYPSIYMPAVLEGTGKSQMYVQHRVAEAFRVAVAAGDPNLPVLPYVQIFYDTTNHFLPLDELEHSLGESAAQGAAGVVLWVSWENTRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCSGHGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSLRGALSLEDQAQMAVEFKCRCYPGWQAPWCERKSMW

[0060]

[0061]

[0062]

[0063]

[0064] Such Hyal1 variants are capable of effectively decomposing hyaluronic acid in a wide pH range from acidic to neutral, and also exhibit higher enzymatic activity than the Hyal1 wild type at acidic pH. In addition, since the Hyal1 variant, which is an embodiment of the present invention, is capable of decomposing hyaluronic acid in an acidic to neutral pH range, it is also capable of effectively delivering drugs to various sites. This is because when hyaluronic acid in the extracellular matrix is ​​hydrolyzed, the viscosity of hyaluronic acid is reduced and its permeability into tissues (skin) is increased. In particular, since the subcutaneous region of the skin is neutral with a pH of approximately 7.0 to 7.5, permeability can be increased by an embodiment of the present invention that can decompose hyaluronic acid at a neutral pH. Therefore, the drug delivery agent, which is an embodiment of the present invention, can effectively deliver drugs to various sites by including the variant, which is an embodiment of the present invention.

[0065] Here, the drug is a component that exhibits pharmacological activity, and may be, but is not limited to, an ophthalmic relaxant, anesthetic, antibody therapeutic agent, anticancer agent, etc. The drug may be formed into a composition together with the variant, which is an embodiment of the present invention. That is, the variant, which is an embodiment of the present invention, may be in the form of a pharmaceutical composition, either by itself or together with the drug.

[0066] The Hyal1 variant, which is an embodiment of the present invention, may be included in formulations that can be applied to various sites. For example, a subcutaneous formulation, an intravenous formulation, or an ophthalmic formulation, which is an embodiment of the present invention, may include the Hyal1 variant, which is an embodiment of the present invention. In this case, the ophthalmic formulation may be an eye drop, and the eye drop may be used to promote the diffusion of an anesthetic during ophthalmic surgery. Furthermore, the subcutaneous formulation may be specifically a subcutaneous injection. The subcutaneous injection may be used to hydrolyze hyaluronic acid during filler treatment. Furthermore, the intravenous formulation may be used to increase the accessibility of an anticancer agent to tumor cells. This is because the intravenous formulation can transport the Hyal1 variant, which is an embodiment of the present invention, through the bloodstream, hydrolyze hyaluronic acid overexpressed on the surface of tumor cells, thereby increasing the accessibility of the anticancer agent to tumor cells. In this way, the Hyal1 variant, which is an embodiment of the present invention, may be used for cosmetic purposes in addition to therapeutic purposes. Therefore, the formulation of one embodiment of the present invention may be a pharmaceutical formulation and / or a cosmetic formulation.

[0067] The dosage of the Hyal1 variant, which is an 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 divided into several times, and this dosage may be based on an adult (body weight 60 kg), but it is understandable that it may vary depending on body weight, physical condition, etc. The Hyal1 variant, which is an embodiment of the present invention, is mainly administered parenterally, for example, by subcutaneous injection or intravenous injection, or by eye drops.

[0068] The Hyal1 variant, which is one embodiment of the present invention, can be formulated with pharmaceutically acceptable additives and manufactured in the form of an injection, eye drop, transdermal patch, etc.

[0069] Pharmaceutically acceptable excipients may be selected based on a number of factors well known to those skilled in the art, including, but not limited to, the specific bioactive agent employed, its concentration, stability, and intended bioavailability; the disease and disorder or condition to be treated; the individual, age, size, and general condition to be treated; and factors such as nasal, oral, ocular, topical, transdermal, and intramuscular routes of administration. Pharmaceutically acceptable excipients commonly used for administration of bioactive agents other than oral include D5W (5% glucose in water), dextrose, and aqueous solutions containing physiological salts up to 5% by volume. For topical injection, various injectable hydrogels can be used to enhance the effect and prolong the duration of action. Pharmaceutically acceptable excipients may also include additional ingredients that enhance the stability of the active ingredients, such as preservatives and antioxidants. The variant, which is an embodiment of the present invention, can be formulated using a method appropriate to the field, and can be formulated preferably according to each disease or condition or according to the ingredient.

