Pharmaceutical composition for subcutaneous injection, comprising growth hormone receptor antagonist and long-acting carrier

Abstract

The present invention relates to: a fusion protein comprising a growth hormone receptor antagonist and a long-acting carrier, the growth hormone receptor antagonist comprising a growth hormone variant in which one or more amino acids in the amino acid sequence of growth hormone are substituted with another amino acid; and a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, comprising the fusion protein. A pharmaceutical composition comprising a growth hormone receptor inhibitor, a carrier, and hyaluronidase, according to the present invention, may have strong binding affinity to the growth hormone receptor and may exhibit sustained antagonism. A pharmaceutical composition for subcutaneous injection, comprising a growth hormone receptor inhibitor, a carrier, and hyaluronidase, according to the present invention, may increase the amount of a drug for subcutaneous injection, thereby increasing the amount of the drug absorbed into the human body without a rapid increase in the concentration of the growth hormone receptor inhibitor.

Description

Subcutaneous injection pharmaceutical composition comprising a growth hormone receptor antagonist and a long-acting carrier

[0001] The present invention relates to a fusion protein comprising a growth hormone receptor antagonist comprising a growth hormone variant in which one or more amino acids in the amino acid sequence of growth hormone are substituted with other amino acids, and a long-acting carrier, and a pharmaceutical composition comprising the fusion protein, preferably a pharmaceutical composition for subcutaneous injection.

[0002]

[0003] Growth hormone is an endocrine hormone that promotes growth. Human growth hormone (hereinafter referred to as "hGH") is secreted by the pituitary gland and acts on various tissues, including the liver. This hGH binds to the extracellular domain of the human growth hormone receptor (hGHR), which belongs to the class I cytokine receptor superfamily (JF Bazan, Haemopoietic receptors and helical cytokines, Immunol. Today. 11 (1990) 350-354.), and this binding leads to subsequent signaling via the dimerized hGHR, which increases the expression of insulin-like growth factor I (IGF-1) (F.F. Casanueva, Physiology of growth hormone secretion and action, Endocrinol. Metab. Clin. North Am. 21 (1992) 483-517.; H. Li, P.M. Bartold, C.Z. Zhang, R.W. Clarkson, W.G. Young, M.J. Waters, Growth hormone and insulin-like growth factor I induce bone morphogenetic proteins 2 and 4: a mediator role in bone and tooth formation?, Endocrinology. 139 (1998) 3855-3862.).

[0004] Excessive hGH secretion and subsequent increased IGF-1 production lead to acromegaly, a chronic condition characterized by enlarged hands and feet. Surgical removal of both pituitary glands, radiation therapy, and dopamine agonists are among the treatments for acromegaly. However, there is a group of patients who do not respond to surgical treatment (B. Swearingen, FG Barker, L. Katznelson, BM Biller, S. Grinspoon, A. Klibanski, N. Moayeri, PM Black, NT Zervas, Long-term mortality after transsphenoidal surgery and adjunctive therapy for acromegaly, J. Clin. Endocrinol. Metab. 83 (1998) 3419-3426.; S. Ahmed, M. Elsheikh, IM Stratton, RC Page, CB Adams, JA Wass, Outcome of transphenoidal surgery for acromegaly and its relationship to surgical experience, Clin. Endocrinol. (Oxf.). 50 (1999) 561-567.), and radiotherapy is not only ineffective but also has a delayed effect (AL Barkan, I. Halasz, KJ Dornfeld, CA Jaffe, RD Friberg, WF Chandler, HM Sandler, Pituitary irradiation is ineffective in normalizing plasma insulin-like growth factor I in patients with acromegaly, J. Clin. Endocrinol. Metab. 82 (1997) 3187-3191. doi:10.1210 / jcem.82.10.4249.; A.J. van der Lely, WWde Herder, SW Lamberts, The role of radiotherapy in acromegaly, J. Clin. Endocrinol. Metab. 82 (1997) 3185-3186.). hGH receptor antagonists (hereafter, “hGHRAs”) are an alternative treatment option because they occupy the hGH receptor instead of hGH, thereby preventing hGH from binding to the hGH receptor (JJ Kopchick, C. Parkinson, EC Stevens, PJ Trainer, Growth Hormone Receptor Antagonists: Discovery, Development, and Use in Patients with Acromegaly, Endocr. Rev. 23 (2002) 623-646. doi:10.1210 / er.2001-0022.; AF Muller, JJ Kopchick, A. Flyvbjerg, VD Lely, A. Jan, Growth Hormone Receptor Antagonists, J. Clin. Endocrinol. Metab. 89 (2004) 1503-1511.).

[0005] hGHRA, which requires long-term administration for treatment, can improve patients' quality of life by reducing the frequency of administration. Pegvisomant is a commercially available pegylated version of the hGH antagonist (MO Thorner, CJ Strasburger, Z. Wu, M. Straume, M. Bidlingmaier, SS Pezzoli, K. Zib, JC Scarlett, WF Bennett, Growth hormone (GH) receptor blockade with a PEG-modified GH (B2036-PEG) lowers serum insulin-like growth factor-I but does not acutely stimulate serum GH, J. Clin. Endocrinol. Metab. 84 (1999) 2098-2103.; V. Goffin, P. Touraine, Pegvisomant. Pharmacia, Curr. Opin. Investig. Drugs London. Engl. 2000. 3 (2002) 752-757.). Pegvisomant's long-term mechanism of action is to attach a polyethylene glycol polymer to the therapeutic protein, thereby increasing its molecular size and allowing it to bypass the kidneys and remain in the bloodstream (RJ Ross, KC Leung, M. Maamra, W. Bennett, N. Doyle, MJ Waters, KK Ho, Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimer, J. Clin. Endocrinol. Metab. 86 (2001) 1716-1723.).Another advantage of PEGylation is that it protects the protein from proteases (S. Jevsevar, M. Kunstelj, VG Porekar, PEGylation of therapeutic proteins, Biotechnol. J. 5 (2010) 113-128). However, PEGylation of therapeutic proteins may have limited utility. The PEGylation process requires a series of chemical reactions, which adds to the production cost, and it is difficult to obtain a homogeneous reaction product, necessitating additional purification steps. Furthermore, although PEGylated proteins appear to be safe when using small polyethylene glycols (e.g., 5 kDa), safety concerns remain, as animal studies have reported renal vacuolation and the appearance of antibodies to PEGylation. Furthermore, PEGylated proteins tend to have reduced binding affinity to protein receptors compared to native proteins (RP Garay, R. El-Gewely, J.K. Armstrong, G. Garratty, P. Richette, Antibodies against polyethylene glycol in healthy subjects and in patients treated with PEG-conjugated agents, Expert Opin. Drug Deliv. 9 (2012) 1319-1323. ; A. Bendele, J. Seely, C. Richey, G. Sennello, G. Shopp, Short communication: renal tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins, Toxicol. Sci. Off. J. Soc. Toxicol. 42 (1998) 152-157.; VL Elliott, GT Edge, MM Phelan, L.-Y. Lian, R. Webster, RFFinn, BK Park, NR Kitteringham, Evidence for metabolic cleavage of a PEGylated protein in vivo using multiple analytical methodologies, Mol. Pharm. 9 (2012) 1291-1301.). Although the exact reason is not known, Somavert (Pfizer, USA), a pegvisomant formulation approved as a PEGylated hGHRA, is administered as a daily subcutaneous injection, which is insufficient in terms of improving the quality of life of patients.

[0006]

[0007] Human growth hormone is typically administered via subcutaneous injection once to seven times a week. However, repeated subcutaneous injections into the same area can cause side effects such as adipose tissue atrophy. However, subcutaneous administration (or subcutaneous injection) of hyaluronidase together with therapeutic drugs causes the hyaluronidase to hydrolyze hyaluronic acid distributed in the extracellular matrix, reducing the viscosity of the subcutaneous tissue and increasing substance permeability. This reduces side effects and facilitates the delivery of growth hormone into the body.

[0008] There are six types of hyaluronidase genes in humans: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20 / SPAM1. Hyal1 and Hyal2 are expressed in most tissues, and PH20 / SPAM1 (hereinafter referred to as PH20) is expressed in the plasma membrane and acrosome membrane of sperm. HyalPS1 is a pseudogene and is not expressed. PH20 is an enzyme (EC 3.2.1.35) that cleaves the β-1,4 bond between N-acetylglucosamine and glucuronic acid, which are constituent sugars of hyaluronic acid. Human hyaluronidase PH20 has an optimal pH of 5.5 but shows some activity at pH 7-8, whereas other human hyaluronidases, including Hyal1, have an optimal pH of 3-4 and are very weakly active at pH 7-8. Since the human subcutaneous area is approximately neutral at pH 7.4, among the various types of hyaluronidase, PH20 is widely used in clinical practice. Examples of clinical uses of PH20 include subcutaneous injection of antibody therapeutics, as an ocular relaxant and anesthetic injection additive during ophthalmic surgery, hydrolyzing hyaluronic acid in the extracellular matrix of tumor cells to increase the accessibility of anticancer therapeutics to tumor cells, and promoting the reabsorption of excessive body fluids and blood within tissues.

[0009] Meanwhile, the PH20 currently widely used commercially is extracted from the testes of cows or sheep. Examples include Amphadase (bovine hyaluronidase) and Vitrase (sheep hyaluronidase). Repeated administration of high doses of animal-derived hyaluronidase to humans may produce neutralizing antibodies, and other animal-derived biological substances included as impurities in addition to PH20 may cause allergies. In particular, the use of PH20 extracted from cows is restricted due to concerns about mad cow disease. To improve these problems, research on recombinant human PH20 proteins has been conducted.

