Hyaluronidase hyal2 variant with improved properties

Abstract

The present invention provides: an Hyal2 variant comprising an amino acid substitution at one or more amino acids associated with a catalytic amino acid in wild-type human Hyal2, which comprises glutamic acid as a catalytic amino acid; a method for preparing the Hyal2 variant; a nucleic acid, an expression vector and a host cell, which are applicable to the preparation of the Hyal2 variant; and a formulation or use of the Hyal2 variant. The Hyal2 variant of the present invention can degrade hyaluronic acid more effectively than the wild type, and thus exhibits high utility.

Description

Hyaluronidase HYAL2 variants with improved properties

[0001] The present invention relates to a hyaluronidase Hyal2 mutant, and more particularly, to a Hyal2 mutant having improved properties compared to the wild type.

[0002] Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid. Human hyaluronidases include Hyal1, Hyal2, Hyal3, Hyal4, and PH20. Hyal2 deficiency can lead to serious consequences, such as cleft lip and palate, facial deformities, ocular abnormalities, and thrombocytopenia. Furthermore, the Hyal2 gene is also used as a biomarker for cancer diagnosis (see International Patent Application Publication No. WO 2020 / 169826 A1).

[0003] Meanwhile, hyaluronidases such as PH20 have been used to increase subcutaneous drug delivery due to their ability to hydrolyze hyaluronic acid in the extracellular matrix. However, wild-type Hyal2 has low enzymatic activity, and improvements in its enzymatic activity-related properties are necessary for its effective use.

[0004] The inventors of the present invention completed the present invention as a result of research to improve the usability of Hyal2.

[0005] [Prior Art Literature]

[0006] [Patent Document]

[0007] (Patent Document 1) International Patent Application Publication WO 2020 / 169826 A1, August 27, 2020, Specification

[0008] One problem that the present invention seeks to solve is to provide a Hyal2 variant having improved characteristics.

[0009] In addition, another problem that the present invention seeks to solve is to provide a nucleic acid, an expression vector, and a host cell that can be applied to the method for producing a Hyal2 mutant of the present invention.

[0010] In addition, another problem that the present invention seeks to solve is to provide a method for producing the Hyal2 variant of the present invention.

[0011] In addition, another problem that the present invention seeks to solve is to provide a formulation or use of the Hyal2 variant of the present invention.

[0012] The problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0013] The present invention provides a Hyal2 variant comprising an amino acid substitution in one or more amino acids associated with the catalytic amino acid, based on a wild-type human Hyal2 comprising glutamic acid (Glu, E) as a catalytic amino acid.

[0014] Additionally, the present invention provides a nucleic acid encoding the Hyal2 variant.

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

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

[0017] In addition, the present invention provides a method for producing a Hyal2 variant, which comprises a step of culturing a host cell.

[0018] In addition, the present invention provides a hyaluronic acid decomposing agent comprising the above Hyal2 variant.

[0019] In addition, the present invention provides a drug delivery agent comprising the above Hyal2 variant.

[0020] In addition, the present invention provides an intravenous injection preparation comprising the above Hyal2 variant.

[0021] In addition, the present invention provides a subcutaneous administration formulation comprising the above Hyal2 variant.

[0022] In addition, the present invention provides an ophthalmic preparation comprising the above Hyal2 variant.

[0023] In addition, the present invention provides a pharmaceutical composition for treating or preventing hyaluronidase deficiency, comprising the above Hyal2 variant as an active ingredient.

[0024] The Hyal2 variant of the present invention has improved enzymatic activity, and thus can effectively decompose hyaluronic acid, resulting in high utility. That is, the Hyal2 variant of the present invention enables more effective hyaluronic acid decomposition and drug delivery using the same, and can be utilized in various formulations and applications (including those for treating and / or preventing Hyal2 deficiency), such as subcutaneous formulations, intravenous formulations, and ophthalmic formulations. Furthermore, the Hyal2 variant of the present invention can be effectively produced using the nucleic acid, expression vector, host cell, and method of the present invention.