[0070] The Hyal1 variant, which is an embodiment of the present invention, can be stored in a physiological saline solution, and after adding mannitol or sorbitol, can be freeze-dried in an ampoule, and when used for administration, can be dissolved in physiological saline or the like. At this time, the physiological saline solution may include a buffer, stabilizer, and / or surfactant.

[0071] In addition, the hyaluronic acid degradation and / or drug delivery method, which is an embodiment of the present invention, includes a step of administering a Hyal1 variant, which is an embodiment of the present invention, to a mammal, including a human, in need of administration. In this case, the Hyal1 variant, which is an embodiment of the present invention, administered may be an effective amount of the variant.

[0072] In addition, one embodiment of the present invention is a use for manufacturing a hyaluronic acid decomposition agent or drug delivery agent of the Hyal1 variant of one embodiment of the present invention.

[0073] The Hyal1 variant, which is an embodiment of the present invention, can be produced by genetic engineering technology. For example, a fusion gene encoding a fusion protein consisting of a fusion partner and a Hyal1 variant protein, which is an embodiment of the present invention, can be produced through genetic engineering, and then a host cell can be transformed with the fusion gene. The fusion protein can then be expressed in the host cell. Then, the Hyal1 variant, which is an embodiment of the present invention, can be cleaved and separated from the fusion protein using a protease or a compound, thereby producing the desired protein. 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, a cell lysate, or in a partially purified or substantially pure form. The nucleic acid can be, for example, DNA or RNA.

[0074] In addition, an expression vector according to an embodiment of the present invention comprises a nucleic acid according to an embodiment of the present invention. In order to express the Hyal1 variant according to an embodiment of the present invention, DNA encoding the Hyal1 variant according to an embodiment of the present invention can be obtained by molecular biology techniques (e.g., PCR amplification, cDNA cloning using a hybridoma expressing the Hyal1 variant according to an embodiment of the present invention), and such DNA can be operably linked to transcription and translation control sequences and inserted into an expression vector, thereby producing an expression vector according to an embodiment of the present invention. In this case, "operably linked" may mean that a gene encoding the Hyal1 variant according to an embodiment of the present invention is ligated into the vector so that the transcription and translation control sequences within the vector perform the intended function of regulating transcription and translation of the gene encoding the Hyal1 variant according to an embodiment of the present invention. The expression vector and expression control sequences are selected to be compatible with the host cell for expression used. A gene encoding a Hyal1 variant, which is an embodiment of the present invention, is inserted into an expression vector by a standard method (e.g., ligation of complementary restriction enzyme sites on a gene fragment encoding a Hyal1 variant, which is an embodiment of the present invention, and a vector, or blunt-end ligation if no restriction enzyme site exists at all). The recombinant expression vector has a regulatory sequence that controls the expression of the gene encoding a Hyal1 variant, which is an embodiment of the present invention, in a host cell. The "regulatory sequence" may include a promoter, an enhancer, and other expression control elements (e.g., a polyadenylation signal) that control the transcription or translation of the gene encoding a 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 vary by selecting a different regulatory sequence depending on factors such as the selection of the host cell to be transformed, the expression level of the protein, etc.

[0075] Additionally, a host cell, which is an embodiment of the present invention, may include a nucleic acid, which is an embodiment of the present invention, or an expression vector, which is an embodiment of the present invention. The host cell is not limited thereto, but may be selected from the group consisting of, for example, animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0076] The nucleic acid or expression vector is transfected or transformed into a host cell. A variety of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for "transfection" or "transfection" can be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. Various expression host / vector combinations can be used to express the Hyal1 variant of the present invention. Suitable expression vectors for eukaryotic hosts include, but are not limited to, expression control 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 with a wider host range, such as RP4; phage DNA, exemplified by the numerous 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 the 2μm plasmids and their derivatives. A useful vector for insect cells is pVL941.

[0077] In addition, the method for producing a Hyal1 variant, which is an embodiment of the present invention, may include a 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, may be produced by culturing the host cell for a period of time sufficient to be expressed in the host cell, more preferably, for a period of time sufficient to cause 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.

[0078] If necessary, the expressed Hyal1 variant, which is an embodiment of the present invention, can be isolated from the host cell and purified to a uniform concentration. The isolation or purification of the Hyal1 variant, which is an embodiment of the present invention, can be performed using a separation or purification method commonly used for proteins, such as chromatography. Chromatography can be, for example, a combination of one or more selected from affinity chromatography, ion exchange chromatography, or hydrophobic chromatography, but is not limited thereto. In addition to chromatography, filtration, ultrafiltration, salting out, dialysis, and the like can also be used in combination.