[0010] Recombinant human PH20 proteins have been reported to be expressed in yeast (P. pastoris), DS-2 insect cells, and animal cells (Chen et al., 2016; Hofinger et al., 2007). Recombinant PH20 proteins produced in insect cells and yeast differ from human PH20 in the pattern of N-glycosylation during posttranslational protein modification.

[0011] A structure-function relationship study on human PH20 reported that the C-terminal region of PH20 is important for protein expression and enzyme activity, and in particular, the C-terminal region ending at amino acids 477-483 is reported to be important for enzyme expression and activity (Frost, 2007). The activity of full-length PH20 (amino acids 1-509) or a PH20 mutant with a C-terminal truncation after amino acid 467 was less than 10% of the enzyme activity of a PH20 mutant with a C-terminal truncation at either amino acid 477 or 483 (Frost, 2007). Halozyme Therapeutics developed rHuPH20 (amino acids 36-482), a recombinant protein in which the C-terminus of mature PH20 was truncated at Y482 (Bookbinder et al., 2006; Frost, 2007).

[0012] Research is being conducted to develop various therapeutic drugs in the form of subcutaneous injection formulations using human PH20, and under this technical background, the applicant has confirmed that a human PH20 variant having one or more amino acid substitutions in the amino acid sequence of wild-type hyaluronidase PH20 at the alpha helix 8 site (S347-C381) and the junction site of alpha helix 7 and alpha helix 8 (A333-R346), and in which some of the amino acids located at the N-terminal and / or C-terminal portions of PH20 are truncated, has excellent enzyme activity and thermal stability, and has filed a patent application (WO 2020-022791 A1).

[0013] In addition, the applicant has confirmed that the PH20 variant according to the applicant's prior invention can be applied to a pharmaceutical composition or formulation containing various different drugs, and thus, the pharmaceutical composition and formulation containing the PH20 variant together with various drugs can be used for subcutaneous administration, and the activity of the drugs and the PH20 variant is very stable and can be maintained for a long period of time (WO 2020-197230 A1, WO 2021-150079 A1).

[0014]

[0015] In order to solve the above problems, the present invention aims to provide a subcutaneous pharmaceutical composition of a growth hormone receptor antagonist fusion protein that fuses a more effective growth hormone receptor antagonist with a carrier as a cost-effective and active alternative candidate to hGHRA.

[0016] In addition, the present invention aims to provide a pharmaceutical composition for subcutaneous injection that increases the content of the injection drug and / or increases the subcutaneous absorption rate of the drug in order to eliminate the inconvenience of having to inject a conventional growth hormone receptor antagonist every day.

[0017] An example of a candidate growth hormone receptor antagonist is the antagonist disclosed in Korean Patent Publication No. 10-2019-0074958. The present invention aims to provide a suitable carrier and hyaluronidase through research and improvement of such a candidate, and to provide a pharmaceutical composition for subcutaneous injection that is suitable for use in terms of pharmacokinetics and pharmacodynamics. In particular, the present invention aims to provide a method for increasing the amount of drug absorbed into the body without a rapid increase in drug concentration by using hyaluronidase as a method for increasing the amount of drug absorbed into the body.

[0018]

[0019] One object of the present invention is to provide a pharmaceutical composition for subcutaneous injection comprising a fusion protein of a growth hormone receptor antagonist and a carrier, and hyaluronidase, which comprises a growth hormone variant in which one or more amino acids in the amino acid sequence of natural human growth hormone are substituted with other amino acids.

[0020] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by human growth hormone, comprising a growth hormone receptor antagonist fusion protein and hyaluronidase.

[0021] Another object of the present invention is to provide a pharmaceutical composition comprising a carrier capable of increasing the biological activity of the growth hormone receptor antagonist and improving the pharmacodynamic activity and / or pharmacodynamic activity.

[0022]

[0023] Below, the configuration of the present invention to achieve the above purpose is described in more detail.

[0024]

[0025] Each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, it will be apparent to those skilled in the art that the scope of the present invention is not limited by the specific descriptions set forth below. Furthermore, those skilled in the art will recognize or be able to ascertain, through routine experimentation, numerous equivalents to the specific embodiments of the present invention described in this application. Furthermore, such equivalents are intended to be encompassed by the present invention.

[0026]

[0027] As an aspect of the present invention for solving the above problem, the present invention provides an antagonist-carrier fusion protein suitable for increasing the biological activity of a growth hormone receptor antagonist, which comprises a growth hormone variant in which one or more amino acids in the amino acid sequence of a natural human growth hormone are substituted with other amino acids, and provides a subcutaneous injection formulation capable of improving pharmacokinetic / pharmacodynamic activity.

[0028] The term "growth hormone" used herein refers to a protein hormone secreted by the pituitary gland that promotes body growth. In addition to promoting body growth, it also has metabolic regulatory functions. Specifically, growth hormone may be human growth hormone (hGH; used interchangeably with "hGH" in the specification and drawings below). Natural hGH, as is known, is composed of 191 amino acids.

[0029] The term "growth hormone mutant" herein refers to a growth hormone in which one or more amino acids in the amino acid sequence of the growth hormone are substituted with other amino acids. In other words, it refers to a growth hormone having one or more amino acid substitutions.

[0030] Specifically, the substitution may include a substitution of the 120th amino acid in the amino acid sequence of the growth hormone (more specifically, a substitution with lysine or arginine). It may also include a substitution of the 46th amino acid (more specifically, a substitution with lysine).

[0031] Additionally, the substitution may include a substitution of amino acid position 174 (more specifically, a substitution with serine) in the amino acid sequence of the growth hormone, and may include a substitution of amino acid position 21 (more specifically, a substitution with asparagine).

[0032] Specifically, the substitution may include a substitution at any one or more positions selected from the group consisting of the 18th amino acid, the 21st amino acid, the 46th amino acid, the 54th amino acid, the 64th amino acid, the 120th amino acid, the 167th amino acid, the 168th amino acid, the 171st amino acid, the 172nd amino acid, the 174th amino acid, the 176th amino acid, and the 179th amino acid in the amino acid sequence.

[0033] More specifically, the substitution may include one or more substitutions selected from H18D, H21N, Q46K, F54P, R64K, G120K, R167N, K168A, D171S, K172R, E174S, F176Y, and I179T in the amino acid sequence of the growth hormone.

[0034] In some cases, the above variants may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, or amidation.

[0035] More specifically, the above variant may include those described in Republic of Korea Patent Publication No. 10-2019-0074958.

[0036]

[0037] As a non-limiting example herein, the growth hormone variant may be a protein having an amino acid sequence of any one of SEQ ID NOs: 1 to 9 disclosed in Table 1 below. SEQ ID NO: 10 represents a human growth hormone sequence.

[0038] 서열번호 1FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF서열번호 2FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEERIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF서열번호 3FPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVSTFLRIVQCRSVEGSCGF서열번호 4FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVSTFLRTVQCRSVEGSCGF서열번호 5FPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVSTYLRIVQCRSVEGSCGF서열번호6FPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVSTYLRIVQCRSVEGSCGF서열번호 7FPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRIVQCRSVEGSCGF서열번호 8FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGF서열번호 9FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLKNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVSTFLRTVQCRSVEGSCGF서열번호 10FPTIPLSRLFQNAMLRAHRLHQLAFDTYEEFEEAYIPKEQKYSFLQAPQASLCFSESIPTPSNREQAQQKSNLQLLRISLLLIQSWLEPVGFLRSVFANSLVYGASDSDVYDLLKDLEEGIQTLMGRLEDGSPRTGQAFKQTYAKFDANSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0039]

[0040] The term "Growth Hormone Receptor" (hereinafter, "GHR" and interchangeably used in this specification and drawings) used herein refers to a receptor to which growth hormone binds and transmits signals into cells. It has a structure that penetrates the cell membrane once, and when the receptor is activated, STAT dimers regulate the transcription of various genes within the nucleus through the JAK / STAT pathway. Growth hormone receptors are found in tissues throughout the body, including the liver, muscle, fat, kidney, and tissues of early embryos and fetuses. When growth hormone binds to the receptor, subsequent signaling increases the secretion of IGF (insulin-like growth factor)-1. Excessive secretion of hGH and the resulting increase in IGF-1 production can cause acromegaly, a chronic disease with a typical symptom of enlarged hands and feet.

[0041] The term "growth hormone receptor antagonist" as used herein refers to an agent that antagonizes the binding of growth hormone to the growth hormone receptor, thereby suppressing the side effects that arise from excessive binding of growth hormone to the growth hormone receptor. Specifically, the growth hormone receptor antagonist may be a growth hormone variant that has high binding affinity to the growth hormone receptor and can competitively antagonize the effects of growth hormone.

[0042] Additionally, the growth hormone receptor antagonist may be a fusion protein fused with a long-acting carrier.

[0043] As used herein, the term "long-acting carrier" refers to a substance capable of increasing the in vivo half-life. By incorporating various known long-acting carriers capable of increasing the in vivo half-life into the growth hormone variant according to the present invention, a long-acting agent can be used that antagonizes the growth hormone receptor while also increasing the in vivo half-life.

[0044] As examples of persistent carriers that are not limited in the present invention, various carriers capable of reducing renal clearance, specifically, at least one selected from the group consisting of polyethylene glycol, fatty acids, albumin or fragments thereof, albumin-binding substances, alpha-1 antitrypsin or variants thereof, immunoglobulin Fc or fragments thereof, repeating unit polymers of a specific amino acid sequence, antibodies or fragments thereof, FcRn binding substances, in vivo connective tissues or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and high molecular weight polymers, may be used. More specifically, alpha-1 antitrypsin (hereinafter, interchangeably referred to as “A1AT” in the specification and drawings) or variants thereof may be used as the persistent carrier.