[0025] Figure 1 shows the results of an experiment confirming the enzyme activity of human Hyal2 wild type and mutant using a substrate-gel assay.

[0026] Figure 2 is a graph showing the quantification of the bands in Figure 1 using the ImageJ program.

[0027] Figure 3 is a diagram showing the results of protein 3D structure analysis of Hyal2.

[0028] 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.

[0029] 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. In this specification, 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 in addition to the mentioned elements.

[0030] One embodiment of the present invention is a Hyal2 variant comprising an amino acid substitution in one or more of the amino acids associated with the catalytic amino acid, based on wild-type human Hyal2, which comprises glutamic acid (Glu, E) as a catalytic amino acid.

[0031] Wild-type human Hyal2 may be composed of a sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. Additionally, the catalytic amino acid may be Glu135.

[0032] Additionally, the wild-type human Hyal2 may include Asp133 as a substrate stabilizing amino acid.

[0033] Here, the amino acid associated with the catalytic amino acid refers to an amino acid that is directly or indirectly physically and / or chemically associated with the catalytic amino acid. For example, an amino acid physically associated with the catalytic amino acid may be an amino acid adjacent to the catalytic amino acid, and includes amino acids adjacent to the catalytic amino acid in terms of primary and / or tertiary structure. Furthermore, an amino acid chemically, or more specifically, electrochemically associated with the catalytic amino acid, includes an amino acid that forms an ionic bond with another amino acid.

[0034] By replacing amino acids directly or indirectly related to catalytic amino acids with other amino acids, enzyme properties are improved, such as increased enzyme activity compared to the wild type.

[0035] The amino acid associated with such a catalytic amino acid may be specifically one or more selected from Arg80, Asp81, Arg82, Arg88, Asp90, Ser91, Ala92, Arg94, or Asp136. More specifically, Arg80, Asp81, Arg88, Asp90, Arg94, or Asp136 may be an amino acid that forms an ionic bond, and Arg80, Asp81, Arg82, Arg88, Asp90, Ser91, Ala92, or Asp136 may be an amino acid that is physically adjacent to the catalytic amino acid. By substituting an amino acid that forms an ionic bond, the structure of the wild-type Hyal2 can be modified to be more flexible, and thus, the catalytic activity of the enzyme appears to increase. In addition, by substituting an amino acid physically adjacent to the catalytic amino acid, the acid dissociation constant (pKa) of the catalytic amino acid is affected, so that catalytic activity can be exhibited even at acidic pH where it exhibits low activity or at neutral pH where the wild type does not exhibit enzyme activity.

[0036] Specifically, the amino acid substitution may be, but is not limited to, one or more selected from among R80A, R80D, R80E, R80H, R80Y, D81E, R82D, R82E, R88Y, D90E, S91D, S91E, A92D, A92E, R94A, D136E, D136H, D136N, D136Q, D136S, D136T, or D136Y. That is, the variant may be a variant in which one or more amino acids are substituted, and may be referred to as a double variant, a triple variant, etc., depending on the number of mutated amino acids. Among the amino acid substitutions exemplified, R80A, R80D, R80E, R80H, R80Y, R88Y, R94A, D136H, D136N, D136Q, D136S, D136T, or D136Y may be examples of substitutions of amino acids that form ionic bonds. Additionally, R80D, R80E, D81E, R82D, R82E, R88Y, D90E, S91D, S91E, A92D, A92E, D136E, D136H, D136N, D136Q, D136S, D136T, or D136Y may be examples of substitutions of amino acids that are physically adjacent to the catalytic amino acid.

[0037] More specifically, the Hyal2 variant, which is an embodiment of the present invention, may be composed of one amino acid sequence selected from SEQ ID NOs: 5 to 33.

