Soluble PH20 polypeptide with cleaved N-terminus and / or C-terminus and its uses

Abstract

The present invention provides a soluble recombinant PH20 polypeptide with improved productivity, in which the N-terminus and / or C-terminus are cleaved. [Solution] The present invention provides a recombinant PH20 polypeptide in which 1 to 7 amino acid residues are deleted from the N-terminus of mature animal wild-type PH20, and its uses. The PH20 polypeptide with N-terminus and / or C-terminus deletions presented in this invention exhibits superior expression levels and enzyme activity compared to existing recombinant PH20 polypeptides, and may lead to a reduction in treatment costs by reducing production costs in industrial applications.

Description

Technical Field

[0001] The present invention relates to a soluble PH20 polypeptide with its N-terminus and / or C-terminus truncated and its uses.

Background Art

[0002] When attempting to produce industrially useful proteins using genetic engineering methods, attempts have continued to increase the expression level of proteins expressed within recombinant cells or secreted from recombinant cells. Various existing methods have been tried, and each method has its own advantages and disadvantages. For example, a scheme of appropriately selecting a promoter at the cloning stage is commonly used. However, in this case, if protein folding is the rate-limiting step within the cell, the expression level of the recombinant protein often does not increase as predicted by the promoter used (Su Xiao et al., Curr Opin Struct Biol. 2014, June 32 - 38.).

[0003] To increase the expression level of proteins secreted extracellularly, a method has also been used in which the native signal peptide located at the N-terminus of the protein to be expressed is replaced with the signal peptide of another protein to increase the expression of the protein (Kober, L., Zehe, C. & Bode, J., 2013. Optimized Signal Peptides for the Development of High Expressing CHO Cell Lines. Biotechnology and bioengineering, 110(4), pp. 1164 - 1173.). In this case, the protein expression may increase more compared to when using its own signal peptide, but sometimes when the protein is expressed and passes through the cell membrane of the endoplasmic reticulum within the cell and is cleaved by signal peptidase, it may occur that the cleavage between the signal peptide and the N-terminal amino acid of the mature protein cannot be accurately performed.

[0004] Other attempts to increase protein expression levels include expressing the protein to be expressed together with a chaperone, which helps with recombinant protein folding and prevents the expressed protein from aggregating, thereby increasing expression levels. However, this method is not commonly used industrially due to technical problems such as the need to simultaneously express a membrane-bound chaperone in recombinant cells, as well as physiological problems within cells caused by chaperone expression.

[0005] Another attempt to increase protein expression levels involves inducing site-directed mutations (SMT) in specific sites of recombinant proteins to replace amino acids. Proteins, composed of polypeptides, acquire a tertiary structure through folding during cell production. Replacing amino acids in specific sites of the protein with other amino acids creates conditions favorable for folding, which can subsequently lead to increased protein expression. While there are many successful examples, this method faces practical challenges, such as the risk of increased immunogenicity due to amino acid modification.

[0006] Hyaluronan (hyaluronic acid: HA) is a polypeptide found in the extracellular matrix of many cells, particularly in soft connective tissue. Hyaluronan is also primarily found in mammalian skin, cartilage, and synovial fluid. Furthermore, hyaluronan is a major component of the vitreous fluid of the eye. Hyaluronan plays a role in various physiological processes, such as in the homeostasis of water and plasma proteins (Laurent TC et al. (1992) FASEB J6:2397-2404). Certain diseases are associated with the expression and / or production of hyaluronan. Hyaluronidase is an enzyme that breaks down hyaluronan. By catalyzing the hydrolysis of hyaluronan, hyaluronidase may be used to treat diseases or disorders associated with the accumulation of hyaluronan or other glycosaminoglycans. Furthermore, since hyaluronan is a major component of the subcutaneous tissue or barrier, hyaluronidase may be used to increase tissue permeability and therefore enhance the dispersion and delivery of therapeutic agents during subcutaneous injection.

[0007] Various hyaluronidases based on natural pH20 polypeptides (e.g., Hydase) TM Vitrase TM Wydase TM It is commonly used as a dispersant or spray in combination with other therapeutic agents. Many of these are in forms containing PH20 extracted from sheep or bovine testicles.

[0008] The human PH20 protein is composed of a total of 509 amino acids and is known as a glycophospholipid-anchored protein found in the plasma membrane of sperm. Unlike hyaluronidases such as Hyal1, Hyal2, Hyla3, and Hyal4, which are present in the blood and are only active at acidic pH, PH20 is active at neutral pH as well. For this reason, it has been developed for subcutaneous injection in combination with drugs and is used industrially.

[0009] Recently, Hylenex, a recombinant human PH20 polypeptide composed of 447 amino acids in which some of the amino acids constituting the glycophospholipid anchor position at the C-terminus of PH20 have been cleaved and converted to soluble form, has been developed. TM This is being used, and separately, a substance using a human PH20 polypeptide variant has also been developed.

[0010] On the other hand, human PH20 is a glycoprotein with six N-linked glycosylations and possesses a very complex tertiary structure. To produce an industrially useful enzyme, it must be expressed in animal cells such as CHO cells and then undergo a complex purification process. Therefore, improving productivity by increasing the expression level in fermentation media of animal cells containing the human PH20 gene is crucial for the industrial use of human PH20.

[0011] Against this technical backdrop, the inventors confirmed that cleavage of the N-terminus, or cleavage of both the N-terminus and C-terminus, can improve the productivity of soluble recombinant PH20 polypeptides, and thus completed the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0012] The object of the present invention is to provide a soluble recombinant PH20 polypeptide with improved productivity, in which the N-terminus and / or C-terminus are cleaved.

[0013] The object of the present invention is to provide nucleic acids encoding a soluble recombinant PH20 polypeptide in which the N-terminus and / or C-terminus are cleaved.

[0014] The object of the present invention is to provide a recombinant expression vector containing the nucleic acid.

[0015] The object of the present invention is to provide host cells transformed with the recombinant expression vector.

[0016] The object of the present invention is to provide a method for producing a soluble recombinant PH20 polypeptide with cleaved N-terminus and / or C-terminus, which includes the step of culturing the host cells. [Means for solving the problem]

[0017] The present invention provides recombinant PH20 polypeptides in which one to seven amino acid residues are deleted from the N-terminus of mature animal wild-type PH20.