[0045] Alpha-1 antitrypsin or its variants are disclosed in Korean Patent Publication Nos. 10-2013-0136883 and 10-2013-0029713. Specifically, A1AT is one of the most abundant proteins in human plasma, with a concentration of 1.5-3.5 grams per liter, and is primarily synthesized in hepatocytes and secreted into the blood. The alpha-1 antitrypsin variants are engineered to have additional mutations in A1AT to increase glycosylation and eliminate intrinsic activity. The alpha-1 antitrypsin variants can be fused to target proteins to extend their half-life.

[0046] One of the key advantages of alpha-1 antitrypsin variant technology is its non-immunogenicity. In particular, human plasma A1AT has already been used to treat patients with A1AT deficiency due to emphysema and other lung diseases, at a very high weekly dose of 60 mg per kg of body weight. No serious side effects have been reported, demonstrating the safety of A1AT as a therapeutic agent.

[0047] As an example of a growth hormone receptor antagonist that is not limited to the present invention, a long-lasting hGHRA fusion protein is provided by fusing an alpha-1 antitrypsin variant to a growth hormone variant. The alpha-1 antitrypsin variant may comprise at least one substitution among amino acids at positions 1 to 25 in the sequence of alpha-1 antitrypsin, and the growth hormone variant may comprise any one or more substitutions selected from H18D, H21N, Q46K, F54P, R64K, G120K, R167N, K168A, D171S, K172R, E174S, F176Y, and I179T in the amino acid sequence of growth hormone.

[0048] More specifically, as an example, the alpha-1 antitrypsin variant may have the amino acid sequence of SEQ ID NO: 11 disclosed in Table 2 below. In this specification, SEQ ID NO: 11 is used interchangeably with “NexP.”

[0049] sequence number 11EDPQGDAANKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWER PFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASSLHLPKLSITG TYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAINMSIPPEVKFNKPFVFLMIDQNTKSPLFMGKVVNPTQK

[0050]

[0051] To increase plasma half-life, a growth hormone receptor antagonist fusion protein in which an alpha-1 antitrypsin variant is fused to a growth hormone variant as a carrier not only has a higher binding affinity to the growth hormone receptor compared to a pegylated growth hormone variant, but can also more potently antagonize the effect of growth hormone.

[0052] Although it is preferable to use an alpha-1 antitrypsin variant as a persistent carrier, it is also predicted that natural alpha-1 antitrypsin exhibits an effect similar to that of the alpha-1 antitrypsin variant, and furthermore, some fragments of alpha-1 antitrypsin exhibit the same effect. Therefore, natural alpha-1 antitrypsin, alpha-1 antitrypsin variants, fragments of natural alpha-1 antitrypsin, and fragments of alpha-1 antitrypsin variants can also be used as persistent carriers.

[0053] Another example of a persistent carrier is immunoglobulin Fc and fragments thereof. Immunoglobulin Fc exists as a homodimer or heterodimer. While heterodimers of immunoglobulin Fc are expected to be more advantageous for acting as antagonists against growth hormone receptor dimers, the scope of the present invention is not limited thereto.

[0054] The heterodimer of the immunoglobulin Fc comprises a variant CH3 domain containing amino acid mutations that promote heterodimer formation. These mutations include mutations at positions 366, 394, 405, and 407 of the amino acid sequence, and may further include mutations at positions 390 and 400. Examples of heterodimers that may include, but are not limited to, a Knop-in-Hole structure, as described in U.S. Patent No. 7,695,936.

[0055] Specific examples of such heterodimers of immunoglobulin Fc include the amino acid sequences disclosed in Table 3 below.

[0056] 서열번호 12TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK서열번호 13TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENDYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK서열번호 14TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENDYKTTPPVLDDDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK서열번호 15TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENDYKTTPPVLDKDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK서열번호16TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENDYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO. 17TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0057]

[0058] In a growth hormone receptor antagonist fusion protein in which a persistent carrier is fused to a growth hormone mutant, the persistent carrier may be fused to at least one of the N-terminus and the C-terminus of the growth hormone mutant.

[0059] When the persistent carrier is a heterodimer of immunoglobulin Fc, the growth hormone receptor antagonist can bind to the N-terminus and C-terminus of both chains or one chain of the immunoglobulin Fc heterodimer, respectively. For example, the growth hormone receptor antagonist can include, but is not limited to, a dimer in which the N-terminus of SEQ ID NO: 12 and SEQ ID NO: 13 are bound to and expressed simultaneously, a dimer in which the N-terminus of SEQ ID NO: 12 is bound only to and expressed simultaneously with SEQ ID NO: 13, a dimer in which the N-terminus of SEQ ID NO: 13 is bound only to and expressed simultaneously with SEQ ID NO: 12, a dimer in which the C-terminus of SEQ ID NO: 12 and SEQ ID NO: 13 are bound only to and expressed simultaneously with SEQ ID NO: 13, and a dimer in which the C-terminus of SEQ ID NO: 13 is bound only to and expressed simultaneously with SEQ ID NO: 12.

[0060] In particular, in certain growth hormone mutants, growth hormone receptor antagonist fusion proteins fused to the N-terminus of the mutant have significantly higher binding affinity to the growth hormone receptor or can potently antagonize the effects of growth hormone compared to those fused to the C-terminus.

[0061] In the present invention, the persistent carrier may be directly fused with the growth hormone receptor antagonist, or may be fused via a linker.

[0062] The linker is used for covalent bonding of the long-acting carrier and the growth hormone variant, and can be used without limitation as long as it does not affect the activity. Specifically, it can be a non-peptide linker of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), lipid polymers, chitins, hyaluronic acid, and combinations thereof, and can be a peptide linker in which two or more amino acids are connected, but is not limited thereto. A non-limiting example is GGGGS, and it can be a linker with variously controlled lengths (2X, 3X, 4X, etc.).

[0063]

[0064] The candidate materials exemplified in the present invention are as shown in Table 4, but are not limited thereto.

[0065] 후보 물질서열번호아미노산 서열후보 물질 1서열번호 18EDPQGDAANKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAINMSIPPEVKFNKPFVFLMIDQNTKSPLFMGKVVNPTQKFPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRIVQCRSVEGSCGF후보 물질 2서열번호19FPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRIVQCRSVEGSCGFGGGGSTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK서열번호 13[표 3]에 기재후보 물질 3서열번호 20TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSFPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRIVQCRSVEGSCGF서열번호 13[표 3]에 기재후보 물질 4서열번호21TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRIVQCRSVEGSCGF서열번호 14[표 3]에 기재후보 물질 5서열번호 22TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENDYKTTPPVLDKDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRIVQCRSVEGSCGF서열번호 15[표 3]에 기재후보 물질 6서열번호23TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENDYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRIVQCRSVEGSCGF서열번호 12[표 3]에 기재후보 물질 7서열번호 24TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRIVQCRSVEGSCGF

[0066]

[0067] The PH20 variant or fragment thereof of the present invention comprises a substitution of amino acid residues L354I and / or N356E in the amino acid sequence of wild-type PH20, preferably mature wild-type PH20, and additionally preferably comprises a substitution of amino acid residues at one or more positions selected from T341 to N363, particularly at one or more positions selected from the group consisting of T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361 and N363, but is not limited thereto.

[0068] The substitution of amino acid residues at one or more positions selected from the group consisting of T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361 and N363 is more preferably, but not limited to, one or more selected from the group consisting of T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T and N363G.

[0069] Preferably, the novel PH20 variant or fragment thereof according to the present invention is characterized by comprising substitutions of amino acid residues M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and may further be characterized by comprising substitutions of one or more amino acid residues selected from the group consisting of T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N and N363G, but is not limited thereto.

[0070] More preferably, the PH20 variant or fragment thereof according to the present invention may be any one selected from the group consisting of, but is not limited thereto.

[0071] (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0072] (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0073] (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G;

[0074] (d) T341G, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0075] (e) T341A, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0076] (f) T341C, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0077] (g) T341D, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0078] (h) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; and

[0079] (i) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T.

[0080] In the present invention, an expression in which a one-letter amino acid residue name and a number are written together, such as “S347,” refers to an amino acid residue at each position in the amino acid sequence according to SEQ ID NO: 222. For example, “S347” means that the amino acid residue at position 347 in the amino acid sequence of SEQ ID NO: 222 is serine. In addition, “S347T” means that the 347th serine in SEQ ID NO: 222 is substituted with threonine.

[0081] The PH20 variant or fragment thereof according to the present invention is interpreted to mean also a variant or fragment thereof in which an amino acid residue is conservatively substituted at a specific amino acid residue position.

[0082] As used herein, “conservative substitution” means a modification of a PH20 variant that includes replacing one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the PH20 variant.

[0083] A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues with similar side chains are well-defined and known in the art. These classes 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 non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0084] It is expected that the PH20 variants of the present invention may still retain activity even if they have conservative amino acid substitutions.

[0085] In addition, the PH20 variant or fragment thereof according to the present invention is interpreted to mean also including PH20 variants or fragments thereof that have substantially the same function and / or effect as the PH20 variant or fragment thereof according to the present invention and have an amino acid sequence homology of 80% or 85% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more.

[0086] The PH20 variants or fragments thereof according to the present invention exhibit increased expression in animal cells compared to mature wild-type PH20, increased protein folding, and thus increased thermal stability. Furthermore, despite the increased thermal stability, their enzymatic activity is increased or similar to that of mature wild-type PH20.

[0087] Meanwhile, it is known that enzyme activity decreases when the C-terminal portion of mature wild-type PH20 is truncated, but the PH20 mutants according to the present invention maintained enzyme activity that was increased or similar to that of mature wild-type PH20 despite truncating the C-terminus due to increased protein folding and increased thermal stability. In addition, enzyme activity was maintained even when up to 5 amino acids of the N-terminus were truncated, showing that the P41 residue at the N-terminus plays an important role in protein expression and enzyme activity.