[0038] In this specification, expressions such as "Asp136" or "D136" 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, "Asp136" or "D136" represents aspartic acid, which is the amino acid at position 136 based on the amino acid sequence of SEQ ID NO: 1. In addition, "D136E" represents a mutant in which aspartic acid, which is the amino acid at position 136, is substituted with glutamic acid. In addition, wild-type human Hyal2 in this specification also means a mutant in which an amino acid is conservatively substituted at a specific amino acid position. In this case, "conservative substitution" means a modification of a mutant that includes substituting one or more amino acids with amino acids having similar biological or biochemical properties that do not cause loss of the biological or biochemical function of the mutant. Specifically, a "conservatively substituted variant" may be a variant having a sequence identity of, for example, at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% with Hyal2 consisting of the amino acid sequence of SEQ ID NO: 1, and having substantially the same function and / or effect. Thus, for example, wild-type human Hyal2 may be comprised of a sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1.

[0039] In one embodiment of the present invention, the Hyal2 variant has increased enzyme activity compared to the Hyal2 wild type. In this case, increased enzyme activity means that the Hyal2 wild type exhibits higher enzyme activity than the wild type at low acidic pH, or exhibits enzyme activity even at neutral pH, where the Hyal2 wild type exhibits no enzyme activity at all.

[0040] As can be seen from the experimental examples, the Hyal2 variant of the present invention has improved enzyme properties, including enzyme activity, compared to the Hyal2 wild type.

[0041] Such Hyal2 variants can degrade hyaluronic acid more effectively than the wild type. Therefore, the hyaluronic acid degrading agent, which is an embodiment of the present invention, can degrade hyaluronic acid more effectively by including the Hyal2 variant, which is an embodiment of the present invention. In addition, since the Hyal2 variant, which is an embodiment of the present invention, can degrade hyaluronic acid more effectively, it can also deliver drugs effectively. This is because when hyaluronic acid in the extracellular matrix is ​​hydrolyzed, the viscosity of hyaluronic acid decreases and the permeability into tissues (skin) increases. Therefore, the drug delivery agent, which is an embodiment of the present invention, can effectively deliver drugs by including the Hyal2 variant, which is an embodiment of the present invention.

[0042] At this time, the drug is a component that exhibits pharmacological activity, and may be, but is not limited to, an ophthalmic relaxant, an anesthetic, an antibody therapeutic agent, an anticancer agent, etc. The drug may be formed into a composition together with the Hyal2 variant, which is an embodiment of the present invention. That is, the Hyal2 variant, which is an embodiment of the present invention, may be in the form of a pharmaceutical composition by itself or together with a drug. In addition, the Hyal2 variant of the present invention may be included as an active ingredient in a pharmaceutical composition for treating or preventing hyaluronidase deficiency. That is, the Hyal2 variant of the present invention may be used for treating or preventing hyaluronidase deficiency. The hyaluronidase deficiency is preferably Hyal2 deficiency, and the Hyal2 deficiency includes at least one of cleft lip and palate, facial deformity, ocular abnormality, or thrombocytopenia caused by Hyal2 deficiency.

[0043] In addition, by applying the Hyal2 variant of the present invention together with another type of hyaluronidase (e.g., Hyal1 wild type or variant), hyaluronic acid can be decomposed more effectively. Since Hyal2 hydrolyzes hyaluronic acid to a size of 20 kDa, by applying a hyaluronidase capable of hydrolyzing hyaluronic acid to a smaller size (e.g., 0.8 kDa) (e.g., Hyal1 variant previously discovered by the present inventors) together with the variant of one embodiment of the present invention, hyaluronic acid can be decomposed more effectively.

[0044] Furthermore, the Hyal2 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 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 hyaluronidase, which is an embodiment of the present invention, through the bloodstream, thereby hydrolyzing hyaluronic acid overexpressed on the surface of tumor cells, thereby increasing the accessibility of an anticancer agent to tumor cells. In this way, the Hyal2 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.

[0045] The dosage of the Hyal2 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 Hyal2 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.

[0046] The Hyal2 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.

[0047] 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 or condition to be treated; the subject, age, size, and general condition to be treated; and the route of administration, such as nasal, oral, ocular, topical, transdermal, and intramuscular. 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 Hyal2 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 ingredients.

[0048] The Hyal2 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.

