Novel hyaluronidase variants and pharmaceutical compositions comprising same

Abstract

The invention relates to the technical field of protein engineering, which improves the enzymatic activity of human hyaluronidase at neutral pH, the hyaluronidase is an enzyme for hydrolyzing hyaluronic acid, and relates to novel hyaluronidase variants or fragments thereof, the present invention relates to hyaluronidase Hyal1 comprising one or more amino acid residue substitutions in the enzymatically active region in the amino acid sequence of the native hyaluronidase Hyal1 of SEQ ID NO: 1 and in the vicinity thereof, and wherein amino acid residues that are selectively additionally truncated are present at the N-terminus and / or C-terminus.

Description

Technical Field

[0001] This invention relates to a novel human hyaluronidase variant, which is a hyaluronic acid hydrolase with enhanced activity at neutral pH, and more specifically, to a hyaluronidase variant or fragment thereof comprising one or more amino acid residue substitutions at or adjacent to the active site of a native hyaluronidase having an amino acid sequence selected from any one of SEQ ID NO:1 to SEQ ID NO:4, and further truncated at an N-terminus or C-terminus. The invention also relates to a method for producing the hyaluronidase variant or fragment thereof, and pharmaceutical compositions comprising the hyaluronidase variant or fragment thereof. Background Technology

[0002] Human skin consists of the epidermis, dermis, and subcutaneous fat layer, and contains six types of glycosaminoglycans. Glycosaminoglycans include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparin sulfate, heparin, and keratin sulfate.

[0003] Glycosaminoglycans are structures composed of repeating disaccharide sugars. The length of the sugar chains varies among different glycosaminoglycans, ranging from hundreds to thousands of units. More than half of the hyaluronic acid in the body is found in the skin. Hyaluronic acid is synthesized by hyaluronic acid synthase, present on cell membranes, exists independently, and is not bound to proteoglycans; it is also the only glycosaminoglycan without a sulfate group. Other glycosaminoglycans are bound to proteoglycans and contain sulfate groups. Hyaluronic acid is composed of glucuronic acid and N-acetylglucosamine, linked by β-1,3 and β-1,4 bonds, and these disaccharides repeat approximately 5,000 times. It is known that about one-third (5g) of the hyaluronic acid in the human body is broken down daily.

[0004] Hyaluronidase is an enzyme that breaks down hyaluronic acid, which is located in the extracellular matrix. Six classes of hyaluronidases are known to exist in humans. These six classes are Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20 / SPAM1. In humans, Hyal1 and Hyal2 are hyaluronidases expressed in most tissues and are known to be present in plasma. Naturally occurring PH20 / SPAM1 (hereinafter referred to as PH20) is expressed in the cell membrane and acrosomal membrane of sperm. However, HyalPS1, as a pseudogene, is not expressed. Naturally occurring human Hyal1 is expressed in lysosomes within cells and most tissues (including the liver, kidneys, heart, etc.), and consists of the amino acid sequence of SEQ ID NO:1. The mature form consists of amino acids F22 to W435 of the amino acid sequence of SEQ ID NO:1 and is a protein with excellent enzymatic activity at acidic pH. Natural human Hyal2 is expressed in most tissues and consists of the amino acid sequence of SEQ ID NO:2. The mature form consists of amino acids M21 to G448 of the sequence of SEQ ID NO:2 and possesses enzymatic activity capable of hydrolyzing high molecular weight hyaluronic acid to approximately 20 kDa. Natural human Hyal3 is found in tissues such as skin, bone marrow, and testes. It consists of the amino acid sequence of SEQ ID NO:3, and the mature form consists of amino acids Q21 to V417 of the sequence of SEQ ID NO:3. It exhibits very weak enzymatic activity under in vitro conditions. Natural human Hyal4 is distributed in the placenta and muscle. It consists of the amino acid sequence of SEQ ID NO:4 and functions as a membrane protein. Natural human PH20 is expressed in testes and sperm. It consists of the amino acid sequence of SEQ ID NO:5, and the mature form consists of amino acids L36 to S490 of the sequence of SEQ ID NO:5. It exhibits excellent enzymatic activity even at neutral pH.

[0005] Based on the different ways in which hyaluronidase cleaves hyaluronic acid, hyaluronidase can be divided into three types. These three types are: enzymes that use H2O to cleave the β-1,4 bond between N-acetylglucosamine and glucuronic acid (EC 3.2.1.35); enzymes that cleave the β-1,3 bond (EC 3.2.1.36); and bacterial hyaluronidase that does not use H2O to cleave the β-1,4 bond (EC 4.2.99.1).

[0006] Hyal1 catalyzes the amino acids D129 and E131, hydrolyzing hyaluronic acid via substrate-assisted catalysis. Hyal1 exhibits optimal activity at acidic pH 3–4, showing no enzymatic activity at pH 4.5 or higher. Unlike Hyal1, PH20 exhibits enzymatic activity across a wide pH range from 3 to 8.

[0007] Arming et al. found that the catalytic amino acids of PH20 are D111 and E113 (Arming et al., 1997). Arming et al. designated the first amino acid Leu of the mature PH20 protein as position 1, so the catalytic amino acids in the full-length PH20 correspond to D146 and E148.

[0008] Hyaluronidase hydrolyzes hyaluronic acid, thereby reducing its viscosity in the extracellular matrix and increasing tissue (skin) permeability to external substances. Since the subcutaneous region of the skin is neutral, with a pH of approximately 7.0 to 7.5, PH20 is widely used in clinical practice among various types of hyaluronidase (Bookbinder et al., 2006). Examples of clinical use of PH20 include ocular relaxants and anesthetic injection additives in ophthalmic surgery, and it is also co-administered with subcutaneous antibody therapy agents (Bookbinder et al., 2006). Furthermore, by utilizing the overexpression of hyaluronidase in tumor cells, PH20 is used to hydrolyze hyaluronic acid in the extracellular matrix of tumor cells, thereby increasing the accessibility of anticancer drugs to tumor cells. Additionally, PH20 promotes the reabsorption of excess fluid and blood within tissues.