[0018] Specifically, the recombinant PH20 polypeptide according to the present invention, in which an amino acid residue is deleted at the N-terminus of mature animal wild-type PH20, (a) Recombinant PH20 polypeptide having the amino acid sequence of SEQ ID NO: 1, in which the N-terminal amino acid residue is deleted by cleavage before an amino acid residue selected from the group consisting of N37 to P42; (b) Recombinant PH20 polypeptides in which a primate wild-type PH20 having one of the amino acid sequences from SEQ ID NOs. 2 to 8 is cleaved before an amino acid residue selected from the group consisting of N37 to P42, resulting in the deletion of the N-terminal amino acid residue; (c) Recombinant PH20 polypeptide having the amino acid sequence of Sequence ID No. 9, wherein the N-terminal amino acid residue is deleted by cleavage before an amino acid residue selected from the group consisting of D37 to P42; and (d) Recombinant PH20 polypeptide having the amino acid sequence of Sequence ID No. 10, in which the N-terminal amino acid residue is deleted by cleavage before an amino acid residue selected from the group consisting of D37 to P42; It may be characterized by being selected from a group consisting of, but is not limited to, this.

[0019] The present invention provides a nucleic acid encoding the recombinant PH20 polypeptide and a recombinant expression vector containing the nucleic acid.

[0020] The present invention also provides a method for producing a soluble recombinant PH20 polypeptide with a cleaved N-terminus and / or C-terminus, which includes a host cell transformed with the recombinant expression vector and a step of culturing the host cell.

Brief Description of Drawings

[0021] [Figure 1] This is a diagram showing the enzyme activity in the culture broth of PH20 polypeptides starting with L36, N37, F38, R39, and A40 at the N-terminus when the C-terminus of SEQ ID NO: 1 is Y482 or F468, respectively, according to specific examples of the present invention. Each activity is shown as a relative activity percentage based on L36-Y482 having the longest amino acid length. [Figure 2] This is a diagram showing the enzyme activity at pH 5.3 of the purified PH20 polypeptides starting with L36, N37, F38, R39, and A40 at the N-terminus when the C-terminus of SEQ ID NO: 1 is Y482 or F468, respectively, according to specific examples of the present invention, in terms of specific activity. Each activity is shown as a relative activity percentage based on L36-Y482 having the longest amino acid length. [Figure 3] This is a diagram showing the enzyme activity at pH 7.0 of the purified PH20 polypeptides starting with L36, N37, F38, R39, and A40 at the N-terminus when the C-terminus of SEQ ID NO: 1 is Y482 or F468, respectively, according to specific examples of the present invention, in terms of specific activity. Each activity is shown as a relative activity percentage based on L36-Y482 having the longest amino acid length. [Figure 4] This is the result of performing multiple sequence alignment (Multiple Sequence Alignment) on the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 10 using Clustal Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ). [Figure 5]According to specific embodiments of the present invention, it is a diagram showing the enzyme activity of the culture solution of the PH20 polypeptide starting with L36, N37, or D37, F38, R39, A40, P41 at the N-terminus in the cases of SEQ ID NO: 2 (C-terminus is Y483), SEQ ID NO: 9 (C-terminus is H478), and SEQ ID NO: 10 (C-terminus is H477), respectively. Each activity is shown as a measured value. [Figure 6] According to specific embodiments of the present invention, it is a diagram showing the specific activity of the enzyme activity of the purified PH20 polypeptide starting with L36, N37, F38, R39, A40, P41 at the N-terminus in the cases of SEQ ID NO: 2 (C-terminus is Y483), SEQ ID NO: 9 (C-terminus is H478), and SEQ ID NO: 10 (C-terminus is H477) at pH 5.3. Each activity is shown as a measured value. [Figure 7] According to specific embodiments of the present invention, for L36-Y482, N37-482, F38-Y482 of SEQ ID NO: 1, different signal peptides were used, and the results of measuring the enzyme activity of the PH20 polypeptide in the culture solution and the purified state at pH 5.3 are shown. The enzyme activities of the culture solution and the purified protein when using the signal peptide of Human Serum Albumin, the enzyme activities of the culture solution and the purified protein when using the signal peptide of human PH20, and the enzyme activities of the culture solution and the purified protein when using the signal peptide of Human Immunoglobulin kappa are shown. The scale of the enzyme activity of the culture solution is shown on the main Y-axis in a bar graph, and the scale of the activity of the purified protein is shown on the secondary Y-axis in a line graph.

Modes for Carrying Out the Invention

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by skilled experts in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0023] To expand the industrial applicability of recombinant PH20 polypeptide converted to solubility by cleaving at the C-terminus of PH20, the present invention presents N-terminal and / or C-terminal cleaved PH20 polypeptides with significantly increased productivity due to further cleavage of the N-terminus of PH20. In the present invention, "soluble" means a three-dimensional structural form that is active and does not undergo aggregation in aqueous solution.

[0024] Accordingly, the present invention relates to recombinant PH20 polypeptides in which one to seven amino acid residues, preferably one to six amino acid residues, more preferably one to five amino acid residues, even more preferably one to four amino acid residues, most preferably one to three amino acid residues, particularly preferably one to two amino acid residues, and especially preferably one amino acid residue, are deleted from the N-terminus of mature animal wild-type PH20.

[0025] In this invention, "mature animal wild-type PH20" can mean a recombinant PH20 polypeptide in a form that exhibits the desired function. According to the present invention, mature animal wild-type PH20 can mean, for example, a state in which a signal peptide that promotes the secretion of mature animal wild-type PH20 outside the cell has been cleaved during the secretion process.

[0026] The aforementioned animals may be, for example, mammals, rodents, etc., and may be, but are not limited to, humans, rats, mice, hamsters, rabbits, pigs, cows, deer, sheep, monkeys, etc.