[0049] In addition, a method for decomposing hyaluronic acid, a method for delivering drugs, and / or a method for treating or preventing hyaluronidase deficiency, which is an embodiment of the present invention, comprises administering a Hyal2 variant, which is an embodiment of the present invention, to a mammal, including a human, in need thereof. In this case, the variant, which is an embodiment of the present invention, administered may be an effective amount of the variant.

[0050] In addition, one embodiment of the present invention is a use for manufacturing a hyaluronic acid decomposition agent or drug delivery agent of the Hyal2 variant of one embodiment of the present invention, or a treatment or prevention agent for hyaluronidase deficiency.

[0051] The Hyal2 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 Hyal2 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. After the fusion protein is expressed in the host cell, the Hyal2 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 Hyal2 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 may be present in a cell, a cell lysate, or in a partially purified or substantially pure form. The nucleic acid may be, for example, DNA or RNA.

[0052] 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 a variant according to an embodiment of the present invention, DNA encoding a variant according to an embodiment of the present invention can be obtained by molecular biology techniques (e.g., PCR amplification, site-directed mutagenesis), 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" can mean that a gene encoding a 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 variant according to an embodiment of the present invention. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. A gene encoding a variant according to 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 variant according to an embodiment of the present invention and a vector, or blunt-end ligation if no restriction enzyme site is present at all). The recombinant expression vector has a regulatory sequence that controls the expression of the gene encoding the variant according to 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 the variant according to 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.

[0053] 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.

[0054] 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 electroporation, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. Various expression host / vector combinations can be used to express the variants 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, pGEX, pUC, col E1, pCR1, pBR322, pMB9 and their derivatives; plasmids with a wider host range, such as RP4; phage DNA, exemplified by the wide variety of lambda phage derivatives, such as λgt10 and λgt11; and other DNA phages, such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells are the 2 μm plasmids and their derivatives. A useful vector for insect cells is pVL941.

[0055] In addition, the method for producing a variant according to an embodiment of the present invention may include a step of culturing a host cell according to an embodiment of the present invention. When a recombinant expression vector capable of expressing a variant according to an embodiment of the present invention is introduced into a mammalian host cell, the variant according to 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 variant according to an embodiment of the present invention to be secreted into the culture medium in which the host cell is cultured.

[0056] If necessary, the expressed variant of one embodiment of the present invention can be isolated from the host cell and purified to a uniform state. The isolation or purification of the variant of one embodiment of the present invention can be performed by 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, size exclusion chromatography, or hydrophobic chromatography, but is not limited thereto. In addition to chromatography, filtration, ultrafiltration, salting out, dialysis, etc. can also be used in combination.

[0057] Unless otherwise stated, matters mentioned in the Hyal2 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.

[0058] 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.

[0059] 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.

[0060] <Example 1> Hyal2 mutant

[0061] 1-1. Production of Hyal2 mutants

[0062] Human Hyal2 cDNA (Clone ID: hMU006389) of sequence number 1 was purchased from the Korean Human Gene Bank. Human Hyal2 cDNA was amplified by PCR (Bioneer, AllInOneCycler PCR system) using pOTB7-human Hyal2 plasmid. The amplified cDNA was inserted into a vector (named pSGHV1 vector) in which human growth hormone (hGH) and TEV protease cleavage sites were removed from the pSGHV0 vector using restriction enzymes XhoI and NotI. The sequence corresponding to Ala449 to Leu473 of the C-terminus, which is removed during the GPI-anchor binding process after Hyal2 protein translation, was cut out (Hyal2Δ449, SEQ ID NO: 2). At this time, Ni 2+ - For protein purification using a column, a DNA sequence of six His residues was positioned at the 3'-end of the Hyal2 structure.

[0063] Additionally, to increase gene expression of Hyal2, the Hyal2 signal sequence was replaced with the Hyal1 signal sequence using NEBuilder HiFi DNA Assembly (New England Biolabs) (Hyal2Δ449-SP1, SEQ ID NO: 3).

[0064] Wild-type Hyal1 and Hyal2 have 21 and 20 signal peptide amino acids, respectively. Hyal2Δ449-SP1 has the signal peptide of Hyal1, so it has one more signal peptide amino acid than Hyal2. However, since the signal peptide is removed during protein secretion, the mature form of Hyal2Δ449-SP1 has the same sequence as the wild-type Hyal2 mature form (SEQ ID NO: 4). In this specification, the amino acid number of Hyal2 is based on the wild-type Hyal2 of SEQ ID NO: 1.

