Hyaluronidase Hyal1 variants exhibiting activity at neutral ph

Abstract

According to the present invention, the wild type human hyaluronidase Hyal1 containing asparaginic acid and glutamic acid as catalytic amino acids is adopted as the reference; the invention provides a Hyal1 variant in which more than one amino acid adjacent to a catalytic amino acid is replaced by an acidic or polar amino acid, a preparation method of the Hyal1 variant, nucleic acid, an expression vector and a host cell which can be applicable to preparation of the Hyal1 variant, and a preparation and even application of the Hyal1 variant. Moreover, the invention provides a Hyal1 variant for catalyzing substitution of more than one amino acid in amino acid and amino acid adjacent to a tertiary structure into basic amino acid, and a preparation method of the Hyal1 variant, which can be suitable for nucleic acid prepared from the Hyal1 variant, an expression vector and a host cell, and a preparation and even application of the Hyal1 variant. The hyaluronidase Hyal1 variant disclosed by the invention has the technical effects that hyaluronic acid can be effectively decomposed in neutral pH, and the applicability is high.

Description

Technical Field

[0001] The present invention is a technology for changing the action pH of human hyaluronidase, and specifically relates to a variant of human hyaluronidase Hyal1 that plays a catalytic role in the acidic to neutral pH range. Background Art

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

[0003] Hyaluronidase PH20 has the property of hydrolyzing hyaluronic acid of the extracellular matrix, and therefore, it is used clinically as a subcutaneous injection formulation of an antibody therapeutic agent, and can be used to increase subcutaneous drug delivery. However, the hydrolysis of hyaluronic acid currently depends on an enzyme, i.e., PH20. That is, among human hyaluronidases other than PH20, Hyal1 mainly plays a catalytic role under acidic conditions of pH 3 to 4, and therefore, is limited in application. For example, when Hyal1 is applied to the subcutaneous part showing neutrality of pH 7.0 to 7.5, it is difficult to expect the decomposition effect of hyaluronic acid. In addition, wild-type Hyal2, Hyal3, Hyal4, etc. also show weak neutrality in the neutral region, and therefore, are not actually adopted.

[0004] In view of this, the present inventors conducted studies to improve the applicability of the above-mentioned wild-type human Hyal1, and thus completed the present invention. Summary of the invention

[0005] Issues to be solved

[0006] The technical 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.

[0007] Problem Solution

[0008] The human hyaluronidase Hyal1 variant of the present invention has Asp129 and Glu131 as catalytic amino acids. In the primary structure or tertiary structure of Hyal1, the present invention replaces the amino acid adjacent to the catalytic amino acid with an acidic or polar amino acid so that Hyal1 can also play a catalytic role at neutral pH.

[0009] Furthermore, in the tertiary structure of Hyal1, any one of the amino acids adjacent to the catalytic amino acid is substituted with a basic amino acid, and the substituted basic amino acid can form an ionic bond with the acidic or polar amino acid adjacent to the catalytic amino acid.

[0010] The catalytic amino acid and the amino acid adjacent to the primary structure or tertiary structure of Hyal1 are amino acids located in a loop consisting of Ala132 or Ser76 to Leu98 amino acids, or amino acids adjacent to acidic or polar amino acids that may affect the activity of the catalytic amino acid in the tertiary structure of Hyal1.

[0011] Ala132 can be substituted with an acidic or polar amino acid. In this case, the acidic amino acid is Asp or Glu, and the polar amino acid is Ser, Thr, Asn, Gln or Tyr. In the ring composed of Ser76 to Leu98 amino acids, the catalytic amino acid and the adjacent tertiary structure amino acid are Ser, Thr, or Pro, and these amino acids are substituted with Asp or Glu.

[0012] In addition, in the tertiary structure of Hyal1, another adjacent amino acid that may affect the activity of the catalytic amino acid is the acidic amino acid Asp206 and the polar amino acid Tyr210. The amino acid adjacent to Asp206 or Tyr210 is substituted with the basic amino acid Arg, Lys or His.

[0013] 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 also hydrolyze hyaluronic acid at neutral pH.

[0014] Furthermore, the present invention provides a nucleic acid encoding the hyaluronidase Hyal1 variant.

[0015] Furthermore, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0016] Furthermore, the present invention provides a host cell transformed with the expression vector.

[0017] Furthermore, the present invention provides a method for preparing a hyaluronidase Hyal1 variant, comprising a host cell culture step.

[0018] Furthermore, the present invention provides a hyaluronan decomposing agent comprising the hyaluronidase Hyal1 variant.

[0019] Furthermore, the present invention provides a drug delivery agent comprising the hyaluronidase Hyal1 variant.

[0020] Furthermore, the present invention provides a preparation for intravenous injection comprising the hyaluronidase Hyal1 variant.

[0021] Furthermore, the present invention provides a preparation for subcutaneous administration comprising the hyaluronidase Hyal1 variant.

[0022] Furthermore, the present invention provides an ophthalmic preparation comprising the hyaluronidase Hyal1 variant.