[0027] The wild-type animal PH20 according to the present invention is, for example, (a) Recombinant PH20 polypeptide having the amino acid sequence of SEQ ID NO: 1, in which the N-terminal amino acid residue is deleted by cleavage before an amino acid residue selected from the group consisting of N37 to P42; (b) Recombinant PH20 polypeptides in which a primate wild-type PH20 having one amino acid sequence from SEQ ID NOs. 2 to SEQ ID NOs. 8 is cleaved before an amino acid residue selected from the group consisting of N37 to P42, resulting in the deletion of the N-terminal amino acid residue; (c) Recombinant PH20 polypeptide having the amino acid sequence of Sequence ID No. 9, wherein the N-terminal amino acid residue is deleted by cleavage before an amino acid residue selected from the group consisting of D37 to P42; or (d) Recombinant PH20 polypeptides having the amino acid sequence of Sequence ID No. 10, in which the N-terminal amino acid residue is deleted by cleavage before an amino acid residue selected from the group consisting of D37 to P42, are obtained from wild-type sheep PH20.

[0028] [Table 1]

[0029] [Table 2] TIFF2026065068000004.tif255136TIFF2026065068000005.tif49137

[0030] Hyaluronidases present in the human body include Hyal1, Hyal2, Hyal3, and Hyal4, which are active at acidic pH levels, and PH20, which is expressed in the acrosome of sperm and plays a role in the fertilization process.

[0031] Of these, the human PH20 polypeptide having the amino acid sequence of SEQ ID NO: 1 consists of a signal sequence (M1-T35), a hyaluronidase active site (L36-S490), and a C-terminal GPI-binding sequence (Glycosyl-phosphatidyl inositol-anchored sequence) (A491-L509). Recombinant PH20 polypeptides in which the entire C-terminal GPI-binding sequence is absent and converted to a soluble form are used in vivo as therapeutic agents, dispersants, or sprays. In particular, there are reports that cleavage of any part of the C-terminal amino acid sequence, i.e., any part of I465-L509, maintains both solubility and enzyme activity. When developed as a commercial product, the C-terminus is set to 482. However, in this case, the N-terminal sequence starting at L36, excluding the signal sequence, is maintained (e.g., WO2010 / 077297A).

[0032] In recent years, studies have shown that human PH20 polypeptide variants, when the N-terminus is cleaved, also possess enzymatic activity, starting not only with L36 but also with N37, F38, R39, A40, P41, and P42. Based on this, hyaluronidase variants with superior thermal stability and enzymatic activity have been developed (e.g., WO2020 / 022791A).

[0033] The monkey (Nasalis larvatus) PH20 polypeptide has 93.1% sequence homology with the human PH20 polypeptide. Therefore, based on the multiple sequence analysis results in Figure 4, it can be predicted that the C-terminus of the monkey (Nasalis larvatus) PH20 polypeptide terminates at Y483.

[0034] The PH20 polypeptide of cattle (Bos taurus) and sheep (Ovis aries) has 90.6% sequence homology with each other, and 63.6% and 63.5% sequence homology, respectively, with human PH20 polypeptide. According to Meyer et al. (1997), the soluble polypeptide of cattle (Bos taurus) PH20 polypeptide terminates at H478 in the C-terminus. Based on these results, and based on the multiple sequence analysis results in Figure 4, it can be predicted that the PH20 polypeptide of sheep (Ovis aries) terminates at H477 in the C-terminus.

[0035] This invention presents N-terminal and C-terminal cleaved recombinant PH20 polypeptides with increased productivity, achieved by further cleaving the N-terminus of mature, naturally occurring PH20 polypeptides, i.e., by further deleting amino acid residues. In this invention, N-terminal and C-terminal cleaved recombinant PH20 polypeptides are used with substantially the same concept as "PH20 mutants."

[0036] The recombinant PH20 polypeptide according to the present invention is cleaved before an amino acid residue selected from the group consisting of N37 to P42 in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, resulting in the deletion of the N-terminal amino acid residue.

[0037] Specifically, the recombinant PH20 polypeptide according to the present invention may contain sequences that are cleaved before an amino acid residue selected from the group consisting of N37 to P42, and that begin with N37, F38, R39, A40, P41, or P42 in the amino acid sequence of SEQ ID NO: 1. More specifically, the recombinant PH20 polypeptide according to the present invention may contain sequences that begin with N37 or F38 in the amino acid sequence of SEQ ID NO: 1.

[0038] The fact that the molecule was cleaved before an amino acid residue selected from the group consisting of N37-P42 means that the molecule was cleaved and deleted up to the amino acid residue immediately preceding the N37-P42 amino acid residue at the N-terminus.

[0039] For example, the statement that cleavage occurred before the amino acid residues N37, F38, R39, A40, P41, and P42 means that, in the amino acid sequence of Sequence ID No. 1, cleavage and removal occurred up to residue 36, which is immediately preceding N37; residue 37, which is immediately preceding F38; residue 38, which is immediately preceding R39; residue 39, which is immediately preceding A40; residue 40, which is immediately preceding P41; and residue 41, which is immediately preceding P42.

[0040] The recombinant PH20 polypeptide according to the present invention may further have some amino acid residues deleted at the C-terminus. Specifically, the recombinant PH20 polypeptide according to the present invention may be cleaved after an amino acid residue selected from the group consisting of F468 to Y482 in the amino acid sequence of SEQ ID NO: 1, resulting in the deletion of an amino acid residue at the C-terminus. More specifically, the recombinant PH20 polypeptide according to the present invention may contain a sequence ending at F468 or Y482 in the amino acid sequence of SEQ ID NO: 1.

[0041] The deletion of the C-terminal amino acid residue in the amino acid sequence of SEQ ID NO: 1, where the cleavage occurred after an amino acid residue selected from the group consisting of F468 to Y482, means that the cleavage and deletion began at the amino acid residue immediately following the amino acid residue selected from the group consisting of F468 to Y482. For example, cleavage after the F468 or Y482 residue means that the cleavage and removal began at the residue following F468 or Y482 in the amino acid sequence of SEQ ID NO: 1.