[0009] PH20 was initially discovered in guinea pig sperm by Lathrop et al., and is known to be expressed in sperm of many other species. The human PH20 gene was cloned by Lin et al. and Gmachl et al. The native human PH20 gene of SEQ ID NO:5 shares 60% amino acid sequence homology with the guinea pig PH20 gene. The human PH20 enzyme is encoded by the SPAM1 (sperm adhesion molecule-1) gene, and exists as PH20 Ser490 bound to GPI (glycosylphosphatidylinositol) on the sperm cell membrane surface and the inner side of the acrosome membrane. PH20 hydrolyzes hyaluronic acid as sperm penetrates the hyaluronic acid-rich cumulus oophorus to enter the oocyte. PH20 exists in sperm as a protein at a content of less than 1% and has six N-glycosylation sites (N82, N166, N235, N254, N368, and N393).

[0010] Currently, commercially available PH20 is extracted from bovine or ovine testes, including Amphadase (bovine hyaluronidase) and Vitrase (ovine hyaluronidase).

[0011] Bovine testicular hyaluronidase (BTH) is the natural bovine PH20 form, with its signal peptide and C-terminal 56 amino acids removed through post-translational modification. BTH is also a glycoprotein, comprising 5% mannose and 2.2% glucosamine in its total amino acid composition. Repeated administration of high doses of animal-derived hyaluronidase to humans may induce neutralizing antibodies. Furthermore, animal-derived hyaluronidase contains other biological substances besides PH20, which may induce allergic reactions when administered to humans (Bookbinder et al., 2006). In particular, the production and use of bovine PH20 are restricted due to concerns about mad cow disease. To alleviate these issues, recombinant human PH20 proteins are being investigated.

[0012] It has been reported that recombinant human PH20 proteins have been expressed in yeast (Pichia pastoris), DS-2 insect cells, and animal cells. The recombinant PH20 proteins produced in insect cells and yeast differ from human PH20 in their N-glycosylation patterns during post-translational modification.

[0013] Among hyaluronidases, the protein structures of Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidases (PDB ID: 1FCQ, 1FCU, 1FCV) have been elucidated. Hyal1 consists of two domains: a catalytic domain and an EGF-like domain. The catalytic domain forms an (β / α)8 conformation, with each α-helix and β-chain repeated eight times, characterizing the secondary structure of the protein (Chao et al., 2007). The EGF-like domain is conserved in all Hyal1 variants with different C-terminal splicing. Hyal1 and PH20 share 35.1% amino acid sequence identity, but the protein structure of PH20 has not yet been elucidated.

[0014] The recombinant protein of human PH20 was developed by Halozyme Therapeutics and sold under the trade name Hylenex (Bookbinder et al., 2006; Frost, 2007).

[0015] When the catalytic amino acids D146 and E148 of PH20 are mutated to asparagine (D146N) and glutamine (E148Q), respectively, enzyme activity is lost (Arming et al., 1997). Furthermore, replacing R246 of PH20 with glycine leads to a 90% reduction in enzyme activity, while replacing E319 with glutamine and R322 with threonine results in loss of enzyme activity. A PH20 variant with a 36-amino acid deletion at the C-terminus (amino acid truncation from 474 to 509) exhibits 75% less enzyme activity compared to native PH20. This mutant is not secreted extracellularly but remains within HeLa cells. A PH20 variant with a 134-amino acid deletion at the C-terminus lacks the enzyme activity of PH20 and is not secreted extracellularly. According to Frost et al., the C-terminal region 477 to 483 of PH20 is crucial for soluble expression (Frost, 2007). The activity of full-length PH20 (1 to 509) or PH20 variants truncated at C-terminus 467 (1 to 467) is only 10% of that of PH20 variants truncated at any of the positions from C-terminus 477 to 483 (Frost, 2007).

[0016] Meanwhile, recombinant PH20 still lacks sufficient thermostability or expression levels, thus creating a significant demand for hyaluronidases with superior properties. However, using proteins found only in human testes or sperm in skin or other tissues carries the potential risk of safety-related issues. Therefore, recombinant proteins of Hyal1, expressed in most human tissues, are needed; however, these enzymes are only active at acidic pH conditions, limiting their availability.

[0017] [List of Citations]

[0018] [Non-patent literature]

[0019] Arming,S.,Strobl,B.,Wechselberger,C.,and Kreil,G.(1997).In vitromutagenesis of PH-20hyaluronidase from human sperm.Eur J Biochem 247,810-814.

[0020] Bookbinder, LH, Hofer, A., Haller, MF, Zepeda, ML, Keller, GA, Lim, JE, Edgington, TS, Shepard, HM, Patton, JS, and Frost, GI (2006). Arecombinant human enzyme for enhanced interstitial transport of therapeutics. JControl Release 114, 230-241.

[0021] Chao,KL,Muthukumar,L.,and Herzberg,O.(2007).Structure of humanhyaluronidase-1,a hyaluronan hydrolyzing enzyme involved in tumor growth andangiogenesis.Biochemistry 46,6911-6920.

[0022] Frost, GI (2007). Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin DrugDeliv 4, 427-440. Summary of the Invention

[0023] One object of the present invention is to provide a novel hyaluronidase variant or fragment thereof, having a structure similar to that of natural hyaluronidase (preferably mature natural hyaluronidase) and being active at neutral pH.

[0024] Another object of the present invention is to provide a therapeutic composition comprising the novel hyaluronidase variant or a fragment thereof, and a treatment method using the composition.