[0023] Effects of the Invention

[0024] The hyaluronidase Hyal1 variant of the present invention can not only be at acidic pH, but also at neutral pH, effectively hydrolyzing hyaluronic acid, with a technical effect with high applicability. That is, using the hyaluronidase of the present invention, hyaluronic acid can also be decomposed at neutral pH, and it is used to realize drug delivery, and can also be used as a variety of preparations and even purposes, such as, subcutaneous preparations, intravenous preparations, ophthalmic preparations. In addition, the hyaluronidase of the present invention can be effectively prepared by nucleic acid, expression vector, host cell and method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 (A) shows the results of an experiment to investigate whether the Hyal1 wild type and Ala132 variant exhibit enzyme activity depending on pH.

[0026] Figure 1 (B) shows the results of an experiment to confirm the expression of Hyal1 wild-type and Ala132 variant in CHO-K1 cells.

[0027] Figure 2 (A) shows the results of an experiment to confirm whether the Hyal1 wild type and Ser77, Thr86 or Pro87 variants exhibit enzyme activity depending on pH.

[0028] Figure 2 (B) shows the results of experiments confirming the expression of Hyal1 wild-type and Ser77, Thr86 or Pro87 variants in CHO-K1 cells.

[0029] Figure 3 (A) shows the results of an experiment to confirm whether a variant in which two or more substitutions are simultaneously made at Ala132, Ser77 or Thr86 exhibits enzyme activity, depending on pH.

[0030] Figure 3 (B) shows the results of an experiment to confirm the expression of variants in which two or more substitutions were simultaneously achieved at Ala132, Ser77, or Thr86 in CHO-K1 cells.

[0031] Figure 4 The enzyme active site structures of human Hyal1 and human PH20 were compared.

[0032] Figure 5(A) shows the results of an experiment to confirm whether the Hyal1 wild type and Phe139, Tyr210, or Phe139 / Ile225 variants exhibit enzyme activity depending on pH.

[0033] Figure 5 (B) shows the experimental results of the expression of Hyal1 wild type and Phe139, Tyr210 or Phe139 / Ile225 variants in CHO-K1 cells.

[0034] Figure 5 (C) shows the positions of Phe139, Asp206, Tyr210, and Ile225 adjacent to the catalytic amino acid in the tertiary structure of Hyal1. DETAILED DESCRIPTION

[0035] The advantages and features of the present invention and methods for achieving the same can be clearly seen with reference to the accompanying drawings and the embodiments described in detail below. However, the present invention is not limited to the embodiments described below, but can be implemented in a variety of different forms. However, this embodiment can make the disclosure of the present invention complete. This embodiment is only used to enable a person skilled in the art to which the present invention belongs to fully understand the scope of the present invention. The present invention is only defined by the scope of the claims.

[0036] Throughout the content of this specification, "and / or" includes all combinations of each or more than one of the components involved. The terms used in this specification are used to describe the embodiments and are not used to limit the present invention. In this specification, unless otherwise defined in the context, the singular includes the plural. The components involved in the specification using "comprises" and / or "comprising" do not exclude the existence or addition of one or more other components.

[0037] According to one embodiment of the present invention, the hyaluronidase Hyal1 variant (hereinafter, 'Hyal1 variant') as the catalytic amino acid refers to the wild-type human Hyal1 comprising aspartic acid (e.g., Asp129) and glutamic acid (e.g., Glu131), wherein the catalytic amino acid and one or more of the amino acids in the adjacent primary structure or tertiary structure are substituted with acidic or polar amino acids.

[0038] According to yet another embodiment of the present invention, in the tertiary structure of wild-type Hyal1, any one of the amino acids adjacent to the catalytic amino acid is substituted with a basic amino acid, wherein the substituted basic amino acid can form an ion bond with the acidic or polar amino acid adjacent to the catalytic amino acid.

[0039] That is, according to one embodiment of the present invention, the hyaluronidase Hyal1 variant is equivalent to a variant of wild-type human Hyal1, and the catalytic amino acid and the amino acid adjacent to the primary structure or tertiary structure are replaced by specific acidic amino acids or polar amino acids, or the catalytic amino acid and the amino acid adjacent to the tertiary structure are replaced by basic amino acids, so that hyaluronic acid can be effectively hydrolyzed even at neutral pH, thereby improving applicability.

[0040] In this specification, expressions such as "Ala132" or "A132" that record three-letter or one-letter amino acid names and numbers at the same time mean the amino acids at each position based on the amino acid sequence of sequence number 1. For example, "Ala132" and "A132" represent the amino acid at the 132nd position based on the amino acid sequence of sequence number 1, that is, Ala. In addition, in this specification, a variant of the wild-type human hyaluronidase means a variant that also includes a substitution with a reserved amino acid at a specific amino acid position. In this case, "reserved substitution" refers to an abnormal modification that includes replacing one or more amino acids with amino acids having similar biological or biochemical properties that do not cause damage to the biological or biochemical function of the variant. Specifically, a "conservatively substituted variant" may be: a Hyal1 variant consisting of an amino acid sequence selected from the group consisting of amino acid sequences of sequence numbers 2 to 20; a variant having, for example, a sequence homology of more than 80%, preferably more than 90%, further preferably more than 95%, and further preferably more than 99%, and having substantially the same function and / or effect.