[0042] In specific examples, the recombinant PH20 polypeptide according to the present invention may be selected from the group configured as follows: (1) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 1 that starts at N37 and ends at Y482 (N37-Y482); (2) PH20 polypeptide containing the amino acid sequence of SEQ ID NO: 1, which begins at N37 and ends at F468 (N37-F468); (3) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 1 that begins at F38 and ends at Y482 (F38-Y482); (4) PH20 polypeptide containing the amino acid sequence of SEQ ID NO: 1, which begins at F38 and ends at F468 (F38-Y468); (5) A PH20 polypeptide containing one of the amino acid sequences from SEQ ID NOs. 2 to SEQ ID NOs. 8, which begins at N37 and ends at L490 (N37-L490); (6) A PH20 polypeptide containing one of the amino acid sequences from SEQ ID NOs. 2 to SEQ ID NOs. 8, which begins at F38 and ends at L490 (F38-L490); (7) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 9 that begins at D37 and ends at H478 (D37-H478); (8) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 9 that begins at F38 and ends at H478 (F38-H478); (9) PH20 polypeptide containing the amino acid sequence of SEQ ID NO: 10, which begins at D37 and ends at H477 (D37-H477); (10) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 10 that begins at F38 and ends at H477 (F38-H477); (11) PH20 polypeptide containing the amino acid sequence of SEQ ID NO: 10, which begins at R39 and ends at H477 (R39-H477); and (12) A PH20 polypeptide containing the amino acid sequence of Sequence ID No. 10, which starts at A40 and ends at H477 (A40-H477).

[0043] In other aspects, the present invention provides a cancer treatment composition comprising the recombinant PH20 polypeptide and a cancer treatment method using the same.

[0044] The aforementioned cancers are not particularly limited and include both solid tumors and hematological cancers. Examples of such cancers may be selected from, but are not limited to, the group consisting of skin cancers such as melanoma, liver cancer, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma. Preferably, cancers treatable with the composition of the present invention may be selected from, but are not limited to, the group consisting of colorectal cancer, breast cancer, lung cancer, and kidney cancer.

[0045] The composition may be a pharmaceutical composition. The pharmaceutical composition may further contain a pharmaceutically acceptable carrier, which is commonly used in the formulation of drugs and may be, but is not limited to, one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The pharmaceutical composition may further contain one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives commonly used in the manufacture of pharmaceutical compositions.

[0046] The pharmaceutical composition may be administered orally or parenterally. Parenterally, it may be administered by intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, endothelial infusion, local infusion, intranasal infusion, intrapulmonary infusion, or rectal infusion. Since proteins or peptides are digested during oral administration, the oral composition may be coated with the active agent or formulated to protect it from gastric degradation. Furthermore, the composition may be administered by any device that allows the active substance to be transferred to target cells.

[0047] The pharmaceutical composition may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or it may be formulated in the form of an extract, powder, granule, tablet, or capsule, and may further contain a dispersant or stabilizer for dosage formulation.

[0048] In particular, the cancer treatment composition according to the present invention is characterized by its use in combination therapy with other anticancer agents.

[0049] The anticancer agents that can be used for the aforementioned combination therapy are preferably, but not limited to, chemoanticancer agents, antibody-based anticancer agents, RNAi, and cell therapy agents.

[0050] The anticancer agents that can be used for the aforementioned combination therapy are immunosuppressants, particularly preferably immune checkpoint inhibitors, but are not limited thereto.

[0051] In other words, the present invention relates to a nucleic acid encoding a recombinant PH20 polypeptide in which amino acid residues at the N-terminus and / or C-terminus of mature animal wild-type PH20 according to the present invention are deleted.

[0052] The nucleic acids used herein may be present in cells, cell lysates, or in partially purified or substantially pure forms. The nucleic acids are “isolated” or “substantially purified” when purified from other cellular components or other contaminants, such as nucleic acids or proteins from other cells, by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. The nucleic acids of the present invention may be, for example, DNA or RNA.

[0053] In further aspects, the present invention relates to vectors comprising the nucleic acid, particularly recombinant expression vectors. For the expression of the recombinant PH20 recombinant polypeptide according to the present invention, the DNA encoding the PH20 recombinant polypeptide can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the PH20 recombinant polypeptide), and the DNA may be "operationally bound" to transcriptional and translational regulatory sequences and inserted into the expression vector.

[0054] As used herein, the term "binding in an operational manner" can mean that the gene encoding the PH20 recombinant polypeptide is ligated into the vector such that the transcriptional and translational regulatory sequences within the vector perform the intended function of regulating the transcription and translation of the gene encoding the PH20 recombinant polypeptide. The expression vector and expression regulatory sequences are selected to suit the host cells used for expression. The gene encoding the PH20 recombinant polypeptide is inserted into the expression vector by a standard method (e.g., ligation of the gene fragment encoding the PH20 recombinant polypeptide and complementary restriction enzyme sites on the vector, or blunt-terminus ligation if no restriction enzyme sites are present).

[0055] Furthermore, the recombinant expression vector has a regulatory sequence that controls the expression of the gene encoding the PH20 recombinant polypeptide in the host cell. The "regulatory sequence" may include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals) that control the transcription or translation of the gene encoding the PH20 recombinant polypeptide. A typical technician can recognize that the design of the expression vector may change by separately selecting the regulatory sequence according to factors such as the selection of host cells to be transformed and the level of protein expression.

[0056] In further aspects, the present invention relates to a host cell comprising the nucleic acid or the vector. The host cell according to the present invention is preferably selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells, but is not limited thereto.

[0057] Specifically, the host cells according to the present invention may be prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, or Staphylococcus sp. Alternatively, they may be eukaryotic cells such as fungi like Aspergillus sp., yeasts like Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp., and Neurospora crassa, other lower eukaryotic cells, and cells of higher eukaryotes such as insect cells.

[0058] Furthermore, the host cells according to the present invention may be derived from plants or mammals. Preferably, monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT78 cells, and HEK293 cells are available, but are not limited thereto. Particularly preferably, CHO cells may be used.