[0088] Accordingly, the PH20 variant or fragment thereof according to the present invention is characterized by, but is not limited to, substitution of some amino acid residues in the alpha helix 8 region (S347 to C381) and / or the connecting region between alpha helices 7 and 8 (A333 to R346) of wild-type PH20, and additional deletion of some amino acid residues in the C-terminus and / or N-terminus.

[0089] In one aspect, the PH20 variant or fragment thereof according to the present invention comprises a truncation before an amino acid residue selected from the group consisting of M1 to P42 at the N-terminus of the amino acid sequence of SEQ ID NO: 222, preferably before an amino acid residue of L36, N37, F38, R39, A40, P41 or P42, so that some amino acid residues at the N-terminus are deleted, and / or after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus, preferably after an amino acid residue selected from the group consisting of V455 to S490, most preferably V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, It may be characterized by deletion of some amino acid residues at the C-terminus due to truncation at amino acid residues P486, T488 or S490.

[0090] The expression that cleavage occurred in front of an amino acid residue selected from the group consisting of M1 to P42 of the N-terminus means that the amino acid residue immediately preceding the amino acid residue selected from M1 to P42 of the N-terminus was cleaved and deleted.

[0091] For example, the expression that cleavage occurred before amino acid residues L36, N37, F38, R39, A40, P41 or P42 means that in the sequence of SEQ ID NO: 222, the amino acid residues M1 to T35 immediately preceding L36, M1 to L36 immediately preceding N37, M1 to N37 immediately preceding F38, M1 to F38 immediately preceding R39, M1 to R39 immediately preceding A40, M1 to A40 immediately preceding P41, and M1 to P41 immediately preceding P42 were cleaved and removed.

[0092] Additionally, the expression that cleavage occurred after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus means that cleavage occurred and deletion occurred starting from the amino acid residue immediately following the amino acid residue selected from V455 to L509 at the C-terminus.

[0093] For example, the expression that cleavage occurred at an amino acid residue of V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 of the C-terminus means that cleavage occurred at an amino acid residue of V455, C458, D461, C464, I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 in the sequence of SEQ ID NO: 222, respectively. This means that the next amino acid residue was cut and removed.

[0094] Preferably, the PH20 variant or fragment thereof according to the present invention may be selected from the group consisting of amino acid sequences of SEQ ID NO: 25 to SEQ ID NO: 70, but is not limited thereto.

[0095] Most preferably, the PH20 variant or fragment thereof according to the present invention is characterized by having an amino acid sequence of SEQ ID NO: 64. The PH20 variant having the amino acid sequence of SEQ ID NO: 64 is characterized by having 15 amino acid residues substituted as T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and being cleaved before the F38 residue at the N-terminus and cleaved after the F468 residue at the C-terminus.

[0096] The sequences of amino acids substituted or truncated in the PH20 mutants produced by the applicant are as described in Table 5. In the prior invention, the applicant produced PH20 mutants having higher stability than wild-type PH20 by replacing a specific alpha helix forming the secondary structure of PH20 with an alpha helix of another human hyaluronidase, and these mutants are characterized in that they are mutants having constant enzymatic activity regardless of the C-terminal truncation position, since the interaction of the substituted alpha helix domain with other parts forming the secondary structure of PH20 is different from that of wild-type PH20. In a specific embodiment of the applicant's prior invention, the novel PH20 variant or fragment thereof according to the present invention, which has increased enzyme activity and thermal stability compared to mature wild-type PH20, comprises a substitution of one or more amino acid residues selected from the group consisting of T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E and I361T in the amino acid sequence of wild-type PH20, and is characterized in that some amino acids located in the alpha helix 8 region (S347 to C381) and / or the junction region of alpha helix 7 and alpha helix 8 (A333 to R346) are substituted with other amino acids.

[0097] Specifically, amino acid substitutions at the junctions of alpha helix 8 and alpha helix 7 and alpha helix 8 include substitutions of some amino acids in the T341~N363, T341~I361, L342~I361, S343~I361, I344~I361, M345~I361, or M345~N363 sections.

[0098] To investigate the effect of C-terminal truncation in PH20 mutants with substitutions at the junction of alpha helix 8 and alpha helix 7 and alpha helix 8, three PH20 mutants, HM6, HM10, and HM21, were selected as templates.

[0099] HM6 is a mutant in which the amino acids in the M345 to N363 region are substituted with the amino acid sequence of Hyal1 (M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T are substituted in SEQ ID NO: 222), and is a mutant with the fewest substitutions in the alpha helix 8 region and the junction region of alpha helix 7 and alpha helix 8 among the PH20 mutants according to the present invention that do not contain additional C-terminal truncation (i.e., in the form in which the C-terminal amino acid residue is S490, like the mature wild-type PH20).

[0100] HM10 is a mutant in which the amino acids in the L342~I361 section are substituted with the amino acid sequence of Hyal1 (L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T are substituted in SEQ ID NO: 222). It also does not include additional C-terminal truncation and is the mutant with the highest thermal stability among the PH20 mutants according to the present invention while having an enzymatic activity similar to that of the mature wild-type PH20.

[0101] HM21 is a mutant in which the amino acids in the T341~I361 section are substituted with the amino acid sequence of Hyal1 (T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T are substituted in SEQ ID NO: 222). It also does not contain additional C-terminal truncation, and is a mutant with an enzyme activity approximately twice as high as that of the wild-type PH20 at pH 7.0.

[0102] The HM6-based PH20 variant produced in the applicant's prior invention has the N-terminus truncated at L36 and the C-terminus truncated at I465, F468, or P471 as shown in Table 5.

[0103] As shown in the examples of mutants containing amino acid substitutions in the alpha helix 8 region and the L342 to I361 region of the alpha helix 7 and alpha helix 8 junction region using HM10 as a template, and having the N-terminus truncated before residue F38 and the C-terminus truncated at residue I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486 or T488, the PH20 mutants according to the present invention exhibited enzymatic activity similar to that of mature wild-type PH20, regardless of the truncation position at the C-terminus.

[0104] Two HM21-based PH20 mutants (HP34: 38-470, HP46: 38-468) had in common that the N-terminus was truncated before the F38 residue and the C-terminus was truncated after the F468 or K470 residue. They contained amino acid substitutions in helix 8 and the T341-I361 region at the junction of helices 7 and 8, using HM21 as a template, which has approximately twice the enzymatic activity of mature wild-type PH20. The mutants, which were truncated before the F38 residue at the N-terminus and truncated after the F468 or K470 residue at the C-terminus, retained the high enzymatic activity of HM21 regardless of the C-terminal truncation position.

[0105] In a study by Frost et al., when the C-terminus of PH20 was truncated before 477 and thus shorter, the enzyme activity was reduced to 10% compared to a mutant in which the C-terminus was truncated after 477 (Frost, 2007). However, in the prior invention of the present applicant, when the amino acid at the junction with alpha helix 8 of PH20 was substituted, the enzyme activity was maintained regardless of the C-terminal truncation position due to increased protein stability. This result is significant in that it solved the problem of decreased enzyme activity due to C-terminal truncation of wild-type PH20.

[0106] In addition, in the prior invention of the present applicant, the influence of amino acids in the N-terminal region of PH20, which were not known in the existing research, was studied, and some amino acid residues in the alpha helix 8 region and the alpha helix 7 and alpha helix 8 connection region (M345 to I361) of the wild-type PH20 were substituted with amino acid residues in the corresponding alpha helix 8 region and the alpha helix 7 and alpha helix 8 connection region of Hyal1. In order to investigate the effect of N-terminal truncation amino acids in the HM6 mutant (substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T in SEQ ID NO: 222, and not including additional C-terminal truncation), amino acid residues from L36 to V47 in the amino acid sequence of SEQ ID NO: 222 were replaced with FRGPLLPNR using HM6 as a template. Mutants were constructed in which amino acid residues L36 to A52 were substituted with FRGPLLPNRPFTTV. In addition, mutants HM40, HM13, HM41, HM24, HM42, and HM25 in which the N-terminus was truncated before residues N37, F38, R39, A40, P41, or P42 of the amino acid sequence of SEQ ID NO: 222 were constructed using HM6 as a template.

[0107] As a result, when the N-terminus of HM6 was cleaved before residues N37, F38, R39, A40, or P41, the enzyme activity was not significantly affected. However, when the N-terminus was cleaved before residue P42, the enzyme activity was significantly reduced, indicating that the N-terminal portion of PH20 after P41 is important for protein expression and enzyme activity. In addition, when the amino acids in the N-terminal L36~V47 or L36~A52 section of HM6 were substituted with the amino acids of Hyal1, it was not expressed in ExpiCHO cells, indicating that the N-terminal portion is important for protein expression.