[0041] According to one embodiment of the present invention, Hyal1 variant is equivalent to a hyaluronidase that can decompose hyaluronic acid in a wide pH range of acidic to neutral. In the hyaluronidase described in one embodiment of the present invention, the wild-type human hyaluronidase is Hyal1, and Hyal1 is composed of the amino acid sequence of sequence number 1, and may include substitutions of one or more amino acids selected from the group consisting of S77D, S77E, T86D, T86E, P87E, A132D, A132E, F139R, Y210H and F139R / I225D. That is, the variant described in one embodiment of the present invention is based on the wild-type Hyal1 composed of the amino acid sequence of sequence number 1, and may include substitutions of one or more amino acids selected from the group consisting of S77D, S77E, T86D, T86E, P87E, A132D, A132, F139R, Y210H and F139R / I225D.

[0042] Here, S77D means that the 77th serine in sequence number 1 is substituted with aspartic acid.

[0043] Hyal1 variants are partial amino acid variations in the sequence of wild-type human Hyal1, specifically, amino acid substitutions, including substitutions of one or more amino acids selected from the group consisting of S77D, S77E, T86D, T86E, P87E, A132D, A132E, F139R, Y210H and F139R / I225D. Among them, the meaning of F139R / I225D substitution means that the amino acid substitutions of F139R and I225D are achieved simultaneously.

[0044] The amino acid sequence of wild-type human Hyal1 is shown in Table 1 below. As shown in the sequence in Table 1, Ala132 is located adjacent to Asp129 and Glu131, which are catalytic amino acids. Thus, Hyal1 variants in which the amino acids adjacent to the catalytic amino acids are substituted with specific amino acids appear to show characteristics different from those of the wild type while maintaining the hydrolase activity of hyaluronic acid. That is, as confirmed in the test examples, when Ala132 is substituted with Asp or Glu, hyaluronic acid can be hydrolyzed even at neutral pH.

[0045] That is, based on the amino acid sequence of wild-type human Hyal1, the 132nd amino acid, i.e., Ala, is substituted with Asp or Glu, so that hyaluronic acid can be hydrolyzed at neutral pH. However, Hyal1 variants in which Ala132 is substituted with other types of amino acids do not show this technical effect. It can be seen that, in particular, the Hyal1 variant described in one embodiment of the present invention can also effectively hydrolyze hyaluronic acid at neutral pH.

[0046] Furthermore, Ser77 is 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, based on the amino acid sequence of wild-type human Hyal1, the 77th amino acid, i.e., Ser, is substituted with Asp or Glu, so that hyaluronic acid can be hydrolyzed at neutral pH.

[0047] Furthermore, Thr86 and Pro87 are arranged adjacent to the catalytic amino acid in the tertiary structure of Hyal1, and when Thr86 is substituted with Asp or Glu, or when Pro87 is substituted with Glu, hyaluronic acid can be immediately hydrolyzed at neutral pH. That is, based on the amino acid sequence of wild-type human Hyal1, the 86th and 87th amino acids, that is, Thr and Pro, are substituted with Asp or Glu, so that hyaluronic acid can also be hydrolyzed at neutral pH.

[0048] In addition, Asp206 and Tyr21 are arranged adjacent to the catalytic amino acid in the tertiary structure of Hyal1. At this time, Phe139 adjacent to Tyr210 is substituted with a basic amino acid, thereby forming an ionic bond with Tyr210. When Tyr210 adjacent to Asp206 is substituted with a basic amino acid, it can form an ionic bond with Asp206.

[0049] Specifically, when Phe139 is substituted with Arg, the hydroxyl group of Tyr210 exists in a dehydrogenated state through the ionic bonding between Arg139 and Tyr210, which can increase the pKa value of the catalytic amino acid Glu131. In addition, when Phe139 and Ile225 are substituted with Arg and Asp respectively (F139R / I225D), due to the ionic bonding between Arg139 and Asp225, the positive charge of Arg139 is stabilized, and Arg139 can form an ionic bond with Tyr210 again, thereby dehydrogenating the hydroxyl group of Tyr210.

[0050] In addition, when Tyr210 is substituted with His, the pKa value of Asp206 decreases due to ionic bonding between Asp206 and His210, and as a result, the pKa value of the catalytic amino acid Glu131 can be increased.

[0051] The amino acid sequence of wild-type Hyal1 is recorded in Table 1, the Ala132 variant is recorded in Table 2, the amino acid sequence of the loop variant is recorded in Table 3, the variant amino acid sequences in which one or more of Ala132 or the loop variant is replaced in the amino acid sequence are recorded in Table 4, and the amino acid sequences of the Phe139 and Tyr210 variants are recorded in Table 5, respectively.

[0052]

Table 1

[0053]

[0054]

Table 2

[0055]

[0056]

[0057]

[0058]

Table 3

[0059]

[0060]

[0061]

[0062]

Table 4

[0063]

[0064]

[0065]

[0066]

Table 5

[0067]

[0068]

[0069] The Hyal1 variant can decompose hyaluronic acid in a wide pH range of acidic to neutral. In addition, in one embodiment of the present invention, the Hyal1 variant can decompose hyaluronic acid in an acidic to neutral pH range, so the drug can be effectively delivered to a variety of parts. This is because when the hyaluronic acid of the extracellular matrix is ​​hydrolyzed, the viscosity of the hyaluronic acid will decrease, and the permeability to the tissue (skin) will increase. In particular, the pH of the subcutaneous part of the skin is neutral, about 7.0 to 7.5, so one embodiment of the present invention can decompose hyaluronic acid at a neutral pH, which can increase permeability. Thus, the drug delivery agent according to one embodiment of the present invention includes a variant of one embodiment of the present invention, and the drug can be effectively delivered to a variety of parts.