[0065] The Hyal2 mutants described in Table 2 were constructed using PCR using Hyal2Δ449-SP1 (SEQ ID NO: 3) as a template. The amino acid sequences of the Hyal2 mutants are shown in the amino acid sequence of the Hyal2 mature form (SEQ ID NO: 4) excluding the signal peptide sequence of Hyal1. The PCR-amplified products were treated with DpnI restriction enzyme at 37°C for 1 hour to remove the template plasmid. The DNA sequences of the constructed Hyal2 wild-type and mutants were confirmed by DNA sequencing. The primers used for PCR are as described in Table 3.

[0066] 야생형서열번호아미노산 서열Hyal2 WT (Full length)1MRAGPGPTVTLALVLAVSWAMELKPTAPPIFTGRPFVVAWDVPTQDCGPRLKVPLDLNAFDVQASPNEGFVNQNITIFYRDRLGLYPRFDSAGRSVHGGVPQNVSLWAHRKMLQKRVEHYIRTQESAGLAVIDWEDWRPVWVRNWQDKDVYRRLSRQLVASRHPDWPPDRIVKQAQYEFEFAAQQFMLETLRYVKAVRPRHLWGFYLFPDCYNHDYVQNWESYTGRCPDVEVARNDQLAWLWAESTALFPSVYLDETLASSRHGRNFVSFRVQEALRVARTHHANHALPVYVFTRPTYSRRLTGLSEMDLISTIGESAALGAAGVILWGDAGYTTSTETCQYLKDYLTRLLVPYVVNVSWATQYCSRAQCHGHGRCVRRNPSASTFLHLSTNSFRLVPGHAPGEPQLRPVGELSWADIDHLQTHFRCQCYLGWSGEQCQWDHRQAAGGASEAWAGSHLTSLLALAALAFTWTLHyal2 Δ4492MRAGPGPTVTLALVLAVSWAMELKPTAPPIFTGRPFVVAWDVPTQDCGPRLKVPLDLNAFDVQASPNEGFVNQNITIFYRDRLGLYPRFDSAGRSVHGGVPQNVSLWAHRKMLQKRVEHYIRTQESAGLAVIDWEDWRPVWVRNWQDKDVYRRLSRQLVASRHPDWPPDRIVKQAQYEFEFAAQQFMLETLRYVKAVRPRHLWGFYLFPDCYNHDYVQNWESYTGRCPDVEVARNDQLAWLWAESTALFPSVYLDETLASSRHGRNFVSFRVQEALRVARTHHANHALPVYVFTRPTYSRRLTGLSEMDLISTIGESAALGAAGVILWGDAGYTTSTETCQYLKDYLTRLLVPYVVNVSWATQYCSRAQCHGHGRCVRRNPSASTFLHLSTNSFRLVPGHAPGEPQLRPVGELSWADIDHLQTHFRCQCYLGWSGEQCQWDHRQAAGGHyal2Δ449-SP13MAAHLLPICALFLTLLDMAQGMRAGPGPTVTLALVLAVSWAMELKPTAPPIFTGRPFVVAWDVPTQDCGPRLKVPLDLNAFDVQASPNEGFVNQNITIFYRDRLGLYPRFDSAGRSVHGGVPQNVSLWAHRKMLQKRVEHYIRTQESAGLAVIDWEDWRPVWVRNWQDKDVYRRLSRQLVASRHPDWPPDRIVKQAQYEFEFAAQQFMLETLRYVKAVRPRHLWGFYLFPDCYNHDYVQNWESYTGRCPDVEVARNDQLAWLWAESTALFPSVYLDETLASSRHGRNFVSFRVQEALRVARTHHANHALPVYVFTRPTYSRRLTGLSEMDLISTIGESAALGAAGVILWGDAGYTTSTETCQYLKDYLTRLLVPYVVNVSWATQYCSRAQCHGHGRCVRRNPSASTFLHLSTNSFRLVPGHAPGEPQLRPVGELSWADIDHLQTHFRCQCYLGWSGEQCQWDHRQAAGGHyal2 WT (Mature form)4MELKPTAPPIFTGRPFVVAWDVPTQDCGPRLKVPLDLNAFDVQASPNEGFVNQNITIFYRDRLGLYPRFDSAGRSVHGGVPQNVSLWAHRKMLQKRVEHYIRTQESAGLAVIDWEDWRPVWVRNWQDKDVYRRLSRQLVASRHPDWPPDRIVKQAQYEFEFAAQQFMLETLRYVKAVRPRHLWGFYLFPDCYNHDYVQNWESYTGRCPDVEVARNDQLAWLWAESTALFPSVYLDETLASSRHGRNFVSFRVQEALRVARTHHANHALPVYVFTRPTYSRRLTGLSEMDLISTIGESAALGAAGVILWGDAGYTTSTETCQYLKDYLTRLLVPYVVNVSWATQYCSRAQCHGHGRCVRRNPSASTFLHLSTNSFRLVPGHAPGEPQLRPVGELSWADIDHLQTHFRCQCYLGWSGEQCQWDHRQAAGG