[0059] The nucleic acids or vectors described above are transfected or transfected into host cells. To perform transfecting or transfection, exogenous nucleic acids (DNA or RNA) are introduced into prokaryotic or eukaryotic host cells using various commonly used techniques, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. Various host / vector combinations may be used to express the PH20 recombinant polypeptide according to the present invention. Suitable expression vectors for eukaryotic hosts include, but are not limited to, SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus-derived regulatory sequences. Expression vectors usable in bacterial hosts include pET, pRSET, pBluescript, pGEX2T, pUC vectors, bacterial plasmids obtained from Escherichia coli such as col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids with a broader host range such as RP4, phage DNA which can be exemplified as a wide variety of phage lambda derivatives such as λgt10, λgt11, NM989, and other DNA phages such as M13 and filamentous single-strand DNA phages. Useful expression vectors for yeast cells are 2 μm plasmids and their derivatives. A useful vector for insect cells is pVL941.

[0060] In another aspect, the present invention relates to a method for producing a recombinant PH20 recombinant polypeptide according to the present invention, comprising the step of culturing host cells and expressing the recombinant PH20 recombinant polypeptide according to the present invention.

[0061] When a recombinant expression vector capable of expressing the recombinant PH20 recombinant polypeptide is introduced into mammalian host cells, the PH20 recombinant polypeptide may be produced by culturing the host cells for a period of time sufficient for expression in the host cells, or more preferably for a period of time sufficient for the PH20 recombinant polypeptide to be secreted into the culture medium in which the host cells are cultured.

[0062] Depending on the circumstances, the expressed PH20 recombinant polypeptide may be isolated from the host cell and purified to a homogeneous state. The isolation or purification of the PH20 recombinant polypeptide may be carried out by separation and purification methods commonly used for proteins, such as chromatography. The chromatography may be, but is not limited to, one or more combinations selected from affinity chromatography, ion exchange chromatography, or hydrophobic chromatography. In addition to chromatography, filtration, ultrafiltration, salting out, dialysis, etc., may be used in combination.

[0063] The present invention will be described in more detail below with reference to examples. It will be obvious to those ordinary skill in the art that these examples are merely illustrative and should not be construed as limiting the scope of the present invention.

[0064] Example 1. Cloning of recombinant PH20 hyaluronidase with cleaved N-terminus and C-terminus. For the expression of PH20 polypeptide, the following sequences were synthesized by Genscript (South Korea): the sequence encoding amino acids L36 to S509 of the natural human PH20 polypeptide; the DNA sequence encoding amino acids M1 to L510 for the monkey (Nasalis larvatus) PH20 polypeptide (Uniprot ID: H2DJA7); the DNA sequence encoding amino acids L36 to H478 for the bovine (Bos taurus) PH20 polypeptide (Uniprot ID: F1MTV1); and the DNA sequence encoding amino acids L36 to H477 for the sheep (Ovis aries) PH20 polypeptide (Uniprot ID: W5NSU1).

[0065] Each synthesized PH20 polypeptide gene was amplified by polymerase chain reaction (PCR) and inserted into the XhoI and NotI restriction enzyme sites of the pcDNA3.4-TOPO vector. Human serum albumin signal peptide was used for expression in ExpiCHO cells. For protein purification using a HisTrap column, the His-Tag DNA sequence was positioned at the 3' end of the PH20c DNA. N-terminal and C-terminal cleavage of PH20 polypeptides was performed using PCR, and amino acid substitutions were confirmed by DNA sequencing analysis.

[0066] To create mutants of the human PH20 polypeptide with the N-terminus cleaved, a plasmid containing L36-Y482 was prepared and used as a template to sequentially create four mutants in which one more amino acid was removed from the N-terminus. Additionally, four mutants with the C-terminus ending in F468 were created for each of these mutants.

[0067] To create mutants of the monkey (Nasalis larvatus) PH20 polypeptide with the N-terminus cleaved, a plasmid containing L36-Y483 was constructed. Using this as a template, five mutants were sequentially created by removing one more amino acid from the N-terminus.

[0068] To create mutants of bovine (Bos Taurus) PH20 with the N-terminus cleaved, a plasmid containing L36-H478 was prepared and used as a template to sequentially create five mutants in which one amino acid was removed from the N-terminus.

[0069] To create mutants of sheep (Ovis aries) PH20 with the N-terminus cleaved, a plasmid containing L36-H477 was prepared and used as a template to sequentially create five mutants in which one amino acid was removed from the N-terminus.

[0070] Example 2. Expression and purification of recombinant PH20 hyaluronidase with cleaved N-terminus and C-terminus. Expression of PH20 polypeptides with cleaved N-terminus and C-terminus was performed using the ExpiCHO expression system. The cell density of ExpiCHO cells was 6 × 10⁶. 6 When the cell count reached cells / mL, a plasmid containing cDNA of the N-terminal and C-terminal cleaved PH20 polypeptide inserted into the pcDNA3.4-TOPO vector was used to transfect ExpiCHO cells using ExpiFectamine CHO reagent. ExpiCHO expression medium (100-500 mL) was used as the cell culture medium. After transfecting, ExpiCHO cells were cultured with shaking at 130 rpm for a total of 6 days, during which time the cells were cultured at 37°C for 1 day and then at a lower temperature of 32°C for an additional 5 days. After completion of culture, the cell supernatant was collected by centrifugation at 10,000 rpm for 30 minutes.

[0071] PH20 polypeptides with a His-tag attached to the C-terminus, produced in ExpiCHO cells, were purified by two-step column chromatography using AKTA prime equipment or similar equipment (GE Healthcare). For human PH20 and Nasalis larvatus PH20, since the pI was close to 6, anion exchange chromatography (Q Sepharose) was used. For bovine and sheep PH20 polypeptides, since the pI was 8 or higher, one-step purification was performed using cation exchange chromatography (Capto S column). Each protein was then purified in two steps using His-Tag affinity chromatography (HisTrap HP column).

[0072] For protein purification using a Q Sepharose column, buffer A (20 mM sodium phosphate, pH 7.5) and buffer B (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl) were prepared. Proteins were bound to the Q Sepharose column, buffer A was passed through for 5 CV to remove nonspecifically bound proteins, and then buffer B was passed through for 5 CV with a concentration gradient from 0 to 100% to elute the proteins.