[0070] In this case, the drug is a component that exhibits pharmacological activity, but is not limited thereto, and may be, for example, an eye relaxant, an anesthetic, an antibody therapeutic agent, an anticancer agent, etc. The drug may be combined with the variant in one embodiment of the present invention to form a composition. That is, the variant described in one embodiment of the present invention may be in the form of a pharmaceutical composition together with itself or the drug.

[0071] The Hyal1 variant described in one embodiment of the present invention can be included in a preparation that can be applied to multiple parts. For example, the preparation for subcutaneous administration, the preparation for intravenous injection, or the ophthalmic preparation described in one embodiment of the present invention can contain the Hyal1 variant described in one embodiment of the present invention. At this time, the ophthalmic preparation can be eye drops, and the eye drops can be used to promote the diffusion of anesthetics during ophthalmic surgery. In addition, the preparation for subcutaneous administration can specifically be an injection for subcutaneous administration. The injection for subcutaneous administration can be used to fill the hydrolysis of hyaluronic acid during surgery. In addition, the preparation for intravenous injection is used to improve the tumor cell accessibility of anticancer agents. This is because the intravenous injection preparation moves the Hyal1 variant described in one embodiment of the present invention with the blood, and hydrolyzes the hyaluronic acid overexpressed on the surface of tumor cells to improve the tumor cell accessibility of anticancer agents. In this way, the Hyal1 variant described in one embodiment of the present invention can be used for beauty in addition to treatment. Therefore, the preparation described in one embodiment of the present invention can be a pharmaceutical preparation and / or a cosmetic preparation.

[0072] The dosage of the Hyal1 variant described in one embodiment of the present invention is 10 ng / ml to 10 mg / ml per subcutaneous injection, preferably 10 ng / ml to 100 μg / ml. The dosage can be once or several times a day. Of course, the dosage can be based on an adult (weight 60 kg), but it will change according to body weight, physical condition, etc. The dosage of the Hyal1 variant described in one embodiment of the present invention is mainly a non-oral method, for example, subcutaneous injection or intravenous injection or eye drop administration.

[0073] The Hyal1 variant described in one embodiment of the present invention can be formulated together with pharmaceutically acceptable additives to form injections, eye drops, transdermal patches, and the like.

[0074] Pharmaceutically acceptable additives can be applied based on a variety of factors known to those skilled in the art, and factors need to be considered, such as the specific bioactive substance used and its concentration, stability and expected bioavailability; the disease and condition or state to be treated; the individual, age, size and general state to be treated, such as nasal, oral, ocular, topical, transdermal and intramuscular, etc., but not limited thereto. Pharmaceutically acceptable additives commonly used for administration of bioactive substances other than oral routes include aqueous solutions, etc., in which the content of D5W (5% glucose in water), glucose and physiological salt is within 5% of the volume. When locally injected, in order to enhance the effect and increase the duration, a variety of injectable hydrogels can be used. In addition, pharmaceutically acceptable additives can include additional ingredients that can enhance the stability of the active ingredient, such as preservatives and antioxidants. The variants described in one embodiment of the present invention can be formulated using appropriate methods in the field, preferably, formulated according to each disease or state or component.

[0075] The Hyal1 variant described in one embodiment of the present invention can be stored as a physiological saline solution, and after adding mannitol or sorbitol, it can be put into an ampoule for freeze-drying, and when used for administration, it can be dissolved in physiological saline, etc. In this case, the physiological saline solution can contain a buffer, a stabilizer and / or a surfactant.

[0076] Furthermore, the hyaluronic acid decomposition and / or drug delivery method described in one embodiment of the present invention includes the step of administering to mammals including humans, and humans at this time need to be administered with the Hyal1 variant described in one embodiment of the present invention. The Hyal1 variant described in one embodiment of the present invention administered at this time can be an effective amount of the variant.

[0077] Furthermore, the use of one embodiment of the present invention is to prepare a hyaluronic acid decomposition preparation or a drug delivery preparation of the Hyal1 variant described in one embodiment of the present invention.

[0078] The Hyal1 variant described in one embodiment of the present invention can be produced by genetic engineering technology. For example, a fusion gene for encoding a fusion protein can be prepared by genetic engineering, and the fusion protein is composed of a fusion partner and the Hyal1 variant protein described in one embodiment of the present invention, which is sequentially transformed into a host cell, and then expressed in the host cell as a fusion protein form, and then, using a protease or a compound, the Hyal1 variant described in one embodiment of the present invention is cut and divided from the fusion protein to prepare the desired protein form. Therefore, the Hyal1 variant described in one embodiment of the present invention can be effectively prepared by the nucleic acid, expression vector, host cell and / or method of one embodiment of the present invention. Specifically, the nucleic acid described in one embodiment of the present invention encodes the variant described in one embodiment of the present invention. The nucleic acid can be present in a cell, a cell lysate, or in a partially purified form or a substantially pure form. For example, the nucleic acid can be DNA or RNA.