[0079] Unless otherwise stated, matters mentioned in the Hyal1 variant, hyaluronic acid decomposer, drug delivery agent, composition, use, formulation, and method of the present invention are equally applicable to each other to the extent of identity unless they are contradictory.

[0080] Hereinafter, the present invention will be described in more detail by examples, comparative examples, and manufacturing examples. However, the following examples and manufacturing examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples or manufacturing examples.

[0081] The reagents used in the examples below are commercially available and of the highest quality, and unless otherwise stated, those purchased from Sigma-Aldrich were used.

[0082] <Example 1> Production of Hyal1 mutant

[0083] The human Hyal1 gene (Clone ID: hMU005315) was purchased from the Korean Human Gene Bank. The Hyal1 gene was amplified by PCR (Bioneer, AllInOneCycler PCR system) using the pCMV-SPORT6-Hyal1 plasmid. The amplified gene was inserted into a vector (named pSGHV1 vector) in which the human growth hormone (hGH) and TEV protease cleavage sites were removed from the pSGHV0 vector, using restriction enzymes Xho I and Not I. At this time, a DNA sequence of six His residues was positioned at the 3'-terminus of the Hyal1 cDNA for protein purification using a Ni2+-column. Hyal1 mutants were also constructed using a PCR reactor, and the DNA sequences of the constructed Hyal1 mutants were confirmed by DNA sequencing. The primers used for PCR are listed in Table 6.

[0084]

[0085] <Example 2> Confirmation of enzyme activity and expression of Hyal1 Ala132 mutant

[0086] CHO-K1 cells (purchased from the Korea Cell Line Bank) were transfected with the plasmids containing the Hyal1 wild-type or mutant genes prepared in Example 1 using polyethylenimine (PEI, Sigma-Aldrich) or Lipofectamine 3000 (Thermo Fisher) reagents. When 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 and 150 μL of DMEM medium containing 6 μg of PEI were mixed. After 30 minutes, the PEI-DNA mixture was transferred to a 6-well plate and cultured in a 5% CO2 incubator.

[0087] After 48 hours of transformation, the cell culture medium was harvested and centrifuged at 10,000 Х g to obtain the supernatant. The enzyme activities of the Hyal1 wild type and mutants were measured using 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) at 4 °C for 2 hours. 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 a phosphate buffer with a pH of 4 to 7, and the enzyme reaction was performed for 4 to 17 hours while shaking at 50 rpm at 37 °C. After the enzymatic reaction, hyaluronic acid in the SDS gel was stained with 1.0% Alcian blue dye. Since the purpose of the present invention was to investigate the enzymatic activity of Hyal1 mutants at neutral pH, pH 4 was used as a control, and the enzymatic activity at pH 6 and pH 7 was investigated.

[0088] Figure 1(A) shows the results of an experiment confirming whether Hyal1 variants exhibit enzymatic activity depending on pH. Hyaluronic acid hydrolyzed by Hyal1 variants using the substrate-gel assay method is not stained by Alcian blue and therefore 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 variants.

[0089] As shown in Fig. 1(A), at pH 4, all mutants except A132Y hydrolyzed hyaluronic acid and exhibited enzymatic activity, but at pH 6, A132E, A132D, and A132H mutants exhibited enzymatic activity, and at pH 7, only A132E and A132D exhibited enzymatic activity. Among these Hyal1 mutants, A132E exhibited the highest enzymatic activity at pH 7. A132N and A132S, which were substituted with polar amino acids, exhibited higher enzymatic activity than the wild type only at pH 4. The basic amino acid His exhibited enzymatic activity at pH 6 but not at pH 7.

[0090] In addition, to investigate 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 the Hyal1 wild-type and Ala132 mutant was examined using a Hyal1 monoclonal antibody (1D10, Santa Cruze Biotechnology, Dallas, TX, US) and an anti-mouse IgG binding protein (m-IgGk BP-HRP, Santa Cruze Biotechnology). The results are shown in Fig. 1(B). Fig. 1(B) shows the results of an experiment to confirm the expression of the Hyal1 mutant. 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 in Fig. 1(B), the 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 is inferred that the negative result of the A132Y mutant shown in Fig. 1(A) is due to non-expression of the protein. Fig. 1(C) shows the experimental results comparing the enzyme activities of the Hyal1 wild-type and A132G mutant. The A132G mutant showed a decrease in enzyme activity compared to the Hyal1 wild-type.