[0073] For protein purification using a Capto S column, buffer A (20 mM sodium phosphate, 15 mM NaCl, pH 6.0) and buffer B (20 mM sodium phosphate, 500 mM NaCl, pH 6.0) were prepared. The pH and conductivity of the culture medium were adjusted to match those of buffer A, and the culture medium was filtered through a 0.22 μm pore size membrane. Next, proteins were bound to the Capto S column, and buffer A was passed through the column for 3 CVs to remove non-specifically bound proteins. Buffer B was then passed through the column for 4 CVs to elute the target protein.

[0074] For protein purification 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% buffer B was passed through for 7 CV to remove nonspecifically bound proteins, and 40% buffer B was passed through for 3 CV to elute the target protein. The column eluate was dialyzed using dialysis buffer (20 mM sodium phosphate, 100 mM NaCl, pH 7.0).

[0075] Example 3. N-terminal sequence analysis of recombinant PH20 hyaluronidase with cleaved N-terminus and C-terminus. Purified PH20 polypeptides with cleaved N-terminus and C-terminus were loaded at a rate of 10 μg per lane onto a 7.5% SDS-PAGE gel and subjected to electrophoresis (150 V, 1 hour). The gel containing the developed proteins was then placed in a blotting kit along with a PVDF membrane and transferred at 100 V for 90 minutes. Ponceau S staining was used to confirm successful transfer. Finally, samples obtained by dissecting each protein band were sequenced using a PPSQ-53A protein sequencer (Shimadzu, Japan) to analyze the five amino acids at the N-terminus.

[0076] N-terminal sequence analysis confirmed the expected sequences in other PH20 polypeptides. However, in the human PH20 polypeptides N37-Y482 and N37-F468, the N-terminal amino acid was partially found aspartate instead of asparagine, indicating de-amidation. Based on these results and the multiple sequence analysis results in Figure 4, since this amino acid is found aspartate in cattle or sheep, it can be inferred that aspartate plays a role in stabilizing the protein structure at this position.

[0077] Example 4. Measurement of recombinant PH20 hyaluronidase activity with cleaved N-terminus and C-terminus. Hyaluronidase activity was measured by turbidity analysis, which measures the absorbance of the turbidity generated by the precipitate formed when hyaluronic acid is mixed with albumin (BSA). When hyaluronic acid is hydrolyzed by a pH20 polypeptide, the turbidity / absorbance of the precipitate formed when mixed with albumin decreases. This analysis is generally performed at pH 5.3 as follows: Hyaluronidase standards with known activity (units) were diluted to 6, 8, 10, 12, 15, and 20 units / mL and prepared in separate test tubes. The purified protein samples were diluted with buffer (20 mM sodium phosphate, pH 5.3, 77 mM sodium chloride, and 0.01% (w / v) bovine serum albumin) by adjusting the dilution factors to fall within the standard curve range. 50 μl of the diluted sample was dispensed into each well of a 96-well plate and incubated at 37°C for 10 minutes. 50 μl of 0.06% hyaluronic acid was further dispensed into each well. The 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 was complete, 40 μl of the enzyme-substrate reaction solution was dispensed into 200 μl of acidic albumin solution and left at room temperature for 19 minutes. Then, the absorbance was measured at 600 nm using a spectrophotometer. The acidic albumin solution was a solution in which 0.1% albumin (BSA) was dissolved in 24 mM sodium acetate, 79 mM acetic acid, pH 3.75 buffer. The measured absorbance values ​​of the sample were converted to activity using a standard curve with an activity standard.

[0078] When the above process was performed at pH 7.0, the protein sample buffer was 20 mM sodium phosphate, pH 7.0, 77 mM sodium chloride, and 0.01% (w / v) bovine serum albumin. The 0.06% hyaluronic acid aqueous solution was prepared by dissolving it in 20 mM sodium phosphate buffer, pH 7.0, 70 mM sodium chloride, and the same process was carried out.

[0079] While this type of activity measurement is also possible in culture medium, values ​​below 300 units / mL are unreliable in this case. Therefore, the Limit of Quantification (LOQ) was set to 300 units / mL, and values ​​below this were indicated as no activity or 0. Furthermore, when measuring activity using purified protein samples, the Limit of Quantification (LOQ) was set to 15 units / μg.

[0080] The N-terminus of the natural PH20 polypeptide of Sequence ID No. 1 was adjusted to L36, N37, F38, R39, and A40 in the portion excluding the M1-T35 signal sequence, and F468 and Y482 were selected as the C-terminus. A total of 10 PH20 polypeptides with cleaved N-terminus and C-terminus were prepared using the method of Example 1. Each of the clones thus prepared was purified in a culture medium obtained by transient transfection of animal cells, and the enzyme activity was compared at pH 5.3 and pH 7.0 using the method of Example 2.

[0081] As shown in Figure 1, when comparing the human PH20 activity of each culture medium, the activity in the N37-Y482 culture medium was surprisingly more than three times higher than the activity in the mature L36-Y482 culture medium which has a natural N-terminus. Furthermore, even in the case of F38-Y482, which has two more amino acid residues deleted from the N-terminus, the human PH20 activity in the culture medium was more than twice as high.

[0082] However, enzyme expression was almost nonexistent in R39-Y482, which lacked three N-terminal amino acid residues, and even when four N-terminal amino acid residues were missing, enzyme expression in the culture medium was almost nonexistent.

[0083] In conclusion, we confirmed that mutant human hyaluronidase PH20, in which one or two N-terminal amino acid residues are missing in mature human hyaluronidase PH20, shows a significant increase in expression levels in culture medium. In particular, considering the industrial potential of human hyaluronidase PH20 as an injectable drug, we demonstrate that producing mutant enzymes in a form with one or two N-terminal amino acid cleavage can yield industrially superior economic viability.

[0084] Furthermore, as shown in Figures 2 and 3, when the specific activity of L36-Y482, N37-Y482, and F38-Y482 was measured, there was almost no difference in specific activity. Therefore, it was confirmed that the difference in human hyaluronidase PH20 activity shown in Figure 1 was due to differences in productivity. Moreover, among the PH20 mutants, which have 14 fewer C-terminal amino acids compared to the protein ending at Y482, only N37-F468, which lacks one N-terminal amino acid, showed activity in the culture medium.