[0079] Furthermore, the expression vector described in one embodiment of the present invention comprises the nucleic acid described in one embodiment of the present invention. In order to express the Hyal1 variant described in one embodiment of the present invention, a DNA encoding the Hyal1 variant described in one embodiment of the present invention can be obtained by molecular biological techniques (e.g., PCR amplification, cDNA cloning using a hybridoma expressing the Hyal1 variant described in one embodiment of the present invention), and the DNA is initiated to be bonded to the transcription and translation control sequences, and embedded in the expression vector to prepare the expression vector described in one embodiment of the present invention. At this time, "initiate bonding" means that the gene encoding the Hyal1 variant described in one embodiment of the present invention is ligated into the vector so that the transcription and translation control sequences in the vector play the following expected functions, that is, the function of the gene encoding the Hyal1 variant described in one embodiment of the present invention to adjust the transcription and translation memory. An expression vector and expression control sequence that match the host cell to be used for expression are selected. The gene encoding the Hyal1 variant described in one embodiment of the present invention is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites on the gene fragment encoding the Hyal1 variant described in one embodiment of the present invention and the vector, or ligation of blunt ends when the restriction enzyme sites are completely absent). The recombinant expression vector has a regulatory sequence, which controls the expression of the gene encoding the Hyal1 variant described in one embodiment of the present invention in the host cell. "Regulatory sequence" may include: a promoter, which controls the transcription or translation of the gene encoding the Hyal1 variant described in one embodiment of the present invention; an enhancer; and other expression control factors (e.g., a polyadenylation signal). At this time, the regulatory sequences are selected according to factors such as the selection of the host cell to be transformed and the expression level of the protein, so it is obvious that the design of the expression vector will be different.

[0080] Furthermore, the host cell described in one embodiment of the present invention may contain the nucleic acid of one embodiment of the present invention or the expression vector of one embodiment of the present invention. The host cell is not limited thereto, for example, it may be selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli and insect cells.

[0081] Nucleic acid or expression vector is transformed and introduced or transfected into host cells. In order to achieve "transformation and introduction" or "transfection", exogenous nucleic acid (DNA or RNA) is introduced into prokaryotic or eukaryotic host cells, and various techniques are usually used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection, etc. In order to express the Hyal1 variant described in one embodiment of the present invention, a variety of expression host / vector combinations can be used. Expression vectors suitable for eukaryotic hosts are not limited thereto and include expression regulatory sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. Expression vectors that can be used for bacterial hosts include: bacterial plasmids 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 that can be represented as many derivatives of λ-shaped phages (phage lambda); such as λgt10, λgt11, NM989; and other different DNA phages, such as M13 and filamentous single-stranded DNA phages. Expression vectors useful for yeast cells are 2μm plasmids and their derivatives. Vectors useful for insect cells are pVL941.

[0082] Furthermore, the method for preparing the Hyal1 variant described in one embodiment of the present invention may include a step of culturing the host cell described in one embodiment of the present invention. When the recombinant expression vector capable of expressing the Hyal1 variant described in one embodiment of the present invention is embedded in a mammalian host cell, the variant described in one embodiment of the present invention is prepared by culturing the host cell for a period sufficient to achieve expression in the host cell, more preferably, for a period sufficient to secrete the Hyal1 variant described in one embodiment of the present invention into the culture medium of the host cell.

[0083] If necessary, the expressed Hyal1 variant described in one embodiment of the present invention can be separated from the host cell to achieve uniform purification. The separation or purification of the Hyal1 variant described in one embodiment of the present invention can be implemented by a separation and purification method generally used for proteins, for example, chromatography. For example, the chromatography can be a combination of one or more selected from environmental chromatography, ion exchange chromatography or hydrophobic chromatography, but is not limited thereto. In addition to chromatography, filtration, ultrafiltration, salting out, dialysis, etc. can be further combined for use.

[0084] Unless otherwise described, the contents of the Hyal1 variant, hyaluronan decomposing agent, drug delivery agent, composition, use, preparation, and method described in one embodiment of the present invention are within the same scope and are equally applicable to each other, except when they are contradictory.

[0085] Hereinafter, the present invention will be further described in detail based on examples, comparative examples and preparation examples. However, the following examples and preparation examples are only used to illustrate the present invention, and the content of the present invention is not limited to the following examples or preparation examples.

[0086] The reagents used in the following examples etc. can be purchased from the market. The best quality reagents used here are those purchased from Sigma-Aldrich Company unless otherwise specified.

[0087] <Example 1> Preparation of Hyal1 variants

[0088] The human Hyal1 gene (Clone ID: hMU005315) was purchased from the Korean Human Gene Bank. The human Hyal1 gene was amplified using the pCMV-SPORT6-human Hyal1 plasmid by PCR (AllInOneCycler PCR system, Baiye). 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. 2+ -column for protein purification, and a DNA sequence of 6 His residues was configured at the 3′-end of Hyal1 cDNA. Hyal1 variants were also prepared using PCR reaction bases, and the DNA sequence of the prepared Hyal1 variants was confirmed by DNA sequencing. The primers used for PCR are listed in Table 6.