[0025] To achieve the above objectives, the present invention provides a novel hyaluronidase variant or fragment thereof, which exhibits improved activity at neutral pH, comprising one or more amino acid residues substituted at or adjacent to the enzyme active site and / or substrate binding site in the amino acid sequence of a natural hyaluronidase (preferably a mature natural hyaluronidase), and having truncated amino acid residues at the N-terminus and / or C-terminus.

[0026] The present invention also provides a composition for treating cancer, comprising a novel hyaluronidase variant or a fragment thereof according to the present invention, and a treatment method using the composition.

[0027] This invention relates to a novel hyaluronidase variant or fragment thereof, comprising an enzyme active site, a substrate binding site and / or one or more amino acid residue substitutions in the adjacent region of the native hyaluronidase, and exhibiting hyaluronidase activity at neutral pH.

[0028] Furthermore, the present invention relates to novel hyaluronidase variants or fragments thereof, wherein the natural hyaluronidase may be a mature human natural hyaluronidase.

[0029] Furthermore, the present invention relates to novel hyaluronidase variants or fragments thereof, wherein mature human natural hyaluronidase may be in the form of human natural hyaluronidase with the signal peptide removed.

[0030] Furthermore, the present invention relates to novel hyaluronidase variants or fragments thereof, wherein the natural hyaluronidase may be human natural Hyal1.

[0031] Preferably, the human natural Hyal1 or a fragment thereof has the amino acid sequence of SEQ ID NO:1, or has the amino acid sequence F22 to W435 of the SEQ ID NO:1 sequence, but is not limited thereto.

[0032] Furthermore, the present invention also relates to novel hyaluronidase variants or fragments thereof, wherein the novel hyaluronidase variants exhibit hyaluronidase activity at neutral pH.

[0033] This invention relates to novel hyaluronidase variants or fragments thereof, wherein the enzyme active site may be D129 and / or E131 of SEQ ID NO:1.

[0034] Furthermore, the present invention relates to novel hyaluronidase variants or fragments thereof, which may include amino acid substitutions at one or more positions selected from the group consisting of: Q41, W42, G63, S76, S77, Q78, T81, F93, R108, F110, Q111, A115, A116, A132, F139, D142, T143, R148, V170, Q172, G177, A181, Q188, R191, Y210, F212, L213, S214, N216, T2 in the amino acid sequence of SEQ ID NO:1. 18, Q220, Q228, P249, V251, Q263, A267, Q288, D292, T293, T294, H296, H305, L307, L356, R372, L379, G396, A403, M412, E415, and K417, or amino acid substitutions at one or more positions corresponding to amino acids in the mature human Hyal1 amino acid sequence.

[0035] Preferably, the present invention relates to novel hyaluronidase variants or fragments thereof, comprising amino acid substitutions at one or more positions selected from the group consisting of: S76, S77, Q78, A132, D142, T143, Y210, F212, L213, P249, V251, Q288, and D292 in the amino acid sequence of SEQ ID NO:1, or amino acid substitutions at one or more positions corresponding to amino acids in the mature human Hyal1 amino acid sequence.

[0036] More preferably, the present invention relates to novel hyaluronidase variants or fragments thereof, comprising an amino acid residue substitution at D142 or P249 in the amino acid sequence of SEQ ID NO:1, and optionally further comprising an amino acid substitution at one or more positions selected from the group consisting of S77, Q78, A132, D142, T143, Y210, F212, P249 and V251, or an amino acid substitution at one or more positions corresponding to an amino acid in the mature human Hyal1 amino acid sequence.

[0037] Specifically, the present invention relates to novel hyaluronidase variants or fragments thereof, which may include one or more amino acid substitutions selected from the group consisting of: Q41E, W42F, G63R, S76V, S77D, Q78R, T81Y, F93N, R108K, F110K, Q111K, A115F, A116Y, A132E, F139R, D142K, T143P, R148K, V170K, Q172K, G177K, A181D, Q188K, R191K, Y210H, F212Y, L213K, S214K, N216 in the amino acid sequence of SEQ ID NO:1. G, T218N, Q220S, Q228R, P249N, V251Q, Q263R, A267R, Q288R, D292T, T293D, T294Q, H296K, H305Y, L307F, L356K, R372K, L379Y, G396K, A403K, M412F, E415K, and K417Y, or amino acid substitutions at one or more positions corresponding to amino acids in the mature human Hyal1 amino acid sequence.

[0038] Preferably, the present invention relates to novel hyaluronidase variants or fragments thereof, comprising one or more amino acid substitutions selected from the group consisting of: S76V, S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, L213K, P249N, V251Q, Q288R, and D292T in the amino acid sequence of SEQ ID NO:1, or amino acid substitutions at one or more amino acid positions corresponding to amino acids in the mature human Hyal1 amino acid sequence.

[0039] More preferably, the present invention relates to novel hyaluronidase variants or fragments thereof, comprising an amino acid residue substitution of D142K or P249N in the amino acid sequence of SEQ ID NO:1, and optionally further comprising one or more amino acid substitutions selected from the group consisting of S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, P249N and V251Q, or an amino acid substitution at one or more amino acid positions corresponding to amino acids in the mature human Hyal1 amino acid sequence.

[0040] Furthermore, the present invention relates to a composition for treating diseases (preferably cancer), comprising the aforementioned novel hyaluronidase variant or a fragment thereof.

[0041] Furthermore, the present invention relates to a nucleic acid encoding the aforementioned novel hyaluronidase variant or a fragment thereof.

[0042] Furthermore, the present invention relates to a recombinant expression vector comprising the nucleic acid.

[0043] Furthermore, the present invention relates to a host cell transformed with the recombinant expression vector.