[0067]

[0068]

[0069] 1-2. Confirmation of Hyal2 mutant expression and enzyme activity

[0070] 1-1. CHO-K1 cells (purchased from the Korea Cell Line Bank) were transfected with the plasmids containing the Hyal2 wild-type or mutant genes prepared in step 1 using polyethylenimine (PEI, Sigma-Aldrich) or Lipofectamine 3000 (Thermo Fisher) reagents. When CHO-K1 cells grew to 90–95% confluence 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 carefully transferred to a 6-well plate and cultured in a 5% CO2 incubator.

[0071] 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 Hyal2 wild type and mutants were measured using the substrate-gel assay method. Since Hyal2 has weak activity, a 10% SDS gel containing a low concentration of hyaluronic acid (0.15 mg / mL) was used. After electrophoresis, 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. A phosphate buffer containing 100 mM NaCl and adjusted to pH 4 with disodium phosphate and monosodium phosphate was prepared. The SDS gel was placed in the pH 4 phosphate buffer, and the enzyme reaction was performed for 24 hours at 37°C with shaking at 50 rpm. After the enzymatic reaction, the amount of hydrolyzed hyaluronic acid was examined using 1.0% Alcian Blue dye. The results are shown in Figs. 1 and 2. Fig. 1 shows the experimental results confirming the enzyme activity of human Hyal2 wild type and mutant using a substrate-gel assay, and Fig. 2 is a graph showing the quantification of the bands in Fig. 1 using the ImageJ program. In Fig. 2, the x-axis represents the experimental group, and the y-axis represents the relative intensity (%).

[0072] As shown in Fig. 1, hyaluronic acid hydrolyzed by the Hyal2 wild type and mutants in the substrate-gel assay method is not stained by Alcian Blue and therefore appears as a white band. In Fig. 1, Control represents the control group transfected with only the vector, WT represents wild-type Hyal2 (Hyal2Δ449-SP1), and R80A, S91E, R80A / R88Y, R80A / D136E, and R80A / S91E / D136E represent the respective Hyal2 mutants.

[0073] As shown in Figures 1 and 2, when Ser91 was substituted with Glu (S91E), the enzyme activity increased by 30% compared to the WT. In addition, in the case of the R80A / D136E mutant, the enzyme activity increased by 31%. In addition, the R80A mutant alone showed enzyme activity similar to the WT, and the double mutant, such as R80A / R88Y, showed an 11% increase in enzyme activity. In addition, the R80A / S91E / D136E triple mutant showed an 8% increase in enzyme activity.

[0074] It appears that such an effect was achieved by amino acid substitution in an amino acid associated with the catalytic amino acid (Glu135).

[0075] 1-3. Analysis of the tertiary structure of human Hyal2

[0076] The protein tertiary structure of human Hyal2 was generated using AlphaFold and analyzed using the PyMOL program. Figure 3 shows the results of the protein tertiary structure analysis of Hyal2.