[0089]

Table 6

[0090]

[0091]

[0092] <Example 2> Confirmation of enzyme activity and expression of Hyal1 Ala132 variant

[0093] CHO-K1 cells (purchased from the Korean Cell Line Bank) were transformed into plasmids with the gene of the wild type or variant of Hyal1 prepared in Example 1 using polyethyleneimine (PEI, Sigma-Aldrich) or Lipofectamine 3000 (Thermofisher) reagent. When the CHO-K1 cells grew to 90-95% relative to the area of ​​the 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 carefully moved to the 6-well plate and cultured in a 5% CO2 medium.

[0094] After 48 hours of transformation, the cell culture medium was recovered and centrifuged at 10,000 xg to obtain the supernatant. The enzyme activity of the wild type and variant of Hyal1 was detected by the substrate-gel assay method. Specifically, after electrophoresis using a 10% SDS gel containing hyaluronic acid (1.0 mg / mL), SDS was removed at 4°C using a buffer containing Triton X-100 (3% Triton X-100, 50mM Tris, 100mM NaCl, pH 7.5) for 2 hours. Phosphate buffers containing 100mM NaCl and adjusted to pH 4, 5, 6 and 7 using disodium phosphate and monosodium phosphate were prepared respectively. Each SDS gel was poured into a pH 4-7 phosphate buffer, shaken at 37°C and 50rpm, and the enzyme reaction was allowed to proceed for 4 to 17 hours. After the enzyme reaction, hyaluronic acid in the SDS gel was stained with 1.0% Alcian blue. The purpose of the present invention was to investigate the enzyme activity of Hya1 variants at neutral pH, so the enzyme activity at pH 6 and pH 7 was investigated using pH 4 as a control group.

[0095] Figure 1 (A) shows the results of an experiment to confirm whether the Hyal1 variant exhibits enzyme activity according to pH. According to the substrate-gel assay method, hyaluronic acid hydrolyzed by the Hyal1 variant is not stained by Alcian blue and thus appears as a white band. Figure 1In (A), Control shows a control group in which only the vector was transfected (transfection), WT shows wild-type Hyal1, and A132E, A132N, A132D, A132Y, A132S, and A132H show Hyal1 variants, respectively.

[0096] Figure 1 As shown in (A), at pH 4, all variants except A132Y hydrolyzed hyaluronic acid and showed enzyme activity. At pH 6, A132E, A132D and A132S variants showed enzyme activity. At pH 7, only A132E and A132D showed enzyme activity. Among the Hyal1 variants, A132E showed the highest enzyme activity at pH 7. A132N and A132S substituted with polar amino acids had higher enzyme activity than the wild type at pH 4, but, at pH 6, only A132S showed partial enzyme activity. The basic amino acid His showed enzyme activity at pH 6, but did not show enzyme activity at pH 7.

[0097] Furthermore, to understand the protein expression in CHO-K1 cells, e.g. Figure 1 In the experiment shown in (A), protein electrophoresis was performed and then Western blotting was performed. Specifically, 10% SDS gel was transferred at 100 V for 1 hour using nitrocellulose membrane, and then the expression of Hyal1 wild type and Ala132 variant was investigated using Hyal1 monoclonal antibody (1D10, Santa Cruze Biotechnology, Dallas, TX, US) and anti-mouse IgG secondary antibody (m-IgGk BP-HRP, Santa Cruze Biotechnology). The results are shown in Figure 1 (B) Chinese. Figure 1 (B) shows the results of experiments confirming the expression of Hyal1 variants. Figure 1 In (B), Control shows a control group in which only the vector was transfected, WT shows wild-type Hyal1, and A132E, A132N, A132D, A132Y, A132S, and A132H show the respective variants. Figure 1 As shown in (B), WT, A132E, A132N, A132D, A132S, and A132H variant proteins were detected by Hyal1 antibody, but the A132Y variant was not detected. Figure 1 (A) Negative results for the A132Y variant are shown resulting from non-expression of the protein.

[0098] 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 provides a proton for the hydroxyl leaving group, so the protonation of Glu131 is important for the catalytic reaction. Figure 1 The results shown in (A) mean that when the catalytic amino acid Glu131 and the tertiary structure replace the adjacent amino acids with acidic amino acids, the pKa value of Glu131 increases, and Glu131 can play a role in providing protons in acidic to neutral pH. However, even if the acidic amino acids are the same, A132E has higher enzyme activity than A132D at pH 7. In particular, it can be seen from this that the Hyal1 variant A132E described in one embodiment of the present invention can also effectively decompose hyaluronic acid at neutral pH.

[0099] <Example 3> Confirmation of the enzyme activity and expression of Hyal1 loop variants

[0100] In order to investigate the enzyme activity of the prepared Hyal1 ring variants (S77D, S77E, T86D, T86E and P87E) in the pH 4-7 region, CHO-K1 cells were transformed into plasmids into which the genes of the Hyal1 ring variants were embedded using the PEI or lipofectamaine method as described in Example 1. 48 hours after the transformation, the cell culture medium was recovered and centrifuged at 10,000 x g to obtain the supernatant. Using the substrate-gel assay method, the enzyme activity of the Hyal1 WT and the ring variant contained in the supernatant was allowed to react with phosphate buffer at 37°C in the pH 4-7 region for 4 to 17 hours, and the amount of hydrolyzed hyaluronic acid was investigated using 1.0% Alcian blue dye, and the results are shown in FIG. Figure 2 (A) Medium. Figure 2 (A) shows the results of a test to confirm whether Hyal1WT and loop variants exhibit enzyme activity according to pH. Figure 2 As shown in (A), Control shows a control group in which only the vector was transfected (transfection), WT shows wild-type Hyal1, and S77D, S77E, T86D, T86E, and P87E show respective Hyal1 variants.