[0108] NameSequenceNumberSubstitutionSequenceHM12512 amino acids are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLGPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM2267 amino acids are substituted with Y365F, I367L, L371S, A372G, K374L, M375L, and V379A from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPFILNVTSGALLCSQALCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM32719 amino acids are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, N363G, Y365F, I367L, L371S, A372G, K374L, M375L, and V379A from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM42817 amino acids are substituted with G340V, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWVSWENTRTKESCQAIKEYMDTTLGPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM62911 amino acid residues are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM73016 amino acids are substituted with G340V, T341S L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWVSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM83112 amino acids are substituted with I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSNTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM93213 amino acids are substituted with S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHMl03314 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHMl13413 amino acid residues are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, Y365F, and I367L from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPFILNVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM123515 amino acid residues are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, Y365F, I367L, L371S, and A372G from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPFILNVTSGAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM133611 amino acid residues are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM143711 amino acids are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed after the carboxyl group of I465 from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIHM153811 amino acids are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed after the carboxyl group of F468 from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM163911 amino acids are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed after the carboxyl group of P471 from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPHM1740Amino acids L36 to V47 are substituted with FRGPLLPNR(SEQ ID NO:52), and 11 amino acids are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T from SEQ ID NO: 222.FRGPLLPNRPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM1841Amino acids L36 to A52 are substituted with FRGPLLPNRPFTTV (SEQ ID NO:53), and 11 amino acids are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T from SEQ ID NO: 222.FRGPLLPNRPFTTVWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM194214 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue K470 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKHM204314 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue F468 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM214415 amino acid residues are substituted with T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM244511 amino acid residues are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue A40 at the N-terminus from SEQ ID NO: 222.APPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM254611 amino acids are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue P42 at the N-terminus from SEQ ID NO: 222.PVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM294714 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue L36 at the N-terminus and after residue A467 at the C-terminus from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAHM304814 amino acid resides are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue L36 at the N-terminal and after residue C464 at the C-terminus from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCHM314914 amino acid resi from SEQ ID NO: 222dues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue L36 at the N-terminus and after residue D461 at the C-terminus from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADHM325014 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue L36 at the N-terminus and after residue C458 at the C-terminus from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCHM335114 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue L36 at the N-terminus and after residue V455 at the C-terminus from SEQ ID NO: 222.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVHP345215 amino acid residues are substituted with T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue K470 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKHM355314 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue P472 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPHM365414 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue M473 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMHM375514 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue E474 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMEHM385614 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue T475 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETHM395714 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue E476 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEHM405811 amino acid residues are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue N37 at the N-terminus from SEQ ID NO: 222.NFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM415911 amino acid residues are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue R39 at the N-terminus from SEQ ID NO: 222.RAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM426011 amino acid residues are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue P41 at the N-terminus from SEQ ID NO: 222.PPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSITRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM436114 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue I465 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIHM446214 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue D466 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDHM456314 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue A467 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAHP466415 amino acid residues are substituted with T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue F468 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM476514 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue P478 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPHM486614 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminal and after residue I480 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIHM496714 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue Y482 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYHM506814 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue A484 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNAHM516914 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue P486 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPHM527014 amino acid residues are substituted with L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T, and cleavage is performed before residue F38 at the N-terminus and after residue T488 at the C-terminus from SEQ ID NO: 222.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEW RPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSI YLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMC SQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPST.

[0109]

[0110] In addition, as another example of the PH20 variant of the present invention, there may be mentioned a PH20 variant or fragment thereof comprising a substitution, deletion and / or insertion of one or more amino acid residues in the hyaluronidase variant having the amino acid sequence of SEQ ID NO: 64, and optionally a deletion of a portion of the amino acid residues at the N-terminus and / or C-terminus. The PH20 variants or fragments thereof usable in the present invention, exemplified in Table 6, showed increased protein expression levels when expressed in ExpiCHO cells compared to mature wild-type PH20, and the protein aggregation temperature increased by about 4 to 11.5 °C, thereby having high thermal stability and being able to be produced efficiently.

[0111] In addition, the PH20 variants or fragments thereof exemplified in Table 6 that can be used in the present invention have the effect of improving protein refolding and renaturing faster than the mature wild-type PH20 in the substrate-gel assay, which is one of the experiments measuring the activity of hyaluronidase, and have the effect of maintaining the original enzyme activity regardless of the truncation position of the C-terminus.

[0112] Furthermore, the PH20 variants or fragments thereof according to the examples in Table 6 that can be used in the present invention have low immunogenicity, and thus can be repeatedly administered to the human body.

[0113] NameSEQ ID NOSubstitutionSequenceHM6371One amino acid residue R346M is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTMTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM6472One amino acid residue T347Q is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRQKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM6573One amino acid residue K348Q is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTQESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM6674One amino acid residue S350Q is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKEQCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM6775One amino acid residue K355Q is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIQEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM6976One amino acid residue M358V is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20 and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYVDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM7077One amino acid residue L362A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTANPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM7178One amino acid residue E343V is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWVNTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM7279One amino acid residue N344F is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWEFTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM7380One amino acid residue D359K is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMKTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM7481One amino acid residue T360Y is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDYTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM7582One amino acid residue T361M is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTMLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM7683One amino acid residue Q352E is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCEAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM7784One amino acid residue N363M is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLMPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM7885One amino acid residue T84N is substituted from SEQ ID NO: 64, residue cleavage occurs before F38 amino acid at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINANGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM7986One amino acid residue N166K is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKKRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM8287One amino acid residue I354E is substituted from SEQ ID NO: 64, residue cleavage occurs before F38 amino acid at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAEKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM8388One amino acid residue I354Q is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAQKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM8489One amino acid residue I354S is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQASKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM8590One amino acid residue I354V is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAVKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM8691One amino acid residue I354A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAAKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM8892One amino acid residue I354N is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQANKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM8993One amino acid residue I354T is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQATKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM9094One amino acid residue E356M is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKMYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM9195One amino acid residue E356F is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKFYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM9296One amino acid residue E356I is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKIYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM9397One amino acid residue E356L is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKLYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM9498One amino acid residue E356Q is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKQYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM9599One amino acid residue E356V is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKVYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM961003 amino acid residues N166K, E343V and T361M are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKKRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWVNTRTKESCQAIKEYMDTMLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM97101One amino acid residue G340Q is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWQSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM98102One amino acid residue S341H is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGHWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM99103One amino acid residue W342I is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSIENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM100104One amino acid residue E343Y is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWYNTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM101105One amino acid residue T345E is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENERTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM102106One amino acid residue R346F is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTFTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM103107One amino acid residue T347E is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTREKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM104108One amino acid residue E349L is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKLSCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM105109One amino acid residue S350I is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKEICQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM106110One amino acid residue Q352G is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCGAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM107111One amino acid residue I354R is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQARKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM110112One amino acid residue M358R is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYRDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM111113One amino acid residue D359V is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMVTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM112114One amino acid residue T360R is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDRTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM114115One amino acid residue T345K is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENKRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM115116One amino acid residue R346L is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTLTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM116117One amino acid residue T347V is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRVKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM117118One amino acid residue E349W is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKWSCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM118119One amino acid residue I354W is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAWKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM121120One amino acid residue D359Y is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMYTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM125121One amino acid residue T347W is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRWKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM126122One amino acid residue Y357W is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEWMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM130123One amino acid residue W342D is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSDENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM131124One amino acid residue E343Q is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWQNTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM132125One amino acid residue T347H is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRHKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM133126One amino acid residue K348F is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTFESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM134127One amino acid residue S350D is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKEDCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM135128One amino acid residue Q352Y is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCYAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM136129One amino acid residue A353E is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQEIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM138130One amino acid residue M358Y is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYYDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM139131One amino acid residue D359Q is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMQTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM140132One amino acid residue T360L is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDLTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM141133One amino acid residue T361E is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTELNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM142134One amino acid residue N363E is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLEPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM143135One amino acid residue W342H is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSHENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM144136One amino acid residue K348D is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTDESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM145137One amino acid residue T361H is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTHLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM146138No additional substitution occurs, cleavage occurs before R39 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.RAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM147139No additional substitution occurs, cleavage occurs before A40 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.APPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM149140No additional substitution occurs, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after D456 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDHM1501412 amino acid residues S350Q and T360R are substituted from SEQ ID NO: 64, cleavage occurs before R39 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.RAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKEQCQAIKEYMDRTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM152142One amino acid residue D65A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFAEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM153143One amino acid residue E66A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDAPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM154144One amino acid residue P67A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEALDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM155145One amino acid residue L68A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPADMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM156146One amino acid residue Q311A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDAVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM157147One amino acid residue V312A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQALKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM158148One amino acid residue L313A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVAKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM159149One amino acid residue K314A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLAFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM160150One amino acid residue N266A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLATQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM161151One amino acid residue T267A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNAQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM162152One amino acid residue Q268A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTAQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM163153One amino acid residue Q269A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQASPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM164154One amino acid residue P271A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSAVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM165155One amino acid residue V272A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPAAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM166156One amino acid residue I102A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYADSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM167157One amino acid residue D103A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIASITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM168158One amino acid residue S104A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDAITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM169159One amino acid residue I105A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSATGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM170160One amino acid residue T132A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDIAFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM171161One amino acid residue F133A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITAYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM172162One amino acid residue Y134A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFAMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM173163One amino acid residue V241A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNAEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM174164One amino acid residue E242A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVAIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM175165One amino acid residue I243A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEAKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM176166One amino acid residue K244A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIARNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM177167One amino acid residue L179A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQASLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM178168One amino acid residue S180A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLALTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM179169One amino acid residue L181A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSATEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM180170One amino acid residue T182A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLAEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM181171One amino acid residue T185A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEAAEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM182172One amino acid residue E186A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATAKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM183173One amino acid residue K187A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEAAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM184174One amino acid residue K290A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSAIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM185175One amino acid residue I291A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKAPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM186176One amino acid residue P292A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIADAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM190177One amino acid residue L441A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTASCKEKADVKDTDAVDVCIADGVCIDAFHM191178One amino acid residue S442A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLACKEKADVKDTDAVDVCIADGVCIDAFHM192179One amino acid residue D451A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKATDAVDVCIADGVCIDAFHM193180One amino acid residue T452A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDADAVDVCIADGVCIDAFHM194181One amino acid residue D453A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTAAVDVCIADGVCIDAFHM195182One amino acid residue D461A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIAAGVCIDAFHM196183One amino acid residue G462A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADAVCIDAFHM197184One amino acid residue V463A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGACIDAFHM198185One amino acid residue N82A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRIAATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM199186One amino acid residue N166A is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKARSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM203187One amino acid residue S104N is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDNITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM204188One amino acid residue I105Q is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSQTGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM205189One amino acid residue Q268D is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTDQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM208190One amino acid residue Q268I is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTIQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM210191One amino acid residue I291G is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKGPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM211192One amino acid residue P292D is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIDDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM212193One amino acid residue T452D is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDDDAVDVCIADGVCIDAFHM213194One amino acid residue T452H is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDHDAVDVCIADGVCIDAFHM214195One amino acid residue T452K is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDKDAVDVCIADGVCIDAFHM216196One amino acid residue T452G is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDGDAVDVCIADGVCIDAFHM217197One amino acid residue T452P is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDPDAVDVCIADGVCIDAFHM218198One amino acid residue T452M is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDMDAVDVCIADGVCIDAFHM219199One amino acid residue T452F is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDFDAVDVCIADGVCIDAFHM220200One amino acid residue D461R is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIARGVCIDAFHM231201One amino acid residue V463Y is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGYCIDAFHM232202One amino acid residue S180T is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLTLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM233203One amino acid residue D451S is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKSTDAVDVCIADGVCIDAFHM234204One amino acid residue L313P is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVPKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM235205One amino acid residue L313M is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVMKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM243206One amino acid residue L179S is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQSSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM245207One amino acid residue L179I is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQISLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM246208One amino acid residue L179F is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQFSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM2542093 amino acid residues N344F, K348Q and K355Q are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWEFTRTQESCQAIQEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM2612107 amino acid residues T132S, L181A, E186D, Q268N, I291L, V312A, and T452D are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDISFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSATEATDKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTNQSPVAATLYVRNRVREAIRVSKLPDAKSPLPVFAYTRIVFTDQALKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDDDAVDVCIADGVCIDAFHM262211No additional substitution occurs, cleavage occurs before N37 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.NFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM263212No additional substitution occurs, cleavage occurs before L36 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM2662139 amino acid residues R39K, I105A, T132S, L181M, E186D, I291L, Q268A, V312A and T452D are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FKAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSATGVTVNGGIPQKISLQDHLDKAKKDISFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSMTEATDKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTAQSPVAATLYVRNRVREAIRVSKLPDAKSPLPVFAYTRIVFTDQALKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDDDAVDVCIADGVCIDAFHM2682147 amino acid residues T132A, L181A, E186A, Q268A, I291L, V312A, and T452D are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDIAFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSATEATAKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTAQSPVAATLYVRNRVREAIRVSKLPDAKSPLPVFAYTRIVFTDQALKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDDDAVDVCIADGVCIDAFHM2712152 amino acid residues N344I and K348M are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWEITRTMESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM27521610 amino acid residues S341D, W342L, E343S, N344I, T345S, R346S, K348M, K355D, D359E and T361I are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGDLSISSTMESCQAIDEYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM2762179 amino acid residues S341D, W342L, E343S, N344I, T345S, K348M, K355D, D359E, and T361I are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGDLSISRTMESCQAIDEYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFHM2792188 amino acid residues T132S, L181A, E186D, Q268N, I291L, V312A, T452D and K348M are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDISFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSATEATDKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTNQSPVAATLYVRNRVREAIRVSKLPDAKSPLPVFAYTRIVFTDQALKFLSQDELVYTFGETVALGASGIVIWGSWENTRTMESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDDDAVDVCIADGVCIDAFHM2802199 amino acid residues T132S, L181A, E186D, Q268N, I291L, V312A, T452D, N344I, and K348M are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDISFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSATEATDKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTNQSPVAATLYVRNRVREAIRVSKLPDAKSPLPVFAYTRIVFTDQALKFLSQDELVYTFGETVALGASGIVIWGSWEITRTMESCQAIKEYMDTTLNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDDDAVDVCIADGVCIDAFHM28722017 amino acid residues T132A, L181A, E186A, Q268A, I291L, V312A, S341D, W342L, E343S, N344I, T345S, R346S, K348M, K355D, D359E, T361I, and T452D are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDIAFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSATEATAKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTAQSPVAATLYVRNRVREAIRVSKLPDAKSPLPVFAYTRIVFTDQALKFLSQDELVYTFGETVALGASGIVIWGDLSISSTMESCQAIDEYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDDDAVDVCIADGVCIDAFHM28822116 amino acid residues T132A, L181A, E186A, Q268A, I291L, V312A, S341D, W342L, E343S, N344I, T345S, K348M, K355D, D359E, T361I and T452D are substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGIPQKISLQDHLDKAKKDIAFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSATEATAKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTAQSPVAATLYVRNRVREAIRVSKLPDAKSPLPVFAYTRIVFTDQALKFLSQDELVYTFGETVALGASGIVIWGDLSISRTMESCQAIDEYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDDADVVCIADGVCIDAF.

[0114]

[0115] MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL

[0116]

[0117] The amino acid sequence in Table 7 above is the amino acid sequence of wild-type human PH20 consisting of 509 amino acids (SEQ ID NO: 222).

[0118]

[0119] As another aspect of the present invention for solving the above problem, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by human growth hormone, comprising a growth hormone receptor antagonist. The terms used herein are as described above.

[0120] The term "human growth hormone-induced disease" as used herein refers to a disease caused by causes such as the pituitary gland not being able to normally regulate the secretion of growth hormone and secreting excessively. Examples may include acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, lung inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).

[0121] Additionally, diseases induced by human growth hormone include diseases that occur due to increased secretion of IGF (insulin-like growth factor)-1 caused by excessive growth hormone action.

[0122]

[0123] As another aspect of the present invention for solving the above problem, the present invention provides a method for producing a growth hormone receptor antagonist, comprising the step of culturing a cell containing a polynucleotide encoding a growth hormone variant in which one or more amino acids in the amino acid sequence of a native human growth hormone having the amino acid sequence represented by SEQ ID NO: 10 are substituted with other amino acids. The terms used herein are as described above.

[0124] The above cells may be cells into which an expression vector of a growth hormone variant has been transferred, and non-limiting examples thereof include CHO (Chinese hamster ovary)-K1.

[0125]

[0126] As another aspect of the present invention for solving the above problem, the present invention provides a method for preventing or treating a disease caused by human growth hormone, comprising administering to a subject a pharmaceutical composition comprising the growth hormone receptor antagonist described above. The terms used herein are as described above.

[0127] The term "administration" used herein refers to introducing the pharmaceutical composition of the present invention into a patient by any suitable method. The administration route of the composition of the present invention may be administered through various oral or parenteral routes as long as it can reach the target tissue, and preferably refers to subcutaneous administration. The formulation according to the present invention can be prepared in various dosage forms depending on the intended administration method.

[0128] In the present invention, administration may be performed prophylactically or therapeutically. The frequency of administration of the formulation of the present invention is not particularly limited, but may be administered once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days, or may be administered in divided doses several times a day.

[0129] The subject of administration of the formulation according to the present invention may refer to any animal, including humans. The animal may include, but is not limited to, mammals such as cows, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar symptoms, as well as humans.

[0130] In particular, the administration of a pharmaceutical composition containing a growth hormone receptor antagonist, the subject of the present invention, is carried out periodically over a long period of time. Therefore, for the convenience of the patient, it is necessary to administer it by subcutaneous injection in a sustained-release formulation that can limit the number of administrations. While the use of the above-mentioned carrier allows the formulation to function as a sustained-release formulation, a large amount of the pharmaceutical composition must be administered at once to provide a dosage suitable for the sustained-release formulation. To achieve this, methods include using a pharmaceutical composition containing a high concentration of the antagonist or using hyaluronidase to contain a high content of the antagonist. The present invention proposes this through specific experimental examples.

[0131]

[0132] As another aspect of the present invention for solving the above problem, the present invention provides a pharmaceutical composition comprising the growth hormone receptor antagonist for the prevention or treatment of diseases caused by human growth hormone. The terms used herein are as described above.

[0133]

[0134] A fusion protein comprising a growth hormone receptor inhibitor and a long-acting carrier according to the present invention has strong binding affinity to the growth hormone receptor and can exhibit a sustained antagonistic effect.

[0135] In addition, a pharmaceutical composition for subcutaneous injection comprising a growth hormone receptor inhibitor according to the present invention, a carrier, and hyaluronidase has strong binding affinity to the growth hormone receptor and can exhibit a sustained antagonistic effect.

[0136] In addition, the pharmaceutical composition for subcutaneous injection comprising a growth hormone receptor inhibitor, a carrier, and hyaluronidase according to the present invention can increase the content of the drug absorbed into the human body without a rapid increase in the concentration of the growth hormone receptor inhibitor by increasing the content of the subcutaneous injection drug.

[0137]

[0138] Figure 1 is a drawing comparing the human growth hormone receptor binding properties of Pegvisomant and growth hormone receptor antagonist - carrier candidate substance 3, where Figure 1A shows the result of confirming binding to the water-soluble portion of the human growth hormone receptor, and Figure 1B shows the result of confirming the biological activity of inhibiting growth using HEK293F cells in which the human growth hormone receptor is overexpressed.

[0139] Figure 2 shows the results of measuring serum IGF-1 levels after subcutaneous injection of Pegvisomant and growth hormone receptor antagonist - carrier candidate substance 3 into NZW rabbits for each cycle.

[0140] Figure 3 shows the results of measuring the IGF-1 level in serum after a single subcutaneous injection of growth hormone receptor antagonist - carrier candidate substance 3 in combination with hyaluronidase “ALT-B4” (SEQ ID No: 99 of WO 2020-022791 A1) at different doses in NZW rabbits.