[0085] In particular, Figure 2 shows the specific activity of each purified polypeptide at pH 5.3. When the C-terminus was Y482, there was almost no difference between L36-Y482, N37-Y482, and F38-Y482. However, when the C-terminus was F468, the specific activity of N37-F468 and F38-F468 was found to be about 1.5 to 2 times higher than that of L36-F468. This trend was also confirmed from the specific activity at pH 7.0.

[0086] In other words, as can be seen in the results in Figures 2 and 3, when the C-terminal amino acid of PH20 was further shortened beyond the 482 position of the currently commercially available PH20 and cleaved to terminate at position 468, L36-F468 was hardly expressed in animal cells. In this case, when the minute amount of expressed L36-F468 mutant was purified and its specific activity was measured, it was found that the specific activity of the enzyme was at about 60% of that of L36-Y482. From these facts, it can be seen that the hyaluronidase activity or productivity of the PH20 polypeptide increases significantly when one or two more amino acid residues are deleted from the N-terminal side of mature human hyaluronidase PH20.

[0087] In conclusion, while the specific activity of the human hyaluronidase PH20 mutant enzyme composed of L36-F468 did not decrease significantly compared to the L36-Y482 PH20 mutant enzyme, its expression in recombinant cells was extremely low, indicating that it is a PH20 mutant in a form unsuitable for industrially useful applications.

[0088] On the other hand, the N37-F468 mutant showed a significant increase in expression in cells, a trend similar to that of the L36-Y482 mutant. However, the intracellular expression level of N37-F468, which has one more N-terminal amino acid cleaved, was found to be at least 100 times higher than that of L36-F468.

[0089] Combining molecular modeling of PH20 with the experimental results of the present invention, it can be seen that the interaction between the N-terminal amino acid and the C-terminal amino acid of PH20 affects the stability and folding rate of the recombinant PH20 protein during transcription and transfer processes when the recombinant PH20 protein is expressed in cells, and further significantly affects the expression level of the recombinant protein secreted from the cell. This has a very significant impact on the productivity of PH20 and its mutants. These results can be applied not only to human PH20 but also to mammalian-derived PH20 extracted from cattle or sheep, and it can be predicted that they can be applied to other PH20 molecules that have structurally or physicochemically similar properties to human PH20.

[0090] Furthermore, as shown in Figures 5 and 6, the characteristics and sequence structure of human PH20 polypeptides were similar in monkeys (Nasalis larvatus), cattle (Bos taurus), and sheep (Ovis aries). This suggests that these characteristics are maintained in mammalian PH20, particularly in the monkeys (Nasalis larvatus), cattle (Bos taurus), and sheep (Ovis aries) listed in Table 1, where the biological functions are identical. In particular, sheep PH20 polypeptides were found to be highly expressed when the N-terminus began with A40.

[0091] In short, according to the results of this invention, in mature PH20 proteins, particularly animal PH20 such as human PH20, the expression rate in recombinant cells was dramatically increased and protein productivity was enhanced by removing one to four, preferably one or two, amino acid residues from the N-terminal region.

[0092] When human PH20 terminates at Y482 in the C-terminus, PH20 with further removal of some N-terminal amino acid residues in mature PH20 showed a significant increase in expression without major changes in the intrinsic activity of hyaluronidase. When the C-terminus terminates at F468, mutants with one or two further N-terminal amino acid residues removed in mature PH20 showed an increase in enzyme specific activity compared to the protein with a mature N-terminus. While there have been attempts to increase the specific activity of human PH20 by substituting specific amino acids with other amino acids, there have been no previous cases of significantly increasing the expression rate in cells while maintaining or increasing protein activity by removing N-terminal amino acids. It can be predicted that N-terminal deletion mutants will be advantageous in terms of immunogenicity even when administered to humans over a long period of time, compared to PH20 with amino acid substitution at specific sites.

[0093] While there are reports of increased expression levels after N-terminal cleavage in mature proteins, such as with Human alpha1-antitrypsin (H. Johansen et al., Mol. Biol. Med. 1987, 4:291-305) and Human papillomavirus L1 protein (M. Wei et al., Emerging Microbes & Infections, 2018, 7:160), it is difficult to find common similarities in the position of amino acids at the N-terminus and the number of cleavage segments in each case. For example, in Human alpha1-antitrypsin, cleavage of 5 to 10 N-terminal amino acids increased intracellular expression levels, but enzyme activity remained unchanged. In Human papillomavirus L1 protein, cleavage of 10 or 15 N-terminal amino acids did not change the total protein expression level, but sometimes increased protein solubility. In particular, no such attempts had been made with hyaluronidases like PH20, and no cases had been found where cleavage of one or two N-terminal amino acids, as in the present invention, dramatically increased expression levels and activity.

[0094] The tertiary crystal structure of human PH20 has not yet been determined. However, using a program to predict the tertiary structure of proteins, the results of predicting the tertiary structure of PH20 show that amino acids such as F38, R39, and Y434 are located close to the asparagine side chain at position 37 at the N-terminus of PH20, forming appropriate charge interactions. In contrast, Leu36, an amino acid of mature proteins, does not form specific bonds with surrounding amino acids, and the exposure of this hydrophobic amino acid in aqueous solution is predicted to negatively affect the rate or stability of binding and, consequently, the expression of the protein during its expression.

[0095] In mature wild-type animal PH20 proteins, the same results as the present invention can be predicted even in mutants in which amino acids are substituted within a range that does not significantly modify the protein's tertiary structure. The effect was confirmed in cattle and sheep PH20 proteins with homology to human PH20 of 63.6% and 63.5%, respectively, as well as in primate PH20 proteins with homology of 93.1% or more. Therefore, the same results as the present invention can be predicted for PH20 proteins with homology to each other of PH20, such as 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, within a range that does not significantly modify the protein's tertiary structure.

[0096] Example 5. Preparation and activity measurement of recombinant PH20 hyaluronidase with different signal peptides and cleaved N-terminus and C-terminus. To confirm the changes induced by signal peptides, nine mutants were created by substituting human PH20, human serum hormones, or Ig kappa signal peptides. The signal peptide sequences are shown in Table 2 below.