[0101] like Figure 2As shown in (A), Hyal1 WT hydrolyzes hyaluronic acid at pH 4 and exhibits enzymatic activity, but the loop variants (S77D, S77E, T86D, T86E and P87E) exhibit enzymatic activity in the entire region of pH 4 to 7. From this result, it can be seen that Hyal1 variants in which Ser77, Thr86 and Pro87 of the loop site of wild-type human Hyal1 are substituted with Asp or Glu can also effectively decompose hyaluronic acid at neutral pH. In particular, according to the Hyal1 variants described in one embodiment of the present invention, at pH 7, S77D, S77E and P87E have higher enzymatic activity than T86D and T86E variants. Figure 2 (B) shows the results of experiments to confirm the expression of Hyal1 variants. Figure 2 (B) As shown, Hyal1 WT and variants achieved normal expression in CHO-K1 cells.

[0102] <Example 4> Confirmation of enzyme activity and expression of mixed variants with substitution of Hyal1 Ala132 or loop amino acids

[0103] The enzyme activity in the pH 4-7 region was investigated by simultaneously mutating the A132E variant and one or more of the loop variants, which had the highest activity among the Ala132 variants. In order to investigate the enzyme activity of the prepared Hyal1 variants (S77D / A132E, S77E / A132E, S77D / T86D, S77D / T86E, and S77D / T86D / A132E) in the pH 4-7 region, CHO-K1 cells were transformed into plasmids into which the genes of the Hyal1 variants were inserted using PEI or lipofectamine as described in Example 1. After 48 hours of transformation, the cell culture medium was recovered and centrifuged at 10,000 xg to obtain the supernatant. The enzyme activity of the Hyal1 variant contained in the supernatant was allowed to react by substrate-gel assay at 37°C in the pH range of 4 to 7 using phosphate buffer for 4 to 17 hours, and the amount of hydrolyzed hyaluronic acid was investigated using 1.0% Alcian blue dye.

[0104] like Figure 3 As shown in (A), the double or triple variants can also show enzymatic activity for hydrolysis of hyaluronic acid not only at pH 4 but also at pH 7. In particular, different degrees of activity were shown at pH 7, and the S77D / A132E variant showed the best activity.

[0105] Figure 3 (B) shows the results of experiments to confirm the expression of Hyal1 variants. Figure 3As shown in (B), Hyal1 variants were normally expressed in CHO-K1 cells.

[0106] <Example 5> Comparison of the tertiary structures of human Hyal1 and PH20 proteins

[0107] In order to compare the sites of the sudden mutation amino acids introduced into Hyal1 of the present invention with those of PH20, the tertiary structures of human Hyal1 and PH20 were compared. The tertiary structure model of human PH20 was prepared 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 displayed in Figure 4 In. Figure 4 As shown, in human Hyal1, Ser76, Ser77, Thr86 and Pro87 are located adjacent to the catalytic amino acid Glu131. It can be seen that in the tertiary structure of human PH20, Asp94, Asp103 and Glu149 are located adjacent to the catalytic amino acid Glu148.

[0108] Therefore, the Hyal1 variant of the present invention is like PH20 having enzymatic activity at neutral pH, in which the amino acid adjacent to the catalytic amino acid in the tertiary structure of human Hyal1 is substituted with an acidic amino acid, thereby being able to effectively decompose hyaluronic acid at neutral pH.

[0109] <Example 6> Confirmation of enzyme activity and expression of F139R and Y210H variants

[0110] Another method to increase the pKa value of the catalytic amino acid Glu131 of Hyal1 is to replace the amino acid adjacent to Glu131 with a basic amino acid, thereby reducing the pKa value of the acidic or polar amino acid that forms an ionic bond with the basic amino acid. In the tertiary structure of Hyal1, Phe139, Asp206, Ile225 and Tyr210 are located at the site adjacent to the catalytic amino acid. When the hydrophobic amino acid Phe139 is replaced with Arg, the pKa value of Tyr210 is reduced due to the ionic bonding between Arg139 and Tyr210, thereby increasing the pKa value of the catalytic amino acid Glu131. Furthermore, when the hydrophobic amino acids Phe139 and Ile225 are substituted with Arg and Asp, respectively, the positive charge of Arg139 is enhanced by the ionic bonding between Arg139 and Asp225, and thus, the ionic bonding between Arg139 and Tyr210 can further promote the catalytic dehydrogenation of the hydroxyl group of Tyr210 compared to the F139R variant alone. Furthermore, when the polar amino acid Tyr210 is substituted with the basic amino acid His, the pKa value of Asp206 is reduced due to the ionic bonding between Asp206 and His210, and as a result, the pKa value of the catalytic amino acid Glu131 can be increased.