[0091] The Hyal1 variant of the present invention hydrolyzes hyaluronic acid by general acid-base catalysis. The carboxyl group (-COOH) of the catalytic amino acid Glu131 serves as a proton donor to the hydroxyl leaving group, so protonation of Glu131 is important for the catalytic reaction. The results of Fig. 1(A) indicate that substituting an adjacent amino acid in the primary or tertiary structure with an acidic amino acid increases the pKa value of Glu131 due to repulsion between negative charges, enabling Glu131 to play a proton donor role at acidic to neutral pH. However, even with the same acidic amino acid, A132E exhibited higher enzymatic activity than A132D at pH 7. Therefore, it can be seen that, particularly with the Hyal1 variant A132E, which is an example of the present invention, hyaluronic acid can be effectively decomposed even at neutral pH.

[0092] Also, as shown in Fig. 1(A), the Ala132 mutants exhibited higher enzyme activity than the Hyal1 wild type at pH 4. These Ala132 mutants have in common that the Ala132 position is substituted with an amino acid larger than Ala. When the Ala132 position was substituted with Gly, which is smaller than Ala, the enzyme activity of the A132G mutant decreased, as shown in Fig. 1(C). Therefore, it can be seen that the Ala132 mutants, excluding the Hyal1 A132G mutant, which is an example of the present invention, can effectively decompose hyaluronic acid more effectively than the Hyal1 wild type even at acidic pH.

[0093] <Example 3> Confirmation of enzyme activity and expression of Hyal1 loop mutants

[0094] To investigate the enzyme activity of the constructed Hyal1 loop mutants (S76D, S76E, S77D, S77E, Y85D, T86D, T86E, P87D, P87E, T88D, and G89D) in the pH range of 4 to 7, CHO-K1 cells were transfected with plasmids containing the genes of Hyal1 loop mutants using PEI or Lipofectamaine as described in Example 1. After 48 hours of transfection, the cell culture medium was harvested and centrifuged at 10,000 Х g to obtain the supernatant. The enzyme activity of Hyal1 WT and loop mutants contained in the supernatant was measured using a substrate-gel assay method. The enzyme reaction was performed for 4 to 17 hours using phosphate buffer at 37°C and pH range of 4 to 7, and the amount of hydrolyzed hyaluronic acid was examined using 1.0% Alcian blue dye. The results are shown in Fig. 2(A). Fig. 2(A) shows the results of an experiment to confirm whether the Hyal1 wild type and loop mutants exhibit enzymatic activity depending on pH. In Fig. 2(A), Control represents the control group transfected with only the vector, WT represents wild-type Hyal1, and S76D, S76E, and A132E represent the respective mutants. In Fig. 2(B), WT represents wild-type Hyal1, and S77D and S77E represent the respective mutants. In Fig. 2(C), Y85D, T86D, T86E, P87D, P87E, T88D, G89D, and A132E represent the respective mutants. In Figs. 2(A) and 2(C), A132E was used as a positive control.

[0095] In one example of the Hyal1 mutants of the present invention, S76D, S77D, S77E, P87D, and P87E showed high enzyme activity at pH 7. T86D showed low enzyme activity at pH 7.

[0096] <Example 4> Confirmation of enzyme activity and expression of mixed mutants with Hyal1 Ala132 or loop amino acids substituted.

[0097] Among the Ala132 mutants, the most active A132E mutant and one or more loop mutants were mutated together, and the enzyme activity in the pH range of 4 to 7 was investigated. To investigate the enzyme activity of the constructed Hyal1 mutants (S77D / A132E, S77E / A132E, S77D / T86D, S77D / T86E, and S77D / T86D / A132E) in the pH range of 4 to 7, CHO-K1 cells were transfected with plasmids containing the genes of these Hyal1 mutants using the PEI or Lipofectamaine method as described in Example 1. After 48 hours of transfection, the cell culture medium was harvested and centrifuged at 10,000 Х g to obtain the supernatant. The enzyme activity of the Hyal1 mutant contained in the supernatant was measured using a substrate-gel assay method. The enzyme reaction was performed for 4 to 17 hours using a phosphate buffer at 37°C and pH 4 to 7, and the amount of hydrolyzed hyaluronic acid was examined using 1.0% Alcian blue dye.