[0077] As shown in Figure 3, wild-type human Hyal2 also contains Asp and Glu, which are acidic amino acids commonly located in the active site of human hyaluronidase. Specifically, wild-type human Hyal2 has Asp133 and Glu135 located in the β4-loop of the tertiary structure. Glu135 is involved in hyaluronic acid hydrolysis as a general acid-base catalyst, and Asp133 plays an auxiliary role in stabilizing the substrate during the catalytic reaction. In addition, Arg80 and Asp81 located in the β3-loop in wild-type human Hyal2 form ionic bonds with Asp136 located in the β4-loop and Arg88 located in the same β3-loop, respectively, thereby stabilizing the β3 and β4-loops. In addition, an ionic bond between Asp90 and Arg94 within the β-hairpin also exists in wild-type human Hyal2.

[0078] As confirmed from the experimental results above, it can be seen that the enzyme activity is increased by increasing the structural flexibility of the active site by changing such ionic bonds (including elimination of ionic bonds) through amino acid substitution. For example, by substituting Arg80 with Ala, Tyr, Asp, or Glu (e.g., R80A, R80Y, R80D, or R80E mutants), the β3-β4 ionic bond between Arg80 and Asp136 can be eliminated. In addition, by substituting Arg88 with Tyr (e.g., R88Y mutant), the ionic bond between Asp81 and Arg88 can be eliminated. In addition, by substituting Arg94 with Ala (e.g., R94A mutant), the ionic bond within the β-hairpin between Asp90 and Arg94 can be eliminated. In addition, examples thereof include double mutants (e.g., R80A / R88Y mutant) or triple mutants (e.g., R80A / R88Y / R94A mutant) in which two or three amino acids among Arg80, Arg88, and Arg94 are substituted to truncate two or three of these ionic bonds. In addition, substituting Asp136, which forms an ionic bond with Arg80, with His, Asn, Gln, Ser, Thr, or Tyr (e.g., D136H, D136N, D136Q, D136S, D136T, or D136Y mutants) can affect the enzyme activity. In addition, substituting Arg80 with His (e.g., R80H mutant) can also affect the enzyme activity.

[0079] In addition, it appears that by substituting an amino acid adjacent to the catalytic amino acid Glu135 of Hyal2 and increasing the pKa value of Glu135, the mutant can be made active even at a pH (e.g., neutral) where the wild-type Hyal2 is inactive. In particular, such amino acid substitutions may be more effective when the structural flexibility of the active site is increased by changing the ionic bond. Specifically, examples include mutants in which the catalytic amino acid Glu135 of Hyal2 is substituted with a different amino acid, using the wild-type Hyal2 or a Hyal2 mutant in which the ionic bond involving the β3 and β4 loops is changed as a template. More specifically, examples include variants in which Asp136, adjacent to the catalytic amino acid Glu135 in the primary structure, is replaced with another acidic amino acid, Glu, to provide negative charge repulsion, or with a polar amino acid (Asn, Gln, Ser, Thr, Tyr) or a basic amino acid (His) (e.g., D136E, D136H, D136N, D136Q, D136S, D136T, D136Y, R80A / D136E, R80Y / D136E, R80H / D136E, R80A / S91E / D136E variants). In this case, depending on the variant, it may also exhibit the function of changing ionic bonding.

[0080] Also, examples of mutants that substitute other amino acids for the catalytic amino acid Glu135 and the adjacent amino acid in the β3-loop in the 3D structure of the Hyal2 wild type are also provided. Specifically, examples of mutants that substitute Arg80 and / or Arg82 in the Arg80-Asp81-Arg82 sequence of the β3-loop with the acidic amino acid Asp or Glu (e.g., R80D, R80E, R82D, R82E mutants) are provided. In addition, since Asp81 is an acidic amino acid, examples of mutants that substitute it with the acidic amino acid Glu with a longer side chain (e.g., D81E mutant) are provided.