[0141] Figure 4 shows the results of pharmacokinetic and pharmacodynamic analyses for the results of Figure 3.

[0142] Figure 5 is a photograph showing the swelling phenomenon observed at the injection site during subcutaneous injection of Figure 3.

[0143]

[0144] The present invention

[0145] a) a fusion protein of a growth hormone receptor antagonist and a long-acting carrier in which one or more amino acids in the amino acid sequence of growth hormone are replaced with other amino acids; and

[0146] b) A pharmaceutical composition comprising hyaluronidase, preferably a pharmaceutical composition for subcutaneous injection.

[0147] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the growth hormone receptor antagonist comprises a substitution of lysine at the 46th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10, a substitution of lysine or arginine at the 120th amino acid, or a combination of these substitutions.

[0148] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the growth hormone receptor antagonist further comprises a substitution at any one or more positions selected from the group consisting of the 18th amino acid, the 21st amino acid, the 54th amino acid, the 64th amino acid, the 167th amino acid, the 168th amino acid, the 171st amino acid, the 172nd amino acid, the 174th amino acid, the 176th amino acid, and the 179th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10.

[0149] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the growth hormone receptor antagonist further comprises a substitution of the 174th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10 with serine, a substitution of the 21st amino acid with asparagine, or a combination of these substitutions.

[0150] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the growth hormone receptor antagonist comprises at least one substitution selected from the group consisting of a substitution of the 18th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10 with aspartic acid, a substitution of the 21st amino acid with asparagine, a substitution of the 46th amino acid with lysine, a substitution of the 54th amino acid with proline, a substitution of the 64th amino acid with lysine, a substitution of the 120th amino acid with lysine or arginine, a substitution of the 167th amino acid with asparagine, a substitution of the 168th amino acid with alanine, a substitution of the 171st amino acid with serine, a substitution of the 172nd amino acid with arginine, a substitution of the 174th amino acid with serine, a substitution of the 176th amino acid with tyrosine, and a substitution of the 179th amino acid with threonine.

[0151] In addition, the present invention also relates to a pharmaceutical composition for subcutaneous injection, wherein the fusion protein has an amino acid sequence of one of SEQ ID NOs: 18 to 24, but it is apparent to a person skilled in the art that the present invention is not limited to this sequence.

[0152] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the persistent carrier is fused to at least one of the N-terminus or C-terminus of the growth hormone receptor antagonist.

[0153] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the persistent carrier is selected from the group consisting of, but not limited to, polyethylene glycol, fatty acid, albumin or a fragment thereof, an albumin-binding substance, alpha-1 antitrypsin or a variant thereof, immunoglobulin Fc or a fragment thereof, a repeating unit polymer of a specific amino acid sequence, an antibody or a fragment thereof, an FcRn binding substance, an in vivo connective tissue or a derivative thereof, a nucleotide, fibronectin, transferrin, a saccharide, and a high molecular weight polymer, although preferred examples thereof are not limited thereto.

[0154] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, comprising immunoglobulin Fc or a fragment thereof as the persistent carrier.

[0155] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the immunoglobulin Fc or a fragment thereof is a heterodimer.

[0156] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the immunoglobulin Fc comprises an amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 17.

[0157] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the growth hormone receptor antagonist is fused with the long-acting carrier directly or through a linker.

[0158] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the linker is a peptidic linker or a non-peptidic linker.

[0159] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the non-peptide linker is not limited to the examples below, but is preferably polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), a lipid polymer, chitin, hyaluronic acid, or a combination thereof.

[0160] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the peptide linker is a pharmaceutical composition in which two or more amino acids are linked.

[0161] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, for the prevention or treatment of a disease selected from the group consisting of diseases induced by human growth hormone, such as acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).

[0162] In addition, the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the hyaluronidase is a human or mammalian hyaluronidase or a variant thereof, having hyaluronidase activity. The hyaluronidase refers to the naturally occurring PH20, Hyal1, Hyal2, Hyal3, Hyal4 of mammals such as humans, sheep, cows, and apes, and their mature forms. These mutants refer to those in which one or more amino acids in the amino acid sequence of the above-described natural and / or mature hyaluronidase are substituted, deleted or added, and specifically, mutants in which amino acids 1-35, 1-36, 1-37, 1-38, 1-39, 1-40 or 1-41 of human PH20 are truncated and / or all amino acids after 480 or after any one of amino acids 468 to 490 are truncated, and the hyaluronidase may be one selected from SEQ ID NOs: 25 to 222, and in addition, WO 2004-078140 A2, WO 2006-091871 A1, WO 2010-077297 A1, WO 2013-102144 A2, WO 2015-003167 A1, WO Examples include, but are not limited to, various hyaluronidase variants disclosed in WO 2020-22791 A1, WO 2021-150079 A1, etc.

[0163]

[0164] In addition, the present invention relates to a fusion protein comprising a growth hormone receptor antagonist in which one or more amino acids in the amino acid sequence of growth hormone are substituted with other amino acids and an immunoglobulin Fc or a fragment thereof as a long-acting carrier.

[0165] In addition, the present invention relates to a fusion protein wherein the immunoglobulin Fc or a fragment thereof is a heterodimer.

[0166] In addition, the present invention relates to a fusion protein, wherein the immunoglobulin Fc comprises an amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 17.

[0167] In addition, the present invention relates to a fusion protein, wherein the growth hormone receptor antagonist comprises a substitution of lysine at the 46th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10, a substitution of lysine or arginine at the 120th amino acid, or a combination of these substitutions.

[0168] In addition, the present invention relates to a fusion protein, wherein the growth hormone receptor antagonist further comprises a substitution at any one or more positions selected from the group consisting of the 18th amino acid, the 21st amino acid, the 54th amino acid, the 64th amino acid, the 167th amino acid, the 168th amino acid, the 171st amino acid, the 172nd amino acid, the 174th amino acid, the 176th amino acid, and the 179th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10.

[0169] In addition, the present invention relates to a fusion protein, wherein the growth hormone receptor antagonist further comprises a substitution of the 174th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10 with serine, a substitution of the 21st amino acid with asparagine, or a combination of these substitutions.

[0170] In addition, the present invention relates to a fusion protein, wherein the growth hormone receptor antagonist comprises at least one substitution selected from the group consisting of a substitution of the 18th amino acid from the N-terminus with aspartic acid, a substitution of the 21st amino acid with asparagine, a substitution of the 46th amino acid with lysine, a substitution of the 54th amino acid with proline, a substitution of the 64th amino acid with lysine, a substitution of the 120th amino acid with lysine or arginine, a substitution of the 167th amino acid with asparagine, a substitution of the 168th amino acid with alanine, a substitution of the 171st amino acid with serine, a substitution of the 172nd amino acid with arginine, a substitution of the 174th amino acid with serine, a substitution of the 176th amino acid with tyrosine, and a substitution of the 179th amino acid with threonine in the human growth hormone amino acid sequence represented by SEQ ID NO: 10.

[0171] In addition, the present invention relates to a fusion protein in which the carrier is fused to at least one of the N-terminus or C-terminus of the growth hormone receptor antagonist.

[0172] Furthermore, the present invention relates to a fusion protein in which the growth hormone receptor antagonist is fused directly or through a linker to the long-acting carrier.

[0173] In addition, the present invention relates to a fusion protein, wherein the linker is a peptidic linker or a non-peptidic linker.

[0174] In addition, the present invention relates to a fusion protein, wherein the non-peptide linker is polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), a lipid polymer, chitin, hyaluronic acid, or a combination thereof.

[0175] In addition, the present invention relates to a fusion protein in which the peptidic linker is composed of two or more amino acids linked together.

[0176] In addition, the present invention relates to a fusion protein, characterized in that the fusion protein is for the prevention or treatment of a disease caused by human growth hormone.

[0177] In addition, the present invention relates to a fusion protein having an amino acid sequence of one of SEQ ID NOs: 18 to 24, but it is obvious to a person skilled in the art to which the present invention pertains that the present invention is not limited to this sequence.

[0178] In addition, the present invention relates to a fusion protein characterized in that the disease induced by the human growth hormone is selected from the group consisting of acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).

[0179] In addition, the present invention relates to a pharmaceutical composition for preventing or treating a disease caused by human growth hormone, comprising the fusion protein.

[0180] Furthermore, the present invention relates to a nucleic acid encoding the fusion protein, a vector for expressing the fusion protein comprising the nucleic acid, a host cell comprising the vector, and a pharmaceutical composition for preventing or treating a disease caused by human growth hormone comprising the nucleic acid, vector, or host cell. Furthermore, the present invention relates to a method for producing a fusion protein using the nucleic acid, vector, or host cell, and a method for producing a pharmaceutical composition for preventing or treating a disease caused by human growth hormone using the nucleic acid, vector, or host cell.

[0181]

[0182] Below, the composition of the present invention is described in more detail with specific examples. However, the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention to the scope described in the examples.

[0183]

[0184] Example 1. hGHA efficacy and receptor binding analysis

[0185] Example 1-1. hGH efficacy analysis method

[0186] For this assay, the hGH receptor gene was introduced into the chromosomes of HEK293F cells containing the luciferase gene, which can be induced by hGH receptor signal. The resulting cell line was designated hGHR / Luc / HEK293F. Serial dilutions of hGH and hGHRA-NexP were added to each of the 96-well white plates containing hGHR / Luc / HEK293F. The plates were incubated for 24 h in a 5% CO2 incubator at 37°C. After incubation, 100 μL of luciferase assay reagent (Steady-Glo ® Luciferase assay system (Promega) was added to each well, and the plate was sealed and protected from light. After 5 min at room temperature, the luminescence of each well was analyzed using a multimode microplate reader (SpectraMax M5, Molecular Devices).