[0098] As shown in Fig. 3(A), the double and triple mutants exhibited enzymatic activity by hydrolyzing hyaluronic acid not only at pH 4 but also at pH 7. In particular, a difference in the degree of activity was observed at pH 7, with the S77D / A132E mutant showing the best activity.

[0099] Figure 3(B) shows the results of an experiment to confirm the expression of the Hyal1 mutant. As shown in Figure 3(B), the Hyal1 mutant was expressed normally in CHO-K1 cells.

[0100] <Example 5> Comparison of the protein tertiary structures of human Hyal1 and PH20

[0101] In order to compare the positions of the mutant amino acids of Hyal1 introduced in the present invention with those of PH20, the 3D structures of human Hyal1 and PH20 were compared. The 3D structure model of PH20 was created using Swiss-Model based on the crystal structure of Hyal1 (PDB code 2PE4). The structures of Hyal1 and PH20 were compared using the PyMol program, and the results are shown in Fig. 4. As shown in Fig. 4, in Hyal1, Ser76, Ser77, Thr86, Pro87, Thr88, and Ala132 are located adjacent to the catalytic amino acid Glu131. In the 3D structure model of PH20, Asp94, Asp103, and Glu149 are also located adjacent to the catalytic amino acid Glu148.

[0102] Therefore, in the present invention, the Hyal1 variant substituted with an acidic amino acid exhibits the effect of effectively decomposing hyaluronic acid even at neutral pH by increasing the pKa value of Glu131 due to the negative charge repulsion between these acidic amino acids and the catalytic amino acid Glu131.

[0103] In addition, the Hyal1 variant of the present invention appears to increase the enzymatic activity of the Hyal1 variant at acidic pH by increasing structural flexibility by destabilizing the loop where the catalytic amino acid Glu131 is located when an amino acid larger than Ala is introduced into the Ala132 position.

[0104] <Example 6> Confirmation of enzyme activity and expression of F139R and Y210H mutants

[0105] Another way to increase the pKa value of the catalytic amino acid Glu131 of Hyal1 is to replace the amino acids adjacent to Glu131 with basic amino acids, 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 acids. Replacing the hydrophobic amino acid Phe139 with Arg decreases the pKa value of Tyr210 due to the ionic bond between Arg139 and Tyr210, which can increase the pKa value of the catalytic amino acid Glu131. In addition, when the hydrophobic amino acids Phe139 and Ile225 are substituted with Arg and Asp, respectively, the positive charge of Arg139 is strengthened by the ionic bond between Arg139 and Asp225, and thus the hydroxyl group dehydrogenation of Tyr210 can be promoted compared to the F139R single mutant in the ionic bond between Arg139 and Tyr210. In addition, when the polar amino acid Tyr210 is substituted with the basic amino acid His, the pKa value of Asp206 decreases 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.

[0106] To investigate the enzyme activity of the constructed Hyal1 mutants (F139R, F139R / I225D, and Y210H) in the pH range of 4 to 7, CHO-K1 cells were transfected with plasmids containing the genes of these Hyal1 mutants using PEI or Lipofectamaine as described in Example 1. After 48 hours of transfection, the cell culture medium was harvested and centrifuged at 10,000 Х g to obtain the supernatant. The enzyme activity of Hyal1 WT and mutants contained in the supernatant was measured using a substrate-gel assay method. The enzyme reaction was performed for 4 to 17 hours using phosphate buffer at 37°C and pH range of 4 to 7, and the amount of hydrolyzed hyaluronic acid was examined using 1.0% Alcian blue dye.

[0107] In Fig. 5(A), Control represents the control group transfected with only the vector, WT represents wild-type Hyal1, and F139R, Y210H, and F139R / I225D represent each Hyal1 mutant. WT exhibited enzymatic activity only at pH 4, whereas F139R, Y210H, and F139R / I225D mutants exhibited enzymatic activity at both pH 4 and pH 7.

[0108] Figure 5(B) shows the results of an experiment to confirm the expression of the Hyal1 mutant. As shown in Figure 5(B), the Hyal1 mutant was expressed normally in CHO-K1 cells.