[0081] Also, examples of mutants that use the Hyal2 wild type or Hyal2 mutants that change the ionic bonds involving the β3 and β4 loops as templates and substitute other amino acids for the catalytic amino acid Glu135 and the amino acids located in the adjacent β-hairpin in the 3D structure can be cited. Specifically, examples of mutants that substitute Ser91 and / or Ala92 in the Asp90-Ser91-Ala92 sequence of the β-hairpin with Asp or Glu (e.g., S91D, S91E, A92D, A92E, R80A / S91E / D136E mutants) can be cited. In addition, since Asp90 is an acidic amino acid, examples of mutants that substitute it with Glu, an acidic amino acid with a longer side chain (e.g., D90E mutant) can be cited.

[0082] From the above results, it can be seen that the Hyal2 variant of the present invention can effectively degrade hyaluronic acid compared to the wild type, thereby demonstrating high utility. In other words, drug delivery using the Hyal2 variant of the present invention is possible, and it can also be utilized in various formulations and applications, such as subcutaneous administration formulations, intravenous injection formulations, and ophthalmic formulations.

[0083] In addition, from the above results, it can be seen that Hyal2 mutants can be effectively produced using the nucleic acid, expression vector, host cell, and method of the present invention.

[0084] <Manufacturing Example 1> Liquid preparation

[0085] 1 μg of the protein, manufactured using the same method as in Example 1, is dissolved in PBS to make a 1 mL solution. The resulting solution is filled into an ampoule for injection or a sterile container to produce an injectable solution or eye drops. The preparation thus manufactured can be utilized as a hyaluronic acid decomposer, drug delivery agent, etc. In particular, the injectable solution can be utilized as a subcutaneous or intravenous preparation, and the eye drops can be utilized as an ophthalmic preparation.

[0086] The Hyal2 variant of the present invention exhibits improved enzymatic activity, enabling it to effectively decompose hyaluronic acid, resulting in high usability. Therefore, the present invention has industrial applicability.

Claims

1. Based on wild-type human Hyal2 containing glutamic acid as a catalytic amino acid, A Hyal2 variant comprising an amino acid substitution in one or more of the amino acids associated with said catalytic amino acid.

2. In paragraph 1, The above wild-type human Hyal2 is a Hyal2 mutant consisting of a sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1, and wherein the catalytic amino acid is Glu135.

3. In paragraph 1, A Hyal2 variant wherein said amino acid associated with said catalytic amino acid is at least one selected from among amino acids that form an ionic bond with another amino acid, or amino acids that are physically adjacent to said catalytic amino acid.

4. In paragraph 2, A Hyal2 variant wherein said amino acid associated with said catalytic amino acid is at least one selected from Arg80, Asp81, Arg82, Arg88, Asp90, Ser91, Ala92, Arg94, or Asp136.

5. In paragraph 2, A Hyal2 variant wherein the amino acid substitution is at least one selected from R80A, R80D, R80E, R80H, R80Y, D81E, R82D, R82E, R88Y, D90E, S91D, S91E, A92D, A92E, R94A, D136E, D136H, D136N, D136Q, D136S, D136T, or D136Y.

6. In paragraph 1, A Hyal2 variant comprising an amino acid sequence selected from among SEQ ID NO: 5 to SEQ ID NO:

33.

7. A nucleic acid encoding the Hyal2 variant of paragraph 1.

8. A recombinant expression vector comprising the nucleic acid of clause 7.

9. A host cell transformed with the expression vector of clause 8.

10. A method for producing a Hyal2 mutant, comprising the step of culturing the host cell of clause 9.

11. A hyaluronic acid decomposer comprising the Hyal2 variant of clause 1.

12. A drug delivery agent comprising the Hyal2 variant of paragraph 1.

13. A subcutaneous formulation comprising the Hyal2 variant of paragraph 1.

14. An intravenous formulation comprising the Hyal2 variant of paragraph 1.

15. An ophthalmic preparation comprising the Hyal2 variant of paragraph 1.

16. A pharmaceutical composition for treating or preventing hyaluronidase deficiency, comprising the Hyal2 variant of paragraph 1 as an active ingredient.

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