[0044] Furthermore, the present invention relates to a host cell selected from the group consisting of: animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0045] Furthermore, the present invention relates to a method for generating novel hyaluronidase variants or fragments thereof, comprising culturing the host cells. Attached Figure Description

[0046] Figure 1 The results of protein structure analysis of hyaluronidase Hyal1 and hyaluronidase PH20 are shown; they are superimposed. Figure 1 A shows the results confirming that Hyal1 and PH20 have the same active site; Figure 1 B shows the results confirming that the substrate hyaluronic acid in Hyal1 and PH20 has the same binding site; Figure 1 C shows the results confirming the amino acids involved in enzyme activity and / or substrate binding in the adjacent regions of the active sites and / or substrate binding sites of Hyal1 and PH20;

[0047] Figure 2 The results of quantitative analysis using SDS-PAGE of samples obtained by generating and purifying recombinant hyaluronidase Hyal1 and Hyal1-E131H variants are shown, with information about the samples described in Example 1. Figure 2 A shows the SDS-PAGE results of the purified sample. Figure 2 B shows the quantitative curve created using BSA, and Figure 2 C shows the results of quantitative analysis of purified samples 1 and 2.

[0048] Figure 3 The results show the measurement of enzyme activity changes with pH for recombinant hyaluronidase Hyal1 and recombinant hyaluronidase variant Hyal1-E131H.

[0049] (A) The results of measurements showing the change in enzyme activity of Hyal1 with pH.

[0050] (B) Shows the measurement results of the enzyme activity of Hyal1-E131H as a function of pH. Detailed Implementation

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well-known and typical in the art.

[0052] The present invention provides a hyaluronidase variant or fragment thereof, which, in the amino acid sequence of a natural hyaluronidase (preferably a mature natural hyaluronidase), includes one or more amino acid residue substitutions at sites corresponding to its active site and / or linker site, preferably at D129 and E131 of Hyal1 and / or adjacent regions, and also has truncated amino acid residues at the N-terminus and / or C-terminus.

[0053] In this invention, the amino acid residue positions of each variant depend on the amino acid positions of the natural hyaluronidase Hyal1 according to SEQ ID NO:1.

[0054] Furthermore, in this invention, "mature natural hyaluronidase" refers to a protein composed of amino acid residues F22 to W435 of SEQ ID NO:1, obtained by deleting the signal peptide M1-G21 from the amino acid sequence of natural hyaluronidase Hyal1 of SEQ ID NO:1.

[0055] In this invention, based on the protein tertiary structure of human hyaluronidase Hyal1 (SEQ ID NO:1), amino acids located at and adjacent to the active sites D129 and E131 were selected and mutated to induce the enzyme to exert its catalytic activity even at neutral pH. Specifically, the E131H variant showed a trend of enhanced activity at pH 6. Figure 3 In this invention, by making various mutations in the active site and / or substrate binding site and / or adjacent regions of human hyaluronidase Hyal1, the possibility of providing novel hyaluronidase variants or fragments thereof that are different from natural PH20 and its variants has been elucidated.

[0056] Therefore, the novel hyaluronidase variant according to the present invention is characterized in that, in the natural hyaluronidase Hyal1 (hyaluronidase having the amino acid sequence of SEQ ID NO:1), preferably in the mature natural hyaluronidase Hyal1 (a protein composed of F22 to W435 in the amino acid sequence of SEQ ID NO:1), the active site D129 and / or E131 and / or some amino acid residues adjacent thereto are replaced with other amino acid residues, but the present invention is not limited thereto.

[0057] The X-ray diffraction crystal structure of hyaluronidase Hyal1 has been determined, and its protein structure is stored in the public database Protein Data Bank (https: / / www.rcsb.org / ), ID: 2PE4. Furthermore, the X-ray diffraction crystal structure of hyaluronidase PH20, which is active at neutral pH, is not yet available, but its protein structure, estimated by DeepMind (USA) and the European Bioinformatics Institute (EMBL-EBI), is stored in the public database AlphaFold Protein Structure Database (https: / / alphafold.ebi.ac.uk / ). Structural alignment studies were performed on the active sites of these proteins, such as... Figure 1 As shown, possible amino acid substitution variants were obtained through them, and further research was conducted on this basis to achieve the purpose of the present invention.

[0058] The amino acid sequences of natural human Hyal1, Hyal2, Hyal3, Hyal4 and PH20 are shown in Table 1 below.

[0059] [Table 1]

[0060]

[0061]

[0062]

[0063] In this invention, a notation in which a one-letter amino acid residue name is written together with a number, such as "E131", indicates the amino acid residue at each position in the amino acid sequence according to the corresponding sequence number. For example, "E131" in SEQ ID NO:1 means that the amino acid residue at position 131 in the amino acid sequence is glutamic acid.

[0064] In addition, “E131H” in SEQ ID NO:1 means that the amino acid glutamic acid at position 131 in SEQ ID NO:1 is replaced with histidine.

[0065] The amino acids involved in the active site of hyaluronidase Hyal1 are known to be Y75, Y202, Y247, and W321, as well as D129 and E131 (Chao KL2007). These amino acids are also conserved in another hyaluronidase, PH20. Furthermore, the amino acids involved in binding to the substrate hyaluronic acid are conserved in both proteins. The active site and substrate binding site, as well as the amino acids adjacent to these sites, may influence the pH environment for enzyme activity.