[0109] Figure 5(C) is the tertiary structure of Hyal1 with the above amino acids indicated. Asp206 and Tyr210 are located adjacent to the catalytic amino acid Glu 131.

[0110] From the above results, it can be seen that the Hyal1 variant of the present invention can effectively decompose hyaluronic acid not only at acidic pH but also at neutral pH, thus demonstrating high utility. In other words, since the Hyal1 variant of the present invention can decompose hyaluronic acid even at neutral pH, it can be used for drug delivery and can be utilized in various formulations and applications, such as subcutaneous administration formulations, intravenous injection formulations, and ophthalmic formulations.

[0111] In addition, from the above results, it can be seen that a Hyal1 mutant can be effectively produced using the nucleic acid, expression vector, host cell, and method of the present invention.

[0112] <Manufacturing Example 1> Liquid preparation

[0113] Dissolve 1 μg of the Hyal1 variant protein prepared in Examples 2 to 6 in PBS to make a 1 mL solution. The resulting solution is filled into an injection ampoule or sterile container to prepare an injectable solution or eye drop. The preparation thus prepared can be utilized as a hyaluronic acid decomposer, drug delivery agent, etc. In particular, the injection solution can be utilized as a subcutaneous or intravenous preparation, and the eye drop can be utilized as an ophthalmic preparation.

[0114] Electronic file attached.

Claims

1. Based on human hyaluronidase Hyal1 containing glutamic acid 131 as a catalytic amino acid in sequence number 1, characterized in that at least one of the amino acids adjacent to the catalytic amino acid glutamic acid 131 in the primary structure or tertiary structure is substituted with an amino acid that increases the pKa value of glutamic acid 131. A hyaluronidase Hyal1 mutant exhibiting enzymatic activity at neutral pH.

2. In paragraph 1, The substitution of the above amino acid comprises one or more substitutions selected from S76D and P87D. A hyaluronidase Hyal1 mutant exhibiting enzymatic activity at neutral pH.

3. Based on human hyaluronidase Hyal1 containing glutamic acid 131 as a catalytic amino acid in sequence number 1, Characterized in that it substitutes at least one amino acid among the amino acids adjacent to the above glutamic acid 131 in the primary structure or tertiary structure. Hyaluronidase Hyal1 mutant with increased enzyme activity at acidic pH.

4. In paragraph 3, The above amino acid substitution is characterized by substituting at least one amino acid among the amino acids adjacent to glutamic acid 131 in the primary or tertiary structure with an acidic amino acid. Hyaluronidase Hyal1 mutant with increased enzyme activity at acidic pH.

5. In paragraph 4, The substitution of the above amino acid comprises substituting one or more amino acids among the amino acids located at positions 76, 77, 86, 87, 88, and 89 of the above sequence number 1 with an acidic amino acid. Hyaluronidase Hyal1 mutant with increased enzyme activity at acidic pH.

6. In paragraph 5, The substitution of the above amino acid comprises one or more substitutions selected from S76D, S77D, S77E, T86D, T86E, P87D, P87E, T88D, G89D. Hyaluronidase Hyal1 mutant with increased enzyme activity at acidic pH.

7. In paragraph 3, Characterized in that the 132nd amino acid adjacent to the above glutamic acid 131 is replaced with an amino acid larger than alanine. Hyaluronidase Hyal1 mutant with increased enzyme activity at acidic pH.

8. In paragraph 7, The substitution of the above amino acid comprises one or more substitutions selected from A132E, A132N, A132D, A132S, A132H. Hyaluronidase Hyal1 mutant with increased enzyme activity at acidic pH.

9. A nucleic acid encoding a hyaluronidase Hyal1 variant of paragraph 1.

10. A recombinant expression vector comprising the nucleic acid of clause 9.

11. A host cell transformed with the expression vector of clause 10.

12. A method for producing a hyaluronidase Hyal1 variant, comprising the step of culturing the host cell of clause 10.

13. A hyaluronic acid decomposing agent comprising a hyaluronidase Hyal1 variant of clause 1, clause 5, or clause 7.

14. A drug delivery agent comprising a hyaluronidase Hyal1 variant of claim 1, claim 5, or claim 7.

15. A subcutaneous formulation comprising the hyaluronidase Hyal1 variant of clause 1.

16. An intravenous formulation comprising the hyaluronidase Hyal1 variant of clause 1.

17. An ophthalmic preparation comprising a hyaluronidase Hyal1 variant of clause 1.

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