[0097] In the previous example, the signal peptide used was human serum albumin (HSA). We then investigated whether similar results could be obtained by applying other signal peptides listed in Table 2 to this signal peptide.

[0098] Recombinant human PH20 L36-Y482, N37-Y482, and F38-Y482 were prepared in the same manner as in Examples 1 and 2, except for the application of the signal peptides in Table 2. Activity analysis was performed as in Example 4, and the results are shown in Figure 7.

[0099] As shown in Figure 7, when using human PH20 or Ig kappa signal peptides, a similar trend to the increase in productivity observed when further amino acid deletions occur at the N-terminus of mature PH20 were observed, similar to when using human serum hormones. This confirmed that the phenomenon of increased expression and productivity when further amino acid deletions occur at the N-terminus of mature PH20 is not strongly related to the type of signal peptide. [Industrial applicability]

[0100] The PH20 polypeptides presented in this invention, with increased enzyme activity and productivity, exhibit superior expression levels and higher enzyme activity compared to existing recombinant PH20 polypeptides, potentially leading to reduced treatment costs through lower production costs in industrial applications.

[0101] Having described in detail certain aspects of the present invention, it will be clear to those with ordinary skill in the art that such specific descriptions merely represent preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0102] References 1. H. Johansen, J. Sultiphong, G. Sathe, P. Jacobs, A. Cravador, A. Bollen, M. Rosenberg, and A. Shatzman, “High-level production of fully active human alpha 1-antitrypsin in Escherichia coli.” Mol. Biol. Med. (1987) 4:291-305, 2. J.H. Dunham, R.C. Meyer, E.L. Garcia, and R.A. Hall, “GPR37 Surface Expression Enhancement via N-Terminal Truncation or Protein-Protein Interactions”, Biochemistry (2009) 48:10286-10297 3. M. Wei, D. Wang, Z. Li, S. Song, X. Kong, X. Mo, Y. Yang, M. He, Z. Li, B. Huang, Z. Lin, H. Pan, Q. Zheng, H. Yu, Y. Gu, J. Zhang, S. Li and N. Xia, “N-terminal truncations on L1 proteins of human papillomaviruses promote their soluble expression in Escherichia coli and self-assembly in vitro”, Emerging Microbes & Infections (2018) 7:160 4. M. F. Meyer, G. Kreil, and H. Aschauer, “The soluble hyaluronidase from bull testes is a fragment of the membrane-bound PH-20 enzyme”, FEBS letter (1997) 413:385-388

Claims

1. Recombinant PH20 polypeptide in which one to seven amino acid residues are deleted from the N-terminus of mature animal wild-type PH20.

2. (a) Recombinant PH20 polypeptide having the amino acid sequence of Sequence ID No. 1, wherein the N-terminal amino acid residue is deleted by cleavage before an amino acid residue selected from the group consisting of N37 to P42; (b) Recombinant PH20 polypeptides in which a primate wild-type PH20 having one of the amino acid sequences from SEQ ID NOs: 2 to 8 is cleaved before an amino acid residue selected from the group consisting of N37 to P42, resulting in the deletion of the N-terminal amino acid residue; (c) Recombinant PH20 polypeptide having the amino acid sequence of Sequence ID No. 9, wherein the N-terminal amino acid residue is deleted by cleavage before an amino acid residue selected from the group consisting of D37 to P42; and (d) Recombinant PH20 polypeptide having the amino acid sequence of Sequence ID No. 10, in which the N-terminal amino acid residue is deleted by cleavage before an amino acid residue selected from the group consisting of D37 to P42; The recombinant PH20 polypeptide according to claim 1, characterized by being selected from the group consisting of the following.

3. The recombinant PH20 polypeptide according to claim 1, comprising sequences beginning with N37, F38, R39, A40, P41, and P42 among the amino acid sequences of SEQ ID NO:

1.

4. The recombinant PH20 polypeptide according to claim 1, comprising a sequence beginning with N37 or F38 in the amino acid sequence of SEQ ID NO:

1.

5. The recombinant PH20 polypeptide according to claim 1, wherein some amino acid residues at the C-terminus are further deleted.

6. The recombinant PH20 polypeptide according to claim 5, characterized in that the amino acid sequence of SEQ ID NO: 1 is cleaved after an amino acid residue selected from the group consisting of F468 to Y482, resulting in the deletion of a C-terminal amino acid residue.

7. The recombinant PH20 polypeptide according to claim 6, comprising a sequence ending in F468 or Y482 among the amino acid sequences of SEQ ID NO:

1.

8. The recombinant PH20 polypeptide according to claim 1, characterized by being selected from the following group: (1) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 1 that begins at N37 and ends at Y482 (N37-Y482); (2) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 1 that begins at N37 and ends at F468 (N37-F468); (3) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 1 that begins at F38 and ends at Y482 (F38-Y482); (4) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 1 that begins at F38 and ends at F468 (F38-Y468); (5) A PH20 polypeptide containing one of the amino acid sequences from SEQ ID NOs: 2 to 8, which begins at N37 and ends at L490 (N37-L490); (6) A PH20 polypeptide containing one of the amino acid sequences from SEQ ID NOs: 2 to 8, which begins at F38 and ends at L490 (F38-L490); (7) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 9 that begins at D37 and ends at H478 (D37-H478); (8) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 9 that begins at F38 and ends at H478 (F38-H478); (9) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 10 that begins at D37 and ends at H477 (D37-H477); (10) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 10 that begins at F38 and ends at H477 (F38-H477); (11) PH20 polypeptide containing the amino acid sequence of Sequence ID No. 10 that begins at R39 and ends at H477 (R39-H477); and (12) A PH20 polypeptide containing the amino acid sequence of Sequence ID No. 10, which begins at A40 and ends at H477 (A40-H477).

9. A nucleic acid encoding a recombinant PH20 polypeptide according to any one of claims 1 to 8.

10. A recombinant expression vector comprising the nucleic acid described in claim 9.

11. A host cell transformed with the recombinant expression vector described in claim 10.

12. The host cell according to claim 11, characterized in that it is selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells.

13. A method for producing recombinant PH20 polypeptide, comprising the step of culturing the host cells described in claim 12.

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