[0066] The novel hyaluronidase variants according to the present invention may include variations at any position in the amino acid sequence of hyaluronidase Hyal1 of SEQ ID NO:1, but preferably mutations, particularly amino acid substitutions, which may be performed at one or more positions selected from the group consisting of: Q41, W42, G63, S76, S77, Q78, T81, F93, R108, F110, Q111, A115, A116, A132, F139, D142, T143, R148, V170, Q172, G177, A181, Q188, R191, Y210, F212, L213, S214. N216, T218, Q220, Q228, P249, V251, Q263, A267, Q288, D292, T293, T294, H296, H305, L307, L356, R372, L379, G396, A403, M412, E415, and K417, preferably at one or more positions selected from the group consisting of S76, S77, Q78, A132, D142, T143, Y210, F212, L213, P249, V251, Q288, and D292, but not limited thereto.

[0067] More preferably, the present invention relates to a novel hyaluronidase variant or a fragment thereof, wherein one or more amino acid residue substitutions include an amino acid residue substitution at D142 or P249 in the amino acid sequence of SEQ ID NO:1, and optionally further includes an amino acid substitution at one or more positions selected from the group consisting of S77, Q78, A132, D142, T143, Y210, F212, P249 and V251.

[0068] More preferably, the novel hyaluronidase variant according to the invention comprises one or more amino acid substitutions selected from the group consisting of: Q41E, W42F, G63R, S76V, S77D, Q78R, T81Y, F93N, R108K, F110K, Q111K, A115F, A116Y, A132E, F139R, D142K, T143P, R148K, V170K, Q172K, G177K, A181D, Q188K, R191K, Y210H, F212Y, L213K, S214K, N216G, T218N, Q220S, Q228R, P249N in the amino acid sequence of the native hyaluronidase Hyal1 of SEQ ID NO:1. The amino acid substitutions are selected from the group consisting of V251Q, Q263R, A267R, Q288R, D292T, T293D, T294Q, H296K, H305Y, L307F, L356K, R372K, L379Y, G396K, A403K, M412F, E415K, and K417Y, more preferably from the group consisting of S76V, S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, L213K, P249N, V251Q, Q288R, and D292T, but are not limited thereto.

[0069] More preferably, the novel hyaluronidase variant according to the invention comprises an amino acid residue substitution of D142K or P249N in the amino acid sequence of SEQ ID NO:1, and optionally further comprises one or more amino acid substitutions selected from the group consisting of S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, P249N and V251Q.

[0070] The hyaluronidase variants according to the invention should also be understood to include variants in which amino acid residues at specific amino acid residue positions are conservatively substituted.

[0071] As used in this article, “conservative substitution” refers to a modification of a variant that involves replacing one or more amino acids with other amino acids that have similar biochemical properties, without causing a loss of the variant’s biological or biochemical function.

[0072] "Conservative amino acid substitution" is the substitution of an amino acid residue with another amino acid residue having a similar side chain. Categories of amino acid residues with similar side chains are defined and well-known in the art. These categories include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0073] It is expected that the hyaluronidase variant of the present invention will remain active even with conserved amino acid substitutions.

[0074] The novel hyaluronidase variants according to the invention may be, but are not limited to, fragments in which some amino acids are missing, for example, fragments in which the signal peptide is missing or removed.

[0075] Preferably, the human natural Hyal1 or a fragment thereof has the amino acid sequence of SEQ ID NO:1, or the amino acid sequence of F22 to W435 in the sequence of SEQ ID NO:1, and additionally includes, but is not limited to, the above-mentioned amino acid substitutions.

[0076] Another aspect of the present invention provides a composition for treating a disease comprising a hyaluronidase variant according to the present invention, and provides a method for treating a disease using the composition.

[0077] The composition for treating a disease may be a pharmaceutical composition. The pharmaceutical composition may further include a pharmaceutically acceptable carrier, and the carrier may be one or more of the following groups commonly used in pharmaceutical preparations, but not limited to, such as: lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc. The pharmaceutical composition may further include one or more of the following groups: diluents, excipients, lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives commonly used in the manufacture of pharmaceutical compositions.

[0078] The pharmaceutical composition can be administered orally or parenterally. Parenteral administration can include intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. When administered orally, because proteins or peptides are digestible, the oral composition can be formulated to encapsulate the active agent or protect it from degradation in the stomach. Furthermore, the composition can be administered via any device capable of transporting the active agent to target cells.

[0079] The pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an oily or aqueous medium, or it may be formulated into an extract, fine powder, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer for use in the formulation.

[0080] In particular, the therapeutic compositions according to the invention can be used alone or in combination with other therapeutic agents.

[0081] Another aspect of the invention relates to nucleic acids encoding hyaluronidase variants or variants thereof according to the invention.

[0082] The nucleic acids used herein may be present in cells or cell lysates, or may be in a partially purified or substantially pure form. When nucleic acids are purified from other cellular components or other contaminants such as nucleic acids or proteins of other cells using standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art, the nucleic acids are “isolated” or “obtained substantially pure.” The nucleic acids of this invention may be, for example, DNA or RNA.

[0083] Another aspect of the invention relates to a vector comprising the nucleic acid. To express a hyaluronidase variant or fragment thereof according to the invention, DNA encoding the hyaluronidase variant can be obtained using standard molecular biology techniques (e.g., PCR amplification using a hybridoma expressing the hyaluronidase variant or cDNA cloning), and the DNA can be "operably ligated" to transcription and translation control sequences, thereby being inserted into an expression vector.

[0084] As used herein, the term "operably ligated" can mean ligating a gene encoding a variant of hyaluronidase or a fragment thereof into a vector such that the transcriptional and translational control sequences within the vector perform their intended function, namely, regulating the transcription and translation of the gene encoding the variant of hyaluronidase or a fragment thereof. The expression vector and expression control sequences are selected to be compatible with the host cells used for expression. The gene encoding the hyaluronidase variant is inserted into the expression vector using standard methods (e.g., ligating a gene fragment encoding the variant of hyaluronidase or a fragment thereof to a complementary restriction enzyme site on the vector, or, if no restriction enzyme site is present, performing a blunt-end ligation).