[0111] In order to investigate the enzyme activity of the prepared Hyal1 variants (F139R, F139R / I225D and Y210H) in the pH 4-7 region, CHO-K1 cells were transformed into plasmids in which the genes of the Hyal1 variants were embedded using PEI or lipofectamine as described in Example 1. 48 hours after the transformation, the cell culture medium was recovered and centrifuged at 10,000 x g to obtain the supernatant. The enzyme activity of Hyal1WT and variants contained in the supernatant was subjected to enzyme reaction for 4 to 17 hours using phosphate buffer at 37°C in the pH 4-7 region by substrate-gel assay method, and the amount of hydrolyzed hyaluronic acid was investigated using 1.0% Alcian blue dye.

[0112] like Figure 5 As shown in (A), Control shows a control group in which only the vector is transfected (transfection), WT shows wild-type Hyal1, and F139R, Y210H, and F139R / I225D show each Hyal1 variant. WT shows enzyme activity only at pH 4, while F139R, Y210H, and F139R / I225D variants show enzyme activity at pH 4 and pH 7. The F139R / I225D double variant has higher activity than the F139R single variant at pH 6, and at pH 7, the enzyme activities of the single variant and the double variant are similar.

[0113] Figure 5 (B) shows the results of experiments to confirm the expression of Hyal1 variants. Figure 5 As shown in (B), Hyal1 variants were normally expressed in CHO-K1 cells.

[0114] Figure 5 (C) shows the tertiary structure of Hyal1 showing the aforementioned amino acids. Asp206 and Tyr210 are located adjacent to the catalytic amino acid Glu131.

[0115] 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, and therefore has high applicability. That is, when the Hyal1 variant of the present invention is used, hyaluronic acid can be decomposed even at neutral pH, so it can be used to achieve drug delivery and can also be used as a variety of preparations and even uses, such as preparations for subcutaneous administration, preparations for intravenous injection, and preparations for ophthalmology.

[0116] Furthermore, it can be seen from the above results that the Hyal1 variant can be effectively prepared using the nucleic acid, expression vector, host cell and method described in the present invention.

[0117] <Preparation Example 1> Preparation of liquid preparation

[0118] 1 μg of the Hyal1 variant protein prepared in Examples 2 to 6 was dissolved in PBS to prepare 1 mL of a solution. The prepared solution was filled into an injection ampoule or a sterile container to prepare an injection preparation or eye drops. The preparation prepared in this way can be applied as a hyaluronic acid decomposing agent, a drug delivery agent, etc. In particular, the injection preparation can be applied as a preparation for subcutaneous administration or a preparation for intravenous injection, and the eye drops can be applied as an ophthalmic preparation.

[0119] References:

[0120] 1. Pace CN, Grimsley GR, Scholtz JM. Protein ionizable groups: pK values ​​and their contribution to protein stability and solubility. J Biol Chem. 2009; 284(20): 13285-13289.

[0121] 2. Reitinger S, Mullegger J, Greiderer B, Nielsen JE, LepperdingerG. Designed human serum hyaluronidase 1variant, HYAL1DeltaL, exhibits activityup to pH 5.9. J Biol Chem. 2009; 284(29):19173-19177.

[0122] Sequence Directory Free Text

[0123] Attached with electronic file

Claims

1. A hyaluronidase Hyal1 variant showing activity at neutral pH.

2. The hyaluronidase Hyal1 variant showing activity at neutral pH according to claim 1, characterized in that: The hyaluronidase Hyal1 variant is based on the wild-type human hyaluronidase Hyal1 consisting of the amino acid sequence of SEQ ID NO: 1, and contains aspartic acid (Asp) and glutamic acid (Glu) as catalytic amino acids, The catalytic amino acid and one or more of the amino acids in the adjacent primary structure or tertiary structure are substituted with acidic or polar amino acids, or One or more of the catalytic amino acid and the amino acids adjacent to the tertiary structure are substituted with basic amino acids.

3. The hyaluronidase Hyal1 variant showing activity at neutral pH according to claim 2, characterized in that: The hyaluronidase Hyal1 variant is based on the wild-type human hyaluronidase Hyal1, and comprises substitutions of one or more amino acids selected from the group consisting of S76D, S77D, T86D, P87E, A132D, and A132E.

4. The hyaluronidase Hyal1 variant showing activity at neutral pH according to claim 2, characterized in that: The substituted basic amino acid is ionically bonded to the acidic or polar amino acid adjacent to the catalytic amino acid.

5. The hyaluronidase Hyal1 variant showing activity at neutral pH according to claim 2, characterized in that: The hyaluronidase Hyal1 variant comprises an amino acid substitution selected from the group consisting of F139R, Y210H and F139R / I225D, Among them, F139R / I225D means that F139R and I225D are replaced together.

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

7. A recombinant expression vector comprising the nucleic acid of claim 6.

8. A host cell transformed with the expression vector of claim 7.

9. A method for preparing a hyaluronidase Hyal1 variant comprising a step of culturing the host cell according to claim 8. 10 . A hyaluronan decomposing agent comprising the hyaluronidase Hyal1 variant according to claim 1 .

11. A drug delivery agent comprising the hyaluronidase Hyal1 variant according to claim 1.

12. A preparation for subcutaneous administration comprising the hyaluronidase Hyal1 variant according to claim 1.

13. A preparation for intravenous injection comprising the hyaluronidase Hyal1 variant of claim 1.

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

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