[0187]

[0188] Example 1-2. hGH receptor binding assay

[0189] The binding affinity of hGRA-NexP for the hGH receptor was evaluated in a binding assay using a recombinant hGH receptor Fc chimera. Microplates were coated with the hGH receptor chimera overnight at 25°C. After washing with TPBS buffer (PBS buffer containing 0.05% Tween-20), samples (candidates and Pegvisomant) were loaded into each well. The samples were washed three times and then conjugated with anti-hGH polyclonal antibody-biotin. After an additional washing step, 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well to perform the TMB reaction. The absorbance signal from the reaction was recorded at 450-650 nm.

[0190]

[0191] Example 1-3. hGH competitive inhibitory activity assay

[0192] The inhibitory activity of candidate substances on downstream signal transduction was analyzed as follows by hGH competition assay. Serial dilutions of growth hormone receptor antagonists of Examples 1 to 11 and Pegvisomant were added to each well of a 96-well plate containing hGHR / Luc / HEK293F. The plates were incubated for 24 hours in a 5% CO2 incubator at 37°C. After incubation, 100 μL of luciferase assay reagent (Steady-Glo ® Luciferase assay system (Promega) was added to each well, and the plate was sealed and protected from light. After 5 min at room temperature, the luminescence of each well was analyzed using a multimode microplate reader (SpectraMax M5, Molecular Devices).

[0193]

[0194] Example 2. Analysis of biological activity of growth hormone receptor antagonist-carrier according to the type and location of the carrier.

[0195] As a type of carrier, polyethylene glycol, NexP (SEQ ID NO: 11) presented in the present invention, and immunoglobulin Fc were used to analyze growth hormone receptor antagonist-carriers according to the type of carrier and the binding position of the fusion protein and carrier, using the method of Example 1. The results were compared with those of Pegvisomant (Table 8).

[0196] Candidate 1 is a fusion protein in which a growth hormone receptor antagonist having a sequence number of 7 is linked to the C-terminus of NexP (SEQ ID NO: 11), Candidate 2 is a fusion protein in which a linker (GGGGS) and SEQ ID NO: 12 are sequentially linked to the C-terminus of the growth hormone receptor antagonist having a sequence number of 7, and expressed together with SEQ ID NO: 13, and Candidate 3 is a fusion protein in which a linker (GGGGS) and a growth hormone receptor antagonist having a sequence number of 7 are sequentially linked to the C-terminus of SEQ ID NO: 12, and expressed together with SEQ ID NO: 13. As shown in the results in Table 8 below, Candidate 3 appears to have excellent binding affinity and efficacy.

[0197] MaterialCarrierBinding affinity*(receptor binding ELISA)Inhibition potency*(cell-based assay)PegvisomantPEG11Candidate 1NexP*516Candidate 2Fc149Candidate 3Fc1425

[0198] *NexP: SEQ ID NO: 11.

[0199]

[0200] Example 3. Pharmacokinetic analysis / pharmacodynamic analysis using rabbits

[0201] Example 3-1. Comparative study of administration cycles with Pegvisomant

[0202] Pharmacokinetic analysis was performed by measuring blood IGF-1 levels in NZW rabbits after administering Pegvisomant and candidate substance 3 at different dosing cycles. The experimental results are presented in Fig. 2. Pegvisomant and candidate substance 3 were adjusted to have the same molar amount, i.e., the number of molecules of the corresponding hGHA. Pegvisomant was administered six times every day of the dosing cycle, and candidate substance 3 was administered three times every other day. Serum IGF-1 levels were measured 24, 48, 72, 96, 120, 144, 168, 240, and 336 hours after administration. These results showed that candidate substance 3 reduced blood IGF-1 by more than 50% compared to Pegvisomant, despite the dosing cycle being twice as long.

[0203]

[0204] Example 3-2. Comparative test of candidate substance 3 by dose and comparative test of formulation containing hyaluronidase

[0205] Pharmacokinetic analysis was performed by measuring the serum IGF-1 level after administering candidate substance 3 once at each dose to NZW rabbits and collecting blood samples. The experimental results are presented in Fig. 3. Candidate substance 3 was administered once at 10 mg / kg, 15 mg / kg, and 20 mg / kg, respectively, and each sample was administered with hyaluronidase ALT-B4 and compared. Serum IGF-1 levels were measured 24 and 12 hours before administration, immediately before administration, and 12, 24, 48, 72, 96, 120, 144, 168, 192, and 240 hours after administration. The obtained pharmacokinetic and pharmacodynamic results are presented in Fig. 4, and photographs of some test animals immediately after administration are presented in Fig. 5.

[0206] The results in Figure 4 show that the formulation containing hyaluronidase ALT-B4 showed increased pharmacodynamic indices compared to the same dosage form without hyaluronidase, while the pharmacodynamic indices remained at a constant level. This suggests that the formulation containing hyaluronidase can be a powerful alternative when increased pharmacodynamic indices are required. In addition, the formulation containing hyaluronidase showed reduced swelling, which appeared to be helpful for drug absorption (Figure 5).

[0207]

[0208] Example 4. Analysis of hGH competitive inhibitory activity of additional growth hormone receptor antagonist-carrier variants.

[0209] The function of the growth hormone receptor fusion protein according to the binding position of the fusion protein and the carrier was analyzed using immunoglobulin Fc as a carrier by the method of Example 1, using Candidate 3 of Example 2 as a control (Table 9).

[0210] Candidate 4 is a fusion protein produced by expressing sequence number 21 together with sequence number 14, Candidate 5 is a fusion protein produced by expressing sequence number 22 together with sequence number 15, Candidate 6 is a fusion protein produced by expressing sequence number 23 together with sequence number 12, and Candidate 7 is a homodimer fusion protein produced by expressing sequence number 24.

[0211] MaterialCarrierInhibition potency*relative to Candidate 3(cell-based assay)Candidate 4Fc0.894Candidate 5Fc1.001Candidate 6Fc1.016Candidate 7-0.286

[0212]

[0213] The present invention can be used for the prevention or treatment of diseases selected from the group consisting of diseases induced by human growth hormone, such as acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).

[0214]

[0215] Electronic file attached

Claims

1. A fusion protein comprising a growth hormone receptor antagonist in which one or more amino acids in the amino acid sequence of growth hormone are replaced with other amino acids and an immunoglobulin Fc or a fragment thereof as a long-acting carrier.

2. In paragraph 1, The above immunoglobulin Fc or fragment thereof is a heterodimer, a fusion protein.

3. In paragraph 1, The above immunoglobulin Fc is a fusion protein comprising any one of the amino acid sequences of SEQ ID NO: 12 to SEQ ID NO:

17.

4. In paragraph 1, The above growth hormone receptor antagonist is a fusion protein comprising a substitution of lysine at the 46th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10, a substitution of lysine or arginine at the 120th amino acid, or a combination of these substitutions.

5. In paragraph 1, A fusion protein wherein the growth hormone receptor antagonist further comprises a substitution at any one or more positions selected from the group consisting of the 18th amino acid, the 21st amino acid, the 54th amino acid, the 64th amino acid, the 167th amino acid, the 168th amino acid, the 171st amino acid, the 172nd amino acid, the 174th amino acid, the 176th amino acid, and the 179th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO:

10.

6. In paragraph 1, The above growth hormone receptor antagonist is a fusion protein further comprising a substitution of the 174th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10 with serine, a substitution of the 21st amino acid with asparagine, or a combination of these substitutions.

7. In paragraph 1, The growth hormone receptor antagonist is a fusion protein comprising at least one substitution selected from the group consisting of a substitution of the 18th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10 with aspartic acid, a substitution of the 21st amino acid with asparagine, a substitution of the 46th amino acid with lysine, a substitution of the 54th amino acid with proline, a substitution of the 64th amino acid with lysine, a substitution of the 120th amino acid with lysine or arginine, a substitution of the 167th amino acid with asparagine, a substitution of the 168th amino acid with alanine, a substitution of the 171st amino acid with serine, a substitution of the 172nd amino acid with arginine, a substitution of the 174th amino acid with serine, a substitution of the 176th amino acid with tyrosine, and a substitution of the 179th amino acid with threonine.

8. In paragraph 1, A fusion protein wherein the persistent carrier is fused to at least one of the N-terminus or C-terminus of the growth hormone receptor antagonist.

9. In paragraph 1, A fusion protein wherein the growth hormone receptor antagonist is fused directly or through a linker to the persistent carrier.

10. In paragraph 9, A fusion protein, wherein the linker is a peptidic linker or a non-peptidic linker.

11. In paragraph 10, A fusion protein wherein the non-peptide linker is polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), a lipid polymer, chitin, hyaluronic acid, or a combination thereof.

12. In paragraph 10, A fusion protein wherein the above peptide linker is a fusion protein in which two or more amino acids are linked.

13. In paragraph 1, The above fusion protein is a fusion protein having an amino acid sequence of any one of SEQ ID NO: 18 to SEQ ID NO:

24.

14. The fusion protein according to paragraph 1 is a fusion protein for the prevention or treatment of diseases caused by human growth hormone.

15. A fusion protein according to claim 14, wherein the disease is selected from the group consisting of acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).

16. A fusion protein according to any one of claims 1 to 15; and A pharmaceutical composition for preventing or treating a disease caused by human growth hormone, comprising hyaluronidase.

17. In claim 16, the pharmaceutical composition is for subcutaneous injection.

18. A pharmaceutical composition according to claim 16, wherein the hyaluronidase is a human or mammalian PH20 hyaluronidase or a variant thereof having hyaluronidase activity.

19. A pharmaceutical composition according to claim 16, wherein the hyaluronidase is one selected from among sequence numbers 25 to 222.

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