[0085] In addition, the recombinant expression vector carries a regulatory sequence that controls the expression of the gene encoding the hyaluronidase variant in the host cell. The "regulatory sequence" may include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the gene encoding the hyaluronidase variant or a fragment thereof. Those skilled in the art will recognize that the design of the expression vector can be varied by selecting different regulatory sequences, depending on factors such as the choice of host cells to be transformed and the protein expression level.

[0086] Another aspect of the invention relates to a host cell comprising nucleic acids or a vector. The host cell according to the invention is preferably selected from, but not limited to, the group consisting of: animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0087] Specifically, the host cell according to the invention can be a prokaryotic cell, such as *Escherichia coli*, *Bacillus subtilis*, *Streptomyces* sp., *Pseudomonas* sp., *Proteus mirabilis*, or *Staphylococcus* sp. The host cell can also be a eukaryotic cell, such as cells of higher eukaryotes, for example fungi (such as *Aspergillus* sp.), yeasts (such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces* sp., and *Neurospora crassa* sp.), other lower eukaryotic cells, and cells derived from insects.

[0088] The host cells can also be derived from plants or mammals. Preferred examples include, but are not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138 cells, young hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, with CHO cells being particularly preferred.

[0089] Nucleic acid or vectors are transfected into host cells. Any of a variety of techniques commonly used for “transfection” of exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells can be used, such as electrophoresis, calcium phosphate precipitation, DEAE-glucan transfection, or liposome transfection. Various expression host / vector combinations can be used to express the PH20 variant or fragments thereof according to the invention. Examples of expression vectors suitable for eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retroviruses. Examples of expression vectors for bacterial hosts include bacterial plasmids obtained from *Escherichia coli*, such as pET, pRSET, pBluescript, pGEX2T, pUC vector, colE1, pCR1, pBR322, pMB9 and their derivatives; plasmids with a broader host range, such as RP4; bacteriophage DNA, such as the diverse bacteriophage λ derivatives λ and λNM989; and other DNA bacteriophages, such as M13 and filamentous single-stranded DNA bacteriophages. Examples of expression vectors that can be used in yeast cells include 2°C plasmids and their derivatives. A vector that can be used in insect cells is pVL941.

[0090] Another aspect of the invention relates to a method for producing a hyaluronidase variant or fragment thereof according to the invention, comprising expressing the hyaluronidase variant or fragment thereof according to the invention by culturing host cells.

[0091] When a recombinant expression vector capable of expressing a hyaluronidase variant or a fragment thereof is introduced into a mammalian host cell, the hyaluronidase variant or a fragment thereof can be produced by culturing the host cell for a period of time sufficient for its expression in the host cell, or more preferably for a period of time sufficient for the hyaluronidase variant to be secreted into the culture medium in which the host cell is cultured.

[0092] In some cases, expressed hyaluronidase variants can be isolated from host cells and purified to homogenate. Isolation or purification of hyaluronidase variants can be performed using methods commonly used for protein isolation or purification, such as chromatography. Chromatography can be, but is not limited to, a combination of one or more methods selected from, for example, affinity chromatography, ion exchange chromatography, and hydrophobic chromatography. In addition to chromatography, filtration, ultrafiltration, salting out, dialysis, etc., can also be used in combination.

[0093] The present invention can be better understood through the following embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention, as will be apparent to those skilled in the art.

[0094] [Example 1] Generation of hyaluronidase Hyal1 and its variants

[0095] Example 1-1 Cloning of Hyaluronidase Hyal1 and its variants

[0096] To generate recombinant Hyal1, cDNA of natural Hyal1 (clone ID: hMU005315) was purchased from the Korea Human Gene Bank. Mature natural Hyal1 encodes amino acids F22 to W435. The Hyal1 gene was amplified using polymerase chain reaction (hereinafter referred to as PCR) and inserted into the XhoI and NotI restriction enzyme sites of the pcDNA3.4-TOPO vector. The signal peptide of human Hyal1 was expressed in ExpiCHO cells. Furthermore, a 6xHis tag DNA sequence was placed at the 3' end of the Hyal1 cDNA for protein purification using a HisTrap column. Amino acid substitutions were performed on the Hyal1 variant using PCR, and the substitutions were confirmed by DNA sequencing.

[0097] The primers used for Hyal1 cloning are listed in Table 2 below, and their specific sequences are listed in Table 3 below.

[0098] [Table 2]

[0099]

[0100]

[0101] [Table 3]

[0102]

[0103]

[0104] Examples 1-2: Purification of Hyaluronidase Hyal1 and Hyal1-E131H variant

[0105] The recombinant Hyal1 protein and E131H variant produced in ExpiCHO cells were purified using the AKTA Prime system (Cytiva, USA) via the following three-step column chromatography method.

[0106] The culture supernatant was adjusted to pH 10.0 by adding Tris and then injected into Blue Sepharose 6Fast Flow resin equilibrated with buffer A (10 mM glycine, pH 10). After washing away impurities with buffer A, the target protein was eluted with buffer B (10 mM glycine, 1 M NaCl, pH 10). For purification on a CM-650M column, the collected fraction was dialyzed with buffer C (50 mM NaPi, 0.1% Triton X-100, pH 6.0).

[0107] Fractions collected from the Blue Sepharose 6Fast Flow column were injected into CM-650M resin equilibrated with buffer D (50 mM NaPi, 0.1% Triton X-100, pH 6.0). After washing away impurities with buffer D, the target protein was eluted with buffer E (50 mM NaPi, 0.1% Triton X-100, 0.5 M NaCl, pH 6.0). For DEAE Sepharose Fast Flow column purification, the collected fractions were dialyzed with buffer F (10 mM NaPi, pH 7.0).

[0108] Each fraction collected from the CM-650M column was injected into DEAE Sepharose Fast Flow resin equilibrated with buffer G (5 mM K₂HPO₄, pH 7.1). The process was performed in flow-through (FT) mode, with additional flow of buffer G after loading to ensure complete recovery of the target protein from the column. Impurities were then removed using buffer H (5 mM K₂HPO₄, 1 M NaCl, pH 7.1). Each fraction (#4-6, #8-14) collected during the CM-650M process was purified twice using a DEAE Sepharose Fast Flow column, and the samples obtained from the FT portion of each process were designated Sample 1 and Sample 2, respectively. Each sample collected during the purification step was electrophoresed on a 10% acrylamide gel at 150 V for 1 hour.

[0109] Each of Sample 1 and Sample 2 collected from the DEAE Sepharose Fast Flow column was dialyzed with buffer I (20 mM NaPi, 77 mM NaCl, pH 7.0) and concentrated using a stirred tank. The concentrated samples were then electrophoresed on a 10% acrylamide gel, and the concentration of the target protein was calculated using the band intensity calculated by the CS analyzer program, with bovine serum albumin (BSA) as the standard. Figure 2 ].

[0110] Examples 1-3: Purification of HIS-labeled hyaluronidase Hyal1 and its variants

[0111] The C-terminal His-tagged Hyal1 variant peptide generated in ExpiCHO cells was purified in two steps using the AKTA Prime system or a similar system (GE Healthcare). Q Sepharose was used for anion exchange chromatography, and HisTrap HP column was used for His-Tag affinity chromatography, both of which are column chromatography processes.

[0112] Prepare buffer A (20 mM sodium phosphate, pH 7.5) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl) for protein purification using a Q Sepharose column. Bind the protein to the Q Sepharose column, allow 5 CV of buffer A to flow to remove non-specifically bound proteins, and then allow 5 CV of buffer B to flow at a concentration gradient from 0 to 100% to elute the protein.

[0113] To purify proteins using a HisTrap HP column, buffer A (20 mM sodium phosphate, 500 mM NaCl, pH 7.5) and buffer B (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5) were prepared. After binding the protein sample to the HisTrap HP column, 7 CV of 7% buffer B was allowed to flow to remove non-specifically bound proteins, followed by 3 CV of 40% buffer B to elute the target protein. The column elution buffer (20 mM sodium phosphate, 100 mM NaCl, pH 7.0) was used for dialysis.

[0114] [Example 2] Measurement of Hyaluronidase Variant Activity

[0115] Hyaluronidase activity was measured by turbidity analysis, a method that measures the degree of turbidity caused by the precipitate formed when hyaluronic acid is mixed with albumin (BSA), expressed in absorbance. The turbidity / absorbance of the precipitate formed when hyaluronic acid is hydrolyzed by a PH20 peptide decreases when mixed with albumin. This analysis is typically performed at pH 5.3, as follows: Hyaluronidase standards of known activity (units) were diluted to 6, 8, 10, 12, 15, and 20 units / ml and prepared in individual tubes. The purified protein sample was diluted in buffer (20 mM sodium phosphate, pH 5.3, 77 mM sodium chloride, and 0.01% (w / v) bovine serum albumin) by adjusting the dilution factor to be within the standard curve range. 50 μl of the diluted sample was aliquoted into each well of a 96-well plate, and the plate was incubated at 37°C for 10 min. An additional 50 μl of 0.06% hyaluronic acid was aliquoted into each well. 0.06% hyaluronic acid was dissolved in 300 mM sodium phosphate buffer (pH 5.3). The sample and 0.06% hyaluronic acid were reacted at 37°C for 45 minutes. After the reaction, 40 μL of the enzyme-substrate reaction solution was partitioned into 200 μL of acidic albumin solution and allowed to stand at room temperature for 19 minutes. Subsequently, the absorbance was measured at 600 nm using a spectrophotometer. The acidic albumin solution was a solution containing 0.1% albumin (BSA) dissolved in a buffer solution of 24 mM sodium acetate, 79 mM acetic acid, and pH 3.75. A standard curve was plotted using activity standards to convert the measured absorbance values ​​of the sample into activity.

[0116] When the above process is performed at pH 7.0, the protein sample buffer used consists of 20 mM sodium phosphate (pH 7.0), 77 mM sodium chloride and 0.01% (w / v) bovine serum albumin, and the same process is performed using 0.06% hyaluronic acid aqueous solution dissolved in 20 mM sodium phosphate buffer (pH 7.0) and 70 mM sodium chloride.

[0117] Activity can also be measured in culture medium, but in this case, values ​​equal to or less than 300 units / ml are unreliable. Therefore, the LOQ (limit of quantitation) is set to 300 units / ml, and values ​​equal to or less than this value are considered inactive, i.e., 0. Furthermore, for activity measurements using purified protein samples, the LOQ is set to 15 units / μg.

[0118] [Example 3] Analysis of Hyaluronidase Variant Activity Assay Results

[0119] The pH-dependent enzyme activities of natural Hyal1 (SEQ ID NO:1) and the Hyal1-E131H variant, which includes the amino acid substitution E131H in natural Hyal1, were investigated.

[0120] The results are shown in Figure 3 . Figure 3 A shows the measurement results of the change in enzyme activity of Hyal1 with pH. Figure 3 B shows the measurement results of enzyme activity of the Hyal1-E131H variant as a function of pH. The results confirm that Hyal1 has high enzyme activity at acidic pH but almost no activity at neutral pH. For the Hyal1-E131H variant, although its enzyme activity is improved at neutral pH compared to acidic pH, the enzyme activity is relatively low, thus limiting its industrial applicability.

[0121] Based on these results, the design of variants was extended to variants near the enzyme's active site, and the present invention was achieved through combinations of these variants. The characteristics and sequences of the novel hyaluronidase Hyal1 variants according to the present invention are shown in Tables 4 and 5 below.

[0122] As shown in Table 4, the novel hyaluronidase Hyal1 variant according to the present invention has been shown to exhibit excellent enzyme activity at pH 5.3 and pH 7.0, particularly when the amino acid substitutions include D142K, or further include one or more amino acid substitutions selected from the group consisting of S77D, Q78R, A132E, T143P, Y210H, F212Y, P249N and V251Q.

[0123] [Table 4]

[0124]

[0125]

[0126] [Table 5]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] Industrial applicability

[0133] According to the present invention, the natural hyaluronidase variant has a structure similar to that of the mature natural PH20 and exhibits the effect of increasing protein expression levels and enzyme activity at neutral pH.

[0134] Sequence List Free Text

[0135] Electronic documents are attached.

Claims

1. A novel hyaluronidase variant or fragment thereof, comprising amino acid residue substitutions at the enzyme active site, substrate binding site and / or one or more sites adjacent to the natural hyaluronidase, and exhibiting hyaluronidase activity.

2. The hyaluronidase variant or fragment thereof according to claim 1, wherein, The natural hyaluronidase mentioned is mature human natural hyaluronidase.

3. The hyaluronidase variant or fragment thereof according to claim 1, wherein, The mature human natural hyaluronidase is the form in which the signal peptide is missing from the human natural hyaluronidase.

4. The hyaluronidase variant or fragment thereof according to claim 1, wherein, The natural hyaluronidase is human natural Hyal1.

5. The hyaluronidase variant or fragment thereof according to claim 4, wherein, The natural hyaluronidase comprises the sequence of SEQ ID NO:1 or the amino acid sequence F22 to W435 of the sequence of SEQ ID NO:

1.

6. The hyaluronidase variant or fragment thereof according to any one of claims 1 to 5, wherein, The novel hyaluronidase variant exhibits hyaluronidase activity at neutral pH.

7. The hyaluronidase variant or fragment thereof according to claim 1, wherein, The enzyme active site is D129 and / or E131 in SEQ ID NO:

1.

8. The hyaluronidase variant or fragment thereof according to claim 1, comprising amino acid substitutions at one or more positions selected from the group consisting of: Q41, W42, G63, S76, S77, Q78, T81, F93, R108, F110, Q111, A115, A116, A132, F139, D142, T143, R148, V170, Q172, G177, A181, Q188, R191, Y210, F212, L213, S214, N216, T2 in the amino acid sequence of SEQ ID NO:

1. 18, Q220, Q228, P249, V251, Q263, A267, Q288, D292, T293, T294, H296, H305, L307, L356, R372, L379, G396, A403, M412, E415, and K417, or amino acid substitutions at one or more positions corresponding to amino acids in the mature human Hyal1 amino acid sequence.

9. The hyaluronidase variant or fragment thereof according to claim 8, comprising amino acid substitutions at one or more positions selected from the group consisting of: S76, S77, Q78, A132, D142, T143, Y210, F212, L213, P249, V251, Q288, and D292, or amino acid substitutions at one or more positions corresponding to amino acids in the mature human Hyal1 amino acid sequence.

10. The hyaluronidase variant or fragment thereof according to claim 8, comprising an amino acid residue substitution at D142 or P249, and optionally further comprising an amino acid substitution at one or more positions selected from the group consisting of S77, Q78, D142, A132, T143, Y210, F212, P249 and V251, or an amino acid substitution at one or more positions corresponding to an amino acid in the mature human Hyal1 amino acid sequence.

11. The hyaluronidase variant or fragment thereof according to claim 8, comprising one or more amino acid substitutions selected from the group consisting of: Q41E, W42F, G63R, S76V, S77D, Q78R, T81Y, F93N, R108K, F110K, Q111K, A115F, A116Y, A132E, F139R, D142K, T143P, R148K, V170K, Q172K, G177K, A181D, Q188K, R191K, Y210H, F21 2Y, L213K, S214K, N216G, T218N, Q220S, Q228R, P249N, V251Q, Q263R, A267R, Q288R, D292T, T293D, T294Q, H296K, H305Y, L307F, L356K, R372K, L379Y, G396K, A403K, M412F, E415K, and K417Y, or amino acid substitutions at one or more positions corresponding to amino acids in the mature human Hyal1 amino acid sequence.

12. The hyaluronidase variant or fragment thereof according to claim 11, comprising one or more amino acid substitutions selected from the group consisting of S76V, S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, L213K, V251Q, P249N, Q288R, and D292T, or amino acid substitutions at one or more amino acid positions corresponding to amino acids in the mature human Hyal1 amino acid sequence.

13. The hyaluronidase variant or fragment thereof according to claim 11, comprising an amino acid substitution of D142K or P249N, and optionally further comprising one or more amino acid substitutions selected from the group consisting of S77D, Q78R, A132E, D142K, T143P, Y210H, F212Y, P249N and V251Q, or an amino acid substitution at one or more amino acid positions corresponding to amino acids in the mature human Hyal1 amino acid sequence.

14. A therapeutic composition comprising a hyaluronidase variant or a fragment thereof according to any one of claims 1 to 13.

15. A nucleic acid encoding a variant or fragment of hyaluronidase according to any one of claims 1 to 13.

16. A recombinant expression vector comprising the nucleic acid according to claim 15.

17. A host cell transformed with the recombinant expression vector according to claim 16.

18. The host cell according to claim 17, wherein, The host cells are selected from the group consisting of: animal cells, plant cells, yeast, Escherichia coli, and insect cells.

19. A method for producing a variant of hyaluronidase or a fragment thereof, comprising culturing a host cell according to claim